Methods for producing and purifying recombinant alpha-L-iduronidase
The present invention provides a recombinant human α-L-iduronidase and biologically active fragments and muteins thereof with a purity greater than 99%. The present invention further provides large-scale methods to produce and purify commercial grade recombinant human α-L-iduronidase enzyme thereof.
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8 . A method of purifying human recombinant α-L-iduronidase, or a biologically active fragment or mutein thereof, comprising the steps of
a) obtaining culture medium from a culture of Chinese Hamster Ovary (CHO) cells that have been transformed with a nucleic acid that encodes said human recombinant α-L-iduronidase;
b) adjusting the pH of the culture medium to an acidic pH;
c) subjecting said pH-adjusted medium to ultrafiltration;
d) subjecting the filtered medium produced by step (c) to a first dye-affinity chromatography purification step;
e) subjecting the eluant from step (d) to a first metal-ion chelate chromatography step;
f) subjecting the eluant from step (e) to a hydrophobic interaction chromatography (HIC) step; and
g) concentrating and diafiltering the eluant from step (f), to yield a purified preparation of purified human recombinant α-L-iduronidase which has a greater than 99% purity as determined by quantity of contaminating CHO protein per mg of total protein in said preparation.
9 . The method of claim 8 , wherein said first dye-affinity chromatography purification step is performed on a Cibacron-Blue affinity chromatography matrix.
10 . The method of claim 8 , wherein said first metal-ion chelate chromatography step is performed on a copper-chelating Sepharose FF matrix.
11 . The method of claim 8 , wherein said HIC step is performed on a phenyl-Sepharose High Performance chromatography matrix,
12 . The method of claim 9 , wherein said purification on said Cibacron-Blue dye interaction chromatography column produces a seven to ten fold purification of said human α-L-iduronidase as compared to the initial medium applied to said chromatography column.
13 . The method of claim 8 , wherein said method comprises using 10% glycerol in all buffers to increase the quantitative recovery of said human α-L-iduronidase.
14 . The method of claim 8 , wherein step (b) results in the pH of the fluid adjusted to pH 5.3.
15 . The method of claim 8 , wherein said purified human recombinant α-L-iduronidase has a specific activity greater than 200,000 units per milligram protein.
16 . The method of claim 12 , wherein said purified human recombinant α-L-iduronidase has a specific activity greater than 240,000 units per milligram protein.
17 . The method of claim 8 , wherein said purified human recombinant α-L-iduronidase comprises one or more mannose-6-phosphate residues.
18 . The method of claim 17 , wherein said purified human recombinant α-L-iduronidase comprises a mannose-6-phosphate residue attached at position 3 and a mannose-6-phosphate residue attached at position 6.
19 . The method of claim 8 , wherein said purified human recombinant α-L-iduronidase has a half-life inside a cell of approximately 5 days.
20 . The method of claim 8 , wherein said culture of CHO cells is a culture of cell line 2.131 CHO cells.
21 . The method of claim 8 , wherein said CHO cells are cultured in a protein-free culture medium having a pH of between 6.8 and 7.0, said medium being supplemented with 7.6 mg/L thymidine, 13.6 mg/L hypoxanthine, 375 μg/mL G418 and 5% fetal bovine serum
22 . The method of claim 21 , wherein said CHO cells are grown to confluence at a density of between 2.0×105 to 2.5×105 cells per ml.
23 . The method of claim 23 , wherein the medium of said cells at confluence is harvested for said purification of human recombinant α-L-iduronidase, or a biologically active fragment or mutein thereof.
24 . The method of claim 23 , wherein said medium of said cells at confluence is harvested by continuous perfusion.
25 . The method of claim 25 , wherein said continuous perfusion comprises exchanging between 2 to 3.5 culture volumes of said medium over 24 hours.
26 . The method of claim 22 , wherein production of said human recombinant α-L-iduronidase is enhanced by supplementing said medium with sodium butyrate for 12 hours to induce α-L-iduronidase gene expression.
27 . The method of claim 26 , wherein said sodium butyrate is removed from said medium 12 hours after initial induction with said sodium butyrate.
28 . The method of claim 27 , wherein said production of human recombinant α-L-iduronidase is reinduced with sodium butyrate every 48 hours over a 21 day protein production period.