Peptide therapeutics demand more precise analytical chemistry as GLP-1 drugs reshape pharma development

As glucagon-like peptide-1 drugs have moved peptide therapeutics into the mainstream, analytical scientists have faced growing pressure to look beyond conventional purity testing and adopt workflows capable of resolving structural microheterogeneity, low-level impurities and formulation-related complexity

Peptide therapeutics have moved rapidly into mainstream pharmaceutical development, driven largely by the success of glucagon-like peptide-1 (GLP-1) receptor agonists. Their rise has also exposed the limits of analytical approaches originally designed for small molecules – according to Dr. Kelly Broster, senior manager of pharma and biopharma market development and collaborations at Thermo Fisher Scientific.

Broster has spent more than 15 years at the interface between mass spectrometry and biopharmaceutical development, with experience in cancer research, quantitative proteomics and biomarker assay development. Her current work focuses on analytical strategies that can keep pace with increasingly complex drug modalities, including peptide therapeutics whose quality cannot be assessed adequately through simple pass-or-fail metrics.

“The biggest misconception is to assume that liquid chromatography purity and intact mass confirmation are sufficient, when peptide therapeutics demand deep structural and microheterogeneity characterisation, not just chemical purity,” Broster said.

She said that assumption had come from small-molecule workflows, where a high reversed-phase high-performance liquid chromatography purity value, supported by mass confirmation, often indicated acceptable control. However, GLP-1 analogues have shown why that framework can fail when applied to peptides. Material can appear clean by conventional chromatography while still containing sequence variants, deamidation, oxidation, acylation variants or other subtle modifications that could affect potency, stability and potentially immunogenicity.

Many of these molecular species are isobaric, meaning that they have the same nominal mass, or are otherwise closely related. As a result, a single purity percentage cannot fully describe molecular integrity. Broster said peptide programmes required orthogonal, structure-informing analytics, particularly high-resolution accurate-mass mass spectrometry coupled with ultra-high-performance liquid chromatography, to resolve, assign and track variants with confidence.

Broster said traditional small-molecule assumptions tended to fall short first at the level of the primary analytical readout. Teams accustomed to small molecules may expect one chromatogram and one purity value to describe a molecule adequately. With peptides, multiple structurally related species can co-elute, partially resolve or sit beneath the main peak, and a primary assay may quantify those species without truly identifying them.

GLP-1 analogues illustrate this difficulty clearly. Beyond classical process impurities, developers must manage sequence-level variants, isomerisation, epimerisation and side-chain modifications that may occur at low levels yet still influence potency, stability or immunogenicity. A second pressure point arises when conventional liquid chromatography conditions prove inadequate for confident structural assignment. Methods optimised for chromatographic appearance may not be compatible with high-confidence mass spectrometry interrogation.

Tactical choices, such as the selection of formic acid or difluoroacetic acid, could affect the balance between separation efficiency, mass spectrometry sensitivity and mass accuracy. The key shift occurs when a development programme must explain what has changed and why, rather than simply show whether a batch has met a numerical limit.

As GLP-1 programmes progress from early research and development into process development and quality control, the analytical focus also changes. Laboratories must move from deep one-off characterisation towards consistent measurement, intelligent trend analysis and decisions that can withstand regulatory scrutiny. One major challenge is to detect and identify impurities at very low concentrations in a way that is both structurally confident and operationally scalable.

GLP-1s – like many peptides – are prone to impurity types that include deletions, insertions, substitutions, isomerisation and chemical modifications such as oxidation or deamidation. Even small quantities can affect biological activity, which narrows the margin for analytical uncertainty. For that reason, modern workflows increasingly rely on ultra-high-performance liquid chromatography coupled to high-resolution accurate-mass mass spectrometry, which combines separation with accurate mass information and informative fragmentation.

In practice, this may include complementary tandem mass spectrometry activation methods, such as higher-energy collisional dissociation alongside electron-transfer dissociation or electron-transfer and higher-energy collision dissociation, to avoid blind spots for labile modifications and to localise molecular changes with confidence. As peptide programmes mature, developers may also need to understand self-association or aggregation, with size-exclusion chromatography mass spectrometry under native and denaturing conditions used to distinguish reversible association from covalent aggregates.

According to Broster, the boundary between analytically ideal methods and deployable methods depends on risk and actionability. High-end characterisation is essential to establish critical quality attributes, understand degradation or impurity mechanisms, build comparability arguments or investigate process excursions. Routine quality control, however, must be robust, validated, transferable and efficient.

“The key mindset shift is to move from a purity-centric view to a structure-and-heterogeneity-centric view,” she said.

She said microheterogeneity in peptides should not be treated as a rare exception, but as a fundamental characteristic of the product that must be measured, understood and controlled. That shift changes control strategies. Rather than ask only whether a product is pure enough, developers must ask whether they have orthogonal evidence to confirm identity, assign impurities and protect the critical quality attributes linked to safety and efficacy.

Oral peptides add further analytical demands because they must pass through the harsher and more variable environment of the gastrointestinal tract. Oral delivery can introduce enzymatic degradation, chemical instability, permeability limits and formulation effects, which can make systemic exposure lower, more variable and harder to interpret analytically. Developers must therefore assess the intact fraction after gastrointestinal-like exposure, identify dominant degradation pathways and measure very low levels reliably when bioavailability is limited.

Broster said the most significant change in peptide analytics during the next three to five years was likely to be the continued movement of mass spectrometry from high-end characterisation into more routine use across regulated end-to-end workflows. As peptide modalities become more complex, the most important capability will be confident impurity identification and structural verification at low abundance.

For analytical chemistry more broadly, GLP-1s show that progress is not simply about the ability to measure more. It is about the need to measure the right attributes earlier, with decision-grade certainty, and to translate those insights into scalable control.