title: Elucidation of degradants in acidic peak of cation exchange chromatography in an IgG1 monoclonal antibody formed on long-term storage in a liquid formulation.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Formulation and Analytical Resources Process and Product Development, Amgen Inc., Thousand Oaks, California 91320, USA.
authors.forename: Sejal
authors.initials: S
authors.lastname: Gandhi
authors.forename: Da
authors.initials: D
authors.lastname: Ren
authors.forename: Gang
authors.initials: G
authors.lastname: Xiao
authors.forename: Pavel
authors.initials: P
authors.lastname: Bondarenko
authors.forename: Christopher
authors.initials: C
authors.lastname: Sloey
authors.forename: Margaret Speed
authors.initials: MS
authors.lastname: Ricci
authors.forename: Sampathkumar
authors.initials: S
authors.lastname: Krishnan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D016466:CHO Cells; D002626:Chemistry, Pharmaceutical; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D022801:Complementarity Determining Regions / Q000737:chemistry; D006224:Cricetinae; D004355:Drug Stability; D004356:Drug Storage; D005973:Glutamine / Q000737:chemistry; D006031:Glycosylation; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry*; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D010449:Peptide Mapping; D011961:Receptors, Fc / Q000737:chemistry*; D013696:Temperature; D014357:Trypsin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; D005973:Glutamine; D001224:Aspartic Acid; D001216:Asparagine; D014357:Trypsin; C100688:Fc receptor, neonatal