Compositions and Methods for Crystallizing Antibodies

Abstract

The present invention relates to a batch crystallization method for crystallizing anti-human TNFalpha (hTNFalpha) antibody and antibody fragments which allows the production of said antibody on an industrial scale; a method of controlling the size of antibody crystals, for example, crystals of anti-hTNFalpha antibody fragments, compositions containing said crystals as well as methods of use of said crystals and compositions.

1 . A method for the preparation of antibody crystals of a desired substantially uniform size, the method comprising the steps of:
 a) providing an aqueous crystallization mixture comprising an antibody and at least one crystallization agent under conditions that enable the formation of antibody crystals; and   b) agitating said crystallization mixture under controlled conditions, whereby antibody crystals in a desired average size range are formed.   
     
     
         2 . The method of  claim 1 , wherein said controlled conditions are selected from a group consisting of
 a) agitating said crystallization mixture in a roller container at a speed in a range of from about 1 to about 200 rpm;   b) agitating said crystallization mixture in a roller container having a diameter in a range of about 2 to about 100 cm;   c) agitating said crystallization mixture in a roller container wherein about 1 to about 100% of the total internal volume of said roller container is filled with the crystallization mixture;   d) agitating said crystallization mixture in a roller container wherein about 1 to about 100% of the total internal volume of said roller container is filled with the crystallization mixture;   e) agitating said crystallization mixture in a roller container for about 30 minutes to about 20 days; and   f) agitating said crystallization mixture in a roller container at a temperature in a range of about −15 to about +50° C.   
     
     
         3 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the crystallization agent is a polyalkylene polyol. 
     
     
         10 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the antibody is an antibody fragment selected from the group consisting of
 a) an anti-hTNFalpha antibody binding fragment   b) an Fab or F(ab′)2 fragment; and   c) MAK 195F, an F(ab′)2 fragment of MAK195, which is produced by a hybridoma cell line having deposit number ECACC 87050801.   
     
     
         15 - 18 . (canceled) 
     
     
         19 . Antibody crystals obtainable by the method of  claim 1 . 
     
     
         20 . A batch crystallization method for crystallizing an anti-hTNFalpha antibody binding fragment, the method comprising the steps of:
 a) providing an aqueous crystallization mixture comprising an antibody and at least one polyalkylene glycol as a crystallization agent; and   b) incubating said aqueous crystallization mixture until crystals of said antibody are formed;   wherein said at least one polyalkylene glycol is provided either (a) in one step or (b) in more than one step, wherein said antibody crystals formed in a step are not removed in subsequent steps.   
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 20 , wherein said aqueous crystallization mixture
 a) has a pH in the range of about pH 4 to about 6.5; or   b) comprises a buffer.   
     
     
         23 - 26 . (canceled) 
     
     
         27 . The method of  claim 20 , wherein the polyalkylene glycol is selected from the group consisting of
 a) a polyalkylene glycol having an average molecular weight in the range of about 400 to about 10,000 g/mol;   b) a polyethylene glycol; and/or   c) a polyalkylene glycol present in the crystallization mixture at a final concentration in the range of about 5 to about 30% (w/v) of the total volume.   
     
     
         28 - 30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein at least one of the following additional crystallization conditions are met:
 a) incubation is performed for about 1 hour to about 250 days;   b) incubation is performed at a temperature between about −15° C. and about +50° C.; and   c) the crystallization mixture comprises an antibody fragment at a concentration in the range of about 0.5 to about 280 mg/ml.   
     
     
         32 - 34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the crystallization mixture comprises a batch volume in the range of about 1 ml to about 20,000 liters. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 20 , wherein said controlled conditions are selected from the group consisting of
 a) agitating said crystallization mixture in a roller container at a speed in a range of from about 1 to about 200 rpm;   b) agitating said crystallization mixture in a roller container having a diameter in a range of about 2 to about 100 cm;   c) agitating said crystallization mixture in a roller container wherein about 1 to about 100% of the total internal volume of said roller container is filled with the crystallization mixture;   d) agitating said crystallization mixture in a roller container wherein about 1 to about 100% of the total internal volume of said roller container is filled with the crystallization mixture;   e) agitating said crystallization mixture in a roller container for about 30 minutes to about 20 days; and   f) agitating said crystallization mixture in a roller container at a temperature in a range of about −15 to about +50° C.   
     
     
         38 - 42 . (canceled) 
     
     
         43 . The method of  claim 20 , wherein said agitating comprises rolling, stirring, shaking and/or tumbling said crystallization mixture. 
     
     
         44 . A crystal of an anti-hTNFalpha antibody, or a fragment, thereof, wherein the crystal comprises a needle-shaped morphology. 
     
     
         45 . A crystal of an anti-hTNFalpha antibody, or fragment thereof, obtainable by the method of  claim 1 , wherein the crystal comprises a needle-shaped morphology. 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . The crystal obtainable by the method of  claim 20 , wherein said antibody fragment is selected from a group consisting of
 a) a polyclonal antibody fragment or a monoclonal antibody fragment;   b) an antibody fragment selected from the group consisting of fragments of chimeric antibodies, humanized antibodies, non-glycosylated antibodies, human antibodies, and mouse antibodies;   c) a fragment of an IgG antibody, wherein said antibody is selected from the group consisting of: IgG1, IgG2, IgG3 and IgG4, antibodies;   d) a Fab or F(ab′)2 fragment; and   f) MAK 195F, an F(ab′) 2  fragment of antibody MAK195, produced by a hybridoma cell line having the deposit number ECACC 87050801   
     
     
         49 - 53 . (canceled) 
     
     
         54 . A pharmaceutical composition comprising: (a) crystals of an antibody prepared according to the methods of  claim 1 , and (b) at least one pharmaceutical excipient; wherein the composition is provided as a solid, a semisolid, or a liquid formulation. 
     
     
         55 - 62 . (canceled) 
     
     
         63 . An injectable liquid composition comprising the antibody crystals obtainable by the method of  claim 1 , wherein the antibody is present at a concentration in a range of about 10 to about 400 mg/ml. 
     
     
         64 . A crystal slurry composition comprising the antibody crystals obtainable by the method of a  claim 1 , wherein the antibody is present in a concentration greater than about 100 mg/ml. 
     
     
         65 . A method for treating a mammal, the method comprising the step of administering to the mammal an effective amount of the antibody crystals obtainable by the method of  claim 1 . 
     
     
         66 - 68 . (canceled) 
     
     
         69 . A method of treating a hTNFalpha-related disorder in a subject, the method comprising the step of administering a therapeutically effective amount of the antibody crystals of  claim 19 . 
     
     
         70 - 73 . (canceled)