Glucagon-like peptide 1 receptor agonists (GLP‑1RAs) have changed how type 2 diabetes and weight loss are treated. The success of drugs like liraglutide and semaglutide means global demand is rising fast, putting manufacturers under pressure to maintain high quality, purity and stability for these products.

As peptide therapeutics, GLP-1RAs present characterisation challenges for quality assurance (QA) and quality control (QC) operations.1 These high-value biopharmaceuticals are prone to degradation and aggregation, which can potentially reduce their therapeutic efficacy and may trigger unwanted immune responses.1–3 Thus, structural integrity is crucial for safety, effectiveness and regulatory compliance.

Identifying impurities and aggregates during production, especially during scale-up and manufacturing, requires high sensitivity and specificity. Standard analytical approaches like LC‑MS and other chromatographic methods are effective for measuring chemical purity but lack the ability to directly assess molecular conformation.

This is where infrared spectroscopy, and particularly attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), can deliver meaningful insights. Although its ability to analyse protein secondary structure is well established,4,5 ATR-FTIR remains surprisingly underutilised in biopharmaceutical settings,6 despite its effectiveness for studying protein folding and aggregation.

This article explores how ATR-FTIR could enhance drug identity, stability and purity assessment of GLP-1RAs, and how it could give R&D leads, QA/QC scientists and researchers a practical way to simplify and speed up analytical workflows in this fast-moving space.

ATR-FTIR: A powerful lens on peptide structure

When applied to proteins and peptides, FTIR spectroscopy can reveal details about their secondary structure. Recent studies have found that FTIR results match well with circular dichroism (CD) spectroscopy,7 which is widely used for secondary structure determination but requires specialist equipment and highly purified samples, making it incompatible with many common excipients and buffers.6

This makes FTIR an attractive, easy-to-use and cost-effective alternative for peptide drugs in pharmaceutical settings, where tightly monitoring structural fidelity is critical for safety, efficacy and compliance.

What makes FTIR ideal for GLP-1RAs?

  • Structure-sensitive spectral bands: The amide I (1,700–1,600 cm−1) and amide II (1,600–1,500 cm−1) bands are highly sensitive to peptide backbone conformation. Variations in position and shape provide detailed structural information on α-helix, β-sheet and random coil content.5,8
  • Versatile sample handling: A label-free, non-destructive analysis method that works equally well with aqueous, lyophilised or crystalline peptide samples.
  • Rapid with low input requirements: Scan times are typically under 2 minutes and high-quality data can be generated from 10 µL sample volume, a key advantage when only minimal material is available.
  • Ideal for monitoring aggregation: The amide I band allows discrimination between β-sheet conformations, making FTIR particularly suited to detecting aggregates.4,9

ATR-FTIR: An ideal approach for biopharmaceuticals and complex matrices

Traditional transmission FTIR involves laborious sample preparation and is hampered by strong absorbance of water, which overlaps the crucial amide I band.6,8

To avoid this, peptide and protein analysis typically uses the ATR sampling mode. An IR beam is internally reflected in a high refractive index crystal, generating an evanescent wave that scans a thin layer of sample (<5 µm) adjacent to the crystal surface.6,8 This shortens the path length to reduce water interference, and buffer subtraction can further improve the signal.7 Data processing is then performed to extract secondary structural characteristics, which may include:

  • Second-derivative analysis: A common approach that separates overlapping amide I features to enhance resolution.5
  • Fourier self-deconvolution (FSD): Sometimes used as an alternative method of spectral deconvolution.5,10
  • Gaussian curve fitting: Resolves component peaks (including height, bandwidth and position) of the amide I/II bands to permit structural assignment.5,11
  • Quantitative comparison: Peak areas are translated into structural features, such as α-helix, β-sheet and random coil percentages, or α-helix/β-sheet ratio.

Peptide characterisation: Deeper insights and industry evidence

In pharmaceutical QA/QC workflows, analytical tools must be able to assess structural integrity. This is especially important for GLP-1RAs, where aggregation can lower bioavailability and compromise the drug’s therapeutic potency.1,12

While ATR-FTIR can readily detect changes in secondary structure of peptides and proteins, including impurities and aggregates,4,13 it is still rarely used in QA/QC pipelines due to entrenched reliance on chromatographic techniques.6 Nevertheless, several case studies show why closely monitoring structural integrity is important for GLP-1RAs, and demonstrate the value of ATR-FTIR in process development and manufacturing workflows.

1. Liraglutide aggregation and pH sensitivity

One study showed assembly of liraglutide into micelles of different sizes depending on pH, which can affect pharmacokinetic properties and potentially require formulation adjustments.12

  • Structural changes were tracked using light scattering and CD spectroscopy.
  • ATR-FTIR could complement these methods by revealing changes in β-sheet structure linked to oligomerisation, without requiring demanding sample preparation steps like desalting or buffer exchange.

2. Enhanced self-association of lipidated GLP-1RAs

Other researchers investigated the dynamic aggregation behaviour of native GLP-1 forms and two lipidated analogues, liraglutide and semaglutide. They found that the two peptide drugs form larger and more stable oligomers,14 highlighting the need to monitor aggregation during manufacturing and formulation of lipidated GLP-1RAs.

  • These researchers followed tracked different oligomerisation pathways using CD spectroscopy, size exclusion chromatography (SEC) and thioflavin T fluorescence.
  • ATR-FTIR would be ideal for characterising aggregation kinetics, considering the process was associated with increasing β-sheet content.

3. Aggregation triggered by trace metal contamination

A cross-batch analysis of liraglutide and semaglutide from various manufacturers found that trace metal impurities, especially Fe2+ and Cu2+, can promote the formation of oligomers,15 raising concerns about the stability and safety of the drug product.

  • This study used mass spectrometry, thioflavin T fluorescence and chromatographic techniques.
  • ATR-FTIR could add value as an early screening tool for oligomerisation, by measuring β‑sheet content to flag aggregation-prone batches.

These examples show how ATR-FTIR could provide useful structural insights for pharmaceutical scientists working in QA/QC, analytical R&D or formulation development roles, without adding much time or cost. However, there are still some limitations that could slow down wider adoption.

What are the key limitations of peptide ATR-FTIR?

  • Lower structural resolution than techniques like NMR, but much quicker, cheaper and easier to use in QA/QC labs.
  • Requires expertise in spectral deconvolution to extract structural information.
  • Water absorption interference, though this is usually managed by using a matching buffer for background scans, even during real-time in situ measurements.7
  • Sensitivity limit, where signal quality drops below 1 mg/mL.8,16

Overcoming sensitivity constraints with ConcentratIR2

While the above challenges are generally manageable, ATR-FTIR requires protein samples in the mg/mL range8 and a lower limit of ~0.7 mg/mL has been suggested for antibodies.16 However, some finished drug products contain proteins at much lower concentration, which makes analysis more difficult.

This limitation is largely overcome by high-performance ATR accessories like the Harrick ConcentratIR2™ Multiple Reflection ATR Accessory, which incorporates a 23-bounce silicon ATR crystal to greatly enhance signal-to-noise ratio. As demonstrated in this Application Note, resolvable amide I/II bands were obtained at very low concentrations for two injectable peptide drugs, ziconotide (25 µg/mL) and calcitonin (33 µg/mL), using a commercial FTIR spectrometer (Figure 1). At higher concentration (5 mg/mL), the peptide drug leuprolide acetate gave excellent quality spectra in the amide I/II region.

Figure 1: Post-Subtraction Infrared Spectra of Peptide Therapeutics Showing Amide I and II Bands Using the ConcentratIR2 ATR Accessory

These results show that the ConcentratIR2™ can be used to analyse secondary structure over a wide range of concentrations, including at very low levels. This makes it useful for biopharmaceuticals such as GLP-1RAs, where drug substances may need to be monitored in the µg/mL range.

Implications and future directions

Due to their clinical success, GLP-1RAs have become key treatments for diabetes and obesity. As demand increases, pharmaceutical teams must maintain consistent quality, structural stability and batch-to-batch reproducibility while production increases and new markets grow.

With this comes a renewed focus on reliable, scalable and orthogonal QA/QC frameworks to assess purity and stability. As a rapid, cost-effective technique, ATR-FTIR is ideally positioned to enter this space, especially for detecting aggregates that can compromise therapeutic efficacy and potentially induce immunogenicity.1,3

ATR-FTIR: A strategic complement, not a replacement

Rather than replacing other methods, ATR-FTIR offers complementary analytical insights that pharmaceutical teams can use without substantial resource strain. Especially when paired with solutions like the ConcentratIR2™ ATR Accessory, it opens up strategic opportunities to:

  • Detect early signs of aggregation or formulation instability.
  • Identify structural anomalies before advanced biophysical methods are triggered.
  • Correlate structural data with batch variation, manufacturing scale or storage conditions.
  • Implement specification-based alerts for unusual secondary structure profiles, e.g., during in-process controls (IPCs).
  • Enhance release testing, especially for generic GLP-1RAs.

A practitioner’s perspective

For QA/QC managers, ATR-FTIR offers:

  • Speed to decision: Rapid scan times give structural insights in minutes.
  • Scalability: Amenable to high-throughput implementation.6
  • Affordability: Lower barrier to entry than many biopharmaceutical analytical tools.
  • Simplicity: Reduced training and operational overheads compared with LC-MS, SEC or CD spectroscopy setups.

Bringing ATR-FTIR into the peptide QA/QC mainstream

As GLP-1RA production expands worldwide, it is vital to ensure batch consistency. Although often overlooked in pharmaceutical QA/QC settings, ATR-FTIR is uniquely positioned to provide fast, label-free structural insight that complements conventional assay methods. In particular, it offers a cost-effective way to assess key quality attributes such as secondary structure and aggregation.

Tools like the ConcentratIR2™ ATR Accessory extend the capabilities of ATR-FTIR to trace-level detection (µg/mL) of GLP-1RAs and other therapeutic peptides, elevating this well-established technique to meet the demands of contemporary QA/QC pipelines.

If you’re an R&D lead, QA/QC manger or researcher in peptide therapeutics, consider ATR-FTIR not as a legacy method, but a forward-looking solution to enhanceyour structural analytics.

Read our Application Note and book a demo of the ConcentratIR2™ today, to see how this simple accessory can transform your analytical workflow for GLP-1RAs and beyond.

References

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