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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.

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drug manufacturing

Drug Manufacturing

BIT 230

Walsh Chapter 3

drug manufacturing2
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
  • 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
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
Manufacturing facility
  • Most manufacturing facilities have requirements, but some specifics to biotech products, especially
    • Clean room
    • Water
clean rooms
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 rooms for pharma industry
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
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
clean room clothing
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)
  • 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
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
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
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
  • 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
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
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
Current products from E. Coli
  • tPA (Ekokinase)
  • Insulin
  • Interferon 
  • Interleukin-2
  • Human growth hormone
  • Tumor necrosis factor
heterologous systems
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
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
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
Preventing inclusion bodies
  • Lower growth temperature (from 37C to 30C)
  • Use a fusion protein (thioredoxin) - native in E. coli – protein expressed at high levels and remains soluble
expression in animal cells
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
Current products from animal cells
  • tPA
  • FSH
  • Interferon -
  • Erythropoietin
  • FSH
  • Factor VIIa
disadvantages of animal cells compared to e coli
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
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
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
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
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
Downstream processing
  • Diagram page 135 of chapter 3
  • Detailed steps considered confidential
  • Clean room conditions for downstream
downstream cont d
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 d33
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
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
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
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
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
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
  • 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
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
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
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
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
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
  • 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
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