Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)
- 707 Downloads
Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb).
Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies.
mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried.
The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.
KEY WORDSconformation interactions lyophilization monoclonal antibodies spray drying
Attenuated total reflectance
Bovine serum albumin
Controlled ice nucleation
Fourier transform infrared
Hydrogen deuterium exchange with mass spectrometric analysis
Rate of exchange of amide hydrogen atoms in the fast exchanging pool
Rate of exchange of amide hydrogen atoms in the slow exchanging pool
Number of amide hydrogen atoms in the fast exchanging pool
Number of amide hydrogen atoms in the slow exchanging pool
Powder x-ray diffraction
Scanning electron microscope
Solid-state; indicates that an analytical method has been applied to a solid sample
Solid-state photolytic labeling with mass spectrometric analysis
Uncontrolled ice nucleation
ACKNOWLEDGMENTS AND DISCLOSURES
The IgG1 mAb used in this study was provided by Pfizer, Inc. (Chesterfield, MO). This work was supported in part by a Purdue Research foundation grant and a McKeehan Graduate Fellowship awarded by Purdue University to EM.
- 9.Parker A, Rigby-Singleton S, Perkins M, Bates D, Le Roux D, Roberts CJ, et al. Determination of the influence of primary drying rates on the microscale structural attributes and physicochemical properties of protein containing lyophilized products. J Pharm Sci. 2010;99(11):4616–29.CrossRefPubMedGoogle Scholar
- 13.Moorthy BS, Iyer LK, Topp EM. Mass spectrometric approaches to study protein structure and interactions in lyophilized powders. J Vis Exp. 2015(98).Google Scholar
- 14.Moorthy BS, Zarraga IE, Kumar L, Walters BT, Goldbach P, Topp EM, Allmendinger A. Solid-state hydrogen–deuterium exchange mass spectrometry: correlation of deuterium uptake and long-term stability of lyophilized monoclonal antibody formulations. Mol Pharm. 2017. https://doi.org/10.1021/acs.molpharmaceut.7b00504.
- 16.Fu F, DeOliveria D, Trumble W, Sakar B, Singh B. Secondary structure estimation of proteins using the amide III region of Frourier transform infrared spectroscopy. Application to analyse calcium binding-induced structural changes in calsequestrin. Appl Spectrosc. 1994;48:1432–41.CrossRefGoogle Scholar
- 21.Chang L, Shepherd D, Sun J, Ouellette D, Grant KL, Tang XC, et al. Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix? J Pharm Sci. 2005;94(7):1427–44.CrossRefPubMedGoogle Scholar
- 29.Hsu CC, Nguyen HM, Yeung DA, Brooks DA, Koe GS, Bewley TA, et al. Surface denaturation at solid-void interface--a possible pathway by which opalescent particulates form during the storage of lyophilized tissue-type plasminogen activator at high temperatures. Pharm Res. 1995;12(1):69–77.CrossRefPubMedGoogle Scholar