Fourier Transform Infrared (FTIR) Spectroscopy

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Fourier Transform Infrared (FTIR) Spectroscopy

Method Introduction

Fourier transform infrared (FTIR) spectroscopy is a highly valuable technique for monitoring different characteristics of a biopharmaceutical both in the liquid and the dried states (e.g., in lyophilized state).

While traditionally FTIR spectra has been used for the identification of a wide range of compounds, by comparing the measured spectra to spectral databases, for proteins biopharmaceuticals its most extensive, but not only use, is in the analysis of the secondary structure.

FTIR spectra from wavenumbers 1,700 -1,500 cm-1 is sensitive to the protein conformation (secondary structure and conformational flexibility). Measuring protein absorbance over these wavenumbers gives two absorption bands, conventionally called Amide I and Amide II, lying between wavenumbers 1,700 – 1,600 cm-1 and 1,600 – 1,500 cm-1, respectively.

The Amide I band is due to C=O stretching vibrations of the peptide bonds, modulated by the secondary structure (α-helix, β-sheet, etc.) while the Amide II band is due to C-N stretching vibrations in combination with N-H bending.

Secondary structural content can be obtained by comparing the measured spectrum to those obtained for proteins with known secondary structures, and this type of analysis is done by using mainly the Amide I band.

But the FTIR spectrum is per se a fingerprint of a protein in a defined formulation, and it can be used, as whole, to evaluate both changes in secondary structure relative to a reference spectrum (e.g., reference listed product in biosimilar development; unstressed protein, in a stability study) by using the Amide I and II combined regions or changes in the full spectrum, for example in the glycosylation pattern between different protein batches.

Water can interfere with FTIR measurements of protein samples because it is strongly absorbed in the Amide I region. Consequently, FTIR is best suited for lyophilized (freeze-dried) protein samples. Nevertheless, measurements can be obtained for protein samples in solution, but a relatively high (typically >5 mg/mL) protein concentration is required. Both liquid (transmission, attenuated total reflection (ATR)) and solid (ATR) samples can be analyzed at Coriolis Pharma.

Applications

Fourier Transform Infrared (FTIR) Spectroscopy has several important application areas:

  • FTIR is widely used to study the secondary structure of proteins, including therapeutic monoclonal antibodies and other biopharmaceuticals. It can detect secondary structure elements like α-helices, β-sheets, turns, and random coils, providing protein folding and stability insights. It is frequently used in comparability or biosimilarity studies.

  • FTIR is employed to analyze protein-excipient interactions and compatibility during formulation development. It can detect changes in the protein structure upon adding excipients like sugars, salts, and surfactants, helping identify optimal formulation conditions.

  • Combined with Coriolis’ Particle ID service, FTIR spectroscopy attached to microscopy supports the identification and homogeneity assessment of unknown particles by their FTIR spectrum.

Quality and Biosafety Level

We provide all our analytical services with the highest quality standards. Experienced scientists carry out each project, and a scientific reviewer comprehensively checks every report or data presentation.

We offer this technology with the following quality and biosafety levels:

R&D level

We offer this method under R&D. Our GRP system assures the highest-quality research standards.

Up to biosafety level 1

This method can be applied to proteins, nucleic acids, and most viral vectors, including AAVs and more.

Fourier Transform Infrared (FTIR) Spectroscopy Frequently Asked Questions (FAQs)

  • FTIR spectroscopy is an analytical technique that measures the infrared absorption of molecules to determine their structural and chemical composition. For biopharmaceuticals it is particularly useful for analyzing the secondary structure of proteins by detecting characteristic absorption patterns in the Amide I and Amide II regions.

  • In the context of biologics and biopharmaceuticals, FTIR is employed for protein secondary structure analysis, comparability assessments, and formulation development. It can detect subtle conformational changes and evaluate protein-excipient interactions during formulation screening.

  • The Amide I band (1,700–1,600 cm⁻¹) is primarily due to the peptide bonds C=O stretching and the Amide II band (1,600–1,500 cm⁻¹) arises from C-N stretching and N-H bending. Both bands are sensitive to protein conformation. While the Amide I is more sensitive and generally used to monitor secondary structure changes the Amide II can be useful whenever bands, due to the formulation components (i.e., excipients), overlap with the Amide I band.

  • Yes, but FTIR analysis in solution requires higher protein concentrations (typically >5 mg/mL) and careful consideration of water interference in the Amide I region. Coriolis has experience in developing this kind of measurement and offers both liquid and solid-state (e.g., lyophilized) FTIR measurements using transmission and ATR (attenuated total reflection) modes.

  • Coriolis applies FTIR to a wide range of biologic materials, including monoclonal antibodies, proteins, peptides, viral vectors like AAVs, and nucleic acids. The method is available for R&D studies under BSL-1 conditions.

  • Yes. FTIR spectroscopy is frequently used in biosimilarity assessments to compare or the secondary structure of a biosimilar product or the full spectrum (fingerprint) to its reference biologic. It provides a sensitive and orthogonal method for confirming structural consistency.

  • FTIR can monitor protein structural stability in the presence of excipients such as sugars, salts, or surfactants. It helps to identify optimal formulation conditions by revealing protein unfolding or aggregation under stress.

  • Yes. When combined with microscopy, FTIR can generate a chemical fingerprint of unknown particles. This is particularly helpful in root cause analysis and supports Coriolis’ Particle Identification service.

  • FTIR spectroscopy is performed at the R&D level under a Good Research Practice (GRP) system. Each study is executed and reviewed by experienced scientists to ensure high-quality, reliable data.

  • Coriolis offers expert-guided FTIR analysis integrated with other orthogonal techniques to provide a comprehensive understanding of higher-order structure and formulation behavior. Our flexible sample formats and focus on protein science make us a trusted partner in biopharmaceutical development.

Analytical Method Development, Qualification and Validation

For common sample types, we can often apply standardized methods with little setup effort. However, when needed, our experienced analytical experts create or optimize custom methods tailored to your active pharmaceutical ingredient, product type and development phase.

Method Development

Our method development approach tailors sample preparation, method settings and data analysis to the needs of your project and sample.

We include representative samples and, where available, suitable reference standards and stressed/degraded materials, allowing our analytical scientists to design a highly suitable, stability-indicating, robust and repeatable method. Upon request, we will compile a detailed description of the method for your records.

Method Qualification

Method qualification is the initial assessment of an analytical procedure’s performance to show its suitability for its intended purpose.

During method qualification, our analytical scientists perform documented testing demonstrating that the analytical procedure meets criteria in several categories. Criteria may include factors such as repeatability, specificity and robustness. We compile a qualification plan and report, including all relevant data.

Method Validation

Under GMP conditions, method validation confirms that an analytical procedure’s performance suits its intended purpose. Depending on the method’s scope, a broad range of method characteristics, such as specificity, accuracy, precision, limit of detection/limit of quantification (LOD/LOQ), linearity and range, is considered.

During method validation, our analytical scientists perform documented testing demonstrating that the analytical procedure consistently produces a result that meets the predetermined acceptance criteria. We compile a validation plan and report that includes all relevant data.

Depending on the development phase, a fit-for-purpose validation approach can be offered, adjusting the validation required efforts in a phase-appropriate way to meet the method’s needs.

Method Verification

Compendial method verification confirms that a compendial method (e.g., from Ph. Eur. or USP) is suitable and reliable for its intended purpose under the specific conditions of the laboratory.

Unlike full method validation, compendial method verification is often considered a partial validation since the method has already undergone extensive testing and validation during its inclusion in the compendium. The extent of method verification depends on the type of method.

During method verification, our analytical scientists perform documented testing demonstrating that the developed analytical method performs adequately for the specific product or matrix being tested and within the specific laboratory where the method will be employed.

Talk to Our Experts or Request a Quote

Our expert team is ready to answer your questions and guide you to the services best suited to your program’s modality, stage and challenge. If your needs are well-defined, we’ll begin the quotation process.

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