重组人源抗狂犬病病毒单克隆抗体的质控方法及质量标准的建立

目的建立重组人源抗狂犬病病毒单克隆抗体(rhRMcAb)的质控方法和质量标准。方法采用小鼠中和试验(MNT)和快速荧光灶抑制试验(RFFIT)分别测定6批rhRMcAb样品的中和活性;还原、非还原SDS-PAGE检测rhRMcAb样品的纯度;还原烷基化胰蛋白酶酶切和反相高效液相色谱法分析rhRMcAb的肽图;焦谷氨酸肽酶去除N-端焦谷氨酸封闭后,氨基酸序列分析仪测定rhRMcAb的N-端氨基酸序列;表面等离子共振法(Surface plasmon resonance,SPR)测定rhRMcAb与狂犬病病毒糖蛋白的亲和常数;按《中国药典》三部(2005版)要求检测rhRMcAb的各项其他指标,并建立其质量标准。结果采用MNT和RFFIT2种方法检测6批rhRMcAb的中和活性,批间活性基本一致;3批rhRMcAb原液的还原SDS-PAGE纯度大于99.0%,非还原SDS-PAGE除抗体主带外,在高、低相对分子质量处分别存在一些次带;3批原液样品的肽图图谱与理化测定对照品一致;N-端氨基酸序列与理论序列一致;结合狂犬病病毒糖蛋白抗原的亲和常数为1.86×108/M;其他各项指标均符合《中国药典》三部(2005版)要求;建立的质量标准中,中和活性应不低于500IU/mg,SDS-PAGE及HPLC纯度应不低于98.0%。结论已建立了rhRMcAb的质控方法和质量标准,可用于rhRMcAb产品的检定。

Establishment of Method and Standard for Quality Control of Recombinant Human Monoclonal Antibody against Rabies

Objective To establish the method and standard for quality control of recombinant human monoclonal antibody against rabies (rhRMcAb). Methods The rhRMcAb was analyzed for neutralizing activity by mouse neutralization test(MNT)and rapid fluorescent focus inhibition test(RFFIT), for purity by reduced and non-reduced SDS-PAGE and for peptide map by reduction alkylation and trypsin digestion followed by reverse phase HPLC. The amino acids at N-terminus was sequenced by amino acid sequencer after the pyroglutamate blockage at N-terminus was removed by pyroglutamate aminopeptidase. The affinity constant of rhRM-cAb with rabies virus glycoprotein was determined by surface plasmon resonance(SPR)method. Other quality indexes of rhRMcAb were determined according to the requirements in Chinese Pharmacopeia (2005 edition), based on which a standard for quality control was established. Results The activities of six batches of rhRMcAb determined by MNT and RFFIT were basically in agreement. The reduced SDS-PAGE purities of three batches of rhRMcAb bulks were more than 99. 0%. Except major antibody bands, some minor bands with both high and low relative molecular masses were also observed on the non-reduced SDS-PAGE profile of rhRMcAb. The peptide maps of three batches of bulks were consistent with that of in-house reference. The amino acid sequence at N-terminus was identical to the theoretical value. The affinity constant of rhRMcAb with rabies virus glycoprotein was 1. 86 × 108 / M. All the other quality indexes of rhRMcAb met the requirements in Chinese Pharmacopeia(2005 edition). In the established standard for quality control, the neutralizing activity of rhRMcAb was not less than 500 IU / ml, while both SDS-PAGE and HPLC purities were not less than 98. 0%. Conclusion The method and standard for quality control of rhRMcAb were established.