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High-Sensitivity IR Spectroscopy

Summary

New biological drugs such as peptide drugs and vaccines are emerging to fill pharmaceutical voids. They are typically prepared in water-based buffers at lower concentrations (0.1 mg/mL to 10 mg/mL) with less structural stability than other biologicals (e.g., monoclonal antibodies). However, commonly used infrared (IR) spectroscopy cannot characterize those low concentration drugs due to the interference by strongly absorbing water. We are developing a new IR-based approach that can enhance the sensitivity to quantify these drugs in their final preparations.

Description

Infrared (IR) absorption spectroscopy has been used widely as a non-invasive, label-free characterization method for complex biomolecules’ chemical identification and structure. Fourier-transform IR (FT-IR) technology is commonly used to characterize proteins and other biological molecules produced during biopharmaceutical processes. FT-IR is advantageous because it does not require additional sample preparation steps, such as buffer exchange and dilution, which are needed for other techniques like far-ultraviolet (UV) circular dichroism (CD) spectroscopy.

Extensive studies on IR spectroscopy of proteins have shown that the amide bands can be used to semi-quantitatively characterize the secondary structure (e.g., a-helix and b-sheet) of the protein backbone conformation. However, the interference by strong water absorption keeps FT-IR from characterizing low concentration samples of <10 mg/mL and prevents the usage of a long-pathlength optical cell. Non-invasive optical characterization of low-concentration protein samples is critical for new types of drugs in their original formulation conditions.

Recently, we have developed a new optical technique for quantum cascade laser (QCL)-based mid-IR absorption spectroscopy [1]. By the patented solvent absorption compensation (SAC) technique [2], we can detect the amide I band of a protein solution with 100 times better sensitivity compared to conventional mid-IR spectroscopy. We are working to not only improve the sensitivity but also expand the frequency range and increase the acquisition speed.

Comparison of IR spectra acquired by conventional and solvent absorption compensation (SAC) IR approaches
Figure 1. Comparison of IR spectra acquired by conventional and solvent absorption compensation (SAC) IR approaches. The sample was a bovine serum albumin (BSA, 10 mg/mL) solution with a path length of 25 mm using a home-built QCL-IR spectroscopy system [1].

PUBLICATIONS

[1] B. Chon, S. Xu, Y. J. Lee, Compensation of Strong Water Absorption in Infrared Spectroscopy Reveals the Secondary Structure of Proteins in Dilute Solutions. Anal. Chem. 93, 2215 (2021).

[2] Y. J. Lee, Spectrum Adjuster and Producing a Pure Analyte Spectrum. US Patent Appl. No. 16,164,859. (2018).

Created May 15, 2019, Updated June 2, 2022