Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science
Abstract
:1. Introduction
- Use a recipe database as a basis for knowledge management;
- Automate and standardize detection of the exponential growth phase within shake flask experiments with enhanced data science;
- Automate determination of KPIs based on the detected exponential growth phase and data from the recipe database;
- Store KPIs in the database to simplify and enable comparison with other recipes.
2. Results and Discussion
2.1. Workflow
2.1.1. Initial Phase Fitting and Noise Reduction
2.1.2. Optimization of Phase Start
2.1.3. Optimization of Phase End
2.2. Cultivation Results
- maximum specific growth rate µmax;
- cell-specific oxygen consumption rate qO2;
- biomass and product yield YX/S and YP/S;
- maximum achieved biomass concentration CX,max.
2.2.1. Bacteria—E. coli
2.2.2. Yeast—S. cerevisiae
2.2.3. Plant Cells—Vitis vinifera
2.2.4. Animal Cells
2.3. Evaluation of Measurement Techniques
2.4. Evaluation of KPIs
3. Conclusions and Outlook
4. Materials and Methods
4.1. General Equipment and Online Measurement Systems
4.2. Cultivation Details
4.2.1. Bacteria Cultivation—E. coli
- Lysogeny broth (LB) medium with 5 g L−1 yeast extract (cat. Y1625, Sigma-Aldrich, St. Louis, MI, USA), 10 g L−1 tryptone (cat. 95039, Sigma-Aldrich) and 5 g L−1 sodium chloride (cat. S9888, Sigma-Aldrich) [82];
- Terrific broth (TB) medium with 24 g L−1 yeast extract (cat. Y1625, Sigma-Aldrich), 20 g L−1 tryptone (cat. 95039, Sigma-Aldrich), 4 mL L−1 glycerin (cat. 49770, Sigma-Aldrich) and a phosphate buffer consisting of 0.17 mol L−1 KH2PO4 (cat. P5655, Sigma-Aldrich) and 0.72 mol L−1 K2HPO4 (cat. P3786, Sigma-Aldrich) [83];
- Modified Biener medium consisting of glucose (10 g L−1), a mineral salt solution, a trace element solution and MgSO4 solution [60]. Details can be found in Appendix A.
4.2.2. Yeast Cultivation—S. cerevisiae
4.2.3. Plant Suspension Cultivation—Vitis vinifera
4.2.4. Animal Cell Cultivation—CHO, High Five, HEK293
4.3. Software
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Recipe for 1 L E.coli Medium
- 40 mL of glucose stock solution with 500 g L−1 glucose;
- 480 mL of mineral stock solution, consisting of:
- o
- 2.1 g L−1 (NH4)2 H-citrate;
- o
- 4.2 g L−1 Na2SO4;
- o
- 8.4 g L−1 (NH4)2SO4;
- o
- 1.06 g L−1 NH4Cl;
- o
- 31.6 g L−1 K2HPO4;
- o
- 7.4 g L−1 NaH2PO4·H2O;
- 4.5 mL of trace element solution, consisting of:
- o
- 0.18 g L−1 CoCl2·6 H2O;
- o
- 0.50 g L−1 CaCl2·2 H2O;
- o
- 0.18 g L−1 ZnSO4·7 H2O;
- o
- 0.10 g L−1 MnSO4·H2O;
- o
- 10.05 g L−1 Na2-EDTA·2 H2O;
- o
- 8.35 g L−1 FeCl3·6 H2O;
- o
- 0.16 g L−1 CuSO4·5 H2O;
- 2.2 mL of 1 mol L−1 MgSO4·7 H2O stock solution;
- 473.3 mL of sterile deionized H2O.
Appendix B
Appendix B.1. Additional Cultivation Results
Appendix B.1.1. Bacteria—E. coli
Appendix B.1.2. Animal Cell Cultures
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Attribute | Information | Value |
---|---|---|
O2, threshold | Oxygen limit when it can be assumed that exponential growth is impossible. | 10–20% |
Growth speed | Growth speed of the organism | Fast, medium, slow |
Organism | Flask Type | Backscatter | OUR |
---|---|---|---|
E. coli | Without baffles | Average | Good |
With baffles | Noisy at low filling volumes | Good | |
S. cerevisiae | Without baffles | Average | Good |
With baffles | Noisy at low filling volumes | Good | |
V. vinifera | With and without baffles | Good | Metabolic changes affect signal |
CHO cells | Without baffles | Detection of dead cells | Good |
High Five | Without baffles | Detection of dead cells | Good |
HEK293 | With and without baffles | Detection of dead cells | Good |
Organism | Medium | µoffline | µBSL, man. | µBSL, auto. | µOUR, man. | µOUR, auto. | qO2 | YX/S |
---|---|---|---|---|---|---|---|---|
(Replicates) | [h−1] | [h−1] | [h−1] | [h−1] | [h−1] | [mol g−1 h−1] | [g g−1] | |
E. coli | LB (6) | 1.2982 ± 0.1153 | - | - | 1.2752 ± 0.2049 | 1.2075 ± 0.1511 | 1.65 ± 0.28 × 10−2 | - 1 |
TB (6) | 1.2889 ± 0.0453 | - | - | 1.3421 ± 0.0419 | 1.3778 ± 0.0191 | 1.82 ± 0.33 × 10−2 | 1.679 ± 0.032 2 | |
Biener (4) | 0.6199 ± 0.0194 | - | - | 0.6432 ± 0.0041 | 0.5820 ± 0.0021 | 2.13 ± 0.41 × 10−2 | 0.434 ± 0.065 | |
S. cerevisiae | YPD (10) | 0.4964 ± 0.0344 | 0.6226 ± 0.1294 | 0.5110 ± 0.0907 | 0.4895 ± 0.0446 | 0.4744 ± 0.0424 | 2.59 ± 0.39 × 10−3 | 0.812 ± 0.168 2 |
V. vinifera | MS (12) | 0.0084 ± 0.0009 | 0.0088 ± 0.0009 | 0.0080 ± 0.0008 | - | - | 2.29 ± 0.40 × 10−4 | 0.474 ± 0.037 |
[mol cell−1 h−1] | [106 cells g−1] | |||||||
CHO DP-12 | TC-42 (8) | 0.0349 ± 0.0017 | - | - | 0.0335 ± 0.0018 | 0.0327 ± 0.0011 | 2.88 ± 0.62 × 10−13 | 1.990 ± 0.140 |
ExpiCHO-S | SPM (6) | 0.0390 ± 0.0007 | - | - | 0.0359 ± 0.0005 | 0.0362 ± 0.0007 | 2.28 ± 0.24 × 10−13 | 3.126 ± 0.065 |
High Five | Exp.Five (8) | 0.0445 ± 0.0014 | - | - | 0.0453 ± 0.0017 | 0.0430 ± 0.0010 | 4.18 ± 0.02 × 10−13 | 1.274 ± 0.057 |
HEK293 | FS293 (6) | 0.0315 ± 0.0026 | - | - | 0.0365 ± 0.0010 | 0.0355 ± 0.0005 | 9.86 ± 3.76 × 10−14 | 1.392 ± 0.151 |
Organism | µauto. 1 | µlit | qO2, auto. | qO2, lit. | YX/S | YX/S,lit | References |
---|---|---|---|---|---|---|---|
[h−1] | [h−1] | [mol g−1 h−1] | [mol g−1 h−1] | [g g−1] | [g g−1] | ||
E. coli 2 | 0.5820 ± 0.0021 | 0.54-0.56 | 2.13 ± 0.41 × 10−2 | 1.3–2.2 × 10−2 | 0.434 ± 0.065 | 0.50–0.54 | [54,55,60,61] |
S. cerevisiae | 0.4744 ± 0.0424 | 0.42–0.51 | 2.59 ± 0.39 × 10−3 | 1.0–9.0 × 10−3 | 0.812 ± 0.168 | N.A. 2 | [56,62,63,64,65,66] |
V. vinifera | 0.0080 ± 0.0008 | 0.0065–0.01 | 2.29 ± 0.40 × 10−4 | 1.1–7.1 × 10−4 | 0.474 ± 0.037 | 0.47–0.49 | [57,67,68] |
[mol cell−1 h−1] | [mol cell−1 h−1] | [106 cells g−1] | [106 cells g−1] | ||||
CHO DP-12 | 0.0327 ± 0.0011 | 0.0358–0.0363 | 2.88 ± 0.62 × 10−13 | 3.10 × 10−13 | 1.990 ± 0.140 | 2.063–2.216 | [58,69,70,71,72] |
ExpiCHO-S | 0.0362 ± 0.0007 | 0.0316–0.0422 | 2.28 ± 0.24 × 10−13 | 2.30–2.90 × 10−13 | 3.126 ± 0.065 | 3.3–3.4 | [59,73] |
High Five | 0.0430 ± 0.0010 | 0.028–0.044 | 4.18 ± 0.02 × 10−13 | 2.88–9.00 × 10−13 | 1.274 ± 0.057 | 1.250 | [74,75,76] |
HEK293 | 0.0355 ± 0.0005 | 0.03–0.05 | 9.86 ± 3.76 × 10−14 | 1.30–1.85 × 10−13 | 1.392 ± 0.151 | N.A. | [77,78,79] |
Cell Line | Origin/Source | Media |
---|---|---|
CHO DP-12 #1934 | Subclone, courtesy of Prof. Noll, Bielefeld | TC-42 (cat. 511-0001, Xell AG, Bielefeld, Germany) 1 |
ExpiCHO-S-6H8 | Thermo Fisher Scientific, cat. A29127 | ExpiCHO Stable production medium (cat. A3711001) 2 |
Freestyle 293 (HEK) | Thermo Fisher Scientific, cat. R79007 | FreeStyle Expression 293 (cat. 12338018) |
High Five | Thermo Fisher Scientific, cat. B85502 | Express Five SFM (cat. 10486025) 3 |
Cell Line | Temperature [°C] | Shaking Rate [rpm] | Vrel [%] | CO2 [%] |
---|---|---|---|---|
CHO DP-12 | 37 | 120 | 32 | 7.5 |
EXPI-CHO S | 37 | 130 | 24–32 | 8.0 |
Freestyle 293 | 37 | 130 | 32 | 8.0 |
High Five | 27 | 100 | 24 | 0.0 |
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Maschke, R.W.; Pretzner, B.; John, G.T.; Herwig, C.; Eibl, D. Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science. Bioengineering 2022, 9, 339. https://doi.org/10.3390/bioengineering9080339
Maschke RW, Pretzner B, John GT, Herwig C, Eibl D. Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science. Bioengineering. 2022; 9(8):339. https://doi.org/10.3390/bioengineering9080339
Chicago/Turabian StyleMaschke, Rüdiger W., Barbara Pretzner, Gernot T. John, Christoph Herwig, and Dieter Eibl. 2022. "Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science" Bioengineering 9, no. 8: 339. https://doi.org/10.3390/bioengineering9080339
APA StyleMaschke, R. W., Pretzner, B., John, G. T., Herwig, C., & Eibl, D. (2022). Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science. Bioengineering, 9(8), 339. https://doi.org/10.3390/bioengineering9080339