Assessment of HPLC-FLD Method for Determination of Trastuzumab

Objective & Hypothesis:Trastuzumab (150kDa) is a monoclonal antibody (mAbs), which is directed against the human epidermalgrowth factor receptor-2 (HER2) and is used in patients with metastatic breast cancer treatment.

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Key Findings & Data:Present study describes a bioanalytical method for the totalTrastuzumab determination using high-performance liquid chromatography (HPLC) coupled with fluorescencedetection (FLD).

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Conclusion & Impact:The applicability of the method for the pharmacokinetic studieswas discussed.

🔬Design:Research Article

📍Scope:Comprehensive Sample

⚙️Variables:Domain Parameters

Abstract

Trastuzumab (150kDa) is a monoclonal antibody (mAbs), which is directed against the human epidermalgrowth factor receptor-2 (HER2) and is used in patients with metastatic breast cancer treatment. In order to usethese biosimilar drugs in therapy, bioequivalence studies should be performed and the success of these studiesdepends on the reliability of the analytical method. Present study describes a bioanalytical method for the totalTrastuzumab determination using high-performance liquid chromatography (HPLC) coupled with fluorescencedetection (FLD). Trastuzumab was isolated from rat serum using protein G column and eluted with glycine andthen, the eluent was injected into HPLC system where the excitation and emission wavelengths were set at 278and 343 nm, respectively. Experimental parameters namely the flow rate, type of analytical column, type andcomposition of mobile phase, and the eluent used in sample preparation step were optimized by considering thepeak resolution and recovery percentages. Under optimal conditions, a calibration graph was constructed andthe detection limit was calculated as 5.3 µg/mL. The applicability of the method for the pharmacokinetic studieswas discussed.

Özet

Trastuzumab (150kDa), insan epidermal büyüme faktörü reseptörü-2'ye (HER2) karşı yönlendirilen ve metastatik meme kanseri tedavisi gören hastalarda kullanılan bir monoklonal antikordur (mAb). Bu biyobenzer ilaçların tedavide kullanılabilmesi için biyoeşdeğerlik çalışmalarının yapılması gereklidir ve bu çalışmaların başarısı da analitik yöntemin güvenilirliğine bağlıdır. Bu çalışmada, fluoresans dedektörü (FLD) ile birleştirilmiş yüksek performanslı sıvı kromatografisi (HPLC) kullanarak toplam Trastuzumab analizi için bir biyoanalitik yöntem tanımlanmıştır. Trastuzumab, protein G kolonu kullanılarak sıçan serumundan izole edildikten sonra glisinle elüe edilerek, elüent, uyarma ve emisyon dalga boylarının sırasıyla 278 ve 343 nm'ye ayarlandığı HPLC sistemine enjekte edilmiştir. Akış hızı, kolon türü hareketli faz türü ve bileşimi ve örnek hazırlama aşamasında kullanılan elüent gibi deneysel parametreler, piklerin ayrılması ve geri kazanım yüzdeleri dikkate alınarak optimize edilmiştir. Optimum koşullar altında kalibrasyon grafiği oluşturulmuş ve belirtme alt sınırı 5.3 µg/mL olarak hesaplanmıştır. Bu yöntemin farmakokinetik çalışmalar için uygulanabilirliği tartışılmıştır.

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Cite This Article

ENGİN, E., ERTAŞ, H., KARASULU, E. (2022). Assessment of HPLC-FLD Method for Determination of Trastuzumab. Erzincan Üniversitesi Fen Bilimleri Enstitüsü Dergisi, Cilt: 12 - Sayı: 1, 517–526. https://ajindex.com/articles/505475

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References (3)

Damen et al., 2009a. Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma, Damen, C.W.N., de Groot, E.R., Heij, M., Boss, D.S., Schellens, J.H.M., Rosing, H., Beijnen, J.H., Aarden, L.A., Analytical Biochemistry, 391, 114–120 Damen et al., 2009b. The bioanalysis of the monoclonal antibody trastuzumab by high-performance liquid chromatography with fluorescence detection after immuno-affinity mpurification from human serum, Damen, C.W.N., Derissena, E.J.B., Schellensb, J.H.M., Rosinga, H., Beijnena, J.H., Journal of Pharmaceutical and Biomedical Analysis, 50, 861–866. Elgundi et al., 2017. The state-of-play and future of antibody therapeutics, Elgundi, Z., Reslan, M., Cruz, E., Sifniotis, V., Kayser, V., Advanced Drug Delivery Reviews, 122, 2–19 FDA, 2018. https://www.usa.gov/federalagencies/food-and-drug-administration Access date: 13.12.2018 Geigert, 2013.
Biologics Are Not Chemical Drugs, The Challenge of CMC Regulatory Compliance for Biopharmaceuticals and Other Biologics, J. Geigert, Springer Science+Business Media New York Iwamoto et al., 2015. The development of the validated LCMS bioanalysis of trastuzumab in human plasma using a selective detection method for complementarity determining regions of monoclonal antibodies: nano-surface and molecular-orientation limited (nSMOL) proteolysis, Iwamoto, N., Yamane, N., Umino, Y., Hamada, A., Shimada, T., Anal. Methods, 2015, 7, 9177 Kurt et al., 2013. Complementary/alternative therapies in patients with breast cancer, Kurt, H., Keskek, Ş.Ö., Cil, T., Canataroglu, A., Turkish Journal of Oncology, 28(1):10-15 Liu, 2014. The history of monoclonal antibody development - Progress, remaining challenges and future innovations, Liu, J.K.H., Annals of Medicine and Surgery, 3, 113-116 Merino et al., 2011. Monoclonal antibodies. Basic features, Merino, A.G., Neurología. 26(5):301-306 Nacak et al., 2012.
Biosimilar Drugs, Nacak, M., Sezer, Z., Erenmemişoğlu, A., Clin Anal Med; 3(2): 251-256 Öztop, 2007. The Use of Trastuzumab in Adjuvant Setting for Early Stage Breast Cancer, Öztop, İ., International Journal of Hematology and Oncology, 3(17):184-192 Park et al, 2015. Qualification and application of a liquid chromatography–quadrupole time-offlight mass spectrometric method for the determination of trastuzumab in rat plasma, Park, M.H., Lee, M.W., Shin, Y.G., Biomed. Chromatogr., 30(4):625-631. Rebello and Hale, 2002. Pharmacokinetics of CAMPA