Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function

  • Reed J. Harris
  • Edward T. Chin
  • Frank Macchi
  • Rodney G. Keck
  • Bao-Jen Shyong
  • Victor T. Ling
  • Armando J. Cordoba
  • Melinda Marian
  • Don Sinclair
  • John E. Battersby
  • Andy J. S. Jones
Part of the Biotechnology: Pharmaceutical Aspects book series (PHARMASP, volume XI)


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.


Cation Exchange Chromatography Critical Quality Attribute ADCC Activity Transfected Chinese Hamster Ovary Cell Core Fucose 



Antibody-dependent cellular cytotoxicity


Succiminide at an aspartate residue


Complement-dependent cytotoxicity


Complementarity-determining regions


Chinese hamster ovary


Fragment antigen binding


Fragment crystallizable








Hydrophobic interaction chromatography


Ion exchange chromatography


Immunoglobulin gamma subclass 1




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




Variable region of the heavy chain


  1. Ashwell G, Hartford J (1982) Carbohydrate-specific receptors of the liver. Annu Rev Biochem 51:531–554PubMedCrossRefGoogle Scholar
  2. 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
  3. 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 32:1311–1318PubMedCrossRefGoogle Scholar
  4. 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 35:1897–1903PubMedCrossRefGoogle Scholar
  5. 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 99:754–758PubMedCrossRefGoogle Scholar
  6. 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 21:550–553PubMedCrossRefGoogle Scholar
  7. 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 129:6976–6977PubMedCrossRefGoogle Scholar
  8. Cordoba AJ, Shyong BJ, Breen D, Harris RJ (2005) Non-enzymatic hinge region fragmentation of antibodies in solution. J Chromatogr B 818:115–121CrossRefGoogle Scholar
  9. 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 20:2361–2370PubMedCrossRefGoogle Scholar
  10. Dong X, Storkus WJ, Salter RD (1999) Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells. J Immunol 163:5427–5434PubMedGoogle Scholar
  11. Edelman GM, Cunningham BA, Gall WE, Gottleib PD, Rutishauser U, Waxdal MJ (1969) The covalent structure of an entire γG immunoglobulin molecule. Proc Natl Acad Sci U S A 63:78–85PubMedCrossRefGoogle Scholar
  12. Ellison JW, Berson BB, Hood LE (1982) The nucleotide sequence of a human immunoglobulin Cγ1 gene. Nucleic Acids Res 10:4071–4079PubMedCrossRefGoogle Scholar
  13. Ferrara C, Stuart F, Sondermann P, Brünker 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 281:5032–5036PubMedCrossRefGoogle Scholar
  14. Furth AJ (1988) Methods for assaying nonenzymatic glycosylation. Anal Biochem 175:347–360PubMedCrossRefGoogle Scholar
  15. 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 202:163–171PubMedCrossRefGoogle Scholar
  16. 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 262:785–794PubMedGoogle Scholar
  17. Harris RJ (1995) Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J Chromatogr 705:129–134CrossRefGoogle Scholar
  18. 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 752:233–245CrossRefGoogle Scholar
  19. Hodoniczky J, Zheng YZ, James DC (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21:1644–1652PubMedCrossRefGoogle Scholar
  20. Huang L, Biolsi S, Bales KR, Kuchibhotla U (2006) Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization. Anal Biochem 249:197–207CrossRefGoogle Scholar
  21. Jefferis R, Lund J (2002) Interaction sites on human IgG-Fc for FcγR: current models. Immunol Lett 82:57–65PubMedCrossRefGoogle Scholar
  22. Jefferis R, Lund J, Goodall M (1995) Recognition sites on human IgG for Fcγ receptors: the role of glycosylation. Immunol Lett 44:111–117PubMedCrossRefGoogle Scholar
  23. 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 17:529–540PubMedCrossRefGoogle Scholar
  24. 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 93:5512–5516PubMedCrossRefGoogle Scholar
  25. 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 94:680–688PubMedCrossRefGoogle Scholar
  26. 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 17:104–118PubMedCrossRefGoogle Scholar
  27. Kaneka Y, Nimmerjahn F, Ravetch JV (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313:670–673CrossRefGoogle Scholar
  28. 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 325:979–989PubMedCrossRefGoogle Scholar
  29. 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 11:153–159PubMedCrossRefGoogle Scholar
  30. 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 24:210–215PubMedCrossRefGoogle Scholar
  31. 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 36:41–47PubMedCrossRefGoogle Scholar
  32. 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 37:697–706PubMedCrossRefGoogle Scholar
  33. 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 373:179–191PubMedCrossRefGoogle Scholar
  34. Raju TS, Scallon B (2006) Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain. Biochem Biophys Res Commun 341:797–803PubMedCrossRefGoogle Scholar
  35. 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 26:1113–1123PubMedCrossRefGoogle Scholar
  36. 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 277:26733–26740PubMedCrossRefGoogle Scholar
  37. 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 278:3466–3473PubMedCrossRefGoogle Scholar
  38. Skidgell RA (1988) Basic carboxypeptidases: regulators of peptide hormone activity. Trends Pharmacol Sci 9:299–334CrossRefGoogle Scholar
  39. Sondermann P, Osthuizen V (2002) Mediation and modulation of antibody function. Biochem Soc Trans 30:481–486PubMedCrossRefGoogle Scholar
  40. Stahl PD (1992) The mannose receptor and other macrophage lectins. Curr Opin Immunol 4:49–52PubMedCrossRefGoogle Scholar
  41. Tao M-H, Morrison SL (1989) Studies of aglycosylated chimeric mouse–human IgG. J Immunol 143:2595–2601PubMedGoogle Scholar
  42. 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 16:285–290PubMedGoogle Scholar
  43. 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 17:176–180PubMedCrossRefGoogle Scholar
  44. Weitzhandler M, Farnan D, Rohrer JS, Avdalovic N (2001) Protein variant separations using cation exchange chromatography on grafted, polymeric stationary phases. Proteomics 1:179–185PubMedCrossRefGoogle Scholar
  45. 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 21:3940–3947PubMedCrossRefGoogle Scholar
  46. 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 180:1087–1096PubMedCrossRefGoogle Scholar
  47. Wright A, Morrison SL (1997) Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol 15:26–32PubMedCrossRefGoogle Scholar
  48. 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 160:3393–3402Google Scholar
  49. 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 80:2379–2390PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2010

Authors and Affiliations

  • Reed J. Harris
    • 1
  • Edward T. Chin
    • 2
  • Frank Macchi
    • 1
  • Rodney G. Keck
    • 1
  • Bao-Jen Shyong
    • 1
  • Victor T. Ling
    • 1
  • Armando J. Cordoba
    • 1
  • Melinda Marian
    • 3
  • Don Sinclair
    • 4
  • John E. Battersby
    • 1
  • Andy J. S. Jones
    • 2
  1. 1.Protein Analytical Chemistry Department, Genentech, Inc.South San FranciscoUSA
  2. 2.Analytical Chemistry, Genentech, Inc.South San FranciscoUSA
  3. 3.Clinical and Experimental Pharmacology, Genentech, Inc.South San FranciscoUSA
  4. 4.Clinical PK/PD, Genentech, Inc.South San FranciscoUSA

Personalised recommendations