Designing a strategy to realize the potential of rAAV vectors in gene therapy and genetic vaccinations

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 are needed to help establish a structure-function relationship. Also, to guide the development of manufacturing processes and assess the quality of clinical materials.

In this on-demand SelectScience^® webinar, you will hear from principal scientist Stephan M. Koza and senior scientist Ximo Zhang, both from Waters, as they discuss the challenges associated with analyzing AAV samples. You will also 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 highlights from the Q&A discussion at the end of the live webinar or register to watch the full webinar on demand >>

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

XZ: For lentivirus, I can only speak about the LC-MS part. Just like the AAV, we can use this to dissociate the virus, and then just analyze the proteins throughout the LC-MS and we probably need to do some method development through that. Currently, our lab is Biosafety Level 1, so we will need further development of that part, and in the future, we're going to look into that.

SK: And I would add that for the SEC and the 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 surfactant from AAV samples?

XZ: We recently developed this protocol to remove the surfactants: you use the Amicon zinc filter to remove the surfactant, by buffer exchange. We have a detailed, step-by-step protocol.

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

XZ: For sample preparation, people typically 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 acid solution with a pH of two to three. Without any sample preparation, if you are sure that your sample doesn’t contain any contaminants, you can just directly inject the AAV capsid. It will dissociate. If you treat the samples with the acetic acid for a while, you will have more reproducible results.

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

SK: Yes, there are two examples. The most predominant example, where we saw protein fragments (and remember, we're using fluorescence detection so it's selective for proteins versus DNA) was the AE 9 sample that we looked at. And that's one of the advantages of the 450 Å column, you have the ability to get slightly better resolution on those fragments inside of the intact capsid.

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

XZ: Yes. We looked into the sites for post-translational modifications (PTM) through the pattern mapping method. Through pattern mapping, we found differing PTMs and there were some phosphorylation sites. However, since we are still under a non-disclosure agreement with our collaborators we didn't put any of that information in the slides. But through the method we developed, we can see that there are some sites that are prone to phosphorylation.

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

SK: For the SEC method, we would typically recommend the fluorescence method simply because the sensitivity is much better for the low-abundance aggregate forms or fragment forms that are present in these samples. These concentrations are low, and using 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 empty/full (E/F) analysis instead of AEX?

SK: While it is a reasonable way of estimating the approximate empty-to-full ratio, one of the disadvantages that E/F 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.

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

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