Monitoring of the adenovirus production process by flow cytometry.
Adenovirus (Ad) has become the vector of choice for gene therapy clinical protocols worldwide; it is the only viral vector to date that has been licensed for use in a gene therapy treatment. There is, however, a need to develop a simple, reliable at-line method to monitor the production of virus and recombinant proteins (r-proteins) that have no intrinsic reporter properties. Here we utilize flow cytometry to measure cell size, granularity, and DNA content in a single-step analysis and to correlate these parameters to the production of a type-5 Ad (Ad5) expressing the recombinant green fluorescent protein (GFP). Clear correlations between these parameters and productivity are made, with forward scatter and DNA content showing the highest correlation coefficients, 0.9 and 0.83 for virus production and r-protein production, respectively. Measuring these parameters requires little or no processing of the cells from culture to analysis. These parameters have been used successfully to monitor, at-line, the amount of Ad and r-protein product in a 293-Ad system.
Sandhu
Kalbinder Singh
KS
Department of Chemical Engineering, University of Birmingham, Edgbaston Birmingham, B15 2TT, United Kingdom. kalbinder.sandhu@ucd.ie
Al-Rubeai
Mohamed
M
D000256:Adenoviridae / Q000235:genetics / Q000254:growth & development*; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014776:Virus Cultivation / Q000379:methods
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins