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1. (WO1997018834) THERAPEUTIC FRAGMENTS OF VON WILLEBRAND FACTOR
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CLAIMS

1. A process for preparing an aqueous solution of von Willebrand factor (vWF) fragment which is substantially free of aggregate comprising:
(A) providing an acidic aqueous solution including vWF
fragment, denaturant,. and undesired contaminants;
(B) separating said contaminants from said solution by
contacting said solution with an affinity
chromatography medium to which said vWF fragments
adhere;
(C) eluting said vWF fragment from said affinity
chromatography medium in the preεence of the
denaturant ; and
(D) separating the eluted fragment from said denaturant while maintaining the aqueous solution of the
fragment at a pH of about 2.5 to less than about 5.5 to increase monomerization of, and decreaεe
aggregation of, said fragment, thereby forming an
aqueous solution of vWF fragment which is
substantially free of aggregate.

2. The process of Claim 1 wherein said vWF fragment comprises a cysteine-altered vWF fragment.

3. The process according to Claim 1 wherein the εource of said vWF fragment is a recombinant DNA molecule expressed in a host bacterial cell.

4. The process according to Claim 2 wherein said cysteine-altered vWF fragment is prepared by subjecting an aqueous solution of a recombinant vWF fragment and denaturant to alkylating conditions thereby forming an alkylated vWF fragment .

5. A procesε according to Claim 1 wherein said von Willebrand Factor fragment has an amino terminal residue at about amino acid residue 445 and a carboxy terminal residue at about amino acid 733.

6. The process according to Claim 1 wherein said denaturant is urea.

7. A process according to Claim 1 wherein said aqueous solution of fragment having a pH of about 2.5 and less than about 5.5 has a total ion concentration less than about 75 mM.

8. A process according to Claim 1, wherein said
chromatography medium contains heparin to which the vWF fragment adheres.

9. A procesε for limiting the dimerization of monomeric cysteine-altered von Willebrand factor (vWF) fragment isolated by affinity chromatography wherein said fragment has an amino terminal residue at about amino acid residue 445 and a carboxy terminal residue at about amino acid 733, said dimerization involving one or more of ionic, hydrophobic or hydrogen bonds which facilitate in vivo the association of two or more mature vWF subunits, said process comprising forming an aqueous solution of monomeric cysteine-altered vWF fragment which has a pH of about 2,5 to IP than about 5.5 and which includes therein up to about 10 mg/ml of said fragment and wherein the total concentration in the solution of additional species of ions (if any) iε leεs than about 75 mM.

10. A process for preparing an aqueous solution of von Willebrand factor (vWF) fragment which is substantially free of aggregate comprising:
(A) providing an aqueous solution of urea and vWF
fragment which is prepared by recombinant means and containing undesired bacterial contaminants, said
solution having a pH of less than about 6.5;
(B) separating said contaminants from said solution by
contacting εaid εolution with an affinity
chromatography medium containing heparin to which
the vWF fragment adhereε;
(C) eluting said vWF fragment from said affinity
chromatography medium in the presence of the
denaturant by contacting said vWF fragment with an
aqueous salt solution having a concentration
sufficient to cause said vWF fragment to disassociate from said heparin of said affinity
chromatography medium; and
(D) separating the eluted fragment from the denaturant
while maintaining the aqueous solution of the
fragment at a pH of about 2.5 to less than about 5.5 to increase monomerization of, and decrease
aggregation of, said fragment, thereby forming an
aqueous solution of vWF fragment which is
substantially free of aggregate.

11. A procesε for preparing an aqueouε εolution of cyεteine-altered von Willebrand factor (vWF) fragment which haε an amino terminal reεidue at about amino acid reεidue 445 and a carboxy terminal residue at about amino acid 733 and which is subεtantially free of aggregate compriεing:
(A) providing an aqueous solution of said cysteine- altered vWF fragment and denaturant;
(B) purifying an aqueous solution of the fragment and
denaturant under conditions which promote conversion of aggregated forms of the fragment to the dimeric
and/or monomeric forms thereof to provide purified
fragment;
(C) separating the dissolved, purified, fragment from
the denaturant while maintaining the aqueous
solution of the fragment at a pH of about 2.5 to
leεs than about 5.5 to increase monomerization of,
and decrease aggregation of, said fragment, thereby forming an aqueous solution of cysteine-altered vWF fragment which is subεtantially free of aggregate.