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Accelerated Forced Degradation of Therapeutic Peptides in Levitated Microdroplets

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Abstract

Purpose

Forced degradation is critical to probe the stabilities and chemical reactivities of therapeutic peptides. Typically performed in bulk followed by LC-UV or LC-MS analysis, this traditional workflow consists of a reaction/analysis sequence and usually requires half a day to several days to form and measure the desired amounts of degradants. A faster method is needed to study peptide degradation in a shorter time in order to speed up the drug development process.

Methods

In the new rapid method developed in this study, peptide degradation occurs in levitated aqueous microdroplets using the Leidenfrost effect.

Results

This two-minute reaction/analysis workflow allows major degradation pathways of Buserelin, Octreotide, Desmopressin and Leuprorelin to be studied. The reactions include deamidation, disulfide bond cleavage, ether cleavage, peptide bond hydrolysis, and oxidation.

Conclusions

The accelerated forced degradation method requires a minimal amount of therapeutic peptide per stress condition, and the appropriate extent of degradation can be readily generated in seconds by adjusting the droplet levitation time. Levitated microdroplets should be applicable in pharmaceutical development to rapidly determine the intrinsic stability of therapeutic peptides and to aid formulation development by screening the effects of excipients on the stability of the peptides.

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Abbreviations

LC:

liquid chromatography

UHPLC:

ultra-high-performance LC

MS:

mass spectrometry

nESI:

nanoelectrospray ionization

MS/MS:

tandem mass spectrometry

CID:

collision-induced dissociation

CE:

collision energy

HRMS:

high-resolution mass spectrometry

API:

active pharmaceutical ingredient

RA:

relative abundance

ODB1 :

primary degradant of Octreotide under basic conditions

ODB2 :

secondary degradant of Octreotide under basic conditions

B1 :

Buserelin-NHEt

B2 :

Buserelin-NHNH2

B1DA :

degradant of Buserelin-NHEt under acidic conditions

B2DA :

degradant of Buserelin-NHNH2 under acidic conditions

DDA1(a/b) :

primary deamidation degradants of Desmopressin under acidic conditions

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Acknowledgments

We acknowledge financial support of Merck Sharp & Dohme Corp. a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, under a Master Agreement with Purdue University on Chemical Instrumentation (Project number LKR174368). We also acknowledge the financial support from the Defense Advanced Research Projects Agency (Award number W911NF-16-2-0020) and from the National Science Foundation (Grant number CHE-1905087). Yangjie Li acknowledges support by the Henry Bohn Hass Memorial Fellowship. We thank Randall Replogle of the Jonathan Amy Facility for Chemical Instrumentation for teaching precision machining and Fan Pu for help in machining the metal dice. Finally, we thank Dr. Craig Parish and Dr. Daniel Smith from Merck Research Laboratories for helpful discussions.

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Authors and Affiliations

  1. 560 Oval Drive, Department of Chemistry, Purdue University, West Lafayette, IN, 47907-1393, USA

    Yangjie Li, Yanyang Hu, David L. Logsdon & R. Graham Cooks

  2. Dept. of Analytical Sciences, MRL, Merck & Co., Inc., 126 East Lincoln Ave, Rahway, NJ, 07065, USA

    Yong Liu & Yuejie Zhao

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  1. Yangjie Li
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  2. Yanyang Hu
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  3. David L. Logsdon
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  4. Yong Liu
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  6. R. Graham Cooks
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Correspondence to R. Graham Cooks.

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Li, Y., Hu, Y., Logsdon, D.L. et al. Accelerated Forced Degradation of Therapeutic Peptides in Levitated Microdroplets. Pharm Res 37, 138 (2020). https://doi.org/10.1007/s11095-020-02868-y

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