bcil group 3
TRANSCRIPT
BIOPHARMA PRODUCTION AND BIOPROCESS ENGINEERING
A Training Report Submitted in fulfillment of
BCIL INDUSTRIAL TRAINING PROGRAM (BITP)
BATCH VIII (GROUP- 3)
2014-2015
Executed At
Bangalore Biotech Labs Pvt. Ltd.Bangalore
Written by:
Padmalochan Rout Joan D’Souza
Shubhra Sharma Lakshminadh Meduri
Mukhtar Khan Vivek K
Dananjiul Pandi Sanjay Naik
Manoj Kumar J Savinay
Bangalore Biotech Labs Pvt. Ltd
Bangalore
CERTIFICATE
This is to certify that the training report entitled “BIOPHARMAPRODUCTION AND BIOPROCESS ENGINEERING” being summated in the fulfillment of the requirements of the BCIL Industrial Training Program (BITP 2014-2015) is the bonafide work of BCIL-8 Group-3 under my supervision and guidance.
Place: BangaloreDate:
ACKNOWLEDGEMENT
This work of BCIL batch VIII could not have been accomplished without
the valuable support and cooperation of several people. Hereby we take
immense pleasure in expressing our utmost gratitude towards them.
We are extremely grateful to the staff and the heads of Biozeen for giving
us the opportunity to pursue Biotech Industrial Training Program (BITP
2014-15) in Biozeen and for rendering the best of facilities and support.
We would like to express our gratitude to Mr Harish Prabhakaran,
Training Lead of BCIL training, Biozeen whose guidance was instrumental
during the entire course of this training.
.
We would like to express our utmost gratitude to Dr. H. Nellaiah, Mrs Priya Josson, Mrs. Julie Mathew, Mrs. Elizabeth Eldo, Mr Anish RV our teachers for their round the clock assistance, guidance and for having
taught us every concept pertaining to Microbial Fermentation, Downstream
Processing, Animal Cell Culture and Cleaning, Sterilisation and Filtration.
Their exemplary knowledge and support were instrumental for completion
of our training.
Page | 3
INDEX
CONTENTS PAGE NO.
ABSTRACTMODULE – I: MICROBIAL FERMENTATION
1. INTRODUCTION 2. SHAKE FLASK OPTIMIZATION STUDIES
2.1: OPTIMIZATION OF MEDIA AND PH2.2: OPTIMIZATION OF WORKING VOLUME2.3: OPIMIZATION OF INOCULUM PERCENTAGE
3. MICROBIAL FERMENTATION IN 5L LAB SCALE FERMENTOR 4. MICROBIAL FERMENTATION IN 40L PILOT SCALE FERMENTOR
MODULE – II: CLEANING, STERILIZATION AND FILTRATION1. INTRODUCTION TO CLEANING AND STERILIZATION IN
BIOPHARMA INDUSTRY2. CLEANING
2.1: INTRODUTION TO CLEANING2.2: CLEANING OUT OF PLACE2.3: CLEANING IN PLACE
3. STERILIZATION 3.1: INTRODUCTION 3.2: HEAT STERILIZATION 3.2.1: STERILIZATION OUT OF PLACE (a) STEAM STERILIZATION (AUTOCLAVE) (b) DRY HEAT STERILIZATION 3.2.2: STERILIZATION IN PLACE (a) EMPTY VESSEL STERILIZATION IN PLACE: ESIP (b) FULL VESSEL STERILIZATION IN PLACE: FSIP 3.3: COLD STERILIZATION (FILTRATION) 3.3.1 INTRODUCTION 3.3.2 INTEGRITY TESTING OF F
3.4: FAMILIARISATION OF EQUIPEMENTS5. STANDARD OPERATING PROCEDURES AND VALVE MATRICES 5.1 CIP (CLEANING IN PLACE) 5.1.1 CIP OF MEDIA PREPARATION VESSEL
Page | 4
5.1.2 CIP OF DUAL BIOREACTOR 5.1.3 CIP OF FILTRATION SYSTEM 5.2 SIP (STERILIZATION IN PLACE) 5.2.1 ESIP OF MEDIA PREPARATION VESSEL 5.2.2 SIP OF FILTRATION SYSTEM 5.2.3 ESIP OF DUAL BIOREACTOR 5.2.4 FSIP OF DUAL BIOREACTOR 5.2.5 ESIP OF HARVEST VESSEL 5.3 SOP (STERILIZATION OUT OF PLACE) 5.3.1 AUTOCLAVE (A) STANDARD CYCLE (B) LIQUID CYCLE (C) POROUS CYCLE 5.3.2 DRY HEAT STERILIZER 5.4 INTEGRITY TESTING OF FILTERS 5.4.1 DIFFUSION TEST 5.4.2 BUBBLE POINT TEST 5.4.3 WATER INTRUSION TEST
MODULE - III: ANIMAL CELL CULTURE
1. INTRODUCTION2. ASEPTIC HANDLING TECHNIQUES3. SETTLE PLATE TEST4. MEDIA PREPARATION AND FILTRATION5. SUBCULTURING OF BHK-21 CELLS6. GROWTH CURVE STUDIES7. OPTIMIZATION OF SERUM PERCENTAGE8. CRYOPRESERVATION9. REVIVAL OF CELLS10.MTT ASSAY11.KARYOTYPING12.SCALE UP TECHNOLOGY FOR MONOLAYER CELL LINE USING
MICROCARRIERS13.Giemsa Staining
Page | 5
MODULE – IV: DOWMSTREAM PROCESSING TECHNOLOGY
1. INTRODUCTION2. PRIMARY PURIFICATION -TANGENTIAL FLOW FILTRATION
2.1: MICROFILTRATION2.2: ULTRAFILTRATION2.3: DIAFILTRATION
3. SECONDARY PURIFICATION 3.1: ION EXCHANGE CHROMATOGRAPHY
3.1.1:Anion Exchange Chromatography 3.1.2: CATION EXHANGE CHROMATOGRAPHY 3.2: Hydrophobic Intraction Chromatograpy 3.3: AFFINITY CHROMATOGRAPHY 3.4: GEL FILTRATION CHROMATOGRAPHY
3.5: SDS POLYACRYLAMIDE GEL ELECTROPHORESIS
Page | 6
(Module-1)
MICROBIAL
FERMENTATION
Page | 7
1. INTRODUCTION
Fermentation is the process in which a substance breaks down into a simpler substance, such as
break down of complex carbohydrate like starch into simple products. In these microorganisms,
yeast and bacteria usually play a role in the fermentation process, creating beer, wine, bread etc.
The basic principle in fermentation process involves a group of chemical reactions induced by
living organisms that split complex organic compounds into relatively simple substances.
Microbial cells obtain energy through glycolysis, splitting a sugar molecule, and removing
electrons in the process. The electrons are then passed to an organic molecule such as pyruvic
acid. This results in the formation of products that are excreted from the cell.
At s small scale level, fermentation is often demonstrated in a shake flask with volumes
from a few milliliters to liters, where all the conditions required for better growth of the culture
was optimized. At the production and manufacturing level, the fermentation was usually carried
out in larger vessels called fermenter. The capacity of the fermenter may vary from several liters
to several thousand liters and they are usually equipped with aeration devices as well as stirrers,
and pH and temperature controls in order to get a maximum product yield from the fermentation
batch. The research level scientists develop media and culture growth conditions in a shake
flask and so it needs be scaled up to several times larger than the original shake flask. The scale
up process should provide all the necessary conditions that monitor temperature, pH, and
growth in the fermenter to ensure that conditions are optimum for cell growth and product. Most
industrial microbial processes are aerobic, and are mostly carried out in aqueous medium
containing salts and organic substances, so oxygen plays an important role in the growth of
microorganisms and metabolite production. All the optimized parameters need to be controlled
carefully for the better production of metabolites.
The fermentation industry today is witnessing great change in product spectrum, and the
scale of production. It is now possible to develop far more complex and expensive products
such as therapeutic proteins, antibodies (simple and conjugated), anti-cancer agents, etc. The
quality of these products i.e. the potency, efficacy, stability and immunogenicity has also seen
great improvement due to recent development in the upstream and downstream processing of
the products.
Page | 8
Pichia pastoris
Over the last few decades, geneticists have learned how to manipulate DNA to identify, move
and place genes into a variety of organisms that are quite different from the source organism. A
major use for many of these recombinant organisms is to produce proteins. Since many proteins
are of immense commercial value, numerous studies have focused on finding ways to produce
them efficiently and in a functional form.
Pichia pastoris is a highly successful system for production of a wide variety of
recombinant proteins. Several factors have contributed to its rapid acceptance, the most
important of which include:
A promoter derived from the alcohol oxidase I (AOX1) gene of P. pastoris that is
uniquely suited for the controlled expression of foreign genes
The similarity of techniques needed for the molecular genetic manipulation of P.
pastoris to those of Saccharomyces cerevisiae
The strong preference of P. pastoris for respiratory growth, a key physiological trait that
greatly facilitates its culturing at high-cell densities relative to fermentative yeasts
As yeast, P. pastoris is a single-celled microorganism that is easy to manipulate and culture.
However, it is also a eukaryote and capable of many of the post-translational modifications
performed by higher eukaryotic cells such as proteolytic processing, folding, disulfide bond
formation and glycosylation. Thus, many proteins that end up as inactive inclusion bodies in
bacterial systems are produced as biologically active molecules in P. pastoris. The P. pastoris
system is also generally regarded as being faster, easier, and less expensive to use than
expression systems derived from higher eukaryotes such as insect and mammalian tissue culture
cell systems and usually gives higher expression levels.
The production of a functional protein is intimately related to the cellular machinery of the
organism producing the protein. The yeast Pichia pastoris is a useful system for the expression
of milligram-to-gram quantities of proteins for both basic laboratory research and industrial
manufacture. The fermentation can be readily scaled up to meet greater demands, and
parameters influencing protein productivity and activity, such as pH, aeration and carbon source
feed rate, can be controlled. Compared with mammalian cells, Pichia does not require a
complex growth medium or culture conditions, is genetically relatively easy to manipulate, and
has a eukaryotic protein synthesis pathway.
Page | 9
2. SHAKE FLASK OPTIMIZATION STUDIES
Aim: To optimize the best suitable conditions like type of media, pH of media, working volume
and % of inoculum to be inoculated for shake flask studies.
Materials:
Media used: Tryptone soya broth (TSB) and nutrient broth (NB)
Reagents and chemicals: Purified water, acid, alkali and buffers for calibration of
probes
Equipments: Laminar air flow cabinet, autoclave, orbital shaker
Instruments: Digital balance, pH meter, spectrophotometer
Others: Micro-pipettes and tips, conical flasks, beakers, test tubes, cuvette, cotton plugs
etc.
Cultures used: Pichia pastoris
2.1 Optimization of media type and pH for Pichia pastoris culture:
The type of media and pH were optimized using two different media (Nutrient broth,
Tryptone soya broth) at different pH values (3.0, 4.0, 5.0, 6.0, 7.0 & 8.0) keeping other
parameters like inoculum %, media volume, incubation temperature, agitation speed of shaker
constant.
Procedure:
Tryptone Soya Broth
Composition of Tryptone Soya Broth (TSB) medium:
Tryptone -17.0 gL-1
NaCl -5.0 gL-1
Soya -3.0 gL-1
K2PO4 -2.50gL-1
Dextrose -2.50 gL-1
The TSB medium was prepared by dissolving 18 g in 600 ml of water and divided in 6
flasks containing 100 ml each.
The pH was adjusted to 3.0 in 1st flask, 4.0 in 2nd flask and so on.
Page | 10
Nutrient Broth media
Nutrient Broth (NB) medium consists of (gL-1):
Peptic digest of animal tissue -5.0 gL-1
Beef extract - 1.5gL-1
NaCl -5.0 gL-1
Yeast extract -1.5 gL-1.
The NB medium was prepared by dissolving 7.8 g in 600 ml of water and divided
among 6 flasks containing 100 ml each.
The pH was adjusted to 3.0 in 1st flask, 4.0 in 2nd flask and so on.
Sterilization:
All the flasks were sterilized in the autoclave at 121˚C for 20 min at 1.2 bar pressure
using liquid cycle.
Inoculation:
Overnight grown P. Pastoris culture was used as inoculum. Each flask was inoculated
with 5ml (5%).
Incubation:
The flasks were incubated overnight for 17 hours in the orbital shaker at 30˚C at 180
rpm.
Sampling:
After incubation, 1 ml of sample was taken from each flask and diluted to 10 times with
9 ml of water. Absorbance of the samples was measured at 600nm and the measured absorbance
was multiplied with dilution factor of 10 to get final O.D.
Page | 11
Table: Optimization of media and pH:
Sl.No pH NB medium TSB medium
OD at 600nm
1 3 0.82 3.46
2 4 2.79 6.15
3 5 3.23 6.98
4 6 3.53 7.24
5 7 3.46 7.06
6 8 3.29 6.92
Result:
The absorbance of the sample with TSB medium of pH 6 has shown highest O.D.
compared to sample with nutrient broth. It indicates that the TSB medium of pH 6.0 is optimum for
the growth of the culture.
Page | 12
Optimization of pH for Pichia pastoris in fine tuning (5.5-6.6):
The pH was optimized using TSB medium in wide range, so there is a need for fine tuning
of pH value in very narrow range. So, TSB media of different pH was prepared (5.5 to 6.6) in
separate flasks keeping other parameters like inoculum %, media volume, incubation
temperature, agitation speed, etc. constant.
Procedure:
The TSB medium was prepared by dissolving 36 g in 1200 ml of water
The medium was divided into 12 flasks and pH was adjusted to the above mentioned
values
Sterilization:
All the flasks were sterilized in the autoclave at 121˚C for 20 min at 1.2 bar pressure
using liquid cycle.
Inoculation:
Overnight grown P. Pastoris culture was used as inoculum. Each flask was inoculated
with 5ml (5%).
Incubation:
The flasks were incubated for 17h in the orbital shaker at 30˚C in 180 rpm.
Sampling:
After incubation, 1 ml of sample was taken from each flask and diluted to 10 times with
9 ml water. The absorbance of the samples was measured at 600nm and the absorbance obtained
was multiplied with the dilution factor of 10 to get final O.D
Page | 13
Table: Optimization of pH of medium:
S.No TSB medium pH OD at 600 nm
1 5.5 6.28
2 5.6 7.00
3 5.7 7.07
4 5.8 6.60
5 5.9 7.01
6 6.0 7.34
7 6.1 6.71
8 6.2 6.72
9 6.3 6.71
10 6.4 6.66
11 6.5 6.53
12 6.6 6.90
Result:
The absorbance of the sample from TSB medium at pH 6.0 was found to be higher
compared to other pH values. This, therefore, indicates that the TSB medium at pH 6.0 is
favourable for the culture growth.
2.2 Optimization of working volume of medium in flask:Page | 14
The parameters optimized in the previous experiments such as TSB medium of pH 6.0
was used for optimizing the working volume and inoculum percentage. All the remaining
parameters (incubation temperature and agitation speed) kept constant. For optimization of
working volume, a range of 40 ml to 150ml in 250 ml flask was selected, with an interval of 10
ml difference between the flasks.
Procedure:
Medium preparation:
TSB medium of pH 6.0 was prepared and distributed to 12 flasks according to different
working volumes of (40-150 ml).
Sterilization:
All the flasks were sterilized in the autoclave at 121˚C for 20 mins at 1.2 bar pressure using
liquid cycle.
Inoculation:
Each of the 12 flasks was inoculated with 5% of overnight grown inoculum.
Incubation:
Flasks were incubated for 17h in the orbital shaker at 30˚C at 180 rpm.
Sampling:
After incubation for 17 h, 1 ml of sample was taken from each flask and diluted to 10
times with 9 ml water. Optical density of the samples was measured at 600nm and multiplied
with the dilution factor of 10 to get final O.D.
Table: Optimization of working volume:Page | 15
S.No Vol. of media (ml) /250 ml flask O.D at 600nm
1 40 7.24
2 50 7.14
3 60 7.24
4 70 7.26
5 80 7.19
6 90 6.99
7 100 6.73
8 110 6.92
9 120 6.88
10 130 7.02
11 140 6.44
12 150 6.77
Result:
The absorbance of the sample from TSB medium of working volume 80 ml was found
to be 7.19, which was better at high media volume. So this suggests that TSB medium at
working volume of 80 ml was found to be favourable for the culture growth.
Page | 16
2.3 Optimization of inoculum percentage:
To optimize inoculum %, flasks were inoculated with different % of inoculum (1-10%, 15 &
20%) keeping all other parameters constant.
Medium preparation:
The TSB media of pH 6 was prepared in 12 flasks based on the % of inoculum going to be
added to the flask.
e.g: if % of inoculum is 1 %, then 1% of 80 ml will be 0.8 ml. Therefore, 79.2 ml of media
was poured into the flask to which 0.8 ml of inoculum is going to be added, which makes the
final volume to 80 ml.
Sterilization:
All the flasks were sterilized in the autoclave at 121˚C for 20 mins at 1.2 bar pressure using
liquid cycle.
Inoculation:
Each flask was inoculated according to the inoculum % shown in the table below.
Incubation:
The flasks were incubated for 17 h in the orbital shaker at 30˚C at 180 rpm.
Sampling:
After incubation for 17h, 1 ml of sample was taken from each flask and diluted to 10
times with 9 ml of water. Optical density of the samples was measured at 600nm and multiplied
with the dilution factor of 10 to get final O.D.
Page | 17
Table: Optimization of inoculum %:
S.No Inoculum
%
Inoculum volume
(ml)
Media to be taken
(ml)
OD at 600nm
1 1 0.8 79.2 6.24
2 2 1.6 78.4 6.88
3 3 2.4 77.6 6.93
4 4 3.2 76.8 7.26
5 5 4.0 76 7.18
6 6 4.8 75.2 7.23
7 7 5.6 74.4 7.35
8 8 6.4 73.6 7.38
9 9 7.2 72.8 7.35
10 10 8.0 72.0 7.60
11 15 12.0 68.0 7.64
12 20 16.0 64.0 8.24
Result:
The absorbance of the sample from TSB medium inoculated with 4% of inoculum was
found to be 7.26, which was higher compared to other sample in different low inoculum
percentage. So this suggests that TSB medium inoculated with 4% of inoculum is optimum for
better biomass.
3. MICROBIAL FERMENTATION IN 5L LAB SCALE FERMENTER
Aim:
Page | 18
To prepare the components of 5L fermenter for autoclave and to inoculate the fermenter
and provide the conditions required for the growth of Pichia pastoris in the fermenter.
To determine KLa in the fermenter by dynamic gassing out technique.
Materials required:
Medium: Tryptone soya broth (TSB)
Reagents and chemicals: Purified water, acid and alkali, calibration buffers, antifoam
Equipments: 5L fermenter vessel and control system, Digital balance, pH meter,
spectrophotometer, laminar air flow cabinet, autoclave, orbital shaker
Miscellaneous: Micro-pipettes and tips, conical flasks, beakers, test tubes, cuvette,
cotton plugs, rubber bands, Schott-Duran bottles with 2-hole caps, etc.
Culture: Pichia pastoris
Components and features of 5L fermenter:
Vessel made of Borosilicate Glass
Control Panel, DO probe
0.2 air filters (air-in, exhaust-out, addition bottles)
pH probe
Temperature probe
Transmission lines ( Acid, Alkali & Antifoam)
2 Rushton turbine type impellers on a shaft mounted on a motor
Baffle cage
Condenser
Sparger
Rotameter
Sampling line
Chiller
Peristaltic pumps – 3 for acid, alkali, antifoam dosing
Inoculum transfer bottle + needle inoculation assembly + Inoculum port diaphram
Dosing bottles for above
Silicon tubing for attachments
Page | 19
Procedure:
Media Preparation:
32 g of TSB medium was weighed and dissolved in 3.6 l of water.
The pH of the media was adjusted to optimized pH 6.0.
Inoculum development:
300 ml of TSB media was prepared and pH was adjusted to 6.
These flasks were inoculated with 5% of P. pastoris overnight culture and were
incubated at 30°C at 180 rpm for 17 h.
Reagents preparation:
Page | 20
The following reagents were prepared in 250ml Schott duran bottles:
200 ml of 2N HCl
200 ml of 2N NaOH
200 ml of 1% Antifoam
200 ml of Nutrient shot (25 % glucose solution)
Fermenter setup:
3.6 l of TSB media was charged into the fermenter.
The head plate of the fermenter was closed properly by tightening the opposite screws
simultaneously.
The jacket was filled with water by operating the pump.
The surface and sparger-air line were connected to Y- connector with silicone tubing to
the respective ports on one end and the other end was joined to air supply through an air
filter.
The pH probe was calibrated using standard buffers and inserted into the fermenter.
The acid, base and antifoam lines were connected to addition ports on top plate and the
tubings were clamped properly to avoid loss of water from media.
The exhaust line was connected to the condenser and outlet of condenser was connected
to a bottle with a filter for the exhaust.
The fresh DO electrolyte was added into the DO probe tip cartridge and the probe was
calibrated using KCl before inserting into the fermenter.
The tubing to the sampling line was connected with silicone tubing. The end of the tube
was clamped to prevent backflow of medium during sterilization, and then the end was
covered with cotton dipped in alcohol and aluminium foil.
The motor shaft and DO probes were covered with aluminium foil.
pH probe head was closed with the probe cap.
Inoculation bottle along with needle was sterilized along with the fermenter.
Pressure Leak Test:
Page | 21
The leakage in the vessel or connections was checked by passing a minimal quantity of
surface air into the vessel with all outlets and ports closed, except the exhaust line.
The open end of the exhaust line filter was dipped into a beaker filled with water for
immediate visualizing of the air bubbles. In case of any leakage, the bubbling in the
beaker would commence after some time or will not occur at all (in case of huge
leakage).If there is no leakage, bubbles will rise instantly.
Sterilization of the set-up:
The vessel along with probes, supply lines, sampling line, inoculum bottle with
inoculum needle and addition bottles (Acid, Alkali and Antifoam) was sterilized using
the liquid sterilization cycle at 1.2 bar pressure and 121oC for 20 min.
Post sterilization steps:
After sterilization, the fermenter was brought to the fermentation room
Immediately the surface air was passed at a flow rate of 4 lpm through the surface-air
line of the fermenter to initiate cooling of the vessel and to break the vacuum built-up
due to cooling
The chilled water inlet and outlet supplies were connected to the condenser in the
exhaust line
Glycerol was poured into the thermowell and then temperature sensor was inserted and
connected to the panel
Then DO and pH probe cables were connected to the panel
The chilled water line was connected to the jacket only after the temperature is dropped
to or below 60OC to avoid crack in the joints
Motor was connected to the agitator shaft and the cables were connected to the panel
The temperature of the vessel was maintained at 30°C using the closed loop temperature
control
DO probe calibration:
When the vessel is at the required temperature, air is sparged into the vessel through the
ring sparger at a flow rate of 1 VVM
After 30 mins of continuous sparging, media was completely saturated. Now DO probe
was calibrated taking this saturation level as 100% DO
Page | 22
Inoculation and Fermentation:
The aeration through the sparger line was maintained at a flow rate of 4 lpm
The agitator speed was set at 200 rpm and the temperature was controlled at 30oC
The overnight seed culture (3 seed flasks with 100 ml seed culture each) was pooled into
sterile inoculum transfer bottle under aseptic conditions
The inoculation needle was pierced into the inoculation port septum in presence of flame
and the collar was closed properly and inoculation was carried out using a peristaltic
pump
The fermentation was carried out under the required conditions for the culture to achieve
the maximum growth for following 72 h
All the set conditions in the fermenter were continuously monitored and maintained
pH and foaming of the medium was controlled by manual addition of acid/base and
antifoam using respective peristaltic pumps
When DO drops below 40%, agitation was increased at a slow rate to maintain the DO
above the critical level of 40%. When DO starts dropping even after increasing the
agitation, the aeration rate needs to be increased.
Sampling and Harvesting:
Samples were taken at different time intervals for determining the growth rate of the
organism and all the parameters like DO, pH, aeration and agitation were noted down at
the time of sampling
pH of the sample was checked using pH meter for comparison with the display pH
O.D. of each sample was measured at 600nm and recorded
Based on OD of the sample and DO level in the medium at the time of sampling,
different nutrient shots were given for different time intervals. The nutrient shot may be
25% glucose solution or TSB media enriched with glucose.
Whenever the DO level of the media began to increase, the nutrient shot was given to
study the effect of nutrient shot on DO utilization by the active biomass in the medium
At the end of fermentation batch, the culture was harvested through sampling line by
pressurizing the vessel via surface airline
Table: 5L fermenter batch condition supplied with 25 % glucose solution as nutrient
shot in different time intervalPage | 23
S.No Batch
age
(Hour)
Aeration
(LPM)
Agitation
(RPM)
DO (%) O.D
1 0 4 200 96 0.65
2 1.5 4 200 78.7 0.79
3 3 4 300 70.3 1.16
4 4.5 8 200 62.9 1.88
5 22.5 2 200 66.0 3.50
40 ml of 25 % glucose nutrient shot (23 hr)
6 24 4 250 43.3 4.5
7 26 4 300 41.9 4.06
8 28 4 350 38.5 6.18
9 46.5 4 200 5.4 9.19
10 48.5 7 325 48.1 7.77
40 ml of 25 % glucose nutrient shot (49 hr)
11 50 6 350 56.7 8.4
Table: 5L fermenter batch condition supplied with 25 % glucose solution+ TSB medium
as nutrient shot in different time interval:
S.No Batch age
(Hour)
Aeration
(LPM)
Agitation
(RPM)
DO (%) O.D
1 0 4 200 91.6 0.79
2 1 4 200 72.2 0.96
3 2 4 200 64.3 1.04
4 3 4 300 67.2 1.37
5 4 4 300 58.4 2.01
6 5 4 300 77.8 2.57
7 22 3 200 91.6 3.86
8 23.5 4 200 56.3 3.05
40 ml of 25% glucose shot
9 26 4.5 250 32.2 3.99
10 28 4.5 300 52.8 5.04
Page | 24
11 29 4.5 300 53.7 5.07
12 47 4.5 300 63.2 8.20
13 48 4 300 57.8 7.09
Glucose + TSB medium nutrient shot for 4 hours slowly
14 50.5 4.5 300 39.7 7.12
15 52 4.5 325 36.7 6.82
16 53 4.5 375 46.4 7.23
17 69 4 300 37.8 14.88
18 72 4.5 375 31.1 15.56
Result:
The maximum absorbance of the sample from culture provided with glucose as
nutrient shot was found to be 9.19 but maximum absorbance of the sample from culture
provided with glucose and TSB medium as nutrient shot was found to be 15.56.
Observation:
The above results suggest that, when the growing culture was provided with only
glucose solution as nutrient shot, the maximum absorbance of the sample (9.19) obtained was
low compared to the absorbance of the sample (15.56) after adding nutrient shot containing both
glucose solution and TSB medium. In case of only glucose solution shot, it contains full carbon
source but in case of glucose solution and TSB medium, it is provided with carbon source and
other required energy source for the active growth of the culture. This shows the requirement of
different source of energy for the proper growth of the culture.
KLa determination by dynamic gassing out technique
The dynamic method is one of those based on the measurement of dissolved oxygen
concentration in the medium by absorption or desorption of oxygen. After a step change in the
concentration in the inlet gas, the dynamic change in the dissolved oxygen concentration is
analysed. The dynamic technique of absorption consists of producing the elimination of oxygen in
the liquid phase, for example by cutting off the air supply, until the oxygen concentration is equal to
critical level. Later, the liquid is put in contact again with air, measuring the variation (increase) of
Page | 25
the oxygen concentration with time. The dynamic methods are based on the technique proposed for
measuring the respiratory activity of microorganisms which are actively growing in the bioreactor.
If the gas supply to the bioreactor is turned off, the dissolved oxygen concentration will decrease at
a rate equal to oxygen consumption by the respiration of microorganism. In mechanical agitated
bioreactors, the stirrer is the main gas dispersing tool and stirrer speed and design have both a
pronounced effect on mass transfer. Empirical correlations for the volumetric mass transfer
coefficient depend on several geometrical parameters.
Determination of KLa by gassing out technique:
The KLa of the fermenter was determined by using the gassing out technique, in this
method aeration was stopped and the DO level was allowed to drop till 40%
When DO concentration almost reached the critical oxygen concentration level (40%),
aeration was started again
The increased DO level (CL) was noted for every 10 seconds till it reached a constant
DO level, i.e. saturation oxygen concentration (Cs)
A graph of dCL/dt vs CS-CL was plotted and the slope of the graph was calculated
Using the slope, KLa/hr of the fermenter was calculated
For the determination of KLa of the 5 L fermenter, a different operation condition like
varying agitation rate was used.
Condition:
Aeration: 3.5 LPM
Agitation: 200 rpm
Batch age: 1.45 hr
Time interval: 10 sec
Table: Estimation of KLa:
Page | 26
Time
(Seconds)
Cl dCl/dt Cs-Cl
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
39.2
40.1
40.9
41.7
42.5
43.3
44
44.7
45.3
46
46.5
47.1
47.7
48.2
48.7
49.2
49.7
50.1
50.5
51
51.4
51.7
52.1
52.5
52.8
53.1
53.5
53.7
54.1
54.4
54.6
0.09
0.08
0.08
0.08
0.08
0.07
0.07
0.06
0.07
0.05
0.06
0.06
0.05
0.05
0.05
0.05
0.04
0.04
0.05
0.04
0.03
0.04
0.04
0.03
0.03
0.04
0.02
0.04
0.03
0.02
18.3
17.4
16.6
15.8
15
14.2
13.5
12.8
12.2
11.5
11
10.4
9.8
9.3
8.8
8.3
7.8
7.4
7
6.5
6.1
5.8
5.4
5
4.7
4.4
4
3.8
3.4
3.1
Page | 27
310
320
330
340
350
360
370
380
390
400
410
420
430
440
450
460
470
480
54.8
55
55.3
55.5
55.8
55.9
56.1
56.3
56.5
56.6
56.8
56.9
57
57.1
57.2
57.3
57.4
57.5
0.02
0.02
0.03
0.02
0.03
0.01
0.02
0.02
0.02
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.01
0.01
2.9
2.7
2.5
2.2
2
1.7
1.6
1.4
1.2
1
0.9
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Page | 28
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
4
8
12
16
20
f(x) = 213.241126070991 x − 2.21523459812322
3.5 lpm at 200 rpm
dCl/dt
Cs-C
l
Slope
KLa/
sec
KLa/
min KLa/hr
213.2
0.0046
9
0.28142
6
16.8855
5
The above result depicts the KLa/hr of the 5 L fermenter provided with aeration of 3.5
LPM and agitation of 200 rpm and the KLa/hr was found to be 16.88.
Discussion:
The above two results show that there was an increase in KLa/hr, when the rate of
agitation was increased keeping the aeration at a constant of 3.5 lpm. In the first condition, as
agitation was low, the effective oxygen transfer was low compared to the second condition. In
the first condition, the air bubbles rising up through the sparger are not chopped of properly
because of low agitation. But in the second condition, with the same aeration and higher
agitation at 300 rpm, the rate of oxygen transfer was effectively increased. This shows that
increased agitation tends to chop off the bubbles effectively that tends to increase the KLa/hr
than the first condition.
Conclusion:
The culture of Pichia pastoris was cultured in the 5 L lab scale fermenter. The sterilization
process for the fermenter as well all the other connections was done successfully. Fermentation was
carried out with all the optimized parameters and the culture was sampled at different interval of
time and measured for the absorbance of the sample. The KLa was estimated using different
agitation rate to study the effect of agitation in controlling the DO required for the growth of the
culture. Nutrient dosage was also provided, when DO level of the culture increases and kept
constant to maintain the continuous culture growth for the longer period of time. The effect of
nutrient dosage was also studied by using different nutrient solutions. After the fermentation
process, the culture was harvested and stored for further downstream processing.
Page | 29
4. MICROBIAL FERMENTATION IN 40 L PILOT SCALE FERMENTER
Aim:
To prepare a 40 L pilot scale fermenter for growth of Pichia pastoris batch.
Materials required:
Media: Tryptone Soya Broth (TSB)
Reagents and chemicals: Purified water, acid, alkali, buffers, antifoam
Equipments: 40L fermenter vessel with control system, laminar air flow cabinet,
autoclave, orbital shaker
Instruments: Digital balance, pH meter, spectrophotometer
Miscellaneous: Micro-pipettes and tips, conical flasks, test tubes, cuvettes, cotton plugs,
rubber bands, Schott-Duran bottles of different capacity with 2 port facility etc.
Culture: Pichia pastoris
Procedure:
Preparatory steps:
Inoculum preparation:
As mentioned before, working volume of fermenter is 30L; the final volume should be
30 L including inoculum volume
Based on inoculum percentage to be used for inoculation, inoculum was prepared
2 L of inoculum was prepared one day before inoculation into the fermenter by
inoculating TSB media with Pichia pastoris culture and incubated overnight in shaker
incubator at 180 rpm at 300 C
This grown, pure culture was used as inoculum for pilot scale fermenter
Media preparation:
As mentioned above, the fermentor working volume is 30 L and the media was prepared
considering the total volume after inoculation
2 L Concentrated TSB media of pH 6 was prepared, which was diluted to 28 L later in
the fermenter with the help of CIP station by adding 26 L of water
Page | 30
Probe preparation and calibration:
pH probe:
pH probe was connected to control panel using cable
pH probe was calibrated outside the fermenter using buffers of pH 4 and 7 with the help
of transmitter in the control panel connected to the fermenter
DO probe (pO2 probe)
DO probe was connected to control panel using cable
Before half an hour of calibration, DO probe was kept in aerated water
DO probe was calibrated outside in the water using the transmitter and program
Fermenter preparation:
PHT (Pressure Hold Test):
Before starting any sequence, PHT of vessel should be performed to check any leakages
in the connections.
PHT of 40 L fermenter was performed as follows
All the valves were closed and all the connections were tightened
The vessel was pressurized by passing air through surface air line
The vessel was kept undisturbed for certain time and observed for any leakages i.e drop
in the vessel pressure which can be detected by pressure gauge fitted on the vessel
If there is any pressure drop, places of leakage should be detected and should be tighten
again and perform PHT again. If there is no pressure drop, sequence can be started
In our case, there was no pressure drop so, continued with further steps as follows
Mechanical seal sterilization and cooling:
As mechanical seal come in direct contact with product, it should be sterilized before
starting the fermentation batch.
For sterilization, mechanical seal is connected to thermosyphon.
Page | 31
The two loops of this set up was sterilized for 10 mins each by sending the steam
directly into the thermosyphon pot and condensate was drained by opening the drain
valve below the thermosyphon.
After sterilization was over, it was cooled by circulating the condensate.
Probe attachment and Media charging:
After successful calibration, probes were attached to the fermenter at their sites.
Again leakage test for 10 mins was performed to check any leakages in the connections
of probes.
2 L concentrated TSB media was added directly through one of the extra port on the top
plate of fermenter.
Remaining 28 L water was sent to fermenter from CIP station to make the volume to 30
L.
FSIP of fermenter:
Jacket evacuation:
The water remaining in the jacket from previous run was drained by passing the process
air for 2-3 mins and then closed.
Heating and sterilization:
Total sterilization period was 30 mins which was divided into 3 parts as follows
Heating through jacket:
Steam was passed to the jacket directly and corresponding steam trap was opened at the
same time agitation was started for uniform distribution of heat.
When media started heating, vent needle valve was opened for few seconds to remove
the air on the surface of media as air is the bad conductor of heat.
When media started boiling, foam was produced. To control the foam surface air (15
lpm) was started to break and control the foam.
Heat till the temperature reaches to 1210C.
Page | 32
Steaming through air lines:
When the temperature of media reached to 1210C, heating through jacket was stopped
and steaming through air lines was started by opening the steam valves for air lines and
corresponding filter drain valves and steam traps.
The sterilization through air lines was continued for 12 mins (validated time).
The temperature was maintained at 1210C by throttling the steam entry valve.
After 12 mins of sterilization, filter drains, steam traps and steam entry valves of air
lines were closed.
Steaming through vent and auxiliary lines:
After sterilization through air lines, sterilization through vent and auxiliary lines was
started by opening the corresponding steam valves drains of vent and auxiliary lines and
also filter drain in vent line.
This was continued for 9 mins by throttling the steam valve.
After 9 mins, steam valves and drains were closed.
Steaming through spray ball:
Direct steaming through spray ball was performed by throttling the steam entry valve of
spray ball and corresponding steam traps were opened.
This was continued for 9 mins.
After 9 mins, steam valve and drained were closed.
Till this time total sterilization time of 30 mins was completed.
All drain valves were closed.
Cooling of fermenter:
Pressurization:
Before cooling, vessel should be pressurized through surface air to avoid vacuum
formation and collapsing of fermenter to a certain pressure.
Cooling by open loop:
The fermenter was cooled by open loop using chilled water to room temperature or
temperature required for fermentation process.
Page | 33
At 600C, aeration (15 lpm) and agitation (100 rpm) was started.
When temperature reaches to required temperature, started maintaining the temperature
by closed loop.
During this time back pressure of 0.4 bar was maintained by throttling the vent needle
valve.
After FSIP, SIP of all additive lines was done separately by opening the corresponding
steam valves and drains for 10 mins and vessel was kept under positive pressure.
Parameter settings:
Temperature control:
Control the temperature of broth to 300C using closed loop.
pH control:
Give set point of 6.0 to control the pH.
Foam control:
Antifoam bottle should be connected to the fermenter through peristaltic pump before
starting the agitation. Whenever foam formation is there antifoam should be added as
per the requirement to reduce the formation of foam.
Agitation:
Set the agitation of 200 rpm for proper mixing of nutrients and distribution of heat.
Aeration:
Set the aeration (through sparger) at 15 lpm for microbial growth.
DO calibration:
Before calibration, media was aerated for half an hour for oxygen saturation.
Once the media was saturated, started the DO control panel transmitter.
Entered the DO calibration mode by pressing cal.
Calibrate the DO probe using 100% saturation point (one point method).
Page | 34
DO probe considered that point of saturation as 100% for calibration and further
operation.
Inoculation:
The grown inoculum was transferred to a sterile bottle in laminar air flow.
Once the required conditions are achieved in the fermenter, fermentation media was
inoculated with this culture (7.5% of working volume) using peristaltic pump and
respective valves.
After the complete transfer of inoculum all valves were closed.
Fermentation:
Fermentation was started once the conditions were reached and media was inoculated
with culture.
Proper required conditions were maintained with the help of control loops. i.e,
temperature was maintained with the help of closed temperature loop.
pH was maintained with the help of pH control system (ON/OFF system),
Foam was controlled manually.
Proper agitation (100 rpm) and aeration (15lpm) were maintained.
Fermentation of 48 hrs was continued without disturbing the conditions.
Sampling:
Before taking sample, sterilization of sampling line was done independently by opening
the corresponding steam valve and drain valve for 10 mins.
Sampling was done before inoculation which is called pre inoculation sample and after
inoculation which is called post inoculation sample.
Sample was taken for every hour of fermentation at the same time corresponding pH,
temperature, DO, agitation were recorded which are displayed on the screen.
pH was checked in the lab for comparison and recorded.
Sample was diluted to 10 dilutions and O.D of sample was checked at 600nm and
recorded.
Page | 35
Table:
Sample
time
(Hr)
pH Temperature
(0 C)
DO
(%)
Aeration
(lpm)
Agitation
(rpm)
O.D
At
600 nmDisplay Actual
Pre-
inoculation
sample
Post-
inoculation
sample
0 hr
6.7 6.43 29.1 86.4 15 100 0.71
1 hr 6.6 6.43 31.5 86.1 15 100 0.4
18 ½ hr 7.1 6.91 22.9 60.0 15 100 1.82
19 ½ hr 6.68 6.57 26.6 51.3 15 150 1.76
20 ½ hr 6.28 6.19 37.0 68.0 15 150 2.65
21 ½ hr 6.16 6.06 40 77.3 15 150 3.31
Harvesting:
After 48 hrs of fermentation, the broth was harvested into harvest vessel and sent to
downstream processing.
If not required or contaminated the broth should be heated to 1000 C to kill the cells and
drained.
CIP of fermenter:
After harvesting, the vessel should be cleaned for next process.
Fermenter and CIP skid were connected.
Fermenter and CIP station were connected.
Page | 36
Pre-rinse:
Pre-rinse was done with purified water of ambient temperature to wash larger particles
on the surface of fermenter. The water should not be hot as it may coagulate the
proteins. These coagulated proteins will attach to the surface and difficult to wash. The
water was sent through spray ball and not recirculated.
Hot alkali rinse:
Hot alkali (about 600C) was prepared in CIP station sent to fermenter through spray ball
and recirculated in different paths for validated time as follows by opening and closing
corresponding valves:
Spray ball, auxiliary line 1, 2, 3, 4, 5, exhaust line, surface line, sampling line, sparger
line, spray ball.
After passing all the paths hot alkali was drained through drain 1 and drain 2 by opening
the corresponding valves and drains.
Hot water rinse:
Hot water rinse was carried out to remove alkali traces. Purified water was heated to
about 600 C in CIP station and sent to fermenter through spray ball and recirculated
through paths mentioned above and drained.
Hot acid rinse:
Hot acid rinse was carried out to neutralise the alkali remaining after the hot water wash.
Hot acid (600C) was prepared in CIP station and sent to fermenter through spray ball and
recirculated for validated time and drained.
Hot water rinse:
This water was carried out to remove acid traces form the surface of fermenter. Water
was recirculated through all paths and drained.
WFI rinse:
Page | 37
Final rinses of fermenter were carried out with WFI water because WFI contains very
less or no ions and have least conductivity. WFI can take ions coming in the way. WFI
was not recirculated.
WFI rinse 1: First WFI rinse was carried out through all auxillary lines and air lines
and drained.
WFI rinse 2: Second rinse was same as that of first rinse and drained.
WFI rinse 3: Third rinse was carried out only through spray ball and drained. The
rinsing was carried on until the conductivity of outlet water becomes equal to the inlet
WFI.
Results and discussions:
Pichia pastoris was cultured using 40 L fermenter.
PHT, mechanical seal sterilization, cooling, FSIP unit operations were carried out
successfully.
Broth was inoculated and fermentation was carried out for 48 hours.
Sampling was done for every hour and O.D was measured at 600nm and recorded.
KLa estimation was done and calculated to be 11.77 at 100 rpm and 16.23 at 150 rpm at
15 lpm aeration.
After fermentation, culture was harvested and stored for further downstream processing.
Page | 38
(Module-2)
CLEANING,
STERILIZATION AND
FILTRATION
Page | 39
1. INTRODUCTION TO CLEANING AND STERILIZATION IN BIOPHARMA
INDUSTRY
Sterilization is a term referring to any process that eliminates (removes) or kills all forms of life,
including transmissible agents (such as fungi, bacteria, viruses, spore forms etc) present on
surface, contaminated in a fluid, in medication, or in a compound such as biological culture
media. Sterilization can be achieved by applying the proper combination of heat, chemical,
irradiation, high pressure and filtration.
The efficacy of any sterilization process is contingent on the following three essentials:
1. Conditions must be present to effectively destroy living organisms. In other words, the
sterilant and sterilizing equipment must be validated and appropriate in design and
operation to achieve the correct combination of temperature and sterilant combination to
be lethal to microorganisms.
2. Equipments to be sterilized must be thoroughly cleaned to reduce bio-burden in order to
ensure the effectiveness of the sterilization process. The higher the bio-burden the
greater the challenge to the sterilization parameters may not be adequate rendering the
sterilization process ineffective.
3. There must be intimate and adequate contact between the sterilant and all surfaces and
crevices of the equipment to be sterilized.
Sterilization Methods and Parameters
Sterilization involves the use of a physical or chemical procedure to destroy all microbial life,
including highly resistant bacterial pores. The major sterilizing agents commonly used in
healthcare facilities today are a)saturated steam, b)hydrogen peroxide gas plasma, c)liquid
chemicals. Dry heat is also used, although less commonly. And a new sterilizing agent, ozone,
has recently become available for use in the US. There are two types of sterilizations
Sterilization in Place(SIP)
ESIP FSIP
Page | 40
Sterilization Out Of Place(SOP)
Autoclave Dry heat sterilization (DHS)
Steam:
Saturated steam under pressure is the oldest and most widely used, economical, effective and
reliable method of sterilization available to health care facilities. The steam sterilizer consists of
a pressurized chamber, increasing the pressure in the chamber elevates and holds the
temperature. The sterilizer chamber and all contents must be free of any air entrapment to
ensure direct contact of the steam to all surfaces to be sterilized. Steam sterilizers are designed
to eliminate all air from the chamber during the conditioning phase in the sterilization cycle.
Steam is vaporized water and serves as the conduit to rapidly permeate packaging delivering
high temperature moist heat to all contents and destroying microorganisms. Steam kills
microorganisms and moisture associated with steam sterilization it may only be used with high
heat and moisture stable medical devices, instruments and compatible materials.
Liquid Chemical Sterilization
Liquid chemical sterilization is utilized for the sterilization of heat sensitive devices that can be
immersed. This method employs the use of a germicidal solution and requires the complete
immersion of items in the solution for a prescribed period of time to kill microorganisms.
Parasitic acid is a liquid chemical sterilant used in conjunction with a self-contained automated
processor designed for this method of sterilization. It is commonly used for flexible endoscopes
and components. Devices that are sterilized by this method are intended for just, in time use and
have no shelf life.
Page | 41
Dry Heat:
Dry heat sterilization should be used to sterilize anhydrous (waterless) items that can withstand
high temperatures. Dry heat sterilizers are not commonly found in healthcare facilities today. If
used, it is generally to sterilize talcum powder for surgical procedures. Dry heat sterilization
may be used to sterilize sharp instruments such as dental instruments, burrs, and reusable
needles that would be damaged by the moisture of steam. Dry heat sterilization is accomplished
by conduction where heat is transferred fro molecule to molecule or from the exterior surface of
an item to its internal parts. The destruction of organisms occurs by oxidation, which is a slow
burning up process of coagulating the cells protein. It is a long sterilization process due to the
length of time it takes for objects to reach required temperatures; unlike steam sterilization there
is no moisture present, which speeds up heat penetration.
Cleaning:
Cleaning is essential for the production of a safe, effective product is the application of an
effective cleaning, decontamination and sanitation (CDS) regime in the manufacturing facility.
Cleaning involves the removal of dirt that is miscellaneous organic and inorganic material
which may accumulate in the process areas of equipment during production.
Effective cleaning procedure is routinely applied to:
1. Surfaces in the immediate manufacturing area which do not come into direct contact
with the product(clean room walls and floors, worktops, ancillary equipments)
2. Surfaces coming into direct contact with the product(manufacturing vessels,
chromatographic colums, product filters etc.)
3. The cleaning of a process scale chromatography systems used in the purification of
biopharmaceuticals can also present challenges. Although such systems are
disassembled periodically, this is not routinely undertaken after each production run.
Cleaning and sterilization is an important part of production facility specially in
biopharmaceutical industry where in one manufactures drug or other therapeutics. Each and
every step of production needs sterilization as only this practice can render the safety of drug
upon use.
Cleaning comes into the picture whenever there is a batch change over or a product change
over. Most of the products produced involves a number of vessels (for eg, fermenters
bioreactors, harvest vessels, mixing tanks etc). If cleaning of these vessels is not proper, then
there are chances of cross contamination in the process. The carryover of the previous batch
Page | 42
may affect the quality of the next product. Therefore a proper cleaning procedure also renders
safe drug production. Various steps of the production of the drug need cleaning and sterilization
both in upstream and downstream. The main concern in the biopharmaceutical and the
pharmaceutical industry of the present world is of cleaning. An effective and efficient cleaning
process is much required and is an important part of the production.
2. CLEANING
2.1 Introduction to cleaning:
The cleaning is one of the essential for the production of a sterile, safe and effective
product regime in the biotech manufacturing facility. The cleaning involves the removal of dirt
including both organic dirt and inorganic dirt that are left in the production or preparation vessel
that are used in the previous batch of production. The cleaning can be carried out by using water
and different acids and alkali solutions. The cleaning procedures are mainly applied to the
surfaces that are come into contact with the product during manufacturing, that include mainly
media preparation vessel, harvest vessel and fermenter.
The cleaning plays an important role whenever there is a production batch change over.
Most of the product manufacturing involves many vessels, so each vessel will carry a lot of dirt
from the previous batch of production. So if the cleaning of the vessel is not proper that may
lead to several problem like cross contamination by previous batch dirt and also the previous
batch dirt load also tends to affect the quality of the products, so effective cleaning procedure
should be adopted for the manufacturing of effective products.
There are mainly 2 types of cleaning procedure followed in industry
Cleaning in place
Cleaning out of place
2.2 Cleaning out of place (COP):
Cleaning out of Place, is defined as a method of cleaning equipment items by removing
them from their operational area and taking them to the cleaning station for cleaning. This is the
term used for those cleaning operations involving the removal of small sections of piping, valve
parts, filler parts that are not normally cleaned in place and placing them into a cleaning vat.
The vat is equipped with a heat source, adequate heated cleaner solution and a re-circulating
pump. The main disadvantage of COP is that as the parts for cleaning are disassembled from the
vessel, so after cleaning it has be reassembled and checked, which is more time consuming.
Page | 43
2.3 Cleaning in place (CIP):
CIP is defined as a method of cleaning equipment with minimal dismantling and with
minimal operator involvement. CIP cleaning is mainly utilized to clean interior surfaces of tanks
and pipelines of liquid process equipment without the requirement to dismantle or enter the
equipment. A chemical solution is circulated through a circuit of tanks and or lines then
returned to a central reservoir allowing for reuse of the chemical solution. Time, temperature,
and mechanical force are manipulated to achieve maximum cleaning. It can be carried out with
automated or manual systems and is a reliable and repeatable process that meets the stringent
hygiene regulations.
CIP relies on the principal of applying a suitable detergent or solvent at a suitable flow,
pressure, temperature and concentration for the correct length of time. The science is based on
applying the required amount of energy to the equipment to ensure that it is cleaned. The energy
is primarily provided by the solution temperature (thermal energy), the use of detergent or
solvent (chemical energy) and the application of suitable pipeline velocities or pressures (kinetic
energy).
A cleaning program can be composed of the following steps, The steps included in each
particular case depend on the nature of the dirt to be removed:
Pre-rinsing.
Hot alkali treatment.
Intermediate rinsing.
Hot acid treatment.
Intermediate rinsing.
WFI rinse.
The basic components of any CIP system will include the following:
Permanently installed product piping and air operated valves.
CIP solution make-up tanks.
CIP pumps.
CIP supply and return solution piping.
Spray devices (spray ball).
Solution collection manifolds.
Chemical feed systems and equipment.
The CIP control/monitoring systems and necessary recorders.
Page | 44
Benefits of CIP:
Safety operators are not required to enter plant to clean it.
Difficult to access areas can be cleaned.
Production down time between product runs is minimized.
Cleaning costs can be reduced substantially by recycling cleaning solutions.
Water consumption is reduced as cleaning cycles are designed to use the optimum
quantity of water.
The cleaning system can be fully automated therefore reducing labour requirements.
Automated CIP systems can give guaranteed and repeatable quality assurance.
Automated CIP systems can provide full data logging for quality assurance
requirements.
Hazardous cleaning materials do not need to be handled by operators.
Use of cleaning materials is more effectively controlled using a CIP system.
Page | 45
3. STERILIZATION
3.1 Introduction to sterilization:
Sterilization is a term referring to any process that eliminates (removes) or kills all forms of
microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.)
present on a surface, contained in a fluid, in medication, or in a compound such as
biological culture media. Sterilization can be achieved by applying heat, chemicals, irradiation,
and high end filtration. In practice sterility is achieved by exposure of the object to be sterilized
to chemical or physical agent for a specified time. The success of the process depends upon
thechoice of the method adopted for sterilization.
Sterilization
method
Sterilizing agent Mechanism of sterilization Articles sterilized
Dry heat
sterilization
Hot air free form
water vapour.
Process is accomplished by
conduction. Heat is absorbed by
exterior surface of the item and passes
inward creating a uniform temperature
and a sterile condition. Coagulation of
proteins causes the death of microbes.
Powders, heat stable items,
steel, glass wares etc.
Moist heat
sterilization
Hot air heavily loaded
with water vapour
which plays an
important role in
sterilization.
Water vapour generated by boiling
water has high penetrating power. this
destroys the microbes by causing
coagulation of proteins and also causes
oxidative free radical damage.
Microbial cultures, liquids,
glass wares
Chemical
sterilization
Ethylene oxide,
formaldehyde,
chlorine dioxide,
ozone.
Ethylene penetrates through paper,
cloth, plastic and can kill all known
viruses, bacteria, fungi and even
spores. Ozone has the ability of
oxidizing most organic matter.
Biological materials, fibre
optics, electronics, and many
plastics.
Radiation
sterilization
Radiations such as
electron beams, x-
rays, gamma rays or
subatomic particles.
They have very high penetrating
power and are very effective in killing
microbes.
Syringes, needles, cannulas,
air, plastics, heat labile
materials.
Filtration Filter made of
different materials
such as nitrocellulose
or polyethersulfone.
Bacteria are removed effectively
removed through a pore size of 0.2mm
and for viruses a pore size of around
20nm is required.
Sensitive pharmaceuticals
and protein solutions.
Page | 46
Types of sterilization:
3.2 Heat sterilization
Sterilization is a term referring to any process that eliminate or kills all form of life including
transmissible agents(such as fungi, bacteria, viruses, spore forms etc) present on surface,
contaminated in a fluid, in medication, or in a compound such as biological culture media.
Sterilization can be achieved by applying the proper combination of heat, chemical, irradiation,
high pressure and filtration.
To be effective, sterilization requires time, contact, temperature and with steam sterilization
high pressure. The effectiveness of any method of sterilization is also dependent upon the four
other factors.
1. The type of microorganisms present. Some microorganisms are very difficult to kill.
Others die easily.
2. The number of microorganisms present. It is much easier to kill one microorganisms
than many.
3. The amount and type of organic material that protect the microorganisms. Blood or
tissue removing on poorly cleaned instruments act as a shield to microorganisms during
the sterilization process.
4. The number of cracks and crevises on an instrument that might harbor microorganisms.
Microorganisms collect in, and are protected by, scratches, cracks and crevises such as
the serrated jaws of tissue forceps.
Finally without thorough cleaning, which removes any organic matter remaining on the
instruments that could protect microorganisms during the sterilization process, sterilization
cannot be assured, even with longer sterilization times.
Some of the methods used to achieve sterilization are:
Autoclaves: Highly effective and inexpensive. Unsuitable for heat sensitive objects.
Hot air ovens: Inefficient compared to autoclaves.
Ethylene oxide: Suitable for heat sensitive items but leaves toxic residue on sterilized
items.
Low-temperature steam and formaldehyde: Effective for instruments with cavities or
tubular openings.
Page | 47
Irradiation: Gamma rays and accelerated electrons are excellent at sterilization.
The preferred principle for sterilization is through heat, the autoclave being the most widely
used method of achieving it. In a dry air oven, it takes two hours at 1600C to kill spores of the
bacterium Clostridium botulinum (associated with canned food). Using saturated steam, the
same spores are killed in just five minutes at 1210C, proving that moist heat is more effective
than dry heat.
3.2.1 Sterilization out of place
(a) Steam sterilization:
The widely used method for heat sterilization is the autoclave. Autoclaves commonly use steam
heated to 121–134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C at
100 kPa (15 psi), or 3 minutes at 134 °C at 100 kPa (15 psi) is required. Additional sterilizing
time is usually required for liquids and instruments packed in layers of cloth, as they may take
longer to reach the required temperature. Following sterilization, liquids in a pressurized
autoclave must be cooled slowly to avoid boiling over when the pressure is released. Modern
converters operate around this problem by gradually depressing the sterilization chamber and
allowing liquids to evaporate under a negative pressure, while cooling the contents. Proper
autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which
can be quite resistant. To ensure the autoclaving process was able to cause sterilization, most
autoclaves have meters and chart that record or display pertinent information such as
Page | 48
temperature and pressure as a function of time. Indicator tape is often placed on packages of
products prior to autoclaving. A chemical in the tape will change color when the appropriate
conditions have been attained.
Biological indicators can also be used to independently confirm autoclave performance.
Simple bio-indicator are commercially available based on microbial spores. Most contain spores
of the heat resistant microbe Geobacillus stearothermophilus, among the toughest organisms for
an autoclave to destroy. Typically these devices have a self-contained liquid growth medium
and a growth indicator. After autoclaving an internal glass ampoule is shattered, releasing the
spores into the growth medium. The vial is then incubated (typically at 56 °C) for 24 hours. If
the autoclave destroyed the spores, the medium will retain its original color. If autoclaving was
unsuccessful the G. sterothermophilus will metabolize during incubation, causing a color
change during the incubation. For effective sterilization, steam needs to penetrate the autoclave
load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers
must be left a jar.
(b) Dry heat sterilization:
The Dry-Heat sterilization process is accomplished by conduction; that is where heat is
absorbed by the exterior surface of an item and then passed inward to the next layer. Eventually,
the entire item reaches the proper temperature needed to achieve sterilization. The proper time
and temperature for Dry-Heat sterilization is 160°C for 2 hours or 170°C for 1 hour. Instruments
should be dry before sterilization since water will interfere with the process. Dry heat
coagulates the proteins in any organism, causes oxidative free radical damage, causes drying of
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cells and can even burn them to ashes, as in incineration. Dry heat has the advantage that it can
be used on powders and other heat-stable items that are adversely affected by steam.
Dry heat sterilization of an article is one of the earliest forms of sterilization practiced.
Dry heat, as the name indicates, utilizes hot air that is either free from water vapour, or has very
little of it, and where this moisture plays a minimal or no role in the process of sterilization. Dry
heat sterilization and depyrogenation are pure heat treatment, suitable for glassware and metal
as well as liquids with low moisture content. This method is also very suitable for heat
treatment of powder medicaments. Dry heat sterilization and depyrogenation is a complete
destruction of micro-organisms by means of dry heat for a controlled period of time. Dry heat
sterilization and depyrogenation is environmentally safe. It causes no waste problems or
inconvenience for surroundings and personnel. At the same time this process is designed to
meet the most stringent requirement.
Principle of operation:
The Dry heat sterilization process is accomplished by conduction; that is where heat is
absorbed by the exterior surface of an item and then passed inward to the next layer. Eventually,
the entire item reaches the proper temperature needed to achieve sterilization. The proper time
and temperature for Dry-Heat sterilization is 250°C for 30 minutes (Dehydrogenization), 180°C
for 3 hours and121°C for overnight. Instruments should be dry before sterilization since water
will interfere with the process. Dry-heat destroys microorganisms by causing coagulation of
proteins. Dry heat sterilization takes longer than steam sterilization, because the moisture in the
steam sterilization process significantly speeds up the penetration of heat and shortens the time
needed to kill microorganisms
Advantages of DHS:
It is effective method, as dry heat by conduction reaches all surfaces of instruments, so it
can sterilize even the instruments that cannot be disassembled.
It leaves no chemical residues.
It eliminates “wet pack” problems in humid climate condition.
Disadvantages of DHS:
The plastic and rubber items cannot be dry-heat sterilized because temperatures used are
too high for these materials to withstand.Page | 50
The dry heat penetrates materials slowly and unevenly.
It requires continuous source of electricity for operating.
3.2.2 Sterilization in place
(a) Empty vessel sterilization in place: ESIP
ESIP enables the entire processing system to be sterilized as a single entity, thereby eliminating
or reducing the need for aseptic connection. Fermenter, media preparation vessel, processing
equipment, transfer lines and other large systems are normally sterilized in this manner without
any medium in place, thereby empty vessel sterilization done through saturated steam. The
system must be designed in such a way that condensate can be readily removed. In order to
achieve this objective, it may be necessary to sterilize the system in multiple patterns, in which
each pattern st.erilizes a portion of the larger system. When using this type of an approach,
some portions of the system must be sterilized more than once to assure that all portions of the
system are fully covered. Its main application is in animal cell culture fermentation where
media is not sterilized using steam. It can also be applied in microbial fermentation to reduce
the bio burden level. It is essential to maintain sterile conditions in the vessel from the start of
cool down until the system is ready to use. Maintenance of sterility is often accomplished by the
introduction of a pressurized gas into the system through an appropriate filter at the end of the
steaming step. Passage of steam through the jacket of the empty vessel facilitates if its internal
surfaces by dry heat. ESIP is carried out for 60 minutes. This is done when the sterilization
temperaturein the vessel reaches 1210C, it is done by first sterilizing air lines for 20 minutes,
when it is cooled down, later additional lines are sterilized for 20 minutes and then through
spray ball into the vessel for 20 minutes.
(b)Full vessel sterilization in place: FSIP
Sterilization in place of any equipment can be done conveniently thereby eliminating the need
of subsequent aseptic connections or shut down of the whole process. Typically SIP uses
saturated steam at 15 psi for atleast 30 minutes. For microbial fermentation, FSIP is performed.
The whole sterilization is done after charging the media into the vessel. It is obviously
important to many industries including food, dairy, beverages, nutraceutical, biotechnology,
pharmaceutical, cosmetic, health and personal care industries in which the processing must take
place in a hygienic or aseptic environment. This is done to those media which can generally
withstand the sterilizing temperature. Thus, there should be no influence or alteration in the
media.
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Convenience of SIP
It minimizes the carrying of any heavy vessels to and from the autoclave.
Sterilization of vessel, airline, exhaust can be done without dismantling any of the
connections.
Sterilization sequences are fully automated.
Rapid heat up and cool down.
Sterilization sequences are easily initiated and configurable to match any requirement.
FSIP of a vessel e.g 40 L capacity is done for 40 minutes. This is done when the sterilization
temperature in the vessel reaches 1210C, is done by first sterilizing air lines for 20 minutes.
Then it is cooled down, later additional lines are sterilized for 10 minutes and then through
spray ball into the vessel for 10 minutes.
3.3 COLD STERILIZATION (FILTRATION):
1.3.1 Introduction:
Fluids that would be damaged by heat (such as those containing proteins like large
molecule drug products) irradiation or chemical sterilization, can be only sterilized by using
sterile grade membrane filters. This method is commonly used for heat labile pharmaceuticals
and protein solutions in medicinal drug processing. Usually, a filter with pore size 0.2 µm will
effectively remove microorganisms. In the processing of Biologics, viruses must be removed or
inactivated. Nanofilters with a smaller pore size of 20 -50 nm (nanofiltration) are used. The
smaller the pore size the lower the flow rate. To achieve higher total throughput or to avoid
premature blockage, pre-filters might be used to protect small pore membrane filters.
Sterilization by filtration is employed mainly for thermolabile solutions which usually
contains the thermolabile proteins. These may be sterilized by passage through sterile bacteria-
retaining filters, e.g. membrane filters (cellulose derivatives, etc.), plastic, porous ceramic, or
suitable sintered glass filters, or combinations of these. Asbestos-containing filters should not be
used. Appropriate measures should be taken to avoid loss of solute by adsorption onto the filter
and to prevent the release of contaminants from the filter. Suitable filters will prevent the
passage of microorganisms, but the filtration must be followed by an aseptic transfer of the
sterilized solution to the final containers which are then immediately sealed with great care to
exclude any recontamination. Usually, membranes of not greater than 0.22 μm nominal pore
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size should be used. The effectiveness of the filtration method must be validated if larger pore
sizes are employed. Membrane filters are generally used for filtration are selective barriers that
allow for transmission of certain feed components while retaining unwanted components. The
larger molecules than the pore size of filter are retained on the filters and smaller particles are
passes through. This filtration is also called cold sterilization as heat is not used in actual
filtration process.
The filter used are generally reusable, so it the filter integrity need to be tested before
employing the filter in both before and after filtration, a bubble point or similar test should be
used, in accordance with the filter manufacturer's instructions. This test employs a prescribed
pressure to force air bubbles through the intact membrane previously wetted with the product,
with water, or with a hydrocarbon liquid. All filters, tubes, and equipment used "downstream"
must be sterile. Filters capable of withstanding heat may be sterilized in the assembly before use
by autoclaving at 121 °C for 15 - 45 minutes depending on the size of the filter assembly. The
effectiveness of this sterilization should be validated. For filtration of a liquid in which
microbial growth is possible, the same filter should not be used for procedures lasting longer
than one working day.
Filter set-up with associated pipe work:
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Classification of filters:
Based on function: prefilters and sterilize grade filtrers
Based on nature of filtration: Hydrophilic and Hydrophobic filters
Prefiltration
Prefilters are recommended for removing larger contaminants, which leads to decrease
the efficiency of sterile grade filters by clogging the sterile filter membrane on short term use.
So these types of filters are mainly used to increase the service life of the sterile filters. The
economic advantage of extending a membrane filter's life is far greater than the cost of
prefilters. Prefilters are suited for applications where 100% retention of contaminants above a
specified pore size is not required. A non-fiber releasing, membrane-like prefilter is
recommended when using a clarifying or prefilter as a final filter. Fiber matrix prefilters may
occasionally unload retained contaminants into the filtrate if subjected to shock, that caused by
a rapidly actuated valve. Selection of a clarifying filter or prefilter is based upon the retention
efficiency required for the filtration process at hand. Choosing the proper nominal pore size for
a clarifying filter and/or prefilter will ensure the required dirt-holding capacity in your system.
It will also extend the life of the final filter, and provide an economic advantage. In some cases,
it is practical to use a moderately efficient clarifying filter followed by a more retentive
prefilter. This will allow each filter in the train to be more fully expended, while removing as
much incident contamination as possible.
Prefilters material of construction:
Glass fibers:
Glass fibers have long served as prefilters. They are particularly used in the filtration of
sera, blood products and in wine production. Glass fiber filters coated with cellulose nitrate
polymer are helpful in removing proteinaceous impurities from various preparations. These
filters are biological inert with high chemical and thermal resistance, unaffected by humidity
and can be easily sterilize by autoclave or baking.
Polypropylene fibers:
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They are produced by melting, drawing or extruding the bulk polymer into fibers of
designed thickness and lengths. These are widely used in construction of prefilters of various
sizes, shape and defined porosities. The more upstream prefilters face permits a greater loading
of particles.
Microporous membranes:
These are of usually a larger pore size than that of using in final filters may also be used
in Prefilters. These are commonly of cellulosic material and range from 1.2 to 0.65μm. Such
fine prefiltration is required, when the media to be filtered has a wide spectrum of contaminants
and size range.
Sterile grade filters:
Sterilizing grade filters are used downstream to the prefilters. These can be defined as a
filter that will produce a sterile filtrate when challenged with 107 CFU of Brevundimonas
diminuta. The material of sterile filter can be nylons, PVDF, PES and polypropylene. They can
have a different structure such as isotropic or asymmetric. Asymmetric membranes have a grade
pore structure that provides for high permeability while maintaining the required mechanical
strength. For sterile filtration, filterability and validation experiments are typically conducted at
the laboratory scale to determine the size of the large scale system and verify the absolute
retention of bacteria with the actual process solution and operating condition. A post use
integrity testing is performed after the product solution has been filtered to ensure that the
required retention performance was obtained during the filtration process.
Mechanism of filtration:
Straining:
This phenomenon occurs when the opening between the media members (fibers, screen
mesh, corrugated metal, etc.) is smaller than the particle diameter of the particle the filter is
designed to capture. This principle spans across most filter designs, and is entirely related to the
size of the particle, media spacing, and media density.
In order to be intercepted, a particle must come within a distance from a fiber of one
radius of itself. The particle thus makes contact with the fiber and becomes attached. The
interception mechanism can be contrasted with the impaction mechanism in that a particle Page | 55
which is intercepted is smaller and its inertia is not strong enough to cause the particle to
continue in a straight line. It therefore follows the air stream until it comes into contact with a
fiber.
Diffusion:
Diffusion occurs when the random (Brownian) motion of a particle causes that particle
to contact a fiber. As a particle vacates an area within the media, by attraction and capture, it
creates an area of lower concentration within the media to which another particle diffuses, only
to be captured itself. To enhance the possibility of this attraction, filters employing this principle
operate at low media velocities and/or high concentrations of micro-fine fibers, glass or
otherwise. The more time a particle has in the "capture zone", the greater the surface area of the
collection media (fibers), the greater the chances of capture. Filter manufacturers have two
distinct methods of addressing this principle employ more square footage of fine glass-mat type
media or employ less square footage of high lofted glass media.
Inertial separation:
Inertial separation (impaction) uses a rapid change in air direction and the principles of
inertia to separate mass (particulate) from the air stream. Particles at a certain velocity tend to
remain at that velocity and travel in a continuous direction. This principle is normally applied
when there is a high concentration of course particulate and in many cases as prefiltration mode
to higher efficiency final filters.
Electrostatic attraction:
Filters utilizing large diameter fiber media rely on electrostatic charges to increase their
efficiency of fine particle removal. Large diameter fiber media is normally chosen due to low
cost and air flow resistance. However, these filters often lose their electrostatic charge over time
because the particles captured on their surface occupy charged sites, thereby neutralizing their
electrostatic charge.
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Filter designs:
Flat (disc) filters:
Stacked disc cartridge housing is designed for a wide range of filtration tasks for
applications in the chemical industry and related sectors. In addition to the general benefits of
flat disc cartridge filtration as an enclosed depth filtration system, this system offers the
following specific features.
Features:
Stacked disc cartridge housing parts in contact with the product are made from stainless
steel (ss 316L) with high resistance to corrosion. Pre-compressed stacked disc cartridge with flat
adapter and reliable centering and fixed support ensure safe filtration and offer simple handling.
Safe stacked disc cartridge housing range for operating pressures of 87 to 145 psi (600 kPa/6
bar to 1,000 kPa/10 bar) maximum for liquids and gases.
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Cartridge filters:
Cartridge filters provide high throughput and minimal differential pressure. Cartridges
are robust, resilient, and designed to withstand multiple steam-in-place cycles. Each cartridge
filter is integrity tested during the manufacturing process. Cartridge filters are available in five
cartridge lengths, from five to thirty inches, and provide a full range of filtration areas to suite
very size system. Three connection options are offered for easy adaptation to existing housings.
Disposable Syringe Filter Units:
Minimum sample hold-up: Unit housings are specifically designed to maximize
sample recovery
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High purity: Non-pigmented housing and integral filter sealing assure that filtrates will
not be adulterated due to pigment, dye, or adhesives leaching into the filtrate.
Convenient: Each unit is clearly marked with an identifying code to denote pore size,
membrane material and housing polymer
Sterile: Units can be purchased pre-sterilized and individually packaged, or non sterile
in bulk pack. All polypropylene can be autoclaved Acrylic cannot be autoclave.
Types of filters:
4. Hydrophilic Filters:
Hydrophobic filters are easily wet with water. Hydrophilic filters can be wetted virtually
by any liquid and preferred filters for aqueous solutions, as appropriate by compatibility. Once
wetted hydrophilic filters do not allow the free passage of gases until applied pressure is exceed
the bubble point and liquid is expelled from the membrane.
Material of construction:
PES (Polyethersulfone) Membranes:
Polyethersulfone (PES) membranes are hydrophilic filters constructed from pure
Polyethersulfone and available in pore sizes ranges from 0.03 micron to 5.0 micron. PES
membrane filters are designed to remove particulates during general filtration. Their low protein
and drug binding characteristics also make PES membrane disc filters ideally suited for use in
life science applications. This strong, microporous PES membrane is constructed from a high-
temperature Polyethersulfone polymer that is acid and base resistant. The strength and
durability of our PES membrane filters are especially advantageous during procedures that
require aggressive handling or automated equipment. PES membrane is available with large
pore sizes and high capillary flow rates suitable for lateral flow devices.
Polyethersulfone (PES) membrane for aqueous solutions provides removal of fine
particles, bacteria, viruses, and fungi making it a versatile membrane for applications such as
sample preparation, sterile filtration and infusion therapy. PES is an inherently hydrophilic
membrane that wets out quickly and completely resulting in fast filtration with superior flow
rates and high throughputs. The hydrophilic nature of PES means no added surfactants are used
to increase wettability. PES membrane is also extremely low protein binding minimizing the
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likelihood of target analytes binding. Polyethersulfone membrane is compatible with EtO,
gamma irradiation, and autoclave methods of sterilization.
Cellulose Acetate:
Composition: Mixture of cellulose triacetate and diacetate
Characteristics: Low static charge and high strength
Sterilizable: May be repeatedly sterilized without loss of integrity or change in bubble
point
Clean: Lowest aqueous extractable (0.1 wt. %) of all membranes
Relative to MCE (Mixed Cellulose Esters, Nitrocellulose):– improved solvent resistance to low
molecular weight alcohols
– Better heat resistance
– Lower protein binding
Hydrophilic PTFE:
Characteristics: Maximum chemical and pH resistance
High flow rates with minimal aqueous extractable (<0.3 wt. %)
Optically clear when wet with water
Application:
Ideal for HPLC and other mixtures of aqueous and organic solvents
5. Hydrophobic filters:
Hydrophobic filters do not wet in water but it will be wetted by low surface tension
liquids, for example organic solvents such as alcohols. Once the hydrophobic filter has been
wetted, aqueous solutions also pass through. Hydrophobic filters are best suited for gas
filtration, low surface tension solvents and venting in certain applications. hydrophobic filters
are used for aqueous solutions because of compatibility requirement. Water or aqueous
solutions also passed through the hydrophobic filters once the water breakthrough pressure is
reached.
Material of construction (MOC):
Hydrophobic PTFE
Properties: Thin, highly porous, behaves as an absolute retentive membrane
Supported: polypropylene laminated to one side to improve handling
Inert to most chemically aggressive solvents, strong acids and bases
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Thermostable can be used up to 100ºC
Applications:
Sterilize gases: traps aqueous aerosols
Air and gas venting: allows gases to pass freely while blocking aqueous liquids, protect
vacuum pumps and critical samples
Sterilize and clarify strong acids and many other solvents incompatible with other
membrane
Sterile Filtration:
Available in 0.1 and 0.2 µm pore sizes, the polyethersulfone membrane provides
sterilization of buffers, culture media, additives, and pharmaceutical filtration. If mycoplasma
contamination is a concern, the 0.1 µm PES membrane provides assurance that critical samples
will not be contaminated.
Integrity testing for filters:
Integrity testing is a fundamental requirement of critical process filtration applications in
the pharmaceutical industry. FDA Guidelines require integrity testing of filters used in the
processing of sterile solutions such as large volume parenterals (LVPs) and small volume
parenterals.
There are mainly two class of integrity testing, they are
Destructive testing
Non-destructive testing
Membrane filters have been used successfully for many years to remove yeast, bacteria
and particulate from fluid streams. The ultimate integrity test for a sterilizing grade membrane
filter is the bacterial challenge. Unfortunately this is a destructive test; the filter cannot be used
afterwards to filter a product. As a result, one of the most important aspects of the use of filters
to remove bacteria is to have a non-destructive integrity test, which is correlated to the
production of effluent in a bacterial challenge test. It is this correlation, rather than the
nondestructive integrity test alone, that provides assurance that the filter will perform as
intended. During production of sterile product, the filter should be subjected to such an integrity
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test before and after filtration. This is done to ensure that the filter meets specification, is
properly installed and intact during filtration, and to confirm the rating of the filter.
Destructive testing:
The destructive testing are generally done in order to ensure the ability of the filter to
hold the bacterial cells that are passed through the filters to be tested, generally the bacterial
challenge test are carried out on a lot release criteria of the manufactured filters by the
manufacturer. Destructive challenge testing is the best way to determine a sterilizing filter's
ability to retain bacteria. Bacterial challenge testing provides assurance that the fabricated filter
meets the critical performance criteria of a sterilizing filter. During the bacterial challenge test,
the sterilizing grade filter of 0.22 µm filter are challenged with a solution of culture medium
containing bacteria (Brevundimonas diminuta ATCC 19146) at a minimum challenge
concentration of 107 per cm2 of the filter area. The effluent that passes out of the filter was then
passed through an assay filter disc that is placed on an agar plate and incubated for colony
formation.
Non- Destructive testing:
Non-destructive testing may be done on filters before and after use. Integrity testing
sterilizing filters before use monitors filter integrity prior to batch processing, preventing use of
a non-integral filter. Integrity testing sterilizing filters after a batch has been filtered can detect
if the integrity of the filter has been compromised during the process. Detecting a failed filter
alerts operators to a problem immediately after batch processing, eliminating delay and
allowing rapid reprocessing.
There are three types of non-destructive testing that include mainly
For hydrophilic filters
Bubble point test,
Diffusion test.
For hydrophobic filters
Water intrusion test.
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3.3.2 INTEGRITY TESTING OF HYDROPHILIC FILTERS:
1. Bubble point test:
Micro-porous membranes will fill their pores with wetting fluids by imbibing that fluid
in accordance with the laws of capillary rise. The retained fluid can be forced from the filter
pores by air pressure applied from the upstream side. The pressure is increased in gradually in
increments. When the wetting fluid is expelled from the largest pore, a bulk gas flow will be
detected on the downstream side of the filter system. The Bubble Point measurement determines
the pore size of the filter membrane, i.e. the larger the pore the lower the Bubble Point pressure.
Therefore filter manufacturers specify the Bubble Point limits as the minimum allowable
Bubble Point. During an integrity test the Bubble Point test has to exceed the set minimum
Bubble Point.
Bubble point is based on the fact that liquid is held in the pores of the filter by surface
tension and capillary forces. The minimum pressure required to force liquid out of the pores is a
measure of the pore diameter.
2. Diffusion Test:
At differential gas pressures below the bubble point, gas molecules migrate through the
water-filled pores of a wetted membrane following Fick's Law of Diffusion. The gas diffusional
flow rate for a filter is proportional to the differential pressure and the total surface area of the
filter. At a pressure approximately 80% of the minimum bubble point, the gas which diffuses
through the membrane is measured to determine a filter's integrity. The flow of gas is very low
in small area filters, but it is significant in large area filters.
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Integrity testing of hydrophobic filters:
Water intrusion test:
Water Intrusion test, also known as Water Flow Integrity test, is used for hydrophobic
vent and air membrane filters only. The upstream side of the hydrophobic filter cartridge
housing is flooded with water. The water will not flow through the hydrophobic membrane. Air
pressure is then applied to the upstream side of the filter housing above the water level to a
defined test pressure. This is done by way of an automatic integrity tester. A period of pressure
stabilization takes place over, by the filter manufacturer recommended, time frame, during
which the cartridge pleats adjust their positions under imposed pressures. After the pressure
drop thus occasioned stabilizes, the test time starts and any further pressure drop in the upstream
pressurized gas volume, as measured by the automatic tester, signifies a beginning of water
intrusion into the largest (hydrophobic) pores, water being incompressible. The automated
integrity tester is sensitive enough to detect the pressure drop. This measured pressure drop is
converted into a measured intrusion value, which is compared to a set intrusion limit, which has
been correlated to the bacteria challenge test. As with the Diffusive Flow test, filter
manufacturers specify a maximum allowable water intrusion value. Above this value a
hydrophobic membrane filter is classified as non-integral.
Automated integrity tester:
Most integrity tests are nowadays performed with qualified automated integrity
test systems, which allow an easy and accurate test of choice by the end-user. The end-
user programs the test parameter into the unit and when required chose the test needed
for the specific filter, connect the unit to the pre-wetted filter system and let the machine
run the test. The integrity test machine testing the filter system is by far more accurate
than a manual test. Besides these machines provide a hardcopy print-out and/or have the
ability to store the results in a data storage system.
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3.4 FAMILIARIZATION OF EQUIPMENTS:
3.4.1. Media Preparation Vessel:
The media preparation system is used to prepare media, which promotes cell growth in
the seed and production bioreactors. The media preparation system contains equipment used for
large scale and small scale media preparation. The media prep vessels and associated
equipment, instrumentation and piping are contained within a complete assembly. This
assembly design includes all vessels, agitators, vent filters. The media prepared is transferred to
the fermenter through serious of sterile transfer lines.
The Processes need to be carried out in media preparation vessel includes sterilization in
Place (SIP), Media Preparation, Transfer the media for filtration, Cleaning in place (CIP) of
vessel.
Features and Specifications:
Volume: 65 L
Material of Construction (MOC): SS 316L, SS 304
RA: < 0.8 µm
Surface finish: mechanical and electro polished
Pressure limit: 0-6 bar
Temperature: 0-132 degree Celsius
Cleaning Options: CIP
Sterilization option: SIP
Design Jacketed and Insulated
3.4.2 Sterile Filtration Unit:
The sterile filtration unit is used for the filter sterilization of the media containing heat
labile components, which are generally used in animal cell culture. The filtration system
comprises of all the piping, components and instruments connected to the CIP, Steam, and
Process Air lines. The Processes required to be carried out in the filtration system are SIP,
Filtration and CIP.
Features Specifications:
Type of filter: Hydrophilic membrane
Size: 10”cartridge
Page | 65
Pore size: 0.45 µm and 0.2 µm
Material of Construction: SS 316l, SS 304
RA: < 0.5 µm
Surface finish: mechanical and electro polished
Housing Pressure: 0-8.5 bar
Housing Temperature: 0-175 oC
Cleaning Option: CIP
Sterilization option: SIP
3.4.4 Harvest Vessel:
The harvest vessel is generally used for the sterile harvesting of the fermentation
products from the fermenter or bioreactor. The Processes carried out in the harvest vessel are
SIP, sterile harvesting from 40 l fermenter and transfer the culture for downstream processing
and CIP.
Features and specifications:
Volume: 125L
Material of construction: SS 316l, SS 304
RA: < 0.8
Surface finish: mechanical and electro polished
Pressure: 0-6 bar
Temperature: 0-132 oC
Cleaning Options: CIP
Sterilization options: SIP
Design: Insulated
3.4.5 Horizontal Autoclave:
The equipment comprises of inner chamber, outer chamber, stainless steel SS 304 / SS
316, inner and outer jacket support with horizontal autoclave and outer jacket support channel
type support legs. Gasket made of neoprene and outer cover made of stainless steel SS 304 / SS
316. Radial lock door with safety control device system. Ports of the inner chamber for pressure
gauge, safety valve, steam release valve, water outlet, temperature gauge, vacuum breaker, and
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steam trap. Ports of the steam/outer chamber are pressure gauge, safety valve, steam inlet valve,
water outlet, vacuum breaker, and steam trap, temperature and pressure sensors, strip chart
recorder and HMI.
3.4.6 Dry heat sterilizer:
The DHS equipment’s are suitable for sterilization of Glassware like Ampoules, vials,
metal trays, metal material and containers etc.
General Specifications:
SS 304 dynamically balanced cooling fan, Pre filter and HEPA filter, Damper, Temperature
Sensors, Strip chart recorder and data logger.
5. STANDARD OPERATING PROCEDURES AND VALVE MATRICES
5.1 CIP (Cleaning In Place)
5.1.1 Cleaning in place of media preparation vessel
Aim:
To carry out cleaning in place (CIP) of media preparation vessel.
Procedure:
Connection of mobile CIP Skid to Media preparation vessel to be cleaned:
Flexible pipe from pump was connected to manual flush bottom valve of media
preparation vessel.
Recirculation pipe was connected to CIP return of media preparation vessel. The hose of
the drain was connected to the floor drain.
The power cable of the mobile CIP pump was connected to the miniature Circuit
Breaker in the wall and it was switched on.
The mains in the mobile CIP pump were switched on.
The CIP parameters in the SCADA program as well as in the MPV HMI were set.
The MPV valves were operated as mentioned in the HMI of the MPV.
One of the dummy in the vessel was opened for venting.
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Pre-Rinse:
The spray ball header valves V508 and V501 were opened, the pre-rinse water entered
into the vessel from CIP Station
Once the water is completely filled in MPV close the header valves and open Flush
bottom valve in MPV.
The SIC was operated at 100 rpm and the pump CP02 was switched on in MPV HMI.
Mobile CIP skid drain valve V463 was also opened the pump was switched on till the
flow switch sensed line dry.
The SIC was stopped.
The pump CP02 was stopped.
Mobile CIP pump valve V451 was opened to drain the residual fluid by gravity.
The pump drain valve V451 and the main drain valve V463 was closed.
Hot alkali wash:
The spray ball header valve V508 and V501 was opened, the alkali entered into the
vessel from CIP station.
Wait till alkali flow stops in the line.
The SIC and pump CP02 were operated at 100 rpm
Collection through spray ball and recirculation through spray ball: Open 501V, on
recirculation valve 464V and flush bottom valve. Then close spray ball valve 501V and
recirculation valve 464V.
Recirculation through Auxiliary lines: Open header valve 508A, 402A and 408A, flush
bottom valve then on recirculation valve 464V parameter on HMI Alkali solution gets
recirculated for particular time. The SIC was stopped.
The CIP pump drain V463 was opened and the recirculation valve V464 was closed.
The pump CP02 was stopped when the flow switch sensed the line dry.
The mobile CIP pump drain V451 was turned on to drain traces of fluid in pump.
Hot water rinse:
Repeat the steps of the alkali wash.
Hot Acid wash:
Repeat the same steps as alkali wash.
Page | 68
Hot water rinse:
Repeat the steps of alkali wash.
WFI rinse:
Flow of water through auxiliary lines there is no recirculation
The SIC was operated at 100 rpm for the required time then stopped.
The tank bottom V403 and the mobile CIP pump drain valve V643 was closed.
The pump C02 was operated at 50% speed, till the flow switch sensed dry then the pump
was stopped.
The mobile CIP pump drain valve V451 and the main drain V463 were opened to drain
the residual fluid by gravity.
Close all the valves except header valve and auxiliary lines to perform next wash.
WFI rinse 2:
Repeat the same steps as WFI rinse 1.
WFI rinse 3:
The spray ball header valve V508 and V501 were opened.
The WFI entered into the vessel through the spray ball line.
Wait till the WFI water stops in the line.
The SIC was operated at 100 rpm for the required time then stops the SIC.
The tank bottom V403 and the mobile CIP main drain V463 was opened.
The pump was operated till the flow switch sensed the line dry.
The conductivity was checked. If the conductivity was not reached, the WFI rinse 3 was
repeated.
The mobile CIP pump drain was opened to drain the residual fluid by gravity.
The spray ball header valve V501 and V508, tank bottom valve V403, mobile CIP pump
drain valves V463, V451 and mobile CIP pump outlet valve V464 were closed.
The CIP inlet valve in the header valve was closed.
The air inlet valve in the supply line was closed.
The dummy port which was opened was also closed.
It was ensured all the valves were closed, both the vessel and in the supply lines.
The mains for the mobile CIP pump were switched off.
The miniature circuit breaker in the wall was switched off and the power cable for the
mobile CIP pump was disconnected.
All the connections between mobile CIP pump and the MPV were disconnected.
Page | 69
The vent filter in the housing was fixed.
Valve matrix:
S. No Steps ModeActuated
valve
Manual
valve
Control
valve
1. Pre rinse
Open
On
Open
Close
Open
Close
CP02
463V
CP02
451V
451V
508AV
501V
403AV
2. Hot alkali wash
Open
On
Open
Close
Open
Open
Open
Open
Open
Close
Open
Off
CP02
464V
464V
463V
463V
451V
CP02
508AV
501V
501V
508AV
402AV
403AV
408AV
3. Hot water rinse Open
On
Open
Close
Open
CP02
464V
508AV
501V
501V
508AV
Page | 70
Open
Open
Open
Open
Close
Open
Off
464V
463V
463V
451V
CP02
402AV
403AV
408AV
4. Hot acid rinse
Open
On
Open
Close
Open
Open
Open
Open
Open
Close
Open
Off
CP02
464
464V
463V
463V
451V
CP02
508AV
501V
501V
508AV
402AV
403AV
408AV
5. Hot water rinse
Open
On
Open
Close
Open
Open
Open
Open
Open
Close
Open
Off
CP02
464V
464V
463V
463V
451V
CP02
508AV
501V
501V
508AV
402AV
403AV
408AV
6. WFI Rinse 1 Open 508AV
Page | 71
Open
Open
Open
Open
463V
451V
501V
403AV
7. WFI Rinse 2
Open
Open
Open
Open
Open
463V
451V
508AV
501V
403AV
8. WFI Rinse 3
Open
Open
Open
Open
Open
463V
451V
508AV
501V
403AV
5.1.3 CIP of filtration system
Page | 72
Aim:
To perform cleaning in place (CIP) of filter system.
Procedure:
CIP of the filter consists of following steps:
Pre rinse:
Pass 30 l purified water for 10” filter and drain
Hot Alkali Wash:
Pass 1-4 M 30 l NaOH at 60 degree Celsius and drain
Hot water rinse:
Single passes 60 oC purified water into the filter and drain.
Final rinse shall be checked for the conductivity of the water.
Valve matrix:
S. No Actuated valve Control valve Manual valve
Steps Temp Mode
1
Media
preparation
vessel filling
220C Open V851 V459V450, V316A,
V508A, V501
2Pre rinse with
Purified water220C Open
V403A, V513A,
V514, V318, V515,
V319, V516
3 Alkali rinse 220C Open
V403A, V513A,
V514, V318, V515,
V319, V516, V401B,
V402B,V316B
4Recirculation of
Alkali 220COpen
V315B, V403B,
V513B, V514, V515,
V516, V401B,
V402B
Page | 73
5 Hot water rinse 600C Open Same as pre rinse
6 WFI rinse 220C Open Same as above but without recirculation
5.2 STERILIZATION IN PLACE
5.2.1 ESIP of media preparation vessel
Aim:
To perform ESIP of media preparation vessel
Procedure:
Pressure hold test: It is done to check whether there are any leakages in system. Ensure
the ports are tightened before PHT and close all the valves. Open process air valve 315A
and increase the pressure to 2 bar using pressure regulatory valve. Maintain the pressure
for about 2 hours then observe if there is any drop in pressure if not open the exhaust
line and remove the complete pressure.
Jacket evacuation: Evacuation of jacket by opening 304A and its corresponding drain
line 702A.
Direct steaming of vessel by passing pure steam by opening V508 and V501
When temperature reaches 40°C flush bottom valve V403A and its corresponding steam
trapper V404A sampling valve and its corresponding steam trapper are opened.
At 70°C the first auxiliary valve V402 and its corresponding steam trap V703A are
opened.
At 90°C the vent line V316A is opened after ensuring the steam release it was closed
and filter drain was opened.
At 121 oC1.2bar pressure and V508 was opened for 20 min and then closed.
After 20 mins all the valves were closed.
Pressurization of vessel was done by opening V315A
Cooling of vessel was done by supplying chilled water to the jacket by opening V210A,
to ensure jacket is filled with water V304 was opened and chilled water return valve
V212 was opened.
Page | 74
Valve matrix:
S. No Steps Control loops Manual valves Control valves
Temp Mode
1. Jacket evacuation Open304A
702A
2. Pressure Hold test Open315A
316A
3. Steaming Open508A
501
4. Evacuation 40 oC Open
403A
404A
515A
5. Evacuation 70 oC Open402A
703A
6. Evacuation 90 oC Open704A
316A
7.Sterilization hold for
20minsOpen All above valves
8. After 20mins
Close
Open
All above valves
315A
9.Jacket pressure
reduction
Open
Close
304A
304A
10. Cooling
Open
Close
210A
212A
702A
Page | 75
5.2.2 SIP of filtration system
Aim:
To perform sterilization in place (CIP) of filter system.
Procedure:
Before transferring the media into the fermenter, the media has to be filtered using
sterile filtration unit. SIP of filter comprises of 3 major steps:
Wetting the system
Pressure hold test
SIP of filters:
Connect the SIP header line, airline and supply steam line to the filtration unit.
Wet the filter by passing water.
Open compressed air valve.
Cut off pressure when achieves 1 bar and allow 10 minutes hold time
Open drain valve and ensure the PHT is passed.
Open Steam valve at a pressure 1.2 bar for 20 minutes.
Open fermenter transfer line
Set pressure in PRV about 0.2 bar
Close drain lines and Open the air inlet valve.
Once SIP is done; Integrity testing of filters is recommended and carried out as
explained before.
Valve matrix:
S.
No
Actuated
valve
Control
valveManual valve
Steps T 0C Mode
1Wetting the
filter220C Open
V403A, V513A,
V514,
V318,V515,
V319, V516
2 Pressure Hold
test
220C Open
V317, V515,
Page | 76
V516, V407,
V401, V703
3 Direct Heating 1210C Open
V509, V318,
V515, V711,
V516, V713
V712, V710,
V514
4 Pressurization
2
20C&1.2
bar
pressure
Open V317
5.2.5 ESIP of harvest vessel
Aim: To perform ESIP of harvest vessel.
Procedure:
Establish the airline, Steam line and drain connection.
Pressure Hold test for 10 minutes by pressurizing at 1.5 bar.
Depressurize the vessel by opening the exhaust valve for 5 seconds.
Pass the steam into the vessel for a total time period of 30 minutes.
Pass steam via Spray Ball followed by sampling port finally via Vent line.
Cool the vessel by passing the surface air and throttling exhaust valve.
Valve matrix:
S.No Steps Manual valves
1 Pressure Hold Test V5, V4, V3, V10
2 Depressurization V6
3 Steaming through Spray Ball V5, V7
4 Sampling V11
Page | 77
Port evacuation
5 Vent line evacuation V6, V2
6 Cooling V6,V7, V2
5.3 STERILIZATION OUT OF PLACE
5.3.1 Autoclave
(a) Standard cycle
Aim:
To study the heat penetration in standard cycle of autoclave and its validation.
Materials required:
Chemical indicator strips
Biological indicator ampoule (Geobacillus stearothermophilus)
The material to be sterilized
Temperature recorder
Temperature probe thermocouple
Procedure:
Preparation of load for standard cycle in autoclave:
Four petri plates were used as load for standard cycle of autoclave.
The load was prepared by placing the biological indicator ampoule in middle of the petri
plates and keeping the chemical indicator strips in the load.
The plates were packed using double folds brown paper.
The different temperature sensor is placed in the different parts of the load such as
middle of load, and second temperature sensor were placed top of load.
Standard operating procedure:
The load was prepared.
Temperature sensors were placed inside the autoclave chamber as follows.
O9: Middle of load.
11: top left back
01: right op middle
12: left op front
04: right top back
05: left bottom middle
Page | 78
08: left bottom middle
10: right bottom back
06: bottom plate middle
02: Bottom right front
07: drain
03: bottom of load.
Load was placed inside the chamber, along with the temperature sensors connected to
the temperature logger.
The parameters were set for the standard cycle of the autoclave.
The standard cycle was run as per parameters of 20mintes at 121ᵒc.
Valve matrix:
V1- Jacket steam
V2- Jacket exhaust
V3-Chamber steam
V4-Chamber exhaust
V5-Chamber vacuum
V6-Chamber vent
V7-Air
V8- Drain
steps/valves V1 V2 V3 V4 V5 V6 V7 V8
Jacket pressurization Open Open
Heating Control Open
HoldingContro
lControl
Cooling Open OpenOpe
nOpen
CompletionOpe
nOpen Open
Page | 79
Chart for standard cycle:
(b) Liquid cycle
Page | 80
Aim:
To study the heat penetration and validation of liquid cycle in sterilization.
Materials required:
Biological indicator ampoule (Geobacillus stearothermophilus)
Chemical indicator strips
The material to be sterilized (200ml of Trypticase soya casein broth)
Temperature recorder or logger
Temperature sensors
Procedure:
Preliminary steps:
250 ml conical flask containing 150 ml of Trypticase soya casein broth was used as the
load for liquid cycle.
Then the two temperature sensors were placed in the liquid cycle load, one sensor at the
bottom of the load and second on the surface above the load liquid.
The biological indicator was placed inside the load and the chemical indicator was put
on the surface of the load container.
Operating procedure:
Temperature sensors were placed in the chamber of the autoclave at different position as
follows.
01: right op middle
02: Bottom right front
03: bottom of load.
04: right top back
05: left bottom middle
06: bottom plate middle
07: drain
08: left bottom middle
09: Middle of load.
10: right bottom back
11: top left back
12: left op front
Page | 81
The prepared load was placed inside the chamber, along with the temperature sensors.
The parameters for the liquid cycle were set.
The cycle was continued as per the set parameters of 20mintes at 121ᵒC.
Valve matrix:
V1- jacket steam
V2- Jacket exhaust
V3-Chamber steam
V4-Chamber exhaust
V5-Chamber vacuum
V6-Chamber vent
V7-Air
V8- Drain
Page | 82
Chart for liquid cycle:
a. Porous cycle:
Page | 83
Aim: To study the heat penetration and validation of porous cycle in autoclave.
Materials required:
Biological indicator ampoule (Geobacillus stearothermophilus)
Chemical indicator strip
The material to be sterilized (16 towels)
Temperature recorder or logger
Temperature probe
Procedure:
Preliminary steps:
The towels were used as load for performing the porous cycle in the autoclave.
The towels were placed one above the other and the biological indicator ampoule was
placed in the middle of the load and the strips were also placed on the different places in
the load.
The sensor was placed in different location on the load, such as one sensor in the middle
of the load and the other sensor at the top of the load and kept it in the autoclave.
Operating procedure:
The temperature sensors were placed in the different location in the autoclave chamber
as follow.
01: Right op middle
02: Bottom right front
03: Bottom of load.
04: Right top back
05: Left bottom middle
06: Bottom plate middle
07: Drain
08: Left bottom middle
09: Middle of load.
10: Right bottom back
11: Top left back
12: Left op front
The packed load was placed inside the chamber of the autoclave, along with the
temperature sensors.
Then all the parameters were set for the porous cycle of the autoclave.Page | 84
The cycle was run as set parameters like 20mintes at 121ᵒC.
Valve matrix:
V1- jacket steam
V2- Jacket exhaust
V3-Chamber steam
V4-Chamber exhaust
V5-Chamber vacuum
V6-Chamber vent
V7-Air
V8- Drain
O/C-Open and control
Sr. No. Steps Temp. Mode Pneumatic Valve
1 Jacket steaming 220C Open V1, V2
2 Jacket pressurization 220C Open V1
3 Pre vaccum 600C On/off V1, V5
4 Chamber heating 1000C Open V3
5 Pre vaccum (second Pulse) 1000C Open V5
6 Chamber heating 1210C Open V3
7 Holding 1210C Open V3
8 Chamber cooling 900C Open V4,V6
9 Post vaccum hold 900C On/Off (5 mins) V5
10 Vaccum breaking 600C Open V6
Page | 85
Chart for porous cycle:
Page | 86
5.3.2 Dry heat sterilizer (DHS):
Aim:
To study the heat penetration in the load during DHS.
Procedure:
Preliminary steps:
The pipettes were used as load for the DHS.
The Load was placed inside the canister along with biological indicator ampoule
(Bacillus atropheus).
The parameters for running the DHS were set.
The cycle was started as per as set parameters of 3hours at 180°C.
Operating procedure:
Decontaminate, clean and dry all instruments and other items to be sterilized.
If desired, wrap instruments in aluminum foil or place in a Canister with a tight-fitting,
closed lid.
Wrapping helps prevent recontamination prior to use. Hypodermic or suture needles
should be placed in glass tubes with cotton stoppers.
Place loose (unwrapped) instruments in Canister or on trays in the oven and heat to
desired temperature.
After the desired temperature is reached, begin timing. The following temperature/time
ratios are recommended 180°C for 180 minutes 250°C for 30 minutes 121°C overnight.
After cooling, remove packs and/or Canister and store. Loose items should be removed
with sterile forceps/pickups and used immediately or placed in a sterile container with a
tight fitting lid.
Valve matrix:
Steps Blower Prefilter HEPA filter Fan Heating coil Damper
Drying Open Open Open - Open Open
Heating Close Close Close Open Open Close
Holding - - - - Control -
Page | 87
Cooling Open Open Open Close Close Open
Result:
The load was found sterile after 3hours at 180 oC and the biological indicator used did
not develop in the growth media and the colour change was observed in the chemical indicator
strip place on the load.
5.4 Integrity testing of filters:
Integrity testing sterilizing filters is a fundamental requirement of critical process
filtration applications in the pharmaceutical industry. FDA Guidelines require integrity testing
of filters used in the processing of sterile solutions such as large volume parenterals (LVPs) and
small volume parenterals (SVPs). The FDA also requires corresponding testing documentation
be included with batch product records.
There are three types of non-destructive testing – the bubble point test, the diffusion test,
and water intrusion test to check the integrity of the filters. The pressure hold, forward flow, and
pressure decay tests are variations of the diffusion test. The stringent requirements of the
pharmaceutical industry dictate that nondestructive filter integrity testing must be performed in
each sterilizing application.
5.4.1 Diffusion test
Aim:
To perform integrity testing of hydrophilic filter by diffusion test.
Principle:
At differential gas pressures below the bubble point, gas molecules migrate through the
water filled pores of a wetted membrane following Fick’s Law of Diffusion. The gas diffusional
flow rate for a filter is proportional to the differential pressure and the total surface area of the
filter. At a pressure approximately 80% of the minimum bubble point, the gas which diffuses
through the membrane is measured to determine a filter’s integrity. The flow of gas is very low
in small area filters, but it is significant in large area filters. Maximum diffusional flow
specifications have been determined for specific membranes and devices and are used to predict
bacterial retention test results.
Procedure:
Page | 88
Manual Diffusion Test:
Connect all the air and water inlet line.
Wet the filter according to the size of the filter.
Apply 2.5 bar on upstream line of the filter housing
Take 50 ml measuring cylinder fill it with water and invert the cylinder in beaker filled
with water.
Note down the initial level of water in the measuring cylinder.
Keep the setup for 5 minutes in the beaker.
Note down level increment in air volume and decrement in water volume in the cylinder.
The diffusion volume was calculated by subtracting the initial volume from final
volume.
Result:
The manual diffusion test value obtained and the rate of diffusion across the membrane
was found to be 6 ml/min.
Discussion:
For a 10” filters maximum Diffusion volume should be less than 18 ml/min. Hence the
diffusion rate across the filter was less than the limit, it proves the filter was integral and passed
the Diffusion test.
Automatic Diffusion test:
Connect the strobily connecter to the integrity tester in the filter housing with the
Automatic Sartocheck 3.
Fill the filter housing with water to wet the membrane.
Set the Sartocheck 3 parameters for diffusion test. Such as Test pressure, Stabilization
time, Test time maximum, and Diffusion maximum.
Start the system for carrying out the diffusion test.
The system checks the pressure drop, due to diffusion across the membrane and converts
it in terms of diffusion volume.
Once the system completes the test, the strip recorder indicates the pressure drop, inlet
volume and reference pressure.
Result:
The test result is 8 ml/min. The maximum diffusion volume recommended by
manufacturer for integral filter was 18.0ml/min. Hence the diffusion test was passed indicating
the hydrophilic filter is integral.
Page | 89
5.4.2 Bubble point test:
Aim:
To perform integrity testing of hydrophilic filter by bubble point test.
Principle:
The most widely used non destructive integrity test is the bubble point test. Bubble point
is based on the fact that liquid is held in the pores of the filter by surface tension and capillary
forces. The minimum pressure required to force liquid out of the pores is a measure of the pore
diameter.
Where,
P = bubble point pressure
k = shape correction factor
θ = liquid-solid contact angle
σ = surface tension
Procedure:
Manual Bubble Point Test:
Connect the air line and water line to the filter housing and pressure hold test was
carried out
Wet the filter with 30 l of water.
Keep increasing the pressure slowly at the rate of 0.05 bar/30 seconds.
Take a beaker filled with water and keep the downstream line of the filter dipped in
water.
Increase the pressure uniformly till the bubbles appears continuously in the water filled
beaker.
At one point continuous stream of bubbles starts to flow.
Note down the pressure at which the bubbles arise continuously and the pressure is
called bubble point pressure.
Page | 90
Result:
The manual test value bubble point pressure was found to be obtained was found to be
3.45 bar.
Discussion:
For a 10” filter Minimum bubble point should be more than 3.2 bar. Hence the filter
bubble point was 3.45 bar, it shows the filter was integral.
Automatic Bubble Point test:
Connect the vent line and strobily connector with the Automatic Sartocheck 3.
Wet the filter with water.
Set the Sartocheck 3 parameters like Test pressure, Stabilization time, Test time
maximum,. Maximum Bubble point and Minimum Bubble point.
Start the system.
The system checks the pressure drop in the filter housing and converts it in terms of
bubble point pressure.
Once the system completes the test, the strip recorder indicates the pressure drop, inlet
volume and Bubble point.
Result:
The minimum bubble point pressure is found to be 52.66 psi. The minimum bubble
point pressure recommended by manufacturer for the filter was 46.27psi. So the test is passed
and the filter was integral.
5.4.3 Water intrusion test:
Aim:
To perform integrity testing of hydrophobic filter by water intrusion test.
Principle:
The Water Intrusion test, also known as Water Flow Integrity test, is used for
hydrophobic vent and air membrane filters only. The upstream side of the hydrophobic filter
cartridge housing is flooded with water. The water will not flow through the hydrophobic
membrane. Air or nitrogen gas pressure is then applied to the upstream side of the filter housing
above the water level to a defined test pressure.
Procedure:
Conduct the Pressure Hold Test
Page | 91
Fill the housing with purified water
Drain 30 ml of water
Connect the strobily connector to the integrity tester in the filter housing and Sartocheck
3.
Set all the parameter to carry out the intrusion test in Sartocheck 3.
Start the water intrusion test using Sartocheck 3.
Result:
The water intrusion test gives net volume as 3.6ml/10min. The manufacturer
recommended maximum value is 4ml/10min. Here the test result indicates that filter was
integral.
Page | 92
(Module 3)
ANIMAL CELL
CULTURE
Page | 93
1. INTRODUCTON:
Cell culture is the complex process by which cells are grown under controlled
conditions, generally outside of their natural environment. In practice, the term "cell culture"
now refers to the culturing of cells derived from multi-cellular eukaryotes, especially animal
cells. Animal cell culture became a common laboratory technique in the mid-1900s, but the
concept of maintaining live cell lines (a population of cells derived from a single cell and
containing the same genetic makeup) separated from their original tissue source was discovered
in the 19th century.
History:
The 19th-century English physiologist Sydney Ringer developed salt solution containing
the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the
beating of an isolated animal heart outside of the body. In 1885, Wilhelm Roux removed a
portion of the medullary plate of an embryonic chicken and maintained it in a warm saline
solution for several days, establishing the principle of tissue culture. Ross-Granville Harrison,
working at Johns Hopkins Medical School and then at Yale University, published results of his
experiments from 1907 to 1910, establishing the methodology of tissue culture. Cell culture
techniques were advanced significantly in the 1940s and 1950s to support research in virology.
Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of
vaccines. The injectable polio vaccine developed by Jonas Salk was one of the first products
mass-produced using cell culture techniques. This vaccine was made possible by the cell culture
research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins,
who were awarded a Nobel Prize for their discovery of a method of growing the virus in
monkey kidney cell cultures.
Isolation of cells:
Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily
purified from blood; however, only the white cells are capable of growth in culture.
Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such
as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively,
pieces of tissue can be placed in growth media, and the cells that grow out are available for
culture. This method is known as explant culture.
Page | 94
An established or immortalized cell line has acquired the ability to proliferate indefinitely either
through random mutation or deliberate modification, such as artificial expression of the
telomerase gene. Numerous cell lines are well established as representative of particular cell
types.
Maintaining cells in culture:
Cells are grown and maintained at an appropriate temperature and gas mixture
(typically, 37 °C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary
widely for each cell type, and variation of conditions for a particular cell type can result in
different phenotypes.
Aside from temperature and gas mixture, the most commonly varied factor in culture
systems is the cell growth medium. Recipes for growth media can vary in pH, glucose
concentration, growth factors, and the presence of other nutrients. The growth factors used to
supplement media are often derived from the serum of animal blood, such as fetal bovine serum
(FBS), bovine calf serum, equine serum, and porcine serum. One complication of these blood-
derived ingredients is the potential for contamination of the culture with viruses or prions,
particularly in medical biotechnology applications. Current practice is to minimize or eliminate
the use of these ingredients wherever possible and use human platelet lysate (hPL). This
eliminates the worry of cross-species contamination when using FBS with human cells. hPL has
emerged as a safe and reliable alternative as a direct replacement for FBS or other animal
serum. In addition, chemically defined media can be used to eliminate any serum trace (human
or animal), but this cannot always be accomplished with different cell types.
Cells can be grown either in suspension or adherent cultures. Some cells naturally live in
suspension, without being attached to a surface, such as cells that exist in the bloodstream.
There are also cell lines that have been modified to be able to survive in suspension cultures so
they can be grown to a higher density than adherent conditions would allow. Adherent cells
require a surface, such as tissue culture plastic or Microcarrier, which may be coated with
extracellular matrix (such as collagen and laminin) components to increase adhesion properties
and provide other signals needed for growth and differentiation. Most cells derived from solid
tissues are adherent.
Page | 95
Cell line cross-contamination
Cell line cross-contamination can be a problem for scientists working with cultured cells.
Studies suggest anywhere from 15–20% of the time, cells used in experiments have been
misidentified or contaminated with another cell line. Major cell line repositories, including the
American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC)
and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell
line submissions from researchers that were misidentified by them. Such contamination poses a
problem for the quality of research produced using cell culture lines, and the major repositories
are now authenticating all cell line submissions. ATCC uses short tandem repeat (STR) DNA
fingerprinting to authenticate its cell lines.
Other technical issues
As cells generally continue to divide in culture, they generally grow to fill the available area or
volume. This can generate several issues:
Nutrient depletion in the growth media
Changes in pH of the growth media
Accumulation of apoptotic/necrotic (dead) cells
Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing, known
as contact inhibition.
Cell-to-cell contact can stimulate cellular differentiation.
Genetic and epigenetic alterations, with a natural selection of the altered cells potentially
leading to overgrowth of abnormal, culture-adapted cells with decreased differentiation
and increased proliferative capacity.
Manipulation of cultured cells
Among the common manipulations carried out on culture cells are media changes, passaging
cells, and transfecting cells. These are generally performed using tissue culture methods that
rely on aseptic technique. Aseptic technique aims to avoid contamination with bacteria, yeast, or
other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow
Page | 96
cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and
streptomycin) and antifungals (e.g.amphotericin B) can also be added to the growth media.
As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH
indicator is added to the medium to measure nutrient depletion.
Media changes
In the case of adherent cultures, the media can be removed directly by aspiration, and then is
replaced. Media changes in non-adherent cultures involve centrifuging the culture and
resuspending the cells in fresh media.
Passaging of cells
Passaging (also known as subculture or splitting cells) involves transferring a small
number of cells into a new vessel. Cells can be cultured for a longer time if they are split
regularly, as it avoids the senescence associated with prolonged high cell density. Suspension
cultures are easily passaged with a small amount of culture containing a few cells diluted in a
larger volume of fresh media. For adherent cultures, cells first need to be detached; this is
commonly done with a mixture of trypsin-EDTA; however, other enzyme mixes are now
available for this purpose. A small number of detached cells can then be used to seed a new
culture. Some cell cultures, such as RAW cells are mechanically scraped from the surface of
their vessel with rubber scrapers.
Microcarrier Technology
A microcarrier is a support matrix allowing for the growth of adherent cells in
bioreactors. In 1967, microcarrier development began when van Wezel found that microcarriers
could support the growth of anchorage-dependent cells. Microcarriers are typically 125 - 250
micrometre spheres and their density allows them to be maintained in suspension with gentle
stirring. Microcarriers can be made from a number of different materials including DEAE-
dextran, glass, polystyrene plastic, acrylamide, and collagen, and these microcarrier materials,
along with different surface chemistries, can influence cellular behavior, including morphology
and proliferation. Surface chemistries can include extracellular matrix proteins, recombinant
proteins, peptides, and positively or negatively charged molecules.
Page | 97
Microcarriers are regularly used to grow protein-producing or virus-generating adherent cell
populations in the large-scale commercial production of biologics (proteins) and
vaccines.Microcarrier cell culture is typically carried out in spinner flasks, although other
vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also
support microcarrier-based cultures. The advantages of microcarrier technology in the vaccine
industry include (a) ease of scale-up, (b) ability to precisely control cell growth conditions in
sophisticated, computer-controlled bioreactors, (c) an overall reduction in the floor space and
incubator volume required for a given-sized manufacturing operation, and (d) a drastic
reduction in technician labor.
Applications of cell culture
Toxicity testing
Cancer research
Virology
Cell based manufacturing
Genetic applications
Drug screening and Development.
Page | 98
2. ASEPTIC HANDLING TECHNIQUES
Aim: To check sterility of media transfer and handling techniques.
Principle: This protocol describes basic procedures for aseptic technique for cell culture
technology. One basic concern for successful aseptic technique is personal hygiene. The human
skin harbours a naturally occurring and vigorous population of bacterial and fungal inhabitants
that shed microscopically and ubiquitously. Most unfortunately for cell culture work, cell
culture media and incubation conditions provide ideal growth environments for these potential
microbial contaminants. This procedure outlines steps to prevent introduction of human skin
flora during aseptic culture manipulations. Every item that comes into contact with a culture
must be sterile. This includes direct contact (e.g., a pipette used to transfer cells) as well as
indirect contact (e.g., flasks or containers used to temporarily hold a sterile reagent prior to
aliquoting the solution into sterile media).
Single-use, sterile disposable plastic items such as test tubes, culture flasks, filters, and
pipettes are widely available and reliable alternatives to the laborious cleaning and sterilization
methods needed for recycling equivalent glass items. However, make certain that sterility of
plastic items distributed in multiunit packages is not compromised by inadequate storage
conditions once the package has been opened. Aseptic technique refers to a procedure that is
performed under sterile conditions.
Ideally, all aseptic work should be conducted in a laminar cabinet. However, work space
preparation is essentially the same for working at the bench. Flame sterilization is used as a
direct, localized means of decontamination in aseptic work at the open bench. It is most often
used
(1) To eliminate potential contaminants from the exposed openings of media bottles,
culture flasks, or test tubes during transfers,
(2) To sterilize small instruments such as forceps, or
(3) To sterilize wire inoculating loops and needles before and after transfers. Whenever
possible, flame sterilization should be minimized in laminar-flow environments as the
turbulence generated by the flame can significantly disturb the sterile air stream.
Procedure:
Test-tubes are washed and sterilized by autoclave.
Wear gloves, mask to avoid any contamination.Page | 99
Wipe the hands properly with ethanol.
Wipe the test-tubes, test-tube stand and pipette canister with 70% ethanol and keep it
inside the bio-safety cabinet.
Take nutrient broth flask wipe it with 70% ethanol keepinside the bio-safety cabinet.
Take out pipette form canister without touching the tip with hand.
Attach pipette to pipette controller and take approximately 3 ml and transfer it to test-
tube. Same way do for all the test tubes.
Mop up any spillage immediately and swab the area with 70% alcohol.
Remove everything when you have finished, and swab the work surface down again.
Now allow the test-tubes for incubation at 370 c for 24-48 hrs.
Observe the tubes after 24 and 48 hrs.
Observation:
No growth was observed in test-tubes after 24 and 48 hrs.
Result:
The handling was proper and media transfer was found to be sterile inside the bio-safety
cabinet.
Page | 100
3. SETTLE PLATE TEST
Aim: To identify the amount of bio-burden present in the working environment using settle
plate test.
Introduction:
A major consideration in the operation of clean room technology for aseptic dispensing
is the monitoring of viable contamination within clean environments. The bio-burden analysis is
carried out using common techniques like settle plate test. Settle plate sampling is a direct
method of assessing the likely number of microorganisms depositing onto the product or surface
in a given time. It is based on the fact that, in the absence of any kind of influence, airborne
microorganisms, typically attached to larger particles, will deposit onto open culture plates.
Microorganisms are usually found in the air of occupied rooms grafted onto skin cells with very
few present on their own. The average size of microbial particle will deposit, by gravity, onto
surfaces at a rate of approximately 1 cm/s. In settle plate sampling Petri dishes containing agar
medium are opened and exposed for a given period of time, thus allowing microbe-bearing
particles to deposit onto them. Petri dishes are most commonly used. The number of microbe
bearing particles deposited onto the agar surface of the plate over the period of exposure is
ascertained by incubation of the plate and counting the number of microbial colonies, more
commonly known as colony forming units (cfu). The microbial deposition rate may be reported
as the number depositing in a given area per unit time.
The Petri plates containing media are placed in the different areas where a
microbiologically controlled environment is specified should be monitored. Sample locations of
plates in the critical work zone should be selected with reference to the actual work area and its
availability. Monitoring critical areas should be carried out under "worst case" conditions for
contamination with process equipment running and personnel performing normal operations.
Sample locations for settle plates in clean rooms should include areas where there is little air
movement (i.e. dead spaces) or where airflows converge or are excessively turbulent. The areas
where these conditions are most likely to occur are adjacent to doors and in corners of rooms.
Page | 101
Requirements:
Media : Nutrient agar
Glassware: Petri plates
Procedure:
Nutrient agar and petri plates were prepared and autoclaved at 121 oC for 20 min.
The plates were prepared by pouring the nutrient agar onto the petri plates and cooled
under aseptic condition.
The plates were examined for contamination prior to use.
Then the plates were taken into the lab, where the lab conditions need to be tested for the
bio-burden level.
The plates are properly marked at the base to ensure that the correct location of plates
during exposure and incubation.
The plates were then transferred into the area/room/cabinet where they are to be exposed
into environment.
It was placed in different position of the room as mentioned below in table, with the lids
still on.
The lids are then raised to expose the surface of the medium, the lid was rested on the on
the very edge of the plate so that the entire agar surface is completely exposed.
The plates were exposed for 2 hours in the lab condition.
After exposure the lids of the plates were closed and taken for incubation.
The plates were incubated at 37 oC and observed for number of colonies at different time
interval like 24 and 48 hour.
Observation:
Surface Area of Petri plates
Diameter of petriplate = 9cm Radius of petriplate = 4.5
Therefore Surface Area of Petri plates = πr2 = 3.14 × (4.5) 2 = 63.585 cm2
Surface Area of BSC Cabinet = 33×38.5= 1270.5 cm2
Surface Area of Room= 181460 cm2
Bio-burden = Surface area of the room x Number of colonies
No. of petriplates exposed x surface area of petriplates
Page | 102
Tabular column:
Sr No. LocationNumber of colonies
after 24 hours
Number of colonies
after 48 hours
Bio-safety Cabinet
1. Back Left 0 0
2. Back Right 2 2
3. Front Centre 0 0
4. Front Left 0 0
5. Front Right 0 0
6. Middle Centre 0 0
7. Middle Left 0 0
8. Middle Right 0 0
9. Grill Centre 0 0
Room
1. Near Bioreactor PLC 4 9
2. Door 15 21
3. Centre of the Room 10 18
4. Near Incubator 15 19
5. Near Microscope 6 9
Result:
Bioburden of BSC after 24hrs = (1270.5 × 2)÷(9 × 63.58) = 4.43 cfu
Bioburden of BSC after 48hrs = (1270.5 × 2)÷(8 × 63.58) = 4.99 cfu
Bioburden of Room after 24hrs = (181460× 50)÷(5 × 63.64) = 28.54 × 103 cfu
Bioburden of room after 48hrs = (181460 × 76)÷(5 × 63.64) = 43.381 × 103 cfu
Result :
The bioburden of the room was calculated after 24hrs and 48 hrs was found to be 28.54 × 103
cfu and 43.381 × 103 cfu respectively. Whereas the biosafety cabinet was observed to at
maximum sterility as the bioburden inside the cabinet, was found to be 4.43 cfu and 4.99 cfu for
24 hrs and 48 hrs respectively, hence verifying that the biosafety cabinet is sterile for workPage | 103
4. MEDIUM PREPARATION AND FILTRATION
Aim:
To prepare and filter the GMEM medium
Introduction:
Glasgow's Minimum Essential Medium is a modification of Basal medium Eagle
(BME). Ian Macpherson and Michael Stoker added tryptose phosphate broth and twice the
concentration of amino acids and vitamins to BME. Glasgow's MEM was originally formulated
with 10% tryptose phosphate broth. Glasgow's MEM contains no proteins, lipids, or growth
factors. Therefore, Glasgow's MEM requires supplementation with 10% tryptose phosphate
broth. Glasgow's MEM uses a sodium bicarbonate buffer system (2.75 g/L), and therefore
requires a 5–10% CO2 environment to maintain physiological pH. Glasgow's MEM was
developed for use with kidney cell lines, such as BHK-21.
Composition of GMEM media:
Ingredients (mg/L):
Inorganic Salts:
Calcium chloride dehydrate - 265.0
Ferric nitrate anhydrate - 0.10
Magnesium sulphate anhydrous - 97.720
Potassium chloride - 400.0
Sodium chloride - 6400.0
Sodium dihydrogen phosphate anhydrous- 109.0
Amino Acids:
L-Arginine hydrochloride - 42.000
L-Cystine - 24.000
L-Glutamine - 292.000
L-Histidine hydrochloride - 21.000
L-Isoleucine - 52.400
L-Leucine - 52.400
L-Lysine hydrochloride - 73.100
L-Methionine - 15.000
L-Phenylalanine - 33.000
L-Threonine - 47.600
Page | 104
L-Tryptophan - 8.000
L-Tyrosine disodium salt - 52.000
L-Valine - 46.800
Vitamins:
Choline chloride - 2.000
D-Ca-Pantothenate - 2.000
Folic acid - 2.000
Nicotinamide - 2.000
Pyridoxal hydrochloride - 2.000
Riboflavin - 0.200
Thiamine hydrochloride - 2.000
I-Inositol3.600
Others:
D-Glucose - 4500.000
Phenol red sodium salt - 15.000
Tryptose Phosphate Broth - 2950.000
Composition of media for 1000ml:
S. No Ingredients Quantity
1. GMEM media 12.50 g
2. Sodium bicarbonate 1.2 g
3. Penicillin 75 mg
4. Streptomycin 100mg
5. Neomycin 0.050ug
6. Tryptose phosphate broth 2.95 g
7. Pluronic F68 10 ml
8. Antifoam 2 ml
9. Amphotericin --
Media preparation:
Weigh and dissolve the all the above ingredients of GMEM media in WFI.
As cell culture media are not heat sterilizable, so GMEM media was sterilized using
filter sterilization.
Page | 105
The filter sterilization was carried out using 0.2 µm sterile grade filter cartridge mounted
on filter housing assembly.
Filtration:
Filtration through 0.1- to 0.2-μm microporous filters is the method of choice for
sterilizing heat-labile solutions .Filtration may be carried out by positive pressure from a
pressurized container or with a peristaltic pump High pressure filtration from a pressure vessel
is faster than a peristaltic pump but will require a collecting receiver as the flow cannot be
regulated easily, whereas a peristaltic pump can be switched on and off during collection. High
pressure also tends to compact the filter and is unsuitable for viscous solutions such as serum.
Negative-pressure filtration is often simpler, particularly for small scale operations, and will
collect directly into storage vessels. It may cause an elevation of the pH, however, because of
evolution of CO2.
Procedure:
Media was prepared according to above composition.
Filter housing assembly with 4 inch 0.2 μm cartridge filter was attached to the housing
stand.
Silicone tubing’s were attached at the downstream side and upstream sides of filter
housing.
500 ml Schott Duran bottles with, cut caps having two way port, draw tubes and
hydrophobic disc filters were attached.
Allow the whole assembly to autoclave at 1210c for 20 minutes.
After autoclaving of assembly allow it to cool at room temperature.
Now keep the downstream portion in the bio-safety cabinet and change the cap of
downstream bottle with empty bottle without touching the drawtube anywhere.
From upstream side connect the airline to the silicone tube attached through the filter.
Change the cap of upstream bottle with media bottle. Give the positive pressure.
Filter full media in sterile bottle inside the biosafety cabinet and keep it for charging into
Bioreactor.
After filtration do the CIP of filter with water.
Page | 106
Result:
The GMEM media was prepared and filter sterilized successfully and stored at 4 – 8 oC
for further process.
5. SUB-CULTURING OF BHK-21 CELLS
Aim:
To subculture BHK-21 cells from monolayer in T25 flasks.
Introduction:
The first subculture represents an important transition for culture. The need to subculture
implies that the primary culture has increased to occupy all of the available substrate. Hence,
cell proliferation has become an important feature. Although the primary culture may have a
variable growth fraction, depending on the type of cells present in the culture, after the first
subculture, the growth fraction is usually high (80% or more). From a very heterogeneous
primary culture, containing many of the cell types present in the original tissue, a more
homogeneous cell line emerges. In addition to its biological significance, this process has
considerable practical importance, as the culture can now be propagated, characterized, and
stored, and the potential increase in cell number and the uniformity of the cells open up a much
wider range of experimental possibilities. Once a primary culture is sub-cultured (or passaged),
it becomes known as a cell line.
Procedure:
Prepare the hood, and bring the reagents and materials to the hood to begin the
procedure.
T25 Poly-lysine coated flask having confluent cells was taken.
Take the culture flask to a sterile work area, and discard the medium from the flask.
5 ml of PBS was added as pre-wash to the side of the flasks opposite the cells so as to
avoid dislodging cells, rinse the prewash over the cells, and discard. This step is
designed to remove traces of serum that would inhibit the action of the trypsin and
deplete the divalent cations, necessary for cell adhesion.
1ml trypsin was added to the side of the flasks opposite the cells. Turn the flasks over
and lay them down. Ensure that the monolayer is completely covered. Page | 107
Leave the flasks stationary for 15–30 s and keep the flask for incubation in incubator for
5 minutes.
Flask were taken out after 5 minutes and observed under phase contrast microscope for
dislodging of cells from substrate.
4 ml of fresh medium was added, and disperse the cells by repeated pipetting over the
surface bearing the monolayer. Finally, pipette the cell suspension up and down a few
times, with the tip of the pipette resting on the bottom corner of bottle, taking care not to
create foam.
Then discard 4 ml from the suspension and add 1 ml of the suspension into the same T 25
flask and 4 ml of fresh media was added to it and incubated at 37 oC in CO2 incubator.
The flask was observed for its confluence on the following day for checking the growth
of the cells using 4X magnification phase contrast microscope.
Result:
When the flask was 70-80% confluence, the flask was successfully sub-cultured for
maintaining the cell growth.
Page | 108
6. GROWTH CURVE STUDIES
Aim:
To study the different phases of animal cell growth.
To plot standard growth curve of BHK 21 cells.
Introduction:
The increase in the cell size and cell mass during the development of an organism is
termed as growth. It is the unique characteristics of all organisms. The organism must require
certain basic parameters for their energy generation and cellular biosynthesis. The growth of the
organism is affected by both physical and Nutritional factors. The physical factors include the
pH, temperature, Osmotic pressure, Hydrostatic pressure, and Moisture content of the medium
in which the organism is growing. The nutritional factors include the amount of Carbon,
nitrogen, Sulphur, phosphorous, and other trace elements provided in the growth
medium. When the cells reach a certain size, they divide by binary fission, in which the one cell
divides into two, two into four and continue the process in a geometric fashion. The cell is then
known to be in an actively growing phase. When animal cells are seeded into a T 25 flask and
incubated, cells starts attaching to the substrate and grow. But in case of animal cells this
adaption of cells (lag phase) takes more time compared to microbial cells. When the flask
becomes confluent, subculture of cells should be done.
Growth kinetics is the science that relates growth rates to the nutritional concentrations
on which the cells depend. It can be divided into 2 processes, firstly, the kinetics of
concentrated limited accumulation of nutrients and secondly, the efficiency that those nutrients
provide for growth. The determination of kinetics of growth is important in designing routine
subculture and various experimental protocols. At each stages of growth there are changes in
the cell behaviour and biochemical constituents.
The growth in close system is observed by the rate of proliferation of a population and in
open system growth is observed by the rate at which a population can be removed without
disturbing the steady state or by injected trace movement through it. The shape of growth curve
can also give information on the reproductive potential of the culture where differences in
growth rate, adaption or survival and density limitation of growth. Population of animal cells
cultured in vitro increase in number as the individual cell divides mitotically whether they are
primary cells, cell strains or established cell lines. Multiplication starts only after a period of
Page | 109
adjustment and stops when a number is reached that is saturating for the system. These phases
are best studied by representing it graphically.
If the logarithms of the number of cells present at any time in the culture are plotted
against the time interval in hours, a sigmoid growth curve of the culture can be obtained. The
growth curve of mammalian cell culture displat the same type of growth pattern of micro-
organism and thus similarly the growth can be divide into four phases – lag phase, log phase or
exponential phase, stationary phase and death phase.
Lag phase
Lag phase is the first stage of growth and is the time taken by the cells to adopt itself to
grow in fresh environment in the culture medium. After the culturing the cell population
decreases slightly probably because while attaching to the solid surface some cells are lost due
to incapability of adherence or other factors. Later the cells adapt itself in the new environment
and cells try to adjust ton new conditions like new medium, serum concentration, type of culture
vessels/surface and cell density etc. these factors determine the length of lag phase, however, it
may take 24 to 48 hours. There may be increase in cell size but practically there is no division
of cells in this phase. If the cells are cultured initially at too low density these never enter into
log phase. In general, the duration of this is long but varies according to the following
conditions.
When the inoculum is small and duration is longer
If the medium and temperature are unfavourable, generation time of lag phase prolongs.
If the inoculum is from lag, stationary or decline phase lag phase is long. On the other
hand cells multiply without lag phase when inoculum is from log phase.
Log phase or exponential phase
Lag phase is followed by log phase and during this period, regular and maximum growth
of cells occurs. The number of cells increase exponentially. Here changes in biochemical and
respiratory activities of cells also occur. In this phase the medium is rich in nutrients, space for
growth is enough and thus there is neither competition for nutrients nor contact inhibition. The
population doubling time for cultured cells ranges between 12 to 36 hours. A proportion of cells
stops dividing when the essential nutrient is depleted or an inhibiting substance is produced. At
this stage cells enter the stationary phase.
Stationary phase
Page | 110
In this phase due to exhaustion of nutrients and limiting of growth space as cells
approach confluence, rate of division slows down and thus this phase has a constant number of
cells. At this stage the maximum number of cells that can be grown per unit volume of the
medium can determined. The saturation density represents the density at which the cells can no
longer grow exponentially and it will vary depending upon the conditions used. This prevails
for sometime because either cell division ceases in all cells or some degenerate and die and
other continue to divide.
Death phase
In this phase there is rapid decline in the number of cells as division of cells practically
ceases as nutrients are completely exhausted and there is accumulation of metabolites.
Figure: Growth curve of animal cell
Quantification of culture is also important in routine maintenance, as it is a crucial
element for monitoring the consistency of the culture and knowing the best time to subculture,
optimum dilution, estimated plating efficiency at different cell densities, testing of medium,
serum, new culture vessels or substrates and so forth all the required quantitative assessment.
Requirements:
Monolayer culture of BHK 21 cell line
1X Trypsin
GMEM medium
PBS
6 well plate
Haemocytometer
Equipment’s:
Page | 111
Bio-safety cabinet
CO2 incubator
Phase contrast microscope
Procedure:
T25 flask with confluent BHK21 cells was trypsinized and made into suspension.
From the suspension small amount was taken out and the cell count was performed
using haemocytometer.
The total concentration of cells in the suspension was calculated to plate the desired
concentration of cells.
Based on cell count, 12 ml of suspension was prepared by adding 10% serum containing
GMEM medium and the final cell concentration was made to 2X105 cells/ml.
2ml of this cell suspension was seeded into each well of 6 well plate.
The plate was incubated in CO2 incubator at 370C.
Next day (Day 1) cells from the first well were trypsinized and cell count was
performed.
Similarly, on 2nd day, 2nd well’s cells were trypsinized and cell count was performed.
The above step was continued till last well in the 6 well plate.
The cell concentration was calculated and tabulated for all the days.
The cell concentration against time was plotted to obtain growth curve.
Observations:
Sr.No Days Time (in hrs.) Cell Count (in cells/mL)
1. Day 0 - 1 x105
2. Day 1 22:15 0.58 x105
3. Day 2 46:30 2.95 x105
4. Day 3 70:25 10.4 x105
5. Day 4 98:45 20.8 x105
6. Day 5 164:45 5.8 x105
Page | 112
0 1 2 3 4 5 6 7 80
5
10
15
20
25
1 0.582.95
10.4
20.8
5.8
Growth Curve
Cell Count
Time in days
cell
coun
t(ce
ll/m
l)
0 1 2 3 4 5 6 7 80
5
10
15
20
25
1 0.582.95
10.4
20.8
5.8
Growth curve with generation no. & doubling time
Cell Count
Time in hrs
cell
coun
t(ce
ll/m
l)
Generation-3
Generation-2
Generation-1
Page | 113
Calculation:
Cell count of day 1:
Squares- 1 2 3 4
Cells- 1 0 2 0
1+0+2+0/4 = 3/4 x 2x104 =x 104 = 1.5 x105
Likewise all days cell count done as shown in table.
1. Generation no between day1 and day2
Generation no. = 2n =Nh/Ni
n = 3.32[log Nh – log Ni]
Generation no between day2 and day4 i.e doubling time
n = 3.32[log Nh – log Ni]
Growth curve was established, and generation no., doubling time, different phases of
growth were studied. The initial concentration of cells was 1 x105 cells/mL. After 24hr.
incubation cells population i.e. cell count was 0.58 x105cells/mL, during next 48 hr. &72 hr.the
cell number was increased i.e. 2.95 x105cells/mL, 10.4x105 cells/mL respectively. After next
24hr. incubation the cell number was 20.8 x105 cells/mL, i.e. the highest growth of cells.There
was a decline in the cells population in the last 48hr.(160hr. post incubation) i.e. 5.8
x105cells/mL.The Generation No. we got was 1.793 & doubling time was 24hr.
Page | 114
7. OPTIMIZATION OF SERUM PERCENTAGE
Aim:
To optimize the % of serum required in cell culture media for the effective growth of the
BHK cells.
Introduction:
The ideal characteristic of the cell culture medium is to simulate the exact in vivo
conditions for the growth of animal cells. This exact condition can be only achieved to a limited
extent, as physiological conditions required for the growth of animal cells are generally
complex. The basic requirement for the cell to grow involves complex proteins, vitamins,
growth factors etc. serum provides all of the growth factors, co-factors, hormones, attachment
factor (fibronectin, laminin), transport factors (albumin, globulin, transferrin), nutrients
(nucleosides, amino acids, fatty acids and lipids), trace elements and other factors. Serum is a
very complex product and only a small percentage of the components have been fully identified.
Most sera used in cell culture are from some form of animal, mainly bovine origin such as fetal
bovine serum or fetal calf serum. Fetal bovine serum (FBS) is the blood fraction remaining after
the natural coagulation of blood, followed by centrifugation to remove any remaining red blood
cells. Fetal bovine serum is the most widely used serum-supplement for the in vitro cell
culture of eukaryotic cells. This is due to it having a very low level of antibodies and containing
more growth factors, allowing for versatility in many different cell culture applications. The
rich variety of proteins in fetal bovine serum maintains cultured cells in a medium in which they
can survive, grow, and divide.
The bovine based sera bring some disadvantages such as antibodies which may impair
or damage the cell growth, the possibility of presence of animal viruses and the possible
contamination with endotoxins and mycoplasmas which can damage fragile cell lines. However
modern techniques used both in collection of blood, vastly improved production and filtration
techniques have resulted in much higher quality sera free from endotoxins and mycoplasmas.
The sera are generally used mainly for the two purposes, such as additive to the cell culture
media and for inactivation of the trypsin after trypsinization process. Each cells lines need
different amount of serum for their effective growth in the media, so the amount of serum to be
added to the media need to be optimized for getting good cell growth at effective serum
concentration.
Page | 115
Materials required:
6 well plates
Filter sterilized GMEM media
Fetal bovine serum
Trypsin
Phosphate buffer saline (PBS)
Pipettes
Hemocytometer
Trypan blue
Phase contrast microscope
Procedure:
The T25 flask (< 90% confluent) was used as the seed flask for preparing the 6 well
plates. The T25 flask was trypsinized with 1 ml trypsin for the detachment of cells from
the substrate.
4 ml of media containing serum was added to the trypsinized flask for the inactivation of
the trypsin activity.
The cell suspension was properly mixed and 0.5ml of the cell suspension was taken for
the cell count using Hemocytometer.
The cell count was calculated and the plates need to be inoculated with the initial
seeding density of ~ 1.5 X 105 cells/ml, so the appropriate amount of cell suspension
need to be taken for preparing the each plate was calculated.
Each well in the 6 well plates, need to add with 2 ml media and cell suspension mixture,
so for each 6 well plate 14 ml of media and cell suspension mixture was prepared.
For the optimization studies different % of serum such as (5%, 7% and 10%) was taken,
and the media was prepared with different serum concentration.
The appropriate amount of seed suspension as calculated before was added into the
media containing different serum concentration.
The media and cell suspension mixture was dispersed completely and 2 ml of the
mixture was distributed into all the wells in the 6 well plates, each time before
transferring into the wells mix it thoroughly.
The plates were marked and incubated at 37 oC in 5% CO2 incubator.
Page | 116
After 24 hours, one well from each plates need to be trypsinized for counting the number
of cells in that well of the 6 well plates containing different serum concentration and
noted down.
Then the above step was followed for the remaining 5 wells in the 6 well plates with the
interval of 24 hours and their respective cell count was noted down.
The cell count of all the 6 well plate at different time interval was compared and
tabulated.
The 6 well plates with different serum concentration, which yielded highest number of
cells was taken as optimized serum concentration for the further studies.
Observation:
Sr.No. DaysTime (in
hrs.)Cell Count (in cells/mL)
5% 7% 10%
1. Day 0 - 0.5×105 0.5×105 0.5×105
2. Day 1 24 0.26×105 0.21×105 0.29×105
3. Day 2 48 0.525×105 0.59×105 1.0×105
4. Day 3 72 3.5×105 3.63×105 2.8×105
5. Day 4 96 5.0×105 2.83×105 5.53×105
Page | 117
Graph:
- 24 48 72 960
1
2
3
4
5
65%7%10%
Time in hours
cell
coun
t (ce
lls/m
l)
Result :
Cell count is highest in the wells containing 10% serum, and is least in the wells with
5% serum.
Interpretation :
Since serum promotes all the necessary growth factors and adhesion factors for the cells
to proliferate, cell growth is highest in the wells with 10% FBS. Due to less amount of
FBS in 5%, cell growth is least compare to 7% and 10%. After testing the concentration
of serum, where, the cell growth is more, can be useful in large scale cultures.
Note : Other than preparing aliquots of 5%, 7% and 10% serum, there is one more
method. That is, addition of serum to all 24 wells where, they contain already the cells.
If all the wells contain 1ml of cells, add 20µl of FBS to the wells marked 5%, 50µl FBS
to 7% wells and 100µl FBS to 10% wells. Then, keep the plate for incubation at 370C.
8. CRYOPRESERVATIONPage | 118
Aim:
To cryopreserve cells to obtain maximum survivability upon thawing.
Introduction:
Cryopreservation is a process where cells or whole tissues are preserved by cooling to
low sub-zero temperatures, such as (typically) 77k or -196c(the boiling point of liquid nitrogen).
At these low temperatures, any biological activity, including the biochemical reactions that
would lead to cell death, is effectively stopped. Cryopreservation techniques utilize very low
temperatures to preserve the structure and function of living cells. Various strategies have been
developed for freezing mammalian cells of biological and medical significance.
Principle:
Optimal freezing of cells for maximum viable recovery on thawing depends on
minimizing intracellular ice crystals formation and reducing cryogenic damage from foci of
high concentration solutes formed when intracellular water freezes.This is achieved
By freezing slowly to allow water to leave the cell but not so slowly that ice crystal
growth is encouraged,
By using a hydrophilic cryoprotectant to sequester water,
By storing the cells at the lowest possible temperature to minimize the effects of high
salt concentrations on protein denaturation in micelles within the ice, and
By thawing rapidly to minimize ice crystal growth and generation of solute gradients
formed as the residual intracellular ice melts.
Materials required:
Monolayer BHK culture
Trypsin
GMEM
Cryoprotectant agent - 7.5%DMSO
10% SERUM
GMEM (2X)
Biosafety cabinet
Incubator
Water bath
Nitrogen tank
Page | 119
Refrigerator
Deep freezer
Cryovials
Procedure:
1. Crudemethod:
Preparation of cryoprotective agent of 2X concentration.
Cell suspension with CPA should be kept 15 to 60 min at room temperature. This time is
called equilibration.
Transfer the vial to 40C for 60mins.
Transfer the vial to upper portion of the insulated chamber containing liquid nitrogen.
Then finally store the vial in liquid nitrogen at -1960C.
For revival of cells take a vial from cryocan and transferred for thawing in water bath at
37ºC.
After thawing transfer cell suspension to centrifuge tube and add 8.25ml of GMEM
media.
Centrifuge at 1000 rpm for 10 min.
Discard the supernatant and again add 8.25ml of GMEM and mix well.
Cell counting- Before cryopreservation (Pre freeze count)
After cryopreservation (Post freeze count)
2. Stepwise method:
Preparation of cryoprotective agent of 2X concentration.
Cell suspension with CPA should be kept 15 to 60 min at room temperature. This time is
called equilibration.
Transfer the vial to 40C for 60mins.
Then transfer the vial at -200C for 60mins
Then transfer the vial at -800C for 60mins
Then finally store the vial in liquid nitrogen at -1960C.
For revival of cells take a vial from cryocan and transferred for thawing in water bath at
37ºC.
After thawing transfer cell suspension to centrifuge tube and add 8.25ml of GMEM
media.
Centrifuge at 1000 rpm for 10 min.
Page | 120
Discard the supernatant and again add 8.25ml of GMEM and mix well.
Cell counting: Before cryopreservation (Pre freeze count)
After cryopreservation (Post freeze count)
Result:
The given monolayer culture was successfully cryopreserved at -1960C and on revival
was done.
Discussion:
The cells can be stored for longer time by cryopreservation technique using liquid
nitrogen at -1960C. The cells can be reused simply by thawing the vial.
9. REVIVING OF CELLS
Page | 121
Aim:
Revival of cells.
Materials required:
Cryovial containing frozen cells
Complete growth medium, pre-warmed to 37°C
Disposable, sterile centrifuge tubes
Water bath at 37°C
70% ethanol
Tissue-culture treated flasks
Principle:
Unlike the freezing process, rapid thawing of frozen cells is necessary to maintain
viability. Certain precautions should be exercised when thawing cells. Vials stored in liquid
nitrogen, especially screw capped tubes, often fill with liquid nitrogen while submersed. When
these tubes are removed from the tank, the tubes may pressurize and burst. Thus, a face shield
or goggles should be worn while thawing cells. Vials stored in cryogenic freezers are a reduced
risk of bursting. Directly after removal from storage, vials should be thawed with agitation (but
not for fragile hybridoma cells) in a 37°C water bath. As the last ice crystals are melting, the
vial is removed from the water. Wipe, spray, or submerse the vial with 70% ethanol before
opening it in a biosafety hood. It is prudent when working with an unfamiliar cell line to
determine the percentage of viable cells recovered by Trypan Blue staining. This may serve to
uncover any deficiencies in the cryopreservation process. Note that safety precautions must be
taken when recovering vials from the liquid nitrogen. Insulated gloves should be worn to
protect against burns from the low temperatures. Though specially-designed cryovials are used
to store cells, a face shield and laboratory coat serve to protect against fragments of exploding
vials caused by introduction of liquid nitrogen (an all too common occurrence with leaky vials).
Procedure:
Remove the cryovial containing the frozen cells from liquid nitrogen storage and
immediately place it into a 37°C water bath (gentle agitation).
Transfer the vial it into a laminar flow hood. Before opening, wipe the outside of the
vial with 70% ethanol.
Page | 122
After thawing transfer cell suspension to centrifuge tube and add 8.25ml of GMEM
media.
Centrifuge at 1000rpm for 10 min.
Remove the supernatant, resuspend the pellet with fresh media and take out cell aliquot
for cell counting
Transfer to the monolayer flask.
Calculation:
%Viability = Post freeze count / Pre freeze count X 100
1. Crude method:
Pre freeze count = 2.9X106 cells/ml
Post freeze count= 2.56X106cells/ml
% Viability= 2.56X106/ 2.9X106 X 100
= 88.27%
2. Stepwise method:
%viability of cell suspension which was immediately transferred from -20ºC to -196ºC
Pre freeze count = 8.25X106 cells/ml
Post freeze count = 1.75X104cells/ml
%Viability = 1.75X104/ 8.25X106 X 100
=0.2%
Result:
Survivability during crude method was seen about 88.27%.
10. MTT ASSAY
Aim:
Page | 123
To perform cytotoxicity assay using MTT (3-(4, 5-Dimethyl-2-thiazolyl-2-yl)-2,5-
diphenyl-tetrazolium bromide)
Principle:
This is a colorimetric assay that measures the reduction of yellow 3-(4, 5-
dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate
dehydrogenase.MTT reacts with enzyme in mitochondria where it is reduced to an insoluble,
colored (dark purple formazan crystals. Since reduction of MTT can only occur in metabolically
active cells absorbance measured is directly proportional to viability of the cells.
Materials required:
Monolayer culture
One 6 well plate, centrifuge tubes, pipettes and pipette controller.
UV-Spectrophotometer.
Chemicals required:
GMEM media
MTT (5mg/ml)
DMSO
Solubilizing agent- Isopropanol, concentrated hydrochloric acid and TritonX100.
Procedure:
1. Different cell concentrations were prepared in centrifuge tubes using GMEM as
diluent.
2. Control was plated with only media, devoid of cells.
3. The plate was incubated at 37ºC for overnight to get monolayer culture of cells.
4. MTT solution was prepared and sterile filtration was performed.
5. Next day, about 200µl of MTT solution was added to each well.
6. The six well plate was covered and sealed at 37ºC for 4hrs.
7. After incubation purple color formazan crystals were formed to dissolve this
solubilizing agent was added to each well and mixed.
8. Readings (OD) was taken at 570nm.
9. Standard Graph was plotted by taking cells/ml in x-axis and OD in y-axis.
Observation:
Page | 124
Concentration O.D.
A
(2×106cells/ml)
2.89
B
(1×106cells/ml)1.42
C
(0.5×106cells/ml)0.75
D
(0.25×106cells/ml)0.45
UK
(Unknown)0.62
Standard graph:
The standard curve indicates a linear response between cell number and absorption at 570 nm.
Result:
As per the graph, the cell concentration of the unknown is 0.4×106cells/ml
Page | 125
Figure: dark purple formazan crystals
11. KARYOTYPING
Aim: To perform the karyotyping of the given cell line.
Background information:
The genetic material, DNA, exists within the chromosomes and contains the entire
genetic blueprint for the development of an individual. Most normal human cells contain
identical numbers and types of chromosomes. The analysis of chromosomes has allowed
researchers to identify the cause of abnormalities.Each chromosome pair contains unique
physical attributes, which distinguishes them from the 22 other pairs. The three main criteria
used to identify individual chromosomes include:
1. The length of the chromosome.
2. The position of the centromere.
3. Banding patterns on the chromosome which appear after staining.
Using these criteria, geneticists have set up a classification system, which labels each
chromosome by number. Many genetic disorders have been associated with alterations of the
chromosomes an individual possesses. In some instances, pieces of chromosomes may be
transferred (translocation). On other occasions, pieces of chromosomes may break off and be
lost entirely (deletion). Another possibility is that entire chromosomes may be lost or added to
Page | 126
an individual’s chromosome arrangement. Analyzing an individual’s chromosome by doing
what is called a karyotype can identify any of these situations.
Chromosome banding:
Chromosomes display a banded pattern when treated with some stains. Bands are
alternating light and dark stripes that appear along the lengths of chromosomes.
Unique banding patterns are used to identify chromosomes and to diagnose chromosomal
aberrations, including chromosome breakage, loss, duplication, translocation or inverted
segments.
A range of different chromosome treatments produce a range of banding patterns: Q-
bands, G-bands, R-bands, C-bands, NOR-bands and T-band.
Q-Banding:
Quinacrine mustard, an alkyl ting agent, was the first chemical to band chromosomes
viewed under a fluorescence microscope. Quinacrine dihydrochloride has subsequently been
substituted by Quinacrine mustard. The alternating bands of bright and dull fluorescence are
called Q bands.
Q bands are especially useful for distinguishing the human Y chromosome and various
chromosome polymorphisms involving satellites and centromere of specific chromosomes.
G-banding:
Giemsa has become the most commonly used stain in human cytogenetic analysis.
Unlike Q-banding, G-banding usually requires pre-treating chromosomes with either salt or a
proteolytic (protein-digesting) enzyme. When chromosomes are pre-treated with the proteolytic
enzyme trypsin the process is called GTG banding. Giemsa stains preferentially regions rich in
adenine and thymine. Therefore, G bands correspond closely to Q bands.
Standard G band staining techniques allow between 400 and 600 bands to be seen on
metaphase chromosomes. With high resolution G-banding techniques, as many as two thousand
different bands have been catalogued on the twenty-four human chromosomes.
R-banding:
Page | 127
Reverse banding (R-banding) involves the incubation of slides containing metaphase
chromosomes in hot phosphate buffer and stained with Giemsa. The banding pattern that results
is essentially the reverse of G bands. R bands are GC-rich. The AT-rich regions are selectively
denatured by heat leaving the GC-rich regions intact. Fluorochromes that are GC specific also
produce a reverse chromosome banding pattern. R-banding is helpful for analyzing the structure
of chromosome ends, since these areas usually stain light with G-banding.
C-Banding:
C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA.
C-banding is specifically useful in humans to stain the centromeric chromosome regions and
other regions containing constitutive heterochromatin - secondary constrictions of human
chromosomes 1, 9, 16, and the distal segment of the Y chromosome long arm.
NOR-banding:
NOR-banding involves silver staining (silver nitrate solution) of the "nucleolar
organizing region", which contains rRNA genes.
T-Banding:
T-banding involves the staining of telomeric regions of chromosomes using either
Giemsa or acridine orange after controlled thermal denaturation. T bands apparently represent a
subset of the R bands because they are smaller that the corresponding R bands and are more
strictly telomeric.
Principle:
Karyotyping is a test to examine chromosomes in a sample of cells, which can help
identify genetic problems as the cause of a disorder or disease. This test can:
Count the number of chromosomes
Look for structural changes in chromosomes.
Microtubule distractive drugs like vinblastine, colemide, nocodazole have been reported to
act by two concentrations they suppress microtule dynamics and at higher concentration they
reduce microtubule polymer mass. Recent findings indicate that they also produce microtubule
fragment by stimulating microtubule minus-end detachment from their organizing centers.
Metaphase block:
Colchicine (0.1µg) are used for metaphase arrested.
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During of the metaphase block may be increased to give more metaphase for chromosome
counting or shortened to reduce chromosome condensation and improve banding.
Hypotonic treated:
Substitute KCL alone used for the hypotonic citrate.
The duration of the hypotonic treated may be varied from 5 min to 30 min to reduce lysis or
increase spreading.
Spreading:
There are perhaps more variations at this stage than any other, all designed to improve
the degree and flatness of spread. They include,
Make the slide ultra cold.
Dropping cells onto a slide from a greater height and placing the slide over a beaker of
boiling water.
Materials required:
Sterile or aseptically prepared:
Culture of cells in log phase
Colchicine 0.1µg
PBS
Trypsine
Non sterile:
Hypotonic solution(KCL)
Fixate (Methanol and glacial acetic acid 3:1)
Giemsa stain
Centrifuge tube
Pasteur pipettes
Slides and coverslip
Low speed centrifuge
Preparation:
Make the fixative and keep it in freezer for cooling.
Keep the Glass slides in the freezer for cooling.
Keep the ice crystals ready.
Make the hypotonic solution. And keep all working solution ready.
Page | 129
Procedure:
Clean the BSC. Start the UV for 10-15 min. Switch off the UV. Start the air
flow.
Check for the cell growth in the T flask.
Add 200µl of colchicines into the T-flask. Incubate at 37ºC for 2 hrs.
Add 5ml of PBS. Rinse and decant.
Add 1 ml of Trypsin keep it at 37ºC for 5 min.
Add 4 ml of GMEM + FBS. Harvest the cells in to 15 ml tube.
Take the tube outside BSC.
Add 5 ml of 0.075M KCl to harvest cells. Keep it at 37ºC for 15 min.
Centrifuge at 1000 rpm for 8 min.
Decant the supernatant.
Add 1 ml Cold fixative. Homogenize by pipetting up and down.
Add 4 ml cold fixative.
Incubate it for Ice- crystals for 15 min.
Centrifuge at1000 rpm for 8 min.
Repeat from 10 to 15 steps 3 times.
Add 0.5 ml of cold fixative on the pellet.
Drop the suspension from height on to the chilled slide.
Heat dry. ( on glass burner)
Add 1 drop of giemsa stain. Keep it for 5 min. wash it with tap water allow to air
dry.
Add 1 drop of oil and observe under 100X bright field microscope.
Result : Metaphase chromosome spreads from BHK -21 monolayer culture was
not observed after Karyotyping.
12. SCALE UP TECHNOLOGY – FOR MONOLAYER CELL LINE USING
MICROCARRIERS
Aim:
Page | 130
To scale up monolayer type cell line (BHK 21) by Microcarrier Technology using
Cytodex 1 beads.
Introduction:
Microcarriers are tiny beads or particles that facilitate attachment and growth of
anchorage-dependent cells in cell culture processes. Microcarriers typically range in size from
90-300 μm in diameter and have a specific gravity that can be maintained in suspension with
gentle stirring. They may be composed of animal-derived or synthetic materials, including
collagen, dextran and plastic. Most are spherical in shape and may be either solid or porous.
Surface treatments with biological proteins, synthetic compounds and cationic charges may be
added to further enhance cell attachment and propagation. These differences present unique
advantages depending on the intended application.
The main advantage of using microcarriers is their ability to provide increased surface
area to volume ratios, allowing large-scale culture inside a relatively small footprint. They can
be maintained in a simple spinner flask or in a highly controlled stirred tank bioreactor.
There are numerous types of microcarriers, widely varying in their composition, size, shape and
density.
Properties and Characteristics of Cytodex:
Two types of Cytodex (Cytodex 1, Cytodex 3) are available to support the growth of
anchorage dependent animal cells for use in a multitude of applications. Both types of
Microcarriers are designed to meet the special requirements for this technology. Cytodex bead
size and density are optimized to support maximum cell growth rate and cell yield. The
biologically inert matrix provides a stable, but non-rigid substrate for stirred cultures from
which cells are easily harvested. Cytodex is transparent, allowing microscopic examination of
attached cells.
Page | 131
Table 1. Physical characteristics of Cytodex Microcarriers.
Cytodex 1, formed by substituting a cross-linked dextran matrix with positively charged
DEAE* groups distributed throughout the matrix, is a general purpose microcarrier. It is
particularly suitable for most established cell lines and for production of viruses or cell products
from cultures of primary cells and normal diploid cell strains.
Cytodex 3, formed by chemically coupling a thin layer of denatured collagen to the cross-
linked dextran matrix, is the Microcarrier of choice for cells that may be difficult to culture in
vitro, and particularly for cells with an epithelial like morphology. Because the collagen surface
layer can be digested by a variety of proteolytic enzymes, it provides novel opportunities for
harvesting cells from the Microcarriers while maintaining maximum cell viability and
membrane integrity.
These issues may be critical in developing successful serial sub cultivation protocols required
for scaling-up culture volumes.
Fig.1. Schematic representation of the two types of Cytodex
Page | 132
Table2. Microcarrier Types
Application of Cytodex microcarriers:
The use of Cytodex microcarriersenables most anchorage dependent animal cells to grow in
suspension cultures, in either a batch or perfusion culture format. Additionally, they can be used
to increase the surface area of traditional monolayer cultures. In stirred suspension cultures,
cells grow in a homogeneous environment where the culture parameters are easily monitored
and controlled. Cultures can be sampled periodically to examine cell morphology and to
determine cell viability. Microcarrier techniques are therefore a logical choice in applications
where cells are used for the production of biologicals.
Principle:
Adhesion of cells to culture surfaces
The adhesion of cells to culture surfaces is fundamental to both traditional monolayer
culture techniques and Microcarrier culture. Since the proliferation of anchorage-dependent
cells can only occur after adhesion to a suitable culture surface, it is important to use surfaces
and culture procedures which enhance all of the steps involved in adhesion. Adhesion of cells in
culture is a multistep process and involves.
Adsorption of attachment factors to the culture surface,Page | 133
Contact between the cells and the surface,
Attachment of the cells to the coated surface and finally,
Spreading of the attached cells.
The whole process involves divalent cations and glycoproteins adsorbed to the culture
surface. Under usual culture conditions the attachment proteins vitronectin and fibronectin
originates from the serum supplement in the medium. MHS is synthesized by the cells. CIG -
fibronectin or vitronectin. MHS – multivalent heparin sulphate.
Fig.2. Simplified outline of steps involved in adhesion of animal cells to culture sufaces.
The culture surface must be hydrophilic and correctly charged before adhesion of cells can
occur. All vertebrate cells possess unevenly distributed negative surface charged and can be
cultured on surfaces which are either negatively or positively charged. Examples of suitable
culture surfaces bearing charges of different polarities are glass and plastic (negatively charged)
and polylysine coated surfaces or Cytodex 1 Microcarriers (positively charged).
Since cells can adhere and grow on all of these surfaces, the basic factor governing adhesion
and growth of cells is the density of the charges on the culture surface rather than the polarity of
the charges.
Scale up systems:
Most tissue culture is performed on a small scale where relatively small numbers of cells
are required for experiments. At this scale cells are usually grown in T flasks ranging from
25cm2to 175cm2. However, exact yields will vary depending on the cell line. It is not practical
to produce much larger quantities of cells using standard T flasks, due to the amount of time
required for repeated passaging of the cells, demand on incubator space and cost.Page | 134
When considering scaling up a cell culture processes there are a whole range of parameters to
consider which will need to be developed and optimized if scale-up is to be successful. These
include problems associated with nutrient depletion, gaseous exchange, particularly oxygen
depletion, and the buildup of toxic by-products such as ammonia and lactic acid.
Spinner Flask Culture:
This is the method of choice for suspension lines including hybridomas and attached
lines that have been adapted to growth in suspension e.g. BHK 21, CHO and HeLa S3 etc.
Spinner flasks are either plastic or glass bottles with a central magnetic stirrer shaft and side
arms for the addition and removal of cells and medium, and gassing with CO2 enriched air.
Inoculated spinner flasks are placed on a stirrer and incubated under the culture conditions
appropriate for the cell line.
The culture is stirred by a suspended teflon-coated bar magnet which is driven by a magnetic
stirring base unit. The stirrer bar should never come into contact with the inside surface of the
vessel during culture since this may damage the Microcarriers. Similarly, spinner vessels having
a bearing which is immersed in the culture medium are not suitable, since the Microcarriers can
circulate through the bearing and become crushed. When using spinner vessels the position of
the impeller should be adjusted so as to minimize sedimentation of Microcarriers under the axis
of rotation. This is usually accomplished by positioning the end of the impeller a few
millimeters (approx. 5 mm) from the bottom of the spinner vessel. For attached cell lines the
cell densities obtained are increased by the addition of micro-carrier beads. The range of micro-
carriers available means that it is possible to grow most cell types in this system. A recent
advance has been the development of porous micro-carriers which has increased the surface
area available for cell attachment by a further 10-100 fold. The surface area on 2g of beads
isequivalent to 15 small roller bottles.
Procedure:
Inoculum Preparation:
Eight T-75 flasks were seeded with 3 ml of cell suspension and the volume was made up
to 15 ml with fresh medium.
All the flasks were kept in CO2 incubator at 370 C.
Page | 135
Siliconizing Glassware:
Treat glass surfaces with a silicon solution to avoid microcarrier attachment to
glassware. The best Siliconizing fluids are those based on dimenthyldichlorosilane dissolved in
an organic solvent.
For siliconizatiaon process Sigmacote® product use to be applied and it was swirled to
thoroughly coat the surface of all glassware to be exposed to Microcarriers.
All the glassware was kept at 60 deg in DHS (Dry Heat Sterilization) for 1 hour.
Allowed it for cooling and then rinsed it with purified water.
Keep it for air dry.
Hydration of beads:
Cytodex 1 Microcarriers are added to a suitably siliconized glass bottle and are swollen
in Ca2+, Mg2+-free PBS (1gm/100ml) and keep it for at least 3 hours at room
temperature.
Rinsing Process:
The supernatant was removed and the Microcarriers are washed once with gentle
agitation for a few minutes in fresh Ca2+ Mg2+ -free PBS (1gm/100ml).
The PBS was discarded and replaced with fresh PBS (1gm/100ml) and this process
should be carried out three times.
Microcarriers are sterilized by autoclaving at 121°C for 20 mins.
Equilibration:
Optimum bead density should be used 1-5gms per litre.
Bead density used 3gms per litre for both spinner flask and bioreactor.
Sterilized Microcarriers are allowed to settle, the supernatant (PBS) is removed and
again added the same amount of fresh media.
Then the Microcarriers are allowed to settle down for 5 minutes and again the
supernatant is removed and added same amount of fresh media. This equilibration
process was followed for spinner flask and bioreactor.
Kept for overnight incubation.
Page | 136
Initiation of Microcarrier culture:
Next day the medium was removed from spinner flask without disturbing the beads and
the volume was made up to 150 ml.
Seeding: Cell suspension was prepared from eight sub cultured flasks and seeded into
the spinner flask.
Mixed it properly, aliquot of sample was taken for cell counting and cell count was
performed and recorded.
The content of spinner flask was mixed intermittently for 3-6 hours to allow the proper
cells attachment to microcarrier. Shake for 2 minutes and kept in CO2 incubator for 30
minutes and again sake it for 2 mins. This process was carried out for 6 hrs.
Next day, cell count was performed by nuclei staining.
Composition of Nuclei stain-
0.02% Crystal violet
0.2 M Citric acid
2% Triton X- 100
100µl of sample was mixed with 100µl of stain and incubated for 1 hr at 370 C.
After incubation the supernatant was loaded in the wells of haemocytometer and
observed under 10 X microscope.
Nuclei were counted.
After ensuring the proper growth the volume in spinner flask was made up to 300 ml
by adding fresh media.
Spinner flask was kept in CO2 incubator.
Preparation of 5 L Bioreactor:
5 L bioreactor was autoclaved at 1210 C for 45 minutes with all proper connection by
adding 1 litre purified water.
1 litre of media was prepared with concentration of 2 X.
Equilibration of beads was performed for 5 L bioreactor.
Antifoam (20 ml) and serum (200 ml) was added to prepared media.
2 X media were transferred to sterile bioreactor already having 1 L sterile water which
gives final concentration of 1 X.
Equilibrated beads were transferred to bioreactor.
Page | 137
Seeding:
o Before transferring the culture from spinner flask to bioreactor, partial
trypsinization of cells was carried out (6 ml trypsin/50 ml media).
o Incubate for 15 minutes with trypsin and to stop trypsin activity media was
added (7 ml).
o This trypsinized culure along with beads was transferred to the bioreactor.
Following parameters were maintained as follows
o Agitation - 60 RPM,
o Aeration (surface air) - 0.5 LPM
o Sparger air – Auto mode
Bioreactor was run for 2 days at require conditions.
On harvest day cell count was performed and recorded.
Culture was harvested.
Beads were regenerated by replacing the media with PBS completely and stored for
future use.
Observation:
Cells attached to the microcarriers were observed under 10 X microscope.
Figure: Cells attached to microcarrier
Result:
Scale up for monolayer culture was performed by Microcarrier technology and growing cells
were visualized under microscopy for any contamination/ visual check.
Page | 138
13. Giemsa Staining
Aim: To study the Morphology of Cells in animal cell culture using polychromatic Giemsa
stain.
Principle:
Giemsa stain is used to differentiate nuclear and/or cytoplasmic morphology of cells. It consists
of two components, the acidic component being thr eosine which gives the product azure B-2-
eosinate and the basic component methylene blue which gives azure B. the stain has affinity for
the phosphate groups of DNA and attaches its basic component to regions of DNA where there
are high amount of adenine- thymine bonding, so it stains the nucleolus dark blue and the
cytoplasm pink due to the binding of eosinate to the basic components of the cells.
Materials required:
1. Cell Culture (BHK-21)
2. PBS
3. Anhydrous methanol (absolute)
4. Gimesa stain
Equipment’s:
1. Phase contrast microscope
2. Pipettes (sterile and non-sterile)
Procedure:
1. Clean the BSC. Start the UV for 10-15 min. Switch off the UV. Start the air flow.
2. Check for the cell growth in the T flask.
3. In the BSC the spent media was removed using pipette.
4. Add 5ml of PBS. Rinse and decant.
5. Add 5 ml PBS along with methanol in the ratio 1:1 was added to the cells.
6. It was incubated for 5 min and solution was discarded.
7. 5ml of methanol was added and incubated for 5 min.
8. Methanol was discarded and Giemsa stain was added and incubated for 10 min.
9. After incubation the flask was flush with running water.Page | 139
10. The flask was observed under 20X bright field microscope.
Observation:
The nucleolus was stained dark and the cytoplasm was stained slightly pink.
Result:
Spindle shaped Fibroblastic cell where observed with the nucleolus stained dark blue
and the cytoplasm appeared slightly pink.
Page | 140
(Module-4)
DOWNSTREAM
PROCESSING
TECHNOLOGY
Page | 141
1. INTRODUCTION
Downstream processing refers to the recovery and purification of biosynthetic products,
particularly pharmaceuticals, from natural sources such as animal or plant tissue
or fermentation broth. It is an essential step in the manufacture of pharmaceuticals such as
antibiotics, hormones (e.g. insulin and human growth hormone), antibodies (e.g. infliximab and
abciximab) and vaccines; antibodies and enzymes used in diagnostics; industrial enzymes; and
natural fragrance and flavor compounds. Downstream processing is usually considered a
specialized field in biochemical engineering, itself a specialization within chemical engineering,
though many of the key technologies were developed by chemists and biologists for laboratory-
scale separation of biological products. Purification is accomplished with a wide variety of
technologies including chromatography, membrane techniques, selective precipitation, and
novel hybrid formats.
Downstream processing and analytical bio-separation both refer to the separation or
purification of biological products, but at different scales of operation and for different
purposes. Downstream processing implies manufacture of a purified product fit for a specific
use, generally in marketable quantities, while analytical bio-separation refers to purification for
the sole purpose of measuring a component or components of a mixture, and may deal with
sample sizes as small as a single cell.
The recent Downstream Processing (DSP) is particularly focused on innovations aimed
at improving global healthcare. These include development of novel purification technology to
reduce manufacturing costs, increase productivity, and improve product safety and efficacy.
Some of these innovations involve mechanical systems, such as ultrahigh capacity magnetic
nano-particles, non-column fluidized beds, and simulated moving bed chromatography systems.
Stages in downstream processing:
A widely recognized heuristic for categorizing downstream processing operations
divides them into four groups which are applied in order to bring a product from its natural state
as a component of a tissue, cell or fermentation broth through progressive improvements in
purity and concentration.
Page | 142
Removal of insolubles:
It is the first step and involves the capture of the product as a solute in a particulate-free
liquid, for example the separation of cells, cell debris or other particulate matter from
fermentation broth containing an antibiotic. Typical operations to achieve this
are filtration, centrifugation, sedimentation, precipitation, flocculation, electro-precipitation, and
gravity settling. Additional operations such as grinding, homogenization, or leaching, required
recovering products from solid sources such as plant and animal tissues are usually included in
this group.
Product isolation:
It is the removal of those components whose properties vary markedly from that of the
desired product. For most products, water is the chief impurity and isolation steps are designed
to remove most of it, reducing the volume of material to be handled and concentrating the
product. Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit
operations involved.
Product purification:
It is done to separate those contaminants that resemble the product very closely in
physical and chemical properties. Consequently steps in this stage are expensive to carry out
and require sensitive and sophisticated equipment. This stage contributes a significant fraction
of the entire downstream processing expenditure. Examples of operations include affinity, size
exclusion, reversed phase chromatography, crystallization and fractional precipitation.
Product polishing:
It describes the final processing steps which end with packaging of the product in a form
that is stable, easily transportable and convenient. Crystallization, desiccation,
lyophilization and spray drying are typical unit operations. Depending on the product and its
intended use, polishing may also include operations to sterilize the product and remove or
deactivate trace contaminants which might compromise product safety. Such operations might
include the removal of viruses or depyrogenation.
The figure below explains the general process flow of downstream processing of
different products like vaccines.
Page | 143
The first step of downstream processing starts with harvest of product containing broth
from the fermentor. The broth will contain both cells and other dissolved components, which
need to be removed for obtaining the purified product. The first step is filtration, which is
defined as the process for separating two substances of two different physical states. It is used
for separating solids from turbid liquids (filtrate), pure gases or solids.
The filtration is basically classified into 2 types based on the principle of operation, they
are as follows.
Dead end filtration
Tangential Flow Filtration
In the Dead end filtration, all the flows are directed perpendicular through the membrane
with material building up on the surface of filter. As these particles build up, flow through the
filter is quickly reduced and finally it ceases completely. But in tangential flow filtration, the
flows are directed parallel to the membrane surface. This sweeping action helps to keep the
retained material from settling on the membrane surface and thus will help the membrane to
perform effectively for long periods, so they are so-called depth filters.
The step for removal of suspended cells from liquid generally involves microfiltration. The
micro filtration is defined as the process of removing contaminants in the 0.25 to 10μm range of
particles present in the process fluids, by passing the broth through a micro-porous medium,
such as membrane filter. These filtration techniques are applied in clarification of viral harvest
as well as the bacterial cell mass those are produced by the fermentation techniques or the roller
bottle culture. During this process the desired proteins and products are separated from the
media components which are a part of cultivation.
Page | 144
The broth that is clarified was concentrated by a technique called Ultrafiltration. It is a
technique for separating dissolved molecules in solution on the basis of size rating the particles
will be retained at the surface of the membrane and not through the polymer matrix. In
ultrafiltration, the separation is based on molecular weight of the macro-molecule. The
membrane is a thin semi-permeable polymeric material that will retain macro-molecules and
allow smaller dissolved solutes to pass through the membrane. During this process the desired
proteins and their allied products are separated by their molecular weight, and the volume is
reduced thereby increasing the purity considerably compared to the starting volume, resulting in
the concentrated desired product.
The next step after concentration of the partially purified product is chromatography.
Chromatography is defined as the process of separation of the individual components of a
mixture based on their relative affinities towards stationary and mobile phases. This involves a
sample being dissolved in a mobile phase. The mobile phase is then forced through an
immobile, immiscible stationary phase. The phases are chosen such that components of the
sample have differing solubilities in each phase. A component which is quite soluble in the
stationary phase will take longer to travel through it than a component which is not very soluble
in the stationary phase but very soluble in the mobile phase. As a result of these differences in
mobilities, sample components will become separated from each other as they travel through the
stationary phase. These separation entities are identified by other analytical techniques like UV-
visible, Infrared, NMR (nuclear magnetic resonance), Mass spectroscopy etc.
Based on the different mechanism the chromatography which are generally used,
majorly classified as follows.
Adsorption chromatography:
Adsorption chromatography is probably one of the oldest types of chromatography
around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a
stationary solid phase. The equilibration between the mobile and stationary phase accounts for
the separation of different solutes.
Partition Chromatography:
This form of chromatography is based on a thin film formed on the surface of a solid
support by a liquid stationary phase. Solute equilibrates between the mobile phase and the
stationary liquid.
Page | 145
Ion Exchange Chromatography:
In this type of chromatography, the use of a resin (the stationary solid phase) is used to
covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile
liquid phase are attracted to the resin by electrostatic forces.
Size Exclusion Chromatography:
Also known as gel permeation or gel filtration, this type of chromatography lacks an
attractive interaction between the stationary phase and solute. The liquid or gaseous phase
passes through a porous gel which separates the molecules according to its size. The pores are
normally small and exclude the larger solute molecules, but allow smaller molecules to enter the
gel, causing them to flow through a larger volume. This causes the larger molecules to pass
through the column at a faster rate than the smaller ones.
Affinity Chromatography:
This is the most selective type of chromatography employed. It utilizes the specific
interaction between one kind of solute molecule and a second molecule that is immobilized on a
stationary phase. For example, the immobilized molecule may be an antibody to some specific
protein. When solute containing a mixture of proteins is passed by this molecule, only the
specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later
extracted by changing the ionic strength or pH.
The final step of a downstream processing is formulation of the purified product; it is the
process by which the purified products are converted into the applicable form like tablets or
dermal injections by combining with some chemical substances. The above all the steps explain
the overall flow of different downstream processing steps.
Page | 146
2. TANGENTIAL FLOW FILTRATION
Aim: To clarify the broth and to concentrate the product in the given broth.
Principle:
Tangential flow filtration is a membrane technique used for clarification and
concentration of the product in the broth, where the feed stream passes parallel to the membrane
face as one portion passes through the membrane (permeate) while the remainder (retentate) is
re-circulated back to the feed reservoir. The separation takes place based upon MWCO
(Molecular weight cut off). The cross flow prevents build up of molecules at the surface that
can cause fouling. The TFF process prevents the rapid decline in flux rate seen in direct flow
filtration allowing a greater volume to be processed per unit area of membrane surface.
Cross Flow Rate (CFR)
The cross flow velocity is the rate of the solution flow through the feed channel and across the
membrane. It provides the force that sweeps away molecules that can foul the membrane and
restrict filtrate flow.
Transmembrane Pressure (TMP)
The transmembrane pressure (TMP) is defined as the mean of the applied pressure from the
feed to the concentrate side of the membrane subtracted by the pressure of permeate. This is
applied to dead-end filtration mainly and is indicative of whether a system is fouled sufficiently
to warrant replacement.
Where
PF is the pressure on the Feed Side
PR is the pressure of the Retantate side
PP is the pressure of the Permeate
Page | 147
Materials required:
Membrane cassette, measuring cylinders, purified water etc
2.1 MICROFILTRATION:
Microfiltration (MF) is a type of physical filtration process where a fluid is passed
through a special pore-sized membrane to separate microorganisms and suspended particles
from process liquid. It is commonly used in conjunction with various other separation processes
such as ultrafiltration and reverse osmosis to provide a product stream which is free of
undesired contaminants.
Procedure:
1. Open all the valves.
2. Flushing of filter with 15 lit of purified water. (feed pressure 1.4 bar) to remove storage
buffer.
Clean Water flux
3. Check the Clean water flux.
i. Keep the Feed line Pressure 1.0 bar and Retentate line pressure 0.5 bar.
ii. Stabilize it for 5 mins.
iii. Calculate the flow rate of permeate for 10 sec (3 times).
Optimization of Cross flow rate
4. Optimize the Cross Flow Rate for broth.
i. Keep the feed line pressure 0.2 bar.
ii. Stabilize it for 5 min.
iii. Calculate the flow rate of permeate and retentate as PFR and CFR respectively
for 1 min.
iv. Repeat steps ii and iii by increasing pressure of feed line by 0.2 bar till we get
constant CFR.
Optimization of Transmembrane Pressure
5. Optimize Transmembrane Pressure for broth.
i. Keep the feed line pressure 1 bar and retentate line pressure 0.2 bar.
ii. Stabilize it for 5 min.
iii. Calculate the flow rate of permeate as PFR for 1 min.
iv. Repeat steps ii and iii by increasing pressure of retentate line by 0.2 bar
and keeping feed line pressure constant till we get PFR constant.Page | 148
6. Filtration of broth (3.5- 4 lit) at optimize pressure. Collect permeate.
CIP and Storage
7. Wash with 0.5 M NaOH 500ml for 20 Min. (Recirculation).
8. Wash with 5 lit of water.
9. Check the Clean Water Flux.
10. If CWF is not similar as CWF before filtration wash the filter with 1.0M NaOH 500ml
for 15 min. Repeat Steps 9 & 10.
11. If the Clean Water Flux is same as CWF before filtration pass 0.2M NaOH 500ml
10min.(Recirculation).
12. Close all the valves.
Observation:
Table A. Optimization of CFR (Microfiltration)
Pf
(bar)
PR
(bar)
Pp
(bar)
CFR
(ml/min)
PFR
(ml/min)
Fluxper
(ml/min/m2)
0.4 0 0 255 27 270
0.6 0 0 375 30 300
0.8 0 0 500 34 340
1.0 0 0 620 35 350
1.2 0 0 720 38 380
1.4 0 0 860 41 410
Graph:
200 300 400 500 600 700 800 9000
50100150200250300350400450
Filterate flux Vs CFR
CFR (ml/min)
Filte
rate
flux
(ml/
min
/m2)
Page | 149
Table B. Optimization of TMP (Microfiltration)
Pf
(bar)
PR
(bar)
Pp
(bar)
TMP
(bar)
PFR
(ml/min)
Fluxper
(ml/min/m2)
1.10 0.2 0 0.65 33 330
1.20 0.4 0 0.80 45 450
1.30 0.6 0 0.95 48 480
1.39 0.8 0 1.095 51 510
1.50 1.0 0 1.25 55 550
1.60 1.2 0 1.4 55 550
Graph:
0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.50
100
200
300
400
500
600
Filterate fux Vs TMP
TMP (bar)
Filte
rate
flux
(ml/
min
/m2)
Result: Filtration has been done and permeate collected separately.
2.2 ULTRAFILTRATION:
Page | 150
Ultrafiltration (UF) is a variety of membrane filtration in which forces
like pressure or concentration gradients leads to a separation through a semi-permeable
membrane. Suspended solid and solutes of high molecular weight are retained in the so called
retentate, while water and low molecular weight solutes pass through the membrane in
the permeate. This separation process is used in industry and research for purifying and
concentrating macromolecular (103 - 106 Da) solutions, especially protein solutions.
Ultrafiltration is not fundamentally different from microfiltration, except in terms of the size of
the molecules it retains - it is defined by the Molecular Weight cut off (MWCO) of the
membrane used. Ultrafiltration is applied in cross-flow or dead-end mode.
Procedure:
1. Open all the valves.
2. Flushing of filter with 15 lit of purified water.( feed pressure 1.4 bar) to remove storage
buffer.
Clean Water flux
3. Check the Clean water flux.
i. Keep the Feed line Pressure 1.0 bar and Retentate line pressure 0.5 bar.
ii. Stabilize it for 5 mins.
iii. Calculate the flow rate of permeate for 10 sec (3 times).
Optimization of Cross flow rate
4. Optimize the Cross Flow Rate for broth.
i. Keep the feed line pressure 0.2 bar.
ii. Stabilize it for 5 min.
iii. Calculate the flow rate of permeate and retentate as PFR and CFR respectively
for 1 min.
iv. Repeat steps ii and iii by increasing pressure of feed line by 0.2 bar till we get
constant CFR.
Optimization of Transmembrane Pressure
5. Optimize Transmembrane Pressure for broth.
i. Keep the feed line pressure 1 bar and retentate line pressure 0.2 bar.
ii. Stabilize it for 5 min.
iii. Calculate the flow rate of permeate as PFR for 1 min.Page | 151
iv. Repeat steps ii and iii by increasing pressure of retentate line by 0.2 bar
and keeping feed line pressure constant till we get PFR constant.
6. Filtration of broth (3.5- 4 lit) at optimize pressure. Collect permeate.
CIP and Storage
7. Wash with 0.5 M NaOH 500ml for 20 Min. (Recirculation).
8. Wash with 5 lit of water.
9. Check the Clean Water Flux.
10. If CWF is not similar as CWF before filtration wash the filter with 1.0M NaOH 500ml
for 15 min. Repeat Steps 9 & 10.
11. If the Clean Water Flux is same as CWF before filtration pass 0.2M NaOH 500ml
10min. (Recirculation).
12. Close all the valves.
Observation:
Table A. Optimization of CFR (Ultrafiltration)
Pf
(bar)
PR
(bar)
Pp
(bar)
CFR
(ml/min)
PFR
(ml/min)
Fluxper
(ml/min/m2)
0.4 0 0 226 36 360
0.6 0 0 310 46 460
0.8 0 0 420 56 560
1.0 0 0 500 64 640
1.2 0 0 610 74 740
1.4 0 0 720 84 840
Page | 152
Graph:
200 300 400 500 600 700 8000
100200300400500600700800900
Filterate fux Vs CFR
CFR (ml/min)
Filte
rate
flux
(ml/
min
/m2
)
Table B.Optimization of TMP (Ultrafiltration)
Pf
(bar)
PR
(bar)
Pp
(bar)
TMP
(bar)
PFR
(ml/min)
Fluxper
(ml/min/m2)
1.01 0.2 0 0.65 94 940
1.30 0.4 0 0.85 110 1100
1.40 0.6 0 1.00 120 1200
1.50 0.8 0 1.15 122 1220
1.60 1.0 0 1.30 128 1280
1.70 1.2 0 1.45 130 1300
Graph:
Page | 153
0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.50
200400600800
100012001400
Filterate fux Vs TMP
TMP (bar)
Filte
rate
flux
(ml/
min
/m2
)
Result: Filtration has been done and retentate collected separately
2.3 DIAFILTRATION:
Diafiltration is an ultrafiltration membrane technique for completely removing,
replacing, or lowering the concentration of salts or solvents from solutions containing proteins,
peptides, nucleic acids, and other bio-molecules. The process selectively uses permeable
(porous) membrane filters to separate the components of solutions and suspensions based on
their molecular size. Smaller molecules such as salts, solvents, and water pass freely through the
ultrafiltration membrane, which retains the larger molecules.
Diafiltration can be used for buffer exchange.
There are two ways of diafiltration.
Continuous diafiltration
Discontinuous diafiltration
Continuous diafiltration can be completely automated.
Discontinuous diafiltration can be performed in two modes.
Serial dilution method
Volume reduction method
Procedure:
1. Open all the valves.
2. Flushing of filter with 15 lit of purified water.( feed pressure 1.4 bar) to remove storage
buffer.
Clean Water flux
Page | 154
3. Check the Clean water flux.
a. Keep the Feed line Pressure 1.0 bar and Retentate line pressure 0.5 bar.
b. Stabilize it for 5 mins.
c. Calculate the flow rate of permeate for 10 sec (3 times).
4. Take 500 ml of ultrafiltered media containing protein. Add 6gm of NaCl. Check the
conductivity.
5. Does Filtration of broth (500 ml) at optimize pressure.
6. When 250 ml of media (Half of initial volume) pass through the filter stop the filtration.
7. Check the conductivity of the remaining media. Add equal volume of purified water
(250 ml) so that volume becomes equal to initial volume. Start filtration.
8. Repeat steps 6 & 7 till we get the conductivity negligible.
CIP and Storage
9. Wash with 0.5 M NaOH 500ml for 20 Min. (Recirculation).
10. Wash with 5 lit of water.
11. Check the Clean Water Flux.
12. If CWF is not similar as CWF before filtration wash the filter with 1.0M NaOH 500ml
for 15 min. Repeat Steps 9 & 10.
13. If the Clean Water Flux is same as CWF before filtration pass 0.2M NaOH 500ml
10min. (Recirculation).
14. Close all the valves.
Observation:
Sample No. O.D.
1 4.22
2 5.17
3 3.17
4 2.30
5 1.28
6 0.91
7 0.67
Page | 155
Result: A minimum salt concentration was achieved by diafiltratio
3.1 Ion Exchange Chromatography:
IntroductionThis form of chromatography relies on the attraction between oppositely charged stationary
phase, known as an ion exchangers, and analyte. It is frequently chosen for the separation and
purification of proteins, peptides, nucleic acids, polynucleotides and other charged molecules,
mainly because of its high resolving power and high capacity.
There are two types of ion exchangers, namely cation and anion exchangers. Cation exchangers
possess negatively charged groups and these will attract positively charged cations. These
exchangers are also called acidic ion exchangers because their negative charges result from the
ionisation of acidic groups. Anion exchangers have positively charged groups that will attract
negatively charged anions. The term basic ion exchangers are also used to describe these
exchangers, as positive charges generally result from the association of protons with basic
groups.
Ion exchangers (anion/cation) are polymers that are capable of exchanging particular
ions within the polymer with ions in a solution that is passed through them.
3.1.1: Anion Exchange Chromatography: Aim: Separation of BSA protein based on charge using anion exchange chromatography.
Principle :
Ion exchange chromatography involves the separation of ionisable molecules based on their
total charge. This technique enables the separation of similar types of molecules that would be
difficult to separate by other techniques because the charge carried by the molecules of interest
can be readily manipulated by changing buffer pH.
In anion exchange separation, reversible interactions between charged molecules and
oppositely charged ion exchange media are controlled in order to favors binding or elution of
specific molecules and active separation.
Page | 156
Protein at acidic pH, gains ‘positive’ charge. Protein at basic pH, gains ‘negative’ charge.
Anion exchangers have basic groups with a net positive charge on the matrix and
negatively charged exchangeable counter ions. The protein of interest (Bovine Serum Albumin)
must have a charge opposite that of the functional group attached to the resin in order to bind.
The isoelectric point of protein is 4.7, if we provide pH of 7.4 to it, the protein becomes
negatively charged.
1. Equipments:
Chromatographic column
peristaltic pump
UV spectrophotometer
quartz cuvette
glass beakers and cylinders
2. Reagents :
1M tris HCl of pH 7.4
25mM tris HCl of pH 7.4
7.5 mg/ml BSA
1M NaCl
Procedure :
Column Packing :
30cm column made of acrylic was packed with the matrix polybenzene.
Polybenzene is a base matrix to which trimetylamine functional group is
attached.
Matrix was allowed to settle down due to gravity inside the column.
Then column bed height was measured and found to be 2cm. Therefore
column volume is equal to 3.079ml
CV= πr2h
= 3.142 × (0.7)2 × 2
= 3.07916 ml
Page | 157
Column Equilibration :
The whole column was maintained with a particular pH. Anion exchange
column was equilibrated to pH 7.4 with 25mM Tris HCl of pH 7.4.
Sample Addition :
Sample was prepared by taking 6ml from 10ml BSA stock(7.5mg/ml) with
19ml of 25mM Tris HCl.
Initial sample O.D. at 280nm was noted using 25mM Tris HCl (pH&.4) as a
blank.
25 ml of the sample was then passed into the column by peristaltic pump at a
flow rate of 1ml/minute to column.
About 3ml of flow through was collected in all test tubes and OD at 280nm
was noted.
Unbound wash :
The column was then passed with 25mM tris HCl, pH 7.4, to remove proteins
which are unbound to the matrix.
Unbound fractions were collected till OD at 280m reached nearly zero.
Elution :
Here, increase in the ionic strength brings out the elution of proteins.
Negatively charged proteins bound to the matrix were eluted using different
concentration of NaCl, and fractions were collected till OD at 280nm reached
zero.
For elution, NaCl with different concentration like 100mM, 200mM, 300mM
and 400mM NaCl is used with 25mM tris HCl, pH 7.4 .
Regeneration of the column Matrix:
CV = 3.07916 ml.
Therefore, 4CV = 4×3.079= 12.316ml
And 6CV = 6×3.079=18.474ml.
Page | 158
4CV of 1M NaCl: Around 12ml of 1M NaCl was passed into the column
with the help of peristaltic pump.
6CV of 25mM tris HCl: Then, around 18 ml of 25mM tris HCl was passed
into the column with the help of peristaltic pump.
Storage :
Then the column was stored in 20% ethanol.
Formulas:
Concentration of protein = 1/1.35×OD
Total protein content = concentration × Sample volume
% Recovery of protein = (Total protein eluted/total protein loaded)×100
% of protein loss =(Total protein loaded –Protein recovered in elution)×100
__________________________________________
Total protein loaded
Observation Tables:
Tableno.1
Flowthrough OD at 280nm
1 0.0
2 0.0
3 0.0
4 0.0
5 0.0
6 0.0
7 0.008
8 0.0
9 0.0
Table no. 2
Unbound Wash fractions OD at 280nmPage | 159
1 0.0
2 0.0
3 0.0
4 0.057
5 0.072
6 0.0
7 0.0
8 0.0
9 0.0
10 0.0
Table no. 3
Elution Buffer Fraction OD at 280nm
100mM NaCl in 25mM
tris HCl, pH 7.4
1 1.628
2 2.6000
3 0.688
4 0.349
5 0.209
6 0.119
7 0.059
8 0.026
9 0.004
200mM NaCl in 25mM
tris HCl, pH 7.4
10 0.745
11 0.994
12 0.156
13 0.049
14 0.014
15 0.002
Page | 160
300mM NaCl in 25mM
tris HCl, pH 7.4
16 0.190
17 0.130
18 0.000
19 0.0
400mM NaCl in 25mM
tris HCl, pH 7.4
20 0.0
21 0.0
22 0.0
Table no. 4
SampleOD at
280 nm
Concentrat
ion
Volume in
ml
Total
protein
%
Recovery
Load 1.000 0.7407 25 18.518 100%
Flowthrough 0.005 0.0037 9 0.033 0.17%
Unbound
Wash0.0645 0.047 6 0.2866 1.548%
Elution
100mM0.6313 0.4676 27 12.6252 68.1779%
200mM 0.3266 0.2419 18 4.3542 23.5133%
300mM 0.16 0.1185 6 0.711 3.8395%
400mM - - - - -
Result :
95.53% protein was recovered in elution and, most got eluted using 100mM NaCl in 25mM tris
HCl, pH 7.4
3.1.2: Cation exchange chromatography
Page | 161
Aim: Separation of BSA protein based on charge using cation exchange chromatography.
Principle:
It involves the separation of ionisable molecules based on their total charge. This
technique enables the separation of similar types of molecules that would be difficult to separate
by other techniques because the charge carried by the molecules of interest can be readily
manipulated by changing buffer pH.
In cation exchange separation, reversible interactions between charged molecules and
oppositely charged ion exchange media are controlled in order to favor binding or elution of
specific molecules and active separation.
Here, the protein BSA gains positive charge due to acidic pH (pH 3) created from 20mM
Sodium Acetate.
Cation exchangers have acidic groups with a net negative charge on the matrix and
positively charged exchangeable counter ions. The protein of interest (Bovine Serum Albumin)
must have a charge opposite that of the functional group attached to the resin in order to bind.
The isoelectric point of protein is 4.7, if we provide pH of 3 to it, the protein becomes positively
charged.
1. Equipments:
Chromatographic column
peristaltic pump
UV spectrophotometer
quartz cuvette
glass beakers and cylinders
2. Reagents :
20mM Sodium Acetate of pH 3
7.5 mg/ml BSA
1M NaCl
2M NaCl
0.1M NaOH
Procedure:
Page | 162
Column Packing :
30cm column made of acrylic was packed with the matrix polybenzene, to
which Sulphone (SO3)functional group is attached.
Matrix was allowed to settle down due to gravity inside the column.
Then column bed height was measured and found to be 1.5cm. Therefore
column volume is equal to 3.079ml.
CV= πr2h
= 3.142 × (0.75)2 × 1.5
= 2.6510 ml
Column Equilibration :
The whole column was maintained with a particular pH. Cation exchange
column was equilibrated to pH 3 with 20mM Sodium Acetate of pH 3.
Sample Addition :
Sample was prepared by taking 6ml from 10ml BSA stock(7.5mg/ml) with
19ml of 20mM Sodium Acetate of pH 3.
Initial sample O.D. at 280nm was noted using 20mM Sodium Acetate of pH
3 as a blank.
25 ml of the sample was then passed into the column by peristaltic pump at a
flow rate of 1ml/minute to column.
About 3ml of flow through was collected in all test tubes and OD at 280nm
was noted.
Unbound wash :
The column was then passed with 20mM Sodium acetate of pH 3, to remove
proteins which are unbound to the matrix.
Unbound fractions were collected till OD at 280m reached nearly zero.
Elution :
Here, increase in the ionic strength brings out the elution of proteins.
Page | 163
Negatively charged proteins bound to the matrix were eluted using different
concentration of NaCl, and fractions were collected till OD at 280nm reached
zero.
For elution, NaCl with different concentration like 1M, 1.5M and 2M NaCl is
used with 20mM Sodium Acetate of pH 3.
Regeneration of the column Matrix :
CV = 2.6510
Therefore, 3CV = 3×2.6510 = 7.953ml
5CV = 5×2.6510 = 13.255ml
And 6CV = 6×2.6510 =15.906ml.
3CV of 2M NaCl: Around 8ml of 2M NaCl was passed into the column with the help of
peristaltic pump.
5CV of 20mM Sodium acetate: Then, around 15 ml of 20mM Sodium acetate was
passed into the column with the help of peristaltic pump.
3CV of 0.1M NaOH: Around 8ml of 0.1M NaOH was passed into the column with the
help of peristaltic pump.
6CV of 20mM Sodium acetate: Then, around 17 ml of 20mM Sodium acetate was
passed into the column with the help of peristaltic pump.
Storage :
Then the column was stored in 20% ethanol.
Formulas:
Concentration of protein = 1/1.35×OD
Total protein content = concentration × Sample volume
% Recovery of protein = (Total protein eluted/total protein loaded)×100
% of protein loss =(Total protein loaded –Protein recovered in elution)×100
__________________________________________
Total protein loaded
Page | 164
Observation Tables:
Table no. 1
Flowthrough OD at 280nm
1 0.0
2 0.0
3 0.0
4 0.007
5 0.012
6 0.022
7 0.042
Table no. 2
Unbound Wash fractions OD at 280nm
1 0.023
2 0.008
3 0.002
4 0.0
5 0.0
Table no. 3
Elution Buffer Fraction OD at 280nm
1M NaCl in 20mM
Sodium Acetate of pH 3
1 0.728
2 0.286
3 0.182
4 0.264
5 0.230
6 0.223
7 0.192
8 0.154
9 0.128
10 0.111
Page | 165
11 0.099
1.5M NaCl 20mM Sodium
Acetate of pH 3
12 0.235
13 0.157
14 0.102
15 0.084
2M NaCl in 20mM
Sodium Acetate of pH 3
16 0.048
17 2.195
18 1.318
19 0.560
20 0.285
21 0.178
22 0.086
23 0.061
24 0.563
Table no. 4
SampleOD at 280
nmConcentration
Volume in
ml
Total
protein
%
Recovery
Load 1.178 0.872 25 21.8148 100%
Flowthrough 0.02075 0.01537 12 0.1844 0.845%
Unbound
Wash0.011 0.00814 9 0.0733 0.336%
Elution
1M0.2360 0.1748 33 5.7711 26.455%
1.5M 0.1445 0.1070 12 1.2844 5.8879%
2M 0.58155 0.4307 33 14.215 65.165%
Result : 97.5% protein was recovered in elution and, most got eluted using 2M NaCl in 20mM
Sodium Acetate, pH 3 .
Page | 166
3.2 HYDROPHOBIC INTERACTION CHROMATOGRAPHY
Aim: To separate the proteins according to difference in surface hydrophobicity by reversible
interaction between these proteins and the hydrophobic surface of a HIC medium.
Principle:
The phenomena form the basis for hydrophobic interaction chromatography (HIC) is a
chromatographic matrix containing hydrophobic groups, binds proteins from aqueous solutions
to different extents depending on the hydrophobic area present in the protein structures. The
principle of protein adsorption to HIC media is complementary to ion exchange and SEC.
Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC
column in a high salt buffer. The salt in the buffer reduces the solvation of sample solutes. As
salvation decreases, hydrophobic regions that become exposed are adsorbed by the HIC media.
The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a
decreasing salt gradient is used to elute from the column in order of increasing hydrophobicity.
During HIC, sample components bind to the column in high ionic strength buffer, typically 1 to
2 M ammonium sulfate or 3 M NaCl. High concentrations of salt, especially ammonium sulfate,
may precipitate proteins. Therefore, check the solubility of the target protein under the binding
conditions to be used. Elution is usually performed by decreasing the salt concentration,
stepwise or using a gradient.
HIC is widely used in protein purification as a complement to other techniques that
separate according to charge, size or bio-specific recognition. The technique is an ideal next
step when samples have been subjected to ammonium sulfate precipitation (frequently used for
initial sample concentration and clean-up) or after separation by ion exchange chromatography.
In both situations the sample contains a high salt concentration and can be applied directly to
the HIC column with little or no additional preparation. The elevated salt level enhances the
interaction between the hydrophobic components of the sample and the chromatography
medium. During separation, samples are purified and eluted in smaller volumes, thereby
concentrating the sample so that it can go directly to gel filtration or, after a buffer exchange, to
an ion exchange separation. HIC can be used for capture, intermediate purification or polishing
steps in a purification protocol.
Application of HIC:Page | 167
HIC suits all stages of a purification process. Application examples include high-yield
capture, polishing monoclonal antibodies, removing truncated species from full-length forms,
separating active from inactive forms, and clearing of viruses.
1. Equipments:
Column with matrix
Peristaltic Pump
Spectrophotometer, quartz cuvette
Necessary glassware
2. Reagents:
20mM Tris HCl, pH-7.4 (Stock-1M tris HCL)
1M Ammonium sulfate, (Stock-3M Ammonium sulfate)
Sample (15mg/ml BSA)
Water
Procedure:
Column Packing:
30cm column made of acrylic was packed with methyl HIC matrix. Adaptor was
attached to the column and allowed the matrix to settle down due to gravity
inside the column.
Then column bed height was measured and found to be 2cm. Therefore, one
column volume is approximately equal to 3.5ml.
CV =π r2 h
3.14 x (0.75)2 x 2 = 3.5cm3
Column Equilibration:
HIC column was equilibrated with 1.5M Ammonium sulfate in 25mM tris
HCL (pH-7.4)
Sample Preparation and loading:
1ml of protein sample (BSA) was added 0.25ml of tris HCL plus 5ml of 3M
ammonium sulfate.
Initial sample OD at 280nm using equilibration buffer as a blank was taken.Page | 168
10ml of sample was passed by peristaltic pump at flow rate of 1ml/min. to
column.
Flow through was collected in test tube and OD at 280nm was noted.
Unbound Wash:
The column was washed with equilibration buffer to remove proteins which
are unbound to the HIC matrix.
Unbound wash fractions were collected till OD at 280nm reached nearly
zero.
Elution:
Proteins bound to the matrix were eluted at decreased salt concentration.
Elution started with 1M, 0.75M, 0.5M, 0.25M ammonium sulfate in
25mMtris HCL (pH-7.4). Fractions were collected and OD at 280nm was
taken.
Regeneration of Column Matrix:
5CV, 3ml of 1M acetic acid, 0.5ml of 1% Phosphoric acid were passed
through peristaltic pump at flow rate of 1ml/min. to column to remove
proteins inside the matrix, and then column was stored.
Observation tables:
Table No. 1
Sr.No.Flow
throughUnbound wash
Elution
1M 0.75M 0.5M 0.25M
1. 0.946 1.136 0.009 - - -
2. 1.180 0.199 0.109 - - -
3. - 0.005 - - - -
Table No. 2
Sr.No. OD ConcentrationTotal
Volume
Total
Protein
%
Recovery
1. Load 1.414 1.0474 10 10.474 100%
2. Flow through 1.063 0.787 6 4.7244 45.10%
3. Unbound Wash 0.4466 0.3308 9 2.9777 28.43%
4.Elution
a) 1M 0.059 0.0437 6 0.2622 2.503%
Page | 169
Result:
Proteins were eluted in 1M concentration of Ammonium Sulfate
Page | 170
3.3 AFFINITY CHROMATOGRAPHY
Aim:
To perform affinity chromatography for the separation of given protein using Protein
A matrix.
Principle:
Affinity chromatography is a method of separating biochemical mixtures based on a
highly specific interaction such as that between antigen and antibody, enzyme and substrate,
or receptor and ligands. The starting point is an undefined heterogeneous group of molecules
in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest
will have a well-known and defined property, and can be exploited during the affinity
purification process. The process itself can be thought of as an entrapment, with the target
molecule becoming trapped on a solid or stationary phase or medium. The other molecules in
the mobile phase will not become trapped as they do not possess this property. The stationary
phase can then be removed from the mixture, washed and the target molecule released from
the entrapment in a process known as elution. Possibly the most common use of affinity
chromatography is for the purification of recombinant proteins.
Binding to the solid phase may be achieved by column chromatography whereby the
solid medium is packed onto a column, the initial mixture run through the column to allow
setting, a wash buffer run through the column and the elution buffer subsequently applied to
the column and collected. These steps are usually done at ambient pressure. Alternatively,
binding may be achieved using a batch treatment, for example, by adding the initial mixture
to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase,
washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the elute.
Sometimes a hybrid method is employed such that the binding is done by the batch method,
but the solid phase with the target molecule bound is packed onto a column and washing and
elution are done on the column.
A third method, expanded bed adsorption, which combines the advantages of the two
methods mentioned above, has also been developed. The solid phase particles are placed in a
column where liquid phase is pumped in from the bottom and exits at the top. The gravity of
Page | 171
the particles ensures that the solid phase does not exit the column with the liquid phase.
Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic
strength directly or through a gradient to resolve the particles of interest.
Affinity chromatography can be used to:
Purify and concentrate a substance from a mixture into a buffering solution.
Reduce the amount of a substance in a mixture.
Discern what biological compounds bind to a particular substance.
Purify and concentrate an enzyme solution.
Nucleic acid purification.
Protein purification from cell free extracts.
Separation of mixed recombinant proteins.
Equipments:
Column with matrix
Peristaltic Pump
Spectrophotometer, quartz cuvette
Necessary glassware
Reagents:
10mM PBS (pH-7.4)
Sample (IgG)
0.1M Glycine HCl (pH-3)
1M Acetic acid
Water,
Procedure:
Column Packing:
30cm column made of acrylic was packed with the matrix; Protein A. Adaptor
was attached to the column and allowed the matrix to settle down due to
gravity inside the column.
Then column bed height was measured and found to be 2cm. Therefore, one
column volume is approximately equal to 3.5ml.
CV =π r2 h
Page | 172
3.14 X (0.75)2 x 2 = 3.5cm3
Column Equilibration:
Protein A column was equilibrated to pH-7.4 with 10mM PBS.
Sample Preparation and loading:
1ml of protein sample was added to 9ml of 10mM PBS (pH7.4)
By using 10mM PBS buffer as a blank initial sample OD at 280nm was
taken.
10ml of sample was passed by peristaltic pump at flow rate of 1ml/min. to
column.
Flow through was collected in test tube and OD at 280nm was noted.
Unbound Wash:
The column was washed with 10mM PBS (pH7.4) to remove proteins
which are unbound to the matrix protein A.
Unbound wash fractions were collected till OD at 280nm reached nearly
zero.
Elution:
Proteins bound to the matrix were eluted using 0.1M Glycine HCl.
Elution started with 0.1M Glycine HCl buffer at pH-3 and fractions were
collected till OD at 280nm reached zero.
Regeneration of Column Matrix:
6ml (2CV) of 1M acetic acid was passed through peristaltic pump at flow
rate of 1ml/min. to column to remove proteins inside the matrix.
Then it was followed by 17.5ml (5CV) of 10mM PBS, and then column
was stored.
Observation tables:
Page | 173
Table No. 1
Sr.No. Flow through Unbound wash Elution
1. 0.029 0.015 1.63
2. - - 1.190
3. - - 0.059
Table No. 2
Sr.No. OD ConcentrationTotal
Volume
Total
Protein
%
Recovery
1. Load 1.686 1.248 10 12.48 100%
2. Flow through 0.029 0.0214 3 0.0644 0.51%
3. Unbound Wash 0.015 0.011 3 0.033 0.26%
4. Elution 0.959 0.710 12 8.530 68.35%
Graph:
Elution profile of Gig from Protein A Column.
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.50
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.029 0.015
1.63
1.19
0.0590000000000001
OD
Fractions
OD
at 2
80nm
Result:
IgG got eluted at a lower pH (pH-3).
3.4 GEL FILTRATION CHROMATOGRAPHY
Page | 174
Aim:
To separate the proteins by using gel permeation chromatography based on their
molecular size.
Introduction:
Gel filtration involves the chromatographic separation of molecules of different
dimensions based on their relative abilities to penetrate into a suitable stationary phase or
chromatographic resin. The chromatographic resin has size-exclusion properties and usually
consists of very small, uncharged porous particles in an aqueous solution, which are packed
into a column and then used for the separation. The resin particles have a range of pore sizes
that determine the size of molecules that can be separated. The average or maximum effective
pore size defines what is called the fractionation range or exclusion limit of the resin.
Molecules smaller than the fractionation range can enter the pores of the resin, while
molecules larger than the fractionation range are excluded from entering the pores.
Principle:
To perform a separation, gel filtration medium is packed into a column to form a
packed bed. The medium is a porous matrix in the form of spherical particles that have been
chosen for their chemical and physical stability, and inertness (lack of reactivity and
adsorptive properties). The packed bed is equilibrated with buffer which fills the pores of the
matrix and the space in between the particles. The liquid inside the pores is sometimes
referred to as the stationary phase and this liquid is in equilibrium with the liquid outside the
particles, referred to as the mobile phase. It should be noted that samples are eluted
isocratically, i.e. there is no need to use different buffers during the separation. However, a
wash step using the running buffer is usually included at the end of a separation to facilitate
the removal of any molecules that may have been retained on the column and to prepare the
column for a new run.
Gel filtration is used in group separation mode to remove small molecules from a
group of larger molecules and as a fast, simple solution for buffer exchange. Small molecules
such as excess salt (desalting) or free labels are easily separated. Samples can be prepared for
storage or for other chromatography techniques and assays. Gel filtration in group separation
mode is often used in protein purification schemes for desalting and buffer exchange.
Sephadex G-10, G-25 and G-50 are used for group separations.
Page | 175
Gel filtration is used in fractionation mode to separate multiple components in a
sample on the basis of differences in their size. The goal may be to isolate one or more of the
components, to determine molecular weight, or to analyze the molecular weight distribution
in the sample. The best results for high resolution fractionation will be achieved with samples
that originally contain few components or with samples that have been partially purified by
other chromatography techniques. High resolution fractionation by gel filtration is well suited
for the final polishing step in a purification scheme.
Applications:
One of the principal advantages of gel-filtration chromatography is that separation can
be performed under conditions specifically designed to maintain the stability and activity of
the molecule of interest without compromising resolution.
Separation of proteins and peptides:
Because of its unique mode of separation, gel-filtration chromatography has been
used successfully in the purification of proteins and peptides from various sources. Example-
Recombinant human granulocyte colony stimulating factor (rhG-CSF)
Separation of other bio-molecules:
Gel-filtration chromatography has for many years been used to separate various
nucleic acid species such as DNA, RNA and tRNA as well as their constituent bases, adenine,
guanine, thymine, cytosine, and uracil.
Group separation:
This can be used for example to effect buffer exchanges within samples for desalting
of labile samples prior to concentration and lyophilisation to remove phenol from nucleic
acid preparation and remove inhibitors from enzyme.
Molecular mass estimation:
Gel-filtration chromatography is an excellent alternative to SDS-PAGE for the
determination of relative molecular masses of proteins, since the elution volume of a globular
protein is linearly related to the logarithm of its molecular weight. One can prepare a
calibration curve for a given column by individually applying and eluting at least five suitable
standard proteins the matrix) over the column, determining the elution volume for each
protein standard, and plotting the logarithm of molecular weight versus Ve/V0.
Materials Required:
1. Equipments:
Page | 176
Column with matrix
Peristaltic Pump
Spectrophotometer, quartz cuvette
Necessary glassware
2. Reagents:
25mM Tris HCl (pH 7.4)
Sample (BSA)
Procedure:
Column Packing:
30cm column was packed with gel beads of acrylamide. An adaptor was
attached to the column and allowed the matrix to settle down due to gravity
inside the column.
Then column bed height was measured and was found to be 31.7cm.
CV =π r2 h
3.14 × (0.75)2 x 18 = 31.7cm3
Column Equilibration:
The column was equilibrated with 25 mM tris HCl ( pH 7.4) through the
column beads using peristaltic pump.
The solution was drained down (keep1 mm of buffer above the gel) before
adding the sample mixture.
Sample Preparation and loading:
4 % of column volume sample (BSA) was added to the top of the column
using a micropipette (do not stir up the top of the gel).
4 % of column volume = (4× 31.7) ÷ 100 = 1.268 ml (nearly equals to 1.27
ml).
Then 25 mM tris HCl solution was added to the top, filling the space at the top
of the column.
Fractions (3ml/tube) were collected in the test tubes. (Early fractions contain
large molecules while later fractions contain smaller ones).
The absorbance spectrum of each fraction was measured at 280 nm.
Page | 177
Regeneration of Column Matrix:
25mM Tris HCL pH 7.4, passed through peristaltic pump at flow rate of 1ml/min.
to column to remove proteins inside the matrix, and then column was stored.
Observation:
Observation tables: Table no.1
Sr. no Column volume Absorbance at 280 nm
1. 3 0.012
2. 3 0.011
3. 3 0.503
4. 9 0.99
5 3 0.141
6. 3 0.029
7. 3 0.005
8. 3 0.008
9. 3 0.026
10. 3 0.003
Table 2:
ODconcentr
ationVolume
Total
Protein% percentage
Load 2.720 2.014 20 40.28 100%
Sample
elution1.1133 0.8246 36 29.6856 73.69%
Result: Proteins were separated using gel filtration chromatography.
3.5 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
Aim:
Page | 178
To separate proteins of different molecular weight based on electric mobility using
SDS PAGE.
Principle:
The separation of macromolecules in an electric field is called electrophoresis. A very
common method for separating proteins by electrophoresis uses a discontinuous
polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the
proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). SDS is an anionic detergent, meaning that when dissolved its molecules have a
net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in
proportion to its relative molecular mass. The negative charges on SDS destroy most of the
complex structure of proteins, and are strongly attracted toward an anode (positively-charged
electrode) in an electric field.
Polyacrylamide gels restrain larger molecules from migrating as fast as smaller
molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured
polypeptides, the final separation of proteins is dependent almost entirely on the differences
in relative molecular mass of polypeptides. In a gel of uniform density the relative migration
distance of a protein (Rf) is negatively proportional to the log of its mass. If proteins of
known mass are run simultaneously with the unknowns, the relationship between R f and mass
can be plotted, and the masses of unknown proteins estimated.
Materials Required:
Reagents:
A. Stock solutions:
Deionized water
0.5 M Tris HCl (pH-6.8)
1.5 M Tris HCl (pH-8.8)
10 % SDS
B. Working solutions:
solution A(30%)
Acrylamide and bisacrylamide
Page | 179
10 % APS
Electrophoresis buffer
SDS
Tris HCl
Glycine
Distilled water
C. Sample buffer
0.5M tris HCl pH-6.8
Glycerol
SDS (4%)
Bromophenol blue
Mercaptoethanol
D. Staining solution:
Coomassie brilliant blue R-250
Methanol
Acetic acid
Distilled water
E. Destaining solution:
Methanol
Acetic acid
Distilled water
PROCEDURE:
1. Setting up of the electrophoresis system:
Clean the glass plates with 20% ethanol and assemble them on the clean surface. Then
lay the longer glass plate down the first. Place two spacers of equal thickness along
the rectangular plate. Next place the shorter plate over the spacers so that the bottom
the spacers and the glass plates are aligned.
Loosen the 4 screws on the clamp and fix the plates in such a way that the the screws
are away from you. Firmly grasp the plates and the spacer sandwich in such a way
that the longer plate faces away from you. Then slowly slide it into the gel plate
clamp assembly. Tighten the top two screws of the assembly.
Page | 180
Place the clamp assembly into the casting stand in such a way that the clamp screws
faces away from you. Loosen the top two screws so that the plates and the spacers fit
firmly into the casting stand base. Gently tighten all the screws.
Pull the sandwiched plate from the alignment slot and check that the plates and spacer
are properly aligned. If not then realign the plates and the spacers as in steps 1-3.
Before transferring the clamp into the casting slot, recheck the alignment of the
spacers. Do this by inverting the gel sandwich and make sure that they are properly
aligned.
Transfer the clamp assembly into the casting slot in the casting stand.
2. Preparation of the reagents and the casting gels:
Stock solutions were prepared for the following reagents :
0.5 M Tris HCl pH 6.8 – 50 ml
1.5 M Tris HCl pH 8.8 – 50 ml
10% APS – 10 ml
10% SDS – 10 ml
Sample buffer preparation (2X)
Total volume = 25 ml
0.5M Tris Hcl pH 6.8 – 5 ml
4% SDS – 10 ml
Glycerol – 5ml (50%), 10 ml(100%)
Bromophenol blue – 0.05 g
Mercaptoethanol – 340 µl
Electrophoretic buffer
SDS – 1 g
Tris – 3 g
Glycine – 14.48 g
De-ionised water – 1 l
Staining solution
Coomassie brilliant blue (R250) – 0.25 g
Page | 181
Methanol – 50 ml
Acetic acid – 10 ml
De-ionised water – 40 ml
Destaining solution
Methanol – 50 ml
Acetic acid – 10 ml
De-ionised water – 40 ml
Gel composition
Components Stacking gel
(12%)Resolving gel (4%)
De ionized water 2.8 ml 2.625 ml
0.5 M Tris pH 6.8 1.25 ml ----------
1.5 M Tris pH 8.8 ---------- 1.875 ml
Acrylamide 0.63 ml 3 ml
SDS 50µl 52.5µl
APS 50µl 52.5 µl
TEMED 7 µl 7 µl
Prepare the sealing gel solution and pour it into the casting tray to hold the plates and
the spacer in place. Allow it to solidify.
Combine all the reagents necessary to make the resolving gel solution except APS and
TEMED. Mix it properly and carefully to avoid foam formation. Chances of foam
formation are due to the presence of SDS.
Then add APS and TEMED in appropriate amount mix them well and pour the
solution till the 3/4th of the height of the gap between the two glass plates of the
mould. Allow it to solidify.
Cover the layer of the gel with 1 ml of water to prevent direct contact with air. Water
should be added slowly to prevent mixing with the gel.
Allow the gel to polymerize.
Combine all the reagents necessary to make the stacking gel solution except APS and
TEMED. Mix it properly and carefully to avoid foam formation.
Page | 182
Then add APS and TEMED in appropriate amounts mix them well and pour the
stacking gel solution over the polymerized resolving gel. Then carefully insert the
comb without any bubble formation.
Allow the gel to polymerize for 15 minutes.
Then carefully remove the comb without disrupting the wells formed.
The gel Placed in the buffer chamber and electrophoretic buffer is added into the
chamber.
Samples were prepared as 5mg/ml, 7mg/ml and 10mg/ml by using the sample buffer,
10µl of sample were taken from respective stock concentration to that 10µl sample
buffer were added and concentrated it by slightly heating at 90⁰C, for 10min. Then
20µl sample were added in to the well by using micropipettes. And also the standard
marker was added to it. The electrodes were Connected and switched on the electric
supply.
It was allowed to run for 3-4 hrs at 100 V. After sample was run completely, the
power supply was switched off and gel was removed carefully from the glass plates
and immersed in staining solution for overnight and on next day in distaining solution
for 2-3hr, until the cleared band observation.
Observation:
Result: Resolved bands of BSA were observed
Page | 183