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Peter Holper, Staff Applications Scientist at SCIEX, US, shares his tips and tricks on AAV analysis using CE with the BioPhase 8800 system and the PA 800 Plus system.

Tip 1: Leverage the flexibility in injection modes

When starting out with a new viral vector product, my recommendation is to compare three different modes of injection using UV detection. First, start with a standard electrokinetic injection, which allows for the highest theoretical resolution. Next, use a pressure/ hydrodynamic injection, which will inject the same plug regardless of sample ionic strength and provide a quick estimate of the titer. Finally, use a field-amplified sample stacking (FASS) injection to achieve the highest sensitivity, while understanding it is the most sensitive injection method to the ionic strength of the matrix. Comparing these three peak profiles can give significant insight into the optimal separation conditions for each molecule analyzed.

Tip 2: Deal with low sample amounts

During early-stage development of AAV vectors, oftentimes only a few micrograms of proteins or less are available for analytics. However, most analytical technology is not practical for applications with low protein concentration or small sample volumes. To improve the sensitivity of CE-SDS, my recommendation is to use laser-induced fluorescence (LIF) detection instead of UV absorbance. Comparing the results from the different injection types (tip 1) will help you determine if additional sensitivity and transition to LIF detection is needed.

Tip 3: Optimize fluorescence dye labelling

Labeling procedure can pose challenges and require optimization for each product. Currently, the most common fluorescent dye used in CE-SDS-LIF is Chromeo P503, which has a low quantum yield when not bound to a protein and thus does not require additional cleanup after conjugation. When optimizing the labeling procedure with Chromeo P503, I find the dye-to-protein ratio to be the most important factor. If this ratio is not optimal, low signal or high peak tailing is often observed. I find that estimating the protein titer by referring to the peak area achieved with pressure injection (tip 1) can be highly beneficial, since only the genome titer may be known at this point.