Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques
Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapi...
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Published in | Pharmaceutics Vol. 13; no. 4; p. 586 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
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20.04.2021
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Abstract | Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC-MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS
can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 10
-8.7 × 10
cp/mL for the nominally full rAAV5 samples and 3.4 × 10
-7 × 10
cp/mL for the nominally empty rAAV5 samples with 3-8% and 10-37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC-MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples. |
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AbstractList | Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS
®
can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 10
11
–8.7 × 10
13
cp/mL for the nominally full rAAV5 samples and 3.4 × 10
11
–7 × 10
13
cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples. Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples. Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC-MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 10 -8.7 × 10 cp/mL for the nominally full rAAV5 samples and 3.4 × 10 -7 × 10 cp/mL for the nominally empty rAAV5 samples with 3-8% and 10-37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC-MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples. |
Author | Fernandes, Diogo Stenson, John Hussain, Maryam T Cole, Liam Kaszuba, Michael Markova, Natalia |
AuthorAffiliation | Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK; liam.cole@malvernpanalytical.com (L.C.); diogo.fernandes@malvernpanalytical.com (D.F.); maryam.hussain@malvernpanalytical.com (M.T.H.); john.stenson@malvernpanalytical.com (J.S.); natalia.markova@malvernpanalytical.com (N.M.) |
AuthorAffiliation_xml | – name: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK; liam.cole@malvernpanalytical.com (L.C.); diogo.fernandes@malvernpanalytical.com (D.F.); maryam.hussain@malvernpanalytical.com (M.T.H.); john.stenson@malvernpanalytical.com (J.S.); natalia.markova@malvernpanalytical.com (N.M.) |
Author_xml | – sequence: 1 givenname: Liam surname: Cole fullname: Cole, Liam organization: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK – sequence: 2 givenname: Diogo surname: Fernandes fullname: Fernandes, Diogo organization: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK – sequence: 3 givenname: Maryam T surname: Hussain fullname: Hussain, Maryam T organization: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK – sequence: 4 givenname: Michael surname: Kaszuba fullname: Kaszuba, Michael organization: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK – sequence: 5 givenname: John surname: Stenson fullname: Stenson, John organization: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK – sequence: 6 givenname: Natalia surname: Markova fullname: Markova, Natalia organization: Malvern Panalytical Ltd., Enigma Business Park, Grovewood Road, Malvern, Worcestershire WR14 1XZ, UK |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/33923984$$D View this record in MEDLINE/PubMed |
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Keywords | differential scanning calorimetry (DSC) dynamic light scattering (DLS) high-throughput multiangle dynamic light scattering (MADLS®) SEC–MALS orthogonal techniques recombinant adeno-associated viruses (rAAV) |
Language | English |
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SubjectTerms | Chromatography dynamic light scattering (DLS) Gene therapy Genomes high-throughput Light Molecular weight multiangle dynamic light scattering (MADLS®) orthogonal techniques Particle size Polymerase chain reaction recombinant adeno-associated viruses (rAAV) SEC–MALS Vectors (Biology) Viscosity |
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Title | Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques |
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