PROCESS FOR MAKING RECOMBINANT ANTIDOTE TO FACTOR XA INHIBITOR
Disclosed are methods and isolated cells useful for the improved production of function fXa derivative protein that acts as a fXa inhibitor antidote. One aspect relates to an isolated cell comprising the r-Antidote polynucleotide and Furin polynucleotide. Another aspect relates to a method for preparing the cleaved two-chain r-Antidote by expressing, in a cell, the pre-processed r-Antidote polypeptide and a Furin polypeptide.
1 . An isolated cell comprising:
a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 1 and
a second non-endogenous polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 2.
2 - 4 . (canceled)
5 . The isolated cell of claim 4 , wherein the cell-type is CHO.
6 . The isolated cell of claim 5 , wherein the cell is a CHO cell subtype selected from the group consisting of K, M and DG44.
7 - 8 . (canceled)
9 . The isolated cell of claim 1 , further comprising a selectable marker.
10 . The isolated cell of claim 9 , wherein the selectable marker provides resistance to a compound selected from the group consisting of puromycin, methotrexate, neomycin and hygromycin.
11 . The isolated cell of claim 10 , wherein the selectable marker provides resistance to methotrexate.
12 . The isolated cell of claim 10 , wherein the selectable marker provides resistance to puromycin.
13 . The isolated cell of claim 12 , wherein the selectable marker provides antibiotic resistance to the cell.
14 . The isolated cell of claim 1 , wherein the first or second polynucleotide is on an extrachromosomal DNA construct.
15 . The isolated cell of claim 1 , wherein the first or second polynucleotide is on a DNA construct integrated into the chromosomal DNA of the isolated cell.
16 . The isolated cell of claim 1 , wherein the first and second polynucleotides are on one DNA plasmid.
17 . The isolated cell of claim 1 , wherein the wherein the first and second polynucleotides are on different DNA plasmids.
18 . The isolated cell of claim 1 further comprising
a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 1 and
a second polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 3.
19 . The isolated cell of claim 18 , wherein the ratio of the second polypeptide to the first polypeptide is at least about 8:2.
20 . The isolated cell of claim 18 , wherein the ratio of the second polypeptide to the first polypeptide is at least about 9:1.
21 . The isolated cell of claim 1 further comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 2.
22 . The isolated cell of claim 21 , wherein expression level of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is at least 3 times expression level of endogenous Furin.
23 . A composition comprising the isolated cell of claim 1 and cell culture media.
24 . A method of preparing a cleaved two chain polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 3, wherein the method comprises expressing in an isolated cell:
a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 1 and
a second non-endogenous polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 2.
25 - 52 . (canceled)
53 . A polynucleotide construct that comprises
a first polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 1; and
a second polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 2.