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. 2022 Oct 22;23(21):12715.
doi: 10.3390/ijms232112715.

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors

Christina Wagner  1 , Bernd Innthaler  2 , Martin Lemmerer  1 , Robert Pletzenauer  2 , Ruth Birner-Gruenberger  3
Affiliations

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors

Christina Wagner et al. Int J Mol Sci. .

Abstract

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.

Keywords: adeno-associated virus vectors; critical quality attributes; dynamic light scattering; ion-exchange chromatography; multi-angle light scattering; protein characterization.

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Conflict of interest statement

Authors Christina Wagner, Martin Lemmerer, Bernd Innthaler and Robert Pletzenauer are current full-time employees of Baxalta Innovations GmbH and own company stocks. Ruth Birner-Gruenberger declares no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of the IEC-MALS method. The AAV sample is loaded onto the anion-exchange column, eluted with a salt gradient containing MgCl2 and detected with multi-angle light scattering and UV detectors prior to data analysis using ASTRA software.
Figure 2
Figure 2
Linear correlation of measured % filled AAV capsids using IEC-MALS and expected % filled AAV capsids by AUC.
Figure 3
Figure 3
Linearity of the developed IEC-MALS method. Plots of expected capsid titers obtained by ELISA vs. (a) capsid titer measured with multi-angle light scattering and (b) UV area by integration of the UV profiles at 280 nm.
Figure 4
Figure 4
Evaluation of the robustness of the developed IEC-MALS method. (a) variation of the linear salt gradient. Gradient A: 0–35% buffer E, gradient B: 0–45% buffer E, gradient C: 0–55% buffer E and gradient D: 0–65% buffer E; (b) variation of the flow rate. Flow rates of 0.5 mL min−1, 0.7 mL min−1 and 1.0 mL min−1 were tested using a linear salt gradient from 0–35% buffer E.
Figure 5
Figure 5
Overlay of the LS chromatograms (solid lines) and molar masses of the total capsid, protein and transgene (dashed lines) of serotypes AAV5 (green), AAV6 (pink) and AAV8 (blue).
Figure 6
Figure 6
Evaluation of the method performance of the developed IEC-MALS assay using five different anion-exchange columns. The strong AEX columns ProSwift SAX-1S (blue), UNO Q Polishing (purple) and CIMac AAV full/empty (green) and the weak AEX columns ProSwift WAX-1S (orange) and CIMac PrimaS (yellow). Dashed lines (1)–(6) represent (a) the expected F/E ratio, (b) the absolute molar mass of the protein, the absolute molar mass of the encapsidated ssDNA, (c) the hydrodynamic radius, the radius of gyration of empty AAV capsids and the radius of gyration of filled AAV particles, respectively.
Figure 6
Figure 6
Evaluation of the method performance of the developed IEC-MALS assay using five different anion-exchange columns. The strong AEX columns ProSwift SAX-1S (blue), UNO Q Polishing (purple) and CIMac AAV full/empty (green) and the weak AEX columns ProSwift WAX-1S (orange) and CIMac PrimaS (yellow). Dashed lines (1)–(6) represent (a) the expected F/E ratio, (b) the absolute molar mass of the protein, the absolute molar mass of the encapsidated ssDNA, (c) the hydrodynamic radius, the radius of gyration of empty AAV capsids and the radius of gyration of filled AAV particles, respectively.
Figure 7
Figure 7
Overlay of the LS chromatograms and “viral vector analysis” of AAV8 obtained by a linear pH gradient (red) or linear salt gradient (blue).
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