Patent Publication Number: US-4647454-A

Title: Stable interferon β composition and a method of stabilizing interferon β

Description:
This invention relates to a method of stabilizing Human Fibroblast Interferon (HFIF) also known by the name and hereinafter designated as &#34;Interferon β&#34; and to stabilized Interferon β compositions which are transportable and storable for extended periods in closed vials as well as after resuspension in a suitable diluent. 
     Interferon β is a highly active, antiviral protein substance which is found in and obtained from certain human cells in minute quantities, as described in the pertinent literature. These quantities were insufficient for conducting the manifold clinical and other tests and studies necessary for evaluating the promising therapeutical properties of this substance. 
     Intensive efforts were therefore made by leading research institutes and drug companies the world over to find a method of producing larger quantities of Interferon β, mainly by tissue culture methods. As a result of these efforts, Interferon β is now available in commercial quantities. The Interferon β is purified and admixed with appropriate excipients, filtered, dispensed in glass vials, lyophilized, and sealed in vacuo, then stored at temperatures of -40° C. and shipped in dry ice. 
     It is known that the resulting Interferon β preparation is highly unstable and that it loses its biological activity within a relatively short period. All attempts made hitherto to stabilize Interferon β have failed. 
     It was thus unexpected and surprising when Applicants discovered that Interferon β preparations can be effectively stabilized by means of vinyl pyrrolidone polymer, hereinafter designated P.V.P., a polymer which has been known for a long time exclusively as a clarifying agent in wines and as a dispersing and suspending agent for pharmaceutical compositions. P.V.P. has molecular weights ranging from 10,000 to 700,000 and it is marketed under trademarks such as POVIDONE et al, (see &#34;The Merck Index&#34;, 9th edition, page 7483 under No. 7498). 
     This invention relates to a novel stable Interferon composition comprised of Interferon β, conventional excipients, a buffer and vinyl pyrrolidone polymer, as a stabilizer. 
     This invention also relates to a method of stabilising Interferon β, wherein a highly purified Interferon β solution admixed with known excipients therefor is dialysed against an acetate buffer solution--pH=3.5, for about 48 hours, the resulting Interferon solution is subjected to sterile filtration, the filtrate is admixed with 0.5% to 10% weight/volume of polyvinyl pyrrolidone, dispensed into glass vials, lyophilized, whereupon the vials are sealed in vacuo and stored at 4° C. 
     The preferred excipients are mannitol and human serum albumin (HSA). The acetate buffer used contains sodium acetate and sufficient acetic acid to adjust the pH to 3.5 P.V.P. marketed as POVIDONE, having a molecular weight of about 50,000 is the preferred stabilizer, but P.V.P. having lower or higher molecular weights has also proved to be highly effective as a stabilizer, it is used in a final concentration of about 0.5 to 10% wt/volume of the preparation. 
    
    
     The preparation of the preferred inventive Interferon β composition will now be described in the following example. 
     EXAMPLE 
     20 lts of aqueous acetate buffer solution having a pH=3.5 are prepared by dissolving 21.6 cc of acetic acid and 4.02 gms of sodium acetate in the required volume of distilled water. 
     The inner surface of a sterile dialysis bag is wetted with sufficient concentrated human serum albumin to result in a 1% concentration in a highly purified Interferon β solution, having a specific activity of about 10 7  international units per mg of protein, which is subjected to dialysis therein. 
     The resulting solution is dialysed against the acetate buffer of pH 3.5 and at a temperature of 4° C. for about 48 hours at a ratio of 1:100 Interferon solution to buffer solution with a change of the buffer solution after 24 hours. 
     The dialysed Interferon β preparation is admixed with mannitol 0.5% wt/volume final concentration and with P.V.P. at a 2% final concentration approximately prior to or following filtration through a sterile filter, previously impregnated with sufficient concentrated human serum albumin to raise the albumin concentration in the filtrate to 2% wt/volume. The filtrate is collected in a sterile bottle. 
     The P.V.P. concentration is then finally adjusted to 2% wt/volume and the concentration of mannitol to 0.5% wt/volume, if necessary. The final volume of the solution is adjusted with sterile acetate buffer. 
     2 cc each of the solution obtained are dispensed into sterile glass vials by means of a sterile Cornwall syringe, followed by lyophilization and the vials are then sealed in vacuo and stored at 4° C. The contents of the vials are resuspended by the addition of 2 cc of bidistilled sterile water. 
     The composition of the final product per vial is as follows: 
     
         ______________________________________                                    
Sodium Acetate AG                                                         
                 0.4    mgm                                               
Sodium Chloride AG                                                        
                 1.8    mgm                                               
Human Serum Albumin                                                       
                 40.0   mgm                                               
Fraction V                                                                
Mannitol AG      10.0   mgm                                               
PVP - Stabilizer 40.0   mgm                                               
Human Fibroblast Interferon                                               
                 1.0    10.sup.6 I.U. (approximately)                     
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     The effectiveness of P.V.P. of different molecular weights and in different concentrations on the stability of Interferon in its compositions will now be illustrated by the following tables 1 to 6 of which: Table (1) illustrates the effect of P.V.P. of molecular weight 24,000 at concentration ranging from 0.5% to 5% on the stability of Interferon in compositions immediately before and immediately after lyophilization and after storage for 1 to 4 months in sealed vials at 37° C.; The data in tables (2) and (3) illustrates the stability of the compositions under identical conditions, using P.V.P. of molecular weights 50,000 and 160,000 respectively, and comparative data are reported in these tables for Interferon β compositions without P.V.P. and compositions containing sucrose or Human Serum Albumin in various concentrations instead of P.V.P.; Tables (4), (5) and (6) illustrate the relationship between the titer of inventive Interferon β compositions and their content of P.V.P. of the same molecular weights as in tables (1), (2) and (3); (a) immediately after resuspension as hereinbefore described, and, (b) after storage of the resuspended compositions for 1 month at 4° C. 
     Comparative data for Interferon β compositions admixed with Sucrose or Human Serum albumin are again given. 
     The following data and remarks are essential for the understanding of these tables: 
     (a) The Human Fibroplast Interferon used in the compositions was initially purified to a specific activity ranging from 10 6  -10 7  international units per mg of protein. 
     (b) The data in the Tables relating to the titers of Interferon. in admixture with PVP in different concentrations are the averages of 6 titration results and the data are expressed in megaunits per vial. 
     (c) The difference in the initial titers of Interferon β are due to the use of Interferon from different batches which differ somewhat in their specific activity. 
     It is evident from the data reported in the tables that PVP of different molecular weight have maximal stabilizing effectiveness when used in concentration of from 2 to 4% although the use of PVP in concentration up to 10% also leads to positive stabilization results. Positive stabilization is also attained using PVP having molecular weights from 10,000 to 700,000. 
     Other modifications of the method described hereinabove are known to the man versed in the art and these are included herein provided that they fall within the ambit of the invention defined in the subsequent claims. 
     
                                           TABLE 1                                 
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Polyvinylpyrrolidone 24000                                                
                         Titer after                                      
                               Titer after                                
                                     Titer after                          
                                           Titer after                    
Conc of  Titer before                                                     
                 Titer after                                              
                         1 Month at                                       
                               2 Months at                                
                                     3 Months at                          
                                           4 Months at                    
PVP      Lyophilization                                                   
                 Lyophilization                                           
                         37° C.                                    
                               37° C.                              
                                     37° C.                        
                                           37° C.                  
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0%       1.4     0.8      0.08  0.02 &lt;0.01 &lt;0.01                          
0.5%     1.3     1.0     0.8   0.6   0.2   0.20                           
1%       1.5     1.3     1.2   1.2   1.1   0.9                            
2%       1.3     1.3     1.2   1.2   1.1   0.9                            
3%       1.3     1.2     1.1   1.2   1.1   1.1                            
4%       1.6     1.4     1.2   1.1   1.0   1.0                            
5%       1.4     1.0     0.9   1.0   0.8   0.8                            
0 + Sucrose 5%                                                            
         1.5     0.7      0.05  0.01 &lt;0.01 &lt;0.01                          
0 + Sucrose 10%                                                           
         1.3     0.8      0.04  0.01 &lt;0.01 &lt;0.01                          
0 + HSA 3%                                                                
         1.4     0.8     0.1    0.03 &lt;0.01 &lt;0.01                          
0 + HSA 4%                                                                
         1.3     0.9     0.1    0.05  0.02  0.01                          
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                                           TABLE 2                                 
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Polyvinylpyrrolidone 50000                                                
                         Titer after                                      
                               Titer after Titer after                    
Conc. of Titer before                                                     
                 Titer after                                              
                         1 Month                                          
                               2 Months                                   
                                     Titer after                          
                                           4 Months                       
PVP      Lyophilization                                                   
                 Lyophilization                                           
                         at 37° C.                                 
                               37° C.                              
                                     3 Months                             
                                           at 37° C.               
__________________________________________________________________________
0        1.1     0.7      0.06  0.01 &lt;0.01 &lt;0.01                          
0.5%     1.2     0.9     0.6   0.1    0.07  0.03                          
1%       1.1     1.1     1.1   1.1   1.0   0.8                            
2%       1.2     1.1     1.2   1.2   1.1   1.0                            
3%       1.1     1.2     1.3   1.2   1.1   0.9                            
4%       1.3     1.2     1.2   1.0   1.2   1.0                            
5%       1.0     0.9     1.0   0.8   0.7   0.9                            
0 + Sucrose 5%                                                            
         1.1     0.6      0.07  0.02 &lt;0.01 &lt;0.01                          
0 + Sucrose 10%                                                           
         1.2     0.8      0.05  0.01 &lt;0.01 &lt;0.01                          
0 + HSA 3%                                                                
         1.2     0.8      0.09  0.03 &lt;0.01 &lt;0.01                          
0 + HSA 4%                                                                
         1.1     0.8      0.10  0.03  0.01 &lt;0.01                          
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                                           TABLE 3                                 
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Polyvinylpyrrolidone 160.000                                              
                         Titer after                                      
                               Titer after                                
                                     Titer after                          
                                           Titer after                    
Conc of  Titer before                                                     
                 Titer after                                              
                         1 Month                                          
                               2 Months                                   
                                     3 Months                             
                                           4 Months                       
PVP      lyophilization                                                   
                 lyophilization                                           
                         at 37° C.                                 
                               at 37° C.                           
                                     37° C.                        
                                           37° C.                  
__________________________________________________________________________
0%       1.5     0.8      0.05  0.01 &lt;0.01 &lt;0.01                          
0.5%     1.4     1.0      0.09  0.06  0.05  0.03                          
1%       1.6     1.3     1.0   0.6   0.6   0.6                            
2%       1.4     1.6     1.5   1.3   1.4   1.3                            
3%       1.5     1.5     1.3   1.4   1.3   1.2                            
4%       1.6     1.4     1.3   1.4   1.3   1.2                            
5%       1.7     1.3     1.4   1.3   1.2   1.2                            
0 + Sucrose 5%                                                            
         1.7     0.9      0.04  0.01 &lt;0.01 &lt;0.01                          
0 + Sucrose 10%                                                           
         1.3     0.8      0.05 &lt;0.01 &lt;0.01 &lt;0.01                          
0 + HSA 3%                                                                
         1.4     0.8      0.08  0.04 &lt;0.01 &lt;0.01                          
0 + HSA 4%                                                                
         1.5     1.0     0.1    0.05 &lt;0.01 &lt;0.01                          
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                       TABLE 4                                                     
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Polyvinyl pyrrolidone 24000                                               
                           Titer of IF                                    
                           1 Month after                                  
Conc. of      Titer of IF  resuspension                                   
PVP           with resuspension                                           
                           stored at 4° C.                         
______________________________________                                    
0%            0.8          &lt;0.01                                          
0.5%          1.0          0.3                                            
1%            1.3          0.6                                            
2%            1.3          1.2                                            
3%            1.2          1.1                                            
4%            1.4          1.2                                            
5%            1.0          1.1                                            
0 + Sucrose 5%                                                            
              0.7          &lt;0.01                                          
0 + Sucrose 10%                                                           
              0.8          &lt;0.01                                          
0 + HSA 3%    0.8          &lt;0.01                                          
0 + HSA 4%    0.9          &lt;0.01                                          
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                       TABLE 5                                                     
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Polyvinyl pyrrolidone 50000                                               
                         Titer of IF 1 Month                              
Conc of    Titer of IF   after resuspension stored                        
PVP        with resuspension                                              
                         at 4° C.                                  
______________________________________                                    
0%         0.7           &lt;0.01                                            
0.5%       0.9           0.5                                              
1%         1.1           0.8                                              
2%         1.1           1.0                                              
3%         1.2           1.1                                              
4%         1.2           1.2                                              
5%         0.9           1.0                                              
0 + Sucrose 5%                                                            
           0.6           &lt;0.01                                            
0 + Sucrose 10%                                                           
           0.8           &lt;0.01                                            
0 + HSA 3% 0.8           &lt;0.01                                            
0 + HSA 4% 0.8            0.05                                            
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                       TABLE 6                                                     
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Polyvinylpyrrolidone 160.000                                              
                         Titer of IF 1 Month                              
Conc. of   Titer of IF   after resuspension stored                        
PVP        with resuspension                                              
                         at 4° C.                                  
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0%         0.8           &lt;0.01                                            
0.5        1.0           0.3                                              
1%         1.3           0.8                                              
2%         1.6           1.4                                              
3%         1.5           1.3                                              
4%         1.4           1.4                                              
5%         1.3           1.3                                              
0 + Sucrose 5%                                                            
           0.9           &lt;0.01                                            
0 + Sucrose 10%                                                           
           0.8           &lt;0.01                                            
0 + HSA 3% 0.8           &lt;0.01                                            
0 + HSA 4% 1.0            0.03                                            
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