Analysis of Irreversible Aggregation, Reversible Self-association and Fragmentation of Monoclonal Antibodies by Analytical Ultracentrifugation

  • James D. Andya
  • Jun Liu
  • Steven J. Shire
Part of the Biotechnology: Pharmaceutical Aspects book series (PHARMASP, volume XI)


Since the emergence of recombinant DNA technology from the lab to commercialization, biotechnology protein drugs have garnered an increased share of new molecular entities (NMEs) in development pipelines (Mullin 2004). Monoclonal antibodies have rapidly become one of the major shares of this market with about 25% of biotech products in development. As with any other protein pharmaceutical monoclonal antibodies can degrade both by chemical and physical pathways. One of the most important pathways for protein physical degradation is the generation of protein aggregates. The ability of proteins to aggregate has been recognized from the early beginnings in protein biochemistry, and has become an important degradation pathway that can have a major impact on the safety and efficacy of protein drugs.


Sedimentation Velocity Protein Drug Sedimentation Coefficient Aggregate Species Lamm Equation 
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.


  1. Arthur KK, Babrielson JP, Kendrick BS, Stone MR (2006) Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation (SV-AUC): sources of variability and their relative importance. Presented at workshop on protein aggregation, Breckenridge, CO, 26–27 Sept 2006Google Scholar
  2. Berkowitz SA (2006) Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals. AAPS J 8:E590–E605PubMedCrossRefGoogle Scholar
  3. Byron O (1997) Construction of hydrodynamic bead models from high-resolution X-ray crystallographic or nuclear magnetic resonance data. Biophys J 72:408–415PubMedCrossRefGoogle Scholar
  4. Cantor CR, Schimmel PR (1980) Describing transport in the ultracentrifuge: the Lamm equation. In: Bartlett AC (ed) Biophysical chemistry, Part II techniques for the study of biological structure and function. W.H. Freeman and Company, San Francisco, CA, pp 596–603Google Scholar
  5. Demeler B, Saber H, Hansen JC (1997) Identification and interpretation of complexity in sedimentation velocity boundaries. Biophys J 72:397–407PubMedCrossRefGoogle Scholar
  6. Dintzis RZ, Okajima M, Middleton MH, Greene G, Dintzis HM (1989) The immunogenicity of soluble haptenated polymers is determined by molecular mass and hapten valence. J Immunol 143:1239–1244PubMedGoogle Scholar
  7. Furst A (1997) The XL-I analytical ultracentrifuge with Rayleigh interference optics. Eur Biophys J 35:307–310CrossRefGoogle Scholar
  8. Gabrielson J, Randilph T, Kendrick B, Stoner M (2007) Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system. Anal Biochem 361:24–30PubMedCrossRefGoogle Scholar
  9. Giebler RA (1992) New analytical ultracentrifuge with a novel precision absorption optical system. In: Harding SE, Rowe AJ, Horton JC (eds) Analytical ultracentrifugation in biochemistry and polymer science. Royal Society of Chemistry, Cambridge, England, pp 16–25Google Scholar
  10. Hermeling S, Schellekens H, Maas C, Gebbink MFBG, Crommelin DJA, Jiskoot W (2006) Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregation. J Pharm Sci 5:1084–1096CrossRefGoogle Scholar
  11. Lamm O (1929) Die differentialgleichung der ultazentrifugierung. Ark Mat Astr Fys Part B 21B:1–4Google Scholar
  12. Laue TM (1997) Advances in sedimentation velocity analysis. Biophys J 72:395–396PubMedCrossRefGoogle Scholar
  13. Liu J, Andya JD, Shire SJ (2006) A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation. AAPS J 8:E580–E589PubMedCrossRefGoogle Scholar
  14. McRorie DK, Voelker PJ (1993) Self-associating systems in the analytical ultracentrifuge. Beckman Instruments, Inc., Fullerton, CA 100 ppGoogle Scholar
  15. Meselson M, Stahl FW (1958) The Replication of DNA in Escherichia coli. Proc Natl Acad Sci U S A 44:671–682PubMedCrossRefGoogle Scholar
  16. Minton AP (2005) Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations. J Pharm Sci 94:1668–1675PubMedCrossRefGoogle Scholar
  17. Mullin R (2004) Biopharmaceuticals. Chem Eng News 82:9Google Scholar
  18. Pekar A, Sukumar M (2007) Quantitation of aggregates in therapeutic proteins using sedimentation velocity analytical ultracentrifugation: practical considerations that affect precision and accuracy. Anal Biochem 367:225–237PubMedCrossRefGoogle Scholar
  19. Philo J (1994) Measuring sedimentation, diffusion, and molecular weights of small molecules by direct fitting of sedimentation velocity concentration profiles. In: Schuster TM, Laue TM (eds) Modern analytical ultracentrifugation. Birkhausser, Boston, pp 156–170CrossRefGoogle Scholar
  20. Philpot JSL, Cook GH (1948) Research 1:234Google Scholar
  21. Pickels EG (1950) Mach Des 22:102Google Scholar
  22. Pickels EG (1952) Methods Med Res 5:107Google Scholar
  23. Schachman HK, Edelstein SJ (1966) Ultracentrifuge studies with absorption optics. IV. Molecular weight determinations at the microgram level. Biochemistry 5:2681–2705PubMedCrossRefGoogle Scholar
  24. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619PubMedCrossRefGoogle Scholar
  25. Shire SJ (1994) Analytical ultracentrifugation and its use in biotechnology. In: Schuster TM, Laue TM (eds) Modern analytical ultracentrifugation. Birkhausser, Boston, pp 261–297CrossRefGoogle Scholar
  26. Stafford WFI (1992) Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem 203:295–301PubMedCrossRefGoogle Scholar
  27. Stafford WFI (1997) Sedimentation velocity spins a new weave for an old fabric. Curr Opin Biotechnol 8:14–24PubMedCrossRefGoogle Scholar
  28. Svedberg T, Fåhraeus RA (1926) New method for determining the molecular weight of the proteins. J Am Chem Soc 48:430CrossRefGoogle Scholar
  29. Svedberg T, Rinde H (1924) The ultra-centrifuge, a new instrument for the determination of size and distribution of size of particle in amicroscopic colloids. J Am Chem Soc 46:2677–2693CrossRefGoogle Scholar
  30. Van Holde KE, Weischet W (1978) Boundary analysis of sedimentation-velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 17:1387–1403CrossRefGoogle Scholar
  31. Vogelstein B, Dintzis RZ, Dintzis HM (1982) Specific cellular stimulation in the primary immune response: a quantized model. Proc Natl Acad Sci U S A 79:395–399PubMedCrossRefGoogle Scholar
  32. Williams RCJ (1976) Improvement in precision of sedimentation-equilibrium experiments with an on-line absorption scanner. Biophys Chem 5:19–26PubMedCrossRefGoogle Scholar
  33. Zhou H-X, Rivas GM, Minton AP (2008) Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu Rev Biophys 37:375–397PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2010

Authors and Affiliations

  1. 1.Early Stage Pharmaceutical DevelopmentGenentech, Inc.South San FranciscoUSA
  2. 2.Late Stage Pharmaceutical and Processing DevelopmentGenentech, Inc.South San FranciscoUSA

Personalised recommendations