Adeno-associated viruses (AAV) are one of the most commonly used viral vectors for gene
therapy to deliver therapeutic genes. Quantitation of empty and full AAV capsids is critical to
understand and ensure the product quality, safety, and potential efficacy. Multiple techniques
have been used in this application however all have some shortcomings and challenges:
Time consuming: Analytical ultra-centrifugation (AUC) is the classical and golden
standard technique for this application; however, it requires long hours of processing time
and large amount of sample consumption.
Lack of accuracy: UV absorbance at 260 nm versus 280 nm has also been widely used for
the quick estimation of empty and full capsids, but the accuracy can be compromised.
Traditional anion exchange chromatography (AEX) based on relative surface charge
differences were also reported to provide the potential separation; however, the AEX
analytical products on market often lack accurate quantitative measurement to
demonstrate the full separation of empty and full capsid. Empty capsids may still be
present in the AUC run of the fraction collected from full capsid peak.
By improving both its beads technology and surface coating chemistry, Sepax introduces a new
Proteomix AAV SAX MALS columns which are specifically designed for optimal separation of
empty and full AAV capsids with enhanced precision, delivering a fast and accurate quantitation
method for analytical characterization and quality control.
These columns feature a monosized resin matrix, consisting of 3 µm spherical, highly cross-
linked poly(styrene-divinylbenzene) (PS/DVB) beads. The surface of these beads is modified
with a nanometer-thick hydrophilic, neutral polymer coating to improve selectivity and reduce
non-specific interactions with biological analytes and further enhanced by a uniformly applied
trimethylammonium layer using Sepax’s proprietary chemical attachment methods, topped with
ion-exchange functional groups through an exclusive synthetic route, thereby offering high-
capacity ion-exchange capabilities. The incorporation of the strong anion exchanger with
quaternary ammonium functional groups facilitates effective sample binding and narrow band
broadening by reducing secondary interactions. Such detailed surface engineering ensures
superior resolution, efficiency, and recovery in AAV analyses, significantly reducing sample loss
and promoting more accurate analytical results.
The columns are also designed to tolerate high pressures, offering flexibility to adapt and
optimize operational conditions to meet specific application requirements. Additionally, these
columns are compatible with multi-angle light scattering (MALS) detectors, ensuring low noise
levels and enhancing their suitability for a diverse range of applications that require precise
molecular weight determination. The uniformity in particle size distribution of the monosized
beads, in contrast to traditional polydisperse particles, improves column packing and ensures
consistent lot-to-lot reproducibility. Furthermore, AAV8 standard sample combined with UV-
MALS was employed for each resin batch QC with stringent QC specification to ensure the lot-
to-lot reproducibility of the robust resin and column manufacturing processes, which is crucial in
method development and data interpretation. Additionally, the use of polyether ether ketone
(PEEK) for column hardware, recognized for its bioinert properties, minimizes nonspecific
protein adsorption, contrasting with metal-based alternatives and thus, solidifying the column's
suitability for analyzing complex samples with high resolution and excellent reproducibility.
Proteomix AAV SAX® Technical Specifications
Phases
Proteomix AAV SAX
Resin Matrix
Highly cross-linked PS/DVB monodisperse resin support
with a nanometer-thick hydrophilic coating that chemically
bonded to an optimally uniform trimethylammonium layer,
ensuring robust performance and stability
Particle Size
3 µm
Pore structure
Non-porous
Dynamic binding
capacity
~36 mg/mL for Proteomix AAV SAX
pH Stability
2-9, 9.5*
Resin pressure limit
8000 psi
Column operating
pressure limit
6,000 psi
(a 4.6 x 50 mm PEEK column)
Typical flow rate
0.1-1.0 mL/min
(for a 4.6 x 50 mm PEEK column)
Mobile Phase Compatibility
Compatible with aqueous solution, a mixture of water and
acetonitrile, acetone, or methanol. Typical buffers:
phosphate, tris, acetate, and so on.
*Extended use at higher pH levels (≥ 9.5) may reduce the column's lifetime.
Improving Separation of Empty and Full AAV Capsids: Achieved higher
resolution separation of full capsids from empty capsids, allowing for full
separation and more accurate molecular weight calculations in the applications.
Easy Linear Salt Gradient for User Friendly Method Development: An easy
linear salt gradient method was used as an excellent starting point for method
development and provided a user-friendly and flexible approach for developing
analytical methods across a variety of AAV samples.
MALS Compatibility with Low Shedding and Accuracy: AEX-MALS method
was employed in the application for accurate quantitation of empty and full
capsids with low shedding and baseline noise.
Monosized Base Beads: Precision-engineered monosized base beads
provide excellent higher resolving power and improved column packing and lot-
to-lot reproducibility for consistent and reliable performance techniques.
Low Pressure with High Pressure Limit for Fast Run: The beads have low
pressure with a high-pressure limit, enabling up to 1 mL/min high throughput fast
assay.
Robust Lot-to-Lot Consistency: AAV8 sample combined with UV-MALS with
stringent specification for batch quality control ensures robust lot-to-lot
consistency for product release.
UPLC and HPLC Compatible: This 3 µm HPLC column is compatible with both
UPLC and HPLC use which enables easy adaptability for method transfers
applied across a diverse range of applications, maximizing both performance and
efficiency.
