Seediscussions,stats,andauthorprofilesforthispublicationat:http://www.researchgate.net/publication/282652909

ProcessvalidationofrecombinantHumanGranulocyteColonyStimulatingFactorforbulkproduction

CONFERENCEPAPER·OCTOBER2015

READS

2

14AUTHORS,INCLUDING:

SyedMonim

InceptaPharmaceuticalCompanyLtd.

8PUBLICATIONS6CITATIONS

SEEPROFILE

TarifulIslam

InceptaPharmaceuticalsLimited

3PUBLICATIONS3CITATIONS

SEEPROFILE

Availablefrom:SyedMonim

Retrievedon:08October2015

CHARACTERIZATION RH-GCSF ACCORDING TO EP GUIDELINE & OTHER NON-COMPENDIAL TESTS

Process Validation of Recombinant Human Granulocyte Colony Stimulating Factor for Bulk ProductionSyed Abdul Monim, Md. Ehtashamul Haque, Ataur Rahman, Tariful Islam, Md. Nasim Ferdous Kallol, Md. Rashed Nahid Rana, A. G. M. Rakibuzzaman, Md. Jahidul Islam, Abdul

Wadud Molla, Fahima Akter Mukta, Romana Akter, Mohammad Maududul Huq, Mohammad Shamsul Islam and Md. Iqbal Hassan Khan*Research and Development, Biotechnology Derived Product Facility, Incepta Pharmaceuticals Ltd, Dhaka, Bangladesh.

*Correspondence to iqbal@inceptapharma.com

rh-GCSF was characterized according to the EP (European Pharmacopeia) guideline whichcomplied all the specifications. The formulated rh-GCSF is now under preclinical trail and theclinical trial will be conducted soon.

CONCLUSION

To claim the synthesized drug as a biosimilar, several tests have been executed in accordance with European Pharmacopeia (EP) guideline and other non-compendial tests. Among them physical appearance, biological activity, peptide mapping, related protein, impurities profiling by Size ExclusionChromatography and SDS-PAGE, endotoxin level, host cell protein, host cell DNA, isoelectric focusing, intact mass, amino acid sequence and sterility of theproduct have been mentioned.

Designed by: Syed Abdul Monim, Md. Ehtashamul Haque & Md. Nasim Ferdous Kallol

CLONE CHARACTERIZATION

Host Cell Protein (HCP)

Isoelectric Focusing

REGULATORY CONSIDERATIONS FOR PROCESS VALIDATION

Good Documentation Practices(GDP) Environment, Health & Safety(EHS)

MANUFACTURING PROCESS

Recombinant human Granulocyte Colony Stimulating Factor (rh-GCSF) is a proteinconsisting of 175 amino acid expressed in E. coli which plays a vital role in the treatment ofcancer patients receiving Chemotherapy and patients with different categories ofneutropenia. Rh-GCSF was approved as a drug under the name of Filgrastim (INN) by theEU on August 22, 2002 and the innovator is Amgen. It is a 1.2-1.4 billion dollar per yearproduct for Amgen. It has 8 biosimilars in the market approved by EMA but the price is toohigh for the mass population of developing country like Bangladesh, where Cancer is now agreat concern. In this regard Incepta has taken the challenge to produce Filgrastimbiosimilar in Bangladesh. Following is the roadmap to the validation of rh-GCSF productionprocess.•Clone construction, Master cell bank & Working cell bank preparation•R & D scale production and fixing scale for commercial production• Optimizing process for Commercial scale•Finally process validation done following the Guidelines of ICH and EMA•Accelerated and real time stability study is going on•Pre-clinical study has been passed & commercial production will be done following the

successful clinical trial.

IN-PROCESS QUALITY CONTROL

METHOD OF EXECUTION

Sterility of the Product

State of art Equipments

Host Cell DNA

Tests Specifications results

Identity

Gram's staining Gram negative

Identification of culture as E.Coli (Growth on MacConkey Agar)

Identified as E.Coli

Restriction Digestion Release two fragments of 528bp & 4305bp

DNA sequencing Matched with reference Filgrastim sequence

Purity Fungal or Bacterial contamination No groth on Cetrimide and Mannitol Salt Agar

Stability Not less than 75% of cells with Plasmid DNA 82.80%

Plasmid Copy number

More than 100 copies of plasmid per cell 149 copies

Viability Growth in Kanamycin containing media After overnight culture OD was 1.5

1.32 1.37 1.2

8.4 8.58

0

2

4

6

8

10

Batch 1 Batch 2 Batch 3

Seed culture time and final OD in three validation batches

Final OD at600nm

Timetaken(Hour)

537 531 535

127 115 122

0

100

200

300

400

500

600

Batch 1 Batch 2 Batch 3

Biomass & Inclusion Bodies found in three validation batches

Biomass AfterFermentation(gm)

TotalInclusionBodies (gm)

0

5

10

15

20

0 2 4 6 8 10 12 14 16

OD

at 6

00

nm

Fermentation Hour

Growth Curve of Fermentation (Bath 1)

0

5

10

15

20

0 2 4 6 8 10 12 14 16

OD

at

60

0n

m

Fermentation Hour

Growth Curve of Fermentation (Batch 2)

0

5

10

15

20

0 2 4 6 8 10 12 14 16

OD

at

60

0n

m

Fermentation Hour

Growth Curve of Fermentation (Batch 3)

Fig: Identity of clone checked by DNA sequencing & matched in NCBI(BLAST)

Fig: Purity checked by Plating in Ca & MSA plate

Lane No Sample Name Protein %

1 protein MWmarker NA

2 Standard GCSF NA

3 Blank -

4 Fermentation 4th hour 41.98

5 Fermentation 8th hour 46.30

6 Fermentation 12th hour 62.02

7 Fermentation Final hour 65.87

Fig: SDS PAGE of different hour Fermrntation sample(Batch 1)

Batch No Step Total Protein (gm) Amount of GCSF Percentage of Recovery

1

IB Sol.

17.74 N/A N/A

2 16.63 N/A N/A

3 18.7 N/A N/A

1

Reduction

16.63 N/A N/A

2 16.08 N/A N/A

3 17.20 N/A N/A

1

Refolding

N/A 12.87 72.55

2 N/A 12.18 73.24

3 N/A 13.19 70.53

1

Buffer Exchange-1

N/A 9.8 55.24

2 N/A 9.7 58.33

3 N/A 10.6 56.68

1

Cation Exchange-1

N/A 9.0 50.73

2 N/A 9.1 54.72

3 N/A 9.4 50.27

1

Buffer Exchange-2

N/A 8.7 49.04

2 N/A 8.7 52.32

3 N/A 9.0 48.13

1

Cation Exchange-2

N/A 7.5 42.28

2 N/A 7.6 45.7

3 N/A 7.9 42.25

Data from Upstream

ACKNOWLEDGEMENT

1 2 3 4 5 6 7

116.0

97.466.2

45.0

31.0

21.5

14.4KD

65.87 66.64 70.32

0

20

40

60

80

Batch 1 Batch 2 Batch 3

Percentage of rh-GCSF expression from SDS PAGE

Percentageof rh-GCSFexpression

Data for standard curve

SampleConcentration

(ng/mL)Absorbance

1Absorbance

2Average

AbsorbanceStandard 0 0.096 0.091 0.094Standard 1 0.099 0.097 0.098Standard 3 0.137 0.128 0.133Standard 12 0.267 0.265 0.266Standard 40 0.656 0.664 0.660Standard 100 1.476 1.525 1.501

Host Cell Protein (HCP) calculation of Three BatchesAbsorbanc

eIntercept of Std.

CurveSlope of Std.

CurveConcentration of

HCPBatch 1 0.119 0.091 0.014 0.003Batch 2 0.475 0.071 0.009 0.044Batch 3 0.564 0.071 0.009 0.054

8.6

7.2

6.6

5.9

5.1

4.6

3.6

Lane No. 1 2 3

Analysis Information:

Sample Loading

Information

SI. No. Sample NameLoading Volume

(µL)

Loading Amount

(µg)

1 pI marker, 20 NA

2Standard

Filgrastim(NIBSC)20 5.78

3 rh-GCSF batch 1 20 5.75

Type of Gel pH: 3-10 IEF Gel (Invitrogen)

Sample Buffer

Invitrogen

Type of Staining

Brilliant Blue G Colloidal

Result/Observation: The Isoelectric point of the samples is between 5.7 and 6.3

pIMarker

Bacterial Endotoxin Test

Impurities

110 kDa

80 kDa

50 kDa

30 kDa

20 kDa

15 kDa

10 kDa

Lane No. 1 2 3 4 5 6 7

110 kDa

80 kDa

50 kDa

30 kDa

20 kDa

15 kDa

10 kDa

Lane No 1 2 3 4 5 6 7

Sample : Batch 1

We are very much grateful to the following persons for their cordial help during every step of this bulk production process validation:

Mr. Abdullah Sarker, Mr. Javed Jahangir, Mr. Raquibur Rahman, Mr. Ashadullah Hill Galib, Mr. Hasan Manjur Qadir, Mr. Inan Ishraque, Mr. Syed Morsalin Ali, Mr. Saidul Islam, Ms. Raznin Akter,, Mr. S.K.Rafiqul Islam, Mr. Aminur Talukder, Mr. Siraj Mollah, Mr. Saiful Islam Khan, Mr. Rashidul Islam, Mr. Billal Hossen, Mr. Sayed Hossen, Mr. Hasibul Islam, Mr. Anwar Hosen, Mr. Likhon Islam

Bioassay for potency calculation

L-1: Cell Control; L-2 to 6: Reference Std.; L-7 to 11: R&D rhG-CSF

Analysis Information:

Standard NIBSC-GCSF

Detection method MTT Assay

Cell type NFS-60

Detection filter 570 nm

Statistical method of analysisParallel Line Assay

Data:

Stated % IU/mg%

Estimated

Stated potency - 100000000 -

Estimated potency 81.54 82000000 -

Lower confidence limit

61.25 61000000 75.1

Higher confidence limit

108.20 110000000 132.7

Result/Observation:

Sample activity (IU/mg)

8.2x 107

Sample activity (IU/mL)

-

17.7412.87

72.55

16.6312.18

73.24

18.713.19

70.53

0

10

20

30

40

50

60

70

80

Total Protein for rh-GCSFpurification (gm)

Amount of rh-GCSF (gm) Percentage of Recovery

rh-GCSF Recovery at Refolding

Batch1

Batch2

17.74

9.8

55.24

16.63

9.7

58.33

18.7

10.6

56.68

0

10

20

30

40

50

60

70

Total Protein for rh-GCSFpurification (gm)

Amount of rh-GCSF (gm) Percentage of Recovery

rh-GCSF Recovery at Buffer Exchange 1

Batch1Batch2Batch3

17.74

8.7

49.04

16.63

8.7

52.32

18.7

9.0

48.13

0

20

40

60

80

Total Protein for rh-GCSFpurification (gm)

Amount of rh-GCSF (gm) Percentage of Recovery

rh-GCSF Recovery at Buffer Exchange 2

Batch1

Batch2

Batch3

17.74

9.0

50.73

16.63

9.1

54.72

18.7

9.4

50.27

0

20

40

60

Total Protein for rh-GCSFpurification (gm)

Amount of rh-GCSF (gm) Percentage of Recovery

rh-GCSF Recovery at Cation Exchange 1

Batch1Batch2Batch3

17.74

7.5

42.28

16.63

7.6

45.7

18.7

7.9

42.25

0

10

20

30

40

50

Total Protein for rh-GCSFpurification (gm)

Amount of rh-GCSF (gm) Percentage of Recovery

rh-GCSF Recovery at Cation Exchange 2

Batch1Batch2Batch3

Fig: Data of ESI-TOF MS at CExFig: Chromatogram of Refolding sampleFig: Chromatogram of Reduction sampleFig: Gran staining image of Seed culture and

Fermentation

For all the three consecutive batches, reproducible data have been achieved. The OD of seedculture, duration of seed culture and fermentation, growth pattern, the Yields of upstream(Biomass & Inclusion Bodies), percentage desired protein expression were in a similarfashion.

Fig: HVAC system

Peptide mapping was done using both RP-HPLC and MassSpectrometry. A Reference Standard (here NIBSC standard) isalso processed in a same way of the sample and analyzedsimultaneously.

Peptide Mapping & comparison with CRS

Data of Peptide Mapping (ESI-TOF MS)Chromatograms of Peptide Mapping (RP-HPLC)

Sample

Standard (NIBSC)

We have used NFS-60 Cell line and double parallel line assaymethod. A Reference Standard (here NIBSC standard) wasused with sample for the assay. The data of standard andsample were plotted and the parallelism is observed.

Related Proteins

Reference Standard (NIBSC)

Rh-GCSF

Related protein assay was used todetermine the similar proteins otherthan rh-GCSF. The purity of our rh-GCSF was more than 99.0%

We have used ELISA to determine whetherany Cell wall protein or protein fragment

Isoelectric Point (pI) of all the samples ofour batches complied with the standard(NIBSC )

We have used the Real-Time PCR methodto detect whether host DNA or itsfragment is present in the product or not.

Chromatogram of Size Exclusion Chromatography

Non-Reducing gel

Reducing gel

The rh-GCSF were tested for any otherprotein impurities by Size ExclusionChromatography and SDS-PAGE. Thepurity of our rh-GCSF was more than99.0% for all the validation batches.

•The samples from validation batches are analyzed for sterility according to EP.

The maximum limit of endotoxinis 2.0IU/mg for rh-GCSF and allof our product from validationbatches passed this criteria.

Fig: Cleaning is going on in the ‘C’ area

Fig: Cleaning of the corridor at the entry of ‘C’ area

Fig: Laminar flow Cabinet & Double Door Autoclave

Fig: Area Monitoring

Fig: Simplified diagram of our production area mentioning man & material flow. Red and green arrow for material and man flow respectively.

Production area is fully classified area. The Final purification area is ‘Class B’ (pink marked) andthe all other area is ‘Class C’ (white). Remotely controlled CIP/SIP /DIP system for upstreamarea. Total area is under strict environment monitoring according to regulatory guideline.

Fig: HVAC system

Total logbook

140

Room condition Monitoring- 44

Machine & Equipment- 48

Maintenance - 48

SOP

55

Process SOP

7

Machine & Equipment

48

Upstream

10

Downstream

14

General

24

• Each and every work is documented properly • All the following documents are updated regularly to maintain c-GMP practice.• A screen shot of BMR is given here.

Fig: Snap of BMR

• We are highly aware of EHS policy.• Biological samples are handled under Biosafety Cabinet, and Acids or volatile liquids are dispensed under Fume hood.

Fig: screen shot of MSDS Fig: Carrying acids Fig: Dispensing of acids

Fig: Working under Biosafety cabinet

• We have equipments from world leader brands.• All the equipments are under CMC or AMC to ensure proper function all the time.• Calibration of the equipments are done regularly to get reproducible results. • Employee of Incepta Biotech are sent abroad to be well trained for using all the equipments.

Applied Biosystems DNA sequencer

AB Sciex ESI-TOF MS Dionex UHPLC Akta Ready (GE)

Bioengineering 100L Fermenter Applied Biosystems Real-Time PCR

Akta Pilot (GE)

Working Cell Bank

Kans

,Absent

Screening for best clone using plate containing

Kanamycin

Kanr

Present

Best clone

Grow in flask

Store at -80ºC

Glycerine stock (Master Cell Bank)

Clone characterization

MCBLB plate of transformed cell

Pre Inoculum preparation in

TB mediaFermentation

Optimization of the process, Process Validation followed by risk assessment and cleaning validation

Pre-clinical Study Clinical TrailMarketing of the product

SEED CULTURE

Fig: Fermenter Fig: Fermentation batch monitoring record

The E. Coli cells are cultured in a controlled pH, temperature, air flow & agitation.

FERMENTATION

Fig: Centrifuge Fig: Cell Disruptor

HARVESTING & CELL DISRUPTION

Fig: Centrifuge to collect washed IB

Fig: IB mixing with wash buffer

WASH OF INCLUSION BODIES(IB)

Fig: IB Solubilization

IB SOLUBILIZATION & REDUCTION

Fig: Refolding in 4-6°C with Argon gas flush

REFOLDING

Fig: TFF for buffer exchange

BUFFER EXCHANGE-1CHROMATOGRAPHY -1

Fig: Process Columns

Dataf ile Name:RNDGCSF002-Final.lcdSample Name:RNDGCSF002-FinalSample ID:RNDGCSF002-Final

0.0 5.0 10.0 15.0 20.0 min

-100

0

100

200

300

400

500

600

700

mAU215nm4nm (1.00)

11

.39

2

Fig: Chromatogram of CExsample

CHROMATOGRAPHY -2BUFFER EXCHANGE-2

Fig: TFF SystemFig: Tangential Flow Filtration

(TFF) for buffer exchange

BUFFER EXCHANGE FOR FORMULATION

Fig: Sterile filtration under Laminar air flow

STERILE FILTRATION

The below mentioned process steps were optimized for producing rh-GCSF. These steps were executed repeatedly during the process validation batches.

We have taken 110g Inclusion bodies in every batches for downstream processing. The percentage of recovery in every steps and final yield of protein were almost similar.

According to validation sampling plan, in process samples were taken and analyzed fortracking the process at various time intervals. Gram staining, SDS-PAGE, HPLC, LC-MSanalysis were done in various steps.

INTRODUCTION

Process Development Scale determination

Process Optimization

Process Validation followed by risk assessment & cleaning

validation

Product characterization

Stability studies(Real time & accelerated)

Before starting validation batch the clone has been characterized and the results are shown in following table:

A PARADIGM OF TRANSLATIONAL RESEARCH

Data from DownstreamRESULTS & DISCUSSION

Fig. Sterility test of rh-GCSF

= Pass-box = Door = Material Flow = Man Flow

from host cell ispresent in theproduct or not.

GMP COMPLIANT AREA