Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions

The following report describes the use of hydrophobic-interaction chromatography (HIC) to separate and characterize populations of monoclonal antibodies resulting from variable N- and C-terminal processing, stressed-induced covalent modifications and conformationally altered populations present in t...

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Bibliographic Details
Published in:	Journal of Chromatography A 2008-12, Vol.1214 (1), p.81-89
Main Authors:	Valliere-Douglass, John, Wallace, Alison, Balland, Alain
Format:	Article
Language:	eng
Subjects:	Aggregation Amidation Analytical, structural and metabolic biochemistry Antibodies Antibodies, Monoclonal - chemistry Antibodies, Monoclonal - isolation & purification Biological and medical sciences Biotechnology Chromatography - methods Clipping Fundamental and applied biological sciences. Psychology Glycoproteins Hydrophobic and Hydrophilic Interactions Hydrophobic-interaction chromatography (HIC) Immunoglobulin Fab Fragments - chemistry Immunoglobulin Fab Fragments - isolation & purification Immunoglobulin Fc Fragments - chemistry Immunoglobulin Fc Fragments - isolation & purification Isomerism Isomerization Methods. Procedures. Technologies Others Oxidation Papain - metabolism Potency Protein Stability Proteins Succinimide Succinimides - metabolism Various methods and equipments
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Summary:	The following report describes the use of hydrophobic-interaction chromatography (HIC) to separate and characterize populations of monoclonal antibodies resulting from variable N- and C-terminal processing, stressed-induced covalent modifications and conformationally altered populations present in the drug product. We investigated the use of HIC to characterize heterogeneity in the intact molecule and the Fab and Fc sub-domains resulting from papain cleavage. We found that certain classes of covalent modifications to antibodies are highly amenable to HIC separation. Specific covalent modifications occurring on antibodies could be separated into pure fractions which contained unmodified, singly modified (on 1 heavy or light chain) and doubly modified (on both heavy or light chains) molecules. This report demonstrates the utility of HIC for assessing the heterogeneity, stability and, in some cases, potency of monoclonal antibodies.
ISSN:	0021-9673