Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Dynamic Light Scattering (DLS)
2.2.2. Multi-Angle Dynamic Light Scattering (MADLS)
2.2.3. DLS Thermal Ramps
2.2.4. Nanoparticle Tracking Analysis (NTA)
2.2.5. Electrophoretic Light Scattering (ELS)
2.2.6. Size-Exclusion Chromatography Static Light Scattering (SEC-SLS)
2.2.7. Differential Scanning Calorimetry (DSC)
3. Results
3.1. Size Distribution and Sample Polydispersity
3.1.1. What Sizing Techniques Work for Vectors >50 nm?
3.1.2. What Sizing Techniques Work for Vectors <50 nm?
3.1.3. Viral and Non-Viral Vector Polydispersity by DLS, MADLS, NTA and SEC-SLS
3.1.4. What Can Be Said about Sample Polydispersity?
3.1.5. What Can Be Learnt about the Polydispersity of the Main Population?
3.2. Particle Concentration of Viral Capside Titer
3.2.1. What Concentration Range Can Be Measured for a Particular Vector?
3.2.2. MADLS Extends the Measurement Range for Heterogenous Samples
3.3. Identification of Components and Quantification of Payload
3.3.1. Recombinant Adeno-Associated Viruses
3.3.2. Lipid Nanoparticles
3.4. Electrophoretic Light Scattering
pH Titrations
3.5. Thermal Stability by DLS Thermal Ramps and DSC
3.5.1. Thermal Stability of rAAV5 by DLS Thermal Ramps and DSC
3.5.2. Thermal Stability of mRNA-LNP1 and mRNA-LNP2 by DLS Thermal Ramps and DSC
4. Discussion
4.1. Size Distribution and Sample Polydispersity
4.2. Particle Concentration
4.3. SEC-SLS
4.3.1. AAV
4.3.2. LNPs
4.4. Zeta Potential Discussion
4.5. Thermal Stability by DLS Thermal Ramps and DSC
4.5.1. Thermal Stability of rAAV5
4.5.2. Thermal Stability of mRNA-LNP1 and mRNA-LNP2
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Property | Relevant Technology | References |
---|---|---|
Capsid/particle size | DLS, SEC/AF4-SLS, NTA, cryo-TEM | [7,12,19,20] |
Capsid titer or particle count | MADLS, SEC/AF4-SLS, NTA | [7,12,20] |
Fragmentation | SEC/AF4-SLS | |
Aggregate formation | DLS, MADLS, NTA, TEM, AF4-SLS | [7,12,20] |
Composition: | ||
Percentage of genome-containing virus particles/%full analysis | SEC/AF4-SLS, Anion exchange chromatography, analytical ultracentrifugation, native MS, ELISA, qPCR | [7,21] |
Encapsulation level | LC-UV-Vis, fluorescence assays, gel electrophoresis | [22] |
Lipid quantification | LC coupled with Charged aerosol detector or Evaporative light scattering detector or MS | [7,12,22,23,24] |
Charge | ELS | [7,25] |
Binding interaction | Isothermal titration calorimetry | [26,27] |
Thermal stability | DSC, DLS thermal ramp, DSF | [20,28,29] |
Sample | Camera Level | Detection Threshold | Software Version |
---|---|---|---|
Values used for LNP1 and LNP2 | 16 | 5 | NTA software version 3.4 |
Liposomes | 13–16 * | 4–6 * | NTA software version 3.2 |
Modified Vaccinia Ankara (MVA) | 14 | 5 | NTA software version 3.4 |
Delivery Vector | Formulation Description | Payload/Transgene 1 | Expected Size Range, Diameter |
---|---|---|---|
Full rAAV5 | Recombinant Adeno-associated virus serotype 5 with transgene | pFB-GFP ssDNA 2544 bases | 25–35 nm |
Empty rAAV5 | Recombinant Adeno-associated virus serotype 5 without transgene | - | 25–35 nm |
mRNA-LNP1 | LNP 1 (MC3) 994.4 μg/mL (Total lipid mass) | 0.041 mg/mL Fluc mRNA 1929 bases | 50–120 nm |
mRNA-LNP2 | LNP2 (SM102) 560.7 μg/mL (Total lipid mass) | 0.026 mg/mL FLuc mRNA 1929 bases | 50–100 nm |
Liposome | HSPC/CHOL liposomes | - | 60–110 nm |
MVA | Modified Vaccinia Ankara virus (attenuated) | linear dsDNA ca 180 kbp 2 | 100–400 nm (elongated) |
Sample: (Repeat Measurements per Aliquot) | Z-Average (Cumulants Analysis, nm) | Peak 1 mean (NNLS analysis, nm) | Peak 2 Mean (NNLS Analysis, nm) | Peak 1 Mean (MADLS Analysis, nm) | Peak 2 Mean (MADLS Analysis, nm) | Size Distribution Mode (NTA, nm) | Size Distribution Mean (NTA, nm) |
---|---|---|---|---|---|---|---|
LNP 1 (5) | 87.1 ± 5.5 (6.3%) | 81.7 ± 2.9 (3.5%) | 1930 ± 1922 (99.5%) | 75.0 ± 6.8 (9.1%) | 246 ± 179 (73%) | 64 ± 6 (10%) | 95 ± 2 (2.1%) |
LNP 2 (5) | 104.3 ± 2.3 (2.2%) | 116.4 ± 8.7 (7.5%) | 5021 ± 5 (0.01%) | 105.9 ± 6.3 (5.9%) | 420 ± 88.0 (21%) | 82 ± 25 (31%) | 116 ± 15 (13%) |
Liposomes (5) | 100.1 ± 3.6 (3.6%) | 105.5 ± 4.0 (3.8%) | - | 98.8 ± 3.5 (3.5%) | 452 ± 6 (1.3%) | 85.5 ± 3.6 (4%) | 89.4 ± 0.4 (0.4%) |
Modified Vaccinia Ankara (MVA) (5) | 250 ± 3.0 (1.2%) | 323 ± 15.0 (4.6%) | 4877 ± 42 (0.9%) | 178 ± 11 (6.2%) | 428 ± 11 (2.6%) | 119 ± 14 (11%) | 186 ± 7 (3.6%) |
rAAV5 full 1 (5) | 25.4 ± 0.1 (0.3%) | 26.7 ± 0.2 (0.9%) | - | 25.4 ± 0.1 (0.3%) | - | n/a | n/a |
rAAV5 empty 1 (5) | 29.5 ± 0.2 (0.5%) | 33.2 ± 0.4 (1.3%) | - | 30.7 ± 0.4 (1.3%) | - | n/a | n/a |
Monomer | Mw (g/mol) | Mw/Mn | Frac. of Sample (%) | Peak Conc. (mg/mL) | |
---|---|---|---|---|---|
rAAV5 Empty 1 | Monomer | 3.79 × 106 ± 2.89 × 104 | 1.004 ± 0.0020 | 91.23 ± 1.09 | 0.341 ± 0.0142 |
Dimer | 7.1 × 106 ± 5.58 × 105 | 1.011 ± 0.0049 | 6.55 ± 0.72 | 0.025 ± 0.0018 | |
Aggregates | 2.2 × 107 ± 6.65 × 106 | 1.19 ± 0.0934 | 2.22 ± 0.49 | 0.008 ± 0.0017 | |
rAAV5 Full 1 | Monomer | 4.52 × 106 ± 7.34 × 104 | 1.001 ± 0.0007 | 94.02 ± 1.26 | 0.411 ± 0.0059 |
Dimer | 5 × 106 3 ± 1.34 × 106 | 1.10 ± 0.1133 | 2.48 ± 0.69 | 0.011 ± 0.0030 | |
Aggregates | 7.8 × 106 3 ± 2.63 × 105 | 1.12 ± 0.0442 | 3.49 ± 0.57 | 0.015 ± 0.0025 | |
rAAV5 Empty 2 | Monomer | 3.63 × 106 ± 2.27 × 104 | 1.012 ± 0.0026 | 98.01 ± 0.32 | 0.256 ± 0.0045 |
Dimer | 7.1 × 106 ± 5.39 × 105 | 1.006 ± 0.0049 | 1.99 ± 0.32 | 0.005 ± 0.0009 | |
rAAV5 Full 2 | Monomer | 4.43 × 106 ± 2.09 × 104 | 1.007 ± 0.0019 | 96.53 ± 0.30 | 0.191 ±0.0115 |
Dimer | 7.5 × 106 ± 1.76 × 105 | 1.04 ± 0.0119 | 3.15 ± 0.17 | 0.006 ± 0.0001 | |
Aggregates | 2.7 × 109 3 ± 4.67 × 109 | 1.32 ± 0.4908 | 0.33 ± 0.25 | 0.01 ± 0.0005 |
Delivery Vector (Number of Repeat Measurements) | Polydispersity Index (PdI) 1a (%Pd) | Span (D90–D10)/D50) | Main Population Peak Polydispersity (%Pd) | Mw/Mn 4 | % Monomer 4 |
---|---|---|---|---|---|
rAAV5 full (6) | 0.03 ± 0.01 1a (17) | 0.70 ± 0.01 1c, 0.42 ± 0.02 2c n/a 3 | 24.0 ± 0.6 1b, 15.0 ± 0.8 2 | 1.004 | 94.0 |
rAAV5 empty (6) | 0.12 ± 0.02 1a (34) | 0.90 ± 0.02 1c, 0.61 ± 0.05 2c n/a 3 | 35.5 ± 1.7 1b, 23.2 ± 2.4 2 | 1.001 | 91.2 |
LNP 1 (5) | 0.325±0.004 1a (57) | 6.95 ± 4.24 1c, 0.92 ± 0.17 2c 0.97 ± 0.19 3 | 43.9 ± 5.3 1b, 33.0 ± 9.4 2 | (1.13) | (100) |
LNP2 (5) | 0.159±0.017 1a (40) | 1.29 ± 0.17 1c, 0.70 ± 0.16 2c 0.98 ± 0.32 3 | 39.7 ± 3.4 1b, 24.0 ± 3.9 2 | (1.16) | (100) |
Liposome (5) | 0.032 ± 0.016 1a (18) | 0.75 ± 0.05 1c, 0.46 ± 0.04 2c 0.38 ± 0.02 3 | 24.1 ± 1.7 1b, 16.0 ± 1.9 2 | n/a | n/a |
MVA (10/5 **) | 0.227 ± 0.020 1a (48) | 1.85± 0.14 1c, Excluded *2c 1.04 ± 0.07 3 | 53.6 ± 4.0 1b, excluded *2 | n/a | n/a |
Mw (g/mol) | Mw/Mn | Wt Fr (Capsid) (%) | % Full AAV | vp/vg Ratio | AAV Titer (vp/mL) | |
---|---|---|---|---|---|---|
Empty rAAV5 | 3,860,000 | 1.011 | 99.82 | 3.71 × 1013 | ||
% RSD | 2.1 | 0.34 | 0.01 | 4.0 | ||
Full rAAV5 | 4,502,000 | 1.008 | 83.9 | 77.49 | 1.291 | 3.08 × 1013 |
% RSD | 0.47 | 0.21 | 0.13 | 0.47 | 0.47 | 5.7 |
Empty rAAV9 | 3,798,000 | 1.007 | 99.83 | 4.34 × 1013 | ||
% RSD | 0.79 | 0.18 | 0.12 | 5.1 | ||
Full rAAV9 | 4,526,000 | 1.015 | 84.13 | 76.97 | 1.299 | 5.35 × 1013 |
% RSD | 0.43 | 0.19 | 0.19 | 1.2 | 1.2 | 2.8 |
Empty rAAV2 | 3,619,000 | 1.002 | 99.85 | 4.10 × 1012 | ||
% RSD | 7.3 | 0.22 | 0.18 | 11 | ||
rAAV2 Full | 4,513,000 | 1.005 | 83.28 | 80.04 | 1.249 | 1.05 × 1013 |
% RSD | 0.90 | 0.07 | 0.13 | 0.62 | 0.62 | 3.8 |
Measurement | Zeta Potential (mV) | z-Average Diameters (nm) | ||
---|---|---|---|---|
mRNA-LNP1 | mRNA-LNP2 | mRNA-LNP1 | mRNA-LNP2 | |
1 | −20.0 | −5.35 | 68.9 | 99.9 |
2 | −18.0 | −6.30 | 69.7 | 102.6 |
3 | −20.5 | −8.23 | 70.0 | 102.8 |
4 | - | - | 69.9 | 102.3 |
5 | - | - | 70.2 | 103.1 |
Mean | −19.5 | −6.63 | 69.7 | 102.1 |
Standard Deviation | 1.32 | 1.47 | 0.50 | 1.29 |
Sample | Run # | Tm1, C | Tm2, C | Total Area, mJ |
---|---|---|---|---|
mRNA-LNP1 | 1 | 20.9 | 71.7 | 0.633 |
2 | 21.4 | 71.1 | 0.687 | |
mRNA-LNP2 | 1 | 25.6 | 75.0 | 0.204 |
2 | 24.6 | 73.6 | 0.197 |
Attribute | Measurement Techniques | Measurand—Parameter Abbreviation (unit) | Sample Information Required? | Sample Applicability |
---|---|---|---|---|
PSD | DLS | Z-average diameter—Dh (nm) | Dispersant viscosity and refractive index | From 0.3 nm to 10–20 µm 1. |
NTA | Number based size distribution mean or mode (nm) | Dispersant viscosity | From 10 nm to 2 µm 1. | |
MADLS | Hydrodynamic diameter—Dh (nm) | Dispersant viscosity and refractive index, Particle absorbance and refractive index | From 0.3 nm to 500 nm 1. | |
SEC-SLS/UV or SLS/RI | Molecular weight—(g/mol) | Particle dn/dc and dA/dc | <200 nm | |
Polydispersity | DLS (Cumulants Analysis) | Polydispersity Index—PdI | Dispersant viscosity and refractive index | Same as size measurements |
(DLS NNLS analysis) | Peak polydispersity—%Pd | Dispersant viscosity and refractive index (for volume and number transformations: Particle absorbance and refractive index) | ||
NTA | Span | Dispersant viscosity | ||
MADLS | Span | Particle absorbance and refractive index | ||
SEC-UV or SEC-RI | Mw/Mn | |||
Particle concentration/Viral capsid titer | MADLS | Particle concentration (particles per mL) | Dispersant viscosity and refractive index, Particle absorbance and refractive index | |
NTA | Particle concentration (particles per mL) | |||
SEC-SLS/UV or SLS/RI | Particle concentration (particles per mL) | Particle dn/dc or dA/dc | ||
Surface charge | ELS | Zeta potential (mV) | Dispersant viscosity | |
Thermal stability | DSC | Tm, Tonset, ΔH, thermogram profile | ||
DLS | Trend in light scattering and size | Dispersant viscosity 2 | ||
Drug payload | SEC-SLS/RI/UV | Vehicle and drug’s dn/dc and dA/dc |
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Markova, N.; Cairns, S.; Jankevics-Jones, H.; Kaszuba, M.; Caputo, F.; Parot, J. Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques. Vaccines 2022, 10, 49. https://doi.org/10.3390/vaccines10010049
Markova N, Cairns S, Jankevics-Jones H, Kaszuba M, Caputo F, Parot J. Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques. Vaccines. 2022; 10(1):49. https://doi.org/10.3390/vaccines10010049
Chicago/Turabian StyleMarkova, Natalia, Stefan Cairns, Hanna Jankevics-Jones, Michael Kaszuba, Fanny Caputo, and Jérémie Parot. 2022. "Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques" Vaccines 10, no. 1: 49. https://doi.org/10.3390/vaccines10010049
APA StyleMarkova, N., Cairns, S., Jankevics-Jones, H., Kaszuba, M., Caputo, F., & Parot, J. (2022). Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques. Vaccines, 10(1), 49. https://doi.org/10.3390/vaccines10010049