US 12,269,843 B2
Refolding proteins using a chemically controlled redox state
Joseph Edward Shultz, Santa Rosa Valley, CA (US); Roger Hart, Loveland, CO (US); and Ronald Nixon Keener, III, Newbury Park, CA (US)
Assigned to AMGEN INC., Thousand Oaks, CA (US)
Filed by AMGEN INC., Thousand Oaks, CA (US)
Filed on Feb. 6, 2018, as Appl. No. 15/889,559.
Application 14/611,037 is a division of application No. 12/820,087, filed on Jun. 21, 2010, granted, now 8,952,138, issued on Feb. 10, 2015.
Application 15/889,559 is a continuation of application No. 15/422,327, filed on Feb. 1, 2017, granted, now 9,856,287, issued on Jan. 2, 2018.
Application 15/889,559 is a continuation of application No. 14/793,590, filed on Jul. 7, 2015.
Application 15/422,327 is a continuation of application No. 14/611,037, filed on Jan. 30, 2015.
Claims priority of provisional application 61/219,257, filed on Jun. 22, 2009.
Prior Publication US 2019/0055281 A1, Feb. 21, 2019
This patent is subject to a terminal disclaimer.
Int. Cl. A61K 39/395 (2006.01); C07K 1/113 (2006.01); C07K 1/14 (2006.01); C07K 14/00 (2006.01); C07K 16/00 (2006.01)
CPC C07K 1/1136 (2013.01) [C07K 1/1133 (2013.01); C07K 1/14 (2013.01); C07K 14/00 (2013.01); C07K 16/00 (2013.01); C07K 2319/30 (2013.01)] 9 Claims
 
1. A method for reproducibly obtaining proper refolding of proteins expressed in a non-mammalian expression system, the method comprising:
(a) selecting a preparation that includes:
at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer;
a concentration of oxidant; and
a concentration of reductant;
(b) selecting a desired yield of properly refolded proteins from a first concentration of proteins that is 1.0 g/l or greater;
(c) determining a thiol-pair ratio of the oxidant and the reductant in the preparation based on the desired yield of properly refolded proteins;
(d) determining a thiol-pair buffer strength for the preparation based on the determined thiol-pair ratio of (c), wherein the thiol-pair ratio and thiol-pair buffer strength are coordinated to achieve the desired yield of properly refolded proteins;
(e) incubating the refold mixture to obtain the desired yield of properly refolded proteins;
(f) selecting a second concentration of proteins;
(g) adjusting, if necessary, the thiol-pair ratio for the second concentration of proteins, based on the desired yield of properly refolded proteins; and
(h) adjusting the thiol-pair buffer strength for the second concentration of proteins until a final thiol-pair ratio is achieved, based on the adjusted thiol-pair ratio to achieve the same desired yield of properly refolded proteins for the second concentration.