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High-Sensitivity IR Spectroscopy of Proteins


New biological drugs such as peptide drugs, antibody drug conjugates, 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.


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. Extensive studies on FT-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. In addition to applying the SAC-IR spectroscopy to various applications, we are working to improve the sensitivity and expand the frequency range and increase the acquisition speed to meet the critical needs of protein characterization.

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


CL-IR spectroscopy system [1].
) IR spectra for mixture solutions of BSA and NISTmAb
Figure 2. (a) IR spectra for mixture solutions of BSA and NISTmAb at a total concentration of 2 mg/mL. (b) Classical least squares (CLS) decomposition of a binary protein mixture (0.4 mg/mL BSA and 1.6 mg/mL NISTmAb) using two absorption spectra of the pure BSA and NISTmAb solutions. The bottom plot is the residual of the mixture spectra after the CLS decomposition. (c) CLS analysis results of the mixture solutions [1].


[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 December 7, 2022