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CN-122029198-A - Method for purifying small multi-domain proteins

CN122029198ACN 122029198 ACN122029198 ACN 122029198ACN-122029198-A

Abstract

The present disclosure provides a method for purifying a T cell-engaging peptide-major histocompatibility complex binding domain (TCE-pMHC) molecule produced by a host cell, the method comprising separating a supernatant of a host cell culture comprising the TCE-pMHC molecule to form product (a), subjecting product (a) to affinity chromatography to form product (b), wherein the TCE-pMHC molecule is eluted by the affinity chromatography at a pH of about 3.0 to about 4.0, neutralizing product (b) by adding a MES buffer having a pH of less than 7.0 to form product (c), wherein the pH of product (c) is about 5.0 to about 7.0, and subjecting product (c) to anion exchange chromatography to form product (d), wherein the pMHC binding domain and T cell-engaging immune effector domain are capable of binding pMHC complexes and T cells, respectively.

Inventors

  • V. PATEL
  • E. JOSEPH

Assignees

  • 英美偌科有限公司

Dates

Publication Date
20260512
Application Date
20241029
Priority Date
20231101

Claims (20)

  1. 1. A method for purifying a T cell-engaging peptide-major histocompatibility complex binding domain (TCE-pMHC) molecule produced by a host cell, the method comprising: a) Isolating the supernatant of a host cell culture comprising the TCE-pMHC molecule to form product (a); b) Subjecting product (a) to affinity chromatography to form product (b), wherein the TCE-pMHC molecule is eluted by the affinity chromatography at a pH of about 3.0 to about 4.0; c) Neutralizing product (b) by adding MES buffer having a pH of less than 7.0 to form product (c), wherein the pH of product (c) is from about 5.0 to about 7.0, and D) Subjecting product (c) to anion exchange chromatography to form product (d); wherein the TCE-pMHC molecule comprises: i. A peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region (VC 1) linked to a constant region and a second variable region (VC 2) linked to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; T cell-engaging immune effector domains comprising an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH), and A half-life extending domain comprising a first IgG Fc region (Fc 1) and a second IgG Fc region (Fc 2), wherein the Fc1 region and the Fc2 region dimerize to form an Fc domain; Wherein the T cell engagement immune effector domain is linked to the N-terminus of VC1, VC1 is linked by its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked by its C-terminus to the N-terminus of VC2, and VC2 is linked by its C-terminus to the N-terminus of the FC2 region, and Wherein the pMHC binding domain and the T cell engagement immune effector domain are capable of binding to a pMHC complex and a T cell, respectively.
  2. 2. The method of claim 1, wherein TCE-pMHC subjected to the affinity chromatography in (b) is eluted with sodium acetate.
  3. 3. The method of claim 2, wherein the concentration of sodium acetate is less than 100 mM.
  4. 4. The method of claim 2, wherein the concentration of sodium acetate is from about 25 mM to about 75 mM.
  5. 5. The process of any one of claims 1 to 4, wherein the pH of product (b) is from about 3 to about 4.
  6. 6. The method of any one of claims 1 to 5, wherein the MES buffer added to neutralize product (b) is less than 5M.
  7. 7. The method of any one of claims 1 to 6, wherein the MES buffer added to neutralize product (b) is from about 0.5M to about 1.5M.
  8. 8. The process of any one of claims 1 to 7, wherein the pH of product (c) is from about 5.5 to about 6.5.
  9. 9. The process of any one of claims 1 to 8, wherein product (c) is diluted at least 2-fold after neutralization to form diluted product (c).
  10. 10. The method of any one of claims 1 to 9, wherein product (c) is diluted to form diluted product (c), and the concentration of the MES buffer in the diluted product (c) is less than 20 mM.
  11. 11. The method of any one of claims 1 to 10, wherein the concentration of the MES buffer in the diluted product (d) is from about 5mM to about 25 mM.
  12. 12. The method of any one of claims 1 to 11, wherein the electrical conductivity of product (c) is 5 mS/cm or less.
  13. 13. The method according to any one of claims 1 to 12, wherein the conductivity of the diluted product (c) is 5 mS/cm or less.
  14. 14. The method of any one of claims 1 to 13, wherein the flow-through buffer used with the anion exchange chromatography is an MES buffer.
  15. 15. The method of claim 14, wherein the flow-through buffer is less than 100 mM.
  16. 16. The method of claim 14, wherein the flow-through buffer is from about 25 mM to about 75 mM.
  17. 17. The process of any one of claims 1 to 16, wherein the pH of product (d) is from about 6 to about 7.
  18. 18. The method of any one of claims 1 to 17, wherein the affinity chromatography comprises protein a affinity chromatography.
  19. 19. The method of any one of claims 1 to 18, wherein the TCE-pMHC molecule is eluted by the affinity chromatography at a pH of about 3.2 to about 4.0.
  20. 20. The method of any one of claims 1 to 19, wherein the anion exchange chromatography comprises salt tolerant anion exchange flow-through chromatography.

Description

Method for purifying small multi-domain proteins Technical Field The present disclosure provides a method for purifying a T cell-engaging peptide-major histocompatibility complex binding domain (TCE-pMHC) molecule produced by a host cell, the method comprising separating a supernatant of a host cell culture comprising the TCE-pMHC molecule to form product (a), subjecting product (a) to affinity chromatography to form product (b), wherein the TCE-pMHC molecule is eluted by the affinity chromatography at a pH of about 3.0 to about 4.0, neutralizing product (b) by adding a MES buffer having a pH of less than 7.0 to form product (c), wherein the pH of product (c) is about 5.0 to about 7.0, subjecting product (c) to anion exchange chromatography to form product (d), and subjecting product (d) to ceramic hydroxy-phosphochromatography to obtain product (e), wherein the pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to T cells, respectively. Background Protein-based therapeutics, including antibodies and fusion proteins, may be rapidly cleared from the body after administration. Its short circulation half-life may be due to its small size, which allows for effective clearance by renal filtration, and lack of protection against intracellular degradation. To improve dosing, several strategies have been employed to extend circulation half-life. These strategies include the use of the synthesis of multi-domain proteins by antibody Fc domain or serum albumin ligation through neonatal Fc receptor (FcRn) recycling (Konnteman, 12 months 2011 current opinion of biotechnology (Curr Opin Biotechnol.); 22 (6): 868-76). Multi-domain proteins may have a tendency to aggregate due to electrostatic interactions and self-association between domains, even after standard separation methods including protein a chromatography. Furthermore, these proteins may include product-related impurities in the form of fragment adduct impurities that are difficult to dissipate from the product due to similar physicochemical properties. The small size of these molecules relative to monoclonal antibodies (mabs) may increase the relative host cell impurity burden. In some cases, downstream processes that enable robust aggregation and impurity removal to increase potency levels may be required to meet regulatory expectations. mAb purification was previously performed using either binding and elution chromatography (B/E) or flow-through (F/T) chromatography. The limitation of B/E chromatography is that its load density is limited by the actual resin binding capacity. In some cases, F/T chromatography may allow high loading densities for standard mabs, but may not be practical for non-platform mabs. In some cases, solution conditions that enable F/T operations on these non-platform mabs may render these operations impractical in existing manufacturing facilities. Disclosure of Invention In some aspects, the disclosure provides a method for purifying a T cell-engaging peptide-major histocompatibility complex binding domain (TCE-pMHC) molecule produced by a host cell, wherein the method comprises (a) isolating a supernatant of a host cell culture comprising the TCE-pMHC molecule to form product (a), (b) subjecting the product (a) to affinity chromatography to form product (b), wherein the TCE-pMHC molecule is eluted by the affinity chromatography at a pH of about 3.0 to about 4.0, (c) neutralizing the product (b) by adding a MES buffer having a pH of less than 7.0 to form product (c), wherein the pH of the product (c) is about 5.0 to about 7.0, and (d) subjecting the product (c) to anion exchange chromatography to form product (d), wherein the TCE-pMHC molecule comprises (i) a peptide-major histocompatibility complex (pMHC) binding domain, wherein the variable region comprises a variable region (TCE-VH) and a variable region (VH) comprising a variable region (TCE-VH) and a variable region (VH 1) and a variable region (VH-variable region (VH) comprising a variable region (TCE-VH 1) and a variable region (VH-variable region (VH 2), the half-life extending domain comprises a first IgG Fc region (Fc 1) and a second IgG Fc region (Fc 2), wherein the Fc1 region and the Fc2 region dimerize to form an Fc domain, wherein the T cell-engaging immune effector domain is linked to the N-terminus of VC1, VC1 is linked to the N-terminus of the Fc1 region by its C-terminus, the Fc1 region is linked to the N-terminus of VC2 by its C-terminus, and VC2 is linked to the N-terminus of the Fc2 region by its C-terminus, and wherein the pMHC binding domain and the T cell-engaging immune effector domain are capable of binding to pMHC complex and T cells, respectively. In some aspects, the TCE-pMHC subjected to the affinity chromatography in (b) is eluted with sodium acetate. In some aspects, the concentration of sodium acetate is less than 100 mM. In some aspects, the concentration of sodium acetate is from about 25 mM to about 75 mM. In s