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Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry

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ABSTRACT

Purpose

To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways.

Methods

Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H2 18O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20.

Results

The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via “acyl-cleavage” of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation.

Conclusions

The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

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Abbreviations

AAPH:

2,2′-azobis (2-amidinopropane) dihydrochloride

CMC:

Critical micelle concentration

Di:

Di-laurate

DIA:

Data-independent acquisition

EO:

Ethylene oxide

FFA:

Free fatty acid

Mi :

Polyoxyethylene isosorbide mono-laurate

Ml :

Polyoxyethylene mono-laurate

Mono:

Mono-laurate

Ms :

Polyoxyethylene sorbitan mono-laurate

POE:

Polyoxyethylene

PS:

Polysorbate

PS20:

Polysorbate-20

RIC:

Reconstructed-ion chromatogram

Tetra:

Tetra-laurate

Tri:

Tri-laurate

UPLC-MS:

Ultra-performance liquid chromatography mass spectrometry

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported by Genentech. We wish to thank Dr. Todd D. Williams of the KU Mass Spectrometry/Analytical Proteomics Laboratory for use of their instruments and guidance.

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Author notes

  1. Lin Zhang

    Present address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana, 46285, USA

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, 66047, USA

    Lin Zhang, Olivier Mozziconacci & Christian Schӧneich

  2. Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California, 94080, USA

    Sandeep Yadav, Barthélemy Demeule & Y. John Wang

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  1. Lin Zhang
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Corresponding author

Correspondence to Christian Schӧneich.

Electronic supplementary material

Fig. S1-S5 show supplemental MS/MS spectra of the Peak A6 ~ A8, the fronting/tailing of the Peak B2, and the Peak B6. Fig. S6 and S7 provid isotopic compositions of free lauric acids generated duiring hydrolysis and AAPH-initiated oxidation of all-laurate PS20 at 5°C. Fig. S8 shows the quantitative analysis of the amount of free lauric acid released from all-laurate PS20 at 40°C over 24 h.

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Zhang, L., Yadav, S., Demeule, B. et al. Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry. Pharm Res 34, 84–100 (2017). https://doi.org/10.1007/s11095-016-2041-y

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