Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. Further decreases in LHF separation were seen upon incubation with urea and arginine. The addition of ethylene glycol decreased LHF removal by half. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. The mechanism of fragment removal was investigated in two series of experiments. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing.
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