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Characterization of Grinding-Induced Subvisible Particles and Free Radicals in a Freeze-Dried Monoclonal Antibody Formulation

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Abstract

Purposes

The primary objectives of this study were to investigate the degradation mechanisms of freeze-dried monoclonal antibody (mAb) formulations under mechanical grinding, assess the sensitivity and suitability of various particle analysis techniques, analyze the structure of the collected subvisible particles (SbVPs), and analyze the antioxidant mechanism of methionine (Met) under degradation process to gain a thorough understanding of the phenomenon.

Methods

The freeze-dried mAb-X formulations underwent grinding, and the resultant SbVPs were characterized through visual inspection, flow imaging microscopy, dynamic light scattering, ultraviolet–visible spectroscopy, and size-exclusion high-performance liquid chromatography. We further evaluated the effect of different temperatures and the free radical scavenger Met on SbVP formation. The produced free radicals were detected using electron paramagnetic resonance, and Met S-oxide formation was detected using liquid chromatography–mass spectrometry. In addition, we analyzed the obtained SbVPs using capillary electrophoresis sodium dodecyl sulfate and Fourier transform infrared spectroscopy.

Results

Grinding leads to SbVP formation under high temperature and free radical formation. Free radicals produced during grinding require the participation of a macromolecule. Met could then bind to the produced free radicals, thus partially protecting mAb-X from degradation while itself undergoing oxidation to form Met(O). Sensitivity differences between different particle analysis techniques were evaluated, and the obtained SbVPs showed significant changes in secondary structure and the formation of covalent aggregates and fragments.

Conclusions

Met plays the role of an antioxidant in protecting macromolecules by quenching the free radicals produced during grinding. To thoroughly characterize SbVPs, multiple and orthogonal particle analysis techniques should be used, and if necessary, SbVPs should be processed by enrichment to accurately analyze primary and high order structures.

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Abbreviations

CD:

circular dichroism

CE-SDS:

capillary electrophoresis sodium dodecyl sulfate

DLS:

dynamic light scattering

EPR:

electron paramagnetic resonance

FIM:

flow imaging microscopy

FT-IR:

Fourier transform infrared spectroscopy

LC–MS:

liquid chromatography–mass spectrometry

mAb:

monoclonal antibody

Met:

methionine

Met(O):

methionine S-oxide

OD:

optical density

PES:

polyethylstyrene

SbVP:

subvisible particle

SE-HPLC:

size-exclusion high-performance liquid chromatography

TIC:

total ion chromatogram

UV–Vis:

ultraviolet–visible spectroscopy

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ACKNOWLEDGMENTS AND DISCLOSURES

We appreciate the National Natural Science Foundation of China (Grant No. 81741144) and the Ministry of Science and Technology of China (Grant No. 2018ZX09J18107–002) for their financial support. We also thank Ms. Xinyu Wang at Zhejiang University for performing the EPR experiments and Zhejiang Bioray Biopharmaceutical for providing us with mAb-X used for this study. Special thanks to Fluid Imaging Technologies for providing access to their FlowCam 8100. The authors declare that they have no known competing financial or personal interest conflict.

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Authors and Affiliations

  1. Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

    Zhen-Yi Jing, Guo-Li Huo, Min-Fei Sun, Bin-Bin Shen & Wei-Jie Fang

  2. Hangzhou Institute of Innovative Medicine, Zhejiang University, Hangzhou, 310016, China

    Zhen-Yi Jing, Guo-Li Huo, Min-Fei Sun, Bin-Bin Shen & Wei-Jie Fang

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  1. Zhen-Yi Jing
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Jing, ZY., Huo, GL., Sun, MF. et al. Characterization of Grinding-Induced Subvisible Particles and Free Radicals in a Freeze-Dried Monoclonal Antibody Formulation. Pharm Res 39, 399–410 (2022). https://doi.org/10.1007/s11095-022-03170-9

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