Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids
Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin col...
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| Format: | Article |
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MDPI AG
2023-12-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/16/1/13 |
| _version_ | 1797342421699264512 |
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| author | Angela Valentic Nicola Böhner Jürgen Hubbuch |
| author_facet | Angela Valentic Nicola Böhner Jürgen Hubbuch |
| author_sort | Angela Valentic |
| collection | DOAJ |
| description | Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles. |
| first_indexed | 2024-03-08T10:33:03Z |
| format | Article |
| id | doaj.art-0227ef6e33c44625a7cd96e2f8ab2bed |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-08T10:33:03Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-0227ef6e33c44625a7cd96e2f8ab2bed2024-01-26T18:46:34ZengMDPI AGViruses1999-49152023-12-011611310.3390/v16010013Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic AcidsAngela Valentic0Nicola Böhner1Jürgen Hubbuch2Institute of Process Engineering in Life Sciences—Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, GermanyInstitute of Process Engineering in Life Sciences—Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, GermanyInstitute of Process Engineering in Life Sciences—Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, GermanyEffective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles.https://www.mdpi.com/1999-4915/16/1/13virus-like particlesnucleic acid extractionsilica spin columnRP-HPLCHBcAganalytical characterization |
| spellingShingle | Angela Valentic Nicola Böhner Jürgen Hubbuch Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids Viruses virus-like particles nucleic acid extraction silica spin column RP-HPLC HBcAg analytical characterization |
| title | Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids |
| title_full | Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids |
| title_fullStr | Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids |
| title_full_unstemmed | Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids |
| title_short | Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids |
| title_sort | absolute quantification of hepatitis b core antigen hbcag virus like particles and bound nucleic acids |
| topic | virus-like particles nucleic acid extraction silica spin column RP-HPLC HBcAg analytical characterization |
| url | https://www.mdpi.com/1999-4915/16/1/13 |
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