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