title: Monoclonal antibody fragment removal mediated by mixed mode resins.
abstract: Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. 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. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. 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. 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. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required.
authors.affiliation: Process Purification Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA. Electronic address: oconnore@medimmune.com.
authors.forename: Ellen
authors.initials: E
authors.lastname: O'Connor
authors.affiliation: Process Purification Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Aspelund
authors.affiliation: Process Purification Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Bartnik
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Berge
authors.affiliation: Analytical Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Coughlin
authors.affiliation: Process Purification Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Mutsa
authors.initials: M
authors.lastname: Kambarami
authors.affiliation: Analytical Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: David
authors.initials: D
authors.lastname: Spencer
authors.affiliation: Analytical Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Huiming
authors.initials: H
authors.lastname: Yan
authors.affiliation: Process Purification Sciences, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: William
authors.initials: W
authors.lastname: Wang
mesh_terms: D000085:Acetates / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification; D013388:Succinimides
chemical_list: D000085:Acetates; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; C465543:N-hydroxysulfosuccimide; D013388:Succinimides
keywords: Antibody; Fragment removal; Mixed mode resin