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Research Article Pharmaceutics, Drug Delivery and Pharmaceutical Technology| Volume 110, ISSUE 3, P1292-1301, March 01, 2021

Probing Microenvironmental Acidity in Lyophilized Protein and Vaccine Formulations Using Solid-state NMR Spectroscopy

  • Author Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    Mingyue Li
    Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    Affiliations
    Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA
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  • Author Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    Sampada Koranne
    Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    Affiliations
    Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA
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  • Author Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    Rui Fang
    Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    Affiliations
    Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA
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  • Author Footnotes
    f Current address: Instrumentation and Service Center for Physical Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China.
    Xingyu Lu
    Footnotes
    f Current address: Instrumentation and Service Center for Physical Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China.
    Affiliations
    Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA
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  • Donna M. Williams
    Affiliations
    Vaccine Drug Product Development, Merck & Co., Inc, West Point, PA 19486, USA
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  • Eric J. Munson
    Affiliations
    Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, IN 47907, USA
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  • Akhilesh Bhambhani
    Correspondence
    Corresponding author.
    Affiliations
    Vaccine Drug Product Development, Merck & Co., Inc, West Point, PA 19486, USA
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  • Yongchao Su
    Correspondence
    Corresponding author. Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA.
    Affiliations
    Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA

    Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, IN 47907, USA

    Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA
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  • Author Footnotes
    e M.L., S.K., and R. F. have equal contributions.
    f Current address: Instrumentation and Service Center for Physical Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China.
Published:November 25, 2020DOI:https://doi.org/10.1016/j.xphs.2020.11.017

      Abstract

      Biophysical and biochemical instability of therapeutic proteins in the solution state may necessitate the development of products in the solid form, due to their enhanced stability. Lyophilization is a widely used method to ensure dry state stabilization of biological products. A commonly encountered issue is the pH shifts that can occur due to undesired crystallization of a buffer component, resulting in loss of protein activities. However, it is technically challenging to noninvasively investigate the physicochemical environment in the lyophile matrix. In this work, we demonstrate an approach based on solid-state NMR to investigate the microenvironmental acidity in lyophilized protein formulations, using histidine, a commonly used buffer agent, as a molecular probe. The solid-state acidity in the lyophilized matrix can be assessed by monitoring the chemical shift changes of histidine. The protonation and tautomeric states of histidine lyophilized at a range of pH values from 4.5 to 11.0 were identified from full 13C and 15N resonance assignments in one-dimensional and two-dimensional NMR experiments. The results demonstrated a pH-dependence of histidine chemical shift in the amorphous state. Moreover, we successfully applied this protocol to investigate the microenvironmental pH in lyophilized formulations of the HPV vaccine and lactate dehydrogenase protein.

      Keywords

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