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[Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX]
Kai Gao1, Jun-zhi Wang, Chun-ming Rao
1National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China.
Aim:
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.
Conclusion:
The methods and requirements had been established for quality control of rAAV-2/hFIX.
