A QbD-Guided RP-HPLC Method for Estimation of Repotrectinib: Design, Optimization, and Validation
DOI:
https://doi.org/10.69980/ajpr.v28i3.712Keywords:
Repotrectinib, Quality by Design (QbD), RP-HPLC, Method Development, Validation, Non-Small Cell Lung Cancer (NSCLC), Analytical Method, Forced Degradation, Tyrosine Kinase Inhibitor (TKI)Abstract
Background: Repotrectinib (C₁₈H₁₈FN₅O₂) represents an advanced macrocyclic inhibitor targeting tyrosine kinases, specifically approved for managing ROS1-driven non-small cell lung cancer (NSCLC). The molecule's intricate design and scarcity of established quantification techniques highlight the demand for a dependable, verified analytical procedure. Objective: The goal was to create and confirm an uncomplicated, durable, and economical reverse-phase high-performance liquid chromatography (RP-HPLC) protocol for assessing repotrectinib levels, guided by a Quality by Design (QbD) strategy in line with ICH standards.Methods: Optimization involved a QbD methodology focusing on key factors like mobile phase makeup and acidity level, analyzed via a central composite experimental layout to examine primary, combined, and squared influences. The refined setup incorporated a Phenomenex Kinetex XB-C18 stationary phase (150 mm length × 4.6 mm diameter, 5 µm particles), eluent of 0.1% trifluoroacetic acid mixed with acetonitrile (60:40 volume ratio), elution speed of 0.9 mL per minute, UV detection at 280 nm wavelength, and sample introduction of 10 µL. Assessments covered selectivity, linear range (40–60 µg/mL), recovery rates, repeatability, detection threshold (LOD), quantification threshold (LOQ), durability, and degradation under forced conditions.Results: Strong linear correlation was observed (R² exceeding 0.999), with recovery values between 99.94% and 100.16%. Repeatability tests indicated relative standard deviation (%RSD) under 2%. Detection limit stood at 2.00 µg/mL, and quantification limit at 6.06 µg/mL. The protocol proved stable against slight purposeful adjustments and clearly resolved repotrectinib from breakdown fragments in stress tests. Conclusion: This QbD-based RP-HPLC protocol offers simplicity, reliability, precision, and durability, ideal for standard evaluations of repotrectinib in raw materials and formulated products.
References
(1) Barbato, M. I.; Bradford, D.; Ren, Y.; Aungst, S. L.; Miller, C. P.; Pan, L.; Zirkelbach, J. F.; Li, Y.; Bi, Y.; Fan, J.; Grimstein, M.; Dorff, S. E.; Amatya, A. K.; Mishra-Kalyani, P. S.; Scepura, B.; Schotland, P.; Udoka, O.; Ojofeitimi, I.; Leighton, J. K.; Rahman, N. A.; Pazdur, R.; Singh, H.; Kluetz, P. G.; Drezner, N. FDA Approval Summary: Repotrectinib for Locally Advanced or Metastatic ROS1-Positive Non–Small Cell Lung Cancer. Clin. Cancer Res. 2024, 30 (16), 3364–3370. https://doi.org/10.1158/1078-0432.ccr-24-0949.
(2) Desilets, A.; Repetto, M.; Drilon, A. Repotrectinib: Redefining the Therapeutic Landscape for Patients with ROS1 Fusion‐driven Non‐small Cell Lung Cancer. Clin. Transl. Med. 2024, 14 (10). https://doi.org/10.1002/ctm2.70017.
(3) Dhillon, S. Repotrectinib: First Approval. Drugs 2024, 84 (2), 239–246. https://doi.org/10.1007/s40265-023-01990-6.
(4) Yun, M. R.; Kim, D. H.; Kim, S.-Y.; Joo, H.-S.; Lee, Y. W.; Choi, H. M.; Park, C. W.; Heo, S. G.; Kang, H. N.; Lee, S. S.; Schoenfeld, A. J.; Drilon, A.; Kang, S.-G.; Shim, H. S.; Hong, M. H.; Cui, J. J.; Kim, H. R.; Cho, B. C. Repotrectinib Exhibits Potent Antitumor Activity in Treatment-Naïve and Solvent-Front–Mutant ROS1-Rearranged Non–Small Cell Lung Cancer. Clin. Cancer Res. 2020, 26 (13), 3287–3295. https://doi.org/10.1158/1078-0432.ccr-19-2777.
(5) Almutairi, M. B. F.; Alrouji, M.; Almuhanna, Y.; Asad, M.; Joseph, B. In-Vitro and In-Vivo Antibacterial Effects of Frankincense Oil and Its Interaction with Some Antibiotics against Multidrug-Resistant Pathogens. Antibiotics 2022, 11 (11), 1591. https://doi.org/10.3390/antibiotics11111591.
(6) Barbato, M. I.; Bradford, D.; Ren, Y.; Aungst, S. L.; Miller, C. P.; Pan, L.; Zirkelbach, J. F.; Li, Y.; Bi, Y.; Fan, J.; Grimstein, M.; Dorff, S. E.; Amatya, A. K.; Mishra-Kalyani, P. S.; Scepura, B.; Schotland, P.; Udoka, O.; Ojofeitimi, I.; Leighton, J. K.; Rahman, N. A.; Pazdur, R.; Singh, H.; Kluetz, P. G.; Drezner, N. FDA Approval Summary: Repotrectinib for Locally Advanced or Metastatic ROS1-Positive Non–Small Cell Lung Cancer. Clin. Cancer Res. 2024, 30 (16), 3364–3370. https://doi.org/10.1158/1078-0432.ccr-24-0949.
(7) Yun, M. R.; Kim, D. H.; Kim, S.-Y.; Joo, H.-S.; Lee, Y. W.; Choi, H. M.; Park, C. W.; Heo, S. G.; Kang, H. N.; Lee, S. S.; Schoenfeld, A. J.; Drilon, A.; Kang, S.-G.; Shim, H. S.; Hong, M. H.; Cui, J. J.; Kim, H. R.; Cho, B. C. Repotrectinib Exhibits Potent Antitumor Activity in Treatment-Naïve and Solvent-Front–Mutant ROS1-Rearranged Non–Small Cell Lung Cancer. Clin. Cancer Res. 2020, 26 (13), 3287–3295. https://doi.org/10.1158/1078-0432.ccr-19-2777.
(8) Bebb, D. G.; Agulnik, J.; Albadine, R.; Banerji, S.; Bigras, G.; Butts, C.; Couture, C.; Cutz, J. C.; Desmeules, P.; Ionescu, D. N.; Leighl, N. B.; Melosky, B.; Morzycki, W.; Rashid-Kolvear, F.; Sekhon, H. S.; Smith, A. C.; Stockley, T. L.; Torlakovic, E.; Xu, Z.; Tsao, M. S. Crizotinib Inhibition of ROS1-Positive Tumours in Advanced Non-Small-Cell Lung Cancer: A Canadian Perspective. Curr. Oncol. 2019, 26 (4), 551–557. https://doi.org/10.3747/co.26.5137.
(9) Keddy, C.; Shinde, P.; Jones, K.; Kaech, S.; Somwar, R.; Shinde, U.; Davare, M. A. Resistance Profile and Structural Modeling of Next-Generation ROS1 Tyrosine Kinase Inhibitors. Mol. Cancer Ther. 2022, 21 (2), 336–346. https://doi.org/10.1158/1535-7163.mct-21-0395.
(10) Ou, S.-H. I.; Hagopian, G. G.; Zhang, S. S.; Nagasaka, M. Comprehensive Review of ROS1 Tyrosine Kinase Inhibitors-Classified by Structural Designs and Mutation Spectrum (Solvent Front Mutation [G2032R] and Central β-Sheet 6 [Cβ6] Mutation [L2086F]). J. Thorac. Oncol. 2024, 19 (5), 706–718. https://doi.org/10.1016/j.jtho.2023.12.008.
(11) Pérol, M.; Li, W.; Pennell, N. A.; Liu, G.; Ohe, Y.; De Braud, F.; Nagasaka, M.; Felip, E.; Xiong, A.; Zhang, Y.; Fan, H.; Wang, X.; Li, S.; Lai, R. K.; Ran, F.; Zhang, X.; Chen, W.; Bazhenova, L.; Zhou, C. Taletrectinib in ROS1 + Non–Small Cell Lung Cancer: TRUST. J. Clin. Oncol. 2025, 43 (16), 1920–1929. https://doi.org/10.1200/jco-25-00275.
(12) Zagalo, D. M.; Sousa, J.; Simões, S. Quality by Design (QbD) Approach in Marketing Authorization Procedures of Non-Biological Complex Drugs: A Critical Evaluation. Eur. J. Pharm. Biopharm. 2022, 178, 1–24. https://doi.org/10.1016/j.ejpb.2022.07.014.
(13) Suleman, S.; Belew, S.; Kebebe, D.; Duguma, M.; Teshome, H.; Hasen, G.; Evelien Wynendaele; Duchateau, L.; De Spiegeleer, B. Quality-by-Design Principles Applied to the Establishment of a Pharmaceutical Quality Control Laboratory in a Resource-Limited Setting: The Lab Water. Int. J. Anal. Chem. 2022, 2022, 1–12. https://doi.org/10.1155/2022/2062406.
(14) Duarte, J. G.; Duarte, M. G.; Piedade, A. P.; Mascarenhas-Melo, F. Rethinking Pharmaceutical Industry with Quality by Design: Application in Research, Development, Manufacturing, and Quality Assurance. AAPS J. 2025, 27 (4). https://doi.org/10.1208/s12248-025-01079-w.
(15) Pramod, K.; Tahir, Ma.; Charoo, N.; Ansari, S.; Ali, J. Pharmaceutical Product Development: A Quality by Design Approach. Int. J. Pharm. Investig. 2016, 6 (3), 129. https://doi.org/10.4103/2230-973X.187350.
(16) Simões, A.; Veiga, F.; Vitorino, C. Question-Based Review for Pharmaceutical Development: An Enhanced Quality Approach. Eur. J. Pharm. Biopharm. 2024, 195, 114174. https://doi.org/10.1016/j.ejpb.2023.114174.
(17) D. Patil, H.; B Patil, C.; V. Patil, V.; S. Patil, P. An Overview on Quality by Design. Asian J. Res. Pharm. Sci. 2023, 49–55. https://doi.org/10.52711/2231-5659.2023.00009.
(18) Yang, S.; Hu, X.; Zhu, J.; Zheng, B.; Bi, W.; Wang, X.; Wu, J.; Mi, Z.; Wu, Y. Aspects and Implementation of Pharmaceutical Quality by Design from Conceptual Frameworks to Industrial Applications. Pharmaceutics 2025, 17 (5), 623. https://doi.org/10.3390/pharmaceutics17050623.
(19) Barbato, M. I.; Bradford, D.; Ren, Y.; Aungst, S. L.; Miller, C. P.; Pan, L.; Zirkelbach, J. F.; Li, Y.; Bi, Y.; Fan, J.; Grimstein, M.; Dorff, S. E.; Amatya, A. K.; Mishra-Kalyani, P. S.; Scepura, B.; Schotland, P.; Udoka, O.; Ojofeitimi, I.; Leighton, J. K.; Rahman, N. A.; Pazdur, R.; Singh, H.; Kluetz, P. G.; Drezner, N. FDA Approval Summary: Repotrectinib for Locally Advanced or Metastatic ROS1-Positive Non–Small Cell Lung Cancer. Clin. Cancer Res. 2024, 30 (16), 3364–3370. https://doi.org/10.1158/1078-0432.CCR-24-0949.
(20) Barbato, M. I.; Bradford, D.; Ren, Y.; Aungst, S. L.; Miller, C. P.; Pan, L.; Zirkelbach, J. F.; Li, Y.; Bi, Y.; Fan, J.; Grimstein, M.; Dorff, S. E.; Amatya, A. K.; Mishra-Kalyani, P. S.; Scepura, B.; Schotland, P.; Udoka, O.; Ojofeitimi, I.; Leighton, J. K.; Rahman, N. A.; Pazdur, R.; Singh, H.; Kluetz, P. G.; Drezner, N. FDA Approval Summary: Repotrectinib for Locally Advanced or Metastatic ROS1-Positive Non–Small Cell Lung Cancer. Clin. Cancer Res. 2024, 30 (16), 3364–3370. https://doi.org/10.1158/1078-0432.CCR-24-0949.
(21) Rais, T.; Shakeel, A.; Naseem, L.; Nasser, N.; Aamir, M. Repotrectinib: A Promising New Therapy for Advanced Nonsmall Cell Lung Cancer. Ann. Med. Surg. 2024, 86 (12), 7265–7269. https://doi.org/10.1097/MS9.0000000000002717.
(22) Rothschild, S. I.; Mauti, L. A. ROS1 Fusion-Positive Non-Small Cell Lung Cancer—Repotrectinib as a New Treatment Option. Transl. Cancer Res. 2025, 14 (6), 3272–3276. https://doi.org/10.21037/tcr-2025-263.
(23) Patel, R.; Rathod, D.; Shah, N.; Vaghela, V.; Patadiya, N. Inhibitors as a Therapeutic Frontier in Lung Cancer: Mechanism, Opportunities, and Molecular Docking Studies. Comput. Biol. Med. 2025, 196, 110889. https://doi.org/10.1016/j.compbiomed.2025.110889.
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