Process for purification of recombinant granulocyte colony stimulating factor (rHu GCSF)

摘要

The present invention relates to methods for purification of recombinant human granulocyte colony stimulating factor (rHu GCSF). The present invention particularly relates to methods for purification of rHu GCSF involving techniques such as aqueous two phase extraction and multimodal chromatographic purification to obtain highly purified rHu GCSF. The present invention also provides a pharmaceutical composition comprising the rHu GCSF, purified using the methods described herein.

主权项

1. A method of purifying recombinant human granulocyte colony stimulating factor (rHu GCSF) from a recombinant host cell, wherein the method comprises:
a) solubilizing inclusion bodies using a solubilization buffer to obtain solubilized rHu GCSF; b) refolding the solubilized rHu GCSF using a refolding buffer to obtain refolded rHu GCSF; c) concentrating the refolded rHu GCSF by ultra-filtration to obtain concentrated rHu GCSF; d) subjecting the concentrated rHu GCSF to aqueous two phase extraction to remove host cell proteins and host cell DNA and to obtain rHu GCSF partitioned into top phase and in the form of precipitate between the two aqueous phases; e) resolubilizing the rHu GCSF using a resulobilization buffer to obtain rHu GCSF solution; and f) subjecting the rHu GCSF solution to multimodal chromatography to obtain purified rHu GCSF, wherein the multimodal chromatography comprises:
i) binding the rHu GCSF to multimodal chromatography resin with a buffer comprising 35 mM to 50 mM acetate and 0 mM to 450 mM sodium chloride with pH in the range of 5.10 to 5.70; andii) eluting the rHu GCSF using an elution buffer comprising 10 mM to 50 mM acetate at pH ranging from 3.00 to 4.30 and 0 mM to 100 mM sodium chloride to obtain purified rHu GCSF, wherein the purified rHu GCSF has less than 1% methionine oxidized form of rHu GCSF, less than 0.5% reduced form of rHu GCSF, and less than 0.2% aggregated form of rHu GCSF.