Size Exclusion Chromatography-Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization

Abstract

Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research.

Keywords: AAV titer; Vg/Cp ratio; gene therapy; sample quality.

Figure 1

Chromatogram analysis of AAV samples. In panel (A,B), blue and purple lines represent the chromatograms of 280 nm and 260 nm absorbance, respectively. Red lines in panel (A) show the baselines. Panel (B) shows the chromatograms after baseline correction. Vertical green lines denote the peak boundaries. The output of a chromatogram analysis program implemented in MATLAB is shown in panel (C).

Figure 2

SEC−MP results of single-species AAV samples. Both empty (A) and fully packaged (B) highly pure-genome AAVs were evaluated using the SEC−MP method. The left panels show the absorbance chromatograms of AAV samples obtained with the 7.8 × 300 mm SEC column. Blue and purple lines represent the 280 nm and 260 nm absorbance signals, respectively. Green vertical lines represent the peak boundaries. The right panels show the MP results. Solid lines represent the Gaussian fits of the mass distribution histograms. The chromatogram analysis results are shown in the subpanels.

Figure 3

SEC −MP results of empty and fully packaged AAV mixtures. Both empty (A) and fully packaged (B) highly homogeneous AAVs were reanalyzed using the method described in scenario 2. Panel (C) shows the absorbance chromatograms (left) and the MP mass distribution (right) of the mixture of empty and fully packaged AAVs. Blue and purple lines in the chromatograms represent the 280 nm and 260 nm absorbance signal, respectively. Green vertical lines represent the peak boundaries. Solid blue line in MP mass distribution represents the Gaussian fit, and the chromatogram analysis results are presented in the subpanel.

Figure 4

SEC−MP results of less pure and heterogeneous AAV samples. Panels (A,B) show the results for two different samples, A and B, respectively. A small-diameter, 4.6 × 300 mm SEC column (WTC−050S5) was used in the measurements. Blue and purple lines in the chromatograms represent the 280 nm and 260 nm absorbance signals, respectively. Green vertical lines represent the peak boundaries. Lighter orange shades in MP distributions represent the 3–6 MDa region and darker orange shades represent the 5.1–5.4 MDa region.

Figure 5

Chromatograms of UV280 absorbance signals obtained using the WTC−050S5 column. The blue, orange, yellow, and purple colors represent the sample loading volumes of 10 µL, 1 µL, 0.5 µL, and 0.2 µL, respectively. Green vertical lines represent the peak boundaries.

Figure 6

Comparative analysis of SEC-MP and other AAV characterization technologies. Five distinct technologies (orange) were qualitatively compared to SEC-MP (blue). The comparison is based on five critical attributes: titer determination (titer), where a larger value indicates a more accurate determination; %full determination in a sample containing only empty and fully packaged AAVs (E/F), where a larger value indicates a more accurate determination; %full determination and quantification of partially packaged AAVs (partial), where a larger value represents a more reliable estimation; sensitivity (sensitivity), where a larger value indicates lower material consumption; and simplicity (simplicity) of an experiment, where a larger value implies the lower experimental time and labor required.