More recently, studies have shown that MALS can also be used for quantification of VLPs in solution 19, 20. MALS has been widely used in tandem with SEC or field flow fractionation to determine the absolute molecular weight, size, conformation, and distribution of polymers and protein biotherapeutics 16, 17, 18. Further, the utility of SEC can be enhanced by combining it with multiangle light scattering (MALS) and refractive index (RI) detectors. SEC is widely used as a polishing purification step of viral vectors and has been developed as a characterization method for virus-like particles (VLPs) and influenza particles 11, 12, 13, 14, 15. However, SEC has the advantage of separating monomeric AAV capsids from higher-order aggregates and other extrinsic impurities. Size exclusion chromatography (SEC), which separates species in solution by size, similarly uses a UV-based detection method. Protein and nucleic acid impurities were found to significantly impact titer results, limiting the use of this method for only highly purified samples. Although OD titers were less variable, the bulk-nature of OD measurements did not allow correction for the impact of extrinsic capsid impurities or aggregates on UV absorbance. In 2003, a bulk OD method to determine AAV capsid (Cp) and vector genome (Vg) titer was proposed as an alternative to qPCR and ELISA. Despite their historic and continued use, each method has its own limitations, such as low throughput or high variability 7, 8.īulk optical density (OD) measurements have been used to quantify the vector genome and capsid protein content of adenovirus preparations 9, 10. Various methods, including electron microscopy, dynamic light scattering, analytical ultracentrifugation, ELISA, and PCR are used to independently monitor purified AAV size, aggregation propensity, stability, empty-to-full capsid ratio, capsid (Cp) and vector genome (Vg) titer, respectively. Therefore, a successful gene therapy program depends on accurate vector characterization and titration, which dictates the safety and efficacy in humans.
.jpg "dynamic light scattering size exclusion")
Previous gene therapy programs have shown positive correlation in general between the presence of gene copy numbers (vg/kg patient weight) and protein expression 5, 6. While several successful methods exist for large-scale, clinical AAV production 2, 3, 4, the development of robust analytics to assess variables in the overall process development and final AAV drug products is an iterative process. As of April 2020, 244 gene therapy trials using AAVs are ongoing worldwide, 24 of which are in Phase III clinical trials, ( ). Due to their relative safety and long-term gene expression, multiple serotypes of recombinant AAV vectors are in use across gene therapy programs at the clinical and research stage 1. AAV particles consist of a small ~ 25 nm icosahedral capsid composed of three types of structural proteins (VP1, VP2, and VP3) coating a single-stranded ~ 4.7 kb genome. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.Īdeno-associated virus (AAV) belongs to the genus Dependoparvovirus in the family Parvoviridae.
Dynamic light scattering size exclusion series#
In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Adeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery.
0 kommentar(er)
