Holographic Imaging of Insect Cell Cultures: Online Non-Invasive Monitoring of Adeno-Associated Virus Production and Cell Concentration
Open accessAbstract
The insect cell-baculovirus vector system has become one of the favorite platforms for the expression of viral vectors for vaccination and gene therapy purposes. As it is a lytic system, it is essential to balance maximum recombinant product expression with harvest time, minimizing product exposure to detrimental proteases. With this purpose, new bioprocess monitoring solutions are needed to accurately estimate culture progression. Herein, we used online digital holographic microscopy (DHM) to monitor bioreactor cultures of Sf9 insect cells. Batches of baculovirus-infected Sf9 cells producing recombinant adeno-associated virus (AAV) and non-infected cells were used to evaluate DHM prediction capabilities for viable cell concentration, culture viability and AAV titer. Over 30 cell-related optical attributes were quantified using DHM, followed by a forward stepwise regression to select the most significant (p < 0.05) parameters for each variable. We then applied multiple linear regression to obtain models which were able to predict culture variables with root mean squared errors (RMSE) of 7 × 105 cells/mL, 3% for cell viability and 2 × 103 AAV/cell for 3-fold cross-validation. Overall, this work shows that DHM can be implemented for online monitoring of Sf9 concentration and viability, also permitting to monitor product titer, namely AAV, or culture progression in lytic systems, making it a valuable tool to support the time of harvest decision and for the establishment of controlled feeding strategies.
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Monitoring of rAAV production culture and prediction of intra- and extracellular rAAV titer with in situ FS spectroscopy and online digital holographic microscopy have been reported.
“Recently, the monitoring of rAAV production culture and prediction of the intra- and extracellular rAAV titer, among other process-related quality attributes, with in situ FS spectroscopy110 and online digital holographic microscopy,111 were reported.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [111]
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- Authors
- Pais, D. A. M., Galrão, P. R. S., Kryzhanska, A., Barbau, J., Isidro, I. A., & Alves, P. M.
- Affiliations
- Instituto de Biologia Experimental e Tecnológica (PT)
- OVIZIO Imaging Systems SA/NV, 1180 Brussels, Belgium
- Corresponding author
- Alves, Paula M.
- Venue
- Processes
- Publisher
- MDPI AG
- Published
- 2020-04-22
- Volume
- 8
- Issue
- 4
- Pages
- 487
- ISSN
- 2227-9717 (ISSN-L)
- OpenAlex
- W3019187438
- Cited by
- 25
- References
- 42
- Citations by year
- 2026: 2 · 2025: 1 · 2024: 7 · 2023: 3 · 2022: 6 · 2021: 4 · 2020: 2
- FWCI
- 1.22
- Type
- journal article
- Language
- en
- Open access
- gold
- dx.doi.org
- Licence
- Locations
- Processes (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Fundação para a Ciência e a Tecnologia (PD/BD/105873/2014)
- iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Digital Holography and Microscopy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Keywords
- digital holographic microscopy, Sf9 cells, recombinant adeno-associated virus, cell viability, harvest time prediction, bioreactor cultivation, forward selection, multiple linear regression
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