Process for separating and determining the viral load in a pancreatin sample

Abstract

Processes for separating an infectious viral load from a pancreatin sample and for quantitatively determining the viral load in a pancreatin sample are described herein.

1 - 47 . (canceled) 
     
     
         48 . A method of treating pancreatic exocrine insufficiency in a mammalian subject, the method comprising:
 orally administering a dosage form comprising pancreatin and one or more pharmaceutically acceptable excipients to the subject in an amount sufficient to treat the pancreatic exocrine insufficiency, wherein a viral load of the pancreatin has been determined by a process comprising the steps of:
 (a) producing a liquid pancreatin test sample; 
 (b) centrifugation of the liquid pancreatin test sample at a relative centrifugal force of less than 10,000×g in which viruses with sedimentation constants of greater than about 120 S do not form a pellet whereby the centrifugation of the liquid pancreatin test sample produces a supernatant; 
 (c) ultracentrifugation of the liquid pancreatin test sample supernatant with a discontinuous gradient medium at a relative centrifugal force of greater than 100,000×g to transfer a virus from the liquid pancreatin test sample supernatant into a target fraction of the discontinuous gradient medium, whereby the target fraction is quantitatively separated from the liquid pancreatin test sample supernatant; 
 (d) quantitatively determining a viral load of the pancreatin test sample by culturing the target fraction with a population of cells, assessing the population of cells for viral infection, and calculating a viral titer in the target fraction; 
   wherein steps (a), (b), and (c) are performed without altering the viral load.   
     
     
         49 . The method of  claim 48 , wherein the liquid pancreatin test sample is a suspension comprising pancreatin, a cell culture medium suitable for a cell line used to culture virus, and one or more antibiotics. 
     
     
         50 . The method of  claim 48 , wherein the liquid pancreatin test sample is at a temperature of between about 0° C. and about 15° C. during the centrifugation of step (b). 
     
     
         51 . The method of  claim 48 , wherein the liquid pancreatin test sample is at a temperature of between about 0° C. and about 15° C. during the ultracentrifugation of step (c). 
     
     
         52 . The method of  claim 48 , wherein the centrifugation of step (b) is carried out for longer than five minutes. 
     
     
         53 . The method of  claim 48 , wherein the ultracentrifugation of step (c) is carried out for more than one hour. 
     
     
         54 . The method of  claim 48 , wherein the discontinuous gradient medium is a discontinuous two-phase sucrose gradient. 
     
     
         55 . The method of  claim 48 , wherein the discontinuous gradient medium is prepared from a 50% (wt./vol.) buffered sucrose solution and a 20% (wt./vol.) buffered sucrose solution. 
     
     
         56 . A pharmaceutical composition comprising:
 a pharmaceutically effective quantity of pancreatin and one or more pharmaceutically acceptable excipients in an oral dosage form, wherein the viral load of the pancreatin has been quantitatively determined by a process comprising the steps of:
 (a) producing a liquid pancreatin test sample; 
 (b) centrifugation of the liquid pancreatin test sample at a relative centrifugal force of less than 10,000×g in which viruses with sedimentation constants of greater than about 120 S do not form a pellet whereby the centrifugation of the liquid pancreatin test sample produces a supernatant; 
 (c) ultracentrifugation of the liquid pancreatin test sample supernatant with a discontinuous gradient medium at a relative centrifugal force of greater than 100,000×g to transfer a virus from the liquid pancreatin test sample supernatant into a target fraction of the discontinuous gradient medium, whereby the target fraction is quantitatively separated from the liquid pancreatin test sample supernatant; 
 (d) quantitatively determining a viral load of the pancreatin test sample by culturing the target fraction with a population of cells, assessing the population of cells for viral infection, and calculating a viral titer in the target fraction; 
   wherein steps (a), (b), and (c) are performed without altering the viral load.   
     
     
         57 . The pharmaceutical composition of  claim 56 , wherein the liquid pancreatin test sample is a suspension comprising pancreatin, a cell culture medium suitable for a cell line used to culture virus, and one or more antibiotics. 
     
     
         58 . The pharmaceutical composition of  claim 56 , wherein the liquid pancreatin test sample is at a temperature of between about 0° C. and about 15° C. during the centrifugation of step (b). 
     
     
         59 . The pharmaceutical composition of  claim 56 , wherein the liquid pancreatin test sample is at a temperature of between about 0° C. and about 15° C. during the ultracentrifugation of step (c). 
     
     
         60 . The pharmaceutical composition of  claim 56 , wherein the centrifugation of step (b) is carried out for longer than five minutes. 
     
     
         61 . The pharmaceutical composition of  claim 56 , wherein the ultracentrifugation of step (c) is carried out for more than one hour. 
     
     
         62 . The pharmaceutical composition of  claim 56 , wherein the discontinuous gradient medium is a discontinuous two-phase sucrose gradient. 
     
     
         63 . The pharmaceutical composition of  claim 56 , wherein the discontinuous gradient medium is prepared from a 50% (wt./vol.) buffered sucrose solution and a 20% (wt./vol.) buffered sucrose solution. 
     
     
         64 . A pharmaceutical composition prepared by a process comprising the steps of:
 providing a quantity of pancreatin and one or more pharmaceutically acceptable excipients, wherein the viral load of the pancreatin has been quantitatively determined by a process comprising the steps of:
 (a) producing a liquid pancreatin test sample; 
 (b) centrifugation of the liquid pancreatin test sample at a relative centrifugal force of less than 10,000×g in which viruses with sedimentation constants of greater than about 120 S do not form a pellet whereby the centrifugation of the liquid pancreatin test sample produces a supernatant; 
 (c) ultracentrifugation of the liquid pancreatin test sample supernatant with a discontinuous gradient medium at a relative centrifugal force of greater than 100,000×g to transfer a virus from the liquid pancreatin test sample supernatant into a target fraction of the discontinuous gradient medium, whereby the target fraction is quantitatively separated from the liquid pancreatin test sample supernatant; 
 (d) quantitatively determining a viral load of the pancreatin test sample by culturing the target fraction with a population of cells, assessing the population of cells for viral infection, and calculating a viral titer in the target fraction; 
   wherein steps (a), (b), and (c) are performed without altering the viral load.   
     
     
         65 . The pharmaceutical composition of  claim 64 , wherein the liquid pancreatin test sample is a suspension comprising pancreatin, a cell culture medium suitable for a cell line used to culture virus, and one or more antibiotics. 
     
     
         66 . The pharmaceutical composition of  claim 64 , wherein the liquid pancreatin test sample is at a temperature of between about 0° C. and about 15° C. during the centrifugation of step (b). 
     
     
         67 . The pharmaceutical composition of  claim 64 , wherein the liquid pancreatin test sample is at a temperature of between about 0° C. and about 15° C. during the ultracentrifugation of step (c). 
     
     
         68 . The pharmaceutical composition of  claim 64 , wherein the centrifugation of step (b) is carried out for longer than five minutes. 
     
     
         69 . The pharmaceutical composition of  claim 64 , wherein the ultracentrifugation of step (c) is carried out for more than one hour. 
     
     
         70 . The pharmaceutical composition of  claim 64 , wherein the discontinuous gradient medium is a discontinuous two-phase sucrose gradient. 
     
     
         71 . The pharmaceutical composition of  claim 64 , wherein the discontinuous gradient medium is prepared from a 50% (wt./vol.) buffered sucrose solution and a 20% (wt./vol.) buffered sucrose solution.