I'm Kichi, a PhD student in the Laboratory of Macromolecular Biotechnology at Osaka University

First of all, I'd like to thank the organizers to arrange this wonderful conference and give me such a great opportunity to share my work today.

I'd like to talk about the method, which is density gradient equilibrium analytical ultracentrification for the analysis of A-B.

Let me start with the background. As you know, gene therapy provides promising therapeutic effects against genetic diseases and cancers.

Among various non-viral or viral vectors, adeno-associated virus A-B is a major vector because the number of clinical trials has been increasing and eight gene therapy products using A-B vectors have been approved so far.

So, A-B vectors are one of the leading platforms for gene therapy.

A-B has a high hexahedral capsid and encapsidates a single-stranded DNA of about 4.7 kilobase.

The capsid is composed of six tema of three different viral proteins in the order of its molecular mass.

The ratio of these VP proteins was estimated to 5 to 50.

For the recombinant A-B for vector, the gene of interest is inside instead of the viral type A-B.

A-B is low pathogenicity and long-term gene expression in humans and also numerous A-B serotypes have been identified with variable tissue tropisms.

In the upstream process of A-B vector manufacturing, the vector particles transiently expressed and it is desirable to produce full particles, this one, which encapsidate a design for DNA links.

However, impurities would also be produced such as empty particles, which is without DNA, and partial particles, which encapsidate shorter DNA, and extra-filled particles or over-filled particles, which is longer DNA in total.

Especially, the empty particle has been suggested to lower transaction efficiency and increase the risk of immunogenicity, so these manufacturing steps continue to the following downstream process to remove impurities.

And, cesium chloride density gradient odorase identification, BZUC, enables high purity purification of viruses and biological substances in intact states and has been agreed for the virus isolation, including A-B vector purification, as shown in the previous slide.

And in principle, the gradient medium, cesium chloride itself, generates a density gradient in the centrifugal field and the particle moves over time in the solution, but when it reaches the isopicnic point, where the particle's buoyant density equals the cesium chloride solution density, it holds there and makes a bond.

And the differences in buoyant density between empty and full particles is approximately 0.02, but this BZUC technique is very high density resolution, so that allows recovery of highly pure full particles.

And in previous study, there was two full particles with different buoyant densities were identified in addition to empty particles during this cesium chloride BZUC purification.

And although full particles with lower buoyant density was significantly higher in vitro transduction efficiency compared to the full particles with higher buoyant density, but no clear conclusions was drawn on the differences between its physicochemical properties between these two full particles.

So the objectives as follows.

The first one is establishment of density gradient equilibrium analytical ultracentrification, so called DZEAUC, for the characterization of A-B vectors.

So I applied the cesium chloride DZEAUC for the purification to the DZEAUC for the analysis in the analytical ultracentrifuge.

And the second thing is to elucidate the causes of the differences in buoyant density in two full particles.

And DZEAUC with multi-wavelength detection enables us to obtain this equilibrium profile at detected wavelengths and UV absorption spectrum for each observed component.

So firstly, the results of DZEAUC and SBAUC were compared using A-B8 sample.

As you know, SBAUC with multi-wavelength detection enables us to obtain the UV absorption properties for each component.

From the COS distribution, not only full particles, but also empty, partial, and extra-field particles are included.

On the other hand, DZEAUC showed three major peaks, the peak number 1, 1, 3, and 4, in DZEAUC profile.

And the UV absorption spectra was generated by plotting the each peak top position at each wavelength, then compared to that of empty and full particles in SBAUC, shown here.

And as you can see, the UV spectra was in good agreement, suggesting that the peak 1 in DZEAUC corresponded to empty particles, and peaks 3 and 4 corresponded to full particles.

So peaks 3 and 4 were full particles with different buoyant density.

So this is the observation made in the previous study, also in our DZEAUC results.

In addition, the quantitative evaluation was investigated.

By mixing the different ratios of full and empty, the linear correlation was confirmed between the measured full particle amount by DZEAUC and the expected full amount by the calculation.

So these results indicated that this DZEAUC is a analytical method that allows quantitative evaluation of observed main components.

A workflow was then developed to identify the components observed in the DZEAUC.

And as the low molecular weight species was included in the sample, that makes a concentration gradient like a seismic, right?

So firstly, the baseline was subtracted, and then peak was fitted.

Then the buoyant density was calculated from each peak to position using the theoretical equation describing the concentration gradient at equilibrium.

And here shows the regression line between the Weber partial specific volume and the encapsulated DNA length for empty and full particles with various AV serotypes and different lengths of DNA.

So from this relationship, using this relationship, we can expect the DNA length for each component by the buoyant density values.

And as you know, the Weber of the particle changes depending on the salt type or its concentration in buffer composition.

So we confirmed the hydration effect on the particle's Weber in the seismic solution.

And it should be noted that the hydration effect on full particles, the full particles are blue lines in this figure, has greater influence by the inter-seismic concentration compared to the empty particles, which are orange ones.

And this relation was, this hydration effect is reflected to this relation.

And also UV absorption spectrum of each component, especially the ratio of 260 and 280, was used for the comprehensive identification, including the full particles with different density.

In this way, we have developed a workflow to assess the components of AV with any serotypes or DNA lengths.

And these are the examples of component identification by DZAUC using the AV8 sample containing different DNA lengths.

And as you can see, from the expected DNA lengths and the ratio of 260 and 280, we can identify the observed component as shown in this table.

So the included component can be identified for different AVs using the developed workflow.