Process for concentrating blood coagulation Factor XIII derived from human placentae

Claim 1

A process for concentrating a blood coagulation Factor XIII derived from human placentae consisting essentially of (1) recovering an extract with a physiologically saline solution from human placentae, (2) removing precipitate from the solution by centrifugation, (3) adding ammonium sulfate to the supernatant in a concentration of 20 to 30% (W/V), (4) recovering the supernatant and removing the precipitate therefrom, (5) adding ammonium sulfate to the supernatant in a concentration of 45 to 55% (W/V) to recover precipitate, (6) dissolving the precipitate into a phosphate buffer solution of pH 6 to 9 containing about 0.005 M of EDTA, (7) adding alkylene-oxide polymer of copolymer having a molecular weight of 2,000 to 20,000 which is selected from the group consisting of polyethylene oxide homopolymer, polypropylene oxide homopolymer and ethylene oxidepropylene oxide copolymer in a concentration of 4 to 0% W/V, (8) removing the formed precipitate and recovering the supernatant, (9) adding the above-mentioned alkylene-oxide polymer or copolymer to the supernatant in a concentration of 20 to 30% (W/V), (10) recovering the precipitate formed and dissolving it into a phosphate buffer of pH 6 to 9, (11) heat-treating the collected precipitate for 9 to 11 hours at 55° to 65° C. in an aqueous solution of a neutral amino acid, monosaccharide, sugar alcohol or a mixture of two or more thereof in a concentration of 10 to 20% (W/V) thereby to inactivate hepatitis virus, (12) contacting the resulting solution with a DEAE-crosslinked dextran which had been equilibrated to pH of 6 to 9 thereby to absorb blood coagulation Factor XIII, (13) eluting the blood coagulation Factor XIII with an about 0.05 M phosphate buffer containing about 0.5 M NaCl, (14) subjecting the collected eluate to dialysis, sterilizing filtration and freeze-drying.