At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization
Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prio...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-06-01
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| Series: | Bioengineering |
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| Online Access: | https://www.mdpi.com/2306-5354/8/6/78 |
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| author | Julian Ebner Diana Humer Robert Klausser Viktor Rubus Reinhard Pell Oliver Spadiut Julian Kopp |
| author_facet | Julian Ebner Diana Humer Robert Klausser Viktor Rubus Reinhard Pell Oliver Spadiut Julian Kopp |
| author_sort | Julian Ebner |
| collection | DOAJ |
| description | Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding. |
| first_indexed | 2024-03-10T10:37:45Z |
| format | Article |
| id | doaj.art-13804829808243b9a7d88d961c13c66e |
| institution | Directory Open Access Journal |
| issn | 2306-5354 |
| language | English |
| last_indexed | 2024-03-10T10:37:45Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Bioengineering |
| spelling | doaj.art-13804829808243b9a7d88d961c13c66e2023-11-21T23:09:36ZengMDPI AGBioengineering2306-53542021-06-01867810.3390/bioengineering8060078At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body SolubilizationJulian Ebner0Diana Humer1Robert Klausser2Viktor Rubus3Reinhard Pell4Oliver Spadiut5Julian Kopp6Research Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, 1060 Vienna, AustriaResearch Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, 1060 Vienna, AustriaResearch Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, 1060 Vienna, AustriaResearch Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, 1060 Vienna, AustriaSANDOZ GmbH, Mondseestrasse 11, 4866 Unterach, AustriaResearch Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, 1060 Vienna, AustriaResearch Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, 1060 Vienna, AustriaRefolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.https://www.mdpi.com/2306-5354/8/6/78inclusion bodiesinclusion body solubilizationtailored refoldingreversed phase liquid chromatographyprocess analytical technology toolsin-process monitoring |
| spellingShingle | Julian Ebner Diana Humer Robert Klausser Viktor Rubus Reinhard Pell Oliver Spadiut Julian Kopp At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization Bioengineering inclusion bodies inclusion body solubilization tailored refolding reversed phase liquid chromatography process analytical technology tools in-process monitoring |
| title | At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization |
| title_full | At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization |
| title_fullStr | At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization |
| title_full_unstemmed | At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization |
| title_short | At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization |
| title_sort | at line reversed phase liquid chromatography for in process monitoring of inclusion body solubilization |
| topic | inclusion bodies inclusion body solubilization tailored refolding reversed phase liquid chromatography process analytical technology tools in-process monitoring |
| url | https://www.mdpi.com/2306-5354/8/6/78 |
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