title: Production and purification of a chimeric monoclonal antibody against botulinum neurotoxin serotype A.
abstract: Production of recombinant antibodies against botulinum neurotoxin is necessary for the development of a post-exposure treatment. CHO-DG44 cells were transfected with a plasmid encoding the light and heavy chains of a chimeric monoclonal antibody (S25) against botulism neurotoxin serotype A. Stable cell lines were obtained by dilution cloning and clones were shown to produce nearly equivalent levels of light and heavy chain antibody by an enzyme-linked immunosorbent assay (ELISA). In suspension culture, cells produced 35 microg/ml of chimeric antibody after 6 days, corresponding to a specific antibody productivity of 3.1 pg/cell/day. A method for the harvest and recovery of an antibody against botulism neurotoxin serotype A was investigated utilizing ethylenediamine-N,N'-tetra(methylphosphonic) acid (EDTPA) modified zirconia and MEP-hypercel, a hydrophobic charge interaction chromatography resin. Purification of the S25 antibody was compared to that achieved using rProtein A-Sepharose Fast Flow resin. After the direct load of culture supernatant, analysis by ELISA and gel electrophoresis showed that S25 antibody could be recovered at purities of 41 and 44%, from the EDTPA modified zirconia and MEP-hypercel columns, respectively. Although the purity obtained from each of these columns was low, the ability to withstand high column pressures and nearly 90% recovery of the antibody makes EDTPA modified zirconia well suited as an initial capture step. Combining the EDTPA modified zirconia and HCIC columns in series resulted in both purity and final product yield of 72%.
authors.affiliation: Department of Chemical Engineering, University of Nebraska, 207 Othmer Hall, 820 North 16th Street, Lincoln, NE 68588, USA. mmowry2@unl.edu
authors.forename: Mark C
authors.initials: MC
authors.lastname: Mowry
authors.forename: Mike
authors.initials: M
authors.lastname: Meagher
authors.forename: Leonard
authors.initials: L
authors.lastname: Smith
authors.forename: Jim
authors.initials: J
authors.lastname: Marks
authors.forename: Anu
authors.initials: A
authors.lastname: Subramanian
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D001681:Biological Assay; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000737:chemistry*; D016466:CHO Cells; D002845:Chromatography; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005029:Ethylenediamines / Q000737:chemistry; D063065:Organophosphonates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D015040:Zirconium / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D005029:Ethylenediamines; D063065:Organophosphonates; D011506:Proteins; D011993:Recombinant Fusion Proteins; C418473:ethylenediaminetetrakis(methylphosphonic) acid; D012685:Sepharose; D015040:Zirconium; D019274:Botulinum Toxins, Type A; C028541:zirconium oxide