[Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX].
OBJECTIVE
To establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX).


METHODS
Identification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT.


RESULTS
Identity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0 x 10(15) VG.L-1. hFIX expression was more than 20.0 micrograms.L-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements.


CONCLUSIONS
The methods and requirements had been established for quality control of rAAV-2/hFIX.
Gao
Kai
K
National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China.
Wang
Jun-zhi
JZ
Rao
Chun-ming
CM
Wu
Xiao-bing
XB
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011786:Quality Control; D011897:Random Allocation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D005164:Factor IX