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 Rooms for Pharma industry Class # microrganisms/m 3 of air

A B C D <1 5 100 500 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 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

 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