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Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function

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Current Trends in Monoclonal Antibody Development and Manufacturing

Part of the book series: Biotechnology: Pharmaceutical Aspects ((PHARMASP,volume XI))

Abstract

The basic structural features of antibodies, including their primary structures (Edelman et al. 1969), were established well before the concept of therapeutic antibodies was conceived. These molecules are now known to bear multiple sources of microheterogenity that can have a dramatic effect on in vivo and in vitro properties. Rituximab (Rituxan®), Trastuzumab (Herceptin®) and omalizumab (Xolair®) are three examples of therapeutic IgG1/kappa subclass antibodies produced by Genentech, Inc.; these molecules are the main subject of this discussion on the impacts of common and unique antibody modifications on functional properties.

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Abbreviations

ADCC:

Antibody-dependent cellular cytotoxicity

ASU:

Succiminide at an aspartate residue

CDC:

Complement-dependent cytotoxicity

CDR:

Complementarity-determining regions

CHO:

Chinese hamster ovary

Fab:

Fragment antigen binding

Fc:

Fragment crystallizable

Fuc:

Fucose

Gal:

Galactose

GlcNAc:

N-Acetylglucosamine

HIC:

Hydrophobic interaction chromatography

IEC:

Ion exchange chromatography

IgG1:

Immunoglobulin gamma subclass 1

IsoAsp:

Isoaspartate

MALDI-TOF/MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Man:

Mannose

VH:

Variable region of the heavy chain

References

  • Ashwell G, Hartford J (1982) Carbohydrate-specific receptors of the liver. Annu Rev Biochem 51:531–554

    Article  PubMed  CAS  Google Scholar 

  • 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 927:61–76

    Article  PubMed  CAS  Google Scholar 

  • Boyd PN, Lines AC, Patel AK (1995) The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol Immunol 32:1311–1318

    Article  PubMed  CAS  Google Scholar 

  • Cacia J, Keck R, Presta L, Frenz J (1996) Isomerization fo an aspartic acid residue in the complementarity-determining region of a recombinant antibody to human IgE: identification and effect on binding activity. Biochemistry 35:1897–1903

    Article  PubMed  CAS  Google Scholar 

  • Cartron G, Dacheux L, Salles G, Solal-Geligny P, Bardos P, Colombat P, Watier H (2002) Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgRIIIa gene. Blood 99:754–758

    Article  PubMed  CAS  Google Scholar 

  • Chaderjian WB, Chin ET, Harris RJ, Etcheverry TM (2005) Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed. Biotechnol Prog 21:550–553

    Article  PubMed  CAS  Google Scholar 

  • Cohen SL, Price C, Vlasak J (2007) β-Elimination and peptide bond hydrolysis: two distinct mechanisms of human IgG1 hinge fragmentation upon storage. J Am Chem Soc 129:6976–6977

    Article  PubMed  CAS  Google Scholar 

  • Cordoba AJ, Shyong BJ, Breen D, Harris RJ (2005) Non-enzymatic hinge region fragmentation of antibodies in solution. J Chromatogr B 818:115–121

    Article  CAS  Google Scholar 

  • Deisenhofer J (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of Protein A from Staphylococcus aureus at 2.9- and 2.8-Angstrom resolution. Biochemistry 20:2361–2370

    Article  PubMed  CAS  Google Scholar 

  • Dong X, Storkus WJ, Salter RD (1999) Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells. J Immunol 163:5427–5434

    PubMed  CAS  Google Scholar 

  • Edelman GM, Cunningham BA, Gall WE, Gottleib PD, Rutishauser U, Waxdal MJ (1969) The covalent structure of an entire ÎłG immunoglobulin molecule. Proc Natl Acad Sci U S A 63:78–85

    Article  PubMed  CAS  Google Scholar 

  • Ellison JW, Berson BB, Hood LE (1982) The nucleotide sequence of a human immunoglobulin CÎł1 gene. Nucleic Acids Res 10:4071–4079

    Article  PubMed  CAS  Google Scholar 

  • Ferrara C, Stuart F, Sondermann P, BrĂĽnker P, Umana P (2006) The carbohydrate at FcgRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms. J Biol Chem 281:5032–5036

    Article  PubMed  CAS  Google Scholar 

  • Furth AJ (1988) Methods for assaying nonenzymatic glycosylation. Anal Biochem 175:347–360

    Article  PubMed  CAS  Google Scholar 

  • Gazzano-Santoro H, Ralph P, Ryskamp TC, Chen AB, Mukku VR (1997) A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody. J Immunol Methods 202:163–171

    Article  PubMed  CAS  Google Scholar 

  • Geiger T, Clarke S (1987) Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem 262:785–794

    PubMed  CAS  Google Scholar 

  • Harris RJ (1995) Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J Chromatogr 705:129–134

    Article  CAS  Google Scholar 

  • Harris RJ, Kabakoff B, Macchi FD, Shen FJ, Kwong M, Andya JD, Shire SJ, Bjork N, Totpal K, Chen AB (2001) Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr 752:233–245

    Article  CAS  Google Scholar 

  • Hodoniczky J, Zheng YZ, James DC (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21:1644–1652

    Article  PubMed  CAS  Google Scholar 

  • Huang L, Biolsi S, Bales KR, Kuchibhotla U (2006) Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization. Anal Biochem 249:197–207

    Article  Google Scholar 

  • Jefferis R, Lund J (2002) Interaction sites on human IgG-Fc for FcÎłR: current models. Immunol Lett 82:57–65

    Article  PubMed  CAS  Google Scholar 

  • Jefferis R, Lund J, Goodall M (1995) Recognition sites on human IgG for FcÎł receptors: the role of glycosylation. Immunol Lett 44:111–117

    Article  PubMed  CAS  Google Scholar 

  • Jones AJ, Papac DI, Chin EH, Keck R, Baughman SA, Lin YS, Kneer J, Battersby JE (2007) Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys. Glycobiology 17:529–540

    Article  PubMed  CAS  Google Scholar 

  • Junghans RP, Andersen CL (1996) The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor. Proc Natl Acad Sci U S A 93:5512–5516

    Article  PubMed  CAS  Google Scholar 

  • Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S, Wakitani M, Konno Y et al (2006) Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC. Biotechnol Bioeng 94:680–688

    Article  PubMed  CAS  Google Scholar 

  • Kanda Y, Yamada T, Mori K, Okazaki A, Inoue M, Kitajima-Miyama K, Kuni-Kamochi R, Nakano R, Yano K, Kakita S et al (2007) Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology 17:104–118

    Article  PubMed  CAS  Google Scholar 

  • Kaneka Y, Nimmerjahn F, Ravetch JV (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313:670–673

    Article  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 

  • Lapolla A, Fedele D, Garbeglio M, Martano L, Tonani R, Seraglia R, Favretto D, Fedrigo MA, Traldi P (2000) Matrix-assisted laser desorption ionization mass spectrometry, enzymatic digestion, and molecular modeling in the study of nonenzymatic glycation of IgG. J Am Soc Mass Spectrom 11:153–159

    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 et al (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215

    Article  PubMed  CAS  Google Scholar 

  • Millward TA, Heitzmann M, Bill K, Längle U, Schumacher P, Forrer K (2007) Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice. Biologicals 36:41–47

    Article  PubMed  Google Scholar 

  • Mimura Y, Church S, Ghirlando R, Ashton PR, Dong S, Goodall M, Lund J, Jefferis R (2000) The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms. Mol Immunol 37:697–706

    Article  PubMed  CAS  Google Scholar 

  • Quan C, Alcala E, Petkovska I, Matthews D, Canova-Davis E, Taticek R, Ma S (2008) A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior. Anal Biochem 373:179–191

    Article  PubMed  CAS  Google Scholar 

  • Raju TS, Scallon B (2006) Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain. Biochem Biophys Res Commun 341:797–803

    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 

  • Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SHA, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcgRIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–26740

    Article  PubMed  CAS  Google Scholar 

  • Shinkawa T, Nakamurai K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Sato M, Yamasaki M et al (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role for enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278:3466–3473

    Article  PubMed  CAS  Google Scholar 

  • Skidgell RA (1988) Basic carboxypeptidases: regulators of peptide hormone activity. Trends Pharmacol Sci 9:299–334

    Article  Google Scholar 

  • Sondermann P, Osthuizen V (2002) Mediation and modulation of antibody function. Biochem Soc Trans 30:481–486

    Article  PubMed  CAS  Google Scholar 

  • Stahl PD (1992) The mannose receptor and other macrophage lectins. Curr Opin Immunol 4:49–52

    Article  PubMed  CAS  Google Scholar 

  • Tao M-H, Morrison SL (1989) Studies of aglycosylated chimeric mouse–human IgG. J Immunol 143:2595–2601

    PubMed  CAS  Google Scholar 

  • Tsuchiya N, Endo T, Matsuta K, Yoshinoya S, Aikawa T, Kosuge E, Takeuchi F, Miyamotot T, Kobata A (1989) Effects of galactose depletion from oligosaccharide chains on immunological activities of human IgG. J Rheumatol 16:285–290

    PubMed  CAS  Google Scholar 

  • Umana P, Jean-Mariet J, Moudry R, Amstutz H, Bailey JE (1999) Engineered glycoforms on an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17:176–180

    Article  PubMed  CAS  Google Scholar 

  • Weitzhandler M, Farnan D, Rohrer JS, Avdalovic N (2001) Protein variant separations using cation exchange chromatography on grafted, polymeric stationary phases. Proteomics 1:179–185

    Article  PubMed  CAS  Google Scholar 

  • Weng W-K, Levy R (2003) Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol 21:3940–3947

    Article  PubMed  CAS  Google Scholar 

  • Wright A, Morrison SL (1994) Effect of altered CH2-associated carbohydrate structure on the functional properties an in vivo fate of chimeric mouse–human immunoglobulin G1. J Exp Med 180:1087–1096

    Article  PubMed  CAS  Google Scholar 

  • Wright A, Morrison SL (1997) Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol 15:26–32

    Article  PubMed  CAS  Google Scholar 

  • Wright A, Morrison SL (1998) Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells. J Immunol 160:3393–3402

    Google Scholar 

  • Zhang B, Yang Y, Yuk I, Pai R, McKay P, Eigenbrot C, Dennis M, Katta V, Francissen KC (2008) Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody. Anal Chem 80:2379–2390

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Protein Analytical Chemistry Department, Genentech, Inc., South San Francisco, CA, USA

    Reed J. Harris, Frank Macchi, Rodney G. Keck, Bao-Jen Shyong, Victor T. Ling, Armando J. Cordoba & John E. Battersby

  2. Analytical Chemistry, Genentech, Inc., South San Francisco, CA, USA

    Edward T. Chin & Andy J. S. Jones

  3. Clinical and Experimental Pharmacology, Genentech, Inc., South San Francisco, CA, USA

    Melinda Marian

  4. Clinical PK/PD, Genentech, Inc., South San Francisco, CA, USA

    Don Sinclair

Authors
  1. Reed J. Harris
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  2. Edward T. Chin
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  3. Frank Macchi
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  6. Victor T. Ling
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  7. Armando J. Cordoba
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  8. Melinda Marian
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  9. Don Sinclair
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  10. John E. Battersby
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  11. Andy J. S. Jones
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Corresponding author

Correspondence to Reed J. Harris .

Editor information

Editors and Affiliations

  1. Genentech Inc., DNA Way 1, San Francisco, 94080, U.S.A.

    Steven J. Shire

  2. Omeros Corporation, Fifth Ave Ste 2600 1420, Seattle, 98101, U.S.A.

    Wayne Gombotz

  3. Boehringer Ingelheim Pharma KG, Birkendorfer Str. 65, Biberach, 88397, Germany

    Karoline Bechtold-Peters

  4. Genentech, Inc., DNA Way 1, South San Francisco, 94080, U.S.A.

    James Andya

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© 2010 American Association of Pharmaceutical Scientists

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Harris, R.J. et al. (2010). Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function. In: Shire, S., Gombotz, W., Bechtold-Peters, K., Andya, J. (eds) Current Trends in Monoclonal Antibody Development and Manufacturing. Biotechnology: Pharmaceutical Aspects, vol XI. Springer, New York, NY. https://doi.org/10.1007/978-0-387-76643-0_12

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