Modified kappa light chain-binding polypeptides

Patent metadata

Field	Value
Publication number	US-10208092-B2
Application number	US-201515526895-A
Country	US
Kind code	B2
Filing date	Dec 11, 2015
Priority date	Dec 17, 2014
Publication date	Feb 19, 2019
Grant date	Feb 19, 2019

Abstract

The invention discloses kappa light chain-binding polypeptide comprising a mutated binding domain of Peptostreptococcus protein L, wherein at least one asparagine residue of a parental domain defined by, or having at least 95% or 98% sequence homology with, SEQ ID NO: 2-6 or 2 has been mutated to another amino acid residue which is not asparagine, proline or cysteine.

First claim

The invention claimed is:

1. A kappa light chain-binding polypeptide comprising at least one mutated binding domain of Peptostreptococcus Protein L, in which the domain has been mutated at positions:
N45A and comprising the amino acid sequence defined by SEQ ID NO: 7;
N10Q,N45A and comprising the amino acid sequence defined by SEQ ID NO: 8;
N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 9; or
N10Q,N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 11.

2. The polypeptide of claim 1 , in which the domain has been mutated at position N45A and comprising the amino acid sequence defined by SEQ ID NO: 7.

3. The polypeptide of claim 1 , in which the domain has been mutated at positions N10Q,N45A and comprising the amino acid sequence defined by SEQ ID NO: 8.

4. The polypeptide of claim 1 , in which the domain has been mutated at positions N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 9.

5. The polypeptide of claim 1 , in which the domain has been mutated at positions N10Q,N45A,N60Q and comprising the amino acid sequence defined by SEQ ID NO: 11.

6. The polypeptide of claim 1 , comprising a plurality of domains wherein the domains are linked by elements comprising up to 15 amino acids.

7. The polypeptide according to claim 1 , wherein the alkaline stability is improved relative to a parental polypeptide, as measured by the remaining binding capacity for kappa light chain-containing proteins after 96-100 10 min incubation cycles in 0.1M aqueous NaOH at 22+/−2° C.

8. A nucleic acid or a vector encoding a polypeptide according to claim 1 .

9. An expression system, which comprises a nucleic acid or vector according to claim 8 .

10. A separation matrix, wherein a plurality of polypeptides of claim 1 have been coupled to a solid support.

11. The separation matrix according to claim 10 , wherein the polypeptides have been coupled to the solid support by multipoint attachment.

12. The separation matrix of claim 10 , wherein the binding capacity of the matrix for kappa light chain-containing proteins after 100 10 min incubation cycles in 0.1M NaOH at 22+/−2° C. is at least 40, such as at least 50, or at least 55% of the binding capacity before the incubation.

13. A method of isolating a kappa light chain-containing protein, comprising isolating the kappa light chain-containing protein with the separation matrix of claim 10 .

14. A method of isolating a kappa light chain-containing protein comprising the steps of:
a) contacting a liquid sample comprising a kappa light chain-containing protein with a separation matrix of claim 10 ,
b) washing said separation matrix with a washing liquid,
c) eluting the kappa light chain-containing protein from the separation matrix with an elution liquid, and
d) cleaning the separation matrix with a cleaning liquid.

15. The method of claim 14 , wherein the cleaning liquid is alkaline, such as with a pH of 12-14.

16. The method of claim 14 , wherein the cleaning liquid comprises 0.01-1.0M NaOH or KOH, such as 0.05-1.0M or 0.05-0.1M NaOH or KOH.

17. The method of claim 14 , wherein steps a)-d) are repeated at least 10 times, such as at least 50 times or 50-200 times.

Assignees

• Ge Healthcare Bioprocess R&D Ab
• Ge Healthcare Bio Process R&D Ab

Inventors

• Rodrigo Gustav
• Ander Mats
• Bjorkman Tomas

Classifications

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2319/02

  containing a signal sequence · CPC title

• C07K2317/55

  Fab or Fab' · CPC title