High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor
Abstract
:1. Introduction
2. Materials and Methods
2.1. Maintenance of Huh7.5 Cells
2.2. HCV Virus Stocks
2.3. CelCradle™ Culture and Virus Production
2.3.1. Cell Seed, Attachment, and Expansion in the CelCradle™
2.3.2. HCV Infection of the CelCradle™ Culture in SCM
2.3.3. Virus Harvests under SF Culture Conditions
2.3.4. Maintenance and Monitoring of the CelCradle™ Cultures and Evaluation of Spread of Infection
2.4. Shake Flask Cell Cultures
2.5. Immunostaining of HCV NS5A
2.6. HCV Infectivity Titers, RNA Titers, and Core Titers
2.7. HCV Neutralization Assays
2.8. Equilibrium Density Gradient Centrifugation
2.9. Virus Genome Sequencing
3. Results
3.1. Attachment and Expansion of Huh7.5 Cells Cultured on BioNOCII™ Carriers
3.2. Production of HCV in SCM (CC-DMEx1-SCM)
3.3. Production of HCV in SFM (CC-DMEx1-SFM1)
3.4. Increased Cell Numbers Did Not Enhance HCV Production under SF Conditions (CC*-DMEx2-SFM)
3.5. Evaluation of the Influence of DME Frequency on HCV Yield in Shake Flasks (ShF-DMEx1-SCM, ShF-DMEx2-SCM, ShF-DMEx1-SFM, ShF-DMEx2-SFM)
3.6. Two DME Improved HCV Yields in SFM (CC-DMEx1-SFM2, CC-DMEx2-SFM)
3.7. Selected Characteristics Were Similar for HCV Produced in the CelCradle™ and in Monolayer Cell Cultures
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Experiment | Cells Only SCM | CC-DMEx1-SCM | CC-DMEx1-SFM1 | CC*-DMEx2-SFM | CC-DMEx1-SFM2 | CC-DMEx2-SFM |
---|---|---|---|---|---|---|
SF virus production a | na | no | yes | yes | yes | yes |
DME/harvests per day | 1 | 1 | 1 | 2 | 1 b | 2 b |
BioNOCII™ carriers, g | 5.5 | 5.5 | 5.5 | 10.7 | 5.5 | 5.5 |
Peak cell numbers | ||||||
Day of peak total cell number, dpcs | 9 | 8 | 8 | 12 | 8 | 7 |
Peak total cell number | 1.6 × 109 | 0.96 × 109 | 1.2 × 109 | 3.6 × 109 | 1.8 × 109 | 1.5 × 109 |
cells/mL | 3.2 × 106 | 1.9 × 106 | 2.4 × 106 | 7.1 × 106 | 3.6 × 106 | 2.9 × 106 |
cells/carrier | 2.0 × 106 | 1.2 × 106 | 1.5 × 106 | 2.3 × 106 | 2.3 × 106 | 1.8 × 106 |
Cell culture supernatant parameters | ||||||
Peak GCR, mmol/day (dpcs) | 9 (8) | 10 (6) | 11 (7) | 23 (7) | 9 (6) | 11 (6) |
Glucose range, mM | 5–24 | 10–25 | 11–25 | 5–29 | 11–27 | 10–27 |
Lactate peak concentration, mM (dpcs) c | nd | nd | nd | 48 (7) | 37 (6) | 26 (6) |
Glutamine range, mM c | nd | nd | nd | 1.7–4.3 | 2.1–3.6 | 2.7–3.6 |
Ammonia range, mM c | nd | nd | nd | 0.3–1.0 | 0.3–1.0 | 0.3–1.0 |
pH range | 6.8–8.3 | 6.9–7.7 | 6.9–8.3 | 6.9–7.9 | 6.9–7.9 | 7.1–7.9 |
Virus yield | ||||||
Total harvest volume, L d | na | 2.3 | 5.9 | 10.6 | 8.6 | 18.5 |
Peak infectivity titer, log10 FFU/mL | na | 6.8 | 6.9 | 6.9 | 7.2 | 7.0 |
Days with high- or moderate yield | na | 9–13 | 7–19 | 8–18 e | 7–25 | 6–26 f |
Total accumulated FFU g | na | 0.81 × 1010 | 2.2 × 1010 | 1.7 × 1010 | 4.4 × 1010 | 5.9 × 1010 |
CSVY, FFU/cell | na | 8.5 | 18.5 | 4.8 | 24.5 | 41.0 |
Yield compared to CC-DMEx2-SFM h | na | 0.14 | 0.37 | 0.29 | 0.74 | 1.0 |
Culture | ShF-DMEx1-SCM | ShF-DMEx2-SCM | ShF-DMEx1-SFM | ShF-DMEx2-SFM |
---|---|---|---|---|
SF virus production a | no | no | yes | yes |
DME/harvests per day | 1 | 2 | 1 | 2 |
BioNOCII™ carriers, g | 0.39 | 0.39 | 0.39 | 0.39 |
Peak cell numbers | ||||
Day of peak total cell number, dpcs | 5 | 6 | 9 | 7 |
Peak total cell number | 1.1 × 108 | 0.94 × 108 | 0.95× 108 | 1.4 × 108 |
cells/mL | 3.9 × 106 | 3.5 × 106 | 4.9 × 106 | 5.5 × 106 |
cells/carrier | 2.3 × 106 | 2.1 × 106 | 2.9 × 106 | 3.2 × 106 |
Cell culture supernatant | ||||
Peak csGCR, mmol/carrier/day (dpcs) | 0.014 (6) | 0.017 (6) | 0.016 (6) | 0.019 (5) |
Glucose range, mM | 12–31 | 7–32 | 11–32 | 9–34 |
Lactate peak concentration, mM (dpcs) | 43 (8) | 33 (7) | 39 (5) | 36 (5) |
Glutamine range, mM b | 1.2–3.6 | 1.8–4.0 | 1.7–3.6 | 2.0–3.6 |
Ammonia range, mM b | 0.4–1.5 | 0.3–1.4 | 0.3–1.2 | 0.3–1.0 |
pH range | 6.8–8.1 | 6.9–8.1 | 6.7–7.8 | 6.8–8.1 |
Virus yield | ||||
Peak infectivity titer, log10 FFU/mL | 6.9 | 7.1 | 6.8 | 6.9 |
Days with high- or moderate yield | 7–12 c | 5–12 c | 7–11 c | 6–11 c |
CSVY, FFU/cell | 4.8 | 20.0 | 4.7 | 7.2 |
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Offersgaard, A.; Duarte Hernandez, C.R.; Pihl, A.F.; Venkatesan, N.P.; Krarup, H.; Lin, X.; Reichl, U.; Bukh, J.; Genzel, Y.; Gottwein, J.M. High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor. Vaccines 2022, 10, 249. https://doi.org/10.3390/vaccines10020249
Offersgaard A, Duarte Hernandez CR, Pihl AF, Venkatesan NP, Krarup H, Lin X, Reichl U, Bukh J, Genzel Y, Gottwein JM. High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor. Vaccines. 2022; 10(2):249. https://doi.org/10.3390/vaccines10020249
Chicago/Turabian StyleOffersgaard, Anna, Carlos Rene Duarte Hernandez, Anne Finne Pihl, Nandini Prabhakar Venkatesan, Henrik Krarup, Xiangliang Lin, Udo Reichl, Jens Bukh, Yvonne Genzel, and Judith Margarete Gottwein. 2022. "High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor" Vaccines 10, no. 2: 249. https://doi.org/10.3390/vaccines10020249
APA StyleOffersgaard, A., Duarte Hernandez, C. R., Pihl, A. F., Venkatesan, N. P., Krarup, H., Lin, X., Reichl, U., Bukh, J., Genzel, Y., & Gottwein, J. M. (2022). High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor. Vaccines, 10(2), 249. https://doi.org/10.3390/vaccines10020249