Characterization and quality control of recombinant adenovirus vectors forgene therapy

Highly purified recombinant adenovirus undergoes routine quality controls f or identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expressio n of the DNA binding protein, an early adenoviral protein, in an immunofluo rescence bioassay on permissive cells as a potency determinant. The specifi c particle count, a key quality indicator, is the total number of intact pa rticles present compared to the number of infectious units. Electron micros copic analysis using negative staining gives a qualitative biophysical anal ysis of the particles eluted from anion-exchange HPLC, One purity assessmen t is accomplished via the documented presence and relative ratios of compon ent adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MAL DI-TOF mass spectrometry and subsequent data mining. Verification of the vi ral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replicat ion-competent adenovirus prior to human use. For adenovirus carrying the hu man IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA a nd cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells, Both quantitative and qualitative analyses show good batc h to batch reproducibility under routine test conditions using validated me thods. (C) 2001 Elsevier Science B.V. All rights reserved.