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Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS

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Abstract

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor’s aqueous environment, and vitreous humor’s viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200 µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques.

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Acknowledgements

The authors would like to thank Virginia Commonwealth University School of Pharmacy and Pharmaceutical Product Development’s Bioanalytical Laboratories in Richmond, VA, for their academic expertise. The authors would also like to extend a special thanks to Diane Lebarbenchon for her help in grammatical and formatting editing.

Funding

This study was supported by Virginia Commonwealth University School of Pharmacy and Pharmaceutical Product Development’s Bioanalytical Laboratories in Richmond, VA.

Rabbit vitreous humor, human vitreous humor, and human aqueous humor were collected ethically within FDA standards.

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Authors and Affiliations

  1. Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, USA

    Catherine E. DelGuidice & Matthew S. Halquist

  2. PPD Laboratories, Richmond, VA, USA

    Catherine E. DelGuidice, William R. Mylott Jr & Moucun Yuan

  3. Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt

    Omnia A. Ismaiel

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  1. Catherine E. DelGuidice
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Correspondence to Catherine E. DelGuidice.

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DelGuidice, C.E., Ismaiel, O.A., Mylott, W.R. et al. Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS. Anal Bioanal Chem 414, 4189–4202 (2022). https://doi.org/10.1007/s00216-022-04071-x

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  • DOI: https://doi.org/10.1007/s00216-022-04071-x

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