Two-step purification of a murine monoclonal antibody intended for therapeutic application in man. Optimisation of purification conditions and scaling up.
The murine hybridoma cell line WT31, which produces a monoclonal antibody (Mab) of the IgG1 isotype with specificity for the human T cell receptor, was grown in batch-suspension cultures in the presence of foetal bovine serum (FBS). To acquire a clinical grade product for the reversal of allograft rejection, the clarified and concentrated cell culture supernatant was purified by a two-step chromatographic procedure, involving protein A affinity chromatography and Q Sepharose anion exchange chromatography. After choosing the appropriate conditions on a small scale, the purification process was scaled up. A BioPilot system was used for automated purification of 1 g WT31 Mab in a closed system. In spite of a relatively high initial ratio of bovine IgG to mouse IgG, the residual level of bovine IgG could be reduced to 1% or less with respect to the Mab content. No other serum proteins nor DNA were detected in the purified product. The efficacy of the purification procedure was demonstrated by a combination of several analytical techniques: ELISA (mouse and bovine IgG contents, protein A content), countercurrent immunoelectrophoresis (bovine serum albumin content), fluorescence activated cell sorter analysis (potency), DNA assay, sodium dodecylsulphate polyacrylamide gel electrophoresis, immunoblotting, isoelectric focusing, and gel permeation chromatography.
Jiskoot
W
W
Department of Pharmaceutics, Faculty of Pharmacy, University of Utrecht, The Netherlands.
Van Hertrooij
J J
JJ
Klein Gebbinck
J W
JW
Van der Velden-de Groot
T
T
Crommelin
D J
DJ
Beuvery
E C
EC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000627:therapeutic use; D000918:Antibody Specificity; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006825:Hybridomas / Q000276:immunology* / Q000378:metabolism; D015151:Immunoblotting; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000627:therapeutic use; D007525:Isoelectric Focusing; D051379:Mice; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A