Expert Insight: LC and LC-MS technologies to the analysis of rAAV viral vectors: Watch on demand

Watch this on-demand webinar to discover the challenges associated with analyzing AAV samples, and learn how LC and LC-MS workflows can help solve these setbacks

17 Nov 2020

Ximo Zhang, senior scientist, and Stephan M. Koza, principle scientist, at Waters

Recombinant adeno-associated viral (rAAV) vectors have emerged as the leading gene delivery platform for gene therapy and genetic vaccination. For this promising gene delivery vector to fully realize its therapeutic or prophylactic potential, a rational design strategy is desired. The current challenges in viral vector manufacturing robustness, scale up, and productivity will need to be addressed to turn this mode of treatment into an accessible global benefit. To meet this goal, reliable analytical technologies to help establish a structure-function relationship, guide the development of manufacturing processes, and assess the quality of clinical materials have become increasingly relevant and urgent.

In this free webinar, hear from Stephan M. Koza, principle scientist, and Ximo Zhang, senior scientist, at Waters, to discover the challenges associated with analyzing AAV samples, and learn how LC and LC-MS workflows can help solve these setbacks, including those that employ size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), protein reversed-phase LC-MS, and peptide mapping LC-MS based analyses.

Read on for the highlights of the live Q&A session or register to watch the webinar at any time that suits you.

Q: Are there any examples of successful analytical chromatography of lentivirus?

XZ: For lentivirus, just like the AAV, we can probably use this activity to dissociate the virus, and then analyze the proteins through LC and MS. We need to do some method development through that. Currently, our lab is a biosafety level 1 lab, so we'll need some further development of that part, and in the future we'll probably look into that.

SK: For the SEC and ion exchange, we haven't done any work with lentivirus as an intact molecule for those same reasons.

Q: Which protocols could be applied for removing surfactants from any of these samples?

XZ: We recently developed this protocol to remove the surfactant. So, generally speaking, it's to use the Amicon filter to remove the buffer. They have a step-by-step protocol listed, and it's very inclusive.

Q: Is there any special sample preparation necessary for analyzing the AAV by RP chromatography?

XZ: For sample preparation, typically people use about 10% acetic acid to dissociate the AAV capsid into the viral protein, but I think the reversed-phase chromatography is also in an acidic solution, with a pH 2 to 3. Without any sample preparation, if you're afraid that your sample doesn't contain any messy stuff, you can directly inject the AAV capsid. It will dissociate, but if you treat the samples with the acetic acid for a while, you probably will have more reproducible results.

Q: Does the SEC method detect peaks of AAV clipping forms?

SK: The most predominant method was fluorescence detection where we saw protein fragments. So, its selected for proteins, verses DNA, was the AE9 sample that we looked at. One of the advantages of the 450 is you have the ability to get a little bit better resolution on the fragmented sides of the intact capsid.

Q: Were you able to identify the phosphorylation site by LC-MS/MS?

ZX: For LC, we looked into the sites for peptide mapping (PTM) through the peptide mapping method. Through this method we did find confirmed PTMs. Through the method we developed, we can see that there are some sites that are prone to phosphorylation.

Q: How much material was used for injection on slide 39?

ZX: Slide 39 is the separation of the AAV stereotypes by the optimized LC-MS method. Here the samples are from different vendors, so they have different concentrations. They were roughly on the range of the injections, the method was roughly from the range of 0.02 to 0.05 micrograms, so around 20 to 50 milligrams. Now, you can see, we've still got the different signals with that much of sample.

Q: Under what circumstances would you recommend fluorescence detection over UV-based detection for the SEC assay?

SK: In general, for the SEC method, we would typically recommend the fluorescence method, simply because the sensitivity is going to be that much better for the low abundance for, aggregate forms or fragment forms that are generally present in these samples.

Generally, these concentrations are low and I think using the fluorescence is going to give you an advantage. It's also going to be very selective for monitoring the protein component, versus the DNA component that's in the samples.

Q: The short SEC UV method, for titer measurement, seems to be more efficient. Is it possible to be used on E/F analyses, instead of IEX?

SK: As I mentioned in the presentation, while I think it is a reasonable way of estimating the approximate empty-to-full ratio, one of the disadvantages it is going to have — particularly as you get to very high purities — is that the uncertainty in that empty-to-full ratio is going to get larger and larger if you look at that response curve.

In general, what we position that method for would be if you're looking at samples that are relatively low in purity, there's a fairly high abundance of empty, it may be a decent way of making quick estimates for those types of samples. It's certainly not going to be good for looking at high purity samples. Furthermore, it will be good for estimating empty/full ratio to make response factor corrections for the titer method.

To find out more about analyzing rAAV viral vectors with LC and LC-MS technologies, watch this on-demand webinar>>