Abstract
Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting.
We present here a method using commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2-D electrophoresis procedure gives highly reproducible spot patterns and entire procedure can be completed in less than 2 days. Software analysis enables the straightforward generation of percent coverage values for the antibody when used to probe HCP-containing samples.
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References
Eaton LC (1995) Host cell contaminant protein assay development for recombinant biopharmaceuticals. J Chromatogr A 705:105–114
Dagoussat N, Haeuw J-F, Robillard V, Damien F, Libon C, Corvaïa 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 syncitial virus. J Immunol Methods 251:151–159
Wan M, Wang Y, Rabideau S, Moreadith R, Schrimsher J, Conn G (2002) An enzyme-linked immunosorbent assay for host cell protein contaminants in recombinant PEGylated staphylokinase mutant SY161. J Pharm Biomed Anal 28:953–963
Savino E, Hu B, Sellers J, Sobjak A, Majewski N, Fenton S, Yang T-Y (2011) Development of an in-house, process-specific ELISA for detecting HCP in a therapeutic antibody, Part 1. Bioprocess Int 9:38–45
Wang X, Hunter AK, Mozier NM (2009) Host cell proteins in biologics development: identification, quantitation and risk assessment. Biotechnol Bioeng 103:446–458
Zhu-Shimoni J, Yu C, Nishihara J, Wong RM, Gunawan F, Lin M, Krawitz DC, Liu P, Wandoval W, Vanderlaan M (2014) Host cell protein testing by ELISA and the use of orthogonal methods. Biotechnol Bioeng 111:2367–2379
Tscheliessnig AL, Konrath J, Bates R, Jungbauer A (2013) Host cell protein analysis in therapeutic protein bioprocessing—methods and applications. Biotechnol J 8:655–670
Valente KN, Schaefer AK, Kempton HR, Lenhoff AM, Lee KH (2014) Recovery of Chinese hamster ovary host cell proteins for proteomic analysis. Biotechnol J 9:87–99
Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307–316
Berggren K, Steinberg TH, Lauber WM, Carroll JA, Lopez MF, Cernokalskaya E, Zieske L, Diwu Z, Haugland RP, Patton WF (1999) A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports. Anal Biochem 276:129–143
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Berkelman, T., Harbers, A., Bandhakavi, S. (2015). 2-D Western Blotting for Evaluation of Antibodies Developed for Detection of Host Cell Protein. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_28
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DOI: https://doi.org/10.1007/978-1-4939-2550-6_28
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
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