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Mol Ther. 2017 Jun 7;25(6):1375-1386. doi: 10.1016/j.ymthe.2017.04.001. Epub 2017 Apr 17.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

Author information

1
Grousbeck Gene Therapy Center, 20 Staniford Street, Boston, MA 02114, USA; Atlantic Gene Therapies, INSERM UMR 1089, University of Nantes, Nantes University Hospital, 22 Boulevard Benoni Goullin, 44200 Nantes, France.
2
Atlantic Gene Therapies, INSERM UMR 1089, University of Nantes, Nantes University Hospital, 22 Boulevard Benoni Goullin, 44200 Nantes, France.
3
Grousbeck Gene Therapy Center, 20 Staniford Street, Boston, MA 02114, USA.
4
Grousbeck Gene Therapy Center, 20 Staniford Street, Boston, MA 02114, USA; Biological and Biomedical Sciences Program, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA.
5
Grousbeck Gene Therapy Center, 20 Staniford Street, Boston, MA 02114, USA; Department of Ophthalmology, Ocular Genomics Institute, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA; Schepens Eye Research Institute, 20 Staniford Street, Boston MA 02114, USA; Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address: luk_vandenberghe@meei.harvard.edu.

Abstract

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

KEYWORDS:

AAV vectors; CMC; adeno-associated virus; capsid thermostability; gene therapy; homogeneity; identity; identity assay; manufacturing; quality control

PMID:
28427840
PMCID:
PMC5475248
DOI:
10.1016/j.ymthe.2017.04.001
[Indexed for MEDLINE]
Free PMC Article

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