PREPARATION OF FACTOR XA DERIVATIVES

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 to generate a first eluted sample;

loading the first eluted sample to an ion exchange and mixed mode chromatograph and eluting the polypeptide to generate a second eluted sample; and

loading the second eluted sample to a hydrophobic interaction chromatograph and eluting the polypeptide,

thereby preparing a purified sample comprising the polypeptide product.

2 . The method of claim 1 , wherein the sample to which the detergent is added contains the polynucleotide construct and a polypeptide product expressed from the polynucleotide construct.

3 . The method of claim 1 , wherein the ion exchange and mixed mode chromatograph comprises a ceramic hydroxyapatite type I chromatograph.

4 . The method of claim 1 , further comprising:

i) a purification step with an anion exchange chromatograph; wherein preferably the anion exchange chromatograph comprises a Sartobind™ ion exchange membrane; and/or

ii) subjecting one or more of the samples to filtration with a nanofleece filter; wherein preferably the filtration with the nanofleece filter is prior to loading the sample to the STI-based affinity chromatograph.

5 . The method of claim 1 , wherein the purified sample contains less than about 1% of contaminant proteins not expressed by the polynucleotide construct.

6 . The method of claim 1 , wherein the polypeptide product is expressed in a cell that contains the polynucleotide construct;

wherein preferably the cell is grown in a medium under conditions to produce at least 100 mg or at least 200 mg of the polypeptide product per liter of medium;

wherein more preferably the purified sample contains (i) more than about 50% of the polypeptide product produced in the medium or (ii) more than about 100 mg of the polypeptide product from each liter production of the medium.

7 . The method of claim 1 , wherein the polypeptide product is a two-chain polypeptide comprising a light chain and a heavy chain.

8 . The method of claim 7 , wherein:

i) about 20% to 50% of the polypeptide product in the purified sample has:

(A) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5; or

(B) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5; and about 5%-95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 5 has two 0-linked glycosylations and about 5%-95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 5 has one O-linked glycosylation;

and/or

ii) about 40-80% of the polypeptide product in the purified sample has:

(A) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; or

(B) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; and at least about 90% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 has one O-linked glycosylation;

and/or

iii) 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; and/or

iv) 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; and/or

v) 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; and/or

vi) 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.