Glycopegylated granulocyte colony stimulating factor

摘要

The invention relates to methods of preparing and purifying conjugates between Granulocyte Colony Stimulating Factor and PEG moieties. The conjugates are linked via an intact glycosyl linking group that is interposed between and covalently attached to the peptide and the modifying group. The conjugates are purified using various chromatography methods.

主权项

1. A method of preparing a purified, pegylated GCSF peptide, which method comprises
(a) providing a composition comprising water, a buffer, at least one polysorbate, and 20 mM L-methionine, wherein the polysorbate is polyoxyethylene sorbitan monolaurate or polyoxvethylene sorbitan monooleate, and the composition has a pH of about 6.5; (b) combining the composition of step (a) with reagents comprising GCSF, GalNAeT2, and UDP-GalNAc, wherein the composition of step (b) comprises 0.45 mM UDP-GalNAc; (c) combining the composition of step (b) with MnCl2 wherein the composition of step (c) comprises 1 mM MnCl2; (d) combining the composition of step (c) with ST6GalNAc1 and an aliquot of cytidine monophosphate sialic acid poly(ethylene glycol) (CMP-SA-PEG), wherein the composition of step (d) comprises 1 mM CMP-SA-PEG, and, wherein the composition of step (e) is not purified prior to step (d); (e) combining the composition of step (d) with an aliquot of CMP-SA-PEG, wherein the composition of step (e) comprises 1.5 mM CMP-SA-PEG; (f) combining the composition of step (e) with a 20 mM citrate buffer, wherein no additional L-methionine is added to the composition of step (e) and wherein the resulting composition has a pH of about 4, and (g) applying the composition of step (f) to a cation exchange chromatography column, whereby purified PEG-GCSF is produced.