Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation
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An IgG1 therapeutic monoclonal antibody showed an increase in acidic or pre-peak by cation exchange chromatography (CEX) at elevated temperatures, though stable at 2–8°C long-term storage in a liquid formulation. Characterization effort was undertaken to elucidate the degradants in CEX pre-peak and effect on biological activity.
Purified CEX fractions were collected and analyzed by peptide mapping, size exclusion, intact and reduced-alkylated reversed phase techniques. Biophysical characterization, isoelectric focusing and Isoquant analysis were also performed to determine nature of degradants. Bioassay and surface plasmon resonance experiments were performed to determine the impact on biological activity of the degradants.
No major degradation due to oxidation, clipping or aggregation was detected; conformational differences between purified fractions observed were not significant. Sialic acid, N-terminal glutamine cyclization and glycation differences contributed to the CEX pre-peak in the mAb control sample; increase in CEX pre-peak at 25°C and higher was caused by additive degradation pathways of deamidation, related isomerization and clipping.
The observed CEX pre-peak increase was caused by multiple degradations, especially deamidation and clipping. This elucidation of degradants in CEX peaks may apply to other therapeutic IgG1 monoclonal antibodies.
KEY WORDSacidic-peak cation exchange chromatography deamidation IgG1monoclonal antibody protein formulation
4,4′-dianilino-1,1′-binapthyl-5,5′-disulfonic acid dipotassium salt
cation exchange chromatography
capillary isoelectric focusing
neonatal Fc receptor
Fourier transform infrared spectroscopy
hydrophobic interaction chromatography
high performance liquid chromatography
immunoglobulin gamma 1
protein L-isoaspartyl methyltransferase
size exclusion chromatography
surface plasmon resonance
tangential flow filtration
ACKNOWLEDGMENTS & DISCLOSURES
Our sincere thanks to Scott Smallwood, Jeffrey Reichert, Himanshu Gadgil, Gary Pipes, Ramil Latypov, David Hambly, Jaymi Lee, Lynn Peabody, Renuka Thirumangalathu, Thomas Dillon and process team members all within Amgen for their technical support and discussion.
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