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BiotechnologyJournal DOI 10.1002/biot.201000381 Biotechnol. J. 2011, 6, 16–27

16 © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1 Introduction

Today, therapeutic antibodies are the most well-characterized proteins and also one of the mostsuccessful and powerful weapons that physiciansuse to fight cancer, inflammation and infectiousdiseases. In 1986, the first antibody, Orthochlone(OKT-3), was approved by the United States Foodand Drug Administration (FDA) for the treatmentof allograft rejection in allogeneic renal transplan-tation, and to date, a total of 28 antibody-baseddrugs have been approved by the FDA [1]. Also,over the last three decades, antibodies, includingmonoclonal antibodies and antibody fragments,

have been employed as valuable tools in many ar-eas of biological research, and have been applied innew biological systems, such as biochip/sensors fordiagnoses and kinetic analyses, bio-imaging, andprotein purification. Now, antibody therapeuticscomprise a major portion (almost 50%) of the pro-tein therapeutic market (approx. $81 billion in2010), and they represent the fastest growing sec-tor in the pharmaceutical industry [1]. Worldwidesales of antibodies for therapeutic/diagnostic pur-poses have risen dramatically in recent yearsfrom approximately $26 billion in 2006 to over $30billion in 2008, and it is projected that sales willreach $56 billion dollars by 2012 with a compoundannual growth rate (CAGR) of 13% (http://www.bccresearch.com/report/BIO016G.html).

An immunoglobulin G (IgG), the most abundantantibody in humans, is a heterodimer consisting oftwo identical heavy chains (HC) and two identicallight chains (LC) with a molecular mass of about150 kDa. In general, two variable domains (VH andVL) in the Fab region are responsible for antigen

Review

Recombinant antibodies: Engineering and production in yeast and bacterial hosts

Ki Jun Jeong1,2, Seung Hoon Jang1 and Natarajan Velmurugan1

1Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea2Institute for the BioCentury, KAIST, Daejeon, Korea

After the appearance of the first FDA-approved antibody 25 years ago, antibodies have become ma-jor therapeutic agents in the treatment of many human diseases, including cancer and infectiousdiseases, and the use of antibodies as therapeutic/diagnostic agents is expected to increase in thefuture. So far, a variety of strategies have been devised for engineering of these fascinating mole-cules to develop superior properties and functions. Recent progress in systems biology has pro-vided more information about the structures and cellular networks of antibodies, and, in addition,recent development of biotechnology tools, particularly in regard to high-throughput screening,has made it possible to perform more intensive engineering on these substances. Based on asound understanding and new technologies, antibodies are now being developed as more power-ful drugs. In this review, we highlight the recent, significant progress that has been made in anti-body engineering, with a particular focus on Fc engineering and glycoengineering for improvedfunctions, and cellular engineering for enhanced production of antibodies in yeast and bacterialhosts.

Keywords: Antibody · Fc engineering · Glycosylation · Production · Synthetic library

Correspondence: Professor Ki Jun Jeong, Department of Chemical andBiomolecular Engineering, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon,305-701, KoreaE-mail: [email protected]

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CDC, com-plement-dependent cytotoxicity; HTS, high-throughput screening

Received 29 October 2010Revised 6 December 2010Accepted 8 December 2010

© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 17

Biotechnol. J. 2011, 6, 16–27 www.biotechnology-journal.com

binding with high specificities and affinities. In con-trast, the Fc region consisting of two constant do-mains (CH2-CH3) and a hinge region is responsiblefor antibody stability and pharmarcokinetics viabinding to various receptors (Fig. 1).The Fab and Fcregions have modular structures and these regionsarising from different sources can be separately en-gineered and combined to make new antibodies withdifferent properties.This ‘Divide and Conquer’ strat-egy has served as the basis of general and usefulmethods employed in current antibody engineering.Until now, various antibody engineering approacheshave been developed for the purposes of bringingabout (i) affinity/folding maturation, (ii) improvedthermostabilities, (iii) new antibody platforms in-cluding single-chain Fv (scFv), single-domain mini-bodies, single-chain antibodies (scAb), fragmentantigen binding (Fab or Fab2), bispecific diabodies(dAb) and disulfide-free intrabodies (Fig. 2), (iv)conjugation with chemicals/peptide,and (v) Fc engi-neering for improved efficacy and pharmacokinet-ics. In the last decade, great progress has been madein developing new biotechnology tools, particularlythose applied to high-throughput screening (HTS),which makes it now possible to perform more inten-sive antibody engineering. Based on new structuralinformation and new techniques,antibodies are nowbeing developed as powerful weapons.

In this review, we primarily highlight recent andimportant developments in the areas of Fc engi-neering and glycoengineering as well as cellularengineering aimed at the efficient production ofantibodies in bacterial and yeast hosts. In contrastto engineering of the variable regions of antibodiesto enhance both targeting and blocking, manipulat-ing the Fc region, which is responsible for effectorfunctions and longer half-lives, remains relativelyunexplored. Following earlier efforts focused onengineering variable regions, the numbers of stud-ies of engineering Fc regions of antibodies to mod-ulate of Fc receptors has increased and recent ap-proaches for Fc engineering are discussed in thefirst part of this review. For some time, mammaliancells have served as the major hosts for antibodyproduction, irrespective of their high cost and thelong time periods required for cultivation. Howev-er, as demand for antibody therapeutics increases,the economics associated with production of anti-bodies becomes an important issue. Consequently,continuing interest exists in devising more afford-able processes that employ simple cost-effectivehosts, such as yeast and bacteria, instead of mam-malian cells. Through recent developments in sys-tems biotechnology, useful information has beengained that allows more sophisticated engineeringof yeast and bacterial hosts. Progress in cellular en-

Figure 1. Antibody (IgG) structure and antibody engineering. Immunoglobulin G consists of three domains (Fab, Fc and hinge); in general, Fab is respon-sible for antigen-binding specificity and affinity, and Fc is responsible for antibody stability through Fc receptor bindings. In antibody engineering, variousstrategies can be employed for each domain.

BiotechnologyJournal Biotechnol. J. 2011, 6, 16–27


gineering for enhanced production is discussed inthe second part.

2 Fc engineering and glycosylation

Fc regions of IgG are responsible for highly specif-ic binding to several receptors, which mediates ef-fector functions of antibodies, such as antibody-de-pendent cellular cytotoxicity (ADCC) and comple-ment-dependent cytotoxicity (CDC), as well aspharmacokinetics [2]. Each IgG subtype has a dif-ferent affinity for each receptor, and this means an-tibody activities can be managed by engineeringbinding properties. Various strategies have beendeveloped to improve antibody activities throughalterations of the affinity of the Fc region for thecorresponding receptors.

2.1 Fc engineering for effector functions

Binding of IgG to various Fc receptors (FcγRs), in-cluding FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, andFcγRIIIb, or the first component of complement(C1q), depends on residues located in the hinge re-gion and the CH2 constant domain, and alteration ofsome amino acids in these regions is one major ap-proach used to modulate effector functions. Simplesubstitution of Ala for Glu333, Ser298 or Lys334 in Rit-

uximab, could increase affinity for FcγRIIa and en-hanced both ADCC and CDC [3, 4]. Even though the1-2 positions are critical for receptor binding, a sin-gle mutation at those positions does not ensure en-hancement of receptor binding and effector func-tions, but multiple substitutions may produce morepositive effects. Using a yeast surface display andHTS technology, Stavenhagen et al. [5] identifiedseveral positions at which mutations can enhancethe binding of IgG to the Fc receptor (FcγRIIIa). In-dividual substitution of specific amino acids identi-fied in this screen gave a small increase in receptorbinding.However,multiple mutations (F243L,R292P,T200L, V305I, and P396L) were found to result insignificant increases (~100 fold) in ADCC. In ADCCand CDC, the binding of IgG to FcγRs absolutely re-quires N-glycosylation at Asn297 in the CH2 domain[6]. Even though glycosylation is one factor that pre-vents yeast and prokaryotic hosts from producingrecombinant antibodies, glycosylation can be a use-ful tool to modulate in the effector functions of anti-bodies. Antibody derivatives, having different effec-tor functions, can be produced in engineered hostcells that produce modified glycosylation patterns.Shields et al. [7] showed that CHO cell mutants,which do not add fucose to primary N-acetylglu-cosamine (GlcNAc) and therefore produce IgG with-out fucose, have dramatically increased (40–50-fold)ADCC through their higher affinities for FcγRII.

Figure 2. Schematic structure of various antibody fragments. Each single VH or VL domain is functional as itself without any further pairing with anotherchain. In scFv, scAb and dAb, heavy and light chains are connected by flexible Gly-Ser linkers. In Fab, Fab2 or Fcab molecules, both chains are covalentlylinked via disulfide bonds. Fcab requires glycosylation at Asn297 position.



2.2 Fc Engineering for pharmacokinetics

The pharmacokinetics of antibodies are dependenton their binding to neonatal Fc receptor (FcRn) onthe surface of endothelial cells, and this binding isknown to be important for increasing the serumpersistence of therapeutic antibodies (7–16 days orlonger serum half-lives) [2]. Fc engineering towardbetter pharmacokinetics is another important ap-proach to improve efficacies of antibodies, by en-abling higher circulating levels, less frequent ad-ministration and reduced number of doses. It isknown that several positions (Ile253, His310, andGln311, and His435) located in the CH2-CH3 cleft ofthe Fc region, which are distinct from FcγRs andC1q binding sites, are critical for FcRn binding and,unlike binding to other receptors, glycosylation atAsn297 is not required for FcRn binding [8]. Anti-bodies with specific substitutions in the Fc region,such as I253A, lose their binding affinity for FcRnand are rapidly eliminated through phagocytosis,whereas antibodies with other substitution (N434A)show greatly enhanced affinities for FcRn, and areeliminated more slowly [9]. In contrast, some anti-bodies require lower pharmacokinetic activities forfast clearance from the body. For example, antibodydrugs used to treat autoimmune diseases or IgGimmunoconjugates, such as toxin-conjugated anti-bodies, need to be cleared from the kidney quicklyto diminish side effects caused by excess of un-bound ligands. Vaccaro et al. [10] developed FcRnblockers named ‘Abdegs’, which serve as powerfulmodulators governing the affinities of antibodiesfor FcRn. Abdegs block the function of FcRn andlead to enhancements in the rates of degradation ofIgG.As a result, their use together with IgG, can en-able injection of reduced levels of IgG in treat-ments of antibody-mediated diseases and promoterapid clearance of the IgG-toxin or IgG-drug com-plex.

As described above, Fc region of antibodies hasbeen the main target for modulation of both effec-tor function and half-lives, and variable regions(VH and CL) have been the main targets for engi-neering of specificity and affinity (Fig. 1). However,the results of several recent studies suggest that anopposite approach exists for developing more po-tent therapeutic drugs. Igawa et al. [11] modifiedthe variable region instead of Fc region, which ledto reduced pI values in variable region and a sig-nificant reduction in the clearance of the antibody.These workers suggested the change of pI in vari-able regions causes resistance to fluid-phasepinocytosis and reduced elimination of IgG inde-pendently of the FcRn-dependent mechanism. Incontrast to this finding, Zalevsky et al. [12] engi-

neered the Fc region to improve antibody affinity.Modification of the Fc regions resulted in pro-longed exposure of IgG due to the FcRn-mediatedenhancement of half-life (5-fold), and also an im-proved antitumor activity in an animal test.Very re-cently, Wozniak-knopp et al. [13] developed a newantibody format termed Fcab, which consists of theFc fragments only that binds to the target ligandwith high specificity and without loss of pharmaco-kinetics. To our knowledge, this is the smallest(~50 kDa) and fully functional antibody fragmentdisplaying both antigen binding and effector func-tion. These new approaches can be used to bridgethe demand for dosing convenience (long half-life)with the clinical necessity of maintaining efficacy(high activity).

2.3 Cellular engineering for glycosylation of antibodies

The N-linked glycan structure in the Fc region ofantibodies is indispensible for binding to receptors(FcγRs), but glycosylation has been one of the ma-jor limitations in choosing possible productionhosts. Eukaryotic yeast as well as bacteria, includ-ing E. coli, have not been used as the main hosts forthe production of recombinant antibodies (full-length IgG format) for use in humans because ofglycosylation problems. Even though yeast are eu-karyotic and thus contain glycosylation systems,their completely different glycosylation patterns(too high mannose contents) have served as a ma-jor limitation. Over the past two decades, many re-search groups have attempted to overcome thislimitation and, recently, major advances have beenmade in glycoengineering of yeast that have culmi-nated in the production of glycoproteins for humanuse. Gerngross and coworkers succeeded in pro-ducing human-like glycosylated antibody (ritux-imab) using Pichia pastoris cell lines, which enablespecific human N-glycosylation with high fidelity[14]. The glycoengineered antibody has an activityat least one order of magnitude higher in bindingits target receptor than the current commercialdrug. The most important advantage in humanizedyeast systems is the possibility of bringing aboutuniform glycosylation, which stands in contrast tomost mammalian cell lines that produce mixturesof glycoforms, which lead to reproducibility prob-lems in protein activity and the requirement ofcomplex purifications in downstream processes.Nowadays, many approaches for glycosylation arecoupled with antibody engineering through direct-ed evolution. From this point of view, comparedwith mammalian hosts, much higher efficiency oftransformation in yeast and availability of HTS via



yeast cell surface display are another advantage ofyeast host. Although it has been thought that gly-cosylation is restricted to eukaryote cell lines in-cluding yeast, the discovery of an N-glycosylationpathway in Camphylobacter jejuni and its function-al transfer into E. coli have given rise to exciting op-portunities to produce recombinant glycoproteinsin bacterial hosts. In 2002, the first recombinant E.coli cells that are able to produce glycosylated pro-teins were developed by introducing 12 pgl genesfrom C. jejuni [15]. However, as yet there have beenno successful results for antibody glycosylation inbacterial hosts, and this topic remains challenging.Recent progress, including co-expression of hu-man sialyltransferase ST6GalNAcl in E. coli [16] ora combination of glycan-trimmed protein with pre-assembled eukaryotic N-glycan [17], suggests thatthe use of E. coli as a production host for glycopro-tein and antibodies hold promise in the future.

2.4 Aglycosylated antibodies with high affinity for Fc receptors

Together with the development of new glycosyla-tion strategies in non-mammalian hosts, two recentreports on the development of aglycosylated IgG,which can bind to specific Fc receptors without gly-cosylation, suggest a new solution for the glycosy-lation problem. Using yeast mutants in which gly-cosylation does not occur, Wittrup and coworkers[18] isolated aglycosylated IgG1 variants that hadaffinities for FcγRs similar to those of the wild-typeglycosylated IgG. The aglycosylated IgG variantcontains mutations of two amino acids adjacent toAsn297. These workers have suggested that two in-teractions, including a hydrogen bonding betweenaglycosylated Asn297 and Ser126 of the receptor anda salt bridge between Asp265 on the B chain of theFc dimer and Lys117 of the FcγR, are responsible forbinding Fc receptors. More recently, Georgiou et al.[19] developed an aglycosylated IgG1 variants us-ing an E. coli protein display system. From a ran-dom library containing modifications in Fc region,IgG1 variants binding to FcγRs with affinities sim-ilar to that of Herceptin were successfully isolated.Although the immunogenicity and pharmacokinet-ics in animal systems of antibodies need to bedemonstrated, it is noteworthy because they cansimply bypass the construction of a very complexcircuit for glycosylation pathway, and can also beapplied to other eukaryotic proteins therapeutics.In addition, recent development for high level ex-pression of FcγRs in E. coli system [20] should makeit possible to carry out more systematic studies ofFc engineering. The recent promising progressmade in glycosylation engineering may enable the

use of simple bacteria and yeast cell systems forantibody production, thus, eliminating the need formammalian cell cultures. This will provide im-provements in product uniformity with significant-ly reduced production times and manufacturingcosts.

3 Production of antibodies and antibodyfragments

Over the last two decades, the clinical and com-mercial success of antibody therapeutics has led toa high demand for these substances (more than1000 kg/year) [21]. To meet this need, improve-ments in production yields through the employ-ment of host cell engineering as well as optimiza-tion of cultivation/purification processes are re-quired.Thus far, various eukaryotic and prokaryot-ic hosts have been exploited for antibodyproduction, but owing to the glycosylation and im-munogenicity problems, mammalian cells, such asCHO, NSO, and HEK293 cells, have been the mostdominant hosts used in antibody production. In thelast decade, rapid progress has been made in mam-malian cell cultivation, including the use of serum-free media, efficient gene expression, developmentof more powerful cell lines, and optimization of cul-ture and reactor [21, 22]. Currently, high cell culti-vation yields (more than 107 cells/mL) with anti-body concentrations exceeding 5 g/L (more than10 g/L in some cases) can be achieved in most com-mon bioreactors [22] and it is expected that pro-ductivity levels of 10–20 mg/L/h in a 21-day processmay be achieved in the next 5 years [23]. In contrastto mammalian hosts, yeast and bacterial cells arebetter suited for the production of antibody frag-ments (scFv, Fab, dAb, etc.) because they do not re-quire glycosylation for their biological activitiesand are relatively easily assembled. At present,among the FDA approved antibodies, non-IgG for-mat antibodies, such as Reopro (Fab), Lucentis(Fab), and Cimzia (Fab), are produced in E. colihosts [1]. Since antibody production can beachieved at limited production scales, yeast or bac-terial hosts that can be cultivated at much highercell densities would be an advantage, and would re-sult in a much higher volumetric productivity. Thishas stimulated efforts aimed at improving the pro-ductivity and minimizing the typical limitations as-sociated with yeast or bacterial hosts.

3.1 Production of antibodies in yeasts

Compared with mammalian cells, the main advan-tages of yeast production are that it requires only



cheap, defined media and an ability to generatehigh cell densities up to 100 g dry cell/L in shortcultivation times [24]. Currently, the achievablevolumetric productivities by yeast cultivations areabout 5–10 mg/L/h, which exceeds that of mam-malian cells (~1–2 mg/L/h) [25]. Also, owing to theexistence of fewer problems with glycosylation,higher solubility and simpler purification process-es, yeasts have gained more interest than E. coli andother bacterial hosts as antibody production plat-forms.

Among yeasts and fungi, the preferred host is P.pastoris, which has several advantageous features,including higher cell densities, strong AOX1 pro-moters, and glycosylation patterns similar to thatobserved in human systems. The recent progressmade in the production of various antibodies usingyeast hosts is summarized in Table 1. For the en-hanced production in yeast, several cellular engi-neering strategies can be considered: (i) use ofstrong promoters, efficient signal peptides, andhost preferable codons for efficient gene expres-sion systems, and (ii) the introduction of assistantsystems (co-expression of foldase, molecular chap-eron, and protein fusion) for improved folding/as-sembly of antibodies.The most frequently used andstrongest promoter is the AOX1, which is inducedby alcohols such as methanol (Table 1). However,another promoter GAP, although weaker, is usefulfor the production of antibodies that fold slowlyand, as a result, are not secreted under the strongAOX1 promoter [24]. For the production of anti-bodies that require 16 disulfide bonds for full as-sembly, the rate-limiting steps are secretion andproper folding in the ER and the Golgi apparatus.Leader peptides play a pivotal role in governing thesecretion efficiencies and, thus, the overall produc-tion yield of antibodies. Rakestraw et al. [26] re-cently engineered a popular alpha mating factor Isecretory leader (MFα1pp) used in Saccharomycescerevisiae using an HTS method. By examiningscFv and full-length IgG containing altered leader

peptides having one conserved mutation (Val22),they showed that higher production levels up to 16-fold and 180-fold over wild type could be obtained.Overexpression by strong promoters may causemisfolding of antibodies, which can lead to cellstress responses and consequently lower produc-tion yields. To improve protein folding, folding as-sistants such as chaperons (e.g., BiP) or foldases(e.g., protein disulfide isomerase, PDI) can be co-overexpressed, or a combination of two differentfoldases and chaperones can have a synergistic ef-fect on antibody folding, although there was someexceptional cases [27, 28]. In the production of A33scFv, which recognizes a cell surface differentiationantigen (A33) as a colorectal cancer marker, co-ex-pression of BiP displayed a 3-fold increase in anti-body production with more than 8 g scFv/L beingproduced. However, co-overexpression of PDI/BiPhad no effect on antibody production [27]. Overex-pression of the general unfolded protein response(UPR) transcription factor HAC1, which can facili-tate the expression of PDI or BiP in a cell, has beensuccessfully employed to obtain improved levels ofproduction of antibodies [24, 29]. Gasser et al. [24]reported that co-expression of both S. cerevisiaePDI and HAC1 in P. pastoris led to improved Fabproduction by 1.3- and 1.9-fold, respectively. Valu-able information has come from examination of‘omics’ tools. Recently, Dragosits et al. [30] demon-strated that under the osmolality conditions in theculture medium at which P. pastoris produces anti-body fragments (Fab), an analysis of the proteomeshows that increasing the osmolality results inUPR-like responses that are quite different fromthat of S. cerevisiae. However, no effect on the Fabproduction was observed. In the future, informa-tion derived from omics studies could be used todesign cell and expression systems that have im-proved levels of antibody production.

The glycoengineered P. pastoris, describedabove, has been used successfully for the produc-tion of human-like glycosylated IgG. Using fed-

Table 1. Antibody production by fermentation of yeast

Strains Antibody Promoter Culture Max cell density Production Comments Refformat volume (L) yield (g/L)

P. pastoris scFv AOX1 2.5 ~500 g wet cell/L 4.88 Control of methanol conc. and pH [60]P. pastoris scFv AOX1 6.0 3.5 Control the specific methanol uptake rate [61]P. pastoris scFv AOX1 2.5 OD = ~20 > 8.0 Co-expression of BiP [27]P. pastoris scFv AOX1 1.0 ~ 100 g DCW/L 0.81 Continuous culture [62]P. pastoris Fab GAP 1.75 ~160 g DCW/L 0.41 Co-expression of PDI [24]P. pastoris Fab AOX1 2.0 OD = 501 ~0.45 Induction at high cell density [63]A. awamori scFv Xylanase 3.5 11.5 g DCW/L 0.11 Induction in the late exponential phase [64]P. pastoris IgG AOX1 40 ~490 g wet cell/L 1.4 Human-like glycosylated IgG [32]



batch cultivation for 7 days, more than 1 g/L IgG1could be produced with a highly uniform N-linkedglycan structure (Man5GlcNAc2) [31].Various envi-ronmental conditions, including pH, temperature,dissolved oxygen, methanol feed rate and differentculture scales (0.5–40 L) were examined, and simi-lar volumetric productivity and N-linked glycanuniformity were maintained under most condi-tions. In further studies, they optimized fed-batchculture conditions including oxygen transfer rateand methanol feeding rate based on modeling, andthe improved production yield (1.4 mg/L in 140 h)was obtained [32]. The volumetric productivity(10.1 mg/L/h) obtained in this fermentation wasmuch higher than that of mammalian hosts (cur-rently 1–2 mg/L/h).

3.2 Production of antibody fragments in bacterialhosts

Compared to yeast and other higher organisms in-cluding plants, insects, and mammalian cells, E. colihas several advantages that include fast cell growthin simple inexpensive media, shorter cultivationtimes, easy gene manipulations, availability ofmany genetic tools and easy scale up in fermenta-tion. So far, many results for production of recom-binant proteins, from small peptides to large pro-teins of up to 250 kDa [33], have been achievedthrough E. coli cultivation. E. coli has two mem-branes, providing three compartments for proteinproduction that include the cytoplasm, periplasmand extracellular medium. Depending on the com-partments used for production, different strategiescan be designed for optimal production of func-tional antibodies (Fig. 3). These include: (i) opti-mization of gene expression systems by includingpromoters, signal peptides and expression order ofgenes coding each chains (routes 1, 5, 6, and 10 inFig. 3) [34], (ii) co-expression of foldase (Skp,DsbA/DsbC, Fkp, etc.) for proper folding and as-sembly (routes 2, 4, 8, and 9) [35–37], (iii) fusionwith highly soluble partners such as the maltosebinding protein (MBP) and NusA (route 1) [38], (iv)removal of destabilizing factors in the cell such asRNase E and proteases (degP, ptr, and ompT) (route7) [39], (v) genetic engineering to develop antibodyfragment mutants using a rational or random de-sign followed by HTS [40, 41], and (vi) optimizationof various environmental conditions involving theuse of lower temperatures and amount of inducers.

Among these three compartments, the peri-plasm, which provides an oxidative environmentpreferable for disulfide bond formation, is the mostpopular space for antibody production. In E. coli,the major secretion route into the periplasm is a

‘SEC-dependent pathway’. By fusion to SEC-de-pendent leader peptides such as PelB and OmpA,various antibodies have been successfully secretedinto the periplasm [39, 42–44]. In addition to SEC-dependent pathway, two other pathways includingSRP (Signal Recognition Particle) and TAT (Twin-Arginine Translocation), have been considered forantibody secretion.The SRP pathway, where trans-lation and translocation occur simultaneously, mayprevent possible aggregation of premature anti-body fragments in the cytoplasm and enhance theproduction of soluble forms in the periplasm [45].There are as yet no successful examples for en-hancement of antibody secretion via the SRP path-way, but it is known that the SRP pathway can beengineered by effective factors (trigger factors,FtsY, etc.) [46].Thus, approaches based on the SRPpathway should serve as more competitive routesfor antibody secretion. Another secretion mecha-nism through the TAT pathway enables transloca-tion of folded proteins only. In E. coli mutants,where the cytoplasm is oxidative, antibodies can befolded and secreted into the periplasm via the TATpathway (route 5 in Fig. 3) [40]. Based on proteom-ic studies, new factors affecting secretion and fold-ing have been identified. Aldor et al. [47] analyzedthe proteome of cells that produce a humanizedFab in the periplasm in an industrial fermentationprocess (10 L). Many physiological changes wereobserved to take place during fermentation, and itwas found that synthesis of the stress proteinphage shock protein A (PspA) strongly correlateswith antibody production. The co-expression ofPspA led to a 1.5-fold improvement in productionyield (~1.1 g Fab/L).

Because of its larger space relative to that of theperiplasm, the E. coli cytoplasm is suitable for massproduction of recombinant proteins. However, thereducing condition of cytoplasm is not compatiblewith the production of functional antibodies thatrequire disulfide bond formation. As a result, mostof the antibodies are produced in the cytoplasm asinsoluble inclusion bodies, which require labor-in-tensive refolding processes that may lead to re-duced production yields (route 3).The reducing en-vironment of the cytoplasm can be changed to anoxidative environment by removal of the thiore-doxin reductase (trxB) and glutathione reductase(gor) genes responsible for reducing pathway incytoplasm. Using these E. coli mutants (ex. Ori-gamiB, FA113 strains), various antibody fragments(e.g., Fab, scFv) have been generated in solubleforms in the cytoplasm with higher levels of pro-ductivity than those produced in the periplasm(route 4) [37, 48]. Sonoda et al. [37] used thioredox-in (Trx) fusion for the production of the antibody



fragment scFv in the cytoplasm. In this work, in ad-dition to Trx fusion, co-expression of cytoplasmicmolecular chaperons (GroELS and trigger factors)increased the specific productivity of an antibodyfragment almost threefold.The reducing cytoplasmitself is the preferable space for the production ofintrabodies, which do not require disulfide bondformation for their activity (route 4). Using HTSbased on new protein display (APEx 2-hybrid) sys-tems in which target antibodies are displayed onperiplasmic side of inner membrane via lipopro-tein fusion in E. coli, new intrabodies were isolated,and a three- to fivefold increase in productionyields was exhibited compared to that of the origi-nal antibody fragment [49]. So far, despite the dis-tinctive advantage associated with simple recoveryof antibodies from the culture medium, only fewexamples of antibody production into the extracel-

lular compartment have been described (route 10).Only a few mechanisms for secretion into culturemedium, such as seen for hemolysin (HlyA, type Ipathway) and pulluanase (type II pathway), areknown in E. coli. Fernandez et al. [50] fused anti-body fragment (scFv) to the C terminus of E. coli α-hemolysin (HlyA), and, via three-component chan-nel (TolC-HlyB-HlyD) connecting the inner mem-brane to outer membrane, HlyA-antibody fusedprotein was produced in culture medium. In E. coli,high volumetric productivities can be achieved us-ing high cell density cultivation (HCDC). SinceCarter et al. [43] first reported their results for an-tibody fragment (Fab) production using HCDC,many different antibody fragments have been pro-duced using HCDC in high yields (up to 4 g/L) [42](summarized in Table 2).

Figure 3. Strategies for the production of antibodies in E. coli. For efficient production of functional antibodies in E. coli, several different routes can beconsidered involving (1) control of gene expressions, (2) prevention of insoluble inclusion body (IB) formation, (3) refolding from IB after cell disruption,(4) soluble production in oxidized cytoplasm, (5) secretion into periplasm of folded antibody via TAT pathway, (6) secretion into periplasm of unfolded an-tibody via SEC or SRP pathway, (7) prevention of proteolysis in periplasm, (8) prevention of aggregation in periplasm, (9) soluble antibody production inperiplasm, (10) secretion into extracellular medium, and (11) glycosylation in periplasm. Dashed lines indicate the routes that should be prevented.



3.3 Production of full-length IgG in E. coli

E. coli is a preferred host for the production of var-ious antibody fragments, but it is one of the worstfor full-length IgG production because of lack ofglycosylation machinery and very poor assembly ofIgG. So far, there have been only two reports on thistopic [44, 51]. In 2002, Simmon et al. [44] reportedthe full-length IgG production in E. coli. The PhoApromoter, which is inducible by phosphate starva-tion but operates constitutively during long-termcultivation, was employed, and several translationinitiation region (TIRs) as well as different poly-cistronic expression systems were examined for ef-ficient expression of heavy and light chains. Theyfound that a similar molar ratio of both chains inthe periplasm is an important factor for obtaininghigh yields of full-length IgG. Although the pro-duction yields were not high (~150 mg soluble full-length IgG/L in 72-h cultivation), several consecu-tive engineering events including co-expression ofthe molecular chaperon (Skp, Fkp) and periplasmicfoldase (DsbA, DsbC) led to improved levels of pro-duction of fully assembled IgG up to ~1.3 g/L, whichare quite competitive with those obtained usingmammalian cell cultures [52, 53]. Very recently,Chan et al. [51] optimized the culture conditions forIgG production in recombinant E. coli by employ-ing a low-copy number plasmid, a reduction of in-ducer concentration, and an induction at the latelog phase for better production of full-length IgG.In both cases, purified IgGs had only marginally re-duced avidity compared to mammalian-derivedIgG, but, owing to the lack of glycosylation, no ef-fector functions were detected as expected.

Full-length IgGs are too large and complex to becorrectly assembled in E. coli, and consequentlylow yields are obtained.Thus, another important is-sue that needs to be addressed in engineering IgG

production concerns expression and assembly in E.coli systems. Mazor et al. [54] developed a new HTSmethod for isolation of full-length IgG that is wellexpressed and assembled in E. coli. Using an APEx-based screening system, several IgG derivativesagainst anthrax toxin PA were successfully gener-ated, all of which show improved assembly andhigh expression levels (~1 mg/L) of soluble IgG inflask cultivation of E. coli.

4 Antibody production in other systems

In addition to E. coli cells, other bacterial hosts arepreferable for the production of antibody frag-ments. Several Bacillus sp. strains that are Gram-positive and generally recognized as safe (GRAS),including Bacillus brevis [55], Bacillus subtilis [56]and Bacillus megaterium [57], have been success-fully used for this purpose. The greatest advantageof Bacillus sp. compared with E. coli is that produc-tion of the antibody fragments in the culture medi-um takes place via secretion, which enables simplepurification of products in downstream processing.In some cases, B. megaterium, which has severaladvantages including high plasmid stability andlack of alkaline proteases, showed better produc-tivities than E. coli hosts [57].

Cell-free production systems could be consid-ered as an alternative technology for antibody pro-duction, even though mass production is not simplein these systems [58, 59]. The advantages of cell-free systems are the rapid and economically feasi-ble modification of reaction environments and highpurities after simple purification. In particular, thismethod is well suited for production of patient-specific therapeutics and for recombinant proteinsthat are difficult to produce in other living cells. Aliet al. [59] synthesized about 20 mg Fab from 1 mL

Table 2. Antibody production by fermentation of E. coli

E. coli strains Antibody Promoter Culture Max cell Production yield Comments Refformat volume (L) density (OD) (g/L)

25F2 F(ab’)2 PhoA 10 ~150 1–2 [43]RV308 Dimeric scFv Lac 10 400 Total 4.1 Highest productivity [42]

(soluble 3 g/L)UL635 Fab Trc 5 ~150 0.7 Engineering of heavy chain [65]W3110 F(ab’)2 PhoA 10 ~250 2.45 Periplasmic protease mutant [39]RV308 scFv Pm-xylS 0.85 ~140 Total 2.1 Higher plasmid copy number [66]

(Soluble -1.1~1.3 g/L)59A7 F(ab’)2 PhoA 10 ~250 1.1 PspA co-expression [47]BL21(DE3) pLysS scFv T7 1.0 ~35 g DCW/L Total ~2 Presence of rare codon [67]

(soluble ~0.3 g/L)33D3 IgG PhoA 10 150 mg/L Full-length IgG [44]



solution containing protease-deficient (degP andompT) E. coli cell extracts. In reality, the producedamount is not high compared with those from cellcultivations, but its productivity (g/L/h) is compet-itive, and the simple purification in downstreamprocesses serve as another advantage.

5 Concluding remarks

At the beginning of the 20th century, antibodieswere referred to as ‘magic bullets’ due to their highspecificity and binding affinity for target mole-cules. Through recent progress made in the devel-opment of genetic engineering tools and antibodyengineering strategies, these ‘bullets’ have evolvedinto much more powerful weapons, which couldnow be referred to as ‘guided missiles’. However,despite the recent accumulation of successful re-sults, many people agree that the current evolutionof recombinant antibodies is not at an endpoint butrather marks the beginning of this field. Many an-tibodies candidates are now at clinical stages, and itis projected that more than 135 antibodies will beapproved by the US FDA in next 10 years. Underthis explosive growth, the finding of new antibodyis no longer a big issue, but the development ofmore potent and competitive antibodies with newdesign and manufacturing cost will be a more sig-nificant issue in the next generation drug. From re-cent development in systems biotechnology andprotein structure analysis, many useful ‘-omics’data concerning antibody networks with othercomponents in a cell have been accumulated, andwe can expand the design limits of these fascinat-ing molecules. In the meantime, increased effortsare needed to reduce manufacturing costs since, asthe demand for antibody drugs increase, muchhigher production yields are required. Althoughcellular engineering to increase productivity is oneapproach to solving this problem, it is also neces-sary to develop downstream processes that aremore economical.As described above, developmentof host cells that are able to produce antibodieswith uniform glycan structures represent a prom-ising approach for yielding highly homogeneousproducts, leading to simpler purification in down-stream processes. Many additional fields related toantibody engineering still remain to be explored.Through more sophisticated designs and intensiveengineering, antibodies will keep their highest po-sition in the market of therapeutic drug.

This research was supported by the Conversing Re-search Center Program through the National Re-

search Foundation of Korea funded by the Ministry ofEducation, Science and Technology (Grant no. 2009-0082332) and by Basic Science Research Programthrough the National Research Foundation of Koreafunded by the Ministry of Education, Science andTechnology (Grant no. 2010-0011216).

The authors have declared no conflict of interest

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Ki Jun Jeong is currently Assistant pro-

fessor in the Department of Chemical

and Biomolecular engineering, Korea

Advanced Institute of Science and

Technology (KAIST), Daejeon, Korea.

He obtained his Ph.D. in Chemical en-

gineering from KAIST in 2001 and he

joined Dr. George Georgiou’s research

group in University of Texas at Austin

(USA) as a post-doctoral fellow. In this

position (2002–2008), his research was focused on antibody engineer-

ing via directed evolution. In 2008, he joined KAIST, and currently his

research is focused on protein/antibody engineering, including (i) an-

tibody engineering against diabetes and infectious diseases, (ii) devel-

opment of new scaffold proteins as alternative antibodies, and (iii)

systems/synthetic biology for efficient production of antibody and an-

tibody fragments in bacterial systems, and mass production by high-

cell-density cultivations.

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