Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype.
Recombinant monoclonal antibodies of therapeutic interest were analyzed by a nonreduced CE-SDS (nrCE-SDS) method developed for the evaluation of size-based variants. We found that immunoglobulins analyzed by this technique exhibited different behavior depending on their subclasses. Under nrCE-SDS conditions, IgG1 molecules were separated in a well-resolved, single peak, whereas IgG2 molecules were consistently separated as a doublet. Investigation of these isoforms showed that they were structurally different, and that the difference was not caused by cell culture condition, glycosylation structure, or recombinant expression system. Commercially available IgG2 affinity-purified from human plasma also showed the presence of structural isoforms. The structural isoforms remained present under pH- and temperature-stressed conditions. Application of a mild cysteine/cystine redox potential converted the main peak doublet into a single peak, indicating that these isoforms were disulfide bond-related species. Bioactivity measured before and after application of a redox potential gave similar values, indicating that the structural isoforms have comparable potency. The nrCE-SDS technique described here demonstrated a unique capability to resolve IgGs, leading to the discovery of novel structural isoforms specific to the IgG2 isotype.
Guo
Amy
A
Department of Analytical and Formulation Sciences, Amgen Inc., Seattle, WA, USA. guoa@amgen.com
Han
Mei
M
Martinez
Theresa
T
Ketchem
Randal R
RR
Novick
Shawn
S
Jochheim
Claudia
C
Balland
Alain
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry; D020033:Protein Isoforms / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate