Investigation and elimination of noncovalent artificial aggregates during non-reduced capillary electrophoresis-sodium dodecyl sulfate analysis of a multi-specific antibody.

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is widely used in the biopharmaceutical industry for monitoring purity and analyzing impurities. The accuracy of the method may be compromised by artificial species resulting from sample preparation or electrophoresis separation due to suboptimal conditions. During non-reduced CE-SDS analysis of a multispecific antibody (msAb), named as multispecific antibody C (msAb-C), a cluster of unexpected peaks was observed after the main peak. The corrected peak area ratio of these peaks showed a strong dependence on loaded protein concentration, which affected the accurate assessment of the purity of msAb-C. After investigation, the unexpected peaks were identified as artifacts produced during electrophoresis separation. These artifacts can be mitigated by three different strategies: 1) adding a more hydrophobic surfactant, sodium hexadecyl sulfate (SHS), to the sample and/or sieving gel buffer; 2) reducing the sample loading amount; and 3) increasing the capillary separation temperature to above 40 ℃. We adopted strategy 1) and strategy 3), and successfully developed an optimal non-reduced CE-SDS method for the accurate and reliable purity assessment of msAb-C samples. These strategies of optimizing non-reduced CE-SDS can be used in developing quality control methods for other therapeutic bispecific/multispecific antibodies.