For the past two decades, the application of recom-binant technologies using mammalian cell lines hasopened the door for the availability of a multitude

of new pharmaceuticals for the treatment of many dis-eases, as exemplified by the recently licensed antibodyHerceptin (Ref. 1) raised against a specific form ofmetastatic breast cancer2. However, the manufacture of products using this technology can still be burdenedby the potential risk of introducing foreign agents orcontamination derived from the host cell lines, rawmaterials or by inappropriate process conditions. Never-theless, new scientific knowledge, progress in technol-ogy and improved standards have led to an increase in the safety, quality and predictable integrity of theseproducts. A brief overview is given of the develop-ments and improvements that have been achieved toreduce the risk of contamination and to guarantee high quality and safety standards. These efforts comprisetechnical improvements, as well as developments, in the validation of the materials used and in process organization.

Raw materialsAlthough the term ‘raw material’ is widely used in

the literature, it is not always clearly defined. Here, rawmaterials (RMs) are defined as the chemical, bio-chemical or biological components that are used in aprocess to manufacture a biopharmaceutical product.Although the cell material used for the cultivation process is often considered to be a raw material, it isexcluded from this definition because there are specialaspects to consider regarding cells; these will be discussed separately.

During the course of a production process, RMs areused for many different purposes. They are needed, forexample, for the cultivation of cells, the isolation andpurification of the desired product or as excipients forthe final product formulation, as well as for the clean-ing and maintenance of production equipment. There-fore, different categories of RMs can be distinguished,reflecting their use or purpose during the process andtheir presence in the final product.

In the light of the definition given above, RMsinclude all substances that are introduced into the manu-facturing process of a biopharmaceutical. Therefore,RMs are the most critical source of possible impuritiesor contamination, with the exception of the producercells3. However, not all RMs contribute to this risk tothe same extent. According to their categories, differ-ent safety and quality standards have to be applied forRMs. Components that are present in the final prod-uct formulation, for example, must comply with phar-macopoeial standards, although manufacturer-definedstandards can be applied to RMs that are used for clean-ing purposes. The fate of a given RM throughout theprocess, with respect to its contact with the productcomponents or its presence in the final formulation,should be considered in this context4. As a result ofthese considerations, a procedure for the quality andsafety validation of RMs used for the manufacture ofbiopharmaceuticals should be designed.

Two strategies are used to ensure the quality andsafety of RMs. First, an RM should be tested to verifyits identity, purity and safety, as well as its suitability forthe process; however, this can be difficult to achieve forcomplex RMs of biological origin, such as serum, pro-tein hydrolysates, and so on. The second strategy is theclear determination and validation of the origin andsource of RMs and is of similar importance to the firststrategy. Tracking RMs from their origin not onlyassists in verifying the identity and purity of RMs, butalso enables the characterization of the most likely con-taminants4. This is of great value when deciding whichbiosafety tests should be included in the testing scheme.Knowing the history of production and the source ofRMs is especially important for complex RMs of bio-logical origin because contaminating agents might bepresent that cannot easily be tested for [e.g. transmissi-ble spongiform encepha-lopathies (TSEs)], or areunknown. In these cases, validation of origin, manu-facturers and vendors of RMs is the only way to guar-antee a certain degree of biosafety. For instance, the lackof a reliable and easy assay system to detect TSE inserum has led to the abolition of European sera fromthe production of biopharmaceuticals5. However,serum that is certified to originate from New Zealand,by contrast, is believed to have a high level of biosafetyand is widely accepted; New Zealand is free of many

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Developments and improvements in themanufacturing of human therapeutics withmammalian cell culturesFriedemann Hesse and Roland Wagner

During recent years, biopharmaceutical products manufactured by processes that use mammalian cell cultures have gained

increasing importance. At the same time, a strong awareness of the importance of the safety and quality of such products

has also emerged. This has led to improvements in cultivation and production technology, validation procedures and process

organization.

F. Hesse and R. Wagner (wagner.roland@gbf.de) are at the Cell CultureTechnology Department, Gesellschaft für Biotechnologische ForschungmbH, 38124 Braunschweig, Germany.

contaminating agents that are common in other coun-tries (e.g. rabies, foot-and-mouth disease, TSE, etc.)6.

To trust only in certificates, however, can prove to benaive. In 1994, for example, ~15 000 litres of high-quality serum were produced in New Zealand; how-ever, ~30 000 litres of ‘New Zealand’ serum were soldworldwide7. This problem can only be overcome bythe establishment of a functional vendor-audit pro-gram. Vendor certification combines quality control(QC) testing with validation elements, such as assaycross-validation, change controls (i.e. a system to eval-uate and approve proposed changes to specifications,test procedures, equipment, etc.) and other contract useprocedures, to provide the maximum possible safetyand quality level for RMs (Ref. 4).

Producer cellsThe cell lines used for the production of biophar-

maceuticals represent the other critical source of con-tamination. From past history of the production of biopharmaceuticals using mammalian cell cultures, theinfluence of biosafety concerns regarding producer cellsbecomes evident. Mammalian primary cells were firstused as producers of biopharmaceuticals for the manu-facture of an inactivated polio virus vaccine8. Numer-ous continuous cell lines (CCLs) were already establishedin the 1950s that had apparent advantages over primarycells, including rapid growth, consistency, relativeeconomy and freedom from animal procurement andattendant contamination problems9. For several decades,however, all processes exclusively relied on the use ofprimary cells10. The biological resemblance of CCLsto, and sometimes their derivation from, tumor cellsincluded the possibility that they might contain trans-missible oncogenic agents or possess a certain tumori-genic potential themselves. Because no scientific meansor analytical tools existed to exclude this possibility,CCLs were not permitted for the production of bio-pharmaceuticals.

A related episode occurred in the early 1960s, whendiploid cell lines were established11. These had many ofthe advantages of CCLs, such as consistency and free-dom from contaminants. However, the same absenceof scientific tools to comprehensively evaluate the safetyof CCLs led to the rejection of diploid cell lines as pro-ducers, in 1962, on similar theoretical grounds to thefailure to prove the absence of putative tumorigenicagents12. Approximately 10 years later, the first diploidlines (WI-38 and MRC-5) were finally licensed for themanufacture of an inactivated polio virus vaccine13,14.This only occurred after many studies had demon-strated the safety of normal diploid cells15–17. However,the general prohibition of the use of CCLs had remained.Over the past 30 years, scientific advances in cell biology,virology and molecular genetics have provided consid-erable insight into the factors and components involvedin tumorigenesis18–21. Some of the potentially tumori-genic agents found in CCLs include: cellular DNA (e.g. activated oncogenes) and oncogenic endogenousviruses, such as retroviruses, transforming proteins (e.g. T-antigens) and the intact cells themselves. Vali-dation of the absence of all possible tumorigenic agents,especially of putative human cancer viruses, from CCLs, is difficult. However, the development of purifi-cation techniques and validation procedures enabled

the establishment of methods to prove the absence ofnon-acceptable levels of DNA and proteins, and there-fore of tumorigenic agents, from the final product. Thisfinally led to the licensing of biopharmaceuticals pro-duced in CCLs during the 1980s (Refs 22–24) andtoday the application of CCLs for the manufacture ofhuman therapeutics is widely accepted.

Cell-banking systemsTo obtain a cell source that guarantees a constant and

high level of biosafety and quality of the producer cellline, a cell-seeding system should be established25. Atwo-tier setup consisting of a master cell bank (MCB)and a working cell bank (WCB) was proposed in 1963during a workshop of the Cell Culture Committee(CCC; Ref. 26). Today, this concept is generallyaccepted as the most practical approach for the estab-lishment of a cell-seeding system, and includes docu-mentation and validation of the producer cell line; ithas been widely accepted as a standard.

The clear and thorough documentation of the his-tory of the cell line intended to be used for the pro-duction of the biopharmaceutical is an importantrequirement for the establishment of the cell bank27.This includes details on the origin of the cell line, aswell as its passage history. The original tissue fromwhich the cell line was taken and the method of iso-lation have to be stated. The documentation also has toinclude detailed information regarding the culture andstorage conditions, and the media used for cultivationand cryopreservation. This intensive documentationeffort has two main objectives: (1) to provide all theinformation needed to decide whether a cell line is safeenough to be used for the production of biopharmaceu-ticals; and (2) to provide all the data necessary to esti-mate the validation and testing effort needed to prove thatthe cell line satisfies the required biosafety regulations.

The cell line that is chosen for the manufacture ofthe biopharmaceutical must then be proven to be freeof contamination before the evaluated cells can be usedfor the preparation of a MCB, which contains the cryo-preserved primary stock of the cell line to be used. Cellsfrom this stock are used to generate the working stockof cryopreserved cells called the manufacturer’s work-ing cell bank (MWCB) or WCB (Ref. 28). This stockserves as the only cell source for the production pro-cess. Cells taken from the WCB are only used for a par-ticular production time. This time has to be defined bycareful studies to guarantee that the cell populationremains genetically stable. Cells can be used for severalproduction batches during that time span. When itsend is reached, cells are usually discarded and anothervial is taken from the WCB to establish a new cell population that is used for subsequent productionbatches during the next time span.

To prove the biosafety of the producer cell line, theMCB and/or WCB have to be validated29. For thispurpose, cells are taken from the cell bank and charac-terized with respect to their phenotype and genotypeto document their identity and determine their gen-etic stability. The cells are also subject to tests for thepresence of contaminating agents; these tests have tocover a wide range of possible contaminants such asmycoplasmas, bacteria, fungi, viruses and retroviruses,to ensure a high level of biosafety.

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The establishment of cell-banking systems, in com-bination with a detailed documentation of the cell lineand careful validation procedures, reduces the risk ofintroducing contaminating agents via the cell line usedfor production. It therefore provides a tool for manu-facturers to maintain a high level of biosafety and ahigh-quality standard of the producer cell line duringthe entire life span of the biopharmaceutical product.

Improvement of cultivation techniques: theroute to chemically defined culture media

In the early days of cell cultivation, almost all culturemedia contained serum as a growth-promoting com-ponent. Serum was shown to have several essentialfunctions in culture30: it is a source of nutrients, hor-mones, growth factors and protease inhibitors. Serumfacilitates the attachment and spreading of cells, andprovides nonspecific protection against mechanicaldamage and shear forces. It contains carriers, such asalbumin and transferrin, for several molecules includ-ing fatty acids, lipids, amino acids, hormones and traceelements (especially iron). In addition, serum is able tobind to toxic compounds.

Besides these growth-promoting properties, serumhas some major disadvantages. It is undefined withrespect to its chemical composition and may containsubstances that induce unwanted cell behavior. As a

result of their high protein content, the downstreamprocessing of serum-containing culture media is moredifficult and costly. In fact, this was one of the mainreasons for the first efforts to develop serum-free cul-ture media for the production of biopharmaceuti-cals31–33. More recently, however, the issue of biosafetyhas gained similar importance.

The main objective for the development of serum-free culture media is to replace the undefined serumcomponent with other, more-defined supplements thatwill provide the essential functions of serum. However,many serum replacements suffer from similar problemsto those of serum34. Some serum-substitutes (e.g.hydrolysates) are not clearly defined with respect totheir chemical composition; others are not clearlydefined with respect to origin and source, or containcomponents of animal origin.

These problems have led to the development of pro-tein-free cell culture media consisting only of chemi-cally defined components that are not derived from animals. The main challenge in designing a protein-freemedium composition is to find non-proteinaceous sub-stitutes for the functions provided by proteins in con-ventional culture media. The iron-carrier function oftransferrin, for example, can be mimicked by addingchelating agents, such as EDTA or citrate35. The shear-force protecting property of albumin can be replaced

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Figure 1 Strategy for the adaptation of cell lines to protein-free media. Cells are adapted by repeatedly exchanging 80% of the culture medium againstprotein-free medium (Step 1). During this phase, cells ‘learn’ to proliferate in a protein-deficient environment. Most cells detach during theadaptation phase and form suspended aggregates. However, some cells stay adherent and can be propagated under protein-free cultivationconditions (Step 2a). The aggregates can be used to establish single-cell suspension cultures in protein-free media (Step 2b).

4/5 Volumepre-warmedprotein-free

medium

4/5

Confluent cultureMedium containing

5–10% FCS

Step 1

Cells formaggregatesand detach

Fewadherent

cells

Suspendedaggregates

Protein-freemedium

2 to 3weeks

Step 2b

Transfer of cellaggregates tospinner flask

Slowly increasing stirringspeedRegular medium exchange(2/3 to 6/7 volume)

Single cells and small spheroids

4 to 9 weeks

Step 2aRegular mediumexchangeRemoval ofdetached cells

Adherentlygrowing cells

3 to 5 weeks

Total medium exchange

trends in Biotechnology

by adding surfactants, such as Pluronic F68 (Refs 36,37).However, the carrier functions of serum proteins must be replaced by more-balanced, optimal, nutrient compositions.

Nevertheless, the transition from a serum-containingto a serum-free or protein-free medium remains a crit-ical step for cell growth and physiology. To introducethese media, the cells must be adapted to the newmedium conditions, a process termed ‘conditioning’.This can be achieved by slowly reducing the protein orserum content of the culture medium, thus giving thecells the opportunity to ‘learn’ to proliferate and pro-duce under serum- or protein-free medium conditions38

(Figs 1,2). However, as a result of the total lack of pro-teins in the culture medium, problems with respect togrowth or productivity of some cell lines can occur.This is particularly the case if the cells need specific pro-teins (e.g. growth factors) for proliferation or production.

Given the problems of using protein-free media, themost rational way to establish defined cultivation con-ditions appears to be by the development of chemicallydefined media that are not necessarily protein free butdo not contain any components of animal or humanorigin34. This can be achieved by applying the follow-ing strategy (Fig. 3). The producer cell line should beadapted to a protein-free basal cultivation medium.During this process, the cell line should be monitoredfor critical parameters (e.g. growth, viability, produc-tivity). The medium should be supplemented with therecombinant growth factors or hormones required,according to the results of the monitoring, to optimize

the performance of the producer cell line. This procedureshould be an iterative process because all changes in themedium composition must be investigated for theireffects on the cells39. During the optimization of themedium composition, all components that are of un-defined animal or human origin should be replaced by substances that are clearly defined with respect tochemical composition, origin and source.

Downstream processingThe quality and biosafety of a biopharmaceutical is,

to a great extent, dependent on the extraction proce-dures used to manufacture the purified product. On theone hand, downstream processing has to ensure aneffective and economic isolation of the desired productfrom the culture broth or cellular material obtainedduring the cell culture process. However, on the otherhand, components that would contaminate the finalproduct must be reliably separated40.

During the cultivation process, the cells either secretethe desired protein into the culture medium or theproduct accumulates in the cells. In either case, the firststep of the purification procedure is the separation ofcells and cell debris41. This is achieved by centrifugationor microfiltration techniques. The efficacy of this step isinfluenced by the viability of the cells and by the mediumcomposition. The product-containing fraction (either theculture broth or a crude cell extract) is then concentratedby ultrafiltration or diafiltration, precipitation, high-affinity adsorption or extraction steps, which reduce thevolume and prepare the material for the chromato-graphic steps used for the final product purification.

Different types of components that should not bepresent in the final product formulation have to beremoved during these steps. These can be classified intotwo groups (Box 1). The first group comprises media-derived or process-derived impurities that can be of aproteinaceous or non-proteinaceous nature (e.g. lipids,antifoaming agents, antibiotics). This group alsoincludes host-cell-derived impurities such as proteins,which might induce unwanted immune responses, ornucleic acids, which are a major concern because theymight harbor potentially harmful genetic informationwhen incorporated within healthy human cells. Thesecond group consists of adventitious agents and con-taminants, and comprises viruses, virus-like particles(VLPs), bacteria, fungi, mycoplasmas and so on. Incontrast to the first group, these agents are not an integral part of the manufacturing process, but their presence in the final product must be excluded to guarantee the safety of the biopharmaceutical.

The removal of medium components and protein-aceous impurities is an integral part of product iso-lation. Nevertheless, the removal of medium supple-ments, such as antibiotics or cytotoxic substances (e.g.geniticine or methotrexate) must be guaranteed by thepurification strategy and appropriate tests have to beestablished to validate their efficiency42. Some impuri-ties, such as DNA, can be reduced by a careful choiceof cultivation and harvesting conditions. Impurities aredefined as compounds that are known to be present inthe production process, and their removal can be mon-itored during the manufacture of the product. Because,for practical reasons, it is not possible to manufacture a100% pure product, acceptable concentration levels for

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Figure 2BHK cells (a) during and (b) after the adaptation to growth in protein-free medium.623 magnification.

the presence of impurities in the final product formu-lation have been defined. For example, the World HealthOrganization (WHO) defined the maximal acceptableamount of DNA to be 100 pg per single dose of abiotechnologically derived protein drug43.

A more complex problem is the removal of potentialcontaminants that might or might not be present (e.g.when serum is used for cultivation) or are unknown,and therefore cannot be proven to be absent. Severaltechniques normally used for product purification havea certain potential for removing contaminants. Forexample, viruses or VLPs can be efficiently removedusing filtration or chromatographic techniques42,44. Theefficacy of a given purification process in removing thesecontaminants, however, cannot be confirmed underregular process conditions because these agents are notnormally present. Therefore, several strategies haverecently been developed to validate the potential of aspecific purification strategy for the removal of adventi-tious agents. For example, to investigate the potential forvirus removal, model viruses are added and the efficiencyof the purification step to remove theses viruses is mon-itored, a procedure called ‘spiking’. Spike experimentsare normally not performed in the manufacturingequipment but in a scaled-down replica, to avoid con-tamination of the manufacturing equipment. To enableextrapolation of the results and therefore estimation ofthe potential of the process to reduce viruses, the choiceof the virus species used for this assay is critical. A use-ful model virus should be closely related to the mostprobable viral contaminant. For practical reasons, itshould be possible to grow high titres of the model virusand a simple but sensitive assay for its detection shouldbe available. To ensure removal of a wider spectrum ofpotential viral contaminants, it is necessary to use anappropriate set of model viruses that covers the rangeof physicochemical properties of different virus species(e.g. size of virus, enveloped and/or non-envelopedvirus, RNA, DNA, single stranded or double stranded).

Alternatively, the potential for inactivating adventi-tious agents during the purification step can beexploited or additional inactivation steps can beincluded, within the purification strategy. Viruses andVLPs, for example, can be inactivated by the appli-cation of inactivating chemicals (e.g. N-acetylethylene-imine45, Tri-N-butylphosphate46), organic solvents,chaotropic salts, extreme pH-values47, irradiation, andso on48. Temperature treatment achieved by the appli-cation of microwave technology has also been shownto inactivate viruses49. Notwithstanding the above, thepotential of the chosen technology for inactivationremains to be validated and this validation has also toprove that the inactivation method does not harmproduct integrity.

Regulations, guidelines and GoodManufacturing Practice

During the past 50 years, there has been an increas-ing awareness of the safety concerns surrounding themanufacturing of medicinal products. The realizationthat an independent evaluation of drugs is important

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Figure 3Schematic diagram showing the strategy for the establishment of chemically definedcultures. The producer cell line should first be adapted to protein-free medium toestablish a defined culture system. The parameters that are critical for the processshould be monitored. The culture medium should then be supplemented with recom-binant growth factors or hormones that are needed to improve the cultivation condi-tions. Afterwards, the critical parameters must be monitored again to decide whetheror not the improvement is sufficient to meet the process requirements. Otherwise,culture conditions have to be further optimized.

Adaptation to basalprotein-free medium

Supplementation withrecombinant growth factors,hormones, and so on

Further optimization ofcultivation conditions

Yes

No

Undefined serum-containing

Defined protein-free culture

Improved defined culture

Optimized defined culture

Critical parametersfulfill process

requirements?

trends in Biotechnology

Box 1. Categories of components that should not be present in the final biopharmaceutical product

Components present due to process conditions:• Host-cell-derived components: DNA and proteins• Process-derived components: lipids, proteins, antifoam agents, antibiotics, substances used for product isolation,

cleansing agentsComponents present due to contaminations:• Adventitious agents: viruses, virus-like particles, bacteria, fungi, mycoplasmas, transmissible spongiform

encepha-lopathy agents

before they are made available to the patients wasinduced by some tragic accidents in drug manufactur-ing during the 1930s in the USA (a mistake in the formulation of a children’s syrup) and during the 1960sin Europe (the thalidomide tragedy). This has led tothe establishment of product-authorization systems, for example, the US Food and Drug Administration(FDA) and the implementation of governmental regu-lations. During the 1960s and 1970s, there was a rapidproliferation, in most countries, in the number of laws,regulations and guidelines concerning the evaluation ofmedicinal products.

The growing awareness of the need for high levels ofsafety and therefore higher quality standards also led toseveral technical improvements in the manufacture ofpharmaceuticals. Besides technical developments, therewere also improvements in the organization of manu-facturing processes leading to the implementation ofthe Good Manufacturing Practice (GMP; Ref. 50).The aim of the GMP is to clearly define the system thatis used to manufacture a specific product in a repro-ducible and documented manner, in order to ensurethe highest possible quality, safety and efficacy of theproduct. This can only be guaranteed if all components(e.g. RMs, machinery, personnel) and steps involved inthe manufacturing process are thoroughly documentedand validated. Today, the establishment of manufacturingprocesses for new biopharmaceuticals is only possibleunder GMP conditions.

Regulatory authorities and agencies such as the FDA,the European Medicines Evaluation Agency (EMEA)and the Japanese Ministry of Health and Welfare, pub-lish guidelines interpreting and explaining their regu-lations (Box 2). These guidelines are updated regularly,as are the GMP regulations, to be able to include the latest technical standards, and this has led to thedevelopment of the term and concept of current GMP.

One major problem arose from the increasing num-ber of laws, regulations and guidelines. Although the

pharmaceutical industry was becoming increasinglyinternational and seeking global markets, the regis-tration of medicines remained a national responsibility.Over time, the different regulatory systems diverged tosuch an extent that many time-consuming and expen-sive validation and testing procedures had to be per-formed in duplicate to market the drugs internationally.This led to a waste of time and resources, and generatedan urgent need for rationalization and harmonizationof regulations on an international scale.

The first efforts to harmonize the regulations wereundertaken by the European Community during the1980s when the development of a single Europeanmarket for pharmaceuticals was initiated. The successof these efforts encouraged regulatory agencies andindustry associations in Europe, Japan and the USA toinitialize the International Conference on Harmoni-zation of Technical Requirements for the Registrationof Pharmaceuticals for Human Use (ICH), in 1990.The ICH was established as a joint regulatory andindustry project, with the aim of improving the effi-ciency of the process for developing and registeringnew medicinal products in Europe, Japan and the USA.

Risk assessmentRisk considerations have been a constant theme in

all the topics discussed so far. Therefore, risk assessment(i.e. the systematic estimation and evaluation of risks)should be considered to be applied as a general con-cept when planning a process for the manufacturing ofbiopharmaceuticals.

The main purpose of risk assessment is to evaluatethe risk potential for all the components that are to beused in the course of a manufacturing process. This isimportant for considering which RMs and cells shouldbe used in a certain process because these are the mostcritical components with respect to their potential forthe introduction of contaminating agents. When esti-mating the risk potential of RMs, the fate of the RM

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Box 2. Examples of guidance documents concerning the manufacturing of biopharmaceuticalspublished by the US Food and Drug Administration (FDA), the European Agency for the Evaluation

of Medicinal Products (EMEA) and the International Conference on Harmonization (ICH)

Guidance documents published by the FDA• Guidance for human somatic cell therapy and gene therapy• Content and format of chemistry, manufacturing and controls information and establishment of description information

for a vaccine or related productGuidance documents published by the EMEABiotechnology Working Party (BWP):• CPMP/BWP/328/99: Development of pharmaceuticals for biotechnological and biological products• CPMP/BWP/1230/98: Note for guidance on minimizing the risk of transmitting animal spongiform encephalopathy

agents via medicinal products• CPMP/BWP/477/97: Note for guidance on pharmaceutical and biological aspects of combined vaccines• CPMP/BWP/268/95: Note for guidance on virus-validation studiesQuality Working Party (QWP):• CPMP/QWP/155/96: Note for guidance on development of pharmaceuticals• CPMP/QWP/486/95: Note for guidance on manufacture of the finished dosage formSafety Working Party (SWP):• CPMP/SWP/112/98: Safety studies for the gene-therapy productsGuidance documents published by the ICH• Q5A: Quality of biotechnological products: viral safety evaluation• Q5B: Quality of biotechnological products: genetic stability• Q5C: Quality of biotechnological products: stability of products• Q5D: Quality of biotechnological products: cell substrates

during the process should also be taken into consider-ation. For both RMs and cells, a clear definition of theorigin and the source, as well as their traceability, is crucial in order to evaluate the risks that are associatedwith their use because they might contain unknown ornot easily detectable contaminating agents. These possi-bilities can only be reduced by proving that the materialwas derived from a source with a low potential for carry-ing these contaminants (e.g. using serum of NewZealand origin). The risks associated with the use of otherequipment should also be taken into consideration.

Risk assessment is also necessary to estimate thepotential risks associated with the application of certainprocedures or techniques during the manufacturingprocess. This, for example, includes the estimation ofrisks associated with the applied isolation steps, as wellas those associated with testing or validation. The robust-ness of a procedure and its susceptibility to errors alsohave a major impact on the level of risk associated withits application. This must be taken into considerationwhen planning the overall process structure.

The application of the concept of risk assessment is unable to totally exclude all risks associated with abiopharmaceutical product; some residual risk alwaysremains. This is, in part, as a result of the fact that theremight be unknown contaminating agents present thatcannot be tested for. Further, it is not possible to testfor all known contaminating agents, for practical rea-sons. Nevertheless, the concept of risk assessment willhelp to estimate which contaminants have the highestprobability of being present and therefore will enablethe establishment of a reasonable and economic testingscheme. As previously explained, risks can only be esti-mated on the basis of solid data; thorough documen-tation and validation procedures are therefore necessary.Although risk assessment cannot rule out all possiblerisks, it enables the clear definition of the residual risk.Therefore, the application of risk assessment is a validtool for manufacturers to establish biopharmaceuticalsthat are well characterized, not only with respect totheir structural and pharmacological features, but alsowith respect to their level of risk.

ConclusionA growing awareness for biosafety and quality issues

led to several developments in the manufacturing ofbiopharmaceuticals in recent years. These develop-ments comprised technical improvements, such as thedevelopment of protein-free and chemically definedculture media, as well as improvements in proceduresfor the validation of RMs, producer cell lines or thepotential of downstream processing for the removal ofpotential contaminants. The establishment of GMPregulations led to organizational improvements result-ing in processes wherein every step from the origin ofRMs to the final dosage is fully comprehensible. Allthese developments and improvements contributed tothe general objective of establishing optimized pro-cesses for the manufacturing of biopharmaceuticalswith mammalian cell cultures that guarantee the highest possible safety and quality standards.

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The analysis of complexcarbohydrates and their applicationsin biotechnology have, untilrecently, been hampered by a lack of straightforward yetdefinitive protocols that areamenable to most researchorganizations. Combinations ofenzyme arrays and more-powerfulanalytical and syntheticinstrumentation are rapidlychanging this situation. CarbohydrateBiotechnology Protocols is volume 10in the Methods in Biotechnologyseries and is aimed at the newresearcher to this field who needs a set of basic protocols to analyseand/or synthesize the variousclasses of complex carbohydrates. It largely fulfils this function, andalso provides a valuable resource tomore-experienced practitioners whorequire alternative methodologies.

Throughout this series, closeattention is made to format andstyle: chapter authors must adhereto the defined format of a briefintroduction, a list of materials, anumbered protocol, method notesthat enlarge on individual steps

or rationalize their use, and finallya list of references. The preciseapplication of a defined format andstyle ensures the reader can findinformation quickly, and only themost relevant details are included;this strategy should be used morewidely.

Many of the chapters in thevolume describe the production ofcomplex carbohydrates (e.g. gums,alginates, celluloses and dextrins)either by whole microbes or bythe use of enzymes isolated frommicrobial systems. For microbialtransformations, the fermentationand product-isolation parametersare described, together with thekey analytical techniques requiredto define product yields and purity.

For in vitro enzymic biosynthesisof complex carbohydrates, thereaction conditions and kineticsbecome critical, and these aredescribed in subsequent chapterstogether with the relevantanalytical estimates of purity andyield. Besides more commoncarbohydrates, the syntheses ofmore unusual molecules with

biotechnology and/orpharmaceutical applications aredescribed, for example, fructo-oligosaccharides and isomaltulose(used as artificial sweeteners), sialylepitopes and nucleotide sugars (usedin medical diagnosis), and mannitolor 3-keto-disaccharides (used asexcipients in pharmaceuticals).

Chapters are also included onthe enzyme-based degradation of key carbohydrates, such ashemicelluloses and chitins, andspecific analytical techniques suchas fluorophore-assisted carbohydrateelectrophoresis. Unfortunately,there are few cross references tovolume 76 in the series Methods inMolecular Biology1, which dealscomprehensively with methods ofcarbohydrate analysis that are equallycritical for a successful carbohydrateinvestigation. Nevertheless, thisvolume will prove a usefulresource for researchers of graduatelevel and above, with an interest in investigating carbohydratebiochemistry as applied to thefields of biotechnology andpharmacology.

Reference1 Hounsell, E.F. (1998) Methods in

Molecular Biology: Glycoanalysis Protocols(Vol. 76, 2nd edn), Humana Press

Nigel JenkinsLilly Research Laboratories,

Indianapolis, IN 46285, USA. (E-mail: njenkins@lilly.com)

A new primer for methods in biotechnology

Carbohydrate Biotechnology Protocolsedited by C. Bucke, 1999, Humana Press.

UK£79.50 pbk (xii 1 337) ISBN 0 89 603563 8