Improved method of purifying tfpi and tfpi analogs

Details Improved method of purifying tfpi and tfpi analogs Improved method of purifying tfpi and tfpi analogs

C - Chemistry, Metallurgy

07

K

C07K 14/81 (2006.01) A61K 38/57 (2006.01) C07K 1/36 (2006.01)

Patent

CA 2535562

Highly purified preparations of TFPI or TFPI analogs can be prepared using a method that generally involves the following steps: (1) expression of TFPI or TFPI analog in E. coli, (2) isolation of refractile bodies, (3) dissolution of the refractile bodies and refolding of the expressed TFPI or TFPI analog, (4) SP-Sepharose fast flow (FF) chromatography, (5) a first concentration and diafiltration step, (6) Q-Sepharose high (HP) performance chromatography, (7) butyl hydrophobic interaction chromatography (HIC), (8) SP-Sepharose HP chromatography, and (9) a second concentration/diafiltration step. Less than about 12% of the TFPI or TFPI analog molecules in such preparations are modified TFPI or TFPI analog species (i.e., oxidized, carbamylated, acetylated, deamidated, aggregated, or misfolded species).

L'invention concerne des préparations hautement purifiées d'analogues de TFPI ou TFPI pouvant être obtenues par le procédé comportant généralement les étapes suivantes: (1) expression d'analogue TFPI ou TFPI dans <i>E.</i> <i>coli, </i>(2<i>) </i>isolation de corpuscules réfrangibles, (3) dissolution des corpuscules réfrangibles et repliage de l'analogue TFPI ou TFPI exprimé, (4) chromatrographie SP-sépharose flux rapide (FF), (5) une première étape de concentration et de diafiltration, (6) chromatographie Q-sépharose haute performance (HP), (7) chromatographie d'interaction hydrophobe (HIC) au butyle, (8) chromatographie SP-sépharose HP, et (9) une deuxième étape de concentration/diafiltration. Moins de 12 % environ des molécules d'analogue TFPI ou TFPI de ces préparations sont modifiés par des espèces d'analogue TFPI ou TFPI (<i>i.e</i>., espèces oxydées, carbamylées, acétylées, désamidées, aggrégées ou mal pliés).

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