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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 l.jpg

Drug Manufacturing

BIT 230

Walsh Chapter 3

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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

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  • 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

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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)

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Manufacturing facility

  • Most manufacturing facilities have requirements, but some specifics to biotech products, especially

    • Clean room

    • Water

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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

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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

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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

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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)

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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

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CDS cont’d

  • Done on surfaces that either are direct or indirect contact with the product

  • Examples of surfaces in both categories?

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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)

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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

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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

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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

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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

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Current products from E. Coli

  • tPA (Ekokinase)

  • Insulin

  • Interferon 

  • Interleukin-2

  • Human growth hormone

  • Tumor necrosis factor

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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?

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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)

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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

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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

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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

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Current products from animal cells

  • tPA

  • FSH

  • Interferon -

  • Erythropoietin

  • FSH

  • Factor VIIa

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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

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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

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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

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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)

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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

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Downstream processing

  • Diagram page 135 of chapter 3

  • Detailed steps considered confidential

  • Clean room conditions for downstream

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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

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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)

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Separation methods

  • Page 142,tables 3.18 and 3.19

  • Familiar with:

    • Ion-exchange

    • Gel-filtration

    • Affinity chromatography

      • Protein A chromatography

      • Immunoaffinity chromatography

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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

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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)

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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

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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

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  • 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

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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

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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

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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)

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Freeze drying cont’d

  • Need to add cryoprotectors

    • Glucose or sucrose

    • Serum albumin

    • Amino acids

    • Polyols

  • Freeze drying can be done in many steps

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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

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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

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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