drug manufacturing bit 230 walsh chapter 3. drug manufacturing most regulated of all manufacturing...
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Drug Manufacturing
BIT 230
Walsh Chapter 3
Drug Manufacturing
Most regulated of all manufacturing industries Highest safety and quality standards Parameters include:
– Design and layout of facility– Raw materials– Process itself– Personnel– Regulatory framework
Pharmacopeias
Discussed before in other units and classes Martindale- not a standards book Gives information about drugs
– Physiochemical properties– Pharmacokinetics– Uses and modes of administration– Side effects– Appropriate doses
GMP guidelines
Different publications world wide, but generally have similar information
Go over everything from raw materials to the facility
US guidelines issues publications called “Points to Consider” for additional guidelines for newer biotech products (will go over these later in semester)
Manufacturing facility
Most manufacturing facilities have requirements, but some specifics to biotech products, especially
– Clean room
– Water
Clean Rooms
Clean room views Environmentally controlled areas Critical steps for bio/injectable drugs are
produced in clean rooms Contain high efficiency particulate air (HEPA)
filters in the ceiling Figure 3.1 page 98 of chapter
Classification of Clean Roomsfor Pharma industry
Class # microrganisms/m3 of air
A <1
B 5
C 100
D 500
See table 3.5 page 100 of chapter
Other considerations
Exposed surfaces – smooth, sealed, non-penetrable surface
Chemically-resistant floors and walls Fixtures (lights, chairs, etc.) minimum and
easily cleaned Proper entry of materials and personnel into
clean room to reduce risk of contamination in clean room
Gowned person in Clean room
Clean Room clothing
Covers most of operators body Change in a separate room and enter clean
room via an air lock Clothing made from non-shredding material Number of people in a clean room at once
limited to only necessary personnel (helps with automated processes)
CDS
Cleaning, decontamination and sanitization C- removal or organic and inorganic material
that may accumulate D-inactivation and removal of undesired
materials S- destroying and removing viable
microorganisms
CDS cont’d
Done on surfaces that either are direct or indirect contact with the product
Examples of surfaces in both categories?
CDS of process equipment
Of course trickier because comes in contact with the final product
Clean equipment, then rid equipment of cleaning solution
Last step involves exhaustive rinsing of equipment with pure water – WFI– Followed by autoclaving if possible– If possible use CIP (cleaning in place)
Examples of CIP agents used to clean chromatography columns
0.5-2.0 M NaCl Non-ionic detergents 0.1-1.0 M NaOH Acetic Acid Ethanol EDTA Protease
Water
WFI- talked about this extensively before 30,000 liters of WFI needed for 1kg of a recombinant
protein Use tap water just for non-critical tasks Purified water – not as pure as WFI, but used for
limited purposes (in cough medicines, etc.) WFI used exclusively in downstream processing Will not cover pages 105-112- water and
documentation pages
Sources of Biopharmaceuticals
Genetic engineering of recombinant expression systems
Your talks will be about types of systems and how they are used- mammalian cells, yeast, bacteria etc.
Most approved products so far produced in E. coli or mammalian cell lines
E. coli
Cultured in large quantities Inexpensive (relatively speaking) Generation of quantities in a short time Production facilities easy to construct
anywhere in the world Standard methods (fermentation) used
Current products from E. Coli
tPA (Ekokinase) Insulin Interferon Interleukin-2 Human growth hormone Tumor necrosis factor
Heterologous systems
Expression of recombinant proteins in cells where the proteins do not naturally occur
Insulin first in E. coli Remember the drawbacks of expression in
E. coli?
Other problems with E. coli
Most proteins in E. coli expressed intracellularly
Therefore, recombinant proteins expressed in E. coli accumulate in the cytoplasm
Requires extra primary processing steps (e.g. cellular homogenization) and more purification (chromatography)
Other problems with E. coli, cont’d
Inclusion bodies– Insoluble aggregates of partially folded product– Heterologous expressed proteins overload the
normal protein-folding machinery– Advantage- inclusion bodies are very dense, so
centrifugation can separate them from desired material
Preventing inclusion bodies
Lower growth temperature (from 37C to 30C)
Use a fusion protein (thioredoxin) - native in E. coli – protein expressed at high levels and remains soluble
Expression in animal cells
Major advantage- correct PT modifications Naturally glycosylated proteins produced in:
– CHO - Chinese hamster ovary– BHK - baby hamster kidney– HEK – human embryonic kidney
Current products from animal cells
tPA FSH Interferon - Erythropoietin FSH Factor VIIa
Disadvantages of animal cells(compared to E. coli)
Complex nutritional requirements Slower growth More susceptible to damage Increased costs
WILL NOT cover bottom of page 116 to page 124 (up to biopharmaceuticals)- you will cover these in your presentations
Final Product Production
Focus on E. coli and mammalian systems Process starts with a single aliquot of the
Master Cell Bank Ends when final products is in labeled
containers ready to be shipped to the customer
Production: Upstream and Downstream
Upstream: initial fermentation process; yields initial generation of product
Downstream: purification of initial product and generation of finished product, followed by sealing of final containers
biomanufacturing process overview
Upstream processing
Remove aliquot from MCB Inoculate sterile medium and grow (starter
culture) Starter culture used to inoculate larger scale
production culture Production culture inoculates bioreactor Bioreactors few to several thousand liters See figure 3.13 of chapter (page 129)
Upstream cont’d
Pages 129-133 go over specific details for microbial fermentation
Pages 133-134 go over specific details for animal cell culture Properties of animal cells
– Anchorage dependent– Grow as a monolayer– Contact inhibited– Finite lifespan– Longer doubling times– Complex media requirements
Downstream processing
Diagram page 135 of chapter 3
Detailed steps considered confidential
Clean room conditions for downstream
Downstream cont’d
Steps involved (intracellular products – E. coli.) – mammalian products secreted in media, so easier to isolate)
– Centrifugation or filtration– Homogenization– Removal of cellular debris– Concentration of crude material (by precipitation or ultra
filtration)– High resolution chromatography (HPLC)– Formulation into the final product
Downstream cont’d
Final product formulation– Chromatography yields 98-99% pure product– Add excipients (non active ingredients), which
may stabilize the final product– Filtration of final product, to generate sterile
product– Freeze drying (lyophilization) if product if to be
sold as a powder (dictated by product stability)
Separation methods
Page 142,tables 3.18 and 3.19 Familiar with:
– Ion-exchange– Gel-filtration– Affinity chromatography
Protein A chromatography Immunoaffinity chromatography
Factors that influence biological activity
Denature or modify proteins Results in loss of/reduced protein activity Need to minimize loss in downstream work Problems can be chemical (e.g., oxidizing,
detergents); physical (e.g., pH, temperature); or biological (e.g., proteolytic degradation)
Table 3.20 page 143
Proteolytic degradation
Hydrolysis of one or more peptide bonds Results in loss of biological activity Trace quantities of proteolytic enzymes or chemical
influences Several classes of proteases:
– Serine– Cysteine– Aspartic– Metalloproteases (also in other ppt)
Protease inhibitors
PMSF – serine and cysteine proteases Benzamidine – serine proteases Pepstatin A – aspartic proteases EDTA – metalloproteases
a.a residue known to be present at active site of protein, so disruption of it causes loss of activity
Others (mentioned before)
Deamidation – hydrolysis of side chain of asparagine and glutamine– Happens at high temp and extreme pH
Oxidation and disulphide exchange– Oxidation by air (met and cys in particular)
Alterations of glycosylation patterns in glycoproteins (more than one sugar)– Affect activity or immunological properties
Excipients
Substances added to final product to stabilize it
Serum albumin– Withstands low pH or elevated temps– Keeps final product from sticking to walls of
container– Stabilize native conformation of protein
Excipients cont’d
Amino acids
– Glycine – stabilizes interferon, factor VIII, stabilizes against heat
Alcohols (and other polyols)– Stabilize proteins in solution
Surfactants– Reduces surface tension; proteins don’t aggregate, so don’t denature
Final product fill
See figure 3.27 page 153 Bulk product gets QC testing Passage through 0.22 m filter for final
sterility Aceptically filled into final product containers Uses automated liquid handling systems
Final product fill cont’d
Freeze drying (lyophilization) Yields a powdered product Reduces chemical and biological degradation
of final product Longer shelf life than products in solution Storage for parenteral products (those
administered intravenously or injected)
Freeze drying cont’d
Need to add cryoprotectors– Glucose or sucrose– Serum albumin– Amino acids– Polyols
Freeze drying can be done in many steps
Labeling and Packing
After sealed in final container, product quarantined
Samples are QC’d Check potency, sterility and final volume Detection and quantitation of excipients Highly automated procedures Labeling function critical- biggest error where
many products are made
Label
Name and strength of product Specific batch number Date of manufacture and expiry date Required storage conditions Name of manufacturer Excipients included Correct mode of usage
Other final product items
Biopharmaceutical products undergo more testing than traditional pharma products
Products made in recombinant systems have more potential to be contaminated than synthetic chemical drugs
Larger, more complex molecules