A high throughput dimer screening assay for monoclonal antibodies using chemical cross-linking and microchip electrophoresis.
A high throughput screening assay was developed to determine the total dimer level in antibody samples. This method utilizes high speed microchip electrophoresis separation following chemical cross-linking. Upon reacting with homobifunctional N-hydroxysuccinimide-esters (NHS-esters), covalent linkages can be established between the primary amines of two neighboring antibody molecules. The reaction conditions are optimized to achieve quantitative cross-linking of only physically associated monomers within an antibody dimer. The resulting cross-linked dimers, originating from either covalent or non-covalent antibody dimers, can then be separated from monomers by SDS electrophoresis. A commercial microchip electrophoresis instrument is used for high speed separation, allowing each sample to be analyzed in about 1min. This approach was applied to crude mammalian cell culture samples. Using a 96-well gel filtration spin column format, interfering species in the cell culture media were efficiently removed from the samples. This method is well suited to the purpose of high throughput antibody dimer quantitation during cell culture expression, including clone selection and cell culture development. The total dimer content, both covalent and non-covalent, can be determined for hundreds of crude samples in a few hours. The effects of different cross-linking conditions on the determined dimer levels, as well as of different antibody pI values, are discussed.
Chen
Xiaoyu
X
Analytical and Formulation Sciences, Amgen Inc., Thousand Oaks, CA 91320, USA.
Flynn
Gregory C
GC
D000911:Antibodies, Monoclonal / Q000032:analysis*; D002850:Chromatography, Gel / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D046209:Electrophoresis, Microchip / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis; D007526:Isoelectric Point; D016014:Linear Models; D010316:Particle Size; D055503:Protein Multimerization; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G