40 BioProcess International JUNE 2004

AA ntibody-basedtherapeutics areincreasingly beingdeveloped for use inindications such as

oncology, inflammation, andinfectious disease. Advances inantibody engineering technologieshave facilitated the rapid generationof high-affinity antibodies of definedspecificity and have led to thedevelopment of a diverse range ofantibody-based molecules formattedto address specific applications.High dose requirements fortreatment of chronic indications inlarge markets necessitate thedevelopment of high-yielding, low-cost manufacturing processes thatconsistently deliver high-qualityproduct. Mammalian cell culture iscurrently the system of choice for

producing whole antibodies,whereas the generally preferredexpression host for antibodyfragments is Escherichia coli. Theadvantages of shorter processingtime and increased scale ofoperation associated with E. coliproduction counter some supplyand cost-of-goods constraintsinherent in mammalian cellexpression. Furthermore, processdevelopment times are shorter formicrobial systems, allowing morerapid progression to the clinic.

This article describes atechnology platform fordevelopment and production ofFab-based antibody therapeuticsusing an E. coli expression system.The technology covers all stages oftherapeutic development fromdesign of the therapeutic entity andgeneration of a high-affinityantibody through to production ofclinical-grade material. When themanufacture of clinical material isoutsourced, the processes must berobust, transferable, and compatiblewith operation at a range of scales.Herein is presented an approach toensuring the successfuldevelopment, transfer, and scale-upof production processes whileminimizing development times toensure speed to the clinic. Finally,some examples of commonproblems associated with processtransfer and scale-up are discussed.

FORMATTING FRAGMENTS

FOR SPECIFIC APPLICATIONS

The high affinity and degree ofspecificity shown by antibodies fortheir target antigens can beexploited through a variety ofmechanisms to mediate atherapeutic effect. For example,therapeutic antibodies may act asantagonists (by specifically blockinga receptor: ligand interaction), asagonists (by binding to, for example,a cell-surface receptor and activatinga downstream event), or as deliveryvehicles targeting effector moleculessuch as cytotoxic drugs to specificcell populations. Although full-length immunoglobulins (IgGs) arecommonly used as therapeutic

PRODUCT FOCUS: PEGYLATED

ANTIBODY FRAGMENTS

PROCESS FOCUS: PROCESS

DEVELOPMENT, FERMENTATION, AND

DOWNSTREAM PROCESSING

WHO SHOULD READ: PRODUCT

AND PROCESS DEVELOPMENT

SCIENTISTS

KEYWORDS: ANTIBODIES, MICROBIAL

EXPRESSION, SCALE-UP, PEGYLATION

LEVEL: INTERMEDIATE OVERVIEW

B I O P R O C E S S TECHNICAL

JVNW, INC. (WWW.JVNW.COM)

Microbial Systems for theManufacture of TherapeuticAntibody FragmentsLC Bowering

entities, they do not necessarilyprovide the optimal structure for allsuch mechanisms of action. Hence adiverse range of antibody formatshas been designed to incorporatethe specific properties required foreach intended application.

For many applications (such asrecruitment of effector cells or thecomplement system for cell killing),the effector functions of theantibody Fc region are not requiredfor therapeutic activity.Furthermore, in some circumstancesthe presence of the Fc region isundesirable: e.g., when trying tosuppress an immune response. Insuch cases, a Fab fragmentcontaining a single antigen-bindingdomain can be formatted to providethe required binding activity andoptimal pharmacokinetic properties.

The monovalent Fab provides asuitable alternative structure to thefull-length IgG when functioning asan antagonist. For example, it hasbeen demonstrated that monovalentFabs can neutralize cytokines withequivalent potency to whole IgG(1). If valency influences efficacy,such as in the selective delivery of adrug or radioisotope to a tumorcell, multivalent species can be builtup from a univalent Fab fragment.Trivalent Fab-maleimide constructs(TFMs) show prolonged retentionat a tumor site and increased ratesof internalization compared withwhole IgG (1). In addition, TFMsshow reduced rates of toxicity andmore rapid clearance from theblood because they lack the Fcantibody domain.

The PEG Solution: Rate ofclearance is a key issue that must beaddressed for antibody fragments tobe used therapeutically, where along circulating half-life is beneficialfor efficacy. Whereas full-length IgGhave relatively long serum residencetimes in humans, Fab and di-Fabfragments are cleared rapidly fromthe blood. Their half-life can beimproved through the covalentattachment of polymers such aspolyethylene glycol (PEG). PEG canbe conjugated to antibodies byrandom attachment, typicallythrough lysine residues. However,

this technique produces aheterogeneous product andgenerally leads to a loss in bindingactivity, probably due to theattachment of PEG within or nearthe antigen-binding sites of somefragments.

Fab fragments can be engineeredto contain a single unpaired cysteineresidue in the hinges (the Fab´fragment), which provides a freethiol group for site-specific PEGattachment (2). Because the PEG isthus attached at a specific pointaway from the antigen-binding site,the fragment’s antigen-bindingactivity is maintained. When divalentbinding is important for antibodyfunction, a PEG polymerterminating in two thiol-reactivegroups can be synthesized andreacted with Fab´ to form a site-specific PEGylated di-Fab´.Conjugation of Fab´ or di-Fab´ toPEG molecules of progressivelyincreasing molecular weight resultsin increasingly longer serumresidence times, with 40-kDa PEGconjugates showing pharmacokineticprofiles similar to whole IgG(Figure 1). PEGylation of proteinshas also been shown to reduceimmunogenicity and proteolysis (3).

GENERATING

HIGH-AFFINITY ANTIBODIES

Monoclonal antibodies (MAbs) of

defined specificity have traditionallybeen generated using hybridomatechnology. An antibody-secretinghybridoma cell line is derived byfusing an immune B-cell expressinga specific antibody gene with animmortal myeloma cell. Low fusionefficiency, however, leads toimmortalization of only a smallfraction of the specific antibody-secreting cells available in theimmunized animal. In addition, thescreening processes used to isolate aclonal population that secretes anantibody of interest are often labor-intensive and time consuming; thusthis technology is generallyrestricted to the generation ofrodent MAbs.

An alternative technology thatcan be used to generate high-affinityantibodies from any species —including humans — is the selectedlymphocyte antibody method(SLAM) (4). This technique permitsthe screening of large cellpopulations without the need togenerate a hybridoma, so a muchlarger proportion of the immunecell repertoire is analyzed. B-celllymphocytes isolated from the bloodor spleen are cultured in vitro andthen screened directly usingmethods allowing identification ofsingle cells that produce an antibodyof interest in very large populationsof antibody-secreting cells. Those

JUNE 2004 BioProcess International 41

FFiigguurree 11:: Pharmacokinetics of 125I-labeled IgG, di-Fab’, and di-Fab’-PEG conjugates in rat.Di-Fab’ is conjugated to PEG molecules of differing molecular weights, ranging 5–40 kDa.The graph shows percentage of injected dose (%ID) per mL of blood over time.

individual cells can then be isolatedand their heavy- and light-chainvariable region genes recovered.Because SLAM improves samplingefficiency of the B-cell repertoirecompared with hybridomatechniques, the probability ofrecovering very rare antibodies withspecific functional characteristics andaffinities below the pico-Molar levelgreatly increases.

E. COLI AS AN EXPRESSION HOST

Mammalian cell culture is currentlythe system of choice for producingtherapeutic whole antibodies.However, the long culture timesand scale limitations associated withthis expression system, combinedwith an apparent constraint inworldwide manufacturing capacityand relatively high cost of goods,make cell culture production forlarge-market indicationsproblematic. For most antibodyfragments, the host organism ofchoice is E. coli. Antibody fragmentscan be expressed to high titers of asoluble form within the bacterialperiplasm and purified by simple,efficient means.

As an expression host, E. colioffers a number of advantages overmammalian cell systems. Its fastergrowth rate provides shorter culturetimes, and fermentation processescan be operated at up to five times

the scale of mammalian cell cultures.In addition, E. coli can be grown ina simple, chemically definedmedium free from serumcomponents, without therequirement of expensive viralclearance steps. These factorscombine to make E. coli anattractive host organism forbiopharmaceutical productionsupplying large markets. Microbialcell lines can also be generatedrapidly with no screening for highexpressers, no gene amplification,and no adaptation to serum-freemedium. These facts considerablyreduce process development timesto just 8–10 months from selectionof the high-affinity antibody toGMP manufacture.

Conventionally soluble Fab´fragments are produced in E. coli bysecretion to the periplasmic space,where oxidizing conditions anddisulphide-bond–forming machineryfacilitate correct inter- and intrachaindisulphide bond formation (5, 6).Final fermentation yields of solubleFab´ in the range of 1–2 g/L havebeen reported (7). However,different Fab´ fragments are toleratedby E. coli to different degrees, withpoorly tolerated Fabs tending tomisfold or aggregate within the cells.Soluble yields are thought to beinfluenced principally by the primarysequence of a given protein.

The solubility of low-yieldingFabs can be improved bysubstituting specific amino acids thatincrease solubility but do not affectbinding affinity (8) or by graftingantigen-binding domains onto theframework of fragments known toexpress well in E. coli (9). Yields canbe influenced by the relative rates ofexpression of the heavy and lightchains, with optimal yields obtainedwhen functional expression of thetwo chains are closely matched (10).Optimal balance can be achieved atthe level of transcription ortranslation, e.g., using differentpromoter–inducer systems orthrough modifications to thetranslation initiation region.

MANUFACTURING PEGYLATED FABS

This manufacturing platform wasdeveloped for the production ofPEGylated Fab´ fragments usingproprietary humanization, E. coliexpression, and PEGylationtechnology. Once a therapeuticcandidate is selected and theappropriate format for its intendedmode of action identified, anemphasis is placed on speed to theclinic. Short process-developmenttimes are favored, but the processesdeveloped must be robust andtransferable to facilitate scale-up forthe clinic. Strategies employed tominimize development timesinclude the use of genericmanufacturing processes (optimizedfor each new product) and theparallel development offermentation and purification steps.

Following selection as adevelopment candidate, an antibodyis humanized and its primarysequences optimized for expressionas a Fab´ fragment in E. coli. Toensure that high yields of solubleFab´ are consistently achieved, anexpression vector system has beendeveloped that enables optimizationof the relative rates of light- andheavy-chain expression. HumanizedFab´ fragments are cloned into asuite of expression vectors thatdiffer exclusively in the shortsequence between their Fab´ lightchain (LC) and heavy chain (HC)genes (the intergenic sequence or

42 BioProcess International JUNE 2004

FFiigguurree 22:: Culture growth and Fab’ production during a 1000-L E. coli fermentation.Periplasmic Fab’ was measured by protein G HPLC assay. Periplasmic extracts were preparedby incubating cell pellets in a Tris-EDTA extraction buffer for 16 hours at 60 °C. SupernatantFab’ was measured by ELISA.

IGS). The IGS affects the rate ofFab´ HC translation initiation andhence the relative rates of LC andHC expression. A short IGS gives ahigh rate of HC translationinitiation because ribosomes remainassociated with RNA molecules inbetween light- and heavy-chainsynthesis. When the IGS sequence islonger, ribosomes dissociate fromthe RNA following LC synthesisand must then reassociate to initiateHC translation, which leads to aslower rate of HC accumulation.Further tuning of the HCtranslation initiation rate can beachieved by modifying theconsensus ribosome binding-sitesequence. The IGS providing anoptimal balance of LC and HCexpression along with the highestyield of soluble periplasmic Fab´ isdetermined experimentally for eachnew development candidate. Thiscan be done rapidly because vectorscan be constructed and tested inparallel; fermentation times areshort, and the number of vectorstested can be limited fromexperience to a small number(generally only two or three).

Once an optimal IGS vector hasbeen selected, fermentation anddownstream purification processesare developed in parallel. The E. colistrain W3110 is used for production.The cells are grown to high biomasson a simple, chemically definedgrowth medium before they areinduced to express the Fab´fragment. The Fab´ is expressedfrom a tac promoter, inducible withisopropyl �-D-thiogalactopyranoside(IPTG, C9H18O5S) or lactose, andOmpA signal sequences direct theFab´ heavy and light chains to theperiplasmic space. The soluble Fab´accumulates in the periplasm over a36-hour induction period, with verylittle loss of Fab´ into thesupernatant (Figure 2). Theinduction system (expression vectorand inducer) and culture conditionsduring induction are tuned to therate-limiting step for Fab´production: the rate at which foldingcan take place in the periplasm.Antibiotic selection is not required

for production fermentors, and theexpression plasmid shows near-complete structural andsegregational stability throughoutfermentation.

Upstream Processing: Soluble Fab´is extracted from the E. coli periplasmusing a simple chemical extractionprocedure (11). Cells harvested bycentrifugation are resuspended in aTris(hydroxymethyl)aminomethane–ethylenediaminetetra-acetic acid(Tris–EDTA) extraction buffer atelevated temperature. The Tris andEDTA synergistically permeabilize thebacterial outer membranes, releasingFab´ into the supernatant withoutlysing the cells. Complete, correctlyfolded Fab´ fragments are stable atthe temperature used; their structureand binding affinities are not affectedby heat treatment. However, many E. coli proteins and incomplete orincorrectly assembled antibodyfragments are unstable at hightemperatures, so they will be removedfrom the process stream at this stage.Hence the high-temperatureextraction enriches the process streamfor the product, with Fab´constituting ~50% of total protein atthe end of this step.

Downstream Processing: Fab´ ispurified from clarified periplasmicextract by two- or three-columnchromatography steps. Fortherapeutic antibodies, the cost of

purification can represent asignificant portion of totalproduction costs. This cost isreduced by avoiding the use ofexpensive affinity matrices andinstead using nonaffinity capturemethods such as ion-exchange andhydrophobic-interactionchromatography (which employlow-cost, reusable resins) andappropriately sizing the columns toproduction scale. A key requirementof the purification process for an E. coli expression system is effectiveremoval of endotoxin. An anion-exchange column will remove thebulk endotoxin present in theprocess stream, and the purificationprocess effectively reduces them towell below acceptable levels.

Purified Fab´ is conjugated toPEG in a controlled, site-specificreaction. A mild reduction stepreduces the free hinge thiol, and thereduced Fab´ reacts with PEG-maleimide to yield a PEGylatedFab´. The reaction produces adefined product, which is beneficialfrom a regulatory perspective(Figure 3) and is simple to scale upfor manufacture. Unreacted Fab´and PEG are removed from theprocess stream by one or twofurther chromatography steps.

In summary, the processes to beoptimized include extraction andprimary capture, purification of bulkantibody fragments, Fab´PEGylation, and purification of thePEGylated product. To reducedevelopment times, these activitiesare carried out in parallel wheneverpossible. Affinity-captured Fab´ isused for initial PEGylation studiesand to establish binding and elutionconditions for the column stepsused for Fab´ purification.Meanwhile, the extraction andprimary capture steps are optimizedin sequence. Naturally, certain risksare associated with using genericprocesses and optimizing steps inparallel. Therefore, experiencegained from developing purificationsteps for previous developmentcandidates is used, and the completeprocess is demonstrated andoptimized in sequence before scale-up for clinical manufacture.

FFiigguurree 33:: Reducing SDS-PAGE gel showingsite-specific attachment of PEG to Fab’.Lane 1 is made of up molecular weightmarkers. Lane 2 is the Fab’. Lane 3 is site-specifically attahed Fab’-PEG (40-kDa PEG).

44 BioProcess International JUNE 2004

46 BioProcess International JUNE 2004

SCALE-UP AND

TECHNOLOGY TRANSFER

Successful scale-up and technologytransfer to a contract manufacturingorganization (CMO) requirescareful planning and goodcommunication. Prior knowledge ofthe manufacturing site and intendedscale of operation allows process fitto be discussed with the CMO at anearly stage. Another benefit is thatequipment, scale, and other site-specific limitations are identifiedearly. Any such limitations can betaken into consideration whendesigning the manufacturing processthat will be transferred. Timelinesfor technology transfer should alsobe defined early, and items oractivities with long lead timesshould be identified (e.g., cellbanking and orderingchromatography resins).

A problem frequently associatedwith the scale-up of high–cell-density microbial fermentation is theability to satisfy oxygen demand orcooling requirements at large scale.If it is known in advance that thosefactors may be problematic on scale-up, the fermentation process can bedesigned such that oxygen uptakeand heat generation are reduced towithin achievable levels usingnutrient-limiting feeds.

Knowledge of the key processcontrol parameters can facilitatesuccessful transfer and scale-up. Forexample, Fab´ distribution between

the periplasm and the supernatant issensitive to temperature and theconcentration of mediumcomponents such as inorganicphosphate, calcium, and magnesiumions. Small changes in one or moreparameters can cause loss of Fab´ tothe supernatant, reducing finalproduct yield. Special attention tomaintaining control of thoseparameters on scale-up will maintaincontrol of Fab´ distribution with noloss in periplasmic yield (Figure 4).

Differences in the design andoperation of bench and productionscale equipment can also lead tounexpected problems at thecommercial scale. One such examplerelates to the scale-up ofcentrifugation. Laboratory batchcentrifuges give tight, dry pellets,whereas continuous disk-stackcentrifuges produce a heavy phase“slurry,” either carrying oversupernatant or losing product in theheavy phase. Neither effect isobserved at the small scale.Following scale-up of one process, itwas observed that a large proportionof the Fab´ (about 45%) wasroutinely being lost during the disk-stack centrifugation step thatremoved cells from the processstream. Those losses were greaterthan what was predicted due toremoval of supernatant in the heavyphase alone. The Fab´ appeared topartition preferentially with theheavy phase, an effect that had not

been observed during processdevelopment in the laboratory. Theproblem was resolved by removingthe centrifugation step and insteadcapturing the Fab´ directly fromunclarified cell extract using anexpanded-bed adsorption (EBA)column. The number of processingsteps was consequently reduced, andthe yield of Fab´ from the extractincreased to over 90% using EBAchromatography at the 2500-L scale.

SMALLER CAN BE BETTER

For many therapeutic applications,the properties of an antibodyrequired to mediate a therapeuticeffect reside within the Fab´fragment. This fragment cantherefore be used as a “buildingblock” for a therapeutic entity,allowing far greater flexibility indesign than can be achieved usingstandard IgG. Therapeutics basedon the Fab´ fragment can beformatted to address specificmechanisms of action throughcontrol of valency (by linking anumber of Fab´ units) and half-life(by PEGylation).

The technology platformdescribed here uses an E. coliexpression system for theproduction of PEGylated antibodyfragments. Novel technologies suchas SLAM provide the means for therapid generation of antibodies ofvery high affinity. Humanized Fab´fragments can be produced at hightiters in soluble form within the E. coli periplasm and can be purifiedby simple, cost-effective means.Site-specific PEGylation provides amethod for modifying half-lifewithout loss of binding affinity —and creates a well-defined product.Manufacturing processes for makingPEGylated antibody fragments arerobust, transferable, and compatiblewith large-scale operations. E. coliproduction systems also allow high-volume production and offer shortprocess development times, whichenables rapid progression to theclinic.

FFiigguurree 44:: Scale-up of an E. coli fermentation producing Fab’ antibody fragment. A 10-Lfermentation is compared with three 1000-L fermentations. The graph shows yields ofperiplasmic Fab’ (closed symbols) and supernatent Fab’ (open symbols) during induction.

ACKNOWLEDGMENTSThe author would like to thank the following people for theircontributions to this work: Neil Weir, Andrew Chapman, AlastairLawson, Andy Popplewell, David Humphreys, David Glover, MariSpitali, and Pari Antoniw. Celltech currently has four PEGylated Fab´fragments in clinical or advanced preclinical development.

REFERENCES1 Weir, ANC; et al. Formatting Antibody Fragments to Mediate

Specific Therapeutic Functions. Biochemical Society Transactions 2002,30: 512–516.

2 Chapman, AP; et al. Therapeutic Antibody Fragments withProlonged In Vivo Half-Lives. Nature Biotechnol. 1999, 17: 780–783.

3 Caliceti, P; Veronese, FM. Pharmacokinetic and BiodistributionProperties of Poly(Ethylene Glycol)-Protein Conjugates. AdvancedDrug Delivery Reviews 2003, 55: 1261–1277.

4 Babcook, JS; et al. A Novel Strategy for GeneratingMonoclonal Antibodies from Single, Isolated Lymphocytes ProducingAntibodies of Defined Specificities. Proc. Natl. Acad. Sci. USA 1996,93: 7843–7848.

5 Better, M; et al. Escherichia Coli Secretion of an ActiveChimeric Antibody Fragment. Science 1988, 240: 1041–1043.

6 Skerra, A; Plückthun, A. Assembly of a FunctionalImmunoglobulin Fv Fragment in Escherichia Coli. Science 1988, 240:1038–1041.

7 Carter, P; et al. High-Level Escherichia Coli Expression andProduction of a Bivalent Humanised Antibody Fragment. Bio/Technology 1992, 10: 163–167.

8 Wall, JG; Plückthun, A. The Hierarchy of MutationsInfluencing the Folding of Antibody Domains in Escherichia Coli.Protein Eng. 1999, 12: 605–611.

9 Jung, S; Plückthun A. Improving In Vivo Folding and Stabilityof a Single-Chain Fv Antibody Fragment By Loop Grafting. ProteinEng. 1997, 8: 959–966.

10 Humphreys, DP; et al. A Plasmid System for Optimisation ofFab´ Production in Escherichia Coli: Importance of Balance of Heavy-and Light-Chain Synthesis. Protein Expression and Purification 2002,26: 309–320.

11 Weir, ANC; Bailey, NA. Process for Obtaining AntibodiesUtilising Heat Treatment. US Patent 5,665,866 (9 September 1997). ��

LC Bowering is team leader for microbial fermentation atCelltech R&D Ltd., 216 Bath Road, Slough, Berkshire, SL1 4EN, United Kingdom; 44-1753-534655;leigh.bowering@slh.celltechgroup.com.