Importance of Biophysical Characterization

by Berangere Tissot, Ph.D., Director, Biochem Method Est. & Biologics Characterization; Prashasti Kumar, Ph.D., Senior Scientist, Biochemistry Method Development and Validation

With the ever-increasing number of antibody-based and rAAV-based gene therapies revolutionizing the field of biotherapeutics, new challenges have emerged. Even though the biophysical toolkit is fairly well established for protein therapeutics, the unknown nature of protein aggregates and their wide size range complicate the characterization. In the case of new modalities like rAAV-based therapies, challenges become manifold as an established biophysical toolkit is lacking, and characterization techniques are moving to the cGMP area (see Table 1).

Protein aggregation analysis

Dynamic light scattering (DLS) because of its high sensitivity and relatively high-throughput can be used to rapidly screen candidates/conditions during the pre-formulation stage. However, it is a qualitative technique and suffers from low accuracy and precision. Size exclusion chromatography coupled to multi angle light scattering (SEC-MALS) can be used to quantitatively assess the aggregate species, as well as determine molecular weight. SEC-MALS also has the advantage that it can be used as a release and stability method. But it is limited by its size range and sample dilution effects. Also, it is not suitable for large or reversible aggregates as they can get disrupted during the chromatography, due to shear forces.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is another technique that can be utilized to determine the extent of aggregation by hydrodynamically separating the various oligomers in their native formulation buffers, without requiring any matrix or label and is applicable to a much wider size range. However, it is low throughput, requires long turnaround time, high volumes and extensive data analysis. In addition, relatively higher limit of quantitation (3-5%) and high dependence on the physical components and their alignment render SV-AUC unsuitable to be used as an absolute method. It is much better suited as an orthogonal method to validate other higher throughput methods having poorer resolution.

rAAV-based gene therapies

With the FDA approval of two rAAV-based therapies for treatment of Leber congential amaurosis and spinal muscular atrophy, the clinical potential of the rAAV gene therapies is being increasingly recognized with a lot more therapies being developed or investigated in clinical trials for diseases ranging from neurological disorders to cancer. This necessitates the need for developing robust and reproducible methods to characterize the critical quality attributes (CQAs) through orthogonal techniques. In the context of rAAV-based therapies, determining capsid titer, content of full, partial and empty capsids and aggregation is key for evaluating the potency, purity and safety of the developed rAAV-based drug product.

The applicability of the tools in the biophysical characterization tool box to rAAV analysis remains a challenge due to the limitations of serotype specificity, low throughput or poor accuracy and precision. On top of these scientific considerations, compliance with cGMP is becoming a point of concern as some of these techniques might be considered to integrate a release panel. In spite of the biophysical tools already at our disposal, there is an imminent need to develop new fit for purpose analytical methods that have a rapid turnaround, high throughput and are suitable for qualification, validation and process development for these modalities.

The SMEs of the Eurofins Biophysical team are here to support our clients with the identification of the critical quality attribute(s) to characterize and/or control, the selection of the appropriate instrumentation, and the design and the execution of the suitable analytical strategies. Contact us to learn more: www.eurofins.com/BPT-Contact-Us.