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The AAPS Journal

, Volume 8, Issue 3, pp E564–E571 | Cite as

Is any measurement method optimal for all aggregate sizes and types?

  • John S. PhiloEmail author
Article

Abstract

Protein-based pharmaceuticals exhibit a wide range of aggregation phenomena, making it virtually impossible to find any one analytical method that works well in all cases. Aggregate sizes cover a range from small oligomers to visible “snow” and precipitates, and generally only the smaller species are reversible. It is less widely recognized that aggregates also exhibit a broad spectrum of lifetimes, and the lifetime has important consequences for detection methods. The fact that the measurement itself may destroy or create aggregates poses a major analytical challenge and is a key determinant for method selection. Several examples of some interesting aggregation phenomena and the analytical approaches we have used are presented. In one case, an “aggregate” seen by SEC in stressed samples was shown to actually be a partially denatured monomer using both size-exclusion chromatography with online multiangle laser light scattering (SEC-MALLS) and sedimentation velocity. In a second case, freeze/thaw stress generates transient, metastable oligomers that are extremely sticky and difficult to measure by SEC. By using sedimentation velocity as the “gold standard” a much improved SEC method was developed and used to investigate the temperature-dependent dissociation of these oligomers. For problems with visible particulates, dynamic light scattering has been effective, in our hands, at detecting the precursors to the large, visible particles and tracking the source of stress or damage to particular manufacturing steps.

Keywords

aggregate oligomer analytical ultracentrifugation sedimentation velocity light scattering 

References

  1. 1.
    Belford GG, Belford R. Sedimentation in chemically reacting systems. II. Numerical calculations for dimerization.J Chem Phys. 1962;37:1926–1932.CrossRefGoogle Scholar
  2. 2.
    Oberhanser DF, Bethune JL, Kegeles G. Countercurrent distribution of chemical reacting systems. IV. Kinetically controlled dimerization in a boundary.Biochemistry. 1965;4:1878–1884.CrossRefGoogle Scholar
  3. 3.
    Moore JM, Patapoff TW, Cromwell ME. Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor.Biochemistry. 1999;38:13960–13967.CrossRefPubMedGoogle Scholar
  4. 4.
    Liu J, Lester P, Builder S, Shire SJ. Characterization of complex formation by humanized anti-IgE monoclonal antibody and monoclonal human IgE.Biochemistry. 1995;34:10474–10482.CrossRefPubMedGoogle Scholar
  5. 5.
    Wen J, Arakawa T, Philo JS. Size-exclusion chromatography with online light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.Anal Biochem. 1996;240:155–166.CrossRefPubMedGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2006

Authors and Affiliations

  1. 1.Alliance Protein LaboratoriesThousand Oaks

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