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常见问题解答

Q:什么是宿主细胞蛋白质?
A:Host Cell Proteins are proteins and their modified forms derived from the hosts of biologics expression and co-exist during the biologics purification process.
宿主细胞蛋白质来源于生物纯化过程中宿主细胞产生的蛋白质及其蛋白质衍生物。
 
Q:宿主细胞蛋白质水平怎样表达?
A:HCPs are typically expressed as ng/mg or parts per million (ppm).
通常表示为毫微克/毫克或百万分之几(ppm)。
 
Q:宿主细胞蛋白质常用的分析技术有哪些?
A:Common technologies used for HCP analysis include ELISA (Enzyme-linked Immunosorbent Assay), one-dimensional or two-dimensional gel electrophoresis, Western Blot and Mass Spectrometry.
常见的用于宿主细胞蛋白的分析的技术包括酶联免疫吸附法(酶联免疫吸附试验),一维或二维凝胶电泳,免疫印迹和质谱分析。
 
Q:美国和欧州的药监部门对宿主细胞蛋白质的检测有哪些指导性文件?
A:ICH Q6B section 2.3.1; 4.1.3 and 6.2.1 have detailed descriptions on impurity analysis including HCP analysis.
人用药品注册技术国际协调会Q6B第2.3.1,4.1.3和6.2.1有杂质分析的详细描述,包括宿主细胞蛋白质的分析。
 
Q:宿主细胞蛋白质的检测方法是如何建立的?
A:HCP Cmethods are developed based on the performance of the anti-HCP antibodies including antibody selectivity, sensitivity.  Many formats have been developed for HCP analysis, the most commonly used format is ELISA.
宿主细胞蛋白质的检测方法是以抗宿主蛋白质的抗体,包括抗体的选择,灵敏度为基础。 宿主细胞蛋白质的分析已开发出多种形式,最常用的形式是酶联免疫吸附试验。
 
Q:宿主细胞蛋白质的检测方法是如何确证的?
A:HCP method validation especially the ELISA-based method validation is the result of extensive study of the anti-HCP reagent capability and their performance in a specific biologics product, therefore method validation is product-specific.  ICH Q2 is the most cited guideline for analytical method validation.
宿主细胞蛋白质的检测特别是ELISA方法验证是广泛的研究抗宿主细胞蛋白质抗体的能力和他们在一个特定的生物技术产品中的表现,因此方法的确证是具有产品专一性的。人用药品注册技术国际协调会中的Q2是被引用次数最多的分析方法指导性文件。
 
Q:在确证宿主细胞蛋白质的检测方法时有哪些关键参数?
A:The most important parameters in HCP method validation are quantitation limit (QL), Accuracy, Precision and range.
宿主细胞蛋白质的检测方法中最重要的参数有:定量下限(QL),准确度,精度和方法适用范围。
 
Q:确证宿主细胞蛋白质的检测方法需要多少时间?
A:Typically 3-6 months will be needed for an ICH method validation.
根据人用药品注册技术国际协调会的方法验证,通常将需要3-6个月
 
Q:宿主细胞蛋白质的抗原是怎样准备的?
A:HCP immunogens are prepared using a null cell line, it should be the same cell line that is used for the biologics production without the expression of the biologics. 
宿主细胞蛋白质的抗原使用一个没有相应产品基因但含有相应质粒载体的细胞株,它应该是所开发的生物药物生产用的未表达的个体。
 
Q:宿主细胞蛋白质的抗体是怎样制备的?
A:HCP antibodies are raised in rabbits or other animals that could provide a relatively large pool of antiserum because the biologics under development could have a long development and market life
宿主细胞蛋白质的抗体是用兔或其他动物提取的抗血清,因为生物药有一个长期的开发和市场周期,抗血清的储存量要有一定的规模以支持产品的开发和销售。
 
Q:宿主细胞蛋白质的酶联测定用何种方式?
A:Sandwich ELISA is commonly used for HCP quantitation because the impurities only exist in parts per million level, enrichment step is necessary to achieve the required sensitivity.
因为生物药中通常只有百万分之几的杂质,所以,宿主细胞蛋白质通常用三明治酶联测定,宿主细胞蛋白质杂质的浓缩步骤是必要的,以达到所需的灵敏度。
 
Q:宿主细胞蛋白质的酶联测定有什么局限性?
A:The current sandwich ELISA can only detect HCPs producing two high affinity antibodies during the HCP antibody production, HCPs with low or no immunogenicity in the host animals will not be detected with this assay format.  It is also possible that HCPs with relatively small molecular size will not be detected because they are cleared quickly in the animal host during antibody production.
目前夹心酶联测定只能用于具有两个高亲和力抗体的宿主细胞蛋白的检测,免疫原性低或没有免疫性的宿主细胞蛋白将不会被发现。此外,有的宿主细胞蛋白因为分子量比较小,它们在动物宿主抗体的生产过程中被迅速清除,它也有可能不会被检测到。
 
Q:为什麽要做宿主细胞蛋白质的电泳分析?
A:The value of gel electrophoresis is to complement the limitations of ELISA for HCP analysis, i.e. detection of HCPs that are weak or not immunogenic, small molecular size HCPs or HCPs that could be associated with biologics and not detected by the ELISA. 
凝胶电泳的价值是用于补充宿主细胞蛋白酶联免疫吸附试验的局限性,比如可以检测酶联测定无法检测到的弱的或没有免疫原性的宿主细胞蛋白,分子量小的宿主细胞蛋白。
 
Q:为什麽要做宿主细胞蛋白质的电泳免疫分析?
A:HCP Western Blot could detect HCPs with one or more high affinity antibodies (ELISA only detects HCPs with two high affinity antibodies), therefore provide a semi-quantitative HCP measurement that sometimes could also be more sensitive than ELISA.
HCP的印迹可以检测到一个或多个高亲和力的抗体(酶联免疫吸附试验必须要有两个高亲和力的抗体检测),因此提供了一个半定量的HCP的测量,有时也可能会比ELISA更为敏感
 
Q:生物信息学在宿主细胞蛋白质分析中的作用。
A:Bioinformatices could be used to analyze the function of the HCPs identified, this will provide the basis for a rick assessment.  If the HCPs or some of their domains identified share high homology with important regulatory proteins in humans, more efforts are necessary to further evaluate their risk or reduce their levels in the biologics.
生物信息学可以为分析宿主细胞蛋白质的功能提供了依据,如果宿主细胞蛋白质或它们的衍生物与人类重要的调节蛋白具有同源性,需要进一步的来评估他们的风险或在生产过程中尽量减少其在生物药中的水平。
 
Q:怎样进行不同宿主细胞蛋白质试剂间的延续性分析。
A:Because HCP antibodies are polyclonal and multiple batches are typically produced during the development and marketing of a biologics, a bridging study is necessary to ensure the consistency of the reagents.  All the reagents and methods should be developed under cGMP, and any future production of any components in the ELISA should be evaluated with the HCP reference and biologics sample retains to make sure that the reporting values are consistent after the switch of reagent(s), adjustment of certain reagents will be needed if there is any significant value changes during the bridging studies.
因为宿主细胞蛋白质抗体及相关的分析组分通常是多批次生产,在开发和销售试剂盒的过程中,为了确保分析结果的一致性,各个批次间衔接的研究是必要的。所有的试剂和方法的开发应根据cGMP,在酶联免疫吸附试验的任何组分的未来的生产应用保留的HCP标准和生物药样品进行评估,以确保报告值一致,如果衔接研究过程中有任何重大价值变动,将需要调整某些试剂。
 
 
2.Antibody Conformational Array-related papers
 
 
1.       James LC. et al. 2003.  Antibody multispecificity mediated by conformational diversity.  Science 299:1362-1367.
 
2.       Nobeli, I et al.  2009. Protein promiscuity and its implications for biotechnology.  Nature Biotechnology 27(2):157-167.
 
3.       Shon D-H et al. 1991. Antibodies raised against peptidefragments of bovine αS1-casein cross-react with the native protein, but recognize sites distinct from the  determinants on the protein. Eur. J. Immuno. 21:1475-1480.
 
4.       Harding, SE. 2010. Some observations on the effects of biopressing on biopolymerstability. J. Drug Targeting. 18(10):732-740.
 
5.       Halimi, H et al. 2005. Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity. Biochim. Biophys. Acta. 1753:247-256.
 
6.       Soonthornsata,B et al. 2010. Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41. Virology 405:157-164.
 
7.       Larman HB. et al. 2011. Autoantigen discovery with a synthetic human peptidome. Nature Biotechnology. 29(6)535-541.
 
8.       Reymond, MT. et al. 1997. Folding properties of peptide fragments of myoglobin. Protein Science. 6:706-716.
 
9.       Chang, J-Y. et al. 2005. Conformational impurity of disulfide proteins: Detection, quantification, and properties. Ana. Biochem. 342:78-85.
 
10.   Petty, TJ. et al. 2011. An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity. The EMBO J. 30:2167-2176.
 
11.   Jefferies, R. 2009. Glycosylation as a strategy to improve antibody-based therapeutics. Nature Reviews, Drug Discovery. 8:226-234.
 
12.   So, T. et al. 2001. Contribution of conformational stability of hen lysozyme to induction of type 2 T-helper immune response. Immunology. 104:259-268.
 
13.   Schlellekens, H. 2005. Factors influencing the immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 20:3-9.
 
14.   Schellekens, H. 2002. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clinical Therapeutics. 24(11):1720-1740.
 
15.   Laat, B. et al. 2011. Immune responses against domain I of β2-glycoprotein I are driven by conformational changes.  Arthritis & Rheumatism. 63(12)3960-3968.
 
16.   Ohhuri, T. et al. 2010. A protein’s conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins. J. Immunology. 185:4199-4205.
 
17.   Buttel, IC. et al. 2011. Taking immunogenicity assessment of therapeutic proteins to the next level. Biologicals. 39:100-109.
 
18.   Schellekens, H. 2009. Assessing the bioequivalence of biosimilars. Drug Discovert Today. 14(9/10):495-499.
 
19.   Sharma, B. 2007. Immunogenicity of therapeutic proteins. Part 1: Impact of product handling. Biotechnology Advances. 25:310-317.
 
20.   Porter, S. 2001. Human immune response to recombinant human proteins. J. Pharmaceutical Sciences. 90(1):1-11.
 
21.   Kromminga, A. et al. 2005. Antibodies against erythropoietin and other protein-based therapeutics. Ann. N.Y. Acad. Sci. 1050:257-265.
 
 
3.     Host Cell Protein-related Research Papers
 
Anderson NL, Anderson NG. 2002. The human plasma proteome: history, character, and diagnostic prospects. Molecular & Cellular Proteomics 1:845-867.
Anicetti VR, Fehskens EF, Reed BR, Chen AB, Moore P, Geier MD, Jones AJS. 1986. Immunoassay for the detection of E. coli proteins in recombinant DNA derived human growth hormone. Journal of Immunological methods 91:213-224.
 Belov ME, Anderson GA, Angell NH, Shen Y, Tolic N, Udseth HR, Smith RD. 2001. Dynamic range expansion applied to mass spectrometry based on data-dependent selective ion ejection in capillary liquid chromatography fourier transform ion cyclotron resonance for enhanced proteome characterization. Analytical Chemistry 73:5052-5060.
Blattner FR, III GP, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF and others. 1997. The complete genome sequence of Escherichia coli K-12. Science 277(5331):1453-1474.
Briggs J, Panfili PR. 1991. Quantitation of DNA and protein impurities in biopharmaceuticals. Analytical Chemistry 63:850-859.
Champion K, Madden H, Dougherty J, Shacter E. 2005. Defining your product profile and maintaining control over it, part 2. BioProcess International 3(8):52-57.
Champion K, Nishihara JC, Joly JC, Arnott D. 2001. Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation process. Proteomics 1:1133-1148.
Crowther JR. 2001. The ELISA Guidebook. Walker JM, editor. Vienna: Humana Press.
Dagouassat N, Haeuw J-F, Robillard V, Damien F, Libon C, Corvaia N, Lawny F, Nguyen TN, Bonnefoy J-Y, Beck A. 2001. Development of a quantitative assay for residual host cell proteins in a recombinant subunit vaccine against human respiratory syncytial virus. Journal of Immunological methods 251:151-159.
Dyk DDV, Misztal DR, Wiljkins MR, Mackintosh JA, Poljak A, Varnai JC, Teber E, Walsh BJ, Gray PP. 2003. Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster overy cell line. Proteomics 3:147-156.
Eaton LC. 1995. Host cell contaminant protein assay development for recombinant biopharmaceuticols. Journal of Chromatography A 705:105-114.
EMEA. 1997. CPMP position statement on DNA and Host Cell Proteins (HCP) impurities, routine testing versus validation studies. London.
FDA. 1997. Points to Consider in the manufacture and Testing of Monoclonal Antibody Products for Human use. Rockville, MD.
Follman DK, Fahrner RL. 2004. factorial screening of antibody purification processes using three chromatography steps without protein A. Journal of Chromatography A 1024:79-85.
Gibbs RA, Weinstock GM, Metzker ML, al e. 2004. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428:493-521.
Godovac-Zimmermann J, Brown LR. 2001. Perspectives for mass spectrometry and functional proteomics. Mass Spectrometry Reviews 20:1-57.
Goffeau A. 1997. The Yeast Genome Directory. Nature 387:5.
Groote DD, Zangerle PF, Gevaert Y, Fassotte MF, Bequin Y, Noizat-Pirenne F, Pirenne J, Gathy R, Lopez M, Dehart I and others. 1992. Direct stimulation of cytokines (IL-1β, TNF-α, IL-6, IL-2, IFN-γ and GM-CSF) in whole blood I. Comparison with isolated PBMC stimulation. Cytokine 4(3):239-248.
Hart RA, Rinas U, Bailey JE. 1990. Protein Composition of Vitreoscilla hemoglobin Inclusion Bodies Produced in Escherichia coli. The Journal of Biological Chemistry 265(21):12728-12733.
Hayduk EJ, Choe LH, Lee KH. 2004. A two-dimensional electrophoresis map of Chinese hamster ovary cell proteins based on fluorescence staining. Electrophoresis 25:2545-2556.
Hoffman K. 2000. Strategies for Host Cell Protein Analysis. BIOPHARM 13(6):38-45.
House RV. 2001. Cytokine measurement techniques for assessing hypersensitivity. Toxicology 158:51-58.
Hunter AK, Hoeltzli SD, Johnson GV, Gustafson ME, Ho SV. 2008a. Use of cyclohexanedimethanol as a nonflammable organic solvent for industrial scale reversed phase chromatography. Journal of Chromatography A 1202:107-110.
Hunter AK, Wang X, Suda EJ, Herberg JT, Shell RE, Thomas KE, Gustafson ME, Mozier NM, Ho SV. 2008b. Separation of a product associating E. coli host cell protein from recombinant apolipoprotein A-IMilano in an industrial HIC unit operation. Biotechnology and Bioengineering.
ICH. 1999. Guidance for Industry  Q6B Specifications: test procedures and acceptance criteria for biotechnological/biological products.
Janeway CAJ, Travers P, Walport M, Shlomchik MJ. 2005. Immunobiology. New York and London: Garland Science.
Kelley BD, Switzer M, Bastek P, Kramarczyk JF, Molnar K, Yu T, Coffman J. 2008. High-throughput screening of chromatographic separations: IV. Ion-exchange. Biotechnology and Bioengineering 100(5):950-963.
Krawitz DC, Forrest W, Moreno GT, Kittleson J, Champion KM. 2006. Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities. Proteomics 6:94-110.
Lander ES, Linton LM, Birren B, al e. 2001. Initial sequencing and analysis of the human genome. Nature 409:860-921.
Lebreton B, Brown A, Reis RV. 2008. Application of High-performance tangential flow filtration (HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in escherichia coli. Biotechnology and Bioengineering 100:964-974.
McKusick VA, Ruddle FH. 1987. A new discipline, a new name, a new journal. Genomics 1:1-2.
Meager A. 2006. Measurement of cytokine by bioassays: Theroy and applications. Methods 38:237-252.
Ohmura T, Ohmizu A, Sumi A, Ohtani W, Uemura Y, Arimura H, Nishida M, Kohama Y, Okabe N, Mimura T and others. 1987. Properties of recombinant Hepattis B vaccine. Biochemical and Biophysical Research Communication 149(3):1172-1178.
Phillips M, Cormier J, Ferrence J, Dowd C, Kiss R, Lutz H, Carter J. 2005. Performance of a membrane adsorber for trace impurity removal in biotechnology manusfacturing. Journal of Chromatography A 1078:74-82.
Rammensee H-G, Bachmann J, Emmerich NPN, Bachor OA, Stevannvic S. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50:213-219.
Rathore AS, Sobacke SE, Kocot TJ, Morgan DR, Dufield RL, Mozier NM. 2003. Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells. Journal of Pharmaceutical and Biomedical Analysis 32:1199-1211.
Rinas U, Bailey JE. 1992. Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli. Applied Microbiol Biotechnology 37:609-614.
Rinas U, Boone TC, Bailey JE. 1993. Characterization of inclusion bodies in recombinant Escherichia coli producing high levels of porcine somatotropin. Journal of Biotechnology 28:313-320.
Shukla AA, Hinckley P. 2008. Host cell protein clearance during protein a chromatography: Development of an improved column wash step. Biotechnology Progress 24:1115-1121.
Shukla AA, Hubbard B, Tressel T, Guhan S, Low D. 2007. Downstream processing of monoclonal antibodies-Application of platform approaches. Journal of Chromatography B 848:28-39.
Shukla AA, Jiang C, Ma J, Rubacha M, Flansburg L, Lee SS. 2008. Demonstration of Robust host cell protein clearance in biopharmaceutical downstream processes. Biotechnology Progress 24:615-622.
Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ, Marshall CT, James DC. 2004. Comparative proteomic analysis of GS-NS0 murine myeloma cell line with varying recombinant monoclonal antibody production rate. Biotechnology and Bioengineering 88(4):474-488.
Speicher DW, editor. 2008. Current Protocols in Protein Science, Chapter 10. Electrophoresis. Hobohen, NJ: John Wiley and Sons.
Thalhamer J, Freund J. 1984. cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens. Journal of Immunological methods 66:245-251.
Tobler SA, Noyes A, Rajewski JW, Shpritzer R, Piacenza W, Tannatt M, Coffman J, Vunnum S, Kelly B. Analysis of mAb protein A chromatograpgy peak precipitates and approaches to reduce peak turbidity; 2006; San Francisco, CA. p 151.
Ullenhag G, Bird C, Ragnhammar P, Frodin JE, Strigard K, Osterborg A, Thorpe R, Mellstedt H, Wadhwa M. 2001. Incidence of GM-CSF antibodies in cancer patients receiving GM-CSF for immunostimulation. Clinical Immunology 99(1):65-74.
Veeraragavan K. 1989. Studies on two major contaminating proteins of the cytoplasmic inclusion bodies in Escherichia coli. FEMS Microbiology Letters 61:149-152.
Venkiteshwaran A, Heider P, Matoservic S, Bogsnes A, Staby A, Sharfstein S, Belfort G. 2007. Optimized removal of soluble host cell proteins for the recovery of met-Human growth hormone inclusion bodies from Escherichia coli cell lysate using crossflow microfiltration. Biotechnology Progress 23:667-672.
Venter JC, Adams MD, al e. 2001. The Sequence of the Human Genome. Science 291:1304-1351.
Wang X, Hunter, A, Mozier, NM.  2009. Host cell proteins in biologics development: Identification, Quantitation and Risk Assessment. Biotech. Bioeng. 103(3)446-458.
Wang X, Schomogy, T, Wells, K, Mozier, NM. 2010. Improved HCP Quantitation by minimizing antibody cross-reactivity to terget proteins. BioProcess International.  January:18-24.
Wang X, Morgan, DM, Wang, G, Mozier, NM. 2012. Residual DNA analysis in biologics development: Review of measurement and quantitation technologies and future directions. Biotech. Bioeng. 109(2):307-317.
Waterston RH, Lindblad-Toh K, Birney E, Rogers J, al e. 2002. Initial sequencing and comparative analysis of the mouse genome. Nature 420:520-562.
Weston AD, Hood L. 2004. System Biology, proteomics, and the future of health care: Toward predictive, Preventive, and Personalized Medicine. Journal of Proteome Research 3(2):179-196.
Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez J-C, Yan JX, Gooley AA, Hughes G. 1996. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Bio/Technology 14(1):61-65.
Zhu D, Saul AJ, Miles AP. 2005. A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli. Journal of Immunological methods 306:40-50.
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