AAV Analysis with AUC
Centrifugation
Optima X-Series - Floor-Standing Ultracentrifuges Our Optima XPN/XE ultracentrifuges provide speeds to 100,000 rpm (802,000 x g) and offer intuitive operation coupled with powerful onboard features such as eXPert software for method development. They can be added to your network. You can also enable VNC (Virtual Network Connection) or API (Application Programming Interface) to allow the system to receive instructions from a remote application (Optima XPN only). Need for High-resolution AAV Analysis AAVs are well-known as having variability in the amount of genetic load in the particles, where only those containing the full gene insert can elicit the desired function. Thus, AAV purification to isolate only the full particles is critical. Further, characterizing the ratio of functional (full) and non-functional (empty/partial) particles is paramount to optimize culture productivity and understand production consistency, establish dosing, and ensure patient safety.
Empty
Type 45 Ti 6 x 94 mL
Partial
Full
• Rotor volumes up to 1675 ml • Offers the capability to support 21 CFR Part 11 (only Optima XPN) • Variety of swinging-bucket, fixed-angle, vertical, near-vertical and zonal rotors Solution Air Bottom Meniscus
• Exclusive s afety concept with Rotor-Safety- System, Dynamic Rotor Inertia Check (DRIC) • Innovative concept for the management of voltage fluctuations and power failures • BioSafe*-Concept for laboratory ware, rotor and centrifuge
SW 32 Ti 6 x 38.5 mL AUC: A Unique Characterization Technique Analytical ultracentrifugation (AUC) is a unique approach for particle separation and characterization. With AUC, particles are often separated by varying sedimentation coe " cient (S) – a collection of size, mass, shape, and density. Because AAV particles of varying load di ! er in mass and density, AUC excels at providing high-resolution data for analysis of such particles in solution, without the need or possible side e ! ects of matrix interactions. VTi 50.1 12 x 39 mL
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MLA-150 8 x 2 mL
Buoyant force
Optima MAX-Series - Tabletop Ultracentrifuges The Optima MAX-Series generates speeds of up to 150,000 rpm with rcf of over 1 million x g. With an extended volume range, these space-saving tabletop ultracentrifuges are a perfect alternative to floor standing centrifuges. Direction of Sedimentation Frictional force Centrifugal force r = 0 x
r
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• Sample volumes from 100 μ l - 194.4 ml • BioSafe*-Concept for laboratory ware, rotor and centrifuge
• MAX-XP software enables extended safety concept (user login, password)
MLA-55 8 x 13.5 mL
AUC Provides Higher-resolution Data AUC has become well-known as the gold standard for quantitative assessment of AAV particle quality and fullness. Compared to anion exchange chromatography (AEX), for example, AUC o ! ers much greater resolution and the clear identification of partials, where AEX falls short. These high-res AUC analyses are not only useful in final product
Optima AUC - Analytical Ultracentrifuges Analytical ultrac entrifugation is a versatile, rigorous and accurate technology available for determining the molecular
QC, but also in cell culture development to optimize for productivity of full particles. weight, hydrodynamic and thermodynamic properties of a protein or other macromolecule. AUC is a stand-alone, first-principle technique performed in a matrix-free environment that does not require standards. • Separation and optical measurement in one step • AUC enables the detection of even small amounts of sub-populations (down to even <1%) • With very few buffer constraints, AUC enables characterization of samples in their native state under biologically relevant solution conditions • Characterization of heterogeneous samples • AUC reliably detects low sub-populations, e.g. aggregation proportions of <1%
Analysis of empty (E), full (F), and partially-full (P) AAV particles with aggregates (X). Reproduced with permission from Wang et al. Analysis of empty (E), full (F), and partially-full (P) AAV particles with aggregates (X) via AEX (A) and AUC (B). Reproduced with permission from Wang et al.†
Summary AUC can provide high-resolution information on AAV samples, including opportunities to quantitatively identify empty, full, and partially-full particles along with aggregates. Click here to learn more about getting started with AUC and learn about the new features of the Optima AUC.
Accelerating Answers
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*BioSafe and BioSafety are terms intended to describe the enhanced biocontainment features of our products. Product not for use in diagnostic or therapeutic procedures.
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