PRODUCTION OF FC FRAGMENTS

• US 20170129966A1
• Filed: 06/02/2015
• Published: 05/11/2017
• Est. Priority Date: 06/02/2014
• Status: Abandoned Application

• Current Assignee: Laboratoire francais du Fractionnement et des Biotechnologies
• Original Assignee: Laboratoire francais du Fractionnement et des Biotechnologies
• Inventors: Masiello, Nicholas C.
• Application Number:
  US15/315,809
• Publication Number:
  US 20170129966A1
• Time in Patent Office: Days
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: B01D 15/30 Partition chromatography
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01D 15/426 Specific type of solvent
• CPC Class Codes: B01D 2311/2626 Absorption or adsorption
• CPC Class Codes: B01D 61/145 Ultrafiltration
• CPC Class Codes: C07K 16/04 from milk
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/12 isolated from milk
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/34 : Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12P 21/02 : having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 204/99 : transferring other glycosyl groups (2.4.99)
• CPC Class Codes: C12Y 304/22002 : Papain (3.4.22.2)

Abstract

In one aspect, the disclosure provides cells and transgenic non-human mammals for the production of Fc fragments, as well as compositions and uses thereof.

Claims

1. A method of producing a fragment crystallizable (Fc) fragment, the method comprising

providing a transgenic non-human mammal that has been modified to express an antibody comprising an Fc fragment in the mammary gland;

harvesting the antibody comprising the Fc fragment from milk produced by the mammary gland of the transgenic mammal; and

isolating the Fc fragment from the antibody.

5. The method of claim 1 or 2, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to

(a) hydrophobic interaction chromatography;

and

(b) ultrafiltration.

7. The method of claim 5 or 6, wherein the ultrafiltration is performed in a solution comprising phosphate, NaCl and Tween 80, wherein the phosphate has a concentration between 10 and 100 mM, the NaCl has a concentration between 100 and 500 mM, and the Tween 80 has a concentration between 0 to 0.01%, optionally wherein the solution comprises 20 mM phosphate pH 7.0, 150 mM NaCl and 0.01% Tween 80.

8. The method of claim 1 or 2, wherein isolating the Fc fragment from the antibody comprises

(a) obtaining an antibody comprising an Fc fragment and one or more additional fragments;

(b) digesting the antibody of (a) to produce an Fc fragment and one or more additional fragments;

(c) separating the Fc fragment from the one or more additional fragments by hydrophobic interaction chromatography, comprising

applying the Fc fragment and the one or more additional fragments of (b) to a hydrophobic interaction chromatography column; and

recovering the Fc fragment from the hydrophobic interaction chromatography column; and

(d) further purifying the recovered Fc fragment by ultrafiltration.

9. The method of claim 8, wherein the one or more additional fragments include a fragment antigen-binding (Fab) fragment, a Fab′

fragment, a F(ab′

)2 fragment or a single-chain variable (scFv) fragment.

12. The method of claim 1, 3 or 11, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, or llama.

13. The method of claim 1 or 3, wherein the transgenic non-human mammal is also transgenic for the expression of a sialyl transferase.

14. The method of claim 8, wherein obtaining the antibody comprises purifying the antibody.

16. The method of claim 14, wherein the antibody is purified using affinity chromatography.

18. The method of claim 16 or 17, wherein the affinity chromatography comprises Protein A affinity chromatography.

25. The method of any one of claims 1, 2, 5, 7-9, 11-14, 16, 18-24, wherein the antibody isotype is IgE, IgG, IgA, IgM or IgD.

26. The method of claim 25, wherein the antibody isotype is IgG.

27. The method of claim 26, wherein the antibody is Herceptin.

29. The method of any one of claims 1-28, wherein the purity of the isolated Fc fragment is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.

30. The method of claim 29, wherein the purity of the isolated Fc fragment is assessed by high performance liquid chromatography, SDS-PAGE gel electrophoresis, or contaminant protein ELISA.

36. The method of any one of claims 1-35, wherein the Fc fragment has anti-inflammatory properties.

37. The method of any one of claims 1-36, wherein the Fc fragment is used to treat a subject with an autoimmune condition or an inflammatory condition.

38. A purified Fc fragment produced by the method of any one of the preceding claims.

2. A method of producing an Fc fragment, the method comprising

providing a mammary epithelial cell that has been modified to express an antibody comprising an Fc fragment;

harvesting the antibody comprising the Fc fragment from the mammary epithelial cell; and

isolating the Fc fragment from the antibody.

11. The method of claim 2 or 4, wherein the mammary epithelial cell is of a non-human mammal.

3. A method of producing an Fc fragment, the method comprising

providing a transgenic non-human mammal that has been modified to express an Fc fragment in the mammary gland;

harvesting the Fc fragment from the milk produced by the mammary gland of the transgenic mammal; and

isolating the Fc fragment.

6. The method of claim 3 or 4, wherein isolating the Fc fragment comprises subjecting the Fc fragment sequentially to

(a) hydrophobic interaction chromatography;

and

(b) ultrafiltration.

10. The method of claim 3 or 4, wherein isolating the Fc fragment comprises

(a) obtaining an Fc fragment;

(b) subjecting the Fc fragment to hydrophobic interaction chromatography, comprising

applying the Fc fragment of (a) to a hydrophobic interaction chromatography column; and

recovering the Fc fragment from the hydrophobic interaction chromatography column; and

(c) further purifying the recovered Fc fragment by ultrafiltration.

15. The method of claim 10, wherein obtaining the Fc fragment comprises purifying the Fc fragment.

17. The method of claim 15, wherein the Fc fragment is purified using affinity chromatography.

31. The method of any one of claims 5, 6, 8, 10, 19, 28, wherein the hydrophobic interaction chromatography is performed using a hydrophobic chromatography column comprising an organic polymer resin.

32. The method of claim 31, wherein the organic polymer resin is phenyl organic polymer resin.

33. The method of any one of claims 5, 6, 8, 10, 19, 28, wherein the hydrophobic interaction column is eluted using a salt buffer.

34. The method of claim 33, wherein the elution of the hydrophobic interaction column is performed using a decreasing gradient of the salt buffer concentration.

35. The method of any one of claims 5, 6, 8, 10, 19, 28, wherein ultrafiltration is performed using gel filtration chromatography.

4. A method of producing an Fc fragment, the method comprising

providing a mammary epithelial cell that has been modified to express an Fc fragment;

harvesting the Fc fragment from the mammary epithelial cell; and

isolating the Fc fragment.

19. A method comprising subjecting an antibody sequentially to

(a) hydrophobic interaction chromatography;

and

(b) ultrafiltration,

wherein the ultrafiltration is performed in a solution comprising phosphate, NaCl and Tween 80, wherein the phosphate has a concentration between 10 and 100 mM, the NaCl has a concentration between 100 and 500 mM, and the Tween 80 has a concentration between 0 to 0.01%, optionally wherein the solution comprises 20 mM phosphate pH 7.0, 150 mM NaCl and 001% Tween 80.

20. The method of 19, wherein the antibody is digested prior to hydrophobic interaction chromatography.

21. The method of claim 8 or 20, wherein the digestion is performed by an enzyme.

22. The method of claim 21, wherein the enzyme is a cysteine protease.

23. The method of claim 22, wherein the cysteine protease is papain.

24. The method of claim 23, wherein the papain is immobilized on a solid support.

28. A method comprising subjecting an Fc fragment sequentially to

(a) hydrophobic interaction chromatography;

and

(b) ultrafiltration,

wherein the ultrafiltration is performed in a solution comprising phosphate, NaCl and Tween 80, wherein the phosphate has a concentration between 10 and 100 mM, the NaCl has a concentration between 100 and 500 mM, and the Tween 80 has a concentration between 0 to 0.01%, optionally wherein the solution comprises 20 mM phosphate pH 7.0, 150 mM NaCl and 001% Tween 80.

39. A method comprising administering a therapeutically effective amount of an Fc fragment produced in a transgenic non-human mammal to a subject in need thereof.

40. The method of claim 39, wherein the subject has an inflammatory condition or an autoimmune condition.

41. A transgenic Fc fragment.

42. The transgenic Fc fragment of claim 41, wherein the transgenic Fc fragment is purified.

43. A method comprising administering a therapeutically effective amount of the transgenic Fc fragment of claim 41 or 42 to a subject in need thereof.

44. The method of claim 43, wherein the subject has an inflammatory condition or an autoimmune condition.