Development of methods for quality control of humanized monoclonal antibody agaist vascular endothelial growth factor / 中国药学杂志

ABSTRACT

OBJECTIVE: To develop methods for quality control of humanized monoclonal antiboy (McAb) against vascular endothelial growth factor (VEGF). METHODS: The purity of humanized anti-VEGF McAb was determined by reduced and non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS), while the contents of monomer and polymer were determined by size-exclusive high performance liqud chromatography (SE-HPLC) at the concentration of 0.5 and 25.0 mg·mL-1, respectively. Isoelectric point was determined by isoelectric focusing capillary electrophoresis (cIEF), and the content of charge isomers was controled by cation exchange HPLC(CEX-HPLC). For identity, Lys-C digested peptide map was analyzed by RP-HPLC and LC-MS. The biological potency of anti-VEGF McAb was determined with a human umbilical vein endothelial cells (HUVEC) proliferation inhibitory test. And other quality indexes required by the Ch.P and other relevant requirements were also investigated. RESULTS: The purities of drug substance (DS) and products (DP) determined by reduced CE-SDS were (97.77±0.25)% and (97.43±0.57)%, respectively. The purities of DS and DP determined by non-reduced CE-SDS were (98.97±0.15)% and (98.73±0.06)%, respectively. In the SE-HPLC analysis, when the concentration of McAb was 0.5 mg·mL-1, the monomer contents of DS and DP were (98.07±0.55)% and (98.20±0.52)%, while the contents of polymer in DS and DP were (2.00±0.53)% and (1.93±0.55)% at the concentration of 25.0 mg·mL-1. In the CEX-HPLC analysis, the contents of the acidic, main, and basic components of DS and DP were (18.33±0.64)% and (18.60±0.44)%, (69.03±0.80)% and (69.20±0.44)%, (12.70±1.37)% and (12.20±0.87)%, respectively. In the cIEF and LC-MS tests, the DS and DP showed consistency in isoelectric point and peptide map with reference materials. The other quality indexes all met the requirements in Ch.P and other relevant requirements. CONCLUSION: The methods for quality control of McAb against VEGF is successfully developed, which can be used for the routine quality control of the product.