Method for Purifying Antibodies

• US 20130171095A1
• Filed: 10/10/2012
• Published: 07/04/2013
• Est. Priority Date: 10/10/2011
• Status: Active Grant

• Current Assignee: Xencor, Inc.
• Original Assignee: Xencor, Inc.
• Inventors: Moore, Gregory L., Desjarlais, John, BERNETT, Matthew J., Rashid, Rumana
• Granted Patent: US 10,851,178 B2
• Time in Patent Office: Days
• US Class Current: 424/85.2
• CPC Class Codes: C07K 14/521 Chemokines
• CPC Class Codes: C07K 14/522 Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
• CPC Class Codes: C07K 14/523 Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/5428 IL-10
• CPC Class Codes: C07K 14/5434 IL-12
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/7151 for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C07K 14/745 Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 : multispecific
• CPC Class Codes: C07K 2317/35 : Valency
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention relates generally to compositions and methods for purifying the desired species from a mixture of desired heterodimer and contaminating homodimer immunoglobulin variants by modifying the isoelectric point(s) of the individual chains.

Claims

1. A composition comprising a heterodimer protein comprising:

a) a first monomer comprising;

i) a first variant heavy chain constant region;

ii) a first fusion partner; and

b) a second monomer comprising;

i) a second variant heavy chain constant region;

ii) a second fusion partner;

wherein the pIs of said first and second variant heavy chain constant regions are at least 0.5 logs apart.

2. A composition according to claim 1 wherein the pIs of said first and second monomer are at least 0.5 logs apart.

3. A composition according to claim 1 or 2 wherein said first monomer comprises a third fusion partner.

4. A composition according to claim 1, 2 or 3 wherein said second monomer comprises a fourth fusion partner.

5. A composition according to claims 1, 3 and 4 wherein said fusion partners are independently selected from the group consisting of an immunoglobulin component, a peptide, a cytokine, a chemokine, an immune receptor and a blood factor.

6. A composition according to claim 5 wherein said immunoglobulin component is selected from the group consisting of Fab, VH, VL, scFv, scFv2, dAb.

7. A composition according to claim 5 wherein said cytokine is selected from the group consisting of IL-2, IL-10, IL-12 and GM-CSF.

8. A composition according to claim 5 wherein said chemokine is selected from the group consisting of RANTES, CXCL9, CXCL10 and CXCL12.

9. A composition according to claim 5 wherein said immune receptor is selected from the group consisting of CTLA-4, TNFRI, TNFRII, a TNFSF protein, and TNFRSF

10. A composition according to claim 5 wherein said blood factor is selected from the group consisting of Factor VII, Factor VIII and Factor IX.

11. A composition according any previous claim wherein one of said variant heavy chain constant regions comprises an amino acid substitution selected from the group consisting of Q196K, P217R, P228R, N276K, H435R and Y436F.

14. A composition according any previous claim wherein one of said variant heavy chain constant regions comprises an amino acid substitution selected from the group consisting of Q196K, P217R, P228R, N276K, H435R and Y436F.

18. A composition according to any of the previous claims wherein said first and said second variant heavy chain constant region comprises CH2 and CH3.

19. A composition according to any of the previous claims wherein said first and said second variant heavy chain constant region comprises CH1, CH2 and CH3.

23. A nucleic acid encoding any of the proteins of claims 1-22.

24. A host cell encoding a nucleic acid according to claim 23.

25. A method of purifying a heterodimeric protein comprising:

a) providing a composition according to any of the previous claims;

b) loading said composition onto an ion exchange column; and

c) collecting the heterodimeric fraction.

26. A method of treating an individual in need thereof comprising administering the composition of any previous claim.

12. A composition according to 11 further comprising wherein one of said variant heavy chain constant regions further comprises a variant selected from the group consisting of S119E, K133E, K133Q, R133E (in case of IgG2-4), R133Q (in case of IgG2-4), T164E, K205E, K205Q, N208D, K210E, K210Q, K274E, K320E, K322E, K326E, K334E, R355E, K392E, Deletion of K447, adding peptide DEDE at the c-terminus, G137E, N₂₀₃D, K274Q, R355Q, K392N and Q419E, 349A, 349C, 349E, 349I, 349K, 349S, 349T, 349W, 351E, 351K, 354C, 356K, 357K, 364C, 364D, 364E, 364F, 364G, 364H, 364R, 364T, 364Y, 366D, 366K, 366S, 366W, 366Y, 368A, 368E, 368K, 368S, 370C, 370D, 370E, 370G, 370R, 370S, 370V, 392D, 392E, 394F, 394S, 394W, 394Y, 395T, 395V, 396T, 397E, 397S, 397T, 399K, 401K, 405A, 405S, 407T, 407V, 409D, 409E, 411D, 411E, 411K, 439D, 349C/364E, 349K/351K, 349K/351K/394F, 349K/354C, 349K/394F, 349K/394F/401K, 349K/394Y, 349K/401K, 349K/405A, 349T/351E/411E, 349T/394F, 349T/394F/401K, 349T/394F/411E, 349T/405A, 349T/411E, 351E/364D, 351E/364D/405A, 351E/364E, 351E/366D, 351K/364H/401K, 351K/366K, 364D/370G, 364D/394F, 364E/405A, 364E/405S, 364E/411E, 364E/411E/405A, 364H/394F, 364H/401K, 364H/401K/405A, 364H/405A, 364H/405A/411E, 364Y/370R, 370E/411E, 370R/411K, 395T/397S/405A and 397S/405A.

13. A composition comprising a heterodimeric antibody comprising:

a) a first heavy chain comprising a first variant heavy chain constant region; and

b) a second heavy chain comprising a second variant heavy chain constant region;

wherein the pIs of said first and second variant heavy chain constant regions are at least 0.5 logs apart.

15. A composition according to claim 13 or 14 wherein said antibody further comprises a first and a second light chain.

17. A composition comprising a heterodimeric antibody according to claim 13 wherein the pIs of said first and second variant heavy chain constant regions are at least 0.5 logs apart.

20. A composition according to claim 13 wherein said first heavy chain and said second heavy chain differ by at least 4 amino acids.

22. A composition according to claim 20 wherein said first heavy chain comprises at least two amino acid substitutions and said second heavy chain comprises at least two amino acid substitutions, wherein said substitutions are different.

16. A composition according to 15 wherein one of said light chains is a variant light chain comprising an amino acid substitution selected from the group consisting of K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E, an addition of DEDE at the C-terminus, R108Q, Q124E, K126Q, N138D, K145T and Q199E.

21. A composition according to 20 wherein said first heavy chain comprises 4 amino acid substitutions and said second chain has none.

27. A composition comprising an antibody variant heavy chain constant region comprising an amino acid substitution selected from the group consisting of Q196K, P217R, P228R, N276K, H435R and Y436F.