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“Using O-PTIR allows significantly higher spatial resolution than traditional IR microscopy because the visible laser can be focused down to a submicron, diffraction limited spot. In addition, the visible laser can be used to generate Raman scattered light, which can be collected and analyzed using a Raman spectrometer. This allows simultaneous IR and Raman spectroscopy at the same time and location on the sample providing complementary chemical information.”
Reporting in Molecular Pharmaceutics, researchers at Purdue University and Merck & Co. investigated the critical relationship between excipient molecular weight and protein stability in solid-state biopharmaceutical formulations. Poor stability of biological products during processing and storage represents a major challenge facing the biopharmaceutical industry, with phase separation between proteins and stabilizing excipients often leading to reduced therapeutic efficacy and potential safety risks.
The research team employed Optical Photothermal Infrared (O-PTIR) spectroscopy as a key analytical technique to evaluate miscibility between bovine serum albumin (BSA) and various stabilizers including dextrans of different molecular weights, trehalose, and HPMC. O-PTIR analysis revealed distinct phase separation in BSA-HPMC films, with ratio imaging clearly distinguishing protein-rich domains from polymer-rich regions.
The technique’s submicron IR spatial resolution enabled precise mapping of heterogeneous distributions that would be impossible to detect with conventional IR microscopy. Complementary solid-state NMR measurements confirmed that BSA stability followed the order: trehalose ≥ Dex 2000 kDa > Dex 70 kDa > Dex 6 kDa > HPMC.
Furthermore, O-PTIR offers a number a key advantages over traditional direct IR (FTIR/QCL) based techniques. Beyond the 20-30x better spatial resolution (at submicron), O-PTIR provides for the ability to collect simultaneous IR+Raman spectra, from the same spot at the same time with the same resolution.
This was exploited in this study to provide for simultaneous IR+Raman hyperspectral maps, Additional, with O-PTIR operating primarily in reflection mode, but still delivering FTIR transmission/ATR-like spectral data, measurements of standard glass slides are now possible, with no glass spectral contributions or spectral range limitations.
Stability testing at accelerated conditions validated these miscibility findings, with formulations showing phase separation exhibiting significantly reduced protein monomer content after storage.
The researchers demonstrated that the relative size ratio between polymer and protein determines miscibility behavior, with larger polysaccharides forming protective meshes around protein molecules while smaller ones induce depletion-driven phase separation.
O-PTIR spectroscopy proved transformative for this pharmaceutical research by delivering unprecedented analytical capabilities for characterizing protein-excipient interactions.
The technique’s unique ability to achieve submicron spatial resolution through visible laser focusing, combined with simultaneous collection of both infrared absorption and Raman scattering data, provided comprehensive chemical mapping of phase-separated domains.
This dual-mode capability allowed researchers to construct detailed ratio images using characteristic peaks from both BSA and excipients, definitively confirming phase separation mechanisms.
The high spatial resolution was particularly crucial for analyzing thin film samples where traditional IR techniques lack sufficient sensitivity, making O-PTIR an indispensable tool for understanding fundamental stability mechanisms in biopharmaceutical formulations.
Authors: Hanh Thuy Nguyen, Mennatallah A. Mohamed, Jing Ling, Yong Du, Kevin Kjoller, Yongchao Su, and Lynne S. Taylor
- Hanh Thuy Nguyen, Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University
DOI: 10.1021/acs.molpharmaceut.4c01488
