VIRAL VECTOR ASSAY AND VECTOR
A process for assaying viral vector manufactured by large-scale viral vector manufacturing processes to assure the resulting vector has acceptable purity and potency. The process entails three different types of assays, each one of which is optionally useful on a stand-alone basis, and which together provide the first system able to assure the quality of viral vector produced by large-scale vector manufacturing processes.
1 . An assay to determine the infectivity of a recombinant viral vector, the assay comprising:
a. obtaining a recombinant viral vector; and
b. obtaining cells that support replication of said viral vector and then dividing said cells into a first cell culture and a second cell culture; and then
c. contacting each of said first cell culture and said second cell culture with said recombinant viral vector, whereby the vector particles per cell contacted to said first cell culture is different from the vector particles per cell contacted to said second cell culture; and then
d. determining the percentage of cells in each cell culture which have been infected with the viral vector; and then
e. calculating a linear response curve to measure relative infectivity using the Slope Ratio method.
2 . The assay of claim 1 , where said determining the percentage of infected cells in the cell cultures involves flow cytometry.
3 . The assay of claim 1 , where said determining the percentage of infected cells in the cell cultures involves fluorescence activated cell sorting.
4 . The assay of claim 1 , where said determining the percentage of infected cells in the cell cultures involves utilizing an antibody against a viral vector antigen.
5 . The assay of claim 1 , where the number of particles per cell in said first cell culture and the number of particles per cell in said second cell culture are selected to provide a linear response curve for viral vector infectivity.
6 . The assay of claim 1 , step b further comprising dividing said cells into a negative control cell culture.
7 . The assay of claim 1 , step b further comprising dividing said cells into a reference standard control cell culture, and step c further comprising contacting the reference standard control cell culture with reference standard virus which is different from the recombinant viral vector.
8 . The assay of claim 1 , step b further comprising dividing said cells into a positive control cell culture, and step c further comprising contacting the positive cell culture with a different manufacturing lot of the recombinant viral vector.
9 . The assay of claim 1 , wherein the cell cultures are seeded to produce, at time of contacting the cell cultures with the recombinant viral vector, a cell density of about 80%.
10 . A recombinant viral vector manufactured by the process comprising:
a. from a batch of recombinant viral vector, removing a sample; and then
b. assaying the sample using the assay of claim 1 ; and then
c. if the infectivity of the sample is not acceptable, then discarding said batch of recombinant viral vector.
11 . The recombinant vector of claim 10 , the process further comprising:
d. if the infectivity of the sample is acceptable, then packaging and releasing said batch of recombinant viral vector.
12 . A process for manufacturing a viral vector having a transgene, comprising: obtaining a sample, and then assaying said sample by a. measuring viral vector infectious titer by calculating a linear response curve to measure relative infectivity using the Slope Ratio method; and using at least two assays selected from the group consisting of: b. measuring transgene expression, c. measuring the activity of the polypeptide expressed by said transgene, and d. measuring viral particle titer.
13 . The process of claim 12 , wherein said assaying said sample comprises a. measuring viral vector infectious titer by calculating a linear response curve to measure relative infectivity using the Slope Ratio method, b. measuring transgene expression and c. measuring the activity of the polypeptide expressed by said transgene.
14 . The process of claim 12 , wherein said assaying said sample comprises d. measuring viral particle titer.
15 . The process of claim 12 , wherein said assaying said sample comprises: a. measuring viral vector infectious titer, b. measuring transgene expression, c. measuring the activity of the polypeptide expressed by said transgene, and d. measuring viral particle titer.
16 . A viral vector produced by the manufacturing process of claim 12 .
17 . A viral vector produced by the manufacturing process of claim 13 .
18 . A viral vector produced by the manufacturing process of claim 14 .
19 . A viral vector produced by the manufacturing process of claim 15 .
20 . An assay for quantifying the potency of a transgene-expressed polypeptide in a test solution comprising:
a. obtaining viable cells which are sensitive to a polypeptide of interest, and
b. obtaining host cells, transfecting said cells with a transgene coding for the polypeptide, of interest to form transformed cells, and culturing said cells in media; and then
c. harvesting from said host cells or media a test sample; and then
d. contacting the cells sensitive to the polypeptide of interest with the test sample; and then contacting the cells sensitive to the polypeptide of interest with an imaging dye; and then identifying, using a colorimetric method, cells sensitive to the polypeptide of interest which are viable.
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