| Abstract
| - Hydrophobic interaction chromatography (HIC) was usedto separate populations of recombinant IgG2 antibody thatwere created as a result of prolonged incubation at40 °C. Antibody was separated by HIC into three majorand seven minor fractions. All but one fraction wascomposed of antibody with distinct chemical modificationsthat resulted from exposure to elevated temperature. Theresults of intact and reduced mass analysis as well aspeptide map data derived from the three major HICfractions indicated that the antibody was being chromatographically separated into populations containing a succinimidyl intermediate in complementarity determiningregion 1 (CDR1) on zero, one, and two light chain arms.Lower level species purified by HIC were analyzed byintact and reduced mass analysis and laser-inducedfluorescence capillary electrophoresis (CE-LIF) and consisted of an antibody that was clipped in four differentplaces in the heavy chain as well as misfolded andaggregated antibody. The potency of the recombinantantibody containing a succinimidyl intermediate on zero,one, and two light chain arms was analyzed by LANCEbinding assay and a cell based in vitro bioassay, and theoccurrence of this modification on one or both light chainarms was associated with a reduction in the bindingaffinity of the molecule to the target by approximately 10%.We show that HIC has the unique ability as a first steppurification method to separate populations of antibodywhich are covalently modified under stability programs.The method conditions that have been developed for theHIC assay are ideal for purifying antibodies with labilemodifications for the purpose of further characterization.
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