Procedure for obtaining pituitary gonadotropic hormones from urine

Claim 1

The process of claim 1 which includes: v. dissolving the precipitate from (u) in water; w. passing the dissolved precipitate from (v) at a temperature of 4.5* C. through a column containing small grains of a hydrophilic, insoluble material made by cross-linking polysaccharide dextran wherein the dextran chains are cross-linked to give a three dimensional network and the functional ionic groups are attached to the glucose units of the polysaccharide chains by ether linkages; and x. collecting the first fractions passing through said column to provide a further purified urinary gonadotropic hormone preparation.

Claim 3

A method for the preparation of a urinary gonadotropic hormone preparation containing FSH and LH hormones, the ratio of FSH:LH being between 3:1 and 6:1 which comprises: a. acidifying menopausal or post-menopausal human urine to a pH between 4.0 and 4.5; b. adding thereto an aqueous suspension of activated kaolin present in amounts of 10-30 percent by weight of said suspension; c. agitating the resulting mixture for a period of about 1 hour; d. separating the said activated kaolin adsorbent from the mixture by settling; e. discarding the supernatant liquid; f. centrifuging the separated activated kaolin adsorbent; g. washing the centrifuged activated kaolin adsorbent twice with acidulated water at a pH of about 4.5; h. eluting the washed activated kaolin adsorbent twice with 1 M aqueous ammonia solution so as to obtain an eluate at a pH between 11.0 and 11.3; i. mixing the eluates from (h); j. adjusting the pH of the mixed eluates to about 8.5 thereby forming a precipitate; k. separating the precipitate of (j) from the mixed eluates to provide a relatively clear liquid; l. acidifying said relatively clear liquid to a pH of about 5.0 to 5.5; m. adding to the acidified clear liquid of (l) about 2 volumes of acetone and maintaining the mixture overnight to form a precipitate; n. separating the precipitate of (l) from the acetone treated clear liquid; o. drying the precipitate; p. washing the precipitate with about 95 percent ethanol and ether; q. extracting the washed precipitate with about 70 percent ethanol containing about 10 percent ammonium acetate to obtain a relatively cleAr extract; r. adding to the extract of (q) absolute ethanol containing about 10 percent ammonium acetate to produce a precipitate; s. dissolving the precipitate from (r) with a 0.05 M phosphate buffer at a pH of about 7; t. stirring the dissolved precipitate from (s) with diethylaminoethyl cellulose and thereafter separating said diethylaminoethyl cellulose therefrom to form a relatively clear filtrate; u. acidifying the relatively clear filtrate from (t) to pH 5.4; v. passing said acidified relatively clear filtrate through a column containing synthetic alumino-silicate gel equilibrated with 0.05 M acetate buffer at a pH of about 5.4; w. eluting the column with 40 percent ethanol containing 10 percent ammonium acetate to provide an eluate; x. adding to the eluate of (w) about 95 percent ethanol in amounts sufficient to form a precipitate; and y. washing the precipitate from (x) to provide said urinary gonadotropic hormone preparation.