基因工程药物宿主细胞蛋白的研究进展

根据宿主细胞蛋白（HCPs）的来源、影响因素等背景，综合近年来国内外在HCPs检测方面所取得的进展，重点介绍新型质谱技术在HCPs检测中的应用。HCPs的分析方法有免疫分析法（例如Western Blot和ELISA）或其他方法（例如电泳和质谱），目前最常用的检测方法是ELISA法，但ELISA法存在很多问题与不足。新型检测技术，如邻位连接分析法（PLA）、生物传感器技术等也被用于HCPs检测，但这些方法难以推广使用。HCPs是产品质量控制的关键属性之一，其细胞丰度检测是药物放行并进行临床研究的重要参数。本文详细比较说明了各种HCPs检测方法的优缺点，基于质谱的蛋白质组学技术对HCPs进行定性与定量分析，目前已取得了较好的结果，弥补了ELISA方法的不足。

According to the background of host cell proteins (HCPs) and its influencing factors,recent progress in the detection of HCPs at home and abroad is summarized,and the application of new mass spectrometry in HCPs detection is highlighted. HCPs can be analyzed by immunoassay (such as Western Blot and ELISA) or other methods (such as electrophoresis and mass spectrometry). The most commonly used detection method is ELISA, but there are many problems and deficiencies in ELISA. New detection technologies,such as proximity ligation assay(PLA)and biosensor technology,are also used for HCPs detection,but these methods are difficult to promote. HCPs is one of the key attributes of product quality control. Cell abundance detection of HCPs is an important parameter for drug release and clinical research. In this paper,the advantages and disadvantages of various HCPs detection methods are described in detail. The qualitative and quantitative analysis of HCPs based on mass spectrometry proteomics has achieved good results and made up for the deficiencies of ELISA methods..

[1] 王志明.基因工程药物中宿主细胞蛋白的检测与控制[J].中国新药杂志,2016,25(22):2550 WANG ZM.Detection and control of host cell proteins in genetic engineering drugs[J].Chin J New Drugs,2016,25(22):2550
[2] VALENTE KN,SCHAEFER AK,KEMPTON HR,et al.Recovery of Chinese hamster ovary host cell proteins for proteomic analysis[J].Biochem J,2014,9(1):87
[3] TSCHELIESSNIG AL,KONRATH J,BATES R,et al.Host cell protein analysis in therapeutic protein bioprocessing-methods and applications[J].Biochem J,2013,8(6):655
[4] GAO SX,ZHANG Y,STANSBERRY PK,et al.Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity[J].Biotechnol Bioeng,2011,108(4):977
[5] CLARK KJ,CHAPLIN FW,HARCUM SW.Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA[J].Biotechnol Prog,2004,20(6):1888
[6] TAIT AS,HOGWOOD CE,SMALES CM,et al.Host cell protein dynamics in the supernatant of a mAb producing CHO cell line[J].Bioprocess Eng,2012,109(4):971
[7] HOGWOOD CE,BRACEWELL DG,SMALES CM.Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses[J].Curr Opin Biotechnol,2014,30:153
[8] HOGWOOD CE,TAIT AS,KOLOTEVA LN,et al.The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process[J].Biotechnol Bioeng,2013,110(1):240
[9] REISINGER V,TOLL H,MAYER RE,et al.A mass spectrometry-based approach to host cell protein identification and its application in a comparability exercise[J].Anal Biochem,2014,463:1
[10] WANG X,HUNTER AK,MOZIER NM.Host cell proteins in biologics development:identification,quantitation and risk assessment[J].Biotechnol Bioeng,2009,103(3):446
[11] KRAWITZ DC,FORREST W,MORENO GT,et al.Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities[J].Proteomics,2006,6(1):94
[12] REY G,WENDERLER MW.Full automation and validation of a flexible ELISA platform for host cell protein and protein A impurity detection in biopharmaceuticals[J].J Pharm Biomed Anal,2012,70:580
[13] ZHU SJ,YU C,NISHIHARA J,et al.Host cell protein testing by ELISAs and the use of orthogonal methods[J].Biotechnol Bioeng,2014,111(12):2367
[14] HOGWOOD CEM,CHIVERTON LM,MARK SC.Characterization of host cell proteins (HCPs) in CHO cell bioprocesses[J].Methods Mol Biol,2017,1603:243
[15] XU D,MANE S,SOSIC Z.Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot[J].Electrophoresis,2015,36(2):363
[16] PEZZINI J,JOUCLA G,GANTIER R,et al.Antibody capture by mixed-mode chromatography:a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins[J].J Chromatogr A,2011,1218(45):8197
[17] DONEANU CE,CHEN W.Analysis of host-cell proteins in biotherapeutic proteins by LC/MS approaches[J].Methods Mol Biol,2014,1129:341
[18] DONEANU CE,XENOPOULOS A,FADGEN K,et al.Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry[J].MAbs,2012,4(1):24
[19] AHLUWALIA D,DHILLON H,SLANEY T,et al.Identification of a host cell protein impurity in therapeutic protein,P1[J].J Pharm Biomed Anal,2017,141:32
[20] LIU N,BREVNOV.Host cellular protein quantification:using an automated solid-phase proximity ligation assay[J].BioProcess Int,2012,10:44
[21] MATTIASSON B,TEEPARUKSAPUN K,HEDSTROM M.Immunochemical binding assays for detection and quantification of trace impurities in biotechnological production[J].Trends Biotechnol,2010,28(1):20
[22] TEEPARUKSAPUN K,HEDSTROM M.Capacitive immunosensor for the detection of host cell proteins[J].J Biotechnol,2012,157(1):207
[23] VALENTE KN,LEVY NE,LEE KH,et al.Applications of proteomic methods for CHO host cell protein characterization in biopharmaceutical manufacturing[J].Curr Opin Biotechnol,2018,53:144
[24] KUMAR A,BAYCIN HD,WOLOZNY D,et al.Elucidation of the CHO super-ome (CHO-SO) by proteoinformatics[J].J Proteome Res,2015,14(11):4687
[25] HEISSEL S,BUNKENBORG J,KRISTIANSEN MP,et al.Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins:a case study of a bacterially expressed recombinant biopharmaceutical protein[J].Protein Expr Purif,2018,147:69
[26] KREIMER S,GAO Y,RAY S,et al.Host cell protein profiling by targeted and untargeted analysis of data independent acquisition mass spectrometry data with parallel reaction monitoring verification[J].Anal Chem,2017,89(10):5294
[27] 贾伟,DONEANU C,FADGEN K,et al.液质联用法鉴定、定量蛋白药物中宿主细胞蛋白[C]//2012年中国药学大会暨第十二届中国药师周论文集.2012:793 JIA W,DONEANU C,FADGEN K,et al.LC/MS identification and quantification of host cell proteins in protein drugs[C]//2012 China Pharmaceutical Conference and the 12th China Pharmacist Week Collection.2012:793
[28] XU X,NAGARAJAN H,LEWIS NE,et al.The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line[J].Nat Biotechnol,2011,29(8):735
[29] MADSEN JA,FARUTIN V,CARBEAU T,et al.Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions,LC-MS/MS,and multivariate analysis[J].MAbs,2015,7(6):1128
[30] ROST HL,ROSENBERGER G,NAVARRO P,et al.Open SWATH enables automated,targeted analysis of data-independent acquisition MS data[J].Nat Biotechnol,2014,32(3):219
[31] COLANGELO CM,CHUNG L,BRUCE C,et al.Review of software tools for design and analysis of large scale MRM proteomic datasets[J].Methods,2013,61(3):287
[32] EGERTSON JD,MACLEAN B,JOHNSON R,et al.Multiplexed peptide analysis using data-independent acquisition and Skyline[J].Nat Protoc,2015,10(6):887
[33] COLLINS BC,HUNTER CL,LIU Y,et al.Multi-laboratory assessment of reproducibility,qualitative and quantitative performance of SWATH-mass spectrometry[J].Nat Commun,2017,8(1):291
[34] ZHANG Q,GOETZE AM,CUI H,et al.Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry[J].MAbs,2014,6(3):659
[35] PETERSONE AC,RUSSELL JD,BAILEY DJ,et al.Parallel reaction monitoring for high resolution and high mass accuracy quantitative,targeted proteomics[J].MCP,2012,11(11):1475
[36] KREIMER S,GAO Y,RAY S,et al.Host cell protein profiling by targeted and untargeted analysis of data independent acquisition mass spectrometry data with parallel reaction monitoring verification[J].Anal Chem,2017,89(10):5294
[37] OW SY,CARDONA T,TATON A,et al.Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp.PCC 7120 using 8-plex isobaric peptide tags[J].J Proteome Res,2008,7(4):1615
[38] CHIVERTON LM,EVANS C,PANDHAL J,et al.Quantitative definition and monitoring of the host cell protein proteome using iTRAQ-a study of an industrial mAb producing CHO-S cell line[J].Biotechnol J,2016,11(8):1014
[39] BEE JS,TIE L,JOHNSON D,et al.Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product[J].Biotechnol Prog,2015,31(5):1360
[40] ROBERT F,BIERAU H,ROSSI M,et al.Degradation of an Fc-fusion recombinant protein by host cell proteases:identification of a CHO cathepsin D protease[J].Biotechnol Bioeng,2009,104(6):1132
[41] KAO YH,HEWITT DP,TREXLER SM,et al.Mechanism of antibody reduction in cell culture production processes[J].Biotechnol Bioeng,2010,107(4):622
[42] ZHANG Z,ALBANETTI T,LINKOUS T,et al.Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry[J].Electrophoresis,2017,38(3-4):401
[43] DONEAEANU CE,ANDERSON M,WILLIAMS BJ,et al.Enhanced detection of low-abundance host cell protein impurities in high-purity monoclonal antibodies down to 1 ppm using ion mobility mass spectrometry coupled with multidimensional liquid chromatography[J].Anal Chem,2015,87(20):10283
[44] SCHENAUER MR,FLYNN GC,GOETZE AM.Identification and quantification of host cell protein impurities in biotherapeutics using mass spectrometry[J].Anal Biochem,2012,428(2):150