The Application Of Monocyte Activation Test In The Pyrogen Detection Of Monoclonal Antibody

To evaluate the safety of monoclonal antibody drugs, a novel monocyte activation test(MAT) was established, and the application of MAT in the pyrogen detection of monoclonal antibody drugs was studied, The following was the work of this subject:Firstly, a novel MAT was established. To find a sensitive cell line and corresponding cytokines, we selected the monocytes that were sensitive to pyrogen referring to the European Pharmacopoeia(EP), British Pharmacopeia(BP) and related documents. The important parameters such as cell density, pyrogen incubation time,fetal bovine serum(FBS) concentration were also studied.(1) Five kinds of human monocytic cell lines:SJ1 cell, THP-1 cell, WLIS-2 cell, Raji cell, and K562 cell were assessed. The cytokines interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) were detected after these five kinds of cell lines were stimulated by endotoxin( also known as lipoteichoic acid, LPS), zymosan, and lipoteichoic acid(LTA). The results showed that among the five cell lines, the SJ1 cell line was sensitive to pyrogen and could produce high IL-6 levels in response to traces of pyrogen. Therefore, SJ1/IL-6 method may be a new MAT.(2)Cell density was explored on the assay, and the results showed that the secretion level of IL-6increased with the increasement of cell density when cell density was in the range of 1×105~1×107cells/ml. As a result, we selected 5×106cells/m L as the seeding cell density in the assay;(3)Pyrogen incubation time on the assay was explored. The results showed that the IL-6 amount reached the highest level when SJ1 cells were stimulated by those pyrogens for about 48 h. Therefore, 48 h was chosed as the time paremeter for the assay.(4)FBS concentration on the assay was explored. The results showed that after SJ1 cells were stimulated by LPS, zymosan and LTA, the secretion level of IL-6reduced with the increasement of FBS concentration when FBS concentration was in the range of 0.5%~20%. The cell medium containing 2% FBS was chosed for the further experiment. Through the above study, SJ1/IL-6 monocyte activationexperiment was established as a complement to the pyrogen testing experiment.Secondly, SJ1 cell was identified according to the EP and Chinese Pharmacopoeia(Ch P), including identification of cell morphology, sterility test,mycoplasma test, exogenous virus contamination test, and stability of cellular function test on SJ1 cell. The morphology of SJ1 cells observed in our lab under microscope was the same with that described by American type culture collection(ATCC). The sterility test, mycoplasma test, and exogenous virus contamination test were checked in accordance with the relevant provisions in Ch P2015. The results were all negative. The stability of cellular function test was explored with the methods of the cellular growth curve and pyrogen sensitivity of different generations. The results showed that each generation of SJ1 cell growth curve was basically identical. The SJ1 cell line was the most sensitive to the pyrogen when the cell line was passaged to 10 th generation, and the sensitivity of cells to the pyrogen declined gradually when the cell line was passaged to 20 th generation.Thirdly, the accuracy, repeatability, limit of detection(LOD) and linear of the SJ1/IL-6 assay was validated. The recoveries of LPS, zysoman and LTA tested in influenza vaccine(Split Virion, Inactivated, freeze-dried.), Rabies Vaccine(Vero Cell,for Human Use, Freeze-dried.), Measles and Rubella Combined Vaccine(live, for Human Use, Freeze-dried.), Measles and Mumps Combined Vaccine(live) were all in the range of 50%~200%, indicating that the accuracy of this method was good. The endotoxin concentration of Group A and C Meningococcal Polysaccharide Vaccine were detected using HL-60/IL-6 assay, the RSD was less than 20%(n=6), so the results showed that the repeatability of this method was good. SJ1 cell line was stimulated by a series of LPS solution, zysoman solution and LTA solution. The concentration of LPS, zysoman and LPS was in good linear relationship with the secretion of IL-6 level.The LOD of LPS, zysoman and LPS was 0.06EU/m L,0.06μg/m L and 0.06μg/m L,respectively.Fourthly, the HL-60/IL-6 Assay and Bacterial Endotoxins test(BET)Assay was compared. the endotoxins of 10 batches of Group A and C Meningococcal Polysaccharide Vaccine from two factories were detected by SJ1/IL-6 assay and turbidity method in Ch P2015, respectively. The results of t-test showed that theendotoxins of 10 batches of Group A and C Meningococcal Polysaccharide Vaccine tested by these two methods were basically identical.Fifthly, the application of SJ1/IL-6 assay was inspected in the pyrogen test of adalimumab injection. The application of this method was studied in the pyrogen test of adalimumab injection.(1) We tested the recoveries of three pyrogens in adalimumab diluted solution that was diluted by 2 times、10 times、20 times、50 times、100 times. The results showed that the recoveries of LPS、zysoman、LTA were all in the range of 50%-200% when adalimumab injection diluted to more than 50 times.Therefore, the diluted adalimumab solution that was diluted 50 times was suitable for the pyrogen test using SJ1/IL-6 assay.(2) The recoveries of LPS、zysoman、LTA with high, medium, and low concentration were tested in adalimumab injection. The results showed that the recoveries of LPS、zysoman、LTA with 3 kinds of concentration in adalimumab injection were all in the range of 50%-200%. These illustrated that the accuracy of SJ1/IL-6 assay applied in the pyrogen test of adalimumab injection is good.(3) The SJ1 cells was stimulated by LPS( the final density of LPS was 0.25EU/m L)which was added into the adalimumab injection without endotoxin, and measured the amount of secretion of IL-6. The results showed that the RSD was less than 20%(n=6).These illustrated that the repeatability of this method applied in the pyrogen test of adalimumab injection was good.(4) The pyrogen contents of 5 batches of adalimumab injection were tested, the results were all negative.Sixthly, the application of SJ1/IL-6 assay was inspected in the pyrogen test of other monoclonal antibodies. We tested the pyrogen contents of rh TNFR : Fc 、Nimotuzumab 、 CD25, Ranibizumab injection,and Bevacizumab injection using SJ1/IL-6 assay, and the recoveries of LPS 、 zysoman 、 LTA in the five kinds of monoclonal antibodies. The results showed that the recoveries of LPS、zysoman、LTA in the five kinds of monoclonal antibodies were all in the range of 50%-200%. And the results of pyrogen contents were all negative.