US 12,448,411 B2
Separation method
Annika Kristina Forss, Uppsala (SE); Gustav Jose Rodrigo, Uppsala (SE); Tomas Bjorkman, Uppsala (SE); Jesper Ulf Hansson, Uppsala (SE); and Mats Ander, Uppsala (SE)
Assigned to Cytiva BioProcess R&D AB, Uppsala (SE)
Filed by Cytiva BioProcess R&D AB, Uppsala (SE)
Filed on Mar. 10, 2023, as Appl. No. 18/181,974.
Application 18/181,974 is a continuation of application No. 16/884,475, filed on May 27, 2020, granted, now 11,623,941.
Application 16/884,475 is a continuation of application No. 15/348,699, filed on Nov. 10, 2016, granted, now 10,703,774, issued on Jul. 7, 2020.
Application 15/348,699 is a continuation in part of application No. 15/282,367, filed on Sep. 30, 2016, granted, now 10,654,887, issued on May 19, 2020.
Prior Publication US 2023/0257417 A1, Aug. 17, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 1/22 (2006.01); B01D 15/00 (2006.01); B01J 20/286 (2006.01); B01J 20/32 (2006.01); C07K 14/31 (2006.01); C07K 16/06 (2006.01); C07K 16/12 (2006.01); C07K 17/10 (2006.01)
CPC C07K 1/22 (2013.01) [B01D 15/00 (2013.01); B01J 20/286 (2013.01); B01J 20/3212 (2013.01); B01J 20/3274 (2013.01); C07K 14/31 (2013.01); C07K 16/065 (2013.01); C07K 16/1271 (2013.01); C07K 17/10 (2013.01); B01J 2220/52 (2013.01)] 11 Claims
 
1. A method of isolating an immunoglobulin, comprising the steps of:
a) providing a separation matrix comprising at least 15 mg/ml multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support,
b) contacting a liquid sample comprising an immunoglobulin with said separation matrix,
c) washing said separation matrix with a washing liquid,
d) eluting the immunoglobulin from the separation matrix with an elution liquid, and
e) cleaning the separation matrix with a cleaning liquid comprising at least 0.5 M NaOH,
wherein in step b) at least 40 mg immunoglobulin per ml separation matrix is contacted with said separation matrix, and
wherein the immunoglobulin-binding alkali-stabilized Protein A domains are, or have at least 80% sequence identity to, a sequence comprising any of SEQ ID NOS: 1 or 3-7, wherein N11 is mutated to an amino acid selected from glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, and arginine.