(T1130-09-57) High-Throughput Characterization of Gene Therapy Products by SEC-MAL

Purpose: Successful deployment of the Gene therapy (GTx) products entail detailed characterization to ensure their safety and efficacy. With more than 200 AAV-based gene therapy drugs in development, the titer, potency, and purity of these drug products must be precisely controlled and monitored. Current methods require significantly large amount of sample for multiple quality attribute analysis. In this current study, we demonstrate the utility of XBridgeTM Premier GTx BEHTM SEC 450 Å 2.5 μm columns for size exclusion chromatography (SEC) coupled with multiangle light scattering (MALS)-based biophysical analysis of AAVs and nucleic acids. The 2.5 μm diol bonded 450 Å pore size BEH particles in these columns appropriately matched to give efficient resolution of AAV sample components including monomers, dimers, trimers, and high molecular weight aggregates for size variant analysis. Further, the hydrophilic High-Performance Surfaces (h-HPS) minimizes secondary interactions (electrostatic and hydrophobic) with the column surfaces leading to excellent peak shape, recovery, and reproducible measurements. These SEC columns exhibited little to no particle shedding and low MALS noise even at low flow rates resulting in enhanced sensitivity for low volume samples. We also show that the peak profiles of a 4.6 mm column obtained with a usually larger flow cell MALS detector match well with the wider pore 7.8 mm version allowing analysis with smaller sample amounts. Thus, these columns provide a powerful platform capability to measure multiple attributes such as molar mass, size, empty and full capsid ratios of AAV. Superior resolution and faster analysis of nucleic acids is another hallmark of these SEC columns enabling orthogonal analysis when needed. Such a platform can easily expedite the process and product related impurity monitoring during process development decision making and regulatory submissions.

Methods: The AAV and nucleic acid samples were analyzed under isocratic conditions using 20 mM phosphate buffer, 276 mM NaCl, 5.4 mM KCl pH 7.4 (2x PBS) on a Waters UHPLC H-Class Bio configured with a Waters flow through needle sample manager (SM-FTN), 30-CHA column manager, ACQUITY TUV detector, ACQUITY RI detector, and WYATT DAWN (NEON) multi-angle light scattering detector with online DLS (QELS). The nucleic acids were evaluated at 50 °C and AAV at 30 °C column temperatures. The viral genome to total capsid content (vg/cp) ratios were prepared at a 0:1, 1:0, 1:1, 1:2, 1:5, and 1:10 using AAV9-Empty and AAV9-CMV-GFP capsids.

Results: Accurate molar mass and size measurements were achievable for AAV and nucleic acids due to low MALS noise ( < 30µV for root mean squared (RMS), < 200 µV peak to peak noise), minimal secondary interactions and higher chromatographic efficiency of the sub-2µm particle. Figure 1 illustrates LS trace of XBridge Premier BEH 450Å GTx 2.5µm, 7.8 x 300 mm column-based separation of empty AAV9 and AAV2 serotypes. Table 1 lists biophysical properties of both serotypes with similar elution times and hydrodynamic radius (Rh) of 13.2 and 13.8 nm. The observed monomeric molar masses, 3.7 and 3.6 MDa, matched well with the theoretical molar mass. Chart 1 illustrates an excellent correlation of Vg/Cp ratio of empty and full capsids with an R2 value of 0.9998. Molar mass of the protein capsid was constant across various ratios of the empty and full AAV9. However, these two forms exhibited near identical elution pattern because of similar hydrodynamic radii. Figure 2 illustrates excellent separation of dsDNA (100 to 3000 bp/50 to 1350 bp) and ssRNA ladders (200 to 6000 nt) on a XBridge Premier BEH 450Å GTx 2.5µm 7.8 x 300 mm column. Fluc mRNA stacked in ladder plots showed 18 nm radius of gyration (Rg) with molar mass of 605 kDa.

Conclusion: The XBridge Premier BEH 450Å GTx 2.5µm columns provided excellent resolution and efficient biophysical characterization of AAVs and nucleic acids through SEC-MALS. Coupled with the hydrophilic high-performance surface to minimize the secondary interactions, these columns provide platform capabilities for multiple attribute analysis of gene therapy products in a timely fashion.

References: 1. Kizekai, L., et al, Heightened Characterization of AAVs by SEC-MALS with XBridge Premier GTx BEH SEC 450 Å 2.5 µm Columns, Waters App Note 720007969 June 2023
2. Pam Wang, Michelle Chen, SEC-MALS Method for Characterizing mRNA Biophysical Attributes. AN1616.

SEC-MALS light scattering trace of molar masses for AAV2-Empty and AAV9-Empty separation on XBridge Premier GTx BEH SEC 450 Å 2.5 µm, 7.8 x 300 mm column. Table 1: AAV9-Empty and AAV2-Empty biophysical characterization acquired from Figure 1.

SEC-MALS ultraviolet trace of molar masses of dsDNA and RNA ladder profiling on XBridge Premier GTx BEH SEC 450 Å 2.5 µm, 7.8 x 300 mm column

Chart 1: Empty and full AAV9 Vg/Cp ratio of 0.00, 0.52, 0.69, 0.93, 0.97, and 1.13, correspond well with the 1:0, 1:1, 1:2, 1:5, 1:10, and 0:1 ratio, respectively.