Large-scale purification of a murine antimelanoma monoclonal antibody
Abstract The 9.2.27 monoclonal antibody (murine IgG 2a ), which recognizes a human melanoma-associated 250 kDa glycoprotein/proteoglycan, was produced in batch-suspension cultures and purified by staphylococcal protein A-Sepharose affinity chromatography. Initially, antibody production was performed in sub-lots of eight fermenters (280 1 per run). The cells were removed by centrifugation and the antibody from supernatant was concentrated 10–20-fold using ultrafiltration. The antibody was adsorbed onto a 500-ml protein A-Sepharose column at pH 8.4, followed by extensive washing with phosphate-buffered saline, pH 8.4, to remove endotoxins and other protein contaminants. The antibody was desorbed at pH 4.5. A 60–140-fold purification of antibody with a 100–1000-fold reduction of endotoxins was achieved. 2–4 g of highly purified antibody were obtained from each column run. Pooled fractions were concentrated by ultrafiltration, formulated and sterile filled as final product. Analysis of the final product included but was not limited to sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, high-performance liquid chromatography, as well as evaluations of specific activity, endotoxin level, protein A concentration, bovine immunoglobulin G level, DNA concentration, mycoplasma contamination, sterility and reverse transcriptase activity.