Freeze-Dried Mesenchymal Stem Cell-Secretome Pharmaceuticalization: Optimization of Formulation and Manufacturing Process Robustness
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Design of Experiments
2.3. Sample Preparation
2.3.1. Isolation and Expansion of Human Adipose-Derived MSCs (AD-MSCs)
2.3.2. MSC-Secretome Ultrafiltration
2.3.3. Freeze-Drying
2.4. Sample Characterization
2.4.1. Residual Moisture
2.4.2. Cake Aspect
2.4.3. Total Protein Content
2.4.4. Phospholipid Quantification by Nile Red Assay
2.4.5. EV Particle Size Determination
2.4.6. Anti-Elastase Activity
2.5. D-Optimal Design
2.6. Optimization and Validation of the Manufacturing Process
3. Results and Discussion
3.1. Model for the “Protein Content” Response, YP
3.2. Model for the “Lipid Content” Response, YL
3.3. Model for the “EV Particle Size” Response, YNTA
3.4. Model for the “In Vitro Anti-Elastase Activity” Response, YA
3.5. Optimization and Validation of the Manufacturing Process
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Formulation | V1 | V2 | V1 = Mannitol % | % w/v |
---|---|---|---|---|
1 | −1.0 | −1.0 | 25 | 0.5 |
2 | −0.5 | −1.0 | 45 | 0.5 |
3 | 0.0 | −1.0 | 65 | 0.5 |
4 | 0.6 | −1.0 | 85 | 0.5 |
5 | 1.0 | −1.0 | 100 | 0.5 |
6 | −1.0 | +1.0 | 25 | 1.5 |
7 | −0.5 | +1.0 | 45 | 1.5 |
8 | 0.0 | +1.0 | 65 | 1.5 |
9 | 0.6 | +1.0 | 85 | 1.5 |
10 | 1.0 | +1.0 | 100 | 1.5 |
Phase | Section | Time (h:min) | Shelf’s Temperature (°C) | Pressure (mbar) |
---|---|---|---|---|
Loading | 1 | - | 25 | 1000 |
Freezing | 2-ramp | 0:20 | 5 | |
3-hold | 0:40 | 5 | ||
4-ramp | 0:10 | −5 | ||
5-hold | 1:00 | −5 | ||
6-ramp | 0:45 | −45 | ||
7-hold | 4:30 | −45 | ||
Primary drying | 8 | 0:20 | −45 | 0.08 |
9 | 0:30 | −40 | ||
10 | 79:09 | −40 | ||
Secondary drying | 11 | 2:30 | 22 | |
12 | 8:00 | 22 |
Factors | Levels | Encoding |
---|---|---|
Secretome batch (S) | 1 | S1 = 1, S2 = 0 |
2 | S1 = 0, S2 = 1 | |
3 | S1 = S2 = 0 | |
Lyophilization (L) | Pre | 1 |
Post | +1 | |
Total concentration of excipients (% w/v) | 0.5 | −1 |
1.5 | +1 | |
Mannitol/sucrose concentration ratio (% w/w) | 25/75 | −1 |
100/0 | +1 |
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Mocchi, M.; Bari, E.; Marrubini, G.; Bonda, A.F.; Perteghella, S.; Tartara, F.; Cofano, F.; Perna, G.d.; Giovannelli, L.; Mandracchia, D.; et al. Freeze-Dried Mesenchymal Stem Cell-Secretome Pharmaceuticalization: Optimization of Formulation and Manufacturing Process Robustness. Pharmaceutics 2021, 13, 1129. https://doi.org/10.3390/pharmaceutics13081129
Mocchi M, Bari E, Marrubini G, Bonda AF, Perteghella S, Tartara F, Cofano F, Perna Gd, Giovannelli L, Mandracchia D, et al. Freeze-Dried Mesenchymal Stem Cell-Secretome Pharmaceuticalization: Optimization of Formulation and Manufacturing Process Robustness. Pharmaceutics. 2021; 13(8):1129. https://doi.org/10.3390/pharmaceutics13081129
Chicago/Turabian StyleMocchi, Michela, Elia Bari, Giorgio Marrubini, Andrea Foglio Bonda, Sara Perteghella, Fulvio Tartara, Fabio Cofano, Giuseppe di Perna, Lorella Giovannelli, Delia Mandracchia, and et al. 2021. "Freeze-Dried Mesenchymal Stem Cell-Secretome Pharmaceuticalization: Optimization of Formulation and Manufacturing Process Robustness" Pharmaceutics 13, no. 8: 1129. https://doi.org/10.3390/pharmaceutics13081129