Method to produce an immunoglobulin preparation with improved yield

Abstract

The present invention provides improved methods for the manufacturing of IVIG products. These methods offer various advantages such as reduced loss of IgG during purification and improved quality of final products. In other aspects, the present invention provides aqueous and pharmaceutical compositions suitable for intravenous, subcutaneous, and/or intramuscular administration. In yet other embodiments, the present invention provides methods of treating a disease or condition comprising administration of an IgG composition provided herein.

1 . A method for preparing an enriched IgG composition from plasma, the method comprising the steps of:
 (a) precipitating a cryo-poor plasma fraction, in a first precipitation step, with between about 6% and about 10% alcohol at a pH of between about 7.0 and about 7.5 to obtain a first precipitate and a first supernatant;   (b) precipitating IgG from the first supernatant, in a second precipitation step, with between about 20% and about 25% alcohol at a pH of between about 6.7 and about 7.3 to form a second precipitate;   (c) re-suspending the second precipitate to form a suspension;   (d) precipitating IgG from the suspension formed in step (c), in a third precipitation step, with between about 22% and about 28% alcohol at a pH of between about 6.7 and about 7.3 to form a third precipitate;   (e) re-suspending the third precipitate to form a suspension; and   (f) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition,   wherein the pH of at least one of the first precipitation step, second precipitation step, or third precipitation step is achieved by addition of a pH modifying agent after addition of the alcohol.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the pH of all of the first precipitation step, second precipitation step, and third precipitation step is achieved by addition of a pH modifying solution after addition of the alcohol. 
     
     
         5 . The method of  claim 1 , wherein the addition of a pH modifying solution comprises spray addition of the pH modifying solution. 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the method further comprises an ion exchange chromatography purification step. 
     
     
         9 . The method of  claim 8 , wherein the method comprises both an anion exchange chromatography purification step and a cation exchange chromatography step. 
     
     
         10 . The method of  claim 1 , wherein the method further comprises a nanofiltration step and/or an ultrafiltration/diafiltration step. 
     
     
         11 . The method of  claim 1 , wherein the enriched IgG composition obtained in step (f) contains at least 85% of the IgG content found in the cryo-poor plasma fraction used in step (a). 
     
     
         12 . The method of  claim 11 , wherein the enriched IgG composition obtained in step (f) contains at least 90% of the IgG content found in the cryo-poor plasma fraction used in step (a). 
     
     
         13 . The method of  claim 1 , wherein the purity of γ-globulins in the final IgG composition is at least about 95%. 
     
     
         14 . The method of  claim 13 , wherein the purity of γ-globulins in the final IgG composition is at least about 98%. 
     
     
         15 . The method of  claim 13 , wherein the purity of γ-globulins in the final IgG composition is at least about 99%. 
     
     
         16 . The method of  claim 1 , wherein the method comprises the steps of:
 (a) cooling a mixture of pooled plasma donations to a temperature between about 2° C. and about 10° C.;   (b) separating liquid and precipitate from the mixture of step (i) by centrifugation;   (c) admixing ethanol into the liquid supernatant formed in step (ii) to a final concentration of between about 5% (v/v) to about 10% (v/v) ethanol, thereby forming a mixture;   (d) cooling the mixture formed in step (iii) to between about −4° C. and about 0° C.;   (e) separating liquid and precipitate from the mixture of step (iv) by centrifugation; and isolating the supernatant, thereby forming a first plasma fraction   (f) adjusting the pH of the first plasma fraction to about 7.0;   (g) adjusting the ethanol concentration of the first plasma fraction of step (f) to about 25% (v/v) at a temperature between about −7° C. and about −9° C., thereby forming a mixture;   (h) separating liquid and precipitate from the mixture of step (g);   (i) re-suspending the precipitate of step (h) with a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with between 300 mL and 700 mL of glacial acetic acid per 1000 L of buffer, thereby forming a suspension;   (j) mixing finely divided silicon dioxide (SiO 2 ) with the suspension from step (i) for at least about 30 minutes;   (k) filtering the suspension with a filter press, thereby forming a filtrate;   (l) washing the filter press with at least 3 filter press dead volumes of a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with between 50 mL and 200 mL of glacial acetic acid per 1000 L of buffer, thereby forming a wash solution;   (m) combining the filtrate of step (k) with the wash solution of step (g), thereby forming a solution, and treating the solution with a detergent;   (n) adjusting the pH of the solution of step (m) to about 7.0 and adding ethanol to a final concentration of about 25%, thereby forming a precipitate;   (o) separating liquid and precipitate from the mixture of step (n);   (p) dissolving the precipitate in an aqueous solution comprising a solvent or detergent and maintaining the solution for at least 60 minutes;   (q) passing the solution after step (p) through a cation exchange chromatography column and eluting proteins absorbed on the column in an eluate;   (r) passing the eluate from step (q) through an anion exchange chromatography column to generate an effluent;   (s) passing the effluent from step (r) through a nanofilter to generate a nanofiltrate;   (t) passing the nanofiltrate from step (s) through an ultrafiltration membrane to generate an ultrafiltrate; and   (u) diafiltrating the ultrafiltrate from step (t) against a diafiltration buffer to generate a diafiltrate having a protein concentration between about 8% (w/v) and about 12% (w/v), thereby obtaining a composition of concentrated IgG.   
     
     
         17 . The method of  claim 16 , wherein the ethanol concentration is adjusted in at least one of steps (g) and (n) by introducing ethanol into the fraction by spraying. 
     
     
         18 - 21 . (canceled) 
     
     
         22 . The method of  claim 16 , wherein the method further comprises filtering the diafiltrate through a filter of 0.22 μm or less. 
     
     
         23 . The method of  claim 16 , wherein the plasma is human plasma. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 16 , wherein the temperature of the mixture from step (g) is about −7° C. 
     
     
         26 . The method of  claim 16 , wherein the precipitate formed in step (h) is re-suspended with between about 12 L and about 18 L of buffer per kg precipitate. 
     
     
         27 . The method of  claim 26 , wherein the precipitate is re-suspended with about 15 L of buffer per kg precipitate. 
     
     
         28 . The method of  claim 16 , wherein the amount of silicon dioxide added to the suspension in step (j) is between about 0.02 grams per gram precipitate formed in step (h) and about 0.06 grams per gram precipitate formed in step (c). 
     
     
         29 . The method of  claim 28 , wherein the amount of silicon dioxide added to the suspension in step (j) is about 0.04 grams per gram precipitate formed in step (h). 
     
     
         30 . The method of  claim 16 , wherein a filter aid is added to the mixture prior to filtration in step (k). 
     
     
         31 . The method of  claim 30 , wherein the filter aid is diatomaceous earth. 
     
     
         32 . The method of  claim 16 , wherein the purity of the γ-globulins in the filtrate formed in step (k) is at least about 85%. 
     
     
         33 . The method of  claim 16 , wherein the filtrate formed in step (k) contains less than about 10 mg of fibrinogen per gram total protein. 
     
     
         34 . The method of  claim 16 , wherein the filtrate formed in step (k) contains less than about 500 IU of PKA activity per gram total protein. 
     
     
         35 . The method of  claim 16 , wherein the detergent used in step (m) comprises between about 0.1% (w/v) and about 0.3% (w/v) polysorbate-80. 
     
     
         36 . The method of  claim 16 , wherein the aqueous solution used in step (p) comprises Triton-X 100, polysorbate-80, and TNBP. 
     
     
         37 . The method of  claim 16 , wherein the solution in step (p) is maintained at a temperature between about 18° C. and about 25° C. 
     
     
         38 . The method of  claim 16 , wherein the nanofilter of step (s) has a mean pore size of between about 15 nm to about 72 nm. 
     
     
         39 . The method of  claim 38 , wherein the nanofilter has a mean pore size of between about 19 nm and about 35 nm. 
     
     
         40 . The method of  claim 39 , wherein the nanofilter has a mean pore size of about 35 nm. 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 16 , wherein the solution formed in step (m) contains at least about 85% of the IgG present in the first plasma fraction of step (f). 
     
     
         44 . The method of  claim 43 , wherein the solution formed in step (m) contains at least about 90% of the IgG present in the first plasma fraction of step (f). 
     
     
         45 . An aqueous IgG composition prepared by the method of  claim 1 . 
     
     
         46 - 49 . (canceled) 
     
     
         50 . A pharmaceutical composition comprising an aqueous IgG composition prepared according to the method of  claim 1 . 
     
     
         51 - 57 . (canceled) 
     
     
         58 . A method of treating an immunodeficiency, autoimmune disease, or acute infection in a human in need thereof, the method comprising administering a pharmaceutical composition of  claim 50 . 
     
     
         59 . The method of  claim 1 , wherein the pH of the first precipitation step is achieved by addition of a pH modifying agent after addition of the alcohol. 
     
     
         60 . The method of  claim 59 , wherein the alcohol is ethanol. 
     
     
         61 . The method of  claim 1 , wherein the pH of the first precipitation step is modified before and after the addition of alcohol. 
     
     
         62 . The method of  claim 1 , wherein the pH of the first precipitation step is modified during and after the addition of alcohol. 
     
     
         63 . The method of  claim 1 , wherein the pH of the first precipitation step is modified before, during, and after the addition of alcohol. 
     
     
         64 . The method of  claim 1 , wherein the wherein the pH of the first precipitation step is maintained for the entire precipitation step. 
     
     
         65 . The method of  claim 1 , wherein the pH of the second precipitation step is achieved by addition of a pH modifying agent after addition of the alcohol. 
     
     
         66 . The method of  claim 65 , wherein the alcohol is ethanol. 
     
     
         67 . The method of  claim 1 , wherein the pH of the second precipitation step is modified before and after the addition of alcohol. 
     
     
         68 . The method of  claim 1 , wherein the pH of the second precipitation step is modified during and after the addition of alcohol. 
     
     
         69 . The method of  claim 1 , wherein the pH of the second precipitation step is modified before, during, and after the addition of alcohol. 
     
     
         70 . The method of  claim 1 , wherein the wherein the pH of the second precipitation step is maintained for the entire precipitation step. 
     
     
         71 . The method of  claim 1 , wherein the pH of the third precipitation step is achieved by addition of a pH modifying agent after addition of the alcohol. 
     
     
         72 . The method of  claim 71 , wherein the alcohol is ethanol. 
     
     
         73 . The method of  claim 1 , wherein the pH of the third precipitation step is modified before and after the addition of alcohol. 
     
     
         74 . The method of  claim 1 , wherein the pH of the third precipitation step is modified during and after the addition of alcohol. 
     
     
         75 . The method of  claim 1 , wherein the pH of the third precipitation step is modified before, during, and after the addition of alcohol. 
     
     
         76 . The method of  claim 1 , wherein the wherein the pH of the third precipitation step is maintained for the entire precipitation step.