Patent Application: US-91316297-A

Abstract:
a process for preparing thrombin which comprises treating a mixture comprising prothrombin , factor xa , factor va , and phospholipids with calcium ions , at a ph of 6 . 0 - 7 . 0 is provided . in particular the ph of 6 . 0 - 7 . 0 may be generated by the addition of the calcium ions or by buffering the preparation to a ph of 6 . 0 - 7 . 0 . thrombin preparations so produced may be subjected to further purification and are particularly stable even when substantially free of exogenous stabilizing agents such as proteins , sugars , polyol and mixtures thereof , and may be subject to freeze - drying and a virus inactivation by heat treatment .

Description:
embodiments of the present invention will now be described by way of example , with reference to the attached figures . fig1 shows the thrombin clotting activity in u / ml and specific activity in u / mg of 15 separate batches of thrombin prepared in accordance with one embodiment of the present invention . fig2 shows the amount of thrombin in u / ml generated over a range of added calcium ion concentrations , in accordance with an embodiment of the present invention . preparation of a mixture comprising prothrombin , factor xa , factor va and phospholipid by deae - cellulose 450 liters of cryoprecipitate plasma was adjusted to ph 6 . 9 ± 0 . 05 and diluted with 150 liters of pyrogen free h 2 o to a final volume of 600 liters . 6 kg of deae - cellulose gel ( de - 52 whatman ) was then added to the plasma / water solution and the resulting suspension mixed continuously for one hour to bind the clotting factors to the gel . the gel was then collected by centrifugation and the supernatant discarded . the gel was then resuspended in 30 mm citrate , 30 mm phosphate ph 6 . 9 buffer and the resulting suspension was poured into a chromatography column . the column was then packed by washing with 21 liters of the same buffer . the clotting factors were then eluated from the column with an elution buffer of 30 mm citrate , 30 mm phosphate , 200 mm nacl , ph 6 . 9 . the eluate pool ( 3 . 1 liters ) was then filtered ( 0 . 45 μm pore size ) into sterile bottles and frozen . the eluate pool contains substantial amounts of prothrombin ( factor ii ) ( at 80 μm and about 25 % of the total protein ). it also includes factors ix and x , activated and non - activated ( at about 5 μm ), coagulant - active phospholipid and sufficient trace amounts of factors v and viii to support the physiological conversion of prothrombin to thrombin via the intrinsic clotting pathway . frozen deae - cellulose eluate ( prepared according to example 1 ) was thawed at room temperature or in a 37 ° c . water bath . ( typical values of the eluate were as follows : conductivity = 17 ms ; ph = 7 . 0 ; 30 mm citrate ; 30 mm phosphate ; 200 mm sodium ; 200 mm chloride ; 15 mg / ml total protein and prothrombin 60 u / ml ). 1m cacl 2 solution was then added dropwise to the thawed eluate , with stirring , at a ratio of 75 ml cacl 2 to 1 , 000 ml eluate , at 20 ° c . this resulted in a final calcium concentration of 70 mm and a drop in ph in the mixture to ph 6 . 4 - 6 . 6 . the reaction was allowed to proceed with stirring overnight for 18 hours at 20 ° c ., to convert the prothrombin to thrombin . in 15 experiments , the thrombin clotting activity was 6 , 333 ± 1 , 146 u / ml ( mean ± sd ) and specific activity of 508 ± 110 u / mg ( see fig1 ). sds page indicated that effectively all the prothrombin band was converted into bands co - migrating with thrombin , by the end of the activation period . thrombin clotting activity was measured by fibrinogen clotting time at room temperature with visual detection and duplicate samples . to 200 μ1 of human fibrinogen solution at 5 mg / ml in 50 mm tris - hcl 100 mm nacl ph 7 . 5 was added 100 μl of standard ( 1 - 4 u / ml ) or test solutions of thrombin diluted in 50 mm tris - hcl , 100 mm nacl ph 7 . 5 supplemented with 100 mm cacl 2 and 0 . 1 % w / v bovine serum albumin , whereupon time to subsequent clot formation was recorded . a standard curve was constructed by plotting log 10 thrombin concentration ( u / ml ) against log 10 clotting time ( sec ) using bovine thrombin standardised against the human alpha - thrombin standard 89 / 588 . thrombin clotting activity of test samples was derived by extrapolation from the standard curve ( 15 ). following the procedure described in example 1 , resulted in a mixture with a ph of 7 . 0 - 7 . 2 . this immediately decreased to ph 6 . 5 on addition of cacl 2 to 70 mm . there was then a steady decrease in ph to 6 . 1 - 6 . 3 during the conversion period ( 18 hours ). the fall in ph was a requirement for the successful generation of thrombin of high activity . this was demonstrated by comparative experiments , where the ph of the solution was adjusted to ph 7 . 0 or ph 7 . 5 immediately after the addition of cacl 2 . here the final ph values at the end of the reaction period were ph 6 . 7 and ph 7 . 1 respectively and a much lower amount of thrombin activity was generated ( see table 1 ). table 1______________________________________ ph of the mixture , ph of the mixture immediately after at the end of the clotting cacl . sub . 2 addition conversion period activityexample ( adjusted as necessary ) ( 18 hours ) ( iu / ml ) ______________________________________1 6 . 5 6 . 1 57162 7 . 0 6 . 7 20123 7 . 5 7 . 1 1493______________________________________ in a further experiment , the mixture was buffered ( 20 mm mes ) to ph 6 . 5 immediately after cacl 2 addition . this resulted in an additional small increase in conversion to thrombin , but the - increase in clotting activity was insignificant . effects of varying the length of time or temperature employed for conversion of prothrombin to thrombin studies were carried out to determine the optimum time course for the conversion of prothrombin to thrombin . a comparison of the amount of thrombin generated at 16 and 24 hours indicated that a plateau had been reached by 16 hours . an investigation was also carried out to determine the effect of incubation at 37 ° c . for one hour prior to subsequent room temperature incubation , in view of a report that this step was necessary to obtain useful yields with this type of feedstock ( european patent application no . 92401889 . 8 ). it was found that while the initial rate of thrombin generation exceeded that obtained at room temperature , the final yield of thrombin was no better at 16 or 24 hours as compared to conversion at room temperature . the amount of thrombin generated at 24 hours with a range of added calcium ion concentrations ( seven batches of deae - cellulose eluates ) was determined ( fig2 ). it was found that the addition of 70 mm calcium consistently resulted in efficient conversion of prothrombin to thrombin . thrombin prepared according to example 2 was mixed by stirring with 0 . 3 % tri -( n - butyl ) phosphate and a 1 % solution of tween 80 at a temperature of 20 °- 30 ° c . for 6 to 24 hours . this was sufficient to inactivate any contaminating lipid - enveloped viruses . the solvent / detergent was removed by chromatography . the chromatography step serves to remove solvent and detergent and to purify the intermediate purity thrombin . a 1 . 6 cm diameter chromatography column was packed with 10 ml of s - sepharose ff ( trademark ) at a linear flow rate of 2 . 2 cm / min ( equivalent to 4 . 5 ml / min ) using 40 mm gluconic acid , 20 mm mes , or 20 mm citrate all at ph 6 . 5 . 100 ml of solvent / detergent treated thrombin according to example 6 or intermediate purity thrombin according to example 2 , following a 1 + 3 dilution in equilibrating buffer was filtered at 0 . 45 μm and applied to the column at the same flow rate . the column was then washed with equilibrating buffer until the absorbency at 280 nm returned to baseline and solvent or detergent were detectable only below acceptable low levels , in the column effluent ( typically approximately 150 ml ). thrombin was then eluted from the column by washing with equilibrating buffer containing 0 . 5m nacl . purified thrombin was obtained in about 25 ml at a typical concentration of 4 , 000 u / ml and 2 mg / ml protein . the typical yield of purified thrombin after the chromatography step was 88 ± 16 %. this yield refers to a thrombin preparation which was not subjected to a solvent / detergent virus inactivation step as described in example 6 . thrombin prepared according to examples 2 or 6 was centrifuged at 3 , 000 rpm for 20 minutes at room temperature and then filtered through a millpore prefilter ( ap25 ) followed by a whatman 0 . 2 μm filter ( polydisc as ). the filtered solution was then diluted in a formulation buffer ( 40 mm gluconic acid or 20 mm mes , 20 mm trisodium citrate , 150 mm nacl , ph 6 . 5 ) to a thrombin activity of 600 u / ml and dispensed in 2 ml lots into glass vials for freeze - drying . freeze - drying was performed in a super - modulyo ( edwards , crawley ) freeze - dryer with a freezing temperature setting of - 45 ° c ., followed by a primary drying temperature setting of - 25 ° c . and a secondary drying temperature setting of + 20 ° c . the vials were then heat - treated to inactivate any contaminating viruses , at 80 ° c . for 72 hours . thrombin was formulated in a variety of formulation buffers , in order to determine the optimum formulation buffer for stabilising thrombin during freeze - drying and subsequent heat - treatment ( virus - inactivation ). intermediate purity thrombin prepared according to example 2 and purified thrombin prepared according to example 7 , were diluted with various formulation buffers ( as described in table 2 ) to a thrombin concentration of 600 u / ml . the thrombin preparation was then freeze - dried according to example 8 and a quantity of the freeze - dried thrombin was also subjected to a heat - treatment of 80 ° c . for 72 hours . thrombin clotting activity was determined , as previously described , to determine the percentage clotting activity that remained after freeze - drying and subsequent heat - treatment . the results are shown in table 2 . it can be seen from table 2 that formulation buffers comprising 20 mm tris - hcl buffer at ph 7 . 2 with or without 20 mm trisodium citrate and / or 150 mm sodium chloride , resulted in a recovery of thrombin clotting activity , after freeze - drying , of greater than 74 %. however , large losses in activity were seen post - heat - treatment , particularly in the absence of trisodium citrate . the inclusion of sodium chloride in the formulation buffer gave rise to an intact plug of material , whereas without sodium chloride , the plug retracted and collapsed . protein ( e . g . human albumin ) can also be included in the formulation at concentrations of 0 . 5 g / l - 10 g / l , to act as a bulking agent and improve plug structure and appearance . when the formulation buffer was made acidic by using gluconic acid or mes buffered at ph 6 . 5 , recovery of thrombin clotting activity after dry heat - treatment was substantially improved . long term stability was determined using the gluconic acid buffer formulation ( see table 2 ). these studies were performed by storing several vials at 4 ° c . and 37 ° c ., after freeze - drying and heat treatment . no loss in thrombin clotting activity was observed over a six month period , when comparing the 37 ° c . stored thrombin to the 4 ° c . stored thrombin . table 2______________________________________recovery of clotting activity (%) intermediate high purityformulation purity thrombin thrombinbuffer post - fd post - ht post - fd post - ht______________________________________20 mm tris - hcl 96 12 93 12ph 7 . 220 mm tris - hcl 92 56 91 56ph 7 . 2 + 20 mmtrisodiumcitrate20 mm tris - hcl 87 10 74 4ph 7 . 2 + 150 mmnacl20 mm tris - hcl 91 41 90 51ph 7 . 2 + 20 mmtrisodiumcitrate + 150 mm nacl20 mm gluconic 100 99 93 85acid + 20 mmtrisodiumcitrate + 150 mmnacl ph 6 . 520 mm mes + 20 mm nd 97 nd 86trisodiumcitrate + 150 mm naclph 6 . 5______________________________________ fd = freezedrying ht = heattreatment of 80 ° c . for 72 hours in vial nd = not done 1 . fenton , j . w . 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