BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 3 potx

GUIDES TO GOOD MANUFACTURING PRACTICE All aspects of pharmaceutical manufacture must comply with the most rigorous standards toensure consistent production of a safe, effective product..

manufactured to exacting specifications laid down in publications termed ‘pharmacopoeias’.There are more than two dozen pharmacopoeias published world-wide, most notably the UnitedStates Pharmacopoeia (USP), the European Pharmacopoeia (Eur Ph.) and the JapanesePharmacopoeia The products listed in these international pharmacopoeias are invariably genericdrugs (i.e drugs no longer patent-protected, which can be manufactured in any pharmaceuticalfacility holding the appropriate manufacturing licence) The vast bulk of such substances aretraditional chemical-based drugs, and biological substances such as insulin and various bloodproducts Two sample monographs from the European Pharmacopoeia are reproduced inAppendix 3 Future editions of such pharmacopoeias are likely to include a growing number ofbiopharmaceuticals, particularly as many of these begin to lose their patent protection.

Martindale, The Extra Pharmacopoeia

Martindale, The Extra Pharmacopoeia (often simply referred to as ‘Martindale’) represents anadditional publication of relevance to the pharmaceutical industry Unlike the pharmacopoeiasdiscussed above, Martindale is not a book of standards The aim of this encyclopaedicpublication is to provide concise, unbiased information (largely summarized from the peer-reviewed literature) regarding drugs of clinical interest

The first edition of Martindale was published in 1883 by William Martindale and the 30thedition was published in 1993 It contains information in monograph format on over 5000 drugs

in clinical use The vast bulk of substances described are chemical-based pharmaceuticals, aswell as traditional biological substances such as antibiotics, certain hormones and bloodproducts Recent editions, however, carry increasing numbers of monographs detailingbiopharmaceuticals — a reflection of their growing importance in the pharmaceutical industry.Martindale is largely organized into chapters that detail groups of drugs having similarclinical uses or actions (Table 3.1) The information presented in a monograph detailing anyparticular drug will usually include:

its physiochemical characteristics;

absorption and fate;

uses and appropriate mode of administration;

adverse/side effects;

suitable dosage levels

In addition, summaries of published papers/reviews of the substance in question are included.Because of its clinical emphasis, Martindale represents a valuable drug information source topharmacists and clinicians, but also provides much relevant drug information to personnelengaged in pharmaceutical manufacturing

GUIDES TO GOOD MANUFACTURING PRACTICE

All aspects of pharmaceutical manufacture must comply with the most rigorous standards toensure consistent production of a safe, effective product The principles underlining suchstandards are summarized in publications which detail good manufacturing practice (GMP).Pharmaceutical manufacturers must be familiar with the principles laid down in thesepublications and they are legally obliged to ensure adoption of these principles to their specificmanufacturing process Regulatory authority personnel will assess compliance of themanufacturer with these principles by undertaking regular inspections of the facility The

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subsequent granting/renewing (or refusing/revoking) of a manufacturing licence depends largelyupon the level of compliance found during the inspection.

Although separate guides to pharmaceutical GMP are published in different world regions theprinciples outlined in them all are largely similar In Europe, for example, the European Union(EU) publishes the EU Guide to Good Manufacturing Practice for Medicinal Products This guideconsists of a number of chapters, each of which is concerned with a specific aspect ofpharmaceutical manufacture (Table 3.2) The principles therein often appear little more thancommon-sense guidelines, e.g the principles outlined in the chapter detailing GMP in relation topersonnel could be summarized as:

an adequate number of sufficiently qualified, experienced personnel must be employed by themanufacturer;

key personnel, such as the heads of production and quality control, must be independent ofeach other;

THE DRUG MANUFACTURING PROCESS 95Table 3.1 List of the major headings under which various drugs are described in Martindale, The ExtraPharmacopoeia

Analgesics and anti-inflammatory

Anti-bacterial agents Contrast media Organic solvents

Anti-coagulants Corticosteroids Paraffins and similar basesAnti-depressants Cough suppressants,

expectorants and mucolytics

ParasympathomimeticsAnti-diabetic agents Dermatological agents Pesticides and repellants

Anti-epileptics Diagnostic agents Preservatives

Anti-fungal agents Disinfectants Prophylactic anti-asthma agentsAnti-gout agents Diuretics Prostaglandins

Anti-hypertensive agents Dopaminergic

anti-parkinsonian agents

RadiopharmaceuticalsAnti-malarials Electrolytes Sex hormones

Anti-migraine agents Gases Skeletal muscle relaxants

Anti-muscarinic agents Gastrointestinal agents Soaps and other anionic

surfactantsAnti-neoplastic agents and

immunosuppressants

General anaesthetics Stabilizing and suspending agentsAnti-protozoal agents Haemostatics Stimulants and anorecticsAnti-thyroid agents Histamine H1-receptor

antagonists

Sunscreen agentsAnti-viral agents Hypothalamic and pituitary

hormones

SympathomimeticsAnxiolytic sedatives, hypnotics and

personnel should have well-defined job descriptions, and should receive training such thatthey can adequately perform all their duties;

issues of personal hygiene should be emphasized, so as to prevent product contamination as aresult of poor hygiene practices

The chapter detailing premises and equipment describes similar obvious principles, such as: all premises and equipment should be designed, operated and serviced such that it is capable

of carrying out its intended function effectively;

facility design and equipment use should be such as to avoid cross-contamination or mix-upbetween different products;

sufficient storage area must be provided, and a clear demarcation must exist between storagezones for materials at different levels of processing (i.e raw materials, partially processedproduct, finished product, rejected product, etc.);

quality control labs must be separated from production and must be designed and equipped

to a standard allowing them to fulfil their intended function

Some of the principles outlined in the guide are sufficiently general to render them applicable tomost manufacturing industries However, many of the guidelines are far more specific in nature(e.g guidelines relating to the requirement for dedicated facilities when manufacturing specificproducts, including some antibiotics and hormones) In addition to the main chapters, the EUguide also contains a series of 14 annexes (Table 3.3) These lay down guidelines relating mainly

to the manufacture of specific pharmaceutical substances, such as radioactive pharmaceuticals

or products derived from human blood or human plasma One such annex (manufacture ofbiological medicinal products for human use) is included as appendix 4 of this textbook.Most of the principles outlined in such guides to GMP are equally as applicable to themanufacture of traditional pharmaceuticals as to the newer biopharmaceutical preparations.However, the regulatory authorities have found it necessary to publish additional guidelinesrelating to many of the newer biotechnology-based biopharmaceuticals Examples include the

‘Points to Consider’ series, which contain guidelines relating to safe production, e.g oftherapeutic monoclonal antibodies by hybridoma technology, and recombinant biopharma-ceuticals produced by genetic engineering (Table 3.4)

Guides to GMP and ancillary publications are among the most significant publicationsgoverning the practical aspects of drug manufacture in the pharmaceutical industry The

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Table 3.2 List of chapter titles present in the Guide to Good

Manufacturing Practice in the European Community(i.e Vol IV of the

Rules Governing Medicinal Products in the European Union)

7 Contract manufacture and analysis

8 Complaints and product recall

9 Self-inspection

implementation of the exacting standards laid down in these publications ensures total qualityassurance in the drug manufacturing process.

THE MANUFACTURING FACILITY

Appropriate design and layout of the pharmaceutical facility is an issue central to theproduction of safe, effective medicines In common with many other manufacturing facilities,pharmaceutical facilities contain specific production, quality control (QC) and storage areas,etc However, certain aspects of facility design and operation are unique to this industry, inparticular with regard to manufacturers of parenteral (injectable) products Incorporation ofthese features is rendered mandatory by guides to pharmaceutical GMP Particularlynoteworthy features, including clean room technology and generation of ultra pure water, arereviewed below

While the majority of critical manufacturing operations of injectable pharmaceuticals (e.g.most biopharmaceuticals) occurs in specialized clean areas, proper design and maintenance ofnon-critical areas (e.g storage, labelling and packing areas) is also vital to ensure overallproduct safety Strict codes of hygiene also apply to these non-critical areas

THE DRUG MANUFACTURING PROCESS 97Table 3.3 List of the specific annexes now associated with good manufacturing practice (GMP) formedicinal products (i.e Vol IV of ‘the Rules Governing Medicinal Products in the European Union’)

1 Manufacture of sterile medicinal products

2 Manufacture of biological medicinal products for human use

3 Manufacture of radiopharmaceuticals

4 Manufacture of veterinary medicinal products other than immunologicals

5 Manufacture of immunological veterinary medicinal products

6 Manufacture of medicinal gases

7 Manufacture of herbal medicinal products

8 Sampling of starting and packaging materials

9 Manufacture of liquids, creams and ointments

10 Manufacture of pressurized metered dose aerosol preparations for inhalation

11 Computerized systems

12 Use of ionizing radiation in the manufacture of medicinal products

13 Good manufacturing practice for investigational medicinal products

14 Manufacture of products derived from human blood or human plasma

Table 3.4 Some of the ‘Points to Consider’ publications available from the FDA Many of these cannow be downloaded directly from the FDA Center for Biologics Evaluation and Research (CBER) homepage, the address of which is: http://WWW.fda.gov/cber/

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human UsePoints to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications

Points to Consider in the Manufacture and Testing of Therapeutic Products for Human Use Derived fromTransgenic Animals

Points to Consider in the Characterization of Cell Lines used to Produce Biologicals

Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by RecombinantDNA Technology

Points to Consider in Human Somatic Cell Therapy and Gene Therapy

Clean rooms

Clean rooms are environmentally controlled areas within the pharmaceutical facility in whichcritical manufacturing steps for injectable/sterile (bio)pharmaceuticals must be undertaken Therooms are specifically designed to protect the product from contamination Common potentialcontaminants include microorganisms and particulate matter These contaminants can beairborne, or derived from process equipment, personnel, etc

Clean rooms are designed in a manner that allows tight control of entry of all substances (e.g.equipment, personnel, in-process product, and even air; Figures 3.1 and 3.2) In this way, once aclean environment is generated in the room, it can easily be maintained

A basic feature of clean room design is the presence in their ceilings of high-efficiencyparticulate air (HEPA) filters These depth filters, often several inches thick, are generallymanufactured from layers of high-density glass fibre Air is pumped into the room via the filters,generating a constant downward sweeping motion The air normally exits via exhaust units,generally located near ground level This motion promotes continued flushing from the room ofany particulates generated during processing (Figure 3.1)

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Figure 3.1 Diagrammatic illustration of the flow pattern of HEPA-filtered air through a typical cleanroom Air is pumped into the room through HEPA filters (see text) located in the ceiling, and exits viaextract units, normally located at floor level Although the air flow is non-unidirectional (i.e not truelaminar flow), it generates a constant downward sweeping motion, which helps remove air-borneparticulate matter from the room

HEPA filters of different particulate-removing efficiency are available, allowing theconstruction of clean rooms of various levels of cleanliness (Table 3.5) Such rooms areclassified on the basis of the number of (a) airborne particles and (b) viable microorganismspresent in the room In Europe clean rooms are classified as grade A, B, C or D (in order ofdecreasing cleanliness) In the USA, where approximately similar specifications are used,cleanrooms are classified as class 100 (equivalent to grade A/B), class 10 000 (grade C) or class

100 000 (grade D)

HEPA filters in grade B, C and D clean rooms are normally spaced evenly in the ceiling,occupying somewhere in the region of 20–25% of total ceiling area Generation of class A cleanroom conditions generally requires a modified design The use of high-specification HEPAfilters, along with the generation of a unidirectional downward air distribution pattern (i.e.laminar flow), is essential This is only achieved if filter occupancy of ceiling space is 100%.Most commonly, portable (horizontal or vertical) laminar flow cabinets placed in class Bcleanrooms are used to generate localized class A conditions In more extensive facilities,however, an entire class A room may be constructed

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Figure 3.2 Generalized clean room design Entry of personnel occurs via changing rooms, where theoperators first remove their outer garments and subsequently put on suitable clean room clothing (see e.g.Figure 3.3) All raw materials, portable equipment, etc enters the clean room via a transfer lock Afterbeing placed in the transfer lock, such items are sanitized (where possible) by, for example, being rubbeddown with a disinfectant solution They are then transferred into the clean room proper, by clean roompersonnel Processed product usually exits the clean room via an exit transfer lock and personnel often exitthe room via a changing room separate from the one they entered (in some cases, the same changing room

is used as an entry and exit route) Note that, in practice, product may be processed in a number ofdifferent (adjacent) clean rooms

While an effective HEPA air-handling system is essential to the generation of clean roomconditions, many additional elements are equally important in maintaining such conditions.Clean room design is critical in this regard All exposed surfaces should have a smooth, sealedimpervious finish in order to minimize accumulation of dirt/microbial particles and to facilitateeffective cleaning procedures Floors, walls and ceilings can be coated with durable, chemical-resistant materials, such as epoxy resins, polyester or PVC coatings Alternatively, such surfacesmay be completely overlaid with smooth vinyl-based sheets, thermally welded to ensure asmooth, unbroken surface.

Fixtures within the room (e.g work benches, chairs, equipment, etc.) should be kept to aminimum, and ideally be designed and fabricated from material that facilitates effective cleaning(e.g polished stainless steel) The positioning of such fixtures should not hinder effectivecleaning processes Pipework should be installed in such a way as to allow effective cleaningaround them and the presence of uncleanable recesses must be avoided All corners and jointsbetween walls and ceilings or floors are rounded, and equipment with movable parts (e.g.motors, pumps) should be encased

The transfer of processing materials, or entry of personnel into clean areas, carries with it therisk of reintroduction of microorganisms and particulate matter The principles of GMPminimizes such risks by stipulating that entry of all substances/personnel into a clean room mustoccur via air-lock systems (Figure 3.2) Such air-locks, with separate doors opening into theclean room and the outside environment, act as a buffer zone All materials/process equipmententering the clean area are cleaned, sanitized, (or autoclaved if practicable) outside this area, andthen passed directly into the transfer lock, from where it is transferred into the clean room byclean room personnel

An interlocking system ensures that both doors of the transfer lock are never simultaneouslyopen, thus precluding formation of a direct corridor between the uncontrolled area and theclean area Transfer locks are also positioned between adjacent clean rooms of different grades

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Table 3.5 Specifications laid down (in the EC Guide to Good Manufacturing Practice for MedicinalProducts) for class A, B, C and D clean rooms, as used in the pharmaceutical industry

Grade

Maximum permitted number of particles per m3

of clean room air

Maximum permitted number of viablemicroorganisms per m3of clean room air0.5 mm particle diameter 5.0 mm particle diameter

A high standard of operator personal hygiene is also of critical importance, and all personnelshould receive appropriate training in this regard Only the minimum number of personnelrequired should be present in the clean area at any given time This is facilitated by a high degree

of process automation The installation in clean room walls of windows that serve asobservational decks, coupled with intercom systems, also helps by facilitating a certain degree ofsupervision from outside the clean area

Cleaning, decontamination and sanitation (CDS)

Essential to the production of a safe, effective product is the application of an effective cleaning,decontamination and sanitation (CDS) regime in the manufacturing facility Cleaning involvesthe removal of ‘dirt’, i.e miscellaneous organic and inorganic material which may accumulate inprocess areas or equipment during production Decontamination refers to the inactivation andremoval of undesirable substances, which generally exhibit some specific biological activitylikely to be detrimental to the health of patients receiving the drug Examples includeendotoxins, viruses, or prions Sanitation refers specifically to the destruction and removal ofviable microorganisms (i.e bioburden)

Effective CDS procedures are routinely applied to:

surfaces in the immediate manufacturing area which do not come into direct contact with theproduct (e.g clean room walls and floors, work tops, ancillary equipment);

surfaces coming into direct contact with the product (e.g manufacturing vessels,chromatographic columns, product filters, etc.)

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Figure 3.3 Operator wearing clean room clothing suitable for working under aseptic conditions Notethat his entire body is covered This precludes the possibility of the operator shedding skin,microorganisms or other particulate matter into the product Photo courtesy of SmithKline BeechamBiological Services s.a., Belgium

CDS of the general manufacturing area

Primary cleaning generally entails scrubbing/rinsing the target surface with water or a detergentsolution Subsequent decontamination/sanitation procedures vary, often involving application

of disinfectants or other bacteriocidal agents Thorough cleaning prior to disinfectantapplication is essential, as dirt can inactivate many disinfectants or shield microorganismsfrom disinfectant action A range of suitable disinfectants are commercially available,containing active ingredients including alcohols, phenol, chlorine and iodine Differentdisinfectants are often employed on a rotating basis, to minimize the likelihood of thedevelopment of disinfectant-resistant microbial strains

CDS of clean room walls, floors and accessible surfaces of clean room equipment is routinelyundertaken between production runs The final CDS step often entails ‘fogging’ the room This

is achieved by placing some of the disinfectant in an aerosol-generating device (a ‘foggingmachine’) This generates a fine disinfectant mist, or fog, within the clean room, capable ofpenetrating areas difficult to reach in any other manner

CDS of process equipment

CDS of surfaces/equipment coming into direct contact with the product requires specialconsideration While CDS procedures of guaranteed efficiency must be applied, it is imperativethat no trace of the CDS agents subsequently remain on such surfaces, as this would result inautomatic product contamination The final stage of most CDS procedures, as applied to suchprocess equipment, involves exhaustive rinsing with highly pure water (water for injections;WFI) This is followed if at all possible by autoclaving

CDS of processing and holding vessels, as well as equipment that is easily detachable/dismantled (e.g homogenizers, centrifuge rotors, flexible tubing filter housing, etc.), is usuallyrelatively straightforward However, CDS of large equipment/process fixtures can be morechallenging, due to the impracticality/undesirability of their dismantling Examples include theinternal surfaces of fermentation equipment, large processing/storage tanks, process-scalechromatographic columns, fixed piping through which product is pumped, etc Specific ‘cleaning

in place’ (CIP) procedures can generally be used to accommodate such equipment A detergentsolution can be pumped through fixed pipework, followed by WFI and then the passage ofsterilizing ‘live’ steam generated from WFI Internal surfaces of fermentation/processing vesselscan be scrubbed down Such vessels are generally jacketed (Figure 3.4), thus allowingtemperature control of their contents by passage of cooling water/steam through the jacket, asappropriate Passage of steam through the jacket of the empty vessel facilitates sterilization ofits internal surfaces by dry heat

The cleaning of process-scale chromatography systems used in the purification ofbiopharmaceuticals can also present challenges Although such systems are disassembledperiodically, this is not routinely undertaken after each production run CIP protocols must thus

be applied periodically to such systems The level and frequency of CIP undertaken will dependlargely on the level and type of contaminants present in the product-stream applied Columnsused during the earlier stages of purification may require more frequent attention than systemsused as a final ‘clean-up’ step of a nearly pure protein product While each column is flushedwith buffer after each production run, a full-scale CIP procedure may be required only afterevery 3–10 column runs Most of the contaminants present in such columns are acquired fromthese previous production runs

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Processing of product derived from microbial sources can result in contamination ofchromatographic media with lipid, endotoxins, nucleic acids and other biomolecules.Application of plant-derived extracts can result in column fouling with pigments and negativelycharged polyphenolics, as well as various substances released from plant cell vacuoles (many ofwhich are powerful protein precipitants/denaturants) In addition, some plant-derived enzymesare capable of degrading certain carbohydrate-based (e.g dextran) chromatographic media.Chromatography of extracts from animal/human tissue can result in column contaminationwith infectious agents or biomolecules, such as lipids Furthermore, buffer components maysometimes precipitate out of solution within the column.

Fortunately, most types of modern chromatographic media are resistant to a range of harshphysicochemical influences that may be employed in CIP protocols (Table 3.6) CIP protocolsfor chromatography columns are normally multistep, consisting of sequential flushing of the gelwith a series of CDS agents

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Figure 3.4 Diagram of a typical jacketed processing vessel Such vessels are usually made from highgrade stainless steel By opening/closing the appropriate valves, steam or cold water can be circulatedthrough the jacket In this way, the vessel’s contents can be heated or cooled, as appropriate In addition,passage of steam through the jacket of the empty vessel will effectively sanitize its internal surfaces

Table 3.6 The range of CIP agents often used to clean/sanitize

chromato-graphic columns Most CIP protocols would make use of two or more of

these agents, allowing them to sequentially percolate through the column at a

slow flow rate Contact time can range from several minutes to overnight

NaOH is particularly effective at removing most contaminant types

Application of concentrated solutions of neutral salts (e.g KCl or NaCl) is often effective inremoving precipitated/aggregated proteins, or other material retained in the column via ionicinteraction with the media The use of buffers containing EDTA or other chelating agents helpsremove any metal ions associated with the gel Use of (dilute) detergent solutions is effective inremoving lipid and a whole range of other contaminants Solvent-containing (e.g ethanol,butanol, isopropanol) buffers may also be used in this regard Increasing the columntemperature to 50–608C may sometimes be considered, particularly if lipid appears to be amajor column contaminant (most lipids liquefy at such temperatures).

Sodium hydroxide is one of the most extensively used chromatography CIP agents It isreadily available, inexpensive and effective It is usually applied to a column at strengths of up to1.0 M At such concentrations, it quickly removes/destroys most contaminants, includingmicroorganisms and viruses It will also degrade endotoxin (discussed later) within minutes.Most types of chromatographic media can withstand incubation with NaOH for prolongedperiods This allows CDS efficiency to be maximized by retaining NaOH in the column for timeperiods of the order of 30–60 minutes (silica gel is an exception, as it is quickly destroyed at pHvalues greater than 8) The chromatographic column is subsequently rinsed exhaustively bypumping WFI (or buffer made with WFI) through, until the column effluent is free from alltraces of NaOH Prolonged exposure of the chromatographic media/column parts to residualNaOH could promote column deterioration, and obviously could contaminate/inactivate theprotein stream in the next production run

Chromatographic systems are also designed to facilitate effective CIP Internal surfaces of thecolumn, its valves and piping, are smooth, impervious and devoid of recesses which couldharbour microorganisms or other contaminants

Periodic system disassembly allows more extensive CDS procedures to be undertaken Mostcolumns are manufactured from glass, or more usually, tough plastic or stainless steel After athorough cleaning of all disassembled components, sterilization by autoclaving is usuallyundertaken prior to re-assembly Most chromatographic media likewise can be autoclavedbefore column re-pouring

CIP of the ring main systems used to store and circulate WFI and purified water around thepharmaceutical plant is also routinely undertaken (see next section) This normally entailsemptying the ring main systems (including reservoirs), opening all the outlet valves, andsubsequently pumping sterile steam through all pipework This is generally sufficient tophysically dislodge any traces of trapped particulate matter or biological agents harboured inthe system

Water for biopharmaceutical processing

Water represents one of the most important raw materials used in biopharmaceuticalmanufacture It is used as a basic ingredient of fermentation media, and in the manufacture

of buffers used throughout product extraction and purification It represents the solvent inwhich biopharmaceutical products sold in liquid form are dissolved, and in which freeze-driedbiopharmaceuticals will be reconstituted immediately prior to use It is also used for ancillaryprocesses, such as the cleaning of equipment, piping and product-holding tanks It isadditionally used to clean/rinse the vials into which the final product is filled

It has been estimated that up to 30 000 litres of water is required to support the production of

1 kg of a recombinant biopharmaceutical produced in a microbial system It is not surprising,therefore, that the generation of water of suitable purity for processing is central to the

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successful operation of any (bio)pharmaceutical facility The use of potable water (i.e water ofdrinking standard) is limited to tasks such as routine cleaning of non-critical areas/processequipment Such water must be subject to further in-house purification prior to its use in themanufacturing process Water of two different levels of quality is usually required These aretermed ‘purified water’ and ‘water for injections’ (WFI) These are distinguished on the basis ofthe range and quantity of allowable contaminants, with WFI being the most pure Specificcriteria defining levels and types of contaminants permissible in both (along with guidelines as tohow the water must be produced) are outlined in international pharmacopoeias.

Purified water has a number of limited uses in pharmaceutical manufacturing It is often used

as the solvent in the manufacture of aqueous-based oral products (e.g cough mixtures,veterinary de-wormers, etc.) It is not intended to be used as a solvent in the downstreamprocessing of parenteral products — the category into which almost all biopharmaceuticals fall

It is used for primary cleaning of some process equipment/clean room floors, particularly inclass D or C clean areas, and for the generation of steam in the facilities’ autoclaves Inbiopharmaceutical processing, purified water is often used in the generation of fermentationmedia used to culture biopharmaceutical-producing recombinant microorganisms Its use insubsequent downstream processing is precluded, as its specifications allows the presence ofmany contaminants which downstream processing aims to minimize or eliminate from theproduct

Water for injection finds extensive application in biopharmaceutical manufacturing.Although there is no regulatory requirement to do so, some manufacturers use WFI whenmaking microbial fermentation media It is also commonly used in making culture media used

in the process-scale propagation of biopharmaceutical-producing mammalian cell lines Lowinitial bioburden in WFI renders its sterilization by filtration more straightforward.Furthermore, mammalian cells can be particularly sensitive to some potential watercontaminants The presence of even low concentrations of heavy metals, for example, canadversely effect the growth/product-producing characteristics of these cells

WFI is the grade of water used exclusively in all downstream biopharmaceutical processingprocedures, ranging from making extraction/homogenization/chromatography buffers torinsing process equipment coming into direct contact with the product

Generation of purified water and water for injections (WFI)

Purified water and WFI are generated from potable water While the main techniques by whichthey are produced are specified by pharmacopoeias, pre-treatment of the incoming potablewater will vary, and is often dictated by the range of contaminants found in this water.While some companies may have a private source of potable water, most obtain incomingwater from local municipal authorities This water is sure to contain various levels of severalpotential contaminants (Table 3.7) A multi-step purification procedure is then undertaken,which usually contains some or all of the following steps (see also Figure 3.5):

the incoming water is collected in a storage or ‘break’ tank, from where it is pumped through

a depth filter, organic trap and carbon filter The depth filter often contains a mixture ofgranular anthracite, washed sand, and gravel Solids and colloidal material are retained asthe water percolates through the filter bed The bed may be regenerated every few days bybackwashing (i.e reversing the direction of water flow through the bed) The organic trap

THE DRUG MANUFACTURING PROCESS 105

contains a resin to which much organic matter will bind, while the carbon (activatedcharcoal) will absorb residual organics (e.g humic acid) as well as chlorine and otherdisinfectants often added by the municipal authorities to control drinking water bioburden; a deionization step is normally undertaken next, entailing sequential passage of waterthrough a cation exchanger and anion exchanger Cations in the water (e.g Na+, Ca2+,

Mg2+) are retained on the cation exchanger, by displacing H+ ions off the exchanger.Anions (e.g Cl7, SO327are exchanged with the hydroxyl (OH7)) counter-ions of the anionexchanger In some instances the water will then be fed directly into a smaller, mixed-bed ionexchanger (e.g containing both cation and anion exchangers), as a final polishing step.The efficiency of these steps can be conveniently monitored by continuous in-line measurement

of the resistivity of the water (deionization results in increased resistivity, typically to levels of 1–

10 MO) If the resistivity of the deionized water falls below a value of approximately 1 MO,automatic system shut-off, followed by regeneration of the anion and cation exchange beds(with NaOH and HCl respectively), is initiated

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Table 3.7 Range of impurities found in potable water Even extremely low levels of any such impuritiesrenders this grade of water unacceptable for pharmaceutical processing purposes Reproduced bypermission of John Wiley & Co Ltd from Walsh & Headon (1994)

Particulate matter Soil particles

Dirt particlesParticles derived from decaying organic matter, such as leavesParticles derived from leaching of internal surfaces of water pipesVarious dissolved substances Substances derived from decaying organic matter

Traces of agricultural run-offMinerals, leached into the source waterVarious polluting substances

Viable organisms Various microorganisms

Figure 3.5 Overview of a generalized procedure by which purified water and WFI are generated in apharmaceutical facility Refer to text for specific details

Deionizers fail to remove microbial contaminants, and exchange resin beds can often actuallyharbour microorganisms, contaminating the water For this reason, filtration or UV treatmentoften constitutes the next purification step One such treatment configuration entails pumpingthe deionized water through a 0.45 mm filter, followed by exposure to UV (at 254 nm) This may

be followed by yet another filtration step through a 0.22 mm filter to remove UV-inactivatedmicroorganisms In some systems the 0.22 mm filter may even be replaced by ultrafilters capable

of retaining molecules whose molecular mass is significantly greater than 10 kDa Such systemswill remove not only bacteria but also many bacterial products, including endotoxin andadditional organic and colloidal material

Deionized water often meets the pharmacopoeial criteria laid down for ‘purified water’.Sometimes, however, further purification may be necessary to attain this standard This oftenentails a distillation or reverse-osmosis step Deionized water will, however, not meet thepharmacopoeial requirements for WFI WFI is best generated by distillation of deionized water.Distillation entails converting water to vapour by heat, followed by passing over a condenser,which results in condensation of pure water Dissolved minerals and most organics are notvolatile at 1008C

Reverse osmosis (RO) entails the use of a semi-permeable membrane (permeable to thesolvent, i.e water molecules, but impermeable to solutes, i.e contaminants) Osmosis describesthe movement of solvent molecules across such a membrane from a solution of lower soluteconcentration to one of higher solute concentration The force promoting this movement istermed ‘osmotic pressure’ During reverse osmosis, a pressure greater than the natural osmoticpressure is applied to the system from the higher solute concentration side, causing solventmolecules to flow in the opposite direction

RO membranes are manufactured from polymers such as cellulose acetate or polyamides Asingle pass of water through such membranes can remove 95% of all dissolved solids and 99%

of microorganisms and endotoxins Double RO systems are often employed, increasing theefficiency of the process still further, and providing some element of protection against theconsequences of puncture of one of the membranes While RO systems are less expensive thandistillation, many regard distillation as being a safer option Pin-head punctures of ROmembranes can be hard to detect Membranes are also susceptible to microbial colonization andcannot be exposed to high temperatures, which renders effective sanitation more difficult

Distribution system for WFI

Upon its manufacture, WFI is fed into a sealed storage vessel, often made from stainless steel.The water is circulated via a series of pipework throughout the building, and from which anumber of outlet valves are available The pipework leads back to the storage tank, allowingconstant recirculation of the water throughout the facility Because of this it is known as a ringmain or loop system (Figure 3.6)

As pharmacopoeial specifications preclude the addition of sanitizing agents such as chlorine,maintenance of WFI within microbiological specifications requires special attention Whilecirculating, the WFI is maintained at a flow rate of the order 9 ft/s This ensures constantturbulent flow, discouraging microbial attachment to internal surfaces of the distribution pipes.The WFI is also constantly maintained at temperatures of the order of 858C, again todiscourage microbial growth

Water collected from the WFI hot loop is generally allowed to cool before use forbiopharmaceutical processing (hot buffers would not only potentially denature the protein

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Figure 3.6 (a) Generalized diagram of how WFI is distributed throughout a typical pharmaceuticalfacility One or more outlet valves from the ring main system are in place in the clean rooms in whichproduct manufacture is undertaken Note that no such outlet is present in the clean room where finalproduct fill will take place (i.e the grade B cleanroom housing the grade A laminar flow hood in the aboveexample) (b) Illustration of an acceptable valve design at a WFI outlet point This design preventsstagnation of water, which could occur if such an outlet point was poorly designed, as illustrated in (c).Refer to text for specific details

product, but their pH values could change quite dramatically when cooled from 858C to 258C).

In some instances a separate loop system is constructed in which WFI is circulated at ambienttemperature allowing its immediate use in processing operations To maintain water quality,however, this entire circulating system must be emptied and sanitized every 24 h

Careful design of circulating systems also helps to maintain the microbiological quality ofWFI Circulating pipework lengths are fused together by welding, as opposed to the use ofthreaded fittings, which could harbour bacteria ‘Dead-legs’ (areas where water could stagnate,e.g at water outlet points), are avoided, and bends in pipes are smooth and curving, as opposed

to the use of abrupt T-junctions UV cells are also fitted on-line in the system, subjectingcirculating water to their continual bactericidal influence

Upon initial installation, the pipework is cleaned by passage of detergent or other cleaningagents, followed by a water rinse ‘Passivation’ (exposure of the internal pipework surfaces tochemical agents, rendering the surface less reactive subsequently) is then undertaken, usually byoxidation using nitric acid or certain organic acids

WFI is quite corrosive, especially at 858C, and it can promote leeching from even high-gradestainless steel piping Addition of ozone to the WFI can alleviate this, as the ozone’s microcidalproperties facilitate prolonged storage/circulation of the water at 258C Other innovations inthis field include replacement of stainless steel pipework with chemically inert plastics However,extensive tests need to be undertaken in order to prove that WFI cannot leach potentiallydangerous substances from these plastics before their use will become routine

Loop systems distributing purified water also exist in most facilities, with their design beingsomewhat similar to WFI systems The more relaxed microbiological specifications relating topurified water renders the design and operation of such systems less complex Purified water isgenerally circulated at ambient temperatures, via stainless steel or sometimes plastic-basedtubing

Samples of WFI and purified water are usually collected daily by quality control personnel,and tested for conformance to specification Failure to meet specification results in the systembeing emptied and fully sanitized, before generation of fresh water

Documentation

Adequate documentation forms an essential part of good manufacturing practice For thisreason, every aspect of pharmaceutical manufacture is characterized by the existence ofextensive associated documentation This is essential in order to:

help prevent errors/misunderstandings associated with verbal communication;

facilitate the tracing of the manufacturing history of any batch of product;

ensure reproducibility in all aspects of pharmaceutical manufacture

Most documents associated with pharmaceutical manufacturing fall into one of fourcategories:

standard operating procedures (SOPs);

and inspected by senior technical personnel (often the production or QC manager or both),before their final approval for general use Such documents are reviewed regularly, and updated

SOPs relating directly to personnel (e.g step-by-step procedures undertaken when

gowning-up before entering a clean room);

SOPs relating to testing/analysis (e.g procedures detailing how to properly sample rawmaterials/finished product for QC analysis, SOPs relating to the routine sampling and testing

of WFI from the ring main system, etc.)

Specifications

Specifications are normally written by QC personnel They detail the exact qualitative andquantitative requirements to which individual raw materials or product must conform Forexample, specifications for chemical raw materials will set strict criteria relating to thepercentage active ingredients present, permitted levels of named contaminants, etc Specifica-tions for packing materials will, for example, lay down exact dimensions of product packagingcartons; specifications for product labels will detail label dimensions and exact details of labeltext, etc Specifications for all raw materials are sent to raw material suppliers and, upon theirdelivery, QC personnel will ensure that these raw materials meet their specifications before beingreleased to production (the raw materials are held in ‘quarantine’ prior to their approval) Finalproduct specifications will also be prepared As most products are manufactured to conformwith pharmacopoeial requirements, many of the specifications set for raw materials/finishedproduct are simply transcribed from the appropriate pharmacopoeia

Manufacturing formulae, processing and packaging instructions

Manufacturing formulae should clearly indicate the product name, potency or strength, andexact batch size It lists each of the starting raw materials required, and the quantity in whicheach is required The processing instructions should contain step-by-step manufacturinginstructions The detail given should be sufficient to allow a technically competent person,unfamiliar with the process, to successfully undertake the manufacturing procedure

The processing instructions should also indicate the principal items of equipment utilizedduring manufacture, and the precise location in which each step should be undertaken (e.g in aspecific clean room, etc.) It will also list any specific precautions which must be observed duringmanufacture (e.g precautions to protect the product from, perhaps, excessive heating, or toprotect the operator from any potentially dangerous product constituent) Each product willalso have its own labelling and packing instructions, indicating:

the label to be used, and its exact text;

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exact packing instructions (e.g how many units of product per pack, how may packs pershipping carton, etc.).

A copy of the label to be used is generally attached to the documents, to allow the supervisorand operators to verify easily that the correct label has been dispensed for the product inquestion

Records

Maintenance of adequate and accurate records forms an essential part of GMP For any givenbatch of product, records relating to every aspect of manufacture of that batch will be retained.These records will include:

specification results obtained on all raw materials;

batch manufacturing, processing and packaging records;

QC analysis results of bulk and finished product

These records, along with samples of finished product, must be retained in the facility for atleast 1 year after the expiry of that batch Should any difficulty arise regarding thefinished product, the records should allow tracing back of all direct manufacturing steps, aswell as indirect procedures which might influence the quality/safety of the product.During inspections, regulatory inspectors usually examine the records relating to a number ofrandomly chosen batches in detail, in order to help them assess ongoing adherence to GMP inthe facility

Generation of manufacturing records

Prior to commencement of the manufacturing of a specific product, production personnel willprint/photocopy the manufacturing formulae, processing and packaging instructions associatedwith that product The responsible individual fills in the batch number in the space provided (abatch number is a unique combination of numbers and/or letters assigned to that batch, in order

to distinguish it from all other batches) This photocopied document forms the blueprint formanufacture of that batch A space is provided after each manufacturing/packing instruction.When that step is completed, the responsible operator initials the space and includes the exacttime and date undertaken This forms a detailed record of manufacture

Additional supporting documents are also included in the manufacturing records These mayinclude computerized print-outs from weighing equipment used to dispense chemical rawmaterials, or recorder charts obtained, e.g from a freeze-drier upon completion of freeze-dryingthat batch of product

QC records relating to raw materials, in-process and final product are generated in much thesame way — by printing/photocopying originals and filling in the test results obtained

Advances in information technology are now impacting upon the pharmaceutical industry.Many documents are now maintained in electronic format In fact, some regard it as likely that

in the future ‘paperless facilities’ will become commonplace, with all documentation beingcomputerized Several aspects of such electronic document maintenance deserve specialattention Adequate back-up files should always be retained Also, restricted access tocomputerized systems is required to ensure that data/documentation is only entered/amended

by persons authorized to do so

THE DRUG MANUFACTURING PROCESS 111

SOURCES OF BIOPHARMACEUTICALS

The bulk of biopharmaceuticals currently on the market are produced by genetic engineeringusing various recombinant expression systems While a wide range of potential proteinproduction systems are available (Table 3.8), most of the recombinant proteins that have gainedmarketing approval to date are produced either in recombinant Eschericia coli or inrecombinant mammalian cell lines (Table 3.9) Such recombinant systems are invariablyconstructed by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of the chosen producer cell Examples include E coli K12 and Chinesehamster ovary strain K1 (CHO-K1) Gene/cDNA transfer is normally achieved by using anappropriate expression plasmid, or other standard gene-manipulating techniques Eachrecombinant production system displays its own unique set of advantages and disadvantages,

as described below

E coli as a source of recombinant, therapeutic proteins

Many microorganisms represent attractive potential production systems for therapeuticproteins They can usually be cultured in large quantities, inexpensively and in a short time,

by standard methods of fermentation Production facilities can be constructed in any worldregion, and the scale of production can be varied as required

The expression of recombinant proteins in cells in which they do not naturally occur is termed

‘heterologous protein production’ By far the most common microbial species used to produce

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Table 3.8 Expression systems that are/could potentially be used for

the production of recombinant biopharmaceutical products (CHO¼Chinese

hamster ovary; BHK¼baby hamster kidney)

E coli(and additional prokaryotic systems, e.g Bacilli)

Yeast (particularly Saccharomyces cerevisiae)

Fungi (particularly Aspergillus)

Animal cell culture (particularly CHO and BHK cell lines)

Transgenic animals (focus thus far is upon sheep and goats)

Plant-based expression systems (various)

Insect cell culture systems

Table 3.9 Some biopharmaceuticals currently on the market that are produced by genetic engineering

in either E coli or animal cells CHO=Chinese hamster ovary cells; BHK=baby hamster kidney cellsBiopharmaceutical product Source Biopharmaceutical product SourceTissue plasminogen activator (tPA) E coli, CHO Follicle-stimulating hormone (FSH) CHOInsulin E coli Interferon-b CHO

Erythropoietin CHOInterferon-a E coli Glucocerebrosidase CHOInterferon-g E coli Factor VIIa BHKInterleukin-2 (IL-2) E coli

Granulocyte colony-stimulating factor

(G-CSF)

E coliHuman growth hormone (hGH) E coli

heterologous proteins of therapeutic interest is Eschericia coli The first biopharmaceuticalproduced by genetic engineering to gain marketing approval (in 1982) was recombinant humaninsulin (trade name Humulin), produced in E coli An example of a more recently approvedbiopharmaceutical which is produced in E coli is that of Ecokinase, a recombinant tissueplasminogen activator (tPA, Chapter 9), approved for sale in the EU in 1996 Many additionalexamples are provided in subsequent chapters.

As a recombinant production system, E coli displays a number of advantages, which include: E coli has long served as the model system for studies relating to prokaryotic genetics Itsmolecular biology is thus well characterized;

high levels of expression of heterologous proteins can be achieved in recombinant E coli(Table 3.10) Modern, high-expression promoters can routinely ensure that levels ofexpression of the recombinant protein reach up to 30% total cellular protein;

E coli cells grow rapidly on relatively simple and inexpensive media, and the appropriatefermentation technology is well established

These advantages, particularly the ease of genetic manipulation, rendered E coli the primarybiopharmaceutical production system for many years However, E coli also displays a number

of drawbacks as a biopharmaceutical producer, including:

heterologous proteins accumulate intracellularly;

inability to undertake post-translational modifications (particularly glycosylation) ofproteins;

the presence of lipopolysaccharide on its surface

The vast bulk of proteins synthesized naturally by E coli (i.e its homologous proteins) areintracellular Few are exported to the periplasmic space, or are released as true extracellularproteins Heterologous proteins expressed in E coli thus invariably accumulate in the cellcytoplasm Intracellular protein production complicates downstream processing (relative toextracellular production) because:

additional primary processing steps are required, i.e cellular homogenization, withsubsequent removal of cell debris by centrifugation or filtration;

more extensive chromatographic purification is required in order to separate the protein ofinterest from the several thousand additional homologous proteins produced by the E colicells

THE DRUG MANUFACTURING PROCESS 113Table 3.10 Levels of expression of various biopharmaceuticals

produced in recombinant E coli cells

Biopharmaceutical

Level of expression(% of total cellular protein)

An additional complication of high-level intracellular heterologous protein expression isinclusion body formation Inclusion bodies (refractile bodies) are insoluble aggregates ofpartially folded heterologous product Because of their dense nature, they are easily observed bydark field microscopy Presumably, when expressed at high levels, heterologous proteinsoverload the normal cellular protein-folding mechanisms Under such circumstances, it would

be likely that hydrophobic patches, normally hidden from the surrounding aqueous phase infully folded proteins, would remain exposed in the partially folded product This, in turn, wouldpromote aggregate formation via intermolecular hydrophobic interactions

However, the formation of inclusion bodies displays one processing advantage — it facilitatesthe achievement of a significant degree of subsequent purification by a single centrifugation step.Because of their density, inclusion bodies sediment even more rapidly than cell debris Low-speed centrifugation thus facilitates the easy and selective collection of inclusion bodies directlyafter cellular homogenization After collection, inclusion bodies are generally incubated withstrong denaturants, such as detergents, solvents or urea This promotes complete solubilization

of the inclusion body (i.e complete denaturation of the proteins therein) The denaturant isthen removed by techniques such as dialysis or diafiltration This facilitates re-folding of theprotein, a high percentage of which will generally fold into its native, biologically active,conformation

Various attempts have been made to prevent inclusion body formation when expressingheterologous proteins in E coli Some studies have shown that a simple reduction in thetemperature of bacterial growth (from 378C to 308C) can significantly decrease the incidence ofinclusion body formation Other studies have shown that expression of the protein of interest as

a fusion partner with thioredoxin will eliminate inclusion body formation in most instances.Thioredoxin is a homologous E coli protein, expressed at high levels It is localized at theadhesion zones in E coli, and is a heat-stable protein A plasmid vector has been engineeredwhich facilitates expression of a fusion protein, consisting of thioredoxin linked to the protein ofinterest via a short peptide sequence, recognized by the protease enterokinase (Figure 3.7) Thefusion protein is invariably expressed at high levels, while remaining in soluble form.Congregation at adhesion zones facilitates its selective release into the media by simple osmoticshock This can greatly simplify its subsequent purification After its release, the fusion protein

is incubated with enterokinase, thus releasing the protein of interest (Figure 3.7)

An alternative means of reducing/potentially eliminating inclusion body accumulation entailsthe high-level co-expression of molecular chaperones, along with the protein of interest.Chaperones are themselves proteins which promote proper and full folding of other proteinsinto their biologically active, native three-dimensional shape They usually achieve this bytransiently binding to the target protein during the early stages of its folding and guiding furtherfolding by preventing/correcting the occurrence of improper hydrophobic associations.The inability of prokaryotes such as E coli to carry out post-translational modifications(particularly glycosylation) can limit their usefulness as production systems for sometherapeutically useful proteins Many such proteins, when produced naturally in the body,are glycosylated (Table 3.11) However, the lack of the carbohydrate component of someglycoproteins has little, if any, negative influence upon their biological activity Theunglycosylated form of interleukin-2, for example, displays essentially identical biologicalactivity to that of the native glycosylated molecule In such cases, E coli can serve as asatisfactory production system

Another concern with regard to the use of E coli is the presence on its surface oflipopolysaccharide (LPS) molecules The pyrogenic nature of LPS (see later) renders essential its

114 BIOPHARMACEUTICALS

THE DRUG MANUFACTURING PROCESS 115

Figure 3.7 High level expression of a protein of interest in E coli in soluble form by using the engineered

‘thiofusion’ expression system Refer to text for specific details

removal from the product stream Fortunately, several commonly employed downstreamprocessing procedures achieve such a separation without any great difficulty.

Expression of recombinant proteins in animal cell culture systems

Technical advances facilitating genetic manipulation of animal cells now allow routineproduction of therapeutic proteins in such systems The major advantage of these systems istheir ability to carry out post-translational modification of the protein product As a result,many biopharmaceuticals that are naturally glycosylated are now produced in animal cell lines(Table 3.9) Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells have becomeparticularly popular in this regard

While their ability to carry out post-translational modifications renders their use desirable/essential for producing many biopharmaceuticals, animal cell-based systems do suffer from anumber of disadvantages When compared to E coli, animal cells display very complexnutritional requirements, grow more slowly and are far more susceptible to physical damage Inindustrial terms, this translates into increased production costs

In addition to recombinant biopharmaceuticals, animal cell culture is used to produce variousother biologically-based pharmaceuticals Chief amongst these are a variety of vaccines andhybridoma cell-produced monoclonal antibodies (Chapter 10) Earlier interferon preparationswere also produced in culture by a particular lymphoblastoid cell line (the Namalwa cell line),which was found to naturally synthesize high levels of several interferon-as (Chapter 4)

Additional production systems: yeasts

Attention has also focused upon a variety of additional production systems for recombinantbiopharmaceuticals Yeast cells (particularly Saccharomyces cerevisiae) display a number ofcharacteristics that make them attractive in this regard These characteristics include:

their molecular biology has been studied in detail, facilitating their genetic manipulation; most are GRAS-listed organisms (‘generally regarded as safe’), and have a long history ofindustrial application (e.g in brewing and baking);

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Table 3.11 Proteins of actual or potential therapeutic use that are glycosylated whenproduced naturally in the body (or by hydridoma technology in the case of monoclonalantibodies) These proteins are discussed in detail in various subsequent chapters

Most interleukins (interleukin-1 being an important exception)

Interferon-b and -g (most interferon-as are unglycosylated)

Colony stimulating factors

Tumour necrosis factors

Gonadotrophins (follicle stimulating hormone, luteinizing hormone and human chorionic

they grow relatively quickly in relatively inexpensive media, and their tough outer cell wallprotects them from physical damage;

suitable industrial scale fermentation equipment/technology is already available;

they possess the ability to carry out post-translational modifications of proteins

The practical potential of yeast-based production systems has been confirmed by the successfulexpression of a whole range of proteins of therapeutic interest in such systems However, anumber of disadvantages relating to heterologous protein production in yeast have beenrecognized These include:

although capable of glycosylating heterologous human proteins, the glycosylation patternusually varies somewhat from the pattern observed in the native glycoprotein (when isolatedfrom its natural source, or when expressed in recombinant animal cell culture systems); in most instances, expression levels of heterologous proteins remain less than 5% of totalcellular protein This is significantly lower than expression levels typically achieved inrecombinant E coli systems

Despite such potential disadvantages, several recombinant biopharmaceuticals now approvedfor general medical use are produced in yeast (S cerevisiae)-based systems (Table 3.12).Interestingly, most such products are not glycosylated The oligosaccharide component ofglycoproteins produced in yeasts generally contain high levels of mannose Such high mannose-type glycosylation patterns generally trigger their rapid clearance from the blood stream Suchproducts, therefore, would be expected to display a short half-life when parenterallyadministered to man

Fungal production systems

Fungi have elicited interest as heterologous protein producers, as many have a long history ofuse in the production of various industrial enzymes, such as a-amylase and glucoamylase.Suitable fermentation technology therefore already exists In general, fungi are capable ofhigh-level expression of various proteins, many of which they secrete into their extracellularmedia The extracellular production of a biopharmaceutical would be distinctly advantageous

in terms of subsequent downstream processing Fungi also possess the ability to carry out

THE DRUG MANUFACTURING PROCESS 117

Table 3.12 Recombinant therapeutic proteins approved for general medical use that are produced inSaccharomycese cerevisiae All are subsequently discussed in the chapter indicated

Trade name Description Use Refer to

ChapterNovolog Engineered short-acting insulin Diabetes mellitus 8Leukine Colony stimulating factor (GM-CSF) Bone marrow

transplantation

6Recombivax, Comvax, Engerix B,

Tritanrix-HB, Infanrix, Twinrix,

post-translational modifications Patterns of glycosylation achieved can, however, differ fromtypical patterns obtained when a glycoprotein is expressed in a mammalian cell line.

Most fungal host strains also naturally produce significant quantities of extracellularproteases, which can potentially degrade the recombinant product This difficulty can bepartially overcome by using mutant fungal strains secreting greatly reduced levels of proteases.Although researchers have produced a number of potential therapeutic proteins in recombinantfungal systems, no biopharmaceutical produced by such means has thus far sought or gainedmarketing approval

Transgenic animals

The production of heterologous proteins in transgenic animals has gained much attention in therecent past The generation of transgenic animals is most often undertaken by directlymicroinjecting exogenous DNA into an egg cell In some instances, this DNA will be stablyintegrated into the genetic complement of the cell After fertilization, the ova may be implantedinto a surrogate mother Each cell of the resultant transgenic animal will harbour a copy of thetransferred DNA As this includes the animal’s germ cells, the novel genetic informationintroduced can be passed on from one generation to the next

A transgenic animal harbouring a gene coding for a pharmaceutically useful protein couldbecome a live bioreactor, producing the protein of interest on an ongoing basis In order torender such a system practically useful, the recombinant protein must be easily removable fromthe animal, in a manner which would not be injurious to the animal (or the protein) A simpleway of achieving this is to target protein production to the mammary gland Harvesting of theprotein thus simply requires the animal to be milked

Mammary-specific expression can be achieved by fusing the gene of interest with thepromoter-containing regulatory sequence of a gene coding for a milk-specific protein.Regulatory sequences of the whey acid protein (WAP), b-casein and a- and b-lactoglobulingenes have all been used to date to promote production of various pharmaceutical proteins inthe milk of transgenic animals (Table 3.13)

One of the earliest successes in this regard entailed the production of human tissueplasminogen activator (tPA) in the milk of transgenic mice The tPA gene was fused to theupstream regulatory sequence of the mouse whey acidic protein — the most abundant proteinfound in mouse milk More practical from a production point of view was the subsequentproduction of tPA in the milk of transgenic goats, again using the murine WAP gene regulatorysequence to drive expression (Figure 3.8) Goats and sheep have proved to be the most attractivehost systems, as they exhibit a combination of attractive characteristics These include: high milk production capacities (Table 3.14);

ease of handling and breeding, coupled to well-established animal husbandry techniques

A number of additional general characteristics may be cited which render attractive theproduction of pharmaceutical proteins in the milk of transgenic farm animals These include: ease of harvesting of crude product — which simply requires the animal to be milked; pre-availability of commercial milking systems, already designed with maximum processhygiene in mind;

low capital investment (i.e relatively low-cost animals replace high-cost traditionalfermentation equipment) and low running costs;

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high expression levels of proteins are potentially attained In many instances, the level ofexpression exceeds 1 g protein/litre milk In one case, initial expression levels of 60 g/l wereobserved, which stabilized at 35 g/l as lactation continued (the expression of the a1-antitrypsin gene, under the influence of the ovine b-lactoglobulin promoter, in a transgenicsheep) Even at expression levels of 1 g/l, one transgenic goat would produce a similarquantity of product in 1 day as would likely be recoverable from a 50–100 l bioreactorsystem;

ongoing supply of product is guaranteed (by breeding);

milk is biochemically well characterized, and the physicochemical properties of the majornative milk proteins of various species are well known This helps rational development ofappropriate downstream processing protocols (Table 3.15)

Despite the attractiveness of this system, a number of issues remain to be resolved before it isbroadly accepted by the industry These include:

variability of expression levels While in many cases expression levels of heterologousproteins exceed 1 g/l, in some instances expression levels as low as 1.0 mg/l have beenobtained;

characterization of the exact nature of the post-translational modifications the mammarysystem is capable of undertaking, e.g the carbohydrate composition of tPA produced in thissystem differs from the recombinant enzyme produced in murine cell culture systems; significant time lag between the generation of a transgenic embryo and commencement ofroutine product manufacture Once a viable embryo containing the inserted desired gene isgenerated, it must firstly be brought to term This gestation period ranges from 1 month forrabbits to 9 months for cows The transgenic animal must then reach sexual maturity beforebreeding (5 months for rabbits, 15 months for cows) Before they begin to lactate (i.e.produce the recombinant product), they must breed successfully and bring their offspring toterm The overall time lag to routine manufacture can, therefore, be almost 3 years in the case

of cows or 7 months in the case of rabbits Furthermore, if the original transgenic embryoturns out to be male, a further delay is encountered as this male must breed in order to pass

THE DRUG MANUFACTURING PROCESS 119Table 3.13 Proteins of actual/potential therapeutic use that have been produced in

the milk of transgenic animals

Protein Animal species Expression levels in milk

Antithrombin III Goat 14 g/l

Human a-lactalbumin Cow 2.5 g/l

Insulin-like growth factor I Rabbit 1 g/l

Growth hormone Rabbit 50 mg/l

on the desired gene to daughter animals — who will then eventually produce the desiredproduct in their milk.

Another general disadvantage of this approach relates to the use of the micro-injectiontechnique to introduce the desired gene into the pronucleus of the fertilized egg This approach

SDS-is inefficient and time-consuming There SDS-is no control over SDS-issues such as if/where in the hostgenomes the injected gene will integrate Overall, only a modest proportion of manipulatedembryos will culminate in the generation of a healthy biopharmaceutical-producing animal.

A number of alternative approaches are being developed which may overcome some of theseissues Replication-defective retroviral vectors are available which will more consistently (a)deliver a chosen gene into cells and (b) ensure chromosomal integration of the gene A secondinnovation is the application of nuclear transfer technology

Nuclear transfer entails substituting the genetic information present in an unfertilized eggwith donor genetic information The best-known product of this technology is ‘Dolly’ the sheep,produced by substituting the nucleus of a sheep egg with a nucleus obtained from an adult sheepcell (genetically, therefore, Dolly was a clone of the original ‘donor’ sheep) An extension ofthis technology applicable to biopharmaceutical manufacture entails using a donor cellnucleus previously genetically manipulated so as to harbour a gene coding for thebiopharmaceutical of choice The technical viability of this approach was proved in the late1990s upon the birth of two transgenic sheep, ‘Polly’ and ‘Molly’ The donor nucleus used togenerate these sheep harboured an inserted (human) blood factor IX gene under the control of amilk protein promoter Both now produce significant quantities of human factor IX in theirmilk

At the time of writing, no therapeutic protein produced in the milk of transgenic animals hadbeen approved for general medical use A number of companies, however, continue to pursuethis strategy These include GTC Biotherapeutics USA (formerly Genzyme Transgenics) andPPL Therapeutics (Scotland) a1-Antitrypsin, antithrombin and a range of monoclonalantibody-based products produced via transgenic technology continue to be evaluated bythese companies

In addition to milk, a range of recombinant proteins have been expressed in various othertargeted tissues/fluids of transgenic animals Antibodies and other proteins have been produced

in the blood of transgenic pigs and rabbits This mode of production is, however, unlikely to bepursued industrially for a number of reasons:

THE DRUG MANUFACTURING PROCESS 121Table 3.14 Typical annual milk yields (litres) as well as time lapse between generation ofthe transgene embryo and first product harvest (first lactation) of indicated species

Species Annual milk yield (l) Time to first production batch (months)

Table 3.15 Some physicochemical properties of the major (bovine) milk proteins

Protein Caseins b-Lactoglobulin a-Lactalbumin Serum albumin IgGConcentration (g/l) 25 (Total) 2–4 0.5–1.5 0.4 0.5–1.0

Isoelectric point Vary 5.2 4.2–4.8 4.7–4.9 5.5–8.3