Patent GB-1027551-A (GB)
Title: Production of physiologically active placental substances
IPC: A61K35/50
Filed: 1963-05-03
Abstract: Physiologically active substances such as gonadatropin and steroid hormones are obtained by contacting viable placental tissue with a culture medium in tissue culture apparatus while maintaining the tissue at a temperature suitable to maintain viability and the pH of the culture medium in a viable range, for sufficient time to produce the active substances in the medium and the active substances are then extracted from the culture medium. Trophoblastic tissue in strips 2 inch wide and 6 inch long or, alternatively, a whole intact placenta disposed on a plate with the umbilical cord depending downwardly through an orifice in the plate, is cultured at a temperature of 37 \sB 0.5 DEG C. in a specified culture medium at a pH in the range of 7.2 to 7.8 within apparatus, as depicted in Figs. 1 to 3 (not shown), comprising a cylindrical body defining a closed chamber, viable tissue support means disposed within the chamber, means for intermittently introducing and withdrawing culture medium and means for maintaining the temperature within the closed chamber at a level suitable to maintain viability of a tissue supported therein.  The culture medium is stirred by a coated magnetic stirring bar 28 or 56.  Semi-permeable membranes of reconstituted cellulose fitted in orifices 101 attached to the walls of the chamber permit the exchange of metabolic products, nutrients and pH-adjusting materials, such as sodium bicarbonate, glutamine or phosphate buffer (as the case may be).  Sterile air is circulated through the apparatus.  The withdrawn culture medium is cooled down to about 5 DEG C., is acidified to about pH 3.5 with glacial acetic acid and is stored at about 5 DEG C. for 48 hours to precipitate the glycoprotein.  The precipitated glycoprotein is vacuum filtered from the medium utilizing activated alumina and a filter aid such as diatomaceous earth, the filter bed being washed with distilled water acidified with glacial acetic acid until the proteinaceious content of the effluent will no longer furnish a precipitate with acetone. The filter cake is thoroughly dispersed in or homogenised with 2N ammonium hydroxide solution and filtered again.  The gonadatropic fraction is recovered from the ammonia filtrate by acidification to pH 4.5 with glacial acetic acid and re-precipitation with acetone. The solution is stored at about 5 DEG C. for 48 hours to assure complete precipitation and then the mixture is centrifuged.  The precipitate is again dissolved in 2N ammonium hydroxide solution, re-precipitated by addition of acetic acid and acetone, and re-centrifuged.  After the removal of residual salts e.g. sodium by dialysis, standardized dosages of the gonadatropin are made up with lactose sodium, hydrogen phosphate (buffer) and freeze dried. Steroid hormones are obtained from the effluent filtrate solution by treating it with diethyl ether, the ether extract is contacted with alcoholic potassium hydroxide to saponify the various fatty acid esters present and soaps are then removed by repeated extraction with distilled water. The ether extract material is then fractionated by passage through a 97%-activated alumina chromatographic column utilizing an elutriant comprising 3% ether in cyclohexane.  The separated components are collected as solids by evaporating the elutriant.  Alternatively, the saponification purification procedure may be replaced by a chromtographic separation wherein the steroids are absorbed on a 100%-activated alumina column, impurities including fatty acid esters being removed by washing the adsorbate with 100% cyclohexane. The ether-extracted filtrate containing alkaline phosphatase, ribonuclease, adenosine triphosphate, choline esterase, desoxyribonuclease and unidentified components, may be subjected to vacuum to remove residual ether, neutralized to pH 7.8 by sodium hydroxide, and then freeze-dried.ALSO:Physiologically active substances such as chorionic gonadatropin and steroid hormones are extracted from a culture medium at pH 7.2-7.8 in which viable placental tissue has been cultured at a temperature of 37 \sB 0.5 DEG C. by cooling the culture medium to about 5 DEG C., acidifying to about pH 3.5 with glacial acetic acid and storing the medium at about 5 DEG C. for 48 hours to precipitate the glycoprotein which is removed by filtration.  The filter cake is thoroughly dispersed in or homogenized with 2N ammonium hydroxide solution and filtered again. The gonadatropic fraction is recovered from the ammonia filtrate by acidification to pH 4.5 with glacial acetic acid and re-precipitation with acetone.  The precipitate is separated and again dissolved in 2N ammonium hydroxide solution, reprecipitated by addition of acetic acid and acetone, and re-centrifuged.  After the removal of residual salts e.g. sodium by dialysis, standardized dosages of the gonadatropin are made up with lactose and sodium hydrogen phosphate (buffer) and freeze dried.  Steroid hormones are obtained from the effluent filtrate solution by treating it with diethyl ether, the ether extract is contacted with alcoholic potassium hydroxide to saponify the various fatty acid esters present and soaps are then removed by repeated extraction with distilled water.  The ether extract material is then fractionated by passage through a 97%-activated alumina chromatographic column utilizing an elutriant comprising 3% ether in cyclohexane.  The separated components are collected as solids by evaporating the elutriant.  Alternatively the saponification purification procedure may be replaced by a chromatographic separation wherein the steroids are absorbed on a 100%-activated alumina column, impurities including fatty acid esters being removed by washing the adsorbate with 100% cyclohexane.  The ether-extracted filtrate containing alkaline phosphatase, ribonuclease, adenosine triphosphate, choline esterase, desoxyribonuclease and unidentified components, may be subjected to vacuum to remove residual ether, neutralized to pH 7.8 by sodium hydroxide and then freeze-dried.