58 BioProcess International FEBRUARY 2003

SS election of the microbialseed culture used to makea recombinant ornonrecombinantbiopharmaceutical product

represents a major commitment inthe early phase of bioprocessdevelopment (Figure 1). Scientificcharacterization and administrativerelease of the seed culture arecritical components of the selectionprocess. Documenting cell substrateand seed bank preparation is oneimportant component of thatcharacterization. Considerableattention in the regulatory literaturehas been given to thecharacterization of mammalian celllines because of greater perceivedrisks associated with them. Therequirements for microbial seeds are

perhaps less clear and more subjectto interpretation.

This article attempts to drawtogether guidance from regulatoryliterature concepts forcharacterization of microbial seeds,addressing some practical seed issuesthat bioprocess organizations faceduring development. The scope islimited to bacterial and yeast seedsused to make protein-, DNA-, orpolysaccharide-based products. Inthis context, the terms cell bank andseed bank are essentiallyinterchangeable and refer to a bankof cells that are capable ofsynthesizing a drug substance (e.g.proteins, nucleic acids, and capsularpolysaccharides) when grown undersuitable conditions.

THE CELLS

Cell Substrate: Characterization of amicrobial seed begins with athorough description of the“source, history, and generation ofthe cell substrate” (1) from whichthe seed bank is derived. The cellsubstrate for a nonrecombinantproduct is a cell from an unmodifiedparental cell line, whereas that for arecombinant product would be aclonally isolated cell bearing anexpression system for the product(1–2). Typically, a small bank of thecell substrate is made by theresearch team. The officialdescription of that cell substrateincludes its species, strain, relevantphenotypic and/or genotypicmarkers, safety information as

Characterization of Microbial Seeds Used in the Manufacture ofBiopharmaceuticalsPaul A. Duncan

PRODUCT FOCUS: VACCINES

PROCESS FOCUS: SEED DEVELOPMENT

WHO SHOULD READ: QA/QC

KEYWORDS: MICROBIAL VACCINE SEED,SEED BANK, GENETIC STABILITY,PRECLINICAL TESTING, REGULATORY

GUIDANCE, LICENSURE

LEVEL: REVIEW/OVERVIEW

PHOTODISC (WWW.PHOTODISC.COM)

appropriate, cultivation history, anddetails of the construction of anyrecombinant hosts and vectors. Thehistory of the cell substrate providesa basis for assessing risks of exposureto infectious or other adventitiousagents, as well as assuring all aspectsof identity (1–2).

Seed banks are made to facilitatelong-term manufacturing of aconsistent biopharmaceuticalproduct. Typically, a master seedbank (MSB) is made from analiquot of the cell substrate or froman intervening pre-MS bank. Aworking seed bank (WSB) madefrom an aliquot of the MSB may beused to inoculate routinemanufacturing lots.

The MSB defines the product, ina sense. The MSB is often the firstproduct intermediate in adevelopment program to beproduced under full GMP (goodmanufacturing practice) conditions,

the first for which a full battery oftests is performed, and it may be thefirst to be formally released.

End-of-Production Cells (EPC):Culture-based manufacturing stepsincrease the number of cells thatproduce the drug substance. Thepopulation of cells at the end of theproduction process reflects adefinable number of doublings,subculture steps, and time in cultureafter the initial MSB. Cells at theend-of-production stage have hadthe greatest opportunity to changetheir characteristics.

THE TESTS

Taken together, all the variousregulatory references describetesting to characterize the purity,identity, quantity, and stability of amicrobial seed. Naturally, testing ofrecombinant microbial seeds is morecomplex that that ofnonrecombinant seeds because ofthe need to characterize keycomponents of the recombinantexpression system.

Purity testing is intended to showthat the culture is “free fromadventitious microbial agents andadventitious cellular contaminants”(1). It should consider “propertiesof the banked cell, the likelycontaminants based upon scientificliterature, source, methods andmaterials used for cultivation, and

other organisms present in thebanking laboratory” (1). Tests maybe limited to an examination ofgrowth on multiple agar media thatvary in selectivity for the productionstrain (1). The specific amount of amicrobial seed bank to be tested forpurity is not defined as it is forbacterial and fungal testing of ametazoan cell bank, for which 1% ofthe containers of the seed bank (notfewer than two containers) must betested (1). Contaminatingbacteriophage might adversely affectseed productivity or consistency, butthe regulatory guidances do notuniformly specify testing bacterialseed banks specifically for phage.For example, of three documentsthat address plasmid DNA made inbacteria as a vaccine or genetherapy, one specifies testing seeds“to ensure they are free frombacteriophage” (3), one says suchtesting “is not required but thepossible presence of bacteriophageshould be considered” (4), and onedoes not specifically mentionbacteriophage testing at all (5).

Routine testing for the prionproteins that are presumed to causeanimal spongiform encephalopathiesis not currently practical. Regulatoryagencies prefer that manufacturersavoid using bovine-derived rawmaterials from regions of the worldwhere such prions are known to

The master cellbank DDEEFFIINNEESSthe product, in asense.

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FFiigguurree 11:: The bioprocess development timeline from the perspective of seed characterization

exist or are at reasonable risk ofexisting (6–7) — or more generallyto avoid any material derived fromanimals known to be naturallysusceptible to transmissiblespongiform encephalopathies (7).The FDA further clarifies a relatedissue specific to microbial seeds:

Working bacterial and viralseed banks and working cell banksthat were established usingbovine-derived materials sourcedfrom countries on the USDA list[of greatest BSE risk] should berederived with bovine-derivedmaterials from countries not onthe USDA list. However, masterbacterial and viral seed banksestablished in a similar manner donot need to be rederived; thepotential risk presented by themaster seed banks is even moreremote than that presented by theworking seed banks and isoutweighed by the risk of alteringthe bacterial or viral vaccinethrough rederivation. (6)

Identity testing is intended “todetermine that the banked cell iswhat it is represented to be” (1).Testing should confirm identity ofthe microbial cells to species andstrain (1). Nonrecombinant strainsare typically identified according toproduct-related characteristics(serotype). Recombinant strainsshould be identified according to“relevant phenotypic and genotypicmarkers which could include theexpression of the recombinantprotein or presence of theexpression construct” (8), or other“special phenotypic features” (5)that may be contributed by plasmidvectors. Strain identity tests can beselected judiciously, though, because“it is not considered necessary to doall the possible tests” (1).

Identity of recombinantexpression systems can be confirmedat a higher level of resolution byrestriction mapping and/or DNAsequencing. For instance, the DNAsequence of the expression cassettein a plasmid may encode the correctpromoter, gene, and terminator, but

an unexpected insertion or deletionin another region of the plasmidwould distinguish it as beingdifferent than expected. Thus, therestriction map of the entire plasmidand the DNA sequence of at leastthe expression cassette should beidentical to that expected (8). Theentire plasmid should be sequencedfor DNA vaccines (3, 5). Thesequence analysis should bevalidated to determine the actuallimit of detection for variantsequences within the population ofDNA molecules, but the sequenceanalysis need not be designed todetect low levels of possible variantsequences (8–9).

For example, validation of DNAsequencing might reveal that 40% ofthe molecules present must have anidentical base change at a position ina sequence to distinguish that twotrue sequences do exist at thatlocation in the total population ofmolecules. On the other hand, it isconsidered unnecessary todetermine the actual number ofmolecules in a population thatmight have an unexpected base at agiven location (e.g. 1 per 106

molecules).Sequence analysis “may also be

useful” (1) for cells producingnatural products (e.g. naturallyoccurring antigens) if the sequencesencoding those products are alreadyknown and well understood, but ingeneral “it is not considerednecessary to carry out investigationsof the sequences encoding complexnatural products” (1).

Quantity of a seed, in the simplestsense, can be understood as ameasure of the cells among a totalpopulation that are capable ofgrowing in suitable conditions.Microbial seeds are expected toshow growth with a characteristiclag phase and exponential rate whenthawed and inoculated into themedium used to manufacture abiopharmaceutical product. Viablecount (or viability) is an indicator ofgrowth potential of the inoculum.In addition, that count istheoretically the best basis fromwhich to calculate the populationdoublings of a culture becausedaughter cells can arise only bydivision of viable cells. Finally, aconsiderable loss in viability mayincrease the chance that asubpopulation of cells with aberrantcharacteristics might proliferate.Thus, measures such as viable countor percent viability help establishthe suitability of the seed formanufacturing (1, 10). Under someconditions, alternative measures ofgrowth potential might be moreuseful than viable count. Forinstance, growth kinetics in brothcould be useful to describe thegrowth potential of inocula withclumpy or filamentous growthhabits.

Two other quantitative measuresare also suggested for recombinantexpression systems: the number ofexpression constructs per cell (copynumber) and the number of cellsretaining extrachromosomalexpression constructs (percentage ofplasmid retention) (8–10).

Stability can be interpreted bothduring cultivation for productionand during the long-term storage ofcryopreserved seeds (1).

Testing to demonstrate stabilityduring cultivation is aimed atdetecting genotypic or phenotypicchanges, using the tests describedabove, which could affect themicrobes’ ability to consistentlymanufacture the product. In generalterms, “extensive identity testing ofthe MCB [master cell bank] shouldbe done once and should include alltests needed to establish allsignificant properties of the cells and

60 BioProcess International MARCH 2003

Measures such asviable count((VVIIAABBIILLIITTYY))help establish thesuitability of a seedfor manufacturinguse.

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the stability of these propertiesthroughout the manufacturingprocess” (11). The study of stabilityduring culture should be performedonce per marketing applicationusing cells grown under “pilot plantscale or commercial scale conditionsto the proposed limit of in vitro cellage for production use or beyond”(1). For recombinant systems, anexpression construct is evaluated “toensure that the correct codingsequence of the product has beenincorporated into the host cell andis maintained during culture to theend of production” (8).

Stability testing during cryopreservedstorage should monitor the“retention of the productioncapacity during storage underdefined conditions” (1). The scopeof testing can probably be limited toviability. “If the viability of the cellsubstrate is not significantlydecreased, generally no furthertesting of the MCB or WCB[working cell bank] is considerednecessary” (1). Other referencesecho that focus on cell viability.“The MCB should be testedperiodically to ensure the viability ofthe cells” (9). “Evidence for viabilityof the bacterial-cell-plasmid systemunder storage and recoveryconditions in the MCB and WCBshould also be provided” (5).

The general monograph onrecombinant DNA products in theEuropean Pharmacopoeia (EP)contains just one phrase regardingstability of seeds, requiringmanufacturers to demonstrate“stability by measuring viability andthe retention of the vector” (10).One is left to interpret this briefphrase: Is it sufficient todemonstrate retention of the vectorby successful production of theintended drug substance (consistentwith ICH [1]), or is it necessary tospecifically test for retention of thevector at time points duringcryopreserved storage? Regardless,all plans for monitoring stabilityduring cryopreserved storage shouldbe documented (1, 10) and includedin the subsequent marketingapplication (1).

IMPLEMENTATION

In the context of the developmenttimeline, GMP seed banks areunique process intermediates. Theymay be used to manufacturelicensed product for many years, butthey are made and initiallycharacterized before phase 1 clinicalstudies even begin (Figure 1). Thereis no question that seeds must becharacterized, but the timing andadministrative strategy for doing somay be negotiable.

To Release or Not to Release: Anearly choice should be made onwhether or not to formally releasethe seeds. Formal release of alicensable GMP product

intermediate, in its simplest sense, isan administrative mechanism bywhich to deem the intermediateacceptable. Formal release mayrequire a higher level of GMPcompliance and organizational buy-in than does simple research-modecharacterization testing. Certainstakeholders would need to agreeon the essential criteria for releasetesting in advance of making theseeds, and the analytical methodswould need to be validated.Validation could be doneprospectively if resources areavailable — or retrospectively if theyare not.

An organization might choose totest the seeds in research mode atthe time of seed manufacture andthen make decisions about theiracceptability without a formal

administrative mechanism that earlyin the development program.Nevertheless, if the seeds ultimatelywill be used to manufacturemarketable product, then methodswould still need to be validated(retrospectively, in this case).Furthermore, the original resultswould have to be reevaluated toensure that they were generated in amanner consistent with theconditions of the validated assay.Finally, the developmentorganization would have to wrestlewith the possibility that regulatoryauthorities may consider informalcharacterization of those seeds (withno documented acceptancemechanism or criteria) to beinconsistent with cGMPs when thelicense application is finallysubmitted.

So several reasons favor formalrelease of seeds. Thorough up-fronttesting leaves less chance forgenuine unexpected seed issues toemerge late in a developmentprogram. Early organizational buy-in to the analytical plan assures asmoother transfer of materials andmethods years later when preparingfor product launch. Thedocumentation associated withGMP-compliant testing facilitates amore organized and perhaps quickerresponse to regulatory questionsfollowing application submission.Formal release may be moreconsistent with current regulatorytrends and therefore less likely topresent as an obstacle to marketapproval years in the future. In fact,the WHO guideline for DNAvaccines states, “New cell banksshould be fully characterized andmeet established acceptance criteria”(5). The EP simply states, “New cellbanks must be fully validated” (10).

Defining Release Tests: Release testscan be defined as those for whichacceptance criteria are clear or areknown to be critical to assureprocess consistency (e.g. purity,identity, and viability). Release testsshould support the critical “go/nogo” decisions for all seed banks.Little argument should come upwithin the development

MARCH 2003 BioProcess International 61

There is noquestion thatseeds must becharacterized, butthe TTIIMMIINNGG andadministrativestrategy for doingso may benegotiable.

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organization if a seed bank truly failsto meet an acceptance criterion.Additional tests (for whichacceptance criteria are not as readilydefined) may be used to furthercharacterize the seed. Such testsprobably do not need to beconsidered as formal release tests.

Getting a Handle on StabilityDuring Culture: Release testing of theMSB is the first time point in aculture-based stability study for anew MSB. At least one other timepoint needs to be included in thestudy — that is, cells at or beyondthe in vitro cell age at the end ofcommercial-scale manufacturing.The in vitro cell age at the end ofproduction can be defined aschronological, populationdoublings, and/or passage level (1).The in vitro cell age and a formaldemonstration of stability duringculture should be reevaluated ifadditional culture expansion stepsare contemplated. In addition, amanufacturer should consider theeffect of allowable changes in seedviability on the number ofpopulation doublings whendesigning the study to demonstratestability during culture.

For instance, if a fixed volume ofseed is always used to inoculate theculture and it is then grown to afixed cell density, then populationdoublings would increase if theviability of the seed decreases.Consequently, stability testingwould take into account the extragenerations that could result if theviability of the seed were to drop.

On the other hand, if a fixednumber of viable cells are alwaysinoculated (based on actual countsat the time of inoculation andcorresponding adjustment of theinoculation volume), then thenumber of population doublings atthe end of the growth period wouldbe unchanged even if the viability ofthe seed bank falls.

It is prudent to make apreliminary assessment of stabilityduring culture very early in thedevelopment program. However,the formal demonstration ofstability during culture should bedone at the pilot or manufacturingscale after a final manufacturingprocess is defined.

FIRST STEPS OF THE JOURNEY

The goals of seed characterizationare to ensure scientific soundness ofthe product (in this case, the seedsthat make the drug substance) andcompliance with pertinentregulatory guidances. Adherence tothese principles, in turn, will helpminimize risk during a bioprocessdevelopment program. The timingof testing and the commitment ofresources to seed characterizationare complicated by the early stage inthe overall development program atwhich seeds are made. Developmentorganizations should makeconscious, informed choices aboutseed characterization to minimizerisks and, ultimately, to contributeto successful market applications.

REFERENCES1 Derivation and Characterization of

Cell Substrates Used for Production ofBiotechnological/Biological Products.International Conference on Harmonisationof Technical Requirements for Registration ofPharmaceuticals for Human Use, ICHTripartite Guideline, 1997;www.ich.org/ich5q.html.

2 Content and Format of Chemistry,Manufacturing and Controls Informationand Establishment Description Information fora Vaccine or Related Product. Guidance forIndustry, Food and Drug Administration,Center for Biologics Evaluation andResearch, Rockville, MD, 1999;www.fda.gov/cber/guidelines.htm.

3 Points to Consider on Plasmid DNAVaccines for Preventive Infectious Disease Indications. Food and Drug Administration,Center for Biologics Evaluation and

Research, Rockville, MD, 1996;www.fda.gov/cber/guidelines.htm.

4 Guidance for Human Somatic CellTherapy and Gene Therapy. Guidance forIndustry. Food and Drug Administration,Center for Biologics Evaluation andResearch, Rockville, MD, 1998;www.fda.gov/cber/guidelines.htm.

5 World Health Organization.Guidelines for Assuring the Quality of DNAVaccines. WHO Technical Report Series No.878, Annex 1; Geneva, Switzerland, 1998;77–90; www.who.int/en.

6 Bovine Spongiform Encephalopathy, I.Introduction: Recommendations for Use ofVaccines Manufactured with Bovine-DerivedMaterials, and III. CBER and FDAGuidance on Sourcing of Bovine-DerivedMaterials. Food and Drug Administration,Center for Biologics Evaluation andResearch, Rockville, MD, http.fda.gov/cber/bse/bse.htm.

7 Minimizing the Risk of TransmittingAnimal Spongiform Encephalopathy Agentsvia Medicinal Products. EuropeanPharmacopoeia, 4th Ed., 2001; GeneralChapter 5.2.8; 423–426.

8 Analysis of the Expression Construct inCells Used for Production of r-DNA-DerivedProtein Products. International Conference onHarmonisation of Technical Requirementsfor Registration of Pharmaceuticals forHuman Use, ICH Tripartite Guideline,1995; www.ich.org/ich5q.html.

9 Supplement to the Points to Consider inthe Production and Testing of New Drugs andBiologicals Produced by Recombinant DNATechnology: Nucleic Acid Characterizationand Genetic Stability. Food and DrugAdministration, Center for BiologicsEvaluation and Research, Rockville, MD, 6 April 1992; www.fda.gov/cber/guidelines.htm.

10 Products of Recombinant DNATechnology. European Pharmacopoeia, 4thEd., 2001; General Monograph 01,2002:0784; 520–522.

11 Points to Consider in theCharacterization of Cell Lines Used to ProduceBiologicals. Food and Drug Administration,Center for Biologics Evaluation andResearch, Rockville, MD, 1993(www.fda.gov/cber/guidelines.htm). ��

Paul A. Duncan is a research fellow inBioprocess and Bioanalytical Researchat Merck Research Laboratories, WP17-101 Sumneytown Pike, West Point, PA19486-0004; 215-652-0498,paul_duncan@merck.com.

Little argumentshould come upwithin theorganization if aseed bank trulyFFAAIILLSS to meet anacceptancecriterion.

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