What is Host Cell Protein (HCP)? Host Cell Proteins are proteins and their modified forms derived from the hosts of biologics expression and co-exist during the biologics purification process.
How to express the level of HCPs? HCPs are typically expressed as ng/mg or parts per million (ppm).
What are the common technologies for HCP analysis? Common technologies used for HCP analysis include ELISA (Enzyme-linked Immunosorbent Assay), one-dimensional or two-dimensional gel electrophoresis, Western Blot and Mass Spectrometry.
What guidelines are related to HCP analysis? ICH Q6B section 2.3.1; 4.1.3 and 6.2.1 have detailed descriptions on impurity analysis including HCP analysis.
How the methods are developed? HCP methods 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.
How the methods are validated? 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.
What are the most important parameters when validating a HCP ELISA? The most important parameters in HCP method validation are quantitation limit (QL), Accuracy, Precision and range.
How much time needs to budget for a HCP method validation? Typically 3-6 months will be needed for an ICH method validation.
How the HCP immunogens are prepared? 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.
How the HCP antibodies are prepared? 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.
What format of ELISA needs to be used? 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.
What are the limitations of HCP ELISA? 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.
What is the value of gel electrophoresis? 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.
What is the value of HCP Western Blot? 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.
What is the value of a bioinformatics analysis? 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.
How to do a bridging study when switching testing reagents? 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.
Antibody Conformational Array-related papers
James LC. et al. 2003. Antibody multispecificity mediated by conformational diversity. Science 299:1362-1367.
Nobeli, I et al. 2009. Protein promiscuity and its implications for biotechnology. Nature Biotechnology 27(2):157-167.
Shon D-H et al. 1991. Antibodies raised against peptidefragments of bovine ¿S1-casein cross-react with the native protein, but recognize sites distinct from the determinants on the protein. Eur. J. Immuno. 21:1475-1480.
Harding, SE. 2010. Some observations on the effects of biopressing on biopolymerstability. J. Drug Targeting. 18(10):732-740
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.
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.
Larman HB. et al. 2011. Autoantigen discovery with a synthetic human peptidome. Nature Biotechnology. 29(6)535-541.
Reymond, MT. et al. 1997. Folding properties of peptide fragments of myoglobin. Protein Science. 6:706-716.
Chang, J-Y. et al. 2005. Conformational impurity of disulfide proteins: Detection, quantification, and properties. Ana. Biochem. 342:78-85.
Petty, TJ. et al. 2011. An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity. The EMBO J. 30:2167-2176
Jefferies, R. 2009. Glycosylation as a strategy to improve antibody-based therapeutics. Nature Reviews, Drug Discovery. 8:226-234.
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.
Schlellekens, H. 2005. Factors influencing the immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 20:3-9.
Schellekens, H. 2002. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clinical Therapeutics. 24(11):1720-1740.
Laat, B. et al. 2011. Immune responses against domain I of ¿2-glycoprotein I are driven by conformational changes. Arthritis & Rheumatism. 63(12)3960-3968.
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.
Buttel, IC. et al. 2011. Taking immunogenicity assessment of therapeutic proteins to the next level. Biologicals. 39:100-109.
Schellekens, H. 2009. Assessing the bioequivalence of biosimilars. Drug Discovert Today. 14(9/10):495-499.
Sharma, B. 2007. Immunogenicity of therapeutic proteins. Part 1: Impact of product handling. Biotechnology Advances. 25:310-317.
Porter, S. 2001. Human immune response to recombinant human proteins. J. Pharmaceutical Sciences. 90(1):1-11.
Kromminga, A. et al. 2005. Antibodies against erythropoietin and other protein-based therapeutics. Ann. N.Y. Acad. Sci. 1050:257-26
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-1¿, TNF-¿, IL-6, IL-2, IFN-¿ 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.