Glycopegylated granulocyte colony stimulating factor
View Patent ↗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.
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 MnCl 2 wherein the composition of step (c) comprises 1 mM MnCl 2 ;
(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.
2. The method of claim 1 , further comprising
(h) combining the composition of step (g) with a buffer mixture comprising 20 mM NaOAc and 600 mM Na 2 SO 4 , and applying the composition to a hydrophobic interaction chromatography column.
3. In a method of preparing a purified, pegylated GCSF peptide, wherein the method comprises a pegylation step and a purification step, the improvement comprising (a) pegylating GCSF in a composition comprising 20 mM L-methionine and at a pH of about 6.5 and (b) purifying the composition comprising the pegylated GCSF by applying the composition comprising the pegylated GCSF to a cation exchange chromatography column after adding a 20 mM citrate buffer and no additonal L-methionine to the composition comprising pegylated GCSF to provide a pH of about 4.0.
4. In the method of claim 3 , the improvement further comprising pegylating GCSF in a composition comprising MnCl 2 at a concentration of 1 mM.