What columns do you recommend for protein and AAV applications?

Introduction

Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) plays a critical role in product and process development of therapeutic proteins and adeno-associated viruses (AAVs). In particular, SEC-MALS combined with multi-wavelength UV detection and differential refractive index (dRI) using an Optilab provides multi-attribute quantitation (MAQ), allowing multiple quality attributes to be monitored simultaneously in a single assay. Of course, successful SEC-MALS quantitation begins with successful separation. Waters XBridge Premier columns combine MaxPeak Premier High-Performance Surfaces and novel BEH-PEO Particle technologies to minimize column interactions and provide enhanced resolution for SEC-MALS measurements of proteins and AAVs. Importantly, they provide ideal light scattering baseline noise (<30 µV, typical) when connected according to our SEC-MALS recommendations.

Figure 1. XBridge Premier SEC column technology.

Proteins: Molar Mass, aggregates, fragments, and conjugation

XBridge Premier Protein SEC columns (250Å, 2.5 µm, 7.8 x 300 mm) are appropriate for both hydrophilic and hydrophobic proteins with minimal optimization. They provide enhanced resolution of aggregates and fragments compared to other columns for improved quantitation of these impurities. Figure 2 shows a typical chromatogram for BSA collected with SEC-UV-MALS-RI using DAWN and Optilab detectors. Baseline separation between the monomer, dimer, and trimer are easily achieved. Moreover, the BSA fragment is readily apparent and separated from the monomer.

Figure 2: Typical BSA chromatogram

AAV MAQ: Identify (molar mass), particle concentration (Cp and Vg), capsid content (Vg/Cp) and purity (aggregation)

XBridge Premier GTx SEC columns (450 Å, 2.5 µm, 7.8 mm x 300 mm) enable robust quantitation of AAV quality attributes by SEC-MALS. When compared to the WTC-050N5 columns previously used for the analysis, the XBridge Premier GTx columns provide superior separation and resolution while maintaining a sensitivity limit as good or better than the previous WTC-050N5 column. In a side-by-side comparison for a given AAV samples, all quality attributes after separation with the XBridge Premier GTx column agreed within 1% of those calculated after separation with the WTC-050N5 column. As shown in Figure 3, the XBridge Premier column provides excellent linearity and sensitivity even with AAV concentrations as low as 5x10¹⁰ AAV/mL.

Figure 3: Linearity and sensitivity of total particle concentration (Cp) for XBridge Premier GTx SEC column.

Conclusion

Waters XBridge Premier SEC columns are now available for purchase from the Wyatt store. They provide excellent resolution and mass recovery, resulting in enhanced detection sensitivity and reducing the minimum sample requirements. The surface and particle technologies minimize interactions, making these platform columns for use with hydrophilic and hydrophobic samples.

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