Recent advances in the field of gene therapy significantly increased the need for lentivirus
vectors. Significant pressure fell on the production where several serious bottlenecks
throttling the expanding field were uncovered. With robust and reproducible production
being a prerequisite for a safe and efficient final product, quick and simple analytical methods
have been recognized as a crucial enabling element in the FDA sponsored Process Analytical
Technology initiative. In this regard, the chromatographic fingerprint method was developed
for deeper process understanding and better process control. The goal is to have a method
that would be able to cover the process from the control of the upstream raw materials,
downstream process steps all the way to the final product quality control. Chromatographic
fingerprints were evaluated through common peak selection, common peak height, relative
height, and whole chromatogram area. The focus was on the bioprocess progression, DNA
removal, broth clarification, concentration, buffer exchange, and chromatography. Change
in the composition of the inflown fresh growth media was detected during the upstream virus
proliferation, presumable DNA peak reduction was observed, while new emerging impurities
were detected and subsequently removed during clarification. The developed method was
also applied to the crossflow filtration cassette test with results indicating significant
differences in their operation. Analysis of the chromatographic step suggested that most of
the impurities, the ones dominating chromatograms in all steps upstream, were removed with
chromatography. The developed chromatographic analytical method enables process
monitoring through a comparison of relative changes between specific process samples. It
does, however, not enable quantification of lentiviral particle concentration.
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