Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold.
Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested.
Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.
Alice R
AR
Mazzer
UCB Celltech, 216 Bath Road, Slough SL13WE, United Kingdom.
Xavier
X
Perraud
UCB Celltech, 216 Bath Road, Slough SL13WE, United Kingdom.
Jennifer
J
Halley
UCB Celltech, 216 Bath Road, Slough SL13WE, United Kingdom.
John
J
O'Hara
Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom. Electronic address: d.bracewell@ucl.ac.uk.
Daniel G
DG
Bracewell
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002021:Buffers; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D066329:Protein Aggregates; D013205:Staphylococcal Protein A / Q000032:analysis*; D038301:Virus Inactivation
D000911:Antibodies, Monoclonal; D002021:Buffers; D007074:Immunoglobulin G; D066329:Protein Aggregates; D013205:Staphylococcal Protein A
Affinity adsorption; Aggregation; IgG; Size exclusion chromatography; Unfolding pH