Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates
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Issue Date
2013-06-19Author
Boylan, Nicholas J.
Zhou, Wen
Proos, Robert J.
Tolbert, Thomas J.
Wolfe, Janet L.
Laurence, Jennifer S.
Publisher
ACS
Type
Article
Article Version
Scholarly/refereed, author accepted manuscript
Rights
Copyright © 2013 American Chemical Society
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Show full item recordAbstract
Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics and tissue distributions. Charge variants arise spontaneously due to degradation and, depending on the type of drug, linker and conjugation site, through drug conjugation. Electrostatic changes in naked antibodies often result in poor performance characteristics, and therefore charge alterations due to degradation are critical to control. Charge properties are expected to be equally important to producing well-behaved ADCs. Charge-based methods of analysis, such as isoelectric focusing and ion exchange chromatography, are capable of probing the underlying complexities within immunoconjugate drug products. Despite the utility of these methods, there are only a few published reports of charge-based assays applied to immunoconjugates. In the present study, we sought to identify the effects of chemical conjugation on the electrostatic properties of Fc-conjugates. In order to minimize the effects of post-translational modifications (e.g. deamidation), a single Fc charge variant was isolated prior to conjugation of a fluorescent probe, Alexa Fluor 350, to the side chains of lysine residues. The resulting Fc-conjugates were assessed by a variety of analytical techniques, including isoelectric focusing and ion exchange chromatography, to determine their charge properties.
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Citation
Boylan, N. J., Zhou, W., Proos, R. J., Tolbert, T. J., Wolfe, J. L., & Laurence, J. S. (2013). Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates. Bioconjugate Chemistry, 24(6), 1008–1016. http://doi.org/10.1021/bc4000564
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