Large-scale recovery and purification of L-asparaginase from Erwinia carotovora

Abstract

A large-scale process was developed to purify gram quantities of a therapeutic enzyme, L-asparaginase, from submerged cultures of Erwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column of L-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. The final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis, N-terminal sequencing, peptide mapping, and immunological comparison.