Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Preparation of Reagent Solutions
2.3. Capillary Electrophoresis System
2.4. Capillary Electrophoresis Methods
2.5. Design of Experiments
2.6. Linearity and Signal-to-Noise Ratio
2.7. Precision
2.8. Evaluation
3. Results
3.1. Design of Experiments
Verification of the Predicted Optimal Factor Settings
3.2. Signal-to-Noise Ratio
3.3. Linearity
3.4. Precision
4. Discussion
4.1. Design of Experiments
4.1.1. Selection of Factors and Responses
4.1.2. Results from the Design of Experiments
4.2. Signal-to-Noise Ratio
4.3. Linearity
4.4. Precision
5. Conclusions and Outlook
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Response | S/N Basic (n = 4) | S/N Main (n = 5) | P/V Acidic (n = 5) | P/V Basic (n = 5) |
---|---|---|---|---|
Mean | 712.0 | 2.410 × 104 | 1.419 | 1.132 |
SD | 10.24 | 177.8 | 0.005327 | 0.007638 |
95% CI | 625.8–753.1 | 2.388 × 104–2.433 × 104 | 1.412–1.425 | 1.123–1.123 |
RSD | 1.439% | 0.7378% | 0.3755% | 0.6745% |
Predicted value | 778.4 | 2.678 × 104 | 1.468 | 1.160 |
Absolute deviation experimental value | −66.37 | −2674 | −0.04938 | −0.02760 |
Relative deviation experimental value | −8.526% | −9.986% | −3.363% | −2.379% |
Matuzumab | NISTmAb | ||||
---|---|---|---|---|---|
Concentration (mg/mL) | UV Detection | LEDIF Detection | Concentration (mg/mL) | UV Detection | LEDIF Detection |
0.01 | 8.7 | 59 | 0.003 | - | 1.7 |
0.02 | 27 | 188 | 0.004 | - | 2.1 * |
0.03 | 42 | 249 | 0.005 | - | 2.0 |
0.04 | 42 | 412 | 0.01 | 2.7 | 28 |
0.05 | 55 | 465 | 0.02 | 14 | 85 |
0.10 | 180 | 1983 | 0.05 | 114 | 452 |
0.25 | 542 | 6745 | 0.10 | 316 | 997 |
0.50 | 1046 | 1.12 × 104 | 0.20 | 828 | 2125 |
1.00 | 1964 | 1.92 × 104 | 0.50 | 2220 | 4241 |
Parameter | Time Interval | Mean | 95% CI | SD | RSD | ||||
---|---|---|---|---|---|---|---|---|---|
UV | LEDIF | UV | LEDIF | UV | LEDIF | UV | LEDIF | ||
Migration time | Week 1, day 1 | 27.86 min | 27.67 min * | 27.78 min–27.93 min | 27.61 min–27.74 min * | 0.1025 min | 0.07062 min * | 0.3681% | 0.2552% * |
Week 1, day 2 | 27.67 min | 27.60 min | 27.57 min–27.77 min | 27.53 min–27.67 min | 0.1405 min | 0.09533 min | 0.5079% | 0.3454% | |
Week 2 | 28.44 min | 27.67 min * | 28.38 min–28.50 min | 27.55 min–27.79 min * | 0.08552 min | 0.1573 min * | 0.3007% | 0.5684% * | |
Week 1 and 2 | 27.99 min | 27.64 min * | 27.86 min–28.12 min | 27.60 min–27.69 min * | 0.3509 min | 0.1161 min | 1.254% | 0.4199% * | |
%area basic | Week 1, day 1 | 7.473% | 7.395% * | 7.215–7.732% | 7.118–7.671% * | 0.3618% | 0.2989% * | 4.841% | 4.042% * |
Week 1, day 2 | 7.278% | 6.521% | 7.152–7.404% | 6.465–6.577% | 0.1762% | 0.07832% | 2.421% | 1.201% | |
Week 2 | 7.296% | 6.215% * | 7.143–7.450% | 6.133–6.297% * | 0.2146% | 0.1063% * | 2.941% | 1.711% * | |
Week 1 and 2 | 7.349% | 6.650% * | 7.249–7.450% | 6.446–6.855% * | 0.2694% | 0.5072% * | 3.666% | 7.627% * | |
%area main 1 | Week 1, day 1 | 18.26% | 16.98% * | 18.20–18.32% | 16.82–17.14% * | 0.08628% | 0.1715% * | 0.4726% | 1.010% * |
Week 1, day 2 | 18.25% | 17.78% | 18.19–18.32% | 17.75–17.82% | 0.09175% | 0.04757% | 0.5026% | 0.2676% | |
Week 2 | 17.96% | 17.48% * | 17.86–18.05% | 17.36–17.60% * | 0.1334% | 0.1568% * | 0.7427% | 0.8972% * | |
Week 1 and 2 | 18.16% | 17.46% * | 18.09–18.22% | 17.32–17.60% * | 0.1762% | 0.3474% * | 0.9702% | 1.990% * | |
%area mid | Week 1, day 1 | 3.615% | 4.009% * | 3.591–3.639% | 3.982–4.036% * | 0.03346% | 0.02957% * | 0.9254% | 0.7376% * |
Week 1, day 2 | 3.559% | 3.945% | 3.542–3.575% | 3.903–3.987% | 0.02317% | 0.05898% | 0.6510% | 1.495% | |
Week 2 | 3.602% | 4.001% * | 3.576–3.628% | 3.975–4.026% * | 0.03644% | 0.03321% * | 1.012% | 0.8301% * | |
Week 1 and 2 | 3.592% | 3.982% * | 3.577–3.607% | 3.961–4.002% * | 0.03918% | 0.05183% * | 1.091% | 1.302% * | |
%area main 2 | Week 1, day 1 | 22.61% | 22.13% * | 22.52–22.71% | 21.95–22.30% * | 0.1363% | 0.1909% * | 0.6027% | 0.8626% * |
Week 1, day 2 | 22.77% | 21.84% | 22.66–22.88% | 21.74–21.95% | 0.1509% | 0.04445% | 0.6626% | 0.6435% | |
Week 2 | 22.45% | 21.70% * | 22.33–22.57% | 21.61–21.79% * | 0.1644% | 0.1172% * | 0.7323% | 0.5400% * | |
Week 1 and 2 | 22.61% | 21.87% * | 22.54–22.68% | 21.78–21.96% * | 0.1972% | 0.2217% * | 0.8722% | 1.013% * | |
%area acidic | Week 1, day 1 | 48.04% | 49.49% * | 47.89–48.18% | 49.23–49.75% * | 0.2054% | 0.2817% * | 0.4277% | 0.5693% * |
Week 1, day 2 | 48.14% | 49.91% | 47.96–48.31% | 49.85–49.96% | 0.2443% | 0.07524% | 0.5075% | 0.1508% | |
Week 2 | 48.67% | 50.60% * | 48.52–48.82% | 50.47–50.73% * | 0.2116% | 0.1693% * | 0.4347% | 0.3345% * | |
Week 1 and 2 | 48.28% | 50.04% * | 48.15–48.41% | 49.84–50.23% * | 0.3526% | 0.4869% * | 0.7303% | 0.9732% * | |
P/V acidic | Week 1, day 1 | 1.784 | 2.084 * | 1.754–1.814 | 2.046–2.121 * | 0.04204 | 0.04075 * | 2.356% | 1.956% * |
Week 1, day 2 | 1.746 | 2.115 | 1.717–1.775 | 2.072–2.158 | 0.04083 | 0.06051 | 2.338% | 2.861% | |
Week 2 | 1.907 | 2.155 * | 1.880–1.933 | 2.094–2.216 * | 0.03727 | 0.07875 * | 1.955% | 3.654% * | |
Week 1 and 2 | 1.812 | 2.120 * | 1.783–1.842 | 2.093–2.148 * | 0.07970 | 0.06722 * | 4.397% | 3.170% * |
Parameter | Time Interval | Mean | 95% CI | SD | RSD | ||||
---|---|---|---|---|---|---|---|---|---|
UV | LEDIF | UV | LEDIF | UV | LEDIF | UV | LEDIF | ||
Migration time | Week 1, day 1 | 18.57 min | 17.35 min | 18.49 min–18.65 min | 17.32 min–17.38 min | 0.1076 min | 0.03669 min | 0.5793% | 0.2115% |
Week 1, day 2 | 18.31 min | 17.35 min | 18.27 min–18.35 min | 17.34 min–17.37 min | 0.05050 min | 0.01996 min | 0.2758% | 0.1150% | |
Week 2 | 18.81 min | 17.52 min | 18.79 min–18.84 min | 17.50 min–17.54 min | 0.03821 min | 0.02855 min | 0.2031% | 0.1630% | |
Week 1 and 2 | 18.57 min | 17.41 min | 18.48 min–18.65 min | 17.37 min–17.44 min | 0.2208 min | 0.08464 min | 1.189% | 0.4863% | |
%area basic | Week 1, day 1 | 16.91% | 15.01% | 16.84–16.97% | 14.87–15.15% | 0.09029% | 0.1927% | 0.5340% | 1.284% |
Week 1, day 2 | 16.55% | 15.61% | 16.45–16.65% | 15.45–15.78% | 0.1350% | 0.2297% | 0.8159% | 1.471% | |
Week 2 | 14.81% | 14.97% | 14.74–14.87% | 14.70–15.24% | 0.09175% | 0.3762% | 0.6196% | 2.513% | |
Week 1 and 2 | 16.09% | 15.20% | 15.74–16.44% | 15.05–15.35% | 0.9385% | 0.4014% | 5.834% | 2.641% | |
%area main | Week 1, day 1 | 67.18% | 67.01% | 67.02–67.34% | 66.74–67.28% | 0.2236% | 0.3794% | 0.3328% | 0.5662% |
Week 1, day 2 | 67.74% | 66.83% | 67.62–67.86% | 66.57–67.08% | 0.1689% | 0.3602% | 0.2494% | 0.5390% | |
Week 2 | 69.26% | 67.44% | 69.10–69.42% | 67.20–67.67% | 0.2212% | 0.3305% | 0.3193% | 0.4901% | |
Week 1 and 2 | 68.06% | 67.09% | 67.71–68.40% | 66.93–67.25% | 0.9169% | 0.4319% | 1.347% | 0.6438% | |
%area acidic | Week 1, day 1 | 15.92% | 17.98% | 15.79–16.05% | 17.60–18.36% | 0.1822% | 0.5298% | 1.144% | 2.947% |
Week 1, day 2 | 15.71% | 17.56% | 15.62–15.81% | 17.37–17.75% | 0.1312% | 0.2611% | 0.8347% | 1.487% | |
Week 2 | 15.93% | 17.59% | 15.81–16.05% | 17.46–17.72% | 0.1682% | 0.1818% | 1.056% | 1.033% | |
Week 1 and 2 | 15.85% | 17.71% | 15.78–15.92% | 17.56–17.86% | 0.1869% | 0.3947% | 1.179% | 2.229% | |
P/V acidic | Week 1, day 1 | 1.353 | 1.405 | 1.311–1.396 | 1.400–1.411 | 0.05928 | 0.007960 | 4.380% | 0.5663% |
Week 1, day 2 | 1.356 | 1.418 | 1.352–1.359 | 1.413–1.423 | 0.005266 | 0.007310 | 0.3885% | 0.5155% | |
Week 2 | 1.385 | 1.424 | 1.379–1.390 | 1.421–1.427 | 0.007782 | 0.004245 | 0.5620% | 0.2982% | |
Week 1 and 2 | 1.365 | 1.416 | 1.351–1.378 | 1.412–1.420 | 0.03646 | 0.01011 | 2.672% | 0.7138% |
mAb | Parameter | Equivalence at 2% | Equivalence at 5% |
---|---|---|---|
Waters mAb | Migration time | Yes | - |
%area basic | - | No | |
%area main 1 | - | Yes | |
%area mid | - | No | |
%area main 2 | - | Yes | |
%area acidic | - | Yes | |
P/V | - | No | |
NISTmAb | Migration time | No | - |
%area basic | - | No | |
%area main | - | Yes | |
%area acidic | - | No | |
P/V | - | Yes |
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Zagst, H.; Hartung, S.; Menges, D.-M.; Wittmann, A.; Wätzig, H. Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection. Separations 2023, 10, 320. https://doi.org/10.3390/separations10050320
Zagst H, Hartung S, Menges D-M, Wittmann A, Wätzig H. Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection. Separations. 2023; 10(5):320. https://doi.org/10.3390/separations10050320
Chicago/Turabian StyleZagst, Holger, Sophie Hartung, Dina-Mareike Menges, Antonia Wittmann, and Hermann Wätzig. 2023. "Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection" Separations 10, no. 5: 320. https://doi.org/10.3390/separations10050320
APA StyleZagst, H., Hartung, S., Menges, D.-M., Wittmann, A., & Wätzig, H. (2023). Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection. Separations, 10(5), 320. https://doi.org/10.3390/separations10050320