Utilization of pharmacological chaperones to improve manufacturing and purification of biologics
View Patent ↗The present invention provides methods for improving the production of recombinant proteins through the use of pharmacological chaperones for the recombinant proteins. As exemplified by the present invention, the binding of a pharmacological chaperone to a recombinant protein expressed by a cell can stabilize the protein and increase export of the protein out of the cell's endoplasmic reticulum, and increase secretion of the protein by the cell.
1. A method for improving the yield of an active recombinant human wild-type acid β-Glucosidase protein expressed by a host cell, wherein the host cell is in media having a pH greater than 5.0, which method comprises
contacting the host cell in vitro with isofagomine, wherein contacting the host cell with isofagomine increases activity of the acid β-Glucosidase secreted out of the cell, and
purifying the secreted acid β-Glucosidase from the media, wherein the isofagomine is contacted to the host cell in an amount effective to increase the yield stability of the active acid β-Glucosidase during purification,
wherein the isofagomine is present at a concentration of at least 3 μM.
2. The method of claim 1 , wherein the host cell is a mammalian cell.
3. The method of claim 1 , wherein the host cell is selected from the group consisting of CHO cells, HeLa cells, HEK-293 cells, 293T cells, COS cells, COS-7 cells, mouse primary myoblasts, and NIH 3T3 cells.
4. The method of claim 1 , wherein contacting the host cell with isofagomine increases the stability of the acid β-Glucosidase in the host cell media.
5. The method of claim 1 , further comprising storing the purified acid β-Glucosidase, wherein the isofagomine increases the stability of the acid β-Glucosidase during storage, and wherein the stability of the acid β-Glucosidase is greater than the stability of a recombinant human wild-type acid β-Glucosidase that is not contacted with isofagomine.
6. The method of claim 1 , wherein the secreted acid β-Glucosidase is purified to be at least 95% pure.
7. The method of claim 1 , wherein purifying the secreted acid β-Glucosidase comprises ammonium sulfate precipitation, column chromatography containing a hydrophobic interaction resin, contacting the secreted acid β-Glucosidase with a cation exchange resin, contacting the secreted acid β-Glucosidase with an anion exchange resin, or contacting the secreted acid β-Glucosidase with a chromatofocusing resin.