| Manufacturing of antibody-related drugs with the large-scale animal cell culture technology have become the mainstream of therapeutic biopharmaceutical industry.With the rapid development of large-scale animal cell culture technology and increasing requirements of drug safety and efficacy in recent years,quality control of therapeutic proteins have become the key issue with great concerns in the large-scale animal cell culture process.Recombinant type ? tumor necrosis factor receptor(TNFR-Fc)with a prosper market prospect,which has a significant curative effect on diseases such as rheumatoid arthritis.In our previously-established high productive bioprocess,the biological activity of TNFR-Fc varied from lot to lot.However,the relationship among the culture condition,protein structure and its biological activity is not clear so far,which greatly hindering the establishment of the efficacy,economic and quality-control bioprocess.Therefore,it is an indispensable step as well as a primary mission in this study to ensure the final product quality and enhance the biological activity of TNFR-Fc through a deeper understanding of the relationship of the Fc fusion protein structure.Firstly,two distinct types of TNFR-Fc protein,which have significantly different biological activities were observed by the hydrophobic interaction chromatography.The biological activity of isomer protein(peak B)is just 14%compared to the active protein(peak A).After the reduction and refolding of the peak B enriched protein with cysteine and other reductants,it was discovered that about 75%of Peak B protein was converted to active Peak A protein.The Peak A and Peak B proteins were then digested with trypsin under non-reducing condition,and the corresponding peptides were further analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry.Results showed that the Peak A and Peak B proteins had distinct connection in the 1338,2004 and 3308 Da molecular weight peptides,which identified the Peak B as a misfold protein caused by disulfide scramble.Comparing the biochemical structures of Peak B and active protein Peak A,it was discovered that both proteins had similar profile in terms of molecular size,charge variant,glycosylation pattern and primary structure.These results indicated that Peak B as a isoform protein induced by the disulfide bond scramble was the main reason to cause the decreasion of the biological activity of TNFR-Fc in the culture process.Secondly,it was determined through the cell-free experiment that the formation of the disulfide bone scramble of the isoform protein occurred during the protein synthesis stage.In addition,the peak B content increased in the time-course of culture process,and the final isoform protein content was 1.3 fold compared with the initial stage.The formation of disulfide bonds during protein synthesis process involved redox conditions,energy supply,enzyme catalysis and many other aspects.Therefore,a dynamic analysis based on cellular level was adopted to investigate the relationship between peak B formation and these three respects variants during the typical CHO fed-batch culture process.It was found that in the late culture stage,the percentage of G1 cell decreased by 15?17%,the intracellular ATP content decreased by 50?63%,and the GSSG/GSH ration decreased by about 30%,compared with the early stage.These results indicated that a high proportion of G1-phase cells composition,a more efficient energy supply and a stronger thiol oxidizing capacity could ensure the correct formation of disulfide bonds of TNFR-Fc in CHO cell.These results also provided a scientific guide to control the isoform protein formation in the large-scale culture process.Finally,in the bioreactor level,the effects of culture parameters such as dissolved oxygen(DO),pH and shift time on Peak B formation were investigated.Results showed that DO was the critical process attribution that influenced the Peak B formation,increasing the DO control level from 10%to 70%could decrease the misfold protein content by 25%.In the same DO level,controlling pH with 6.8 could decrease the Peak B content by 4?6%,early shifting the culture parameters could also decrease the isoform protein by 2.1?3.3%,providing a practical guide to control the Peak B formation in the large-scale culture process.Based on above research results,medium compositions related to cell cycle distribution and intracellular oxidative environment were optimized.Results showed that adding 3 mmol/L sodium butyrate and 3%dimethyl sulfoxide(DMSO)into culture medium could efficiently arrest the cell grow,making more cells entered into the G1 phase,therefore decreased the Peak B content by 6.4%and 8.7%respectively,effectively regulating the correct disulfide bonds formation of TNFR-Fc.This study revealed the reasons for the decreased biological activity of TNFR-Fc in the culture process,and deepened the understanding of the relationship among the protein biological activity,structure and culture process.Hence,this work provided a scientific guide to increase the biological activity of TNFR-Fc in the previous established fed-batch culture process,and also a new methodology for the other antibody protein drugs to establish high-yielding and quality-control culture process under the guidance of quality by design. |
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