Materials and Methods
Sodium dodecyl sulfate (PN L4390-100G) and 2-mercaptoethanol (PN M3148-100ML) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Amicon Ultra-0.5 Centrifugal Filters with 30,000 NMWL were purchased from EMD Millipore (Billerica, MA, U.S.A.). The SDS-MW Analysis Kit (PN 390953) was from SCIEX (Framingham, MA, U.S.A.), which includes the SDS-MW gel buffer(proprietary formulation, pH 8, 0.2% SDS), acidic wash solution (0.1 N HCl), basic wash solution (0.1 N NaOH) and the SDS-MW sample buffer(100 mM Tris-HCl pH 9.0, 1%SDS). EZCE Capillary Cartridge (PN A55625, SCIEX, Framingham, MA, U.S.A) pre-assembled with bare fused-silica capillary (50 µm I.D., 30 cm total length, 20 cm effective length) was used for separation. Universal vials (PN A62251), universal vial caps (PN A62250), PCR vials (PN 144709) and nanoVials (PN 5043467 from SCIEX (Framingham, MA, U.S.A.) were used for sample solution loading.
A PA 800 Plus Pharmaceutical Analysis system (SCIEX, Framingham, MA, U.S.A.) equipped with a PDA detector and 32 Karat software were used for all the experiments. EZ-CE Capillary Cartridge (PN A55625, SCIEX, Framingham, MA, U.S.A.) preassembled with bare fused-silica capillary (50 μm I.D., 30 cm total length, 20 cm effective length) was used for separation.
Data acquisition and analysis were performed using 32 KaratTM Software 10.
Packaged AAV2 of pAV-CMV-GFP with titer at 2.24X1013GC/mL (titer as supplied by vendor) and packaged AAV8 of pAV-CMVGFP with titer at 3.99 X1013GC/mL (titer as supplied by vendor) was purchased from Vigene Biosciences (Rockville, MD, U.S.A.). Both samples were kept in storage solution of PBS (Phosphate Buffered Saline, pH 7.5)/0.001% pluronic F68.
Sample Preparation Procedure. 5µL of AAV8 sample solution with final salt concentration no more than 40mM was mixed with 5µL of 1% SDS and 1.5µL of 2-mercaptoethanol in a 0.65 mL micro-centrifuge tube and incubated at 50°C for 10min. Then, 90µL of DI water was added to the mixture. The diluted mixture was transferred to the sample vial or nanoVial for analysis on the PA 800 Plus Pharmaceutical Analysis system.
Buffer exchange is necessary if salt concentration in AAV sample is higher than 40mM.
For Method Development and Optimization. In this technical note, the method is developed and optimized using AAV8 samples at 1 X 1013 GC/mL. Unless stated otherwise, 1.25 µL of AAV8 sample at 4 X 1013 GC/mL and 3.75 µL of deionized water was used in sample preparation procedure as 5 µL of AAV8 sample solution at 1X1013GC/mL.
All the titer values of AAV samples used in this technical note were provided by the vendor based on results from qPCR.
For Method Evaluation. To evaluate the capability of this method for analysis of AAV8 samples with lower titer, the AAV8 sample at 4 X 1013 GC/mL was diluted to 1 X1012 GC/mL in the storage buffer provided by the vendor to represent the AAV8 samples at 1 X1012 GC/mL.
Buffer exchange not only can be used to exchange the buffer with slat concentration lower than 40mM, it can be also used to concentrate the AAV sample for analysis. In this technical note, four times diluted storage buffer (salt concentration lower than 40mM) was used as elution buffer in buffer exchange procedure. The use of pluronic F68 is to minimize sticking of AAV to hydrophobic surfaces of plastics.1
Comparison of peak profiles of the AAV8 samples with and without buffer exchange was performed and discussed in the Result and Discussion section.