Schering Corporation

David Naveh

John C Tang

Tattanahali L Nagabhushan

Jay Raman

An ion-exchange chromatography technique for purifying crude proteins containing closely related impurities, wherein the isoelectric points of the desired protein and the impurities are determined, is disclosed. In performing this technique, the pH for the ion-exchange chromatography is adjusted to a point between the ranges of isoelectric points of the protein fractions to be separated whereby, upon contacting the crude protein mixture at such pH with an ion exchange resin, the proteins in the first and second fractions are oppositely charged and only one of the fractions binds to the ion exchange resin. The application of the technique to the purification of GM-CSF using cation exchange resin is also disclosed.

Method of purifying protein

Active recombinant human IL-4 is purified from a crude fermentation broth of a secreted E. coli mutant by subjecting said solution to selective chromatogrphy on a zinc-chelating agarose column at pH 7.2 in a buffer containing a high concentration of sodium chloride, i.e., about 1M sodium chloride, washing first with a phosphate buffer at pH 7.2 containing 1.0M sodium chloride then with a buffer containing 10% glycerol and eluting at pH 5.0. The resulting purified active recombinant human IL-4 solution is further treated by cation exchange chromatography, concentration by diafiltration up to 20 mg/mL and size exclusion gel chromatography using a citrate buffer at pH 4.5.

Purification of human interleukin-4 from a secreted escherichia coli

Active recombinant human IL-4 is purified from a crude cell culture medium of a CHO-cell lines mutant by subjecting said crude solution to two sequentialcation exchange chromatographies at pH 7.2 and 0.12M sodium chloride, selective chromatography on a cobalt-chelating agarose column at pH 7.2 in a buffer containing a high concentration of sodium chloride, i.e., about 0.5M sodium chloride, further treated by concentration (diafiltration) up to 20 mg/mL and size exclusion gel chromatography using a citrate buffer at pH 4.5.

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Leiden, Netherlands

David Naveh