fig1 demonstrates one embodiment of the present invention . the twin - chamber syringe comprises a second chamber 1 having a charge is 1a of activity - sensitive human protein which has been introduced into the second chamber and lyophilized therein . the syringe further comprises a first chamber 2 housing a pyrogen - free sterile solvent . the first chamber 2 and second chamber 1 are separated by a rupturable membrane 3 . further , the second chamber 1 is in fluid communication with an injection needle ( arrow in fig1 ). in this way it is now possible to administer activity - sensitive human proteins in the high concentration desired , and within a short period of time . such preparations are in particular urokinase , prourokinase , tpa , streptokinase , factor viii and factor ix , and antithrombin iii . thus , with the usual chamber sizes of such syringes of up to 6 ml for the chamber containing the solution ( i . e . the active substance ) to be lyophilized , and up to 4 . 5 ml for the solvent , at least 2 to 3 million units of urokinase , pro - urokinase , tpa , streptokinase , at least appr . 1000 units of factor viii or factor ix , and at least appr . 500 to 1000 units of antithrombin iii can be introduced into the syringe chamber for the active substance , by charging it with 3 to 3 . 5 cm 3 of the concentrated plasma . a chamber with a volume of 6 ml cannot be charged with more than 3 to 3 . 5 cm 3 of the solution to be lyophilized , because during lyophilization the solution splashes and creeps up the wall . this emphasizes how important it is to use solutions with relatively high concentrations to charge the syringe , in order to accommodate therapeutically adequate amounts in the syringe chamber . a twin - chamber syringe of a known type has the lyophilized active substance in the front , i . e . the second chamber lying next to the needle . the separate sealing cap , onto which the needle is mounted , is positioned on the left side of the neck of this chamber . between the chambers there is a rupturable membrane supplied into small holes which , when the plunger is pushed down , presses the solvent contained in the solvent chamber through the holes . the rupturable membrane is projected ; into the front chamber , where by short shaking , the lyophilized active substance is dissolved , and the contents of the syringe can be administered immediately . because it is not possible to inject unlimited amounts of an injection solution , and the amount to be injected should not be too large , even in an injection i . v ., the volume of chamber 1 can hardly exceed 6 ml . for this reason , it is essential to accommodate as much of the active substance as possible by a single lyophilization step , i . e . at a time , in a chamber of up to this size . charging of the syringe is done in a known way by introducing under sterile conditions the given amount of a solution of the activity - sensitive human protein to be introduced , and then bringing the syringe to the next station where in situ lyophilization is carried out . the syringe with its lyophilized charge is then brought to the next station , where the pyrogen - free solvent is filled into the second chamber , followed by the final assembly and packing under sterile conditions , automated machinery as customary for charging ready - to - use syringes can be used for this , with only slight alterations to allow for the syringe size , inserting the plunger , and the shape of the sealing cap of the syringe , which differs from the usual shape of such stoppers . such syringes are suited not only for use in f . i . surgery and hospitals , but also particularily in first - aid medicine , and for self - treatment by patients at home . the process of concentrating solutions of the activity - sensitive human proteins will be illustrated in a number of examples . in this way , a significantly higher concentration can be obtained in solution than it was possible to obtain until now , so that a syringe chamber holding , f . i ., 6 ml , which can be filled with not more than 3 . 5 ml for lyophilization , so that no spilling - over and creeping occurs during lyophilization , can be provided with one single lyophilization step with a sufficient amount of protein to provide in the charge of one single syringe the amount of protein sufficient for effective treatment . starting with appr . 1500 l urine , a bentonite - activator complex is prepared in a larger number of , f . i . appr . 50 , batches , from which again crude urokinase is isolated in a known way . from the latter , a calcium phosphate - urokinase complex is formed in a number of batches , from which again concentrated urokinase with 500 000 to 600 , 000 i . u ./ ml , contained in a volume of 5 - 10 l is isolated in two batches . urine concentrated with boric acid is mixed with bentonite , the bentonite - activator complex is washed with water suitable for injection , eluted with a solution of 120 kg ammonium sulphate , 6 . 2 kg of a non - ionogenic ether glycol in the form of a bulk polymer , commercially available under the name of pluronic , with water added to give 310 l , and the eluate is salted out with appr . 165 kg ammonium sulphate . the crude urokinase bulkware obtained is eluted with a solution of 0 . 9 kg ethacridinium lactate in 430 l water suitable for injections , and the eluate is absorbed on a calcium phosphate gel , obtained from 11 kg sodium phosphate × 12 h 2 o , 6 . 4 kg calcium chloride × 2 h 2 o , with water added to give 180 1 . the calcium - urokinase complex is eluted with a solution of appr . 12 . 5 kg sodium dihydrogen phosphate × 2 h 2 o , with 3 n sodium hydroxide solution added to give 220 l , and the eluate is purified on cm - cellulose , or cms - sephadex , resp . the cm - cellulose - urokinase complex thus obtained is eluted with a solution of 50 kg sodium dihydrogen phosphate × 2 h 2 o , with 3 n sodium hydroxide solution added to give 165 1 , the eluate is acidified with phosphoric acid , pasteurized at 60 ° c . for appr . 10 h , dialyzed against 0 . 1m phosphate buffer solution , and then filtered . the dialysate is then tested according to standard procedures for purified concentrated urokinase . urokinase concentration usually ranges between 70 , 000 and 160 , 000 i . u ./ ml , and phosphate concentrations are the same as for 0 . 1m phosphate buffer solution for dialysis . optionally , the urokinase solution is diluted to the desired concentration by adding to the dialysate a suitable amount of albumin phosphate - buffer solution of ph 7 . 1 wherein the ratio of nah 2 po 4 × 2 h 2 o to na 2 hpo 4 × 12 h 2 o is 13 / 60 by wt . the solution with the desired concentration is then filled into syringes and lyophilized . the following ion exchangers and membrane filters are used in the preparation procedure : as ion exchanger carboxymethyl cellulose with an ion exchange capacity of 1 . 14 meq / g dry weight , cm - sephadex , type c - 25 , with an ion exchange capacity of 4 . 5 ±- 0 . 5 meq / g dry weight , and as membrane filter a polyamide filter with pore sizes of 0 . 2 μm , and 0 . 8 μm . isolation of antithrombin from blood plasma and blood products , in particular from the cohn plasma fraction vi - 1 - paste , is done in a known way by purification by polyethylene glycol fractionation , affinity - chromatography on heparin - sepharose 4b , and heat treatment at 60 ° c . for 10 h . doing this it is essential not to perform a gradient elution by means of a buffer with an increasing concentration of sodium chloride , but to maintain the concentration of the solvents for washing and elution , i . e . 0 . 4m nacl , 0 . 02m imidazole solution ph 7 . 2 , for washing the column , and 2m nacl , 0 . 02 imidazole solution , ph 6 . 5 , for elution , constant all the time . the antithrombin iii - solution obtained is concentrated through hollow fibres , mixed with sodium citrate , and stirred and pasteurized at 60 ° c . for 10 h . then salts are removed by dialysis on hollow fibres . ( solvent for dialysis : na 3 c 6 h 5 o 7 × 2h 2 o ( appr . 6 g appr . 5 g nacl , and 1 n hcl or 1 n naoh to adjust the ph to 7 . 5 , with water suitable for injection added to give 1000 ml .) the following steps were then performed under septic ( non - sterile ) conditions : after adjusting the titer in the dialysate d - mannitose ( 20 g / l ) is dissolved in it . ph is adjusted to 7 . 5 with 1 n hcl or 1 n naoh followed by filtration through a membrane filter with a pore size of 0 . 45 μm , and the solution is filled into the syringe chamber for lyophilization . the activity of this solution is 300 to 350 i . u ./ ml . preparation of a concentrated solution of human anti - hemophilic factor ( ahf or factor viii , resp .) ahf is prepared from pooled human plasma with the polyethylene glycol method . the frozen plasma is thawed under controlled temperature conditions , and the cryo - precipitate is recovered by centrifugation , resuspended in heparinized distilled water ( 80 u heparin per ml ), and the ph of the solution is adjusted to 7 . 0 with diluted hcl , and then it is resuspended at 28 ° c . by gentle mixing . the volume of the heparinized water is appr . 3 l / kg cryo - precipitate . polyethylene glycol is then added to the ahf solution in a final concentration of 3 %, and admixed well at 25 ° c . the ph of the ahf solution is then adjusted to 6 . 3 with diluted acetic acid . the suspension is then mixed at 25 ° c . for at least appr . 1 / 4 h , followed by centrifugation . the supernatant after centrifugation is filtered to remove all solid particles . then tri - n - butyl phosphate and polysorbat 80 are added to the filtered ahf solution to a final concentration of 0 . 30 % tnbp ( vol ./ wt .) and 1 % polysorbat ( wt ./ wt .). the ph is adjusted to 6 . 3 with diluted acetic acid , and the mixture is left to stand at 27 ° c . over night . then more peg is added to a final concentration of 12 %. the suspension is mixed for some time at 25 ° c ., as described above , and the ahf precipitate formed is recovered by centrifugation . the peg - precipitated ahf is resuspended at 5 ° c . in 1 . 6m glycine solution , containing 0 . 23m citrate and 13 u / l heparin . the volume of the glycine solution is 20 l / kg precipitated ahf . when the suspension is mixed to homogeneity , the ahf precipitate is recovered by centrifugation . the glycine - precipitated ahf is resuspended again in glycine solution , again containing citrate and heparin , at 5 ° c . the volume of the glycine solution is 20 l / kg precipitated ahf . after mixing of the solution to homogeneity the ahf precipitate is again recovered by centrifugation . this precipitate is resuspended in 0 . 4m edta ( ph 7 . 3 - 7 . 5 ) ( 8 l / kg ), and dialyzed against washing buffer ( 0 . 02 m sodium citrate , 0 . 05m sodium chloride . ph 6 . 6 to 7 . 0 ). this can be repeated several times until the desired concentration is reached . as an alternative , this precipitate can be dissolved in glycine - sodium citrate solution at 20 ° c ., where the ph is adjusted with diluted naoh or hcl to 7 . 3 . the volume of the glycine - sodium citrate solution is 20 l / kg glycine - precipitated ahf . this solution is then sterilized by filtration through previously sterilized membrane filters holding back bacteria , filled into the syringes under aseptic conditions , and lyophilized . the concentration is appr . 300 to 350 i . u ./ ml . the factor ix complex - powder ( prothrombin complex ) obtained in the usual way is dissolved in distilled water at a - protein concentration of 1 . 2 %. it is then mixed with diluting buffer ( 0 . 02m sodium citrate , 0 . 25m sodium chloride , ph 7 . 3 ; 4 . 0 l / l ), and 0 . 16 l 1m barium chloride solution per liter is added to precipitate factor ix protein . the precipitated protein is recovered by filtration or centrifugation , then dissolved in a solution of 0 . 4m edta at 8 l / kg , and diafiltered with washing buffer ( 0 . 02m sodium citrate , 0 . 05m sodium chloride ). the - diafiltered factor ix is repeatedly applied to a chromatography column containing dextrane sulphate - sio 2 - resin , and eluted , until the desired concentration is reached . the amount of resin in the column is appr . 2 . 5 l / 100 g processed starting material . the factor ix adsorbed on the resin is washed in the column with 10 or more volumes of washing buffer , which is then discarded . purified factor ix is eluate from the resin with a linear salt gradient by gradually increasing the amount of an elution buffer consisting of 0 . 02m sodium citrate and 0 . 5m sodium chloride , ph 6 . 6 to 7 . at the end of the gradient the column is further eluted with additional elution buffer . the factor ix eluate is pooled , filtered , and , optionally after diafiltration , ph adjusted to 7 , filled into the syringes and lyophilized . the average concentration of the solution is appr . 300 to 350 i . u ./ ml .
a twin - chamber syringe has a pyrogen - free sterile solvent in the chamber averted from the needle , and in the second chamber facing the needle a charge of activity - sensitive human protein , introduced and lyophilized in a single operation in the syringe , where it is stored , in a quantity necessary for therapeutically effective administration . the invention also relates to the filling of the syringe and the potential it offers for immediate use of activity - sensitive human proteins and for self - administration at home .