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Single System for Physicochemical and Biological Minor Variant Characterization of Biotherapeutics: A Proof of Concept
Kozintsev, Alexander
Kozintsev, Alexander
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Abstract
Late stage analytical development activities for recombinant monoclonal antibody therapeuticsnecessitate the physicochemical and biological characterization of product variants and impurities in order to determine their relative risks to patient safety and efficacy. In particular, a minor variant characterization study is performed in order to obtain and demonstrate a thorough understanding of the structure and function of product variants, and the study is achieved through the application of a suite of analytical methods targeting common product and process variants such as charge and size variants. However, the execution of such studies relies primarily on costly and lengthy product variant enrichment processes followed by the execution of many physicochemical and biological assays on an assortment of instruments. In this work, size and charge LC techniques are combined for the first time with functionally relevant Fc receptor affinity chromatographies and mass spectrometry on a single, modular, multi-dimensional system capable of executing online structural and functional characterization of most recombinant monoclonal antibody product variants. Coupling size, charge, and Fc receptoraffinity chromatographies to mass spectrometry in an online, multi-dimensional fashion resulted in significant time savings and enabled direct identification of heterogeneities observed in the chromatographic profiles without the need for additional enrichment or isolation. The sensitivity and efficiency of this system for investigating relevant product variants was demonstrated via online and offline characterization of variants from several pipeline biotherapeutic molecules, including a standard N-glycosylated IgG1 monoclonal antibody and an aglycosylated IgG1-like bispecific antibody. The single-system, physicochemical and biological analytical suite and its applications as described herein demonstrate an important proof of concept and novel direction for rapid and efficient characterization of product variants for biotherapeutics.
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Date
2022-05-31
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University of Kansas
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Keywords
Analytical chemistry, Biochemistry, Biology, chromatography, function, multidimensional, structure