Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Material
2.1.1. Antibodies
2.1.2. Diphtheria Toxin and Toxoid
2.2. mAb Characterization Methods
2.2.1. General BLI Method
2.2.2. Binning Experiment
2.2.3. Inhibition of mAb-DTxn Binding to HB-EGF
2.2.4. Toxin Neutralization Assay
2.2.5. BLI Screening for mAb Sensitivity to Heat-Induced Degradation of DTxd
2.2.6. Western Blot
2.2.7. Single Epitope Antigenicity Testing (SEAT)
SEAT Assay Design
SEAT Qualification
Determination of the Comparability Range
3. Results and Discussion
3.1. mAb Characterization
3.1.1. Determination of Binning Groups and Subunit Binding Sites
3.1.2. Assessment of mAb-DTxn Binding Inhibition by HB-EGF
3.1.3. Determination of Toxin Neutralization Capacity of the mAb Panel
3.1.4. Determination of mAb Ability to Detect Thermally Induced Change in DTxd
3.2. Single Epitope Antigenicity Test (SEAT)
3.2.1. Assay Development
3.2.2. Assay Performance
3.2.3. Sample Comparability Assessment
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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mAb | Clone ID | Binding Site on DTxn (Subunit A or B) | Bin | Distinguishes Native from Heat Stressed DTxd | Inhibits DTxn Binding to HB-EGF Target | Neutralization Capacity |
---|---|---|---|---|---|---|
1-10 | 1-10.3.1.5.10 | B | 2 | Yes | Yes | weak |
1-49 | 1-49.1.2.8.6.3.8 | A | 1 | Yes | No | strong |
1-53 | 1-53.3.2.4.6.6 | B | 2 | Yes | Yes | moderate |
2-1 | 2-1.3.21 | B | 2 | Yes | inconsistent | weak |
2-18 | 2-18.1.8.8.3 | A | 3 | Yes | No | strong |
2-2 | 2-2.2.11 | A | 3 | Yes | No | moderate |
2-25 | 2-25.1.21.9.7.4.7 | B | 2 | Yes | Yes | moderate |
3-11 | 3-11.1.9 | B | 4 | Yes | No | weak |
3-14 | 3-14.1.1 | B | 4 | No | inconsistent | weak |
3-17 | 3-17.1.2 | B | 4 | Yes | No | strong |
3-44 | 3-44.2.7 | A | 3 | Yes | No | weak |
3-45 | 3-45.2.3 | A | 3 | Yes | No | moderate |
mAb | Neutralizing Titre (×10−3 U/mg) | Category | n |
---|---|---|---|
1-10 | <1 | weak | 2 |
1-49 | 10–100 | strong | 4 |
1-53 | 1–9 | moderate | 2 |
2-1 | <1 | weak | 2 |
2-18 | 10–100 | strong | 5 |
2-2 | 1–9 | moderate | 1 |
2-25 | 1–9 | moderate | 4 |
3-11 | <1 | weak | 1 |
3-14 | <1 | weak | 1 |
3-17 | 10–100 | strong | 1 |
3-44 | <1 | weak | 1 |
3-45 | 1–9 | moderate | 1 |
Quality Check | Sample/ Parameter | 1-49 | 1-53 | 2-25 | 2-18 | 3-14 | 3-17 | 1-10 | 2-2 | 3-45 |
---|---|---|---|---|---|---|---|---|---|---|
Intermediate Precision (%CV, n = 3) | 1X | 7% | 8% | 2% | 4% | 4% | 4% | 8% | 9% | 7% |
0.5X | 4% | 3% | 3% | 14% | 6% | 1% | 7% | 5% | 3% | |
0.25X | 9% | 2% | 2% | 10% | 10% | 6% | 5% | 2% | 3% | |
0.1X | 4% | 4% | 6% | 9% | 16% | 9% | 2% 1 | 9% | 4% | |
Accuracy (%Recovery range, n = 3) | 1X | 96–111% | 94–108% | 99–104% | 98–105% | 99–108% | 98–107% | 91–105% | 97–113% | 101–115% |
0.5X | 100–108% | 95–101% | 97–102% | 80–105% | 92–102% | 96–97% | 93–106% | 98–107% | 98–105% | |
0.25X | 93–110% | 93–97% | 93–96% | 84–103% | 86–104% | 90–101% | 93–103% | 96–99% | 97–103% | |
0.1X | 95–103% | 87–93% | 86–96% | 80–95% | 72–100% | 81–96% | 90–92% 1 | 87–103% | 92–99% | |
Linearity (1X, 0.5X, 0.25X, 0.1X) | R2 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
Slope | 1.02 | 1.06 | 1.05 | 1.05 | 1.07 | 1.06 | 1.02 | 1.03 | 1.04 | |
Sensitive to Heat Induced Degradation 2 | N/Ap | Yes | Yes | Yes | Yes | poor 3 | Yes | Yes | Yes | Yes |
Comparability Range (%Recovery) | N/Ap | [80–125%] | [85–118%] | [90–119%] | [80–119%] | [61–141%] | [75–122%] | [82–120%] | [91–112%] | [83–128%] |
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Houy, C.; Ming, M.; Ettorre, L.; Jin, R.; Thangavadivel, N.; Chen, T.; Su, J.; Gajewska, B. Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes. Vaccines 2022, 10, 775. https://doi.org/10.3390/vaccines10050775
Houy C, Ming M, Ettorre L, Jin R, Thangavadivel N, Chen T, Su J, Gajewska B. Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes. Vaccines. 2022; 10(5):775. https://doi.org/10.3390/vaccines10050775
Chicago/Turabian StyleHouy, Camille, Marin Ming, Luciano Ettorre, Robbie Jin, Nemika Thangavadivel, Tricia Chen, Jin Su, and Beata Gajewska. 2022. "Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes" Vaccines 10, no. 5: 775. https://doi.org/10.3390/vaccines10050775