Granted Patent B2

US 12,448,411 · App. 18/181,974 · Granted Oct 21, 2025

Inventors: Annika Kristina Forss (Uppsala, SE); Gustav Jose Rodrigo (Uppsala, SE); Tomas Bjorkman (Uppsala, SE); Jesper Ulf Hansson (Uppsala, SE); Mats Ander (Uppsala, SE)

Assignee: Cytiva BioProcess R&D AB

C07K1/22B01D15/00B01J20/286B01J20/3212B01J20/3274C07K14/31C07K16/065C07K16/1271C07K17/10B01J2220/52

Abstract

Claims (18)

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.

2. The method of claim 1 , wherein said liquid sample is a clarified cell broth and wherein in step d) said immunoglobulin is recovered as an eluate comprising less than 2000 ppm host cell proteins.

3. The method of claim 1 , wherein steps b)-e) are repeated at least 10 times.

4. The method of claim 1 , wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains.

5. The method of claim 1 , wherein in step d) said elution liquid has a pH of 2.5-4.5.

6. The method of claim 1 , wherein said multimers are coupled to said support via thioether links.

7. The method of claim 1 , wherein in step b) the pH is 6-8.

8. The method of claim 1 , wherein in step b) the residence time of said liquid sample on said separation matrix is 2-20 min.

9. The method of claim 1 , wherein said porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml.

10. The method of claim 1 , wherein said cross-linked polymer particles have a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa.

11. The method of claim 1 , wherein said separation matrix has a max pressure of at least 0.58 MPa when packed at 300+/−10 mm bed height in a column.

Assignments (2)

Continuity (4)

References Cited (308)