A Preliminary Study On Critical Quality Attributes Of Recombiant Human Serum Albumin

Human serum albumin(HSA)is a dominant protein in human plasma.Human serum albumin product is a human plasma derived therapeutic drug.Currently,the plasma derived human serum albumin is short of supply because of the shortage of plasma raw material.The advent of recombinant human serum albumin(rHSA)may mitigate the status of human serum albumin's scarcity.Host cell proteins(HCPs)which co-purified with recombinant proteins pose a great challenge to recombinant therapeutic proteins for their potential to cause seriously adverse reactions.Host cells may also bring in non-humanized post-translational modification(PTM),all these structural alter may cause drug safety issues.A single dose of human serum albumin exceeds 10 grams,which far beyong dose of ususal recombinant therapeutic proteins,the high dose of recombinant human serum albumin requires a higher quality control standard.Here is a preliminary study on the detection of host cell proteins which co-purified with recombinant human serum albumin,and we also characterised the structure of different derived human serum albumin.The first two parts of this article are studies on the detection of host cell proteins in recombinant human serum albumin.In the third part of this article,we characterize the structure of recombinant human serum albumin to understand the difference between different derived recombinant human serum albumin.The first part.Object:To figure out whether there was any difference between the component of standard in the procedure specific HCPs ELISA kit which produced by imitating the manufacture procedure of the recombinant human serum albumin and the host cell proteins produced by host cell during the real production of the protein.Methods:We collected standard of the procedure specific HCPs ELISA kit which produced from supernatant of null cell fermentation after the first step of chromatography and recombinant human serum albumin intermediate which also purified after the first step of chromatography.This two sample were all provided by manufacture A.A nanoLC-MS/MS system was applied to identify the standard from the procedure specific HCPs ELISA kit provided by manufacture A.We used the 2DLC-MS/MS system to detect host cell proteins in the intermediate recombinant human serum albumin.Results:We identified 97 kinds of protein in the standard of the procedure specific HCPs ELISA kit,and 32 kinds of host cell proteins in the recombinant human serum albumin intermediate.Note that 8 kinds of protein identified in the intermediate production didn't exsist in the procedure specific HCPs ELISA kit.Conclusion:There was difference between the component of standard in the procedure specific HCPs ELISA kit and host cell proteins in the intermediate production.The second part.Object:To identify host cell proteins in the bulk of the recombinant human serum albumin.Methods:We collected bulk of the recombinant human serum albumin from three different manufactures,and used a 2DLC-MS/MS technology to identify host cell proteins in these samples.Results:In the two batchs of rHSA samples provided by manufacture A,we identified 24 and 23 kinds of HCPs respectively,18 and 10 kinds of HCPs were identified in the sample provided by manufacture B and C respectively.Conclusion:Analysis all the host cell proteins identified in these four samples,we found there was a protein named C4R8K6 exist in all these four samples,this protein can act as marker to develop a universal HCPs ELISA kit for Pichia pastoris derived rHSA.The third part.Object:To understand the structural changes caused by post-translational modification,we characterize the structure of different human serum albumin.Methods:A top-down strategy was applied to characterize the structure of rHSA.We used a LC-qTOF system to characterize four manufactures' HSA in intact molecular weight level and peptide map level.Results:We found sample A and sample B which derived from pichia pastoris were highly glycosylated in peptide K46,and sample A was higher than B in glycosylation level;Sample D derived from Saccharomyces cerevisiae had the lowest glycosylation level;In site M329,the four samples got different level of oxidation,the oxidation level of sample B was far ahead of other samples;sample B got the highest cysteinylation level,followed by sample E.There was glycation in sample E.The deamidation and formylation level were very low in these four samples.Conclusion:Different host cell post-traslational modification caused changes to the structure of human serum albumin,we must take care the alter of immunogenicity and physical and chemical properties of human serum albumin these changes caused to.