Abstract
A method for large scale production and single-step purification of bispecific antibodies is described. Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month. Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step. Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays. Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA. Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step. Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination.
References
Aug 7, 1975·Nature·G Köhler, C Milstein
Sep 1, 1992·Biologicals : Journal of the International Association of Biological Standardization·A R ter AvestC F van Kreyl
Mar 1, 1992·Journal of Biochemical and Biophysical Methods·K Morimoto, K Inouye
May 22, 1992·Journal of Chromatography·L TarditiM Mariani
Jul 1, 1990·Molecular Immunology·P M Lansdorp, T E Thomas
Dec 1, 1990·The Journal of Experimental Medicine·C AnasettiP J Martin
Aug 4, 1989·Journal of Chromatography·A JungbauerH Katinger
Nov 10, 1988·Journal of Immunological Methods·C GranbergT Lövgren
Nov 25, 1988·Journal of Immunological Methods·A DanielssonH Lindblom
Sep 9, 1988·Journal of Chromatography·H H HwangA V Johnston
Apr 1, 1987·Hybridoma·J Bukovsky, R H Kennett
Jul 24, 1986·Journal of Immunological Methods·S H NeohH Zola
Oct 6, 1983·Nature·C Milstein, A C Cuello
Oct 1, 1993·Journal of Biochemical and Biophysical Methods·N Abe, K Inouye
Jul 22, 1995·Lancet·R R FrenchM J Glennie
Jul 12, 1994·Journal of Immunological Methods·H BohlenH Tesch
Feb 26, 1993·Journal of Immunological Methods·C R DarbyK J Wood
May 26, 1993·Journal of Immunological Methods·A GorterG J Fleuren
Oct 1, 1995·Journal of Hematotherapy·C DemanetK Thielemans
Citations
Feb 27, 1999·International Journal of Cancer. Journal International Du Cancer·O ManzkeH Bohlen
Mar 17, 2001·International Journal of Cancer. Journal International Du Cancer·O ManzkeH Bohlen
Feb 28, 2001·Journal of Immunological Methods·D R Husereau, M R Suresh
Nov 6, 2001·Journal of Biochemical and Biophysical Methods·V Kasche
Nov 6, 2001·Journal of Biochemical and Biophysical Methods·E Boschetti
Jun 29, 2002·Journal of Biochemical and Biophysical Methods·Seema Gupta, Mavanur Suresh
Jul 20, 2002·Trends in Biotechnology·Egisto Boschetti
Mar 26, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·An WillemsNico Mertens
May 10, 2000·Drugs·S B Noonberg, C C Benz
Aug 19, 2007·Journal of Chromatography. a·David R H EvansStephen M Notarnicola
Sep 11, 2004·Protein Expression and Purification·Mark C MowryAnu Subramanian
Jan 4, 2006·Journal of Chromatography. a·Raja Ghosh, Lu Wang
Jul 22, 2010·MAbs·Hui F LiuRobert Bayer
Nov 18, 2003·Clinical and Experimental Immunology·M JensenF Berthold
Oct 10, 2018·Biotechnology Progress·Cassia AndradeWai Keen Chung
Jan 9, 2019·MAbs·Gunasekaran DhandapaniGuy Patchornik
Jun 5, 2019·MAbs·Adam R EvansPromod Mehndiratta
Jul 25, 2006·Journal of Biochemistry·Akito NatsumeKenya Shitara
Dec 7, 2020·New Biotechnology·Gunasekaran DhandapaniGuy Patchornik
Dec 7, 2018·Journal of Chromatography. a·Lei Wang, R Kenneth Marcus
Jun 1, 2021·Antibody Therapeutics·Serene W Chen, Wei Zhang
Jan 9, 2007·Journal of Chromatography. a·Brian C S To, Abraham M Lenhoff
Dec 6, 2016·Advanced Drug Delivery Reviews·Zehra ElgundiVeysel Kayser