Patent Application: US-20588188-A

Abstract:
a method of preparing von willebrand factor by disassociating it from a chaotropic agent in solution therewith and preferably treating the same under controlled temperature either in liquid or lyophilized form .

Description:
the main objects of the present invention include : ( a ) the manufacture of a therapeutically effective vwf from waste material which heretofore has been discarded as a bi - product of the process for the manufacturing of viii ; and ( b ) increasing the platelet agglutination activity of vwf by subjecting the same to a controlled temperature environment . the starting material used in the process of the present invention is the waste solution of a chaotropic agent / vwf . said chaotropic agent dissocitates the vwf from the antibodies covalently linked to a solid phase backbone . the preferred chaotropic agent is sodium thiocyanate in the range of from about 0 . 5 m to about 5 . 0 m , however , other agents may be used as well including ethylene glycol , lithium chloride , potassium iodide , urea , ethylamine , ethanolamine , ethylenediamine , diaminohexane , glycerol , dimethylaminopropylamine and combinations thereof . to obtain this starting material , processes known and used by the prior art can be utilized including that disclosed by the above denoted references . the process leading to the chaotropic agent / vwf solution is as follows : cryoprecipitate , obtained from human or animal plasma or commercial concentrate , containing from about 2 , 000 to about 40 , 000 units of viii per liter of column matrix and from about 8 , 000 to about 80 , 000 units of vwf per liter of column matrix is reconstituted in a glycine - sodium chloride solution . the solution is then treated with aluminum hydroxide gel in order to remove vitamin - k dependent coagulation factors . the cryoprecipitate solution is then introduced into a column containing anti - vwf antibodies covalently linked to a solid phase backbone having reactive chemical groups available to bind the antibodies . the amount of antibody used will vary depending on its affinity , however , about 1 gram of antibody per liter of solid phase backbone is required . the solid phase consists of agarose , sephadex , microporous glass , membranes or other solid substances with reactive chemical groups an example of such solid phase is 2b sepharose sold by pharmacia , inc . to . effect the covalant linkage between the solid phase and the antibodies , reagents , such as cyanogen bromide , triazine , and hydrazine may be used . the following defines substances referred to by trademarks : sephadex refers to cross - linked dextrans also known as polidexide and poly [( 2 - diethylamino ) ethyl ] polyglycerylene dextran hydrochloride ( the merck index , tenth edition , # 7426 ). sephadex g - 25 is an extensively cross - linked form of sephadex used for the separation of low - molecular - weight substances , such as sugars and peptides ( molecular biology of human proteins , schultze , h . e . and heremans , j . f ., vol . 1 , elsevier publishing co ., ( 1966 ) 290 - 299 . polysorbate 80 is polyoxyethylene ( 20 ) sorbitan mono - oleate ( the merck index , tenth edition , # 7455 ). from a cryoprecipitate containing 10 , 000 units of viii and 40 , 000 units of vwf per liter of column matrix about 8 - 9 , 000 units of viii and about 30 , 000 units of vwf per liter of column matrix will bound to the column . the unbound proteins , including fibronectin and fibrinogen , are washed from the column using a buffer solution containing about 0 . 0025 to 0 . 05 m histidine ( buffering agent ), 0 . 05 to 2 . 5 m nacl ( solubilizing agent ) and 0 . 05 to 0 . 5 m lysine ( stabilizer against plasmin and plasminogen proteolytic enzymes that tend to degrade viii and vwf ). factor viii then eluted from the column using 0 . 25 to 2 m calcium chloride solution or solutions of other similarly effective chaotropic agents . the column is next treated with a 0 . 50 to 5 m solution of a chaotropic agent , preferably sodium thyocianate , to dissociate vwf from the antibodies and , at the same time , to regenerate the column for further use . having attained the vwf in the chaotropic solution , it is of the utmost importance to immediately separate the vwf to prevent its rapid degradation . separation can be effected by desalting , precipitation and dialyzation . the desalting process comprises : transferring the vwf / chaotropic agent solution onto a desalting column , such as sephadex g - 25 , which has previously been equilibrated with a buffer solution containing from about 0 . 01 to about 0 . 5 m nacl and about 1 to about 10 mm of histidine ; eluting vwf with the same buffer used to equilibrate the column ; sterile filtering the solution ; and lyophilizing the vwf solution . separation of vwf from the chaotropic agent can be effected by dializing against a buffer solution , described under the desalting process , at neutral or close to neutral phs using state of the art techniques . after dialyzation , the vwf is sterile filtered , formulated , and lyophilized . precipitation of vwf can be accomplished by using precipitating agents , such as polyethylene glycol ( peg ) having a molecular weight of from 4 , 000 to 25 , 000 , ammonium sulfate and the like . the concentration of peg is about 6 % to 15 % w / w , while that of ammonium sulfate is about 25 % to 50 % w / w . the precipitate is then isolated by centrifugation and the vwf so obtained dissolved in a buffer containing from about 0 . 01 to about 0 . 5 m tris and 0 . 01 to about 1 . 5 m nacl at a close to neutral ph . the solution is then treated as described above . the lyophilized factor can then be reconstituted when needed for injection . it is preferable to subject the reconstituted vwf to controlled heat treatment to increase its activity as above - described . alternatively , the heat treatment may be carried out prior to lyophilization . the platelet - ristocetin assay being referred to in the examples is essentially the assay method described in thrombos . diathes . haemorrh . ( stuttg . ), 1975 , 34 , 306 - 308 , except for the modification that we use : commerical , lyophilized fixed platelet ; normal pooled plasma at 1 / 2 ( 100 %), 1 / 4 ( 50 %) and 1 / 8 ( 25 %) dilution to prepare a standard curve ; and plasma and vwf preparation in a buffer of ph 7 . 0 . a one liter column of anti - vwf agarose was saturated with a cryoprecipitate solution as described in the prior art . it was washed with 4 volumes of fresh buffer ( 0 . 15 m nacl , 0 . 1 m lysine , 0 . 02 m histidine , ph7 ), then eluted with 2 volumes of 0 . 25 m cacl 2 followed by 2 volumes 3 m nascn . the eluted nascn / protein solution ( 450 ml ) was dialyzed for 18 hours against 5 liters 0 . 05 m nacl , 10 mm histidine , at ph 7 . 2 . the low salt solution containing 0 . 23 mg / ml protein , was heated at 45 ° c . for 4 hours . the agglutination activity increased from 0 . 1 unit / ml to 15 units / ml . the vwf protein was purified as described in example 1 and 480 ml of the eluted nascn protein was desalted over a 10 × 20 cm column of sephadex g - 25 which was previously equilibrated with a solution of 0 . 05 m nacl , and 5 mm histidine at ph 7 . 3 . the protein was then eluted in a 510 ml volume free of nascn . the desalted vwf solution containing 0 . 3 mg / ml was frozen and dried by lyophilization in 30 ml aliquots using 50 ml vials . the dried vials were sealed under vacuum and stored at room temperature for up to 6 months . they were reconstituted with 5 ml water to yield a clear solution containing 1 . 8 mg of vwf / ml and 1 . 5 units of vwf / ml . one of the reconstituted vials was heated at 50 ° c . for 2 hours . the activity of vwf was found to increase the activity to 62 units / ml . vwf protein was purified and desalted as in example 2 . a solution volume of 780 ml , containing 0 . 24 ng of vwf / ml was heated at 50 ° c . for 2 hours to increase the agglutination activity from 0 . 5 to 19 units / ml . the solution was then filtered through 0 . 22 um membranes and 30 ml aliquots were placed into separate 50 ml vials for freeze drying as in example 2 . the dried and sealed vials were stored for 3 months and yielded 90 units of vwf / ml when reconstituted with 5 ml of water . a 1 m solution of the nacl was added to cryosolution prior to purification of the vwf proteins as described in example 1 . the eluted nascn protein was desalted in a 800 ml volume as in example 2 , then formulated with 0 . 5 % polysorbate 80 and 2 % mannitol to aide filtration and lyophilization . the solution was sterilized through a 0 . 22 um membrane filter and freeze dried in 30 ml aliquots . upon reconstitution with 5 ml water , a solution containing 3 . 1 mg of vwf / ml and 2 units of vwf / ml was reactivated by heat treatment at 52 ° c . for 1 . 5 hours to yield 140 units of vwf / ml . sufficient amount of na 2 so 4 was added to cryosolution to give a 0 . 3 m na 2 so 4 solution prior to purification of the vwf proteins described in example 4 . an 850 ml volume was collected from the desalting column and formulated with 0 . 5 % polysorbate 80 and 2 % mannitol . the formulation was then heated at 50 ° c . for 2 hours . a solution containing 0 . 77 mg of vwf / ml and 60 units of vwf / ml was then filtered and lyophized as described in example 4 . sodium thiocyanate eluate from the anti - vwf affinity column was fed directly onto a sephadex g - 25 column equilibrated with 0 . 05m tris ( hydroxymethyl ) aminomethane and 0 . 15m nacl buffer at ph 7 . 1 . to the sephadex g - 25 eluate was added dropwise , saturated ammonium sulfate adjusted to ph 7 . 1 in one case and to ph 4 . 5 in another to bring the final concentration of ammonium sulfate to 50 % in the eluate . after 30 minutes at room temperature both preparations were collected by centrifugation and dissolved in 0 . 05m tris 0 . 15m nacl at ph 7 . 1 . an aliquot of the ph 7 . 1 ammonium sulfate mixture was kept overnight at 4 ° c . and then treated in the same way . all of the reconsituted precipates retained vwf activity as measured by the platelet - ristocetin assay . sodium thiocyanate eluate from anti - vwf affinity column was fed directly onto a sephadex g - 25 column to remove the sodium thiocyanate and effect an exchange for 0 . 05m tris ( hydroxymethyl ) aminomethane , 0 . 15 m sodium chloride buffer , at ph 6 . 3 . 12 grams of polethylene glycol ( peg ) 4000 / 100 ml eluate ( 10 grams of peg 6000 / 100 ml eluate ) was then added and the mixture stirred for 30 minutes at room temperature . the precipitate so formed was isolated by centrifugation , dissolved in 0 . 05m tris - 0 . 15m nacl buffer , at ph 7 . 25 at 1 / 10 the volume of the initial g - 25 eluate . the vwf activity was present in this solution as measured by the platelet - ristocetin assay . analytical characterization of vwf solution showed that platelet agglutination activity gradually increased at room temperature and this increase was amplified by increasing the temperature and prolonging the time of incubation . analytical characterization has also shown that the molecular distribution and binding properties of vwf were unaltered under these conditions . the data shown in tables i and ii are representative of that obtained according to the present invention . table i______________________________________yield and specific activity of vwf vwf specific total activitysample units yield % units / mg______________________________________cryoprecipitate 44 , 451 100 0 . 498solutionunbound pool 10 , 373 23 . 34 0 . 118vwf bound to 34 , 078 76 . 66 -- moab columnlyophilized vwf 18 , 990 42 . 72 48 . 20product______________________________________ table ii______________________________________increase in vwf activityat recovery at incubation specific specific at at unit / activity unit / activity ° c . time / sample ml unit / mg ml unit / mg temp . hrs . ______________________________________1 2 . 49 3 . 77 46 . 50 70 . 45 37 25 34 . 80 52 . 72 rt 25 54 . 36 82 . 31 37 302 1 . 77 -- 12 . 33 -- 37 243 1 . 70 -- 11 . 50 -- 37 244 1 . 50 3 . 00 16 . 75 33 . 50 45 1 20 . 40 40 . 80 45 2 18 . 80 37 . 60 45 4 16 . 80 33 . 60 52 1 21 . 10 42 . 20 52 2 16 . 90 33 . 80 52 55 1 . 05 -- 3 . 45 -- 25 2 23 . 20 -- 25 246 1 . 97 0 . 47 3 . 80 0 . 91 25 4 18 . 80 4 . 48 over - 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