Production of Monoclonal Antibodies in E. coli

  • Dorothea E. Reilly
  • Daniel G. Yansura
Part of the Biotechnology: Pharmaceutical Aspects book series (PHARMASP, volume XI)


The number of monoclonal antibodies approved for use as therapeutic agents by regulatory agencies has increased in the past several years. Monoclonal antibodies are predicted to become an increasingly larger part of biopharmaceutical products, and perhaps dominate the market share by the end of the decade (Walsh 2006). Mammalian expression systems, such as Chinese Hamster Ovary cells (CHO), are currently the preferred system for producing full-length monoclonal antibodies. Fungal systems could become more of a contender for the production of antibodies if titers can be increased (Andersen and Reilly 2004). However, with fungal production systems, there may be concerns about potential non-native mammalian N-linked or O-linked glycosylation that could result in immunogenic responses in humans. Technology developed in recent years (Hamilton et al. 2003) could help to alleviate this concern.


Light Chain Heavy Chain Disulfide Bond Size Exclusion Chromatography Fermentation Culture 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Antibody dependent cellular cytotoxicity


Base pairs


Subcomponent of C1, a complement activator


Complement dependent cytotoxicity


Chinese hamster ovary

E. coli

Escherichia coli


Enzyme-linked immunosorbent assay


Neonatal Fc receptor


Isopropyl β-D-thiogalactopyranoside






Phosphate buffered saline


Size-exclusion chromatography


Translation initiation region



The authors would like to thank Lisa A. Damico, Sean Kelley, and David Xie for the use of unpublished data on the pharmacokinetic analyses of the antibodies, Ralph Schwall for efficacy studies, Gloria Meng and An Song for binding studies, Amy Lim for SEC data, and Michael W. Laird for help with the manuscript preparation.


  1. Andersen D, Reilly DE (2004) Production technologies for monoclonal antibodies and their fragments. Curr Opin Biotechnol 15:456–462PubMedCrossRefGoogle Scholar
  2. Andersen DC, and Simmons LC (2004) A system for antibody expression and assembly. European Patent: EP1427744Google Scholar
  3. Baneyx F, Palumbo JL (2003) Improving heterologous protein folding via molecular chaperone and foldase co-expression. In: Vaillancourt PE (ed) Methods in molecular biology, vol 205, E. coli gene expression protocols. Humana press, Totawa, N J, pp 171–197Google Scholar
  4. Battersby JE, Snedecor B, Chen C, Champion KM, Riddle L, Vanderlaan M (2001) Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth. J Chromatogr A 927:61–76PubMedCrossRefGoogle Scholar
  5. Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci USA 96:13703–13708PubMedCrossRefGoogle Scholar
  6. Bessette PH, Qiu JI, Bardwell JCA, Swartz JR, Georgiou G (2001) Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli. J Bacteriol 183(3):980–988PubMedCrossRefGoogle Scholar
  7. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. Gene 2:95–113PubMedCrossRefGoogle Scholar
  8. Bothmann H, Pluckthun A (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat Biotechnol 16:376–380PubMedCrossRefGoogle Scholar
  9. Bothmann H, Pluckthun A (2000) The periplasmic Escherichia coli peptidylprolyl cis, trans-isomerase FkpA. J Biol Chem 275(22):17100–17105PubMedCrossRefGoogle Scholar
  10. Chen C, Snedecor B, Nishihara JC, Joly JC, McFarland N, Andersen DC, Battersby JE, Champion KM (2004) High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain. Biotechnol Bioeng 85(5):463–474PubMedCrossRefGoogle Scholar
  11. De Boer HA, Comstock LJ, Vasser M (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 80:21–25PubMedCrossRefGoogle Scholar
  12. De Smit MH, van Duin J (1990) Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci USA 87:7668–7672PubMedCrossRefGoogle Scholar
  13. Derman AI, Prinz WA, Belin D, Beckwith J (1993) Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262:1744–1747PubMedCrossRefGoogle Scholar
  14. Friend PJ, Hale G, Chatenoud L, Rebello P, Bradley J, Thiru S, Phillips JM, Waldmann H (1999) Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection. Transplantation 68(11):1632–1637PubMedCrossRefGoogle Scholar
  15. Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T, Nett JH, Rausch S, Stadheim TA, Wischnewski H, Wildt S, Gerngross TU (2003) Production of complex human glycoproteins in yeast. Science 301:1244–1246PubMedCrossRefGoogle Scholar
  16. Hayhurst A, Harris WJ (1999) Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments. Protein Expr and Purif 15:336–343CrossRefGoogle Scholar
  17. Humphreys DP (2007) Periplasmic Expression of Antibody Fragments. In: Ehrmann M (ed) The periplasm. ASM press, Washington DC, pp 361–388Google Scholar
  18. Jeong KJ, Lee SY (2000) Secretory production of human leptin in Escherichia coli. Biotechnol Bioeng 67(4):398–407PubMedCrossRefGoogle Scholar
  19. Joly JC, Laird MW (2007) Practical applications for periplasmic protein accumulation. In: Ehrmann M (ed) The periplasm. ASM Press, Washington DC, pp 345–360Google Scholar
  20. Joly JC, Leung WS, Schwartz JR (1998) Overexpression of Escherichia coli oxidoreductases increases recombinant insulin-like growth factor-I accumulation. Proc Natl Acad Sci USA 95:2773–2777PubMedCrossRefGoogle Scholar
  21. Makrides SC (1996) Strategies for Achieving High-Level Expression of Genes in Escherichia coli. Microbiol. Rev. 60 (3):512–538Google Scholar
  22. Martens T, Schmidt NO, Eckerich C, Fillbrandt R, Merchant M, Schwall R, Westphal M, Lamszus K (2006) A novel one-armed anti-c-Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 12:6144–6152PubMedCrossRefGoogle Scholar
  23. Mazor Y, Blarcom TV, Mabry R, Iverson BL, Georgiou G (2007) Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat Biotechnol 25:563–565PubMedCrossRefGoogle Scholar
  24. McCarthy JEG, Gualerzi C (1990) Translational control of prokaryotic gene expression. Trends Genet 6(3):78–85PubMedCrossRefGoogle Scholar
  25. Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, Carter P (1998) An efficient route to human bispecific IgG. Nat Biotechnol 16:677–681PubMedCrossRefGoogle Scholar
  26. Pluckthun A, Krebber A, Krebber C, Horn U, Knupfer U, Wenderoth R, Nieba L, Proba K, Riesenberg D (1996) Producing antibodies in Escherichia coli: from PCR to fermentation. In: McCafferty J, Hoogenboom HR, Chiswell DJ (eds) Antibody engineering: a practical approach. Oxford university press, New York, N Y, pp 203–251Google Scholar
  27. Prinz WA, Aslund F, Holmgren A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathway in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272(25):15661–15667PubMedCrossRefGoogle Scholar
  28. Qiu JI, Schwartz JR, Georgiou G (1998) Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol 64(12):4891–4896PubMedGoogle Scholar
  29. Routledge EG, Falconer ME, Pope H, Lloyd IS, Waldmann H (1995) The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody. Transplantation 60:847–853PubMedGoogle Scholar
  30. Simmons LC, Yansura DG (1996) Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli. Nat Biotechnol 14:629–634PubMedCrossRefGoogle Scholar
  31. Simmons LC, Reilly D, Klimowski L, Raju TS, Meng G, Sims P, Hong K, Shields RL, Damico LA, Rancatore P, Yansura DG (2002) Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies. J Immunol Methods 263:133–147PubMedCrossRefGoogle Scholar
  32. Tao MH, Morrison SL (1989) Studies of aglycosylated chimeric mouse-human IgG. J Immunol 143:2595–2601PubMedGoogle Scholar
  33. Thome BM, Muller M (1991) Skp is a periplasmic Escherichia coli protein requiring SecA and SecY for export. Mol Microbiol 5(11):2815–2821PubMedCrossRefGoogle Scholar
  34. Walsh G (2006) Biopharmaceutical Benchmarks 2006. Nat Biotechnol 24:769–776PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2010

Authors and Affiliations

  • Dorothea E. Reilly
    • 1
  • Daniel G. Yansura
    • 2
  1. 1.Early Stage Cell Culture, Genentech, Inc.South San FranciscoUSA
  2. 2.Antibody Engineering, Genentech, Inc.South San FranciscoUSA

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