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DELVING DEEPER INTO AAV ATTRIBUTES: ENHANCED CHARACTERIZATION USING MULTIPE TECHNOLOGIES

Malvern Panalytical의 'DELVING DEEPER INTO AAV ATTRIBUTES: ENHANCED CHARACTERIZATION USING MULTIPE TECHNOLOGIES'에 관한 응용자료의 주요 내용은 다음과 같다.

Zetasizer Ultra
The Zetasizer Ultra is an advanced DLS system for the measurement of nanoparticle and viral size, stability, charge, and viral titer or particle concentration. It enables size measurements to be performed up to three times faster compared to previous models, using the powerful new data analysis method Adaptive Correlation, which also improves measurement accuracy, repeatability and tolerance to foreign or large aggregated species. Additionally, it utilizes a unique measurement capability called Multi-Angle Dynamic Light Scattering(MADLS) to measure Particle Concentration.
Dynamic light scattering(DLS) can be used in virus development to measure critical biophysical parameters for a sample, or in a screening function to separate good, stable samples from those with contaminants or aggregates. With the new multi-angle MADLS-based particle concentration measurement available on the Zetasizer Ultra, it is now possible to measure not only hydrodynamic diameter and a size distribution of your samples(including aggregated species), but also particle concentration per population, within a few minutes.

Figure 1. Zetasizer Ultra and an example size distribution

AAV SIZE AND CONCENTRATION MEASUREMENTS
Using Multi-Angle Dynamic Light Scattering(MADLS)
In this study three adeno-associated virus(AAV) samples supplied by a customer were evaluated with Zetasizer Ultra. The concentration results are compared to results from capsid ELISA based virus titre assays.
By using the MADLS measurement on the Zetasizer Ultra, particle size distributions for three AAV samples were measured in triplicate, shown below. Low levels of aggregated species were observed, more so in the standard(A) than in their own AAV samples(B and C). By using the new Particle Concentration measurement, unique to the Zetasizer Ultra and calculated alongside the MADLS measurement, the concentration of AAV and each aggregated species was also measured(D-F) in minutes. Concentrations were compared to those recorded using the established capsid ELISA method(Table 1), showing less than 15% RSD for AAV samples and 45% RSD for the standard.

Figure 2. Intensity distributions of three repeat size measurements each of: graph A - ATCC AAV2 reference sample, graph B - AAV sample 1 and graph C - AAV sample 2. Particle concentration measurements corresponding to the spectra directly above.

CAPSID ELISA MEASUREMENTS
Using AAV capsid ELISA method established in-house
As described in the previous section, AAV Particle Concentration measurements using the Zetasizer Ultra were compared to those recorded by customer following an established capsid ELISA method. Results for monodisperse rAAV samples(RSD 13-15%) show little deviation to the expected rAAV capsid titer, as measured by ELISA. These results show the complimentary nature of the label-free analysis of intact rAAV capsids with other common titer assays. rAAV samples that contained aggregates showed an increase in RSD up to 45% due to a decrease in sizing accuracy for the polydisperse sample.

Table 1. Concentration data from capsid ELISA assay and MADLS-based particle concentration results shown for three samples; ATCC AAV2 reference sample, Sample 1, Sample 2. All concentrations are given as particles/ml. n= shows i how many of the three repeat measurements the peak was identified. Where there is no population peak present it states n/a as in not applicable.

OMNISEC
The OMNISEC system utilizes multiple-detection size exclusion chromatography(SEC) to measure rAAV attributes including capsid titer, absolute molecular weight, aggregate, oligomeric, and fragment distribution, and the full/empty ratio.
In this study conducted by Malvern Panalytical, UK on rAAV5 samples purchased from Virovek, US a mixture of empty and full rAAV samples were measured. The ”filled” rAAV samples had a Mw of 4.49x106g/mol, a protein fraction of 86%, a ssDNA Mw of 6.13x105 g/mol, and a Full AAV % of 78.1, and a total titer of 7.48x1013 (monomer and aggregate). Empty rAAVs had a lower Mw of 3.84x106 g/mol, and a total titer of 5.91x1013(monomer and aggregate). When empty and full rAAV samples were spiked togteher in known ratios, the measured % Full results correlated with expected with an Rsquared value of 0.9912.

Figure 3. OMNISEC system and example chromatogram for a mix of empty rAAV monomers, dimers, aggregates, and fragments.

Figure 4. OMNISEC % Full analysis of mixed empty and full rAAVs.

PEAQ DSC
The PEAQ-DSC automated system utilizes differential scanning calorimetry(DSC) to measure protein, nucleic acid, and viral capsid thermal unfolding and stability. The technique measures the enthalpy(ΔH) and uploaderature(Tm) of thermally-induced structural transitions of molecules in solution. The measurements are direct, solution-phase, and label-free, requiring no additional fluorescent labelling or antibody capture.
Results obtained with a PEAQ-DSC for both empty and full rAAV5 samples(Virovek, US) indicate multiple thermal transition points(Tm), related to capsid disintegrations(Tm1) and ssDNA melting(Tm2). Additionally, Full rAAVs showed a weak thermal signature at 50 degrees, whereas empty rAAVs did not, suggesting the onset of a thermally-induced event likely associated with structural rearrangements in full rAAVs linked to capsid uncoating.

Figure 5. Microcal PEAQ-DSC Automated System and example DSC thermogram for Full rAAV showing distinct thermal transition points(Tm) at 89 and 94℃. Red star indicates early thermal transition at 50℃.

• 15% or lower RSD was observed between the capsid ELISA method established at customer site and particle concentration measurements using the Zetasizer Ultra. 45% RSD was observed for a standard sample containing more aggregates.

• Purity, oligomeric distribution, and % Full for Empty and Full rAAV samples were determined using the OMNISEC multi detection chromatography system.

• Thermal transitions and stability profile were measured for both Empty and Full rAAVs using the automated PEAQ-DSC system.

Malvern Panalytical의 'Zetasizer Ultra, OMNISEC and PEAQ DSC'에 대한 상세한 내용은 Reference(참고자료)를 통하여 확인할 수 있다.

Reference(참고자료): 말번 파날리티칼 응용노트
Model Name(모델명): Zetasizer Ultra, OMNISEC and PEAQ DSC
The Person in Charge(담당자): Hyunjung Gu
Maker(제조사): Malvern Panalytical
Country of Origin(원산지): UK
Mail inquiry: korea.info@malvernpanalytical.com
Data Services(자료제공): Malvern Panalytical Korea

<이 기사는 사이언스21 매거진 2022년 9월호에 게재 되었습니다.>

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