Pharma 4.0 Continuous mRNA Drug Products Manufacturing †
Abstract
:1. Introduction
2. Materials and Methods
2.1. Comprehensive Overview of the Sheet Workflow and Methods Design Strategy
2.2. Inoculation, Upstream Production, and Plasmid Augmentation
2.3. Alkaline Cell Lysis, Plasmid Purification, and DNA Linearization
2.4. Design of IVT Reactor and Purification of mRNA
2.5. mRNA Lipid Nanoparticle (LNP) Formulation Design
2.6. Clean Room Design
2.7. Resource Analysis Method, Total Capital Investment, and Production Cost
2.7.1. Minimum Selling Price of Dose (MSPD) Estimation
2.7.2. Uncertainty Analysis
3. Results
3.1. Upstream Perfusion Reactor Design
3.2. Plasmid Deliverance by Alkaline lysis, Purification, and Linearization
3.3. Purification of the Linearized DNA Template and IVT
3.4. LNP Formulation and Self-Assembly
3.5. Clean Room Related Costs
3.6. Resource Commitment Analysis
3.7. Uncertainty Analysis
4. Discussion
5. Conclusions
- (a)
- We demonstrated the feasibility of continuous end-to-end GMP compliant, mRNA LNP formulated vaccine technology adoption, launching from bacterial culture. Innovations such as perfusion reactors, SMB chromatographic steps, continuous cell lysis modules, and microfluidic formulation were introduced to approach the task. This paves the way for the future actual, modular process line development implementation, under strict quality directives.
- (b)
- The material-driven flowsheet converging mass and energy balances combined or leveraged by bibliographic, empirical, and experimental elements allowed us to link the operational design space framework to the critical quality attributes, hereafter offering a comprehensive mapping of the whole process, accessible for further experimental optimizations. Regarding the processes and the representative material system studied, bleed fraction, residence time, and UTP concentration were identified as the critical parameters for the component blocks of the perfusion bioreactor and the IVT reactors, respectively.
- (c)
- This digital approach furthered allowed us to safely assess the economic aspects of the venture, documenting certain remarkable findings. The MSPD price correlated to single dose regime is expected to range most probably between 1.30 to 1.45 Euros. Indeed, the continuous production of mRNA vaccines overall cost conveys marginally the same price, when compared to batch, being attributed to the limited, applied scale-up grade. MSPD featured reserved dispersion and therefore limited dependence borne by fluctuations. Finally, cell lysis devices including their related purification modules and linearization enzymes ascend as the principal cost factors accounting for 40% and 42% of the equipment and raw material, respectively.
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Experiment | Volume (mL) | Diameter (cm) | Plate Area (cm2) | Length (cm) |
1 | 28 | 1.6 | 2 | 14 |
2 | 334 | 5 | 20 | 17 |
Experiment | Volume Feed (mL) | Retention Time (min) | Flow Rate (mL/min) | Linear Velocity (cm/h) |
1 | 0.5 | 40 | 0.0125 | 0.4 |
2 | 5 | 50 | 0.1 | 0.3 |
Experiment | Plate Height (cm) | Peak Time (min) | Peak Width (min) | Number of Plates |
1 | 0.08 | 7 | 5 | 157 |
2 | 0.1 | 7 | 5 | 157 |
Direct Costs | |
---|---|
Purchased equipment | 100 |
Purchased equipment installation | 39 |
Instrumentation and controls (installed) | 26 |
Piping (installed) | 31 |
Electrical systems (installed) | 10 |
Buildings (including services) | 29 |
Yard improvements | 12 |
Service facilities (installed) | 55 |
Total direct plant cost | 302 |
Indirect Costs | |
Engineering and supervision | 32 |
Construction expenses | 34 |
Legal expenses | 4 |
Contractor’s fee | 19 |
Contingency | 37 |
Total indirect plant cost | 126 |
Total Capital Investment | |
Fixed-capital investment | 428 |
Working capital (15% of total capital investment) | 75 |
Total capital investment | 503 |
Parameter | Lowest Conversion | Lowest Value | Highest Conversion | Highest Value |
---|---|---|---|---|
HIC resin price (€/L) | −10% | 1857 | +30% | 2683 |
AEX membrane price (€/m2) | −10% | 890 | +30% | 1285 |
SEC resin price (€/L) | −10% | 9802 | +30% | 14,159 |
Linearization restriction enzyme price (€/unit) | −10% | 0.00459 | +30% | 0.00663 |
T7 polymerase price (€/mg) | −10% | 34,200 | +30% | 49,400 |
CleanCap Reagent (3’ OMe) AG (€/mg) | −10% | 223 | +30% | 322 |
AFC resin price (€/L) | −10% | 9802 | +30% | 14,159 |
HPLC resin price (€/L) | −10% | 9802 | +30% | 14,159 |
Dimethyldioctadecylammonium (DDAB) price (€/g) | −10% | 498,600 | +30% | 720,200 |
Cholesterol price (€/g) | −10% | 10 | +30% | 14 |
1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) price (€/g) | −10% | 149 | +30% | 215 |
1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) price (€/g) | −10% | 154 | +30% | 223 |
Discount rate (-) | −40% | 0.06 | +40% | 0.4 |
Income tax rate (-) | −40% | 0.126 | +40% | 0.294 |
Source | Sum of Squares | df | Mean Square | F-Value | p-Value | Model A |
---|---|---|---|---|---|---|
Biomass Production | 2.95 × 105 | 3 | 98,287.81 | 19.4 | 0.0003 | significant |
A-Recycling Condensation | 0.0005 | 1 | 0.0005 | 9.84 × 108 | 0.9998 | |
B-Bleed | 1.89 × 105 | 1 | 1.89 × 105 | 37.38 | 0.0002 | |
C-Residence time | 1.06 × 105 | 1 | 1.06 × 105 | 20.81 | 0.0014 | |
Residual | 45,601.9 | 9 | 5066.88 | |||
Cor Total | 3.41 × 105 | 12 | ||||
Coded model equation: Biomass Production Rate = 464 − 0.0079 × A + 153.87 × B − 114.82 × C (14) |
Chromatography Block | ΔP (bar) | u (cm/s) | L (cm) | kO (-) | η (cP) | ε (-) | dp (μm) | N (-) | Lc (cm) | D (cm) |
---|---|---|---|---|---|---|---|---|---|---|
HIC-201 | - | - | - | - | - | - | - | 4 | 42.8 | 21.4 |
AEX-201 | 0.5 | 0.004 | 9 | 150 | 1 | 0.4 | 0.8 | 4 | 12.8 | 6.4 |
SEC-201 | 0.003 | 0.007 | 30 | 180 | 1 | 0.3 | 47 | 4 | 42.8 | 21.4 |
Source | Sum of Squares | df | Mean Square | F-Value | p-Value | Model RP |
---|---|---|---|---|---|---|
Model RNA Production Rate | 1.22 | 9 | 0.1355 | 23.96 | 0.0121 | significant |
A-Polymerase concentration | 0.0017 | 1 | 0.0017 | 0.3032 | 0.6202 | |
B-UTP concentration | 0.8282 | 1 | 0.8282 | 146.47 | 0.0012 | |
C-Magnesium concentration | 0.0016 | 1 | 0.0016 | 0.2916 | 0.6267 | |
AB | 0.0317 | 1 | 0.0317 | 5.6 | 0.0988 | |
AC | 0.0004 | 1 | 0.0004 | 0.0716 | 0.8063 | |
BC | 0.001 | 1 | 0.001 | 0.1843 | 0.6966 | |
A2 | 0.0022 | 1 | 0.0022 | 0.3829 | 0.5799 | |
B2 | 0.2271 | 1 | 0.2271 | 40.16 | 0.0079 | |
C2 | 0.0042 | 1 | 0.0042 | 0.7374 | 0.4536 | |
Residual | 0.017 | 3 | 0.0057 | |||
Cor Total | 1.24 | 12 | ||||
Coded model equation: RNA production rate = 1.03 + 0.0146 × A + 0.3218 × B + 0.0144 × C − 0.08890 × AB − 0.01001 × AC − 0.0161 × BC + 0.0308 × A2 − 0.3152 × B2 + 0.0427 × C2 |
Chromatography Block | ΔP (bar) | u (cm/s) | L (cm) | kO (-) | η (cP) | ε (-) | dp (μm) | N (-) | Lc (cm) | D (cm) |
---|---|---|---|---|---|---|---|---|---|---|
AEX-301 | 0.1 | 0.002 | 5 | 150 | 1 | 0.4 | 0.8 | 4 | 7.2 | 3.6 |
AFC-301 | - | - | - | - | - | - | - | 4 | 23.4 | 11.7 |
HPLC-301 | 0.5 | 0.003 | 25 | 150 | 1 | 0.3 | 2.1 | 4 | 35.6 | 17.8 |
Unit Block | Description | Size | Cp,0 (€) | Year | Lang Factor | References | Simulation Size | Cp (€) | Cp,2021 (€) |
---|---|---|---|---|---|---|---|---|---|
R-101 | Fermentation reactor | V = 0.065 m3 | 34,013 * | 2013 | 0.6 | [38] & assumptions | V = 0.1 m3 | 44,045 | 49,519 |
R-102 | Perfusion reactor | V = 2 m3 | 594,299 * | 2016 | 0.6 | [39] | V = 1 m3 | 392,091 | 461,650 |
V-201 | Horizontal vessel | V = 2.5 m3 | 12,450 | 2013 | 0.2 | [38] & assumptions | V = 0.0005 m3 | 2267 | 2548 |
UF-201 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.023 m2 | 750 | 859 |
HIC-201 | Chromatography | - | - | 2016 | - | [55] | - | 500,000 | 588,702 |
UF-202 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.0016 m2 | 160 | 183 |
AEX-201 | Chromatography | - | - | 2016 | - | [55] | - | 500,000 | 588,702 |
UF-203 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.0016 m2 | 160 | 183 |
SEC-201 | Chromatography | - | - | 2016 | - | [55] | - | 500,000 | 588,702 |
UF-204 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.0016 m2 | 160 | 183 |
C-301 | Container | V = 0.21 m3 | 271 * | 2002 | 0.2 | [40] | V = 0.075 m3 | 221 | 356 |
P-301 | Pump | - | - | 2021 | - | Assumption | - | 500 | 500 |
B-301 | Jacketed mixer | V = 0.2 m3 | 29,931 | 2020 | 0.16 | [55], Pall Corporation | V = 0.075 m3 | 25,584 | 27,369 |
C-302 | Container | V = 0.21 m3 | 271 * | 2002 | 0.2 | [40] | V = 0.075 m3 | 221 | 356 |
P-302 | Pump | - | - | 2021 | - | Assumption | - | 500 | 500 |
UF-301 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.0028 m2 | 221 | 253 |
AEX-301 | Chromatography | - | - | 2016 | - | [55] | - | 500,000 | 588,702 |
UF-302 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.00047 m2 | 79 | 90 |
C-303 | Container | V = 0.21 m3 | 271 * | 2002 | 0.2 | [40] | V = 0,05 m3 | 203 | 328 |
P-303 | Pump | - | - | 2021 | - | Assumption | - | 500 | 500 |
R-301 | Reactor | V = 0.4 m3 | 74,699 * | 2002 | 0.5 | [40] | V = 0.01 m3 | 11,811 | 19,042 |
R-302 | Reactor | V = 0.4 m3 | 74,699 * | 2002 | 0.5 | [40] | V = 0.01 m3 | 11,811 | 19,042 |
R-303 | Reactor | V = 0.4 m3 | 74,699 * | 2002 | 0.5 | [40] | V = 0.01 m3 | 11,811 | 19,042 |
C-304 | Container | V = 0.21 m3 | 271 * | 2002 | 0.2 | [40] | V = 0.05 m3 | 203 | 328 |
P-304 | Pump | - | - | 2021 | - | Assumption | - | 500 | 500 |
UF-303 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.006 m2 | 344 | 394 |
AFC-301 | Chromatography | - | - | 2016 | - | [55] | - | 500,000 | 588,702 |
UF-304 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.005 m2 | 309 | 354 |
HPLC-301 | Chromatography | - | - | 2016 | - | [55] | - | 500,000 | 588,702 |
UF-305 | Ultrafiltration filter | A = 1 m2 | 6686 * | 2015 | 0.58 | [39] | A = 0.005 m2 | 309 | 354 |
B-401 | Non-jacketed mixer | V = 0.2 m3 | 24,994 | 2020 | 0.16 | [55], Pall Corporation | V = 0.0025 m3 | 12,398 | 13,263 |
B-402 | Non-jacketed mixer | V = 0.2 m3 | 24,994 | 2020 | 0.16 | [55], Pall Corporation | V = 0.0075 m3 | 14,780 | 15,812 |
P-401 | Pump | - | - | 2021 | - | Assumption | - | 500 | 500 |
P-402 | Pump | - | - | 2021 | - | Assumption | - | 500 | 500 |
μM-401 | Microfluidic mixer | - | 176,671 * | 2021 | - | Precision Nano-systems Corporation | - | 176,671 | 176,671 |
TF-401 | Tangential flow filtration filter | A = 0.12 m2 | 4504 * | 2018 | 0.8 | [39] | A = 3 m2 | 59,149 | 62,553 |
MS-401 | Molecular sieve | - | - | 2021 | - | Assumption | - | 1800 | 1800 |
Raw Material | Price | References | Total Quantity | Total Price |
---|---|---|---|---|
Glucose | 0.24 * €/kg | [58] | 18,454 kg/year | 4429 €/year |
Diammonium phosphate | 0.83 * €/kg | [59] | 6336 kg/year | 5259 €/year |
Ammonium sulfate | 0.124 * €/kg | [38] | 21,384 kg/year | 2652 €/year |
HIC resin | 2064 €/L (replacement every 100 cycles) | [55,60,61] | 2206 L/year | 4,553,070 €/year |
AEX membrane | 989 €/m2 (replacement every 100 cycles) | [55,60,61] | 6 m2/year | 5505 €/year |
HPLC resin | 10,892 €/L (replacement every 100 cycles) | [55,60,61] | 1476 L/year | 16,076,592 €/year |
SEC resin | 10,892 €/L (replacement every 150 cycles) | [60,61] | 2546 L/year | 27,736,042 €/year |
Linearization restriction enzyme | 0.0051 * €/unit | [62] | 22,464,000,000 units/year | 114,566,400 €/year |
T7 RNA polymerase | 38 €/mg | Merc i | 39 g/year | 1,495,680 €/year |
CleanCap Reagent (3’ OMe) AG | 248 €/mg | Trilink Biotechnologies ii | 216 g/year | CleanCap Reagent (3’ OMe) AG |
AFC resin | 10,892 €/L (replacement every 100 cycles) | [60,61] | 51 L/year | 553,035 €/year |
Ethanol | 6.8 * €/kg | [58] | 41,722 kg/year | 283,707 €/year |
Sodium citrate | 0.7 * €/kg | [38] | 102 kg/year | 72 €/year |
Dimethyldioctadecylammonium (DDAB) | 554 €/g | Merc iii | 8 kg/year | 4,275,947 €/year |
Cholesterol | 11 €/g | Merc iv | 129,015 g/year | 1,419,162 €/year |
1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) | 166 €/g | Merc v | 228,584 g/year | 37,944,885 €/year |
1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) | 172 €/g | Merc vi | 65,996 g/year | 11,351,268 €/year |
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Ouranidis, A.; Davidopoulou, C.; Tashi, R.-K.; Kachrimanis, K. Pharma 4.0 Continuous mRNA Drug Products Manufacturing. Pharmaceutics 2021, 13, 1371. https://doi.org/10.3390/pharmaceutics13091371
Ouranidis A, Davidopoulou C, Tashi R-K, Kachrimanis K. Pharma 4.0 Continuous mRNA Drug Products Manufacturing. Pharmaceutics. 2021; 13(9):1371. https://doi.org/10.3390/pharmaceutics13091371
Chicago/Turabian StyleOuranidis, Andreas, Christina Davidopoulou, Reald-Konstantinos Tashi, and Kyriakos Kachrimanis. 2021. "Pharma 4.0 Continuous mRNA Drug Products Manufacturing" Pharmaceutics 13, no. 9: 1371. https://doi.org/10.3390/pharmaceutics13091371
APA StyleOuranidis, A., Davidopoulou, C., Tashi, R. -K., & Kachrimanis, K. (2021). Pharma 4.0 Continuous mRNA Drug Products Manufacturing. Pharmaceutics, 13(9), 1371. https://doi.org/10.3390/pharmaceutics13091371