Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Sample Preparation Methods for Quantification of tAb, acDrug and Free Payload
2.2.1. Preparation of Calibration Standards, Internal Standards (ISTD) and Quality Control (QC) Samples
2.2.2. Total Antibody (tAb) Quantification Assay by Trypsin Digestion
2.2.3. Antibody-Conjugated Drug (acDrug) Quantification Assay by Linker Cleavable Enzymes Digestion
2.2.4. Free Payload Quantification Assay
2.3. Sample Preparation Methods for Intact Protein Mass Analysis
2.4. Application for In Vitro Linker Stability Studies
2.5. Application for Preclinical Pharmacokinetic Studies in Mice
2.6. LC-MS Conditions
2.7. Softwares
3. Results and Discussion
3.1. In Vitro Linker Stability in Mouse Plasma and IgG Depleted Human Plasma
3.2. In Vivo Pharmacokinetic Study in Mice
3.3. Intact Protein Mass Analysis by Partial Digestion and Reduction
4. Discussion and Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Scan Mode | TOF-MS Scan | Product Ion Scan | |||
---|---|---|---|---|---|
Specific Signature Peptide [M + 2H]2+ | Stable Isotope-Labeled Peptide [M + 2H]2+ | MMAF [M + H]+ | Verapamil [M + H]+ | ||
Mass range (m/z) | 100~1000 | 832~842 | 837~847 | 100~750 | 100~500 |
Parent ion (m/z) | - | 937.5 | 942.3 | 732.5 | 455.3 |
Product ion (m/z) | - | 836.4 | 842.4 | 700.5 | 165.1 |
Declustering potential (V) | 100 | 162 | 162 | 100 | 125 |
Collision energy (V) | 10 | 38 | 38 | 35 | 30 |
Accumulation time (s) | 0.15 | 0.1 | 0.1 | 0.1 | 0.1 |
Scan Mode | TOF-MS Scan |
---|---|
Mass range (m/z) | 1000~3500 |
Declustering potential (V) | 150 |
Collision energy (V) | 20 |
Accumulation time (s) | 0.2 |
Drug | AUClast (µg/day/mL) | CL (mL × day/kg) | α-HL (day) | β-HL (day) | Cmax (µg/mL) | V1 (mL/kg) | Vss (mL/kg) |
---|---|---|---|---|---|---|---|
ITC6103RO | 68.00 ± 18.51 | 44.79 ± 14.13 | 0.44 ± 0.25 | 6.40 ± 3.48 | 60.41 ± 5.75 | 76.45 ± 7.49 | 236.28 ± 40.37 |
ITC6104RO | 71.01 ± 10.13 | 39.40 ± 4.41 | 0.25 ± 0.22 | 4.75 ± 1.63 | 58.83 ± 11.60 | 60.95 ± 14.57 | 203.88 ± 48.68 |
Drug | AUClast (ng/day/mL) | CL (mL × day/kg) | α-HL (day) | β-HL (day) | Cmax (ng/mL) | V1 (L/kg) | Vss (L/kg) |
---|---|---|---|---|---|---|---|
ITC6103RO | 433.30 ± 83.11 | 6839.58 ± 1195.49 | 0.17 ± 0.01 | 2.32 ± 0.23 | 585.38 ± 66.23 | 6.41 ± 0.96 | 15.97 ± 1.24 |
ITC6104RO | 53.55 ± 0.77 | 55514.72 ± 693.39 | 0.06 ± 0.00 | 0.77 ± 0.20 | 518.61 ± 36.82 | 7.18 ± 0.06 | 10.44 ± 0.87 |
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Lee, B.i.; Park, S.-j.; Park, Y.; Shin, S.-H.; Choi, J.-m.; Park, M.-j.; Lim, J.-h.; Kim, S.Y.; Lee, H.; Shin, Y.G. Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay. Pharmaceutics 2021, 13, 125. https://doi.org/10.3390/pharmaceutics13010125
Lee Bi, Park S-j, Park Y, Shin S-H, Choi J-m, Park M-j, Lim J-h, Kim SY, Lee H, Shin YG. Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay. Pharmaceutics. 2021; 13(1):125. https://doi.org/10.3390/pharmaceutics13010125
Chicago/Turabian StyleLee, Byeong ill, Seo-jin Park, Yuri Park, Seok-Ho Shin, Jang-mi Choi, Min-jae Park, Jeong-hyeon Lim, Sun Young Kim, Hyangsook Lee, and Young G. Shin. 2021. "Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay" Pharmaceutics 13, no. 1: 125. https://doi.org/10.3390/pharmaceutics13010125
APA StyleLee, B. i., Park, S.-j., Park, Y., Shin, S.-H., Choi, J.-m., Park, M.-j., Lim, J.-h., Kim, S. Y., Lee, H., & Shin, Y. G. (2021). Assessments of the In Vitro and In Vivo Linker Stability and Catabolic Fate for the Ortho Hydroxy-Protected Aryl Sulfate Linker by Immuno-Affinity Capture Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometric Assay. Pharmaceutics, 13(1), 125. https://doi.org/10.3390/pharmaceutics13010125