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. A method of assaying a batch of recombinant viral vectors to determine if the recombinant viral vectors can be packaged and released, wherein the recombinant viral vectors have a viral transgene that expresses a protein that has biological activity, said method comprising determining a level of infectivity of a sample of the recombinant viral vectors comprising:
a. infecting cultured cells with the sample of the recombinant viral vectors, wherein the cultured cells are infected with more than one concentration of the recombinant viral vector;
b. measuring by flow cytometry a percentage of infected cells that comprise the expressed recombinant viral vector protein;
c. using the percentage of infected cells that comprise the expressed recombinant viral vector protein in a Slope Ratio method to generate a linear response curve; and
d. comparing the linear response curve to a reference standard to determine level of infectivity,
wherein if the level of infectivity is acceptable, then the batch of recombinant viral vectors is packaged and released.
2. The method of claim 1 , wherein the recombinant viral vector is a recombinant adenoviral vector.
3. The method of claim 2 , wherein the recombinant adenoviral vector comprises a nucleotide sequence encoding a human interferon alpha 2b.
4. The method of claim 1 , wherein the recombinant viral vector comprises a nucleotide sequence encoding human interferon.
5. The method of claim 4 , wherein the human interferon is interferon alpha 2b.
6. The method of claim 1 , wherein the cultured cells are HEK293 cells.
7. The method of claim 1 , wherein the recombinant viral vector protein is an adenovirus hexon structural protein.
8. The method of claim 1 , wherein the reference standard was manufactured using the same process as the recombinant viral vector.
9. The method of claim 1 , wherein the cultured cells are infected with two dilution series of the recombinant viral vector.
10. The method of claim 1 , wherein the cultured cells are infected with two dilution series of the reference standard.
11. The method of claim 1 , wherein the infecting step comprises incubating cultured cells for about 15 minutes with an infection medium comprising the recombinant viral vector.
12. The method of claim 11 , further comprising aspirating the infection medium after the about 15 minutes and adding growth media to the cultured cells.
13. The method of claim 1 , further comprising incubating the cultured cells for about 48 hours after the infecting step.
14. The method of claim 1 , wherein the cultured cells are divided into a first cell culture and a second cell culture and wherein a number of recombinant viral vector particles contacted to said first cell culture is different from the number of recombinant viral vector particles contacted to said second cell culture.
15. The method of claim 1 , further comprising measuring transgene expression.
16. The method of claim 1 , further comprising measuring activity of the protein expressed by said transgene.
17. The method of claim 1 , further comprising measuring viral particle titer.