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• W1987448934 endingPage "866" @default.
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• W1987448934 abstract "For the quantification of therapeutic monoclonal antibodies in biological specimens, enzyme-linked immunosorbent assay (ELISA) is the most widely used technique. ELISA's have some limitations and therefore alternative analytical techniques are being explored. In this study we describe the development of a bioanalytical assay using high-performance liquid chromatography (HPLC) coupled with fluorescence detection for the bioanalysis of the monoclonal antibody trastuzumab. Different extraction procedures were explored, like isolation using protein A and protein G. Finally a method using immuno-affinity purification has been developed. Trastuzumab is isolated from human serum using sepharose coupled with anti-trastuzumab idiotype antibodies. After extraction samples are injected onto a Zorbax 300SB C8 column at 75 °C using the organic solvents isopropanol and acetonitrile with high eluotropic strengths. The assay quantifies trastuzumab from 5 to 40 μg/mL in human serum with accuracies <20%. Samples with concentrations above the upper limit of quantification (>ULOQ; >40 μg/mL) can be diluted 5 times with control human serum prior to sample pre-treatment. The assay can now be used to analyse serum samples of patients treated with trastuzumab. The obtained results are comparable to those obtained using ELISA. This is the first report describing a bioanalytical assay using HPLC and fluorescence detection for the quantification of a monoclonal antibody at the intact protein level in human serum. This unique approach has the advantage compared to ELISA that a HPLC separation step is introduced to improve the selectivity. This method is a potential alternative to ELISA to support pharmacokinetic evaluations. However, for purification of trastuzumab from serum anti-idiotype antibodies are necessary. These anti-idiotype antibodies are also used in ELISA and as ELISA is more sensitive and less labor-intensive, ELISA probably remains the analytical technique of first choise." @default.
• W1987448934 created "2016-06-24" @default.
• W1987448934 creator A5000418012 @default.
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• W1987448934 date "2009-12-01" @default.
• W1987448934 modified "2023-10-16" @default.
• W1987448934 title "The bioanalysis of the monoclonal antibody trastuzumab by high-performance liquid chromatography with fluorescence detection after immuno-affinity purification from human serum" @default.
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• W1987448934 doi "https://doi.org/10.1016/j.jpba.2009.04.031" @default.
• W1987448934 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19487098" @default.
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