Claims for Patent: 5,656,459

Summary for Patent: 5,656,459

Title:	Process for the preparation of cyclosporin a from tolypocladium species
Abstract:	The invention relates to a process for the preparation of cyclosporin A from Tolypocladium Sp comprising in the step of preparing a fermented medium of said Tolypocladium sp. The fungal biomass is extracted of said fermented medium to obtain a methanol extract, which methanol is removed by evaporation to obtain a first residue. An ethyl acetate extract is prepared from the aqueous solution of said first residue, which is decolourized and concentrated to obtain a second residue, the second residue is subjected to a step of purification.
Inventor(s):	Balaraman; Kothandapani (Pondieling 605008, IN), Mathew; Nisha (Pondicherry-3, IN)
Assignee:
Application Number:	08/491,566
Patent Claims:	1. A process for the preparation of cyclosporin A from Tolypocladium sp comprising subjecting said Tolypocladium sp to fermentation in a nutrient medium to obtain a fermented medium, extracting the fungal biomass of said fermented medium with methanol to obtain a methanol extract, removing methanol from said extract by evaporation to obtain a first residue, preparing an aqueous solution of said first residue, preparing an ethyl acetate extract of said aqueous solution, decolourizing and concentrating said extract to obtain a second residue and purifying the second residue chromatographically in a first stage on a silica gel column as solid phase with a solvent mixture of hexane, chloroform and methanol as mobile phase and a second stage on a resin column as solid phase and methanol as mobil phase. 2. A process as claimed in claim 1 wherein the fermentation is carried out by static fermentation. 3. A process as claimed in claim 1 wherein the fermentation is carried out by solid substrate fermentation. 4. A process as claimed in claim 1 wherein said Tolypocladium Sp inoculated into the nutrient medium is a master seed. 5. A process as claimed in claim 4, further comprising suspending spores of said Tolypocladium Sp in a medium containing glucose, peptone, casein acid, hydrolysate and sterile water maintained at a pH of between 4-6, inoculating a loopful of spore suspension of the species to agar slopes containing malt extract, yeast extract and agar at a pH of 4-6, and incubating said agar slopes until the culture attends the stage of sporulation to obtain the master seed. 6. A process as claimed in claim 1, which further comprises the step of building up of the inoculum of the species in at least a two stage process for inoculation into the fermentation medium. 7. A process as claimed in claim 6 wherein said first stage comprises transferring the master seed to a medium containing glucose, peptone, casein acid hydrolysate and sterile water at a pH of 4-6, and allowing growth for 3-4 days. 8. A process as claimed in claim 6 wherein said second stage comprises transferring the culture obtained from the first stage to a medium containing glucose, peptone, casein acid hydrolysate and sterile water at a pH of 4-6 and allowing growth for 2-3 days. 9. A process as claimed in claim 5, wherein the step of incubation is carried out for a period of 10-14 days. 10. A process as claimed in claim 1, wherein the nutrient medium comprises glucose, glycerol, casein acid hydrolysate, malt extract, peptone and DL .alpha.-amino butyric acid in sterile water at a pH of 4-6. 11. A process as claimed in claim 10 wherein said medium comprises 2 to 6% glucose, 2 to 5.5% glycerol, 1.5 to 5.5% casein acid hydrolysate, 0.5 to 4.5% malt extract, 0.25 to 2.5% peptone, 0.1 to 3% DL alpha amino burytic acid and sterile water. 12. A process as claimed in claim 1, wherein said step of decolourization is carried out by washing the ethyl acetate extract with sodium bicarbonate solution followed by water. 13. A process as claimed in claim 1, wherein the methanol extract of said biomass is prepared by shaking the biomass with methanol and subjecting the methanol extract so obtained to flash evaporation to obtain a pasty residue. 14. A process as claimed in claim 13, wherein the methanol residue is dissolved in distilled water to obtain an aqueous solution and then extracted with ethyl acetate to obtain an ethyl acetate fraction extract which is treated with sodium bicarbonate and washed with water. 15. A process as claimed in claim 14 wherein, concentration of the ethyl acetate fraction is obtained by flash evaporation. 16. A process as claimed in claim 1, wherein said solvent mixture is made of hexane, chloroform and methanol in the respective ratio of 10:9:1. 17. A process as claimed in claim 2, wherein the nutrient medium comprises glucose, glycerol, casein acid hydrolysate, malt extract, peptone and DL .alpha.-amino butyric acid in sterile water at a pH of 4-6. 18. A process as claimed in claim 17, wherein said medium comprises 2 to 6% of glucose, 2 to 5.5% of glycerol, 1.5 to 5.5% of casein acid hydrolysate, 0.5 to 4.5% of malt extract, 0.25 to 2.5% of peptone, 0.1 to 3% of DL alpha amino butyric acid and remainder sterile water.

Details for Patent 5,656,459

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2039-09-23