Determination of the origin of charge heterogeneity in a murine monoclonal antibody.
OBJECTIVE
The aim of this study was to elucidate the molecular basis of charge heterogeneity found in a purified monoclonal IgG1 antibody, MMA383.


METHODS
Cation exchange chromatography (CEX) and isoelectric focusing (IEF) were used to monitor charge heterogeneity. CEX in conjunction with carboxypeptidase B digests of the antibody was used to determine the contribution of C-terminal lysines to MMA383 charge heterogeneity. Potential chemical degradation sites were identified by peptide mapping of individual chains, with peptide identification by mass spectrometry (MALDI-TOF MS). Peptide sequencing was used to determine specific deamidation sites. Binding constants of predominant isoforms were compared by surface plasmon resonance (SPR).


RESULTS
Extensive charge heterogeneity of purified MMA383 was detected by CEX and IEF. Removal of C-terminal lysines simplified the IEF pattern to nine predominant isoforms. Quantitation of isoaspartate in each of the isoforms indicated deamidation of MMA383 as a major cause of charge heterogeneity. CEX of the individual isoform chains suggested the presence of one deamidation site on each of the heavy and light chains. The two sites of deamidation were identified using peptide mapping, sequencing and mass spectrometry. SPR results showed no significant difference in the binding parameters among the isoforms.


CONCLUSIONS
C-terminal lysine microheterogeneity and deamidation of Asn141 in the heavy chain and Asn161 in the light chain are the major causes of MMA383 charge heterogeneity. Identification of the two deamidation sites will allow replacement of these amino acids in order to create a product less susceptible to degradation.
Perkins
M
M
Department of Pharmaceutical Research, The University of Kansas, Lawrence, USA.
Theiler
R
R
Lunte
S
S
Jeschke
M
M
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003641:Deamination; D007074:Immunoglobulin G / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D020033:Protein Isoforms; D008239:Lysine