Transcript Drug Manufacturing - MCCC Faculty & Staff Web Pages

Drug Manufacturing
BIT 230
Walsh Chapter 3
Drug Manufacturing
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Most regulated of all manufacturing industries
Highest safety and quality standards
Parameters include:
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Design and layout of facility
Raw materials
Process itself
Personnel
Regulatory framework
Pharmacopeias
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Discussed before in other units and classes
Martindale- not a standards book
Gives information about drugs
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Physiochemical properties
Pharmacokinetics
Uses and modes of administration
Side effects
Appropriate doses
GMP guidelines
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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
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Most manufacturing facilities have
requirements, but some specifics to biotech
products, especially
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Clean room
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Water
Clean Rooms
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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 Rooms
for 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
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Exposed surfaces – smooth, sealed, nonpenetrable 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
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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
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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
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Done on surfaces that either are direct or
indirect contact with the product
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Examples of surfaces in both categories?
CDS of process equipment
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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
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WFI
Followed by autoclaving if possible
If possible use CIP (cleaning in place)
Examples of CIP agents used to clean
chromatography columns
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0.5-2.0 M NaCl
Non-ionic detergents
0.1-1.0 M NaOH
Acetic Acid
Ethanol
EDTA
Protease
Water
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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
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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
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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
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tPA (Ekokinase)
Insulin
Interferon 
Interleukin-2
Human growth hormone
Tumor necrosis factor
Heterologous systems
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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
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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
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Inclusion bodies
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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
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Lower growth temperature (from 37C to
30C)
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Use a fusion protein (thioredoxin) - native in
E. coli – protein expressed at high levels and
remains soluble
Expression in animal cells
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Major advantage- correct PT modifications
Naturally glycosylated proteins produced in:
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CHO - Chinese hamster ovary
BHK - baby hamster kidney
HEK – human embryonic kidney
Current products from animal cells
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tPA
FSH
Interferon -
Erythropoietin
FSH
Factor VIIa
Disadvantages of animal cells
(compared to E. coli)
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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
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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
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Upstream: initial fermentation process; yields
initial generation of product
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Downstream: purification of initial product
and generation of finished product, followed
by sealing of final containers
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biomanufacturing process overview
Upstream processing
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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
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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
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Diagram page 135 of chapter 3
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Detailed steps considered confidential
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Clean room conditions for downstream
Downstream cont’d
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Steps involved (intracellular products – E. coli.) –
mammalian products secreted in media, so easier to
isolate)
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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
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Final product formulation
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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
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Page 142,tables 3.18 and 3.19
Familiar with:
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Ion-exchange
Gel-filtration
Affinity chromatography
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Protein A chromatography
Immunoaffinity chromatography
Factors that influence biological
activity
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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
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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:
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Serine
Cysteine
Aspartic
Metalloproteases (also in other ppt)
Protease inhibitors
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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)
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Deamidation – hydrolysis of side chain of
asparagine and glutamine
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Oxidation and disulphide exchange
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Happens at high temp and extreme pH
Oxidation by air (met and cys in particular)
Alterations of glycosylation patterns in
glycoproteins (more than one sugar)
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Affect activity or immunological properties
Excipients
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Substances added to final product to
stabilize it
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Serum albumin
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Withstands low pH or elevated temps
Keeps final product from sticking to walls of
container
Stabilize native conformation of protein
Excipients cont’d
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Amino acids
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Alcohols (and other polyols)
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Glycine – stabilizes interferon, factor VIII, stabilizes against heat
Stabilize proteins in solution
Surfactants
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Reduces surface tension; proteins don’t aggregate, so don’t denature
Final product fill
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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
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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
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Need to add cryoprotectors
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Glucose or sucrose
Serum albumin
Amino acids
Polyols
Freeze drying can be done in many steps
Labeling and Packing
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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
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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
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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