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.
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