Mdck cell lines supporting viral growth to high titers and bioreactor process using the same

Abstract

The present invention relates to novel MDCK cells which can be to grow viruses, e.g., influenza viruses, in cell culture to higher titer than previously possible. The MDCK cells can be adapted to serum-free culture medium. The present invention further relates to cell culture compositions comprising the MDCK cells and cultivation methods for growing the MDCK cells. The present invention further relates to methods for producing influenza viruses in cell culture using the MDCK cells of the invention.

1 . A Madin-Darby Canine Kidney (MCDK) cell, wherein a cell culture composition comprising a plurality of the MDCK cells supports replication of a cold-adapted influenza virus to a base 10 logarithm of the median tissue culture infection dose per milliliter (log 10  TCID 50 /mL) of at least about 7.8 or to a base 10 logarithm of fluorescent focus units per milliliter (log 10  FFU/mL) of at least about 7.8. 
     
     
         2 . The MDCK cell of  claim 1 , wherein the cell culture composition is serum free. 
     
     
         3 . The MDCK cell of  claim 1 , wherein the MDCK cell is adherent. 
     
     
         4 . The MDCK cell of  claim 1 , wherein the MDCK cell is non-tumorigenic and/or non-oncogenic. 
     
     
         5 . The MDCK cell of  claim 1 , wherein the MDCK cell is identified by ATCC Accession No. PTA-7909 or PTA-7910 
     
     
         6 . The MDCK cell of  claim 1 , wherein the influenza virus is also attenuated and temperature sensitive. 
     
     
         7 . The MDCK cell of  claim 1 , wherein the influenza virus comprises one or more gene segments of influenza strain A/Ann Arbor/6/60 or B/Ann Arbor/1/66. 
     
     
         8 . A method for proliferating the MDCK cell of  claim 1  to a cell density of at least about 1×10 6  cells/ml in a Single Use Bioreactor (SUB) system comprising inoculating a serum free cell culture medium with the MDCK cell of  claim 1  at a seeding density of between about 1×10 4  to about 12×10 4  cells/mL and culturing the cells while maintaining one or more culture conditions selected from the group consisting of:
 a. an agitation rate of between about 50 to 150 rpm;   b. a pH of between about 6.0 to about 7.5;   c. dissolved oxygen (DO) between about 35% to about 100%; and   d. a temperature of between about 33° C. to about 42° C.   
     
     
         9 . The method of  claim 8 , wherein the cell culture medium is selected from the group consisting of MediV-105, MediV-105 supplemented with glucose, M-32, M-32 supplemented with glucose, MediV-107, MediV-107 supplemented with glucose. 
     
     
         10 . The method of  claim 8 , wherein the MDCK cell is an adherent MDCK cell and wherein a microcarrier is used for culturing the cells. 
     
     
         11 . A method for producing cold adapted influenza viruses to a log 10  TCID 50 /mL of at least about 7.8 in cell culture, comprising:
 a. proliferating the MDCK cell of  claim 1  to a cell density of at least about 1×10 6  cells/ml in a Single Use Bioreactor (SUB) system comprising inoculating an animal protein free cell culture medium with the MDCK cell of  claim 1  at a seeding density of between about 1×10 4  to about 12×10 4  cells/mL and culturing the cells while maintaining one or more culture conditions selected from the group consisting of an agitation rate of between about 50 to 150 rpm, a pH of between about 6.0 to about 7.5, and dissolved oxygen (DO) between about 35% to about 100%;   b. infecting the proliferated MDCK cells with an influenza virus;   c. incubating the infected proliferated MDCK cells under conditions that permit replication of the influenza virus; and   d. isolating influenza viruses from the cell culture composition.   
     
     
         12 . The method of  claim 11 , wherein fresh medium or additional medium components are added to the cell culture during step (a). 
     
     
         13 . The method of  claim 11 , wherein none or some of the cell culture medium is removed and replaced with fresh medium prior to or during step (b) 
     
     
         14 . The method of  claim 11 , wherein the cell culture medium is selected from the group consisting of MediV-105, MediV-105 supplemented with glucose, M-32, M-32 supplemented with glucose, MediV-107, MediV-107 supplemented with glucose. 
     
     
         15 . The method of  claim 11 , wherein the MDCK cell is an adherent MDCK cell and wherein a microcarrier is used for culturing the cells. 
     
     
         16 . The method of  claim 11 , wherein step (b) is carried out at a Multiplicity Of Infection (MOI) of between about 0.00001 to about 0.003 FFU/cell. 
     
     
         17 . The method of  claim 11 , wherein the conditions of step (c) are selected from the group consisting of an agitation rate of between about 50 to 150 rpm, a pH of between about 6.0 to about 7.5, dissolved oxygen (DO) between about 35% to about 100%, and a temperature of between about 30° C. to about 35° C. 
     
     
         18 . The method of  claim 11 , wherein the MDCK cell is identified by ATCC Accession No. PTA-7909 or PTA-7910. 
     
     
         19 . The method of  claim 11 , wherein the influenza virus is also attenuated and temperature sensitive. 
     
     
         20 . The method of  claim 11 , wherein the influenza virus comprises one or more gene segments of influenza strain A/Ann Arbor/6/60 or B/Ann Arbor/1/66. 
     
     
         21 . A method of eliminating DNA contaminants from a viral preparation comprising:
 a. passing the viral preparation over an affinity chromatography media under conditions wherein the DNA contaminants are not retained on the affinity chromatography media and the virus present in the viral preparation are retained;   b. washing the affinity chromatography media to remove the DNA contaminants; and   c. eluting the virus present in the viral preparation from the affinity chromatography media.   
     
     
         22 . The method of  claim 21 , wherein between steps (a) and (b) a non-specific endonuclease preparation is passed over the affinity chromatography media. 
     
     
         23 . The method of  claim 21 , wherein the viral preparation is an influenza virus preparation. 
     
     
         24 . The method of  claim 23 , wherein the influenza virus preparation was prepared from mammalian cells. 
     
     
         25 . The method of  claim 21 , wherein the affinity chromatography media is Cellufine™ Sulfate resin, and the conditions used in step (a) are 1×SP buffer at about pH 7.2, and the virus are eluted in step (c) in 1×SP buffer containing about 1 M NaCl at about pH 7.2.