Analytical band centrifugation for the separation and quantification of empty and full AAV particles
| Autor: | Greg Kilby, Harshit Khasa, Xiaoyu Chen, Chunlei Wang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Analyte Materials science AUC Separation (statistics) adeno-associated virus QH426-470 sedimentation coefficient Analytical Ultracentrifugation 03 medical and health sciences 0302 clinical medicine Genetics Centrifugation Molecular Biology QH573-671 AAV Sedimentation coefficient Electrophoresis 030104 developmental biology 030220 oncology & carcinogenesis Physical separation analytical band centrifugation Molecular Medicine Particle Original Article Biological system empty and full capsids Cytology ABC analytical ultracentrifugation sedimentation velocity |
| Zdroj: | Molecular Therapy: Methods & Clinical Development, Vol 21, Iss, Pp 585-591 (2021) Molecular Therapy. Methods & Clinical Development |
| ISSN: | 2329-0501 |
| Popis: | Analytical band centrifugation (ABC) was first developed for the separation of macromolecules in centrifugation cells ~60 years ago. Since its development, ABC has been predominantly utilized to study macromolecular interactions or chemical reactions between two solutions in situ upon mixing. In this current study, we evaluated ABC separations on modern analytical ultracentrifugation (AUC) instruments for therapeutic adeno-associated viruses (AAVs). ABC provided sufficient separation between the genome-containing full AAV particle and the empty AAV capsid, which need to be controlled during the manufacturing process. Because ABC produces a physical separation, no complex algorithm or sophisticated software is needed to process the experimental raw data. ABC profiles, dubbed “centrifugrams”, can be analyzed with a similar approach as typically used for electrophoretic separations to produce relative percent area. Sedimentation coefficients (s) of analytes can also be determined from ABC. The relative area percent and s value obtained in ABC experiments were shown to be consistent with those determined by conventional sedimentation velocity AUC (SV-AUC). Additionally, the separation and quantification by ABC were found to be reproducible and did not appear to be sensitive to experimental variations of initial rotor temperature or cell misalignment. The robustness of the separation, ease of data processing, and universal applicability for analysis of different AAV serotypes make ABC a promising technique for routine analysis of empty and full AAV particle composition in therapeutic products. Graphical abstract We demonstrated the use of analytical band centrifugation (ABC) for measuring a critical quality attribute of therapeutical AAV drug candidates, i.e., the empty and full particle ratio. The ABC method produced good linearity and reproducibility and was shown to be more robust than conventional sedimentation velocity analytical ultracentrifugation (SV-AUC). |
| Databáze: | OpenAIRE |
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