Preparation of factor Xa derivatives
View Patent ↗The present disclosure provides methods for manufacturing a fXa derivative protein at large scale leading to high yield of highly pure protein product. The method may include adding a detergent to a sample that contains a polynucleotide construct encoding the protein and purifying the protein through a soybean trypsin inhibitor (STI)-based affinity chromatograph, an ion exchange and mixed mode chromatograph and a hydrophobic interaction.
1. A method for preparing a polypeptide product expressed from a polynucleotide construct comprising the nucleic acid sequence of SEQ ID NO: 7 or a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence encoded by SEQ ID NO: 7, comprising:
adding a detergent to a sample that contains a polypeptide product expressed from the polynucleotide construct;
loading the sample to a soybean trypsin inhibitor (STI)-based affinity chromatograph and eluting the polypeptide with a first elution buffer to generate a first eluted sample, wherein the loaded sample does not contain an organic solvent;
loading the first eluted sample to an ion exchange and mixed mode chromatograph that comprises ceramic hydroxyapatite and eluting the polypeptide with a second elution buffer comprising at least 1M of an inorganic salt to generate a second eluted sample; and
loading the second eluted sample to a hydrophobic interaction chromatograph and eluting the polypeptide with a third elution buffer comprising at least 2 mM sodium chloride,
thereby preparing a purified sample comprising the polypeptide product.
2. The method of claim 1 , wherein the detergent comprises Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether).
3. The method of claim 1 , wherein the first elution buffer comprises 0.5M to 2M arginine.
4. The method of claim 3 , wherein the first elution buffer has a pH of about 5 to 5.4.
5. The method of claim 1 , wherein the ion exchange and mixed mode chromatograph comprises a ceramic hydroxyapatite type I chromatograph.
6. The method of claim 1 , wherein the second elution buffer comprises at least 2M of the inorganic salt.
7. The method of claim 6 , wherein the inorganic salt is sodium chloride.
8. The method of claim 1 , wherein the hydrophobic interaction chromatograph comprises an octyl sepharose chromatograph.
9. The method of claim 1 , further comprising a purification step with an anion exchange chromatograph.
10. The method of claim 9 , wherein the anion exchange chromatograph comprises a SARTOBIND® ion exchange membrane.
11. The method of claim 1 , further comprising subjecting one or more of the samples to filtration with a nanofleece filter.
12. The method of claim 11 , wherein the filtration with the nanofleece filter is prior to loading the sample to the STI-based affinity chromatograph.
13. The method of claim 1 , wherein the purified sample contains less than about 1% of contaminant proteins not expressed by the polynucleotide construct.
14. The method of claim 1 , wherein the polypeptide product is expressed in a cell that contains the polynucleotide construct.
15. The method of claim 14 , wherein the cell is grown in a medium under conditions to produce at least 100 mg of the polypeptide product per liter of medium.
16. The method of claim 15 , wherein the cell is grown in a medium under conditions to produce at least 200 mg of the polypeptide product per liter of medium.
17. The method of claim 15 , wherein the purified sample contains more than about 50% of the polypeptide product produced in the medium.
18. The method of claim 15 , wherein the purified sample contains more than about 100 mg of the polypeptide product from each liter production of the medium.
19. The method of claim 1 , wherein the polypeptide product is a two-chain polypeptide comprising a light chain and a heavy chain.
20. The method of claim 19 , wherein about 20% to 50% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5.
21. The method of claim 20 , wherein about 5%-95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 5 has two O-linked glycosylations.
22. The method of claim 19 , wherein about 40-80% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8.
23. The method of claim 22 , wherein at least about 90% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 has one O-linked glycosylation.
24. The method of claim 19 , wherein about 2%-12% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9.
25. The method of claim 19 , wherein about 0.1%-1.5% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10.
26. The method of claim 19 , wherein about 2%-8% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11.
27. The method of claim 19 , wherein about 35%-60% of the polypeptide product in the purified sample has a light chain consisting of the amino acid sequence of SEQ ID NO: 4.