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Antibody production with yeasts and filamentous fungi: on the road to large scale?

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Abstract

Yeasts and filamentous fungi have gained significant interest for the production of recombinant antibodies and antibody fragments. The opportunities and constraints of antibody (fragment) production in these hosts are highlighted as well as cell engineering strategies to overcome the constraints. Following aspects are addressed: folding, assembly and secretion of antibody related proteins, process optimization to improve productivity and quality, proteolysis, and, as a major point of interest, glycosylation.

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Abbreviations

ADCC:

Antibody dependent cellular cytotoxicity

CH1, CH2, CH3:

Constant domains of heavy chain

C l :

Constant domain of light chain

Fab:

Antigen binding fragment (total light chain VL+CL, VH and CH1 domain of heavy chain, connected by an intermolecular disulfide bond)

Fc:

Cristallizing fragment, consists of CH2 and CH3, glycosylation site, not antigen binding, but responsible for effector functions

HC:

Heavy chain

IgG:

Immunoglobulin G

LC:

Light chain

mAb:

Monoclonal antibody

scFv:

Single chain variable domains fragment (VL and VH connected by a linker peptide)

scFv-Fc:

Gene fusion of scFv to Fc fragment

VH :

Variable domain of heavy chain

VHH :

Heavy chain variable domain of Camelidae derived antibodies (do not possess any light chain; whole antibody is build of VHH domain fused to CH2 and CH3, no CH1 domain)

VL :

Variable domain of light chain

References

  • Abdel-Salam HA, El-Khamissy T, Enan GA, Hollenberg CP (2001) Expression of mouse anticreatine kinase (MAK33) monoclonal antibody in the yeast Hansenula polymorpha. Appl Microbiol Biotechnol 56:157–164

    Article  PubMed  CAS  Google Scholar 

  • Andersen DC, Reilly DE (2004) Production technologies for monoclonal antibodies and their fragments. Curr Opin Biotechnol 15:456–462

    Article  PubMed  CAS  Google Scholar 

  • Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2005) Prokaryotic expression of antibodies. Cancer Metastasis Rev 24:501–519

    Article  PubMed  Google Scholar 

  • Bretthauer RK, Castellino FJ (1999) Glycosylation of Pichia pastoris-derived proteins. Biotechnol Appl Biochem 30:193–200

    PubMed  CAS  Google Scholar 

  • Carter P, Kelley RF, Rodrigues ML, Snedecor B, Covarrubias M, Velligan MD, Wong WL, Rowland AM, Kotts CE, Carver ME, Yang M, Bourell JH, Shepard HM, Henner D (1992) High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Bio/Technology 10:163–167

    Article  PubMed  CAS  Google Scholar 

  • 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 85:463–474

    Article  PubMed  CAS  Google Scholar 

  • Cunha AE, Clemente JJ, Gomes R, Pinto F, Thomaz M, Miranda S, Pinto R, Moosmayer D, Donner P, Carrondo MJ (2004) Methanol induction optimization for scFv antibody fragment production in Pichia pastoris. Biotechnol Bioeng 86:458–467

    Article  PubMed  CAS  Google Scholar 

  • Damasceno LM, Pla I, Chang HJ, Cohen L, Ritter G, Old LJ, Batt CA (2004) An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif 37:18–26

    Article  PubMed  CAS  Google Scholar 

  • Edqvist J, Keränen S, Penttilä M, Straby KB, Knowles JKC (1991) Production of functional IgM Fab fragments by Saccharomyces cerevisiae. J Biotechnol 20:291–300

    Article  PubMed  CAS  Google Scholar 

  • Eldin P, Pauza ME, Hieda Y, Lin G, Murtaugh MP, Pentel PR, Pennell CA (1997) High-level secretion of two antibody single chain Fv fragments by Pichia pastoris. J Immunol Methods 201:67–75

    Article  PubMed  CAS  Google Scholar 

  • Evans D, Das R (2005) monoclonal antibody therapies: evolving into a $ 30 billion market. Datamonitor, London, UK

    Google Scholar 

  • Farid SS (2006) Process economics of industrial monoclonal antibody manufacture. J Chromatogr B (in press)

  • Feige MJ, Walter S, Buchner J (2004) Folding mechanism of the CH2 antibody domain. J Mol Biol 344:107–118

    Article  PubMed  CAS  Google Scholar 

  • Frenken LG, Hessing JG, Van den Hondel CA, Verrips CT (1998) Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms. Res Immunol 149:589–599

    Article  PubMed  CAS  Google Scholar 

  • Frenken LG, van der Linden RH, Hermans PW, Bos JW, Ruuls RC, de Geus B, Verrips CT (2000) Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J Biotechnol 78:11–21

    Article  PubMed  CAS  Google Scholar 

  • Freyre FM, Vazquez JE, Ayala M, Canaan-Haden L, Bell H, Rodriguez I, Gonzalez A, Cintado A, Gavilondo JV (2000) Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris. J Biotechnol 76:157–163

    Article  PubMed  CAS  Google Scholar 

  • Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D (2006) Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng 94:353–361

    Article  PubMed  CAS  Google Scholar 

  • Hackel BJ, Huang D, Bubolz JC, Wang XX, Shusta EV (2006) Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae. Pharm Res 23:790–797

    Article  PubMed  CAS  Google Scholar 

  • 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 301:1244–1246

    Article  PubMed  CAS  Google Scholar 

  • Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2004) Effects of gene dosage, promoters and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85:367–375

    Article  PubMed  CAS  Google Scholar 

  • Horwitz AH, Chang CP, Better M, Hellstrom KE, Robinson RR (1988) Secretion of functional antibody and Fab fragment from yeast cells. Proc Natl Acad Sci U S A 85:8678–8682

    Article  PubMed  CAS  Google Scholar 

  • Joosten V, Gouka RJ, van den Hondel CA, Verrips CT, Lokman BC (2005) Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Appl Microbiol Biotechnol 66:384–392

    Article  PubMed  CAS  Google Scholar 

  • Joosten V, Lokman C, Van Den Hondel CA, Punt PJ (2003) The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. Microb Cell Fact 2:1

    Article  PubMed  Google Scholar 

  • Kauffman KJ, Pridgen EM, Doyle FJ III, Dhurjati PS, Robinson AS (2002) Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae. Biotechnol Prog 18:942–950

    Article  PubMed  CAS  Google Scholar 

  • Khatri NK, Hoffmann F (2006) Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris. Appl Microbiol Biotechnol 72:492–498

    Article  PubMed  CAS  Google Scholar 

  • Kim SJ, Park Y, Hong HJ (2005) Antibody engineering for the development of therapeutic antibodies. Mol Cells 20:17–29

    PubMed  CAS  Google Scholar 

  • Kipriyanov SM, Moldenhauer G, Martin AC, Kupriyanova OA, Little M (1997) Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity. Protein Eng 10:445–453

    Article  PubMed  CAS  Google Scholar 

  • Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol Biol 325: 979–989

    Article  PubMed  CAS  Google Scholar 

  • Lange S, Schmitt J, Schmid RD (2001) High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris. J Immunol Methods 255:103–114

    Article  PubMed  CAS  Google Scholar 

  • Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, Bobrowicz P, Choi BK, Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR, Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Wildt S, Gerngross TU (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215

    Article  PubMed  CAS  Google Scholar 

  • Luo D, Geng M, Schultes B, Ma J, Xu DZ, Hamza N, Qi W, Noujaim AA, Madiyalakan R (1998) Expression of a fusion protein of scFv-biotin mimetic peptide for immunoassay. J Biotechnol 65:225–228

    Article  PubMed  CAS  Google Scholar 

  • Maras M, van Die I, Contreras R, van den Hondel CA (1999) Filamentous fungi as production organisms for glycoproteins of bio-medical interest. Glycoconj J 16:99–107

    Article  PubMed  CAS  Google Scholar 

  • Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135

    Article  PubMed  CAS  Google Scholar 

  • Mayer M, Kies U, Kammermeier R, Buchner J (2000) BiP and PDI cooperate in the oxidative folding of antibodies in vitro. J Biol Chem 275:29421–29425

    Article  PubMed  CAS  Google Scholar 

  • Miller KD, Weaver-Feldhaus J, Gray SA, Siegel RW, Feldhaus MJ (2005) Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Protein Expr Purif 42:255–267

    Article  PubMed  CAS  Google Scholar 

  • Ning D, Junjian X, Qing Z, Sheng X, Wenyin C, Guirong R, Xunzhang W (2005) Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation. J Biochem Mol Biol 38:294–299

    PubMed  CAS  Google Scholar 

  • Nyyssonen E, Keranen S (1995) Multiple roles of the cellulase CBHI in enhancing production of fusion antibodies by the filamentous fungus Trichoderma reesei. Curr Genet 28:71–79

    Article  PubMed  CAS  Google Scholar 

  • Nyyssonen E, Penttila M, Harkki A, Saloheimo A, Knowles JK, Keranen S (1993) Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei. Bio/Technology 11:591–595

    Article  PubMed  CAS  Google Scholar 

  • Ogunjimi AA, Chandler JM, Gooding CM, Recinos A, Choudary PV (1999) High-level secretory expression of immunologically active intact antibody from the yeast Pichia pastoris. Biotechnol Lett 21:561–567

    Article  CAS  Google Scholar 

  • Pavlou AK, Belsey MJ (2005) The therapeutic antibodies market to 2008. Eur J Pharm Biopharm 59:389–396

    Article  PubMed  Google Scholar 

  • Powers DB, Amersdorfer P, Poul M, Nielsen UB, Shalaby MR, Adams GP, Weiner LM, Marks JD (2001) Expression of single-chain Fv-Fc fusions in Pichia pastoris. J Immunol Methods 251:123–135

    Article  PubMed  CAS  Google Scholar 

  • Punt PJ, van Gemeren IA, Drint-Kuijvenhoven J, Hessing JG, van Muijlwijk-Harteveld GM, Beijersbergen A, Verrips CT, van den Hondel CA (1998) Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli. Appl Microbiol Biotechnol 50:447–454

    Article  PubMed  CAS  Google Scholar 

  • Rahbarizadeh F, Rasaee MJ, Forouzandeh M, Allameh AA (2006) Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris. Mol Immunol 43:426–435

    Article  PubMed  CAS  Google Scholar 

  • Ridder R, Schmitz R, Legay F, Gram H (1995) Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris. Biotechnology 13:255–260

    Article  PubMed  CAS  Google Scholar 

  • Robin S, Petrov K, Dintinger T, Kujumdzieva A, Tellier C, Dion M (2003) Comparison of three microbial hosts for the expression of an active catalytic scFv. Mol Immunol 39:729–738

    Article  PubMed  CAS  Google Scholar 

  • Rothman RJ, Perussia B, Herlyn D, Warren L (1989) Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermine-induced alterations of IgG glycosylation. Mol Immunol 26:1113–1123

    Article  PubMed  CAS  Google Scholar 

  • Shi X, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 28:321–330

    Article  PubMed  CAS  Google Scholar 

  • Shusta EV, Raines RT, Pluckthun A, Wittrup KD (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat Biotechnol 16:773–777

    Article  PubMed  CAS  Google Scholar 

  • 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 263:133–147

    Article  PubMed  CAS  Google Scholar 

  • Sotiriadis A, Keshavarz T, Keshavarz-Moore E (2001) Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor. Biotechnol Prog 17:618–623

    Article  PubMed  CAS  Google Scholar 

  • Swennen D, Paul MF, Vernis L, Beckerich JM, Fournier A, Gaillardin C (2002) Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis. Microbiology 148:41–50

    PubMed  CAS  Google Scholar 

  • Takahashi K, Toshifumi Y, Takai T, Ra C, Okumura K, Yokota T, Okumura Y (2000) Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris. Biosci Biotechnol Biochem 64:2138–2144

    Article  PubMed  CAS  Google Scholar 

  • Trentmann O, Khatri NK, Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20:1766–1775

    Article  PubMed  CAS  Google Scholar 

  • Valkonen M, Ward M, Wang H, Penttila M, Saloheimo M (2003) Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl Environ Microbiol 69:6979–6986

    Article  PubMed  CAS  Google Scholar 

  • Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K, Contreras R (2004) In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microbiol 70:2639–2646

    Article  PubMed  CAS  Google Scholar 

  • Ward M, Lin C, Victoria DC, Fox BP, Fox JA, Wong DL, Meerman HJ, Pucci JP, Fong RB, Heng MH, Tsurushita N, Gieswein C, Park M, Wang H (2004) Characterization of humanized antibodies secreted by Aspergillus niger. Appl Environ Microbiol 70:2567–2576

    Article  PubMed  CAS  Google Scholar 

  • Werner RG (2004) Economic aspects of commercial manufacture of biopharmaceuticals. J Biotechnol 113:171–182

    Article  PubMed  CAS  Google Scholar 

  • Xu P, Raden D, Doyle FJ III, Robinson AS (2005) Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding. Metab Eng 7269–7279

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Acknowledgements

Research on antibody expression in yeasts in our laboratory is supported by the Austrian Science Fund (project No. I37-B03), the European Science Foundation (programme EuroSCOPE), the Austrian Research Promotion Agency (programme FHplus), Polymun Scientific GmbH, and Boehringer Ingelheim Austria GmbH.

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Authors and Affiliations

  1. Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190, Vienna, Austria

    Brigitte Gasser & Diethard Mattanovich

  2. School of Bioengineering, University of Applied Science FH-Campus Vienna, Muthgasse 18, A-1190, Vienna, Austria

    Diethard Mattanovich

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  1. Brigitte Gasser
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  2. Diethard Mattanovich
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Correspondence to Diethard Mattanovich.

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Gasser, B., Mattanovich, D. Antibody production with yeasts and filamentous fungi: on the road to large scale?. Biotechnol Lett 29, 201–212 (2007). https://doi.org/10.1007/s10529-006-9237-x

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  • DOI: https://doi.org/10.1007/s10529-006-9237-x

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