A potency assay for a replication incompetent adenovirus type 5 carrying a human fgf-4 gene.
A bioassay that measures the potency of the FGF-4 transgene carried by a replication incompetent adenovirus type 5, Ad5FGF-4, was developed on ARPE-19 cells. The assay is carried out in a microtiter plate format and measures cellular proliferation following infection of ARPE-19 cells with a serial dilution of Ad5FGF-4. Proliferation is measured as a percentage increase in absorbance reading in relation to a mock-infected control. Ad5LacZ and Ad5FGF-4 viruses treated similarly to the test sample are included as negative and positive controls, respectively. The increased absorbance reading resulting from infection with the virus correlates with FGF-4 production as determined by an FGF-4 enzyme-linked immunosorbent assay, an increase in de novo DNA synthesis as measured by BrdU incorporation, and an increase in the total cell number. The assay shows a dose-dependent response and is capable of evaluating the stability of Ad5FGF-4. A sample being tested is compared with a reference standard, and the relative potency value is obtained by a parallel line analysis of the dose-response curve of the test article in relation to the reference standard. Therefore, this procedure can be used as an in vitro efficacy-indicating assay, demonstrating that the FGF-4 transgene product carried by Ad5FGF-4 is biologically active.
Fawaz
Farah S
FS
Berlex Biosciences, Process Development and Product Characterization, Richmond, CA 94806, USA. farah_fawaz@berlex.com
Elsheikh
Moutasem A
MA
Ogawa
Yasushi
Y
Files
Jim G
JG
McCaman
Michael T
MT
Pungor
Erno
E
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D001681:Biological Assay; D002460:Cell Line; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D014779:Virus Replication
C496445:FGF4 protein, human; D051519:Fibroblast Growth Factor 4; D011518:Proto-Oncogene Proteins; D005346:Fibroblast Growth Factors