Development of quality control methods of recombinant anti-EBOV antibodies / 中国药学杂志

ABSTRACT

OBJECTIVE: To establish quality control methods for recombinant anti-EBOV mAbs which are comprised by three anti-EBOV glycoprotein mAbs. METHODS: The binding-activity of recombinant humanized anti-EBOV mAbs to EBOV glycoprotein was evaluated by ELISA. Peptide map by LC-MS was used in the identification tests. Purity was analyzed by CE-SDS and SEC-HPLC. iCIEF was performed to measure the PI value and the charge heterogeneity. 2AB was labeled on the released glycan, and was analyzed by NP-HPLC and mass spectrometry. The concentration of polysorbate 20 was tested by HPLC. RESULTS: The EC50s of recombinant anti-EBOV mAbs were (12.53±1.62), (11.10±0.62) and (6.09±0.35) ng·mL-1, respectively. The theoretical sequence coverage rates of three mAbs were all above 95%. The main peak area percentages shown by non-reduced CE-SDS were (94.41±0.05)%, (95.58±0.17)% and (96.11±0.05)%. The peak area percentages of both heavy and light chain shown by reduced CE-SDS were (98.19±0.06)%, (97.97±0.03)% and (98.57±0.03)%. The main peak area percentages shown by SE-HPLC were (99.59±0.01)%, (99.56±0.01)% and (99.74±0.01)%. The isoelectric points of the main peak shown by iCIEF were (8.70±0.01),(8.26±0.01) and (8.85±0.01). The concentrations of polysorbate 20 were (0.34±0.00),(0.35±0.00) and (0.35±0.01) μg·mL-1, respectively. The glycan mapping analysis was relatively sensitive, and the percentage of fucosylated N linked glycan was less than 0.5%. CONCLUSION: Up-to-date quality control methods for recombinant anti-EBOV mAbs are established in this study, which may be used to ensure the safety, effectiveness and quality controllability of the product. The methods can provide technical assists to Ebola outbreak and be a reference of the quality control of other domestic cocktail monoclonal antibody products.