Patent Abstract:
The invention relates to the use of acridine or acridine derivatives, preferably in combination with benzalkonium chloride, for the inactivation of enveloped or nonenveloped viruses. The process according to the invention is preferably carried out in the presence of proteins whose biological activity is substantially retained.

Full Description:
This application claims foreign priority of Fed. Rep. Germany application P44440456, filed Dec. 10, 1994. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to the use of acridine or acridine derivatives, preferably in combination with benzalkonium chloride, for the inactivation of enveloped or nonenveloped viruses. The process according to the invention is preferably carried out in the presence of proteins whose biological activity is largely retained. 
     BACKGROUND OF THE INVENTION 
     It has been known for years that untreated human plasma or serum can contain human pathogenic viruses, such as HIV, HBV or HCV, which, if transmitted to sensitive recipients, can cause serious diseases, such as AIDS or hepatitis. In order to prevent this potential virus transmission, therapeutics which are obtained from human plasma or serum are prepared, on the one hand, only from preselected starting materials which, as far as anyone can judge, are virus-free and, on the other hand, are subjected to virus-inactivating/eliminating steps in the preparation process. The efficiency of the virus inactivation/elimination method used is established using strict measures and continuously checked. 
     Besides physical virus inactivation steps, chemical virus inactivation steps are also known in the preparation of said therapeutics. A particularly frequently discussed chemical process is the SD (solvent/detergent) method. It is suitable for inactivating enveloped viruses, i.e. viruses which are surrounded by a lipid-containing membrane, but has the crucial disadvantage of being completely ineffective against all known nonenveloped (uncoated) viruses. Moreover, also no other chemical process is known which would be suitable to inactivate nonenveloped viruses while simultaneously retaining the biological activity of the protein constituents of the therapeutic or of the human plasma or serum. 
     Although the chemical virus inactivation processes are only used in a supplementary capacity to the physical methods, and although most viruses potentially transmissible by blood and blood products carry a lipid coat, for reasons of safety there is an exceptional need to make available chemical inactivation processes which also reliably inactivate nonenveloped viruses. This is all the more desirable as recently also HAV and parvoviruses, for example parvovirus B 19, were discussed as viruses which are potentially transmissible by blood fluids or blood products. (Vox Sanguinis 67, Supplement 1, 1994: Proceedings of a Symposium held at the New York Blood Center). 
     The invention was also based on the object of developing an industrially utilizable process for chemical virus inactivation, in which enveloped and nonenveloped viruses, e.g. parvo viruses, are inactivated with retention of the biological activity of proteins present, e.g. therapeutically useful proteins. 
     SUMMARY OF THE INVENTION 
     This object is achieved according to the invention by adding acridine or an acridine derivatives to the protein-containing liquid to be treated. It was surprisingly found that acridine or acridine derivatives inactivate enveloped and nonenveloped viruses. Acridine derivatives are, for example, ethacridine, 9-aminoacridine (=amina-crine), 3,6-acridinediamine (proflavine), acrisorcin, Acrizane chloride (=phenacridane chloride), Acridine Orange, quinacrine, acricide, acridone, acridine-9-carboxylic acid, acranil (1-[(6-chloro-2-methoxy-9-acridinyl)amino]-3-(diethylamino)-2-propanol dihydro-chloride), 3,7-diamino-5-phenylphenazinium chloride (phenosafranin, Safranin B Extra), phenoxazine, phenothiazine and especially acriflavine (3,6-diamino-10-methylacridinium, chloride and 3,6-acridineidiamine) and their salts, e.g. chlorides, sulfates, bromides. 
     Surprisingly, it was additionally found that a combination of acridine or an acridine derivative and benzalkonium chloride displays a synergistic action during virus inactivation, i.e. the magnitude of the virus inactivation of the combination is higher than that of each individual substance. 
     The virus inactivation process according to the invention can be carried out with protein solutions such as blood, serum, plasma, blood products, allantoic fluid or milk. The virus inactivation is carried out at a pH of 3 to 10 or 5 to 9 (in, for example, serum or plasma solutions) and a temperature of 1° C. to 80° C., preferably of 20° C. to 60° C., very preferably from 20° C. to 40° C. or 25° C. to 37° C., and lasts 30 minutes to 10 hours, preferably 2 to 5 hours. For virus inactivation, a concentration of 1.0 g/l-0.00001 g/l or 1.0 g/l-0.004 g/l, preferably 0.1 g/l-0.001 g/l, is used for the acridines or acridine derivatives, and a concentration of 0.1 g/l-0.004 g/l, preferably 0.05 g/l-0.01 g/l, for benzalkonium chloride. 
     The removal of the acridines and of the benzalkonium chloride from the protein solutions, if this is necessary, is possible by means of simple, known methods, such as adsorption on active carbon or dialysis. 
     A further advantage of the virus inactivation process according to the invention is the very extensive protection of the protein constituents of the material to be treated: different biological activities, e.g. antibody activity and clotting-activity, are not reduced or only reduced to a tolerable extent. 
     The virus inactivation process according to the invention can therefore be employed, for example, for the decontamination of the following materials: 
     protein-containing solutions (dilute or concentrated) 
     blood or blood products; both the liquid and the cellular constituents 
     serum, plasma 
     allantoic fluid 
     organ extracts 
     milk 
     buffer solutions 
     antigens for diagnostics 
     vaccines, antigens for vaccines 
     The process according to the invention is furthermore suitable for disinfection in virus contamination of, for example, areas, equipment, effluents, wastes and surfaces of all types. The disinfection of virus-contaminated organ transplants, e.g. cornea, cerebral meninges, liver, heart, lungs or kidneys is also possible. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is furthermore illustrated by the following examples. 
     Methods generally used in the process according to the invention 
     Virus: Was replicated in a known manner in tissue cultures; the virus harvests were centrifuged and used as starting material for the further investigations. 
     The infectiousness titer was determined in microtiter plates—8 replicates of 0.1 ml/-dilution stage—by double titration. 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Stock 
                 Ethacridine 
                 3% strength 
               
               
                   
                 solutions: 
                 lactate (EL) 
                 in dist. water) 
               
               
                   
                   
                 Entozon ® (E) 
                 3% strength Acridines 
               
               
                   
                   
                   
                 in dist. water} 
               
               
                   
                   
                 Acriflavine (A) 
                 1% strength 
               
               
                   
                   
                   
                 in dist. water) 
               
               
                   
                   
                 Benzalkonium 
                 10% in dist. 
               
               
                   
                   
                 chloride (BACI) 
                 water 
               
               
                   
                   
               
             
          
         
       
     
     General experimental procedure: 
     9 parts of buffer or medium or protein solutions were mixed with 1 part of virus. The addition of the amounts of the stock solutions indicated in the individual examples for virus inactivation and renewed mixing with subsequent virus titration were then carried out. After the times and temperatures mentioned in the individual examples, samples were removed and titrated in double determinations in order to be able to measure the process-related virus inactivation. 
     Types of virus investigated 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abbreviation 
                 Name 
               
               
                   
                   
               
             
             
               
                   
                 PI 3 V 
                 Bovine parainfluenza 3 virus 
               
               
                   
                 BPV 
                 Bovine parvovirus 
               
               
                   
                 PPV 
                 Porcine parvovirus 
               
               
                   
                 IBRV 
                 Infectious bovine rhinotracheitis 
               
               
                   
                   
                 virus 
               
               
                   
                 BVDV 
                 Bovine viral diarrhoea virus 
               
               
                   
                 CPV 
                 Canine parvovirus 
               
               
                   
                 BAV-1 
                 Bovine adenovirus type 1 
               
               
                   
                 Reo 3 
                 Reovirus type 3 
               
               
                   
                 Influenza A 
                 Influenza A virus-Shangdong 
               
               
                   
                 Influenza B 
                 Influenza B virus - B Panama 
               
               
                   
                   
               
             
          
         
       
     
     Further abbreviations/names used in the text: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 EME Medium 
                 Eagles Minimum Essential Medium 
               
               
                   
                 Beriate ® P 
                 Clotting factor VIII:C concentrate, 
               
               
                   
                   
                 Pasteurized (Behringwerke AG, Marburg, 
               
               
                   
                   
                 Germany) 
               
               
                   
                 Haemate ® P 
                 Concentrate from clotting factor VIII 
               
               
                   
                   
                 and Von-Willebrand factor, pasteurized 
               
               
                   
                   
                 (Behringwerke AG, Marburg, Germany) 
               
               
                   
                 Beriplex ® P 
                 pasteurized prothrombin complex con- 
               
               
                   
                   
                 centrate (Behringwerke AG, Marburg, 
               
               
                   
                   
                 Germany) 
               
               
                   
                 Venimmun ® 
                 Human polyvalent immunoglobulin 
               
               
                   
                   
                 preparation (7S) for intravenous use 
               
               
                   
                   
                 (Behringwerke AG, Marburg, Germany) 
               
               
                   
                 Entozon ® 
                 Preparation of the following composi- 
               
               
                   
                   
                 tion: 
               
               
                   
                   
                 1 g of granules contains 0.059 g of 
               
               
                   
                   
                 dimethoxy-6-nitro-9-[(3-diethylamino- 
               
               
                   
                   
                 2-hydroxy)propylamino]acridine dihy- 
               
               
                   
                   
                 drochloride and 0.295 g of ethacridine 
               
               
                   
                   
                 lactate (ASID Veterinär Vertriebs 
               
               
                   
                   
                 GmbH) 
               
               
                   
                 QAE cellulose 
                 Diethyle-2-hydroxypropylaminoethyl 
               
               
                   
                   
                 cellulose 
               
               
                   
                   
                 (Ion exchanger for protein purifica- 
               
               
                   
                   
                 tion) 
               
               
                   
                 FCS 
                 Fetal calf serum 
               
               
                   
                   
               
             
          
         
       
     
     Example 1 
     Inactivation of PI 3 V by BACI 
     EME medium was mixed with PI 3  virus and BACI and incubated at 37° C. in a water bath. After the times indicated, samples were taken to test for virus inactivation. The results are shown in Tab. 1. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inactivation of PI 3  virus with BACI at 37° C. 
               
             
          
           
               
                   
                 BACI 
                 Virus titer 
                 Virus inactiva- 
               
               
                   
                 addition 
                 found 
                 tion 
               
               
                 Time (h) 
                 (mg/ml) 
                 (log 10 /ml) 
                 (log 10 /ml) 
               
               
                   
               
             
          
           
               
                 0-0.03 1)   
                 0 (control) 
                 7.4 
                 — 
               
               
                   
                 0.1 
                 ≦1.5 
                 ≧5.9 
               
               
                   
                 0.005 
                 ≦1.5 
                 ≧5.9 
               
               
                   
                 0.025 
                 6.0 
                 1.4 
               
               
                   
                 0.0125 
                 6.9 
                 0.5 
               
               
                 1 
                 0 (control) 
                 6.8 
                 — 
               
               
                   
                 0.1 
                 ≦1.5 
                 ≧5.3 
               
               
                   
                 0.05 
                 ≦1.5 
                 ≧5.3 
               
               
                   
                 0.025 
                 ≦1.5 
                 ≧5.3 
               
               
                   
                 0.0125 
                 5.5 
                 1.3 
               
               
                   
               
               
                   1) Identical after addition of BACI and thorough mixing of the reaction batch  
               
             
          
         
       
     
     As emerges from the results in Table 1, PI 3  virus is very rapidly inactivated by the high doses of BACI (0.1 mg/ml, 0.05 mg/ml), i.e. in the time which was needed to mix the virus-containing sample thoroughly with BACI, to take a sample and to titrate this in order to determine the infectiousness of PI 3  virus, infectious virus was no longer detectable. At the two other BACI concentrations tested (0.025 mg/ml and 0.0125 mg/ml), a clear concentration-time dependence of virus inactivation is discernible. 
     Example 2 
     Virus Inactivation by Means of BACI or Entozon 
     The virus species shown in Tab. 2 were treated with BACI or Entozon and incubated at 45° C. Sample taking for virus titration was-carried out 1 or 2 hours after the start of the test. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Virus inactivation by means of BACI or Entozon at 45° C. 
               
             
          
           
               
                   
                   
                 Substance 
                 Virus titer 
                 Virus inacti- 
               
               
                   
                   
                 Addition 
                 found 
                 vation 
               
               
                 Virus 
                 Time 
                 (mg/ml) 
                 (log 10 /ml) 
                 (log 10 /ml) 
               
               
                   
               
             
          
           
               
                 BPV 
                 1 hour 
                 Control 
                 0 
                 4.7 
                 0 
               
               
                   
                   
                 BACI 
                 0.05 
                 5.2 
                 0 
               
               
                   
                   
                 BACI 
                 0.01 
                 5.2 
                 0 
               
               
                   
                 2 hours 
                 Control 
                 0 
                 4.6 
                 0 
               
               
                   
                   
                 BACI 
                 0.05 
                 4.6 
                 0 
               
               
                   
                   
                 BACI 
                 0.01 
                 4.6 
                 0 
               
               
                   
                 1 hour 
                 Control 
                 0 
                 4.7 
                 0 
               
               
                   
                   
                 Entozon 
                 0.030 
                 ≦1.5 
                 ≧3.2 
               
               
                   
                   
                 Entozon 
                 0.015 
                 ≦1.5 
                 ≧3.2 
               
               
                   
                 2 hours 
                 Control 
                 0 
                 4.6 
                 0 
               
               
                   
                   
                 Entozon 
                 0.030 
                 ≦1.5 
                 ≧3.1 
               
               
                   
                   
                 Entozon 
                 0.015 
                 ≦1.5 
                 ≧3.1 
               
               
                 PPV 
                 1 hour 
                 Control 
                 0 
                 5.6 
                 0 
               
               
                   
                   
                 Entozon 
                 0.030 
                 3.3 
                 2.3 
               
               
                   
                   
                 Entozon 
                 0.015 
                 3.7 
                 1.9 
               
               
                   
                 2 hours 
                 Control 
                 0 
                 5.6 
                 0 
               
               
                   
                   
                 Entozon 
                 0.030 
                 2.2 
                 3.4 
               
               
                   
                   
                 Entozon 
                 0.015 
                 2.7 
                 2.9 
               
               
                   
               
             
          
         
       
     
     As the results in Table 2 show, BPV is not inactivated by BACI under the test conditions selected. Inactivation is possible by means of Entozon, inactivation with BPV taking place substantially more rapidly than with PPV. 
     Example 3 
     Virus Inactivation by Means of BACI and Acriflavine 
     The suspensions of the virus species shown in Tab. 3 were treated with BACI or acriflavine and incubated at 37° C. for 2 hours. Samples were then taken for titration to determine the virus inactivation. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                   
                   
                 Virus titer 
                 Virus inacti- 
               
               
                   
                   
                 Substance addition 
                 found 
                 vation 
               
               
                   
                 Virus 
                 (mg/ml) 
                 (log 10 /ml) 
                 (log 10 /ml) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 IBRV 
                 Control 
                 0 
                 5.1 
                 0 
               
               
                   
                   
                 BACI 
                 0.01 
                 ≦1.5 
                 ≧3.6 
               
               
                   
                   
                 Acriflavine 
                 0.001 
                 1.8 
                 3.3 
               
               
                   
                 PI 3 V 
                 Control 
                 0 
                 5.6 
                 0 
               
               
                   
                   
                 BACI 
                 0.01 
                 ≦1.5 
                 ≧4.1 
               
               
                   
                   
                 Acriflavine 
                 0.001 
                 3.2 
                 2.4 
               
               
                   
                 BVDV 
                 Control 
                 0 
                 6.2 
                 0 
               
               
                   
                   
                 BACI 
                 0.01 
                 2.3 
                 3.9 
               
               
                   
                   
                 Acriflavine 
                 0.001 
                 2.5 
                 3.7 
               
               
                   
                 BPV 
                 Control 
                 0 
                 5.4 
                 0 
               
               
                   
                   
                 BACI 
                 0.01 
                 5.9 
                 0 
               
               
                   
                   
                 Acriflavine 
                 0.001 
                 ≦1.5 
                 ≧3.9 
               
               
                   
                   
               
             
          
         
       
     
     As the results in Table 3 show, the enveloped virus species—IBRV, PI 3 V, BVDV—are inactivated both by BACI and acriflavine, the inactivation by BACI being somewhat greater. The naked virus BPV is inactivated by acriflavine, but not by BACI alone. 
     After virus inactivation by means of acriflavine and BACI was detected in EME medium, it was checked whether these substances can also inactivate virus in protein-containing solutions. The following protein solutions were treated with the virus species indicated: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Beriplex ® 
                 PPV 
               
               
                   
                 Horse serum 
                 BVDV, BPV 
               
               
                   
                 Beriate ® 
                 BVDV, BPV, BAV-1, Reo 3, IBRV 
               
               
                   
                 Haemate ® 
                 PPV, Reo 3 
               
               
                   
                 Venimmun ® 
                 BVDV, PPV, CPV 
               
               
                   
                 FCS 
                 CPV 
               
               
                   
                 Egg allantoic fluid 
                 Influenza A, Influenza B 
               
               
                   
                   
               
             
          
         
       
     
     The results obtained in these experiments are shown in tabular form below. 
     Example 4 
     Virus Inactivation with Acridines and Benzalkonium Chloride in Protein Solutions 
     As emerges from the results in Table 4, it is possible to inactivate completely different viruses in a methodologically simple manner in protein solutions using acridines and/or benzalkonium chloride. It appears clear here, however, that by means of benzalkonium chloride alone only envelope-containing viruses can be, inactivated, whereas by means of acridines both envelope-containing and also nonenveloped virus species are inactivated. Both substances act synergistically in their virucidal action, i.e. the magnitude of the, virus inactivation with the combination acriflavine+benzalkonium chloride is higher than with the two individual substances. 
     The results in Table 4 also show that virus inactivation in protein solutions is dependent on: 
     1. the virus species to be inactivated 
     2. the constituents and nature of the protein solution 
     3. the inactivating agent concentration 
     4. the inactivation time 
     5. the inactivation temperature. 
     The virus inactivation is also pH-dependent—results not shown. At a pH of below 5.5, virus inactivation takes place more slowly than at higher pHs. 
     Tables 5-7 contain the results of the biological activity of the protein solutions after virus inactivation by means of acriflavine and/or benzalkonium chloride. The biological activity was determined in Venimmun® by the content of antibodies before and after virus inactivation, and in Haemate®; Beriate® and Beriplex® by the determination of the clotting-promoting activity—measured in international units. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Virus inactivation with acridines and benzalkonium chloride in protein solutions 
               
             
          
           
               
                 Protein 
                   
                 Concentra- 
                 Inactivation 
                 Inactiva- 
                 Control 
                 Treatment 
                 Titer re- 
               
               
                 solution and 
                 Inactiva- 
                 tion 
                 Temperature 
                 tion time 
                 titer 
                 titer 
                 duction 
               
               
                 virus 
                 ting agent 
                 (mg/ml) 
                 (° C.) 
                 (h) 
                 (log 10 /ml) 
                 (log 10 /ml) 
                 (log 10 /ml) 
               
               
                   
               
             
          
           
               
                 Horse serum + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 5.4 
                 5.4 
                 0 
               
               
                 BPV 
                   
                   
                   
                 1 
                 5.2 
                 ≦1.5 
                 ≧3.7 
               
               
                   
                   
                   
                   
                 2 
                 5.1 
                 ≦1.5 
                 ≧3.6 
               
               
                 Horse serum + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 5.0 
                 5.0 
                 0 
               
               
                 BVDV 
                   
                   
                   
                 1 
                 5.1 
                 ≦1.5 
                 ≧3.6 
               
               
                   
                   
                   
                   
                 2 
                 5.0 
                 ≦1.5 
                 ≧3.5 
               
               
                 Beriate + BPV 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 5.4 
                 5.4 
                 0 
               
               
                   
                   
                   
                   
                 1 
                 5.0 
                 ≦1.5 
                 ≧3.5 
               
               
                   
                   
                   
                   
                 2 
                 5.0 
                 ≦1.5 
                 ≧3.5 
               
               
                 Beriate + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 5.0 
                 5.0 
                 0 
               
               
                 BVDV 
                   
                   
                   
                 1 
                 5.0 
                 ≦1.5 
                 ≧3.5 
               
               
                   
                   
                   
                   
                 2 
                 4.8 
                 ≦1.5 
                 ≧3.5 
               
               
                 Beriate + 
                 Ethacridine 
                 0.3 
                 45 
                 0 
                 5.4 
                 5.1 
                 0.3 
               
               
                 Reo 3 
                 lactate 
                   
                   
                 1 
                 4.7 
                 ≦1.5 
                 ≧3.2 
               
               
                   
                   
                   
                   
                 2 
                 4.7 
                 ≦1.5 
                 ≧3.2 
               
               
                 Beriate + IBR 
                 Ethacridine 
                 0.3 
                 45 
                 0 
                 6.5 
                 6.5 
                 0 
               
               
                   
                 lactate 
                   
                   
                 1 
                 5.7 
                 2.0 
                 3.7 
               
               
                   
                   
                   
                   
                 2 
                 4.3 
                 ≦1.5 
                 ≧2.8 
               
               
                 Haemate + PPV 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 5.4 
                 5.5 
                 0 
               
               
                   
                   
                   
                   
                 1 
                 5.5 
                 3.3 
                 2.2 
               
               
                   
                   
                   
                   
                 2 
                 5.5 
                 1.9 
                 3.6 
               
               
                   
                   
                   
                   
                 3 
                 5.3 
                 ≦1.5 
                 ≧3.8 
               
               
                 Venimmun + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 4.0 
                 4.0 
                 0 
               
               
                 PPV 
                   
                   
                   
                 1 
                 3.9 
                 ≦1.5 
                 ≧2.4 
               
               
                   
                   
                   
                   
                 2 
                 3.9 
                 ≦1.5 
                 ≧2.4 
               
               
                 Venimmun + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 6.9 
                 6.6 
                 0.5 
               
               
                 CPV 
                   
                   
                   
                 1 
                 6.8 
                 4.8 
                 2.0 
               
               
                   
                   
                   
                   
                 2 
                 6.8 
                 3.9 
                 2.9 
               
               
                   
                   
                   
                   
                 3 
                 6.9 
                 3.9 
                 3.0 
               
               
                 Venimmun + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 6.4 
                 6.4 
                 0 
               
               
                 BVDV 
                   
                   
                   
                 1 
                 5.8 
                 4.5 
                 1.3 
               
               
                   
                   
                   
                   
                 2 
                 6.3 
                 1.9 
                 4.4 
               
               
                   
                   
                   
                   
                 3 
                 6.0 
                 0 
                 ≧6.0 
               
               
                 Egg allantoic 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 7.4 
                 7.4 
                 0 
               
               
                 fluid + 
                   
                   
                   
                 1 
                 7.1 
                 6.4 
                 0.7 
               
               
                 Influenza 
                   
                   
                   
                 2 
                 6.9 
                 5.0 
                 1.9 
               
               
                 virus B 
               
               
                 Egg allantoic 
                 BACI 
                 0.02 
                 37 
                 0 
                 7.4 
                 7.4 
                 0 
               
               
                 fluid + 
                   
                   
                   
                 1 
                 7.1 
                 6.0 
                 1.1 
               
               
                 Influenza 
                   
                   
                   
                 2 
                 6.9 
                 3.6 
                 3.3 
               
               
                 virus B 
               
               
                 Egg allantoic 
                 Acriflavine + 
                 0.001 
                 37 
                 0 
                 7.4 
                 7.4 
                 0 
               
               
                 fluid + 
                 BACI 
                 0.02 
                   
                 1 
                 7.1 
                 4.1 
                 3.0 
               
               
                 Influenza 
                   
                   
                   
                 2 
                 6.9 
                 2.5 
                 4.4 
               
               
                 virus B 
               
               
                 Beriplex + 
                 Acriflavine 
                 0.001 
                 30 
                 0 
                 4.8 
                 4.8 
                 0 
               
               
                 PPV 
                   
                   
                   
                 1 
                 4.6 
                 3.6 
                 1.0 
               
               
                   
                   
                   
                   
                 2 
                 4.6 
                 2.9 
                 1.7 
               
               
                   
                   
                   
                   
                 3 
                 4.8 
                 ≦1.5 
                 ≧3.3 
               
               
                 Beriplex + 
                 BACI 
                 0.001 
                 30 
                 0 
                 4.8 
                 4.8 
                 0 
               
               
                 PPV 
                   
                   
                   
                 1 
                 4.8 
                 4.8 
                 0 
               
               
                   
                   
                   
                   
                 2 
                 4.6 
                 4.8 
                 0 
               
               
                   
                   
                   
                   
                 3 
                 4.6 
                 4.6 
                 0 
               
               
                 Beriate + PPV 
                 Acriflavine + 
                 0.001 
                 30 
                 0 
                 4.8 
                 4.8  
                 0 
               
               
                   
                 BACI 
                 0.01 
                   
                 1 
                 4.8 
                 3.5 
                 1.3 
               
               
                   
                   
                   
                   
                 2 
                 4.6 
                 ≦1.5 
                 ≧3.1 
               
               
                   
                   
                   
                   
                 3 
                 4.6 
                 ≦1.5 
                 ≧3.1 
               
               
                 Beriplex + 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 4.8 
                 4.8  
                 0 
               
               
                 PPV 
                   
                   
                   
                 1 
                 4.6 
                 2.0 
                 2.6 
               
               
                   
                   
                   
                   
                 2 
                 4.6 
                 ≦1.5 
                 ≧3.1 
               
               
                   
                   
                   
                   
                 3 
                 4.8 
                 ≦1.5 
                 ≧3.3 
               
               
                 Beriplex + 
                 BACI 
                 0.01 
                 37 
                 0 
                 4.8 
                 4.8 
                 0 
               
               
                 PPV 
                   
                   
                   
                 1 
                 4.6 
                 4.6 
                 0 
               
               
                   
                   
                   
                   
                 2 
                 4.6 
                 4.6 
                 0 
               
               
                   
                   
                   
                   
                 3 
                 4.8 
                 4.6 
                 0.2 
               
               
                 Beriplex + 
                 Acriflavine + 
                 0.001 
                 37 
                 0 
                 4.8 
                 4.8 
                 0 
               
               
                 PPV 
                 BACI 
                 0.01 
                   
                 1 
                 4.6 
                 1.8 
                 2.8 
               
               
                   
                   
                   
                   
                 2 
                 4.6 
                 ≦1.5 
                 ≧3.1 
               
               
                   
                   
                   
                   
                 3 
                 4.8 
                 ≦1.5 
                 ≧3.3 
               
               
                 FCS + CPV 
                 Acriflavine 
                 0.001 
                 37 
                 0 
                 6.6 
                 6.6 
                 0 
               
               
                   
                   
                   
                   
                 1 
                 n.d. 
                 4.9 
                 1.7 
               
               
                   
                   
                   
                   
                 2 
                 n.d. 
                 4.0 
                 2.6 
               
               
                   
                   
                   
                   
                 3 
                 6.9 
                 3.4 
                 3.5 
               
               
                 FCS + CPV 
                 Acriflavine 
                 0.001 
                 45 
                 0 
                 6.8 
                 6.8 
                 0 
               
               
                   
                   
                   
                   
                 1 
                 n.d. 
                 4.8 
                 2.0 
               
               
                   
                   
                   
                   
                 2 
                 n.d. 
                 3.5 
                 3.3 
               
               
                   
                   
                   
                   
                 3 
                 7.0 
                 2.8 
                 4.2 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Determination of the biological activity (antibody titer) 
               
               
                 in protein solutions before and after virus inactivation 
               
               
                 by means of acriflavine and benzalkonium chloride 
               
             
          
           
               
                   
                   
                 Reciprocal neutra- 
               
               
                   
                   
                 lization titer 1   
               
               
                   
                   
                 against polio virus 
               
               
                   
                 Tempera- 
                 Type 1 
               
             
          
           
               
                   
                   
                   
                 ture and 
                 Without 
                 With 
               
               
                   
                   
                   
                 duration 
                 inacti- 
                 inacti- 
               
               
                   
                 Protein 
                   
                 of the 
                 vating 
                 vating 
               
               
                   
                 solution 
                 Name 
                 treatment 
                 agent 
                 agent 2   
               
               
                   
                   
               
               
                   
                 Low-cryo 
                 H1 
                 37° C. 
                  646 
                  646 
               
               
                   
                 human plasma 
                 H2 
                 2 hours 
                  741 
                  562 
               
               
                   
                 (invididual 
                 H3 
                   
                  741 
                  646 
               
               
                   
                 plasma) 
                 H4 
                   
                  646 
                  741 
               
               
                   
                   
                 H5 
                   
                  376 
                  562 
               
               
                   
                 Venimmun ® 
                 V1 
                 37° C. 
                 2234 
                  851 
               
               
                   
                 (Final 
                 V2 
                 2 hours 
                 1950 
                 1698 
               
               
                   
                 product) 
                 V3 
                   
                 1698 
                 1698 
               
               
                   
                 various 
                 V4 
                   
                 1698 
                 1698 
               
               
                   
                 batches 
                 V5 
                   
                 1479 
                 1479 
               
               
                   
                 Low-cryo 
                 H1 
                 45° C. 
                  427 
                  977 
               
               
                   
                 human plasma 
                 H2 
                 2 hours 
                 1288 
                  977 
               
               
                   
                 (individual 
                 H3 
                   
                 1288 
                  977 
               
               
                   
                 plasma) 
                 H4 
                   
                  977 
                 1122 
               
               
                   
                   
                 H5 
                   
                  977 
                 1288 
               
               
                   
                 Venimmun ® 
                 V1 
                 45° C. 
                 1950 
                 2234 
               
               
                   
                 (Final 
                 V2 
                 2 hours 
                 2570 
                 2234 
               
               
                   
                 product) 
                 V3 
                   
                 2234 
                 1479 
               
               
                   
                 various 
                 V4 
                   
                 2234 
                 1950 
               
               
                   
                 batches 
                 V5 
                   
                 2234 
                 1479 
               
               
                   
                   
               
               
                   
                   1 Titer calculation according to Spearman-Karber from log 2  dilution series using 8 replicates/dilution stage  
               
               
                   
                   2 Acriflavine 0.001 mg/ml + BACI 0.02 mg/ml  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Determination of the biological activity (antibody titer 
               
               
                 in protein solutions before and after virus inactivation 
               
               
                 by means of acriflavine and benzalkonium chloride 
               
             
          
           
               
                   
                   
                 Temperature 
                 Rec. HAH anti-PI 3   
                 Rec. HAH anti-reo3 
               
               
                   
                   
                 and duration 
                 virus 
                 virus 
               
               
                 Protein 
                   
                 of the 
                 Antibody titer 1   
                 Antibody titer 1   
               
             
          
           
               
                 solution 
                 Name 
                 treatment 
                 Without I. 2   
                 With I. 2   
                 Without I. 2   
                 With I. 2   
               
               
                   
               
             
          
           
               
                 Low-cryo 
                 H1 
                 45° C. 
                 80 
                 80 
                 320 
                 320 
               
               
                 human 
                 H2 
                 2 hours 
                 80 
                 80 
                 320 
                 320 
               
               
                 plasma 
                 H3 
                   
                 80 
                 80 
                 320 
                 320 
               
               
                 (indivi- 
                 H4 
                   
                 80 
                 80 
                 320 
                 320 
               
               
                 dual 
                 H5 
                   
                 80 
                 80 
                 320 
                 320 
               
               
                 plasma) 
               
               
                 Venimmun ® 
                 V1 
                 45° C. 
                 320 
                 320 
                 320 
                 320 
               
               
                 (Final 
                 V2 
                 2 hours 
                 320 
                 320 
                 320 
                 320 
               
               
                 product) 
                 V3 
                   
                 320 
                 320 
                 320 
                 320 
               
               
                 various 
                 V4 
                   
                 320 
                 320 
                 320 
                 320 
               
               
                 batches 
                 V5 
                   
                 320 
                 320 
                 320 
                 320 
               
               
                 Horsye se- 
                 P1 
                 45° C. 
                 n.d. 
                 n.d. 
                 640 
                 640 
               
               
                 rum (indi- 
                 P2 
                 2 hours 
                   
                   
                 640 
                 640 
               
               
                 vidual 
                 P3 
                   
                   
                   
                 640 
                 640 
               
               
                 sera) 
                 P4 
                   
                   
                   
                 640 
                 640 
               
               
                   
                 P5 
                   
                   
                   
                 640 
                 640 
               
               
                   
               
               
                   1 Titer determination in log 2  dilution series  
               
               
                   2 I. = inactivating agents: acriflavine 0.001 mg/ml + BACI 0.02 mg/ml  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Determination of the biological activity (clotting 
               
               
                 activity) of protein solutions before and after virus 
               
               
                 inactivation by means of acriflavine (A) and benzalkonium 
               
               
                 chloride (BACL) 
               
             
          
           
               
                   
                   
                   
                   
                 Inacti- 
                   
               
               
                   
                   
                 Tempera- 
                   
                 vating 
               
               
                   
                   
                 ture and 
                   
                 agent 
               
               
                   
                   
                 duration 
                 Inacti- 
                 concen- 
               
               
                   
                 Protein 
                 of the 
                 vating 
                 tration 
                 Activity 
               
               
                   
                 solution 
                 treatment 
                 agent 
                 (mg/ml) 
                 (IU/ml) 
               
               
                   
                   
               
             
          
           
               
                   
                 cryo- 
                 3 hours 
                 A 
                 0.01 
                 3.9 
               
               
                   
                 protein 
                 45° C. 
                 A 
                 0.03 
                 4.2 
               
               
                   
                 solution 
                   
                 A 
                 0.001 
                 4.4 
               
               
                   
                 after 
                   
                 BACI 
                 0.1 
                 6.9 
               
               
                   
                 Al(OH) 3  + 
                   
                 BACI 
                 0.05 
                 6.1 
               
               
                   
                 QAE 
                   
                 A + BACI 
                 0.001 + 
                 4.1 
               
               
                   
                 treatment 
                   
                   
                 0.05 
               
               
                   
                   
                   
                 Control 
                 0 
                 5.3 
               
               
                   
                 Haemate ® 
                 3 hours 
                 A 
                 0.002 
                 18.9 
               
               
                   
                 Factor 
                 37° C. 
                 A 
                 0.001 
                 16.8 
               
               
                   
                 VIII 
                   
                 BACI 
                 0.02 
                 20.7 
               
               
                   
                   
                   
                 A + BACI 
                 0.001 + 
                 17.6 
               
               
                   
                   
                   
                   
                 0.02 
               
               
                   
                   
                   
                 Control 
                 0 
                 24.1 
               
               
                   
                 Beriplex ® 
                 3 hours 
                 A 
                 0.002 
                 48.8 
               
               
                   
                 Factor II 
                 37° C. 
                 A 
                 0.001 
                 50.0 
               
               
                   
                   
                   
                 BACI 
                 0.02 
                 61.3 
               
               
                   
                   
                   
                 A + BACI 
                 0.001 + 
                 47.9 
               
               
                   
                   
                   
                   
                 0.02 
               
               
                   
                   
                   
                 Control 
                 0 
                 68.6 
               
               
                   
                   
               
             
          
         
       
     
     As emerges from the results in Tables 5 and 6, the content of neutralizing (Polio Type 1) and hemagglutination-inhibiting (HAH) antibodies (PI 3 —and Reo 3 virus) in Venimmun® is not affected to an extent-exceeding the test variations (as a rule ±1 log 2  stage) during virus inactivation by means of acridines and benzalkonium chloride. The activity decrease with the clotting preparations (Beriate®, Haemate®, Beriplex®) after acridines/benzalkonium chloride, inactivation is tolerable (Table 7).

Technology Classification (CPC): 2