Abstract
Monoclonal antibodies (MAbs)—reagents mass-produced in the laboratory to recognize a specific molecular target (1)—are currently the most widely used proteinbased therapeutic and diagnostic molecules in clinical trials (2). This chapter presents the most effective ready-to-use protocol for MAb purification of clinical-grade standard. Samples of high purity are often produced via a highly selective single step. However, an appropriate combination of chromatographic materials in at least two orthogonal (i.e., based on two different mechanisms) steps is mandatory for obtaining clinical-grade MAbs for the in vitro functional analysis of ligand signals on cell-based assays and protein expression changes on arrayed MAb biochips (3), or in vivo preclinical and therapeutic applications (4).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Köhler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.
Gura, T. (2002) Magic bullets hit the target. Nature 417, 584–586.
Stoll, D., Bachman, J., Templin, M. F., and Joos, T. (2004) Microarray technology: an increasing variety of screening tools for proteomic research. DDT:TARGETS 3, 24–31.
Funaro, A., Horenstein, A. L., Santoro, P., Cinti, C., Gregorini, A., and Malavasi, F. (2000) Monoclonal antibodies and therapy of human cancers. Biotechnol. Adv. 18, 385–401.
Center for Biologics Evaluation and Research. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use. Food and Drug Administration, Rockville, MD, 1997.
Mariani, M., Bonelli, F., Tarditi, R., Calogero, R., Camagna, M., Seccamani, E., and Scassellati, G. A. (1989) Purification of monoclonal antibodies for in vivo use: a two-step HPLC shows equivalent performance to immunoaffinity chromatography. BioChromatography 4, 149–155.
Hahn, R., Schlegel, R., and Jungbauer, A. (2003) Comparison of protein A sorbent. J. Chromat. B 790, 35–51.
Horenstein, A. L., Crivellin, F., Funaro, A., Said, M., and Malavasi, F. (2003) Design and scaleup of downstream processing of monoclonal antibodies for cancer therapy: from research to clinical proof of principle. J. Immunol. Methods 275, 99–112.
Gagnon, P. (1996) Purification Tools for Monoclonal Antibodies. Validated Biosystems, Tucson, AZ.
Horenstein, A. L., Crivellin, F., Durelli, I., and Malavasi, F. Application of Hydroxyapatite and Protein A in the Downstream Processing of MAb for Clinical Use. Presented at the Third International Hydroxyapatite Conference, Lisbon, November 3–5, 2003.
Stanker, L. H., Vanderlaan, M., Juarez-Salinas, H. (1985) One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite. J. Immunol. Methods 76, 157–169.
Juarez-Salinas, H., Ott, G. S., Chen, J. C., Brooks, T. L., and Stanker, L. H. (1986) Separation of IgG idiotypes by high-performance hydroxylapatite chromatography. Methods Enzymol. 121, 615–622.
Franklin, S. G. (2003) Removal of aggregate from an IgG4 product using CHT Ceramic Hydroxyapatite. BioProcess International 1, 50–51.
European Pharmacopea, 3rd ed. Biological Tests. 2.6.8 Pyrogens; 2.6.14 Bacterial Endotoxins, Council of Europe, Strasburg, 1997.
Petsch, D. and Anspach, F. B. (2000) Endotoxin removal from protein solutions. J. Biotechnol. 76, 97–119.
Pearson, F. C. Pyrogens. Marcel Dekker, New York, 1985, p. 119.
Brandwein, H. and Aranha-Creado, H. (2000) Membrane filtration for virus removal. Dev. Biol. Stand. 102, 157–163.
Nau, D. R. (1986) A unique chromatographic matrix for rapid antibody purification. Biochromatography 1, 82–94.
Boschetti, E. (2002) Antibody separation by hydrophobic charge induction chromatography. Trends Biotechnol. 20, 333–337.
Weaver, L. E. Jr. and Carta, G. (1996) Protein adsorption on Cation Exchangers: Comparison of Macroporous and Gel-Composite Media. Biotech. Prog. 12, 342–355.
Follman, D. K. and Fahrner, R. L. (2004). Factorial screening of antibody purification processes using three chromatography steps without protein A. J. Chromatogr. A. 1024, 79–85.
Harlow, E. and Lane, D. Using Antibodies: A Laboratory Manual. New York, Cold Spring Harbor Laboratory Press, 1999.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Horenstein, A.L., Durelli, I., Malavasi, F. (2005). Purification of Clinical-Grade Monoclonal Antibodies by Chromatographic Methods. In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:191
Download citation
DOI: https://doi.org/10.1385/1-59259-922-2:191
Publisher Name: Humana Press
Print ISBN: 978-1-58829-390-9
Online ISBN: 978-1-59259-922-6
eBook Packages: Springer Protocols