| Monoclonal antibody bioactivity detection project is a key index in antibody quality control,it is closely related to pharmacodynamics and provides guarantee for the effectiveness and quality control of products.KM1 13 is a biological analogue of adamumab developed by Beijing Combio Pharmaceutical Co.,Ltd.Establishing the biological activity(binding activity and in vitro cell biological activity)method of KM113 and enterprise quality internal control standard is of great significance to the clinical declaration of KM113 products.The biological activity of KM113(CKP-A20150704)was determined by indirect ELISA(enzyme-linked immunosorbent assay)and L929 cell killing inhibition.Through the establishment and optimization of ELISA method and killing inhibition method,and the verification of the specificity,repeatability,intermediate precision and accuracy of the above two methods,the detection method of the biological activity of KM113 antibody was determined.Three batches of KM113,three batches of commercially drugs and three batches of KM113 long-term stability samples were tested by two activity methods.According to the quality standards of monoclonal antibodies in Chinese pharmacopoeia,the quality standards of commercially available drugs and the determination results of the samples,the quality standards for the biological activities of enterprises were finally established.Firstly,KM113 has a significant dose-effect relationship in both this method,and the four-parameter equation R2 is greater than 0.95.In binding activity,the EC50 of KM113 in the same batch was measured for six times independently,and the average EC50 was(82.83±7.04)ng/ml and RSD was 8.50%,the relative titers of two different dilution groups were(83.74±1.09)%and(120.43± 4.04)%,respectively,and the corresponding recovery rates were(83.74± 1.09)%and(120.43± 4.04)%.The RSD values were(104.68± 1.36)%and(100.36 ± 3.37)%and both of them were less than 10%.In biological activity,the IC50 of KM113 body was determined three times in the same batch of original KM113 antibody standard drug,the average IC50 was(27.63±1.73)ng/ml and RSD was 6.29%,the average IC50 of two experimenters repeated three times was(27.57±0.12)ng/ml and(25.40 ± 1.59)ng/ml,respectively.The RSD was 0.42%and 6.25%,the relative titers of the two groups of samples were(85.83±3.63)%and(115.90±2.93)%.The corresponding recovery rates were(107.29±4.54)%and(96.58±2.44)%,RSD was 4.23%and 2.53%,and RSD wasn't more than 10%.Secondly,three parallel experiments were carried out on the commercial products and reference substances.The results showed that the average relative binding activity of commercial products was(96.67±7.64)%,RSD was(7.90)%,the average biological activity of commercial products was(101.00±7.21)%,RSD was:7.14%.And the average relative binding activity of three batches of KM1 13 and three batches of commercial products was(98.97±4.09)%and(104.63±7.02)%respectivety,and the RSD was 4.13%and 6.71%.The average biological activity was(98.33±2.08)%and(93.00± 2.65)%respectivety,and the RSD was 2.12 and 2.84%.According to the results and the quality standards of monoclonal antibody included in the Chinese Pharmacopoeia,the enterprise internal control standard was established,thich is the relative binding activity of the tested products should be 70-130%of the control products,and the biological activity of the tested products should be 70-130%of the control products.The method for the determination of the binding activity and the biological activity in vitro of KM113 has been established successfully.It has been optimized to verify its specificity,precision and accuracy.And it provides a practical method for the detection of biological activity of KM113.The quality standard of enterprise biological activity has been established successfully. |
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