Fourier-Transform Infrared Spectroscopy
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Fourier-Transform InfraRed spectroscopy (FTIR)
Secondary structure composition is a core quality attribute for biopharmaceutical proteins. Changes in alpha-helix, beta-sheet, or random coil content can indicate misfolding, aggregation onset, or process-induced structural perturbation, none of which are visible by size or activity assays alone. FTIR provides a direct, label-free readout of these structural features through the analysis of characteristic amide band vibrations, requiring no chemical modification and no optical clarity.
How FTIR Works
FTIR measures the absorption of infrared light by a sample across a broad range of wavelengths simultaneously.
A broadband IR source passes through a Michelson interferometer (a configuration of fixed and moving mirrors), which modulates each wavelength at a different frequency as the moving mirror displaces. The resulting beam encodes all measured frequencies at once before reaching the sample.
After interaction with the sample, the transmitted signal is collected by a detector. Fourier transformation converts the raw interferogram into an absorbance spectrum expressed in wavenumber (cm⁻¹), where each peak corresponds to the absorption of IR energy by a specific molecular bond vibration.
The measurement follows a two-step sequence. A background spectrum is first recorded without sample, then repeated with sample present. The sample spectrum is divided by the reference to yield the final transmission spectrum, corrected for instrument and atmospheric contributions.
The key advantage of the Fourier transform approach is multiplexing: all wavelengths are acquired in a single pass, improving signal-to-noise ratio, wavenumber accuracy, and throughput relative to dispersive instruments.
Protein Secondary Structure Analysis
FTIR amid bands The FTIR spectrum of a protein contains multiple amide bands arising from vibrational modes of the peptide backbone. Three are analytically relevant for secondary structure assignment.
The Amide I band is the primary region used for secondary structure quantification. Peak position and shape within this band are directly correlated to helical, sheet, turn, and disordered content. Second derivative and deconvolution analyses resolve overlapping sub-components, allowing quantitative estimation of secondary structure fractions.
Applications
Secondary structure quantification
Determination of alpha-helix, beta-sheet, beta-turn, and disordered content from Amide I band analysis. Provides a complementary, solution-phase measurement to CD spectroscopy, without requiring UV-transparent buffers or low-turbidity samples.
Comparability and biosimilarity
FTIR thermograms and spectral profiles are used as orthogonal structural evidence in ICH Q5E and ICH Q6B comparability packages and biosimilar regulatory submissions. Changes in secondary structure introduced by manufacturing process modifications, cell line changes, or formulation shifts are detectable at the band level.
Thermal stability and structural integrity
Temperature-resolved FTIR experiments track changes in Amide I band shape as a function of temperature, enabling detection of heat-induced unfolding and aggregation. Beta-sheet aggregates produce a characteristic band near 1620 cm⁻¹, distinct from native beta-sheet contributions.
Formulation and excipient screening
FTIR is compatible with a wide range of excipients and buffers at the concentrations used in pharmaceutical formulations. It can be used to detect excipient-induced structural changes and to confirm structural integrity across pH and ionic strength ranges.
Technical Notes
- No labelling, no optical probes required. The measurement is non-destructive and does not require any chemical modification of the sample.
- Compatible with turbid solutions: no optical clarity constraints on sample preparation.
- A buffer-matched reference is mandatory. Background subtraction is critical to isolate the protein signal from solvent contributions.
- All spectral data is acquired in a single pass, covering the full wavenumber range simultaneously. This ensures fast acquisition and consistent signal quality across experiments.
Regulatory Context
FTIR is recognized under ICH Q6B as a method for physicochemical characterization of biotherapeutics, covering secondary structure and conformational integrity. FTIR data is routinely included in IND and BLA submissions as part of the structural characterization package and is expected in biosimilar dossiers by both FDA and EMA, alongside other orthogonal methods.
One CRO, coordinated data
FTIR is part of CovalX’s biophysical characterization offering. It complements the MS-based services available in-house across the same molecule and the same sample.
Running biophysical and structural analyses through a single CRO reduces sample quantity requirement, allows coordinated experimental design, consistent sample handling, and consolidated reporting. For regulatory packages requiring orthogonal characterization data from multiple methods, that continuity matters.