Separation and Characterization of an IgG2 Antibody Containing a Cyclic Imide in CDR1 of Light Chain by Hydrophobic Interaction Chromatography and Mass Spectrometry

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Abstract

Hydrophobic interaction chromatography (HIC) was used to separate populations of recombinant IgG2 antibody that were created as a result of prolonged incubation at 40 degrees C. Antibody was separated by HIC into three major and seven minor fractions. All but one fraction was composed of antibody with distinct chemical modifications that resulted from exposure to elevated temperature. The results of intact and reduced mass analysis as well as peptide map data derived from the three major HIC fractions indicated that the antibody was being chromatographically separated into populations containing a succinimidyl intermediate in complementarity determining region 1 (CDR1) on zero, one, and two light chain arms. Lower level species purified by HIC were analyzed by intact and reduced mass analysis and laser-induced fluorescence capillary electrophoresis (CE-LIF) and consisted of an antibody that was clipped in four different places in the heavy chain as well as misfolded and aggregated antibody. The potency of the recombinant antibody containing a succinimidyl intermediate on zero, one, and two light chain arms was analyzed by LANCE binding assay and a cell based in vitro bioassay, and the occurrence of this modification on one or both light chain arms was associated with a reduction in the binding affinity of the molecule to the target by approximately 10%. We show that HIC has the unique ability as a first step purification method to separate populations of antibody which are covalently modified under stability programs. The method conditions that have been developed for the HIC assay are ideal for purifying antibodies with labile modifications for the purpose of further characterization.

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