Large-scale preparation of purified monoclonal antibody from cell culture supernatant: A case study with a monoclonal antibody to Zeta globin chain
Article Sidebar
Main Article Content
Abstract
Background: Nowadays, monoclonal antibodies have become important tools used in biomedical research, diagnosis and treatment of diseases. The mAbs are isolated from cell culture media, which usually contain different proteins in addition to antibodies. Obviously, antibody purification is becoming critical to guarantee its reliable application. Protein A and G resins are the most efficient and widely-used ligands in chromatographic methods. Nevertheless, some mAbs could not be purified by Protein A or G column.
Objectives: In order to explore the alternative method for purification of an IgG1 mAb, four different types of affinity chromatography were studied and compared in term of efficiency.
Materials and method: Chromatography using four commercial ligands, Protein A, Protein G, Protein L and engineered recombinant Protein A, was employed to purify the anti-zeta globin chain mAb clone PL3. The performance of the chromatography was compared in terms of yield, purity and biological activity of mAbs.
Results: The mAb PL3 could only be purified by using engineered recombinant Protein A column. The biological activity and purity of the purified mAbs were checked by ELISA and SDS-PAGE, respectively. It was found that the obtained purified mAbs were high purity and retained their biological activity.
Conclusion: In conclusion, engineered recombinant Protein A column is an alternative technique for large-scale purification of mAbs produced by unusual hybridoma clone that could not be purified by other columns.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Personal views expressed by the contributors in their articles are not necessarily those of the Journal of Associated Medical Sciences, Faculty of Associated Medical Sciences, Chiang Mai University.
References
[2] Zhang C. Hybridoma technology for the generation of monoclonal antibodies. Methods in molecular biology. 2012;901:117-35.
[3] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. Journal of immunology. 2005;174(5):2453-5.
[4] Goding JW. Antibody production by hybridomas. Journal of immunological methods. 1980; 39(4): 285-308.
[5] Ayyar BV, Arora S, Murphy C, O'Kennedy R. Affinity chromatography as a tool for antibody purification. Methods. 2012;56(2):116-29.
[6] Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR journal. 2005; 46(3): 258-68.
[7] Huse K, Bohme HJ, Scholz GH. Purification of antibodies by affinity chromatography. Journal of biochemical and biophysical methods. 2002; 51(3): 217-31.
[8] Salvalaglio M, Zamolo L, Busini V, Moscatelli D, Cavallotti C. Molecular modeling of protein A affinity chromatography. Journal of chromatography A. 2009; 1216(50): 8678-86.
[9] Roque AC, Silva CS, Taipa MA. Affinity-based methodologies and ligands for antibody purification: advances and perspectives. Journal of chromatography A. 2007; 1160(1-2): 44-55.
[10] Low D, O'Leary R, Pujar NS. Future of antibody purification. Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 2007; 848(1): 48-63.
[11] Gagnon P. Technology trends in antibody purification. Journal of chromatography A. 2012; 1221: 57-70.
[12] Kabir S. Immunoglobulin purification by affinity chromatography using protein A mimetic ligands prepared by combinatorial chemical synthesis. Immunological investigations. 2002; 31(3-4): 263-78.
[13] Choe W, Durgannavar TA, Chung SJ. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials. 2016; 9(12).
[14] Pata S, Khummuang S, Pornprasert S, Tatu T, Kasinrerk W. A simple and highly sensitive ELISA for screening of the alpha-thalassemia-1 Southeast Asian-type deletion. Journal of immunoassay & immunochemistry. 2014; 35(2): 194-206.
[15] Tayapiwatana C, Kuntaruk S, Tatu T, Chiampanichayakul S, Munkongdee T, Winichagoon P, et al. Simple method for screening of alpha-thalassaemia 1 carriers. International journal of hematology. 2009; 89(5): 559-67.
[16] Phunpae P, Chanwong S, Tayapiwatana C, Apiratmateekul N, Makeudom A, Kasinrerk W. Rapid diagnosis of tuberculosis by identification of Antigen 85 in mycobacterial culture system. Diagnostic microbiology and infectious disease. 2014; 78(3): 242-8.
[17] Bonilla FA, Oettgen HC. Adaptive immunity. The Journal of allergy and clinical immunology. 2010; 125(2 Suppl 2): S33-40.
[18] Chaplin DD. Overview of the immune response. The Journal of allergy and clinical immunology. 2010; 125(2 Suppl 2): S3-23.
[19] Kennedy MA. A brief review of the basics of immunology: the innate and adaptive response. The Veterinary clinics of North America Small animal practice. 2010; 40(3): 369-79.
[20] Abbas AK, Lichtman AH, S P. Cellular and Molecular Immunology. Philadelphia: Saunders; 2014.
[21] Kindt TJ, Goldsby RA, Osborne BA., J. K. Kuby Immunology. 6 ed. New York: W. H. Freeman; 2007.
[22] Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification. Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 2007; 848(1): 40-7.
[23] Linhult M, Gulich S, Hober S. Affinity ligands for industrial protein purification. Protein and peptide letters. 2005; 12(4): 305-10.
[24] Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978; 15(7): 429-36.
[25] Moks T, Abrahmsen L, Nilsson B, Hellman U, Sjoquist J, Uhlen M. Staphylococcal protein A consists of five IgG-binding domains. European journal of biochemistry. 1986; 156(3): 637-43.
[26] Graille M, Stura EA, Corper AL, Sutton BJ, Taussig MJ, Charbonnier JB, et al. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proceedings of the National Academy of Sciences of the United States of America. 2000; 97(10): 5399-404.
[27] Akerstrom B, Bjorck L. A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. The Journal of biological chemistry. 1986; 261(22): 10240-7.
[28] Rodrigo G, Gruvegard M, Van Alstine JM. Antibody Fragments and Their Purification by Protein L Affinity Chromatography. Antibodies. 2015; 4: 259-77.
[29] Ishihara T, Nakajima N, Kadoya T. Evaluation of new affinity chromatography resins for polyclonal, oligoclonal and monoclonal antibody pharmaceuticals. Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 2010; 878(23): 2141-4.
[30] Hahn R, Bauerhansl P, Shimahara K, Wizniewski C, Tscheliessnig A, Jungbauer A. Comparison of protein A affinity sorbents II. Mass transfer properties. Journal of chromatography A. 2005; 1093(1-2): 98-110.