How is ultraDAWN used in-line with FPLC to monitor chromatographic purification of AAV?

Positioning an ultraDAWN^® in-line with an AKTA avant or other FPLC system can, with the right setup, provide the same information as the SEC-MALS Viral Vector Analysis (VVA), including Vg/Cp, viral titer, concentrations of full & empty capsids, and molar masses of the capsid, genome and complete virus. The data are acquired in ASTRA^® and processed with ASTRA’s VVA module, giving results immediately following AAV elution.

Here’s what you’ll need:

1. ultraDAWN plumbed in-line with the FPLC, between the UV and conductivity detectors. ultraDAWN tubing should match that of the FPLC downstream of the column. Since AAVs are commonly purified in small batches at flow rates of 1 – 10 mL/min, 0.020” i.d. tubing should be installed. Higher flow rates may necessitate larger i.d. tubing.
2. ASTRA with Viral Vector Analysis module with accurate extinction coefficients entered for the serotype to be run. If the vectors elute above 200-300 mM NaCl (or less of a heavier buffer salt), dn/dc values of protein and DNA may need to be adjusted slightly.
3. Multi-wavelength UV detector set to collect UV1 @ 260 nm and UV2 @ 280 nm. For an AKTA system that would be the triple-wavelength UV. Usually the 1 cm flow cell is preferred, unless your AAV concentrations exceed the detector’s linearity range.
4. Analog output of the two UV signals to the ultraDAWN, for example via an AKTA E9 I/O Box, and appropriate cables feeding into the ultraDAWN’s AUX IN ports.
5. Configure the chromatography software to provide absorbance signals from 260 nm and 280 nm on the analog outputs. In UNICORN, this is done via the I/O Box instructions Configure Analog Out 1 & 2. Be sure to set appropriate output ranges, such as 1000 mV / AU, for each wavelength. The maximum output voltage of an AKTA E9 module is 1 V. Also, configure the FPLC method to produce an autoinject signal upon beginning the elution phase. This is done with a Digital Pulse Out 1 instruction in UNICORN, setting the pulse duration to 0.5 seconds.

Configure ASTRA (with the VVA method) to identify the correspondence between AUX IN ports and UV wavelengths, and match the AU/V factors to those set up in the chromatography software.

If not already done, perform normalization, alignment and band broadening measurements for the ultraDAWN – UV detector pair. Use 40 kDa dextran, filtered to 20 nm, for normalization. Alignment and band broadening can be done with filtered BSA @ 0.5 mg/mL, injected via an injection loop of 200 – 500 µL, bypassing the column.