Serpin fusion polypeptides and methods of purification thereof
This invention relates to molecules, particularly polypeptides, more particularly fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from a serpin and second polypeptide comprising of at least one the following: an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide; a cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide; a WAP domain containing polypeptide or a sequence derived from a WAP containing polypeptide; and an albumin polypeptide or an amino acid sequence that is derived from a serum albumin polypeptide. This invention also relates to methods of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing and purifying such molecules.
1. A method of purifying a fusion protein and preventing oligomerization of AAT in the fusion protein, the method comprising the steps of:
(a) culturing a cell comprising a nucleic acid construct that encodes the fusion protein under conditions that allow for the expression of the fusion protein,
wherein the fusion protein comprises at least one human serpin polypeptide comprising an alpha-1 antitrypsin (AAT) polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 32, 33, 34, and 35 operably linked to:
a human immunoglobulin Fc polypeptide;
(b) contacting a supernatant from the cultured cell with an affinity resin under conditions that allow for binding between the affinity resin and the fusion protein, wherein the affinity resin comprises protein A; and
(c) eluting the fusion protein from the affinity resin using a buffer under conditions that allow for the detachment of the fusion protein from the affinity resin and prevent oligomerization of AAT in the fusion protein, wherein the buffer is at a near-neutral pH,
wherein the purified fusion protein inhibits neutrophil elastase (NE) activity.
2. The method of claim 1 , wherein the cell comprises a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) 293 cell, a COS cell, a PER.C6® cell, a NS0 cell, a SP2/0 cell, or a YB2/0 cell.
3. The method of claim 2 , wherein the cell comprises a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) 293 cell.
4. The method of claim 1 , wherein the nucleic acid construct comprises a secretion signal sequence.
5. The method of claim 1 , wherein the purified fusion protein inhibits neutrophil elastase (NE) activity to a similar or greater extent compared to human serum derived alpha-1 antitrypsin (sdAAT).
6. The method of claim 1 , wherein the human Fc polypeptide comprises a human IgM polypeptide or a human IgG Fc polypeptide.
7. The method of claim 6 , wherein the human IgG Fc polypeptide comprises a human IgG1 polypeptide, a human IgG2 Fc polypeptide, human IgG3 Fc polypeptide, or human IgG4 Fc polypeptide.
8. The method of claim 1 , wherein the immunoglobulin Fc polypeptide comprises an amino acid sequence that is at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, and 7.
9. The method of claim 1 , wherein the human serpin polypeptide and the immunoglobulin Fc polypeptide are operably linked via a hinge region, a linker region, or both a hinge region and a linker region.
10. The method of claim 9 , wherein the hinge region, the linker region, or both the hinge region and the linker region comprise a peptide sequence.
11. The method of claim 1 , wherein the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 36.
12. The method of claim 1 , wherein the fusion protein comprises at least two alpha-1 antitrypsin (AAT) polypeptides and an immunoglobulin Fc polypeptide.
13. The method of claim 12 , wherein the fusion protein comprises two alpha-1 antitrypsin (AAT) polypeptides, and wherein each of the two AAT polypeptides are operably linked to the immunoglobulin Fc polypeptide via a hinge region, a linker region, or both a hinge region and a linker region such that the fusion protein has the structural arrangement from N-terminus to C-terminus as follows: AAT polypeptide-immunoglobulin Fc polypeptide-AAT polypeptide.
14. The method of claim 1 , wherein the immunoglobulin Fc polypeptide comprises at least one of the following mutations: Met252Tyr, Ser254Thr, Thr256G1u, Met428Leu or Asn434Ser.