title: Purification of antibody heteropolymers using hydrophobic interaction chromatography.
abstract: A heteropolymer (HP) is a unique dual antibody conjugate composed of specific, chemically cross-linked monoclonal antibodies (mAbs). In this study we have demonstrated that HPs can be purified using hydrophobic interaction chromatography (HIC). Two propyl HIC resins; [PolyPropyl A and EMD Fractogel Propyl (S)] were evaluated in this study. Phosphate buffers, pH 6.5 containing ammonium sulfate or sodium sulfate were used to bind the HP to the column. A descending sulfate gradient or step gradient was used to elute the bound HP species from the column. The HP reaction mixture typically contains multiple conjugated HP species, as well as unreacted monomer mAbs. Conjugated HP product was successfully separated from unreacted antibody monomers with both propyl resins using buffers with ammonium sulfate. There was no monomer separation from HP using buffers with sodium sulfate. The purification processes, presented in this study allows the non-cross-linked antibodies to pass through the column without being bound to the resin, while the cross-linked antibodies (the HP product) bound to the column were subsequently eluted by decreasing the ammonium sulfate concentration in the running buffer. HP product was efficiently separated from free mAbs using Propyl HIC resins at both analytical and preparative scales.
authors.affiliation: Elusys Therapeutics Inc., 25 Riverside Drive, Pine Brook, NJ 07058, USA. ikostareva@elusys.com
authors.forename: Irina
authors.initials: I
authors.lastname: Kostareva
authors.forename: Finn
authors.initials: F
authors.lastname: Hung
authors.forename: Colin
authors.initials: C
authors.lastname: Campbell
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D002021:Buffers; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D051379:Mice; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D002021:Buffers; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D011108:Polymers; D000645:Ammonium Sulfate