Few topics have captured the analytical chemistry world’s attention quite like glucagon-like peptide-1 (GLP-1) receptor agonists. Medications such as semaglutide (Ozempic, Wegovy) and tirzepatide (Mounjaro, Zepbound) have become household names, driven by their striking efficacy in managing type 2 diabetes and obesity. I was amazed to come across this CNBC article which stated that 1 in 8 American adults are taking one of these drugs. Behind that commercial momentum lies a formidable set of analytical challenges that are reshaping LC-MS workflows, chromatographic column selection, data platforms, and regulatory strategy across the biopharma industry.
In two episodes of Concentrating on Chromatography podcast, I sat down with two seasoned separation science professionals to explore these challenges from complementary vantage points. Sean Orlowicz, Principal Marketing Development Manager for Pharmaceuticals at Phenomenex and a 22-year chromatography veteran, brought a deep column chemistry and method development perspective. Watch full podcast episode here.
Kelly Broster, Senior Manager of Market Development and Collaborations at Thermo Fisher Scientific, whose 15+ years of hands-on LC-MS experience spans cancer biology, quantitative proteomics, and biomarker assays, addressed the high-resolution mass spectrometry and integrated workflow dimensions. Together, their insights cover the analytical lifecycle from sample preparation through regulatory submission. Watch the full podcast episode here.
GLP-1 therapeutics occupy analytically difficult territory between small molecules and large biologics. As Sean explained, this in-between status creates unique problems for the chromatographer: “Peptides really do fall in the gray spaces. Their impurities—whether process-related or degradation products—are so closely related structurally and chemically that it’s a little bit different from what we’re used to when talking about, say, a substituted nitrobenzene, which is a pretty different structure.”
The numbers underscore the point. Sean noted that a typical small-molecule oral tablet USP monograph might list two to six impurities. In Phenomenex’s recent semaglutide work, the team identified 13 known, chromatographically important impurities—and that is “just scratching the surface.” Many of these are deletion analogs (the API minus one amino acid residue), oxidation products, or isomers that share the same molecular formula but differ only in spatial arrangement. As Sean put it: “God forbid it’s isomerization—where it’s the same exact molecular formula, just in a different shape.”
Kelly reinforced the same theme from a mass spectrometry angle: GLP-1 therapeutics are “conformationally dynamic and often chemically modified to improve stability, half-life, and absorption.” They can contain subtle sequence variants and are prone to post-translational modifications such as oxidation and deamidation, all of which are difficult to separate chromatographically and equally challenging to identify spectrometrically. GLP-1 analogs such as semaglutide incorporate a C18 fatty acid via a glutamic acid spacer, while tirzepatide includes dual fatty chains and non-standard amino acids, adding further layers of structural complexity.
On the chromatography side, Sean offered some of the most practically detailed commentary in the series. He organized the column selection problem around two independent variables: selectivity (what chemists call α in the resolution equation) and efficiency (peak shape and plate count). Both matter for GLP-1s, but for different reasons.
Selectivity is governed by the stationary phase surface chemistry. Standard reverse phase separations rely on hydrophobic van der Waals interactions—differences in carbon content. But the structural differences between a GLP-1 drug substance and its key impurities are often ionic or polar in nature, such as a charged amino acid substitution or an oxidation event. “It’s these secondary, or non-van der Waals forces, that make the difference in the ability to separate these compounds on a chromatographic column,” Sean explained. This is why dozens of C18 columns exist in any vendor’s catalog: they are not all equal in their secondary interaction mechanisms.
Phenomenex’s Aeris XB-C18 emerged from peptide mapping work precisely because its unique surface chemistry demonstrated selectivity advantages for peptide separations—a finding that translated directly to synthetic peptide impurity profiling. But Sean was careful to note this is not a universal solution: “I would love to tell you that Aeris XB-C18 works for every peptide. From experience, it doesn’t.” A recent tirzepatide application led Phenomenex to recommend a different chemistry entirely, based on empirical testing.
Efficiency matters for a more quantitative reason. At trace impurity levels—perhaps 0.5% of the API signal—peak symmetry and height become critical to accurate integration. “If my peaks aren’t symmetrical and very tall, I’m going to manually draw baselines, and that leads to precision problems,” Sean said. The Aeris platform’s core-shell (superficially porous) particle construction addresses this directly: the solid silica core minimizes the diffusion path and maximizes efficiency, and the column format scales from conventional HPLC through UHPLC pressures.
One specific caution Sean raised is worth highlighting: TFA (trifluoroacetic acid) is the default ion-pairing agent for many laboratories, and it performs well for many separations. But for some GLP-1 peptides, TFA can be a limiting factor. “I’ve seen some really interesting things in regards to unique organic modifiers… it might not be the best choice for all peptide separations.” The practical takeaway: for peptide work, resist the instinct to simply reuse the mobile phase system sitting on the instrument.
Both guests converged on a theme that goes beyond analytical performance per se: robustness. GLP-1 therapeutics are manufactured, tested, and filed globally, and analytical methods must perform consistently across different laboratories, instrument platforms, and operator skill levels.
Sean used the analogy of a recipe: “We make our SOPs and methods and do our best to write down the ingredients and how much. But if you give a great recipe to a bad cook, the food’s not going to taste very good.” He has seen this play out in real time—conference calls connecting an Indianapolis site to a partner laboratory in China, both trying to run the same semaglutide purity method with different results.
Several instrument-level factors contribute to this challenge in GLP-1 workflows specifically. These methods are often 45 to 100 minutes long with shallow gradients—a combination that amplifies differences between instruments. “Anytime I see a shallow gradient, I see an instrument-to-instrument issue,” Sean said, pointing to gradient delay volumes, flow cell volumes, and injector configuration (loop versus flow-through) as variables that create discrepant retention times or peak asymmetries across systems.
His practical advice: spend the time during development and validation to build robustness in deliberately. Test three batches of columns. Verify mobile phase reagent quality. Explicitly document and bracket the gradient delay volume for your instrument. “You better get the same results with all of your channel partners, CDMOs, and CROs all over the world.”
Temperature control deserves special mention as an underappreciated robustness lever. “A lot of people overlook the temperature control of their HPLC method as a robustness issue,” Sean noted. For GLP-1 peptide separations specifically, tightly controlling both mobile phase and column temperature is one of the most effective ways to improve method transferability.
Kelly echoed the continuity theme from a lifecycle perspective: analytical strategies must transition seamlessly from R&D through QC into manufacturing, without reinventing methods at each stage. Thermo Fisher’s platform philosophy is explicitly designed around this. “Developers can’t afford to reinvent methods at every stage—it’s wasteful,” she said, emphasizing that maintaining data integrity, reproducibility, and scalability without disruption is foundational to their workflow development.
One analytical dimension that receives less attention in general-audience coverage, but is mandatory for GLP-1 drug products, is aggregation testing by size exclusion chromatography (SEC). Sean flagged this as a common area of confusion for laboratories entering the peptide space for the first time.
The FDA requires injectable peptide drug products to be tested for aggregation—specifically the formation of dimers and trimers from monomeric peptide in solution. Aggregation under shipping or storage conditions can significantly reduce potency, and in some cases the aggregated species may become therapeutically harmful. “The monomer peptide to a dimer or trimer in solution—under shipping conditions, is it stable on a shelf?” Sean explained. “As it dimerizes or aggregates, its potency becomes significantly decreased.”
SEC is a fundamentally different technique from the reverse phase and HILIC workflows used for purity and identity testing. Phenomenex recently launched a new SEC column specifically designed for the molecular weight range of GLP-1 peptides, which are smaller and historically more hydrophobic than many biologics. As Sean noted, liraglutide is “one of the most hydrophobic peptides I’ve ever worked on,” and that hydrophobicity is analytically relevant to its SEC behavior. Laboratories new to this space should not assume that a column portfolio designed for monoclonal antibodies will translate directly to GLP-1 aggregation analysis.
Both guests addressed the role of mass spectrometry in GLP-1 workflows, and their perspectives were complementary.
Sean framed the value proposition clearly: “As a chromatographer, it’s very challenging for me to separate chromatographically a 31-residue peptide by the position of a lysine. You know what a triple quad mass spec does really well? It separates them in the spectra.” For process analytical applications and quality control, mass spectrometry’s molecular specificity compensates for cases where chromatographic resolution alone is insufficient.
He also observed that mass spectrometry adoption is growing globally, including in emerging markets like India where it has historically been less prevalent. The impurity complexity of GLP-1 peptides—shallow gradients, method transfer challenges, structural near-identity of related substances—is driving labs toward MS-based solutions precisely because they offer an orthogonal mode of selectivity.
Kelly offered the high-resolution HRAM perspective. Beyond triple quadrupole QC applications, HRAM instruments deliver exact mass, isotopic resolution, and structural confirmation of sequence variants, post-translational modifications, and trace impurities at a level that older platforms cannot achieve. “This capability produces data that can stand up to regulatory scrutiny and global regulatory reviews, regardless of the matrix or background sample,” she said.
She also noted an emerging research application: using HRAM mass spectrometry to understand how GLP-1 drugs are targeting proteins and receptors relevant to therapeutic areas beyond diabetes and obesity. “There’s huge opportunities in other therapeutic areas now, and high-resolution mass spectrometry is being used to understand how these specific drugs are targeting proteins and receptors that are beneficial elsewhere,” Sean added. NDAs and applications in addiction treatment and other indications are already in development.
Cutting-edge analytical literature reinforces these observations. Emerging methods such as two-dimensional LC–HRMS, electron transfer dissociation (ETD), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) are improving impurity detection and structural confirmation, addressing limitations of conventional HRMS for isomeric impurities and complex fragmentation.
Sean raised one dimension that rarely surfaces in discussion of analytical GLP-1 workflows: the commercial-scale purification of the API itself. Most synthetic peptide therapeutics—including GLP-1 drugs—are purified by preparative reverse phase chromatography at manufacturing scale.
He described the tradeoffs using a triangle model: cost, speed, and purity. “Pick two.” This framework captures the practical reality facing API manufacturers, who must balance throughput economics against purity specifications that regulators require.
Phenomenex sees this as a growth area. With the commercial scale and economic weight behind GLP-1 manufacturing, significant investment is flowing into improving preparative column performance—longer stationary phase lifetimes, more effective separations, and greater efficiency at scale. “I take some responsibility for this as a vendor because with as much popularity and frankly as much money behind these drugs, there’s going to be a lot of investment made in improving those processes,” Sean said. He noted that the advancement of solid-phase peptide synthesis (SPPS) is proceeding at something close to Moore’s Law rates, making manufacturing processes simultaneously more capable and more analytically complex.
Despite the technical complexity of the preceding discussion, both guests kept returning to the same anchor: the analytical bar is high because the stakes for patients are high.
Kelly was direct: “The difference between safe and unsafe can really exist at the trace level. Detecting impurities even at parts-per-billion levels, and identifying low-abundance variants, is crucial—and it’s crucial that it’s done early, so developers can mitigate risk before scale-up.”
The technical literature confirms that inadequate analytical work carries real consequences. Impurities such as truncated peptides, degradation products, or process-related impurities, if not properly identified and controlled, may lead to adverse immune responses, toxicity, or reduced therapeutic efficacy.
Sean referenced the scale of the patient population that makes this responsibility urgent: “The safety and efficacy of these drugs is paramount—it affects a huge population. And then throw on top of this that these are sterile injectables.” Sterile injectables carry higher risk than oral dosage forms, with stricter manufacturing controls and analytical release specifications to match.
Sean also drew a historical analogy that puts GLP-1s in their proper context: “I’ve been fortunate to be around pharma for a long time and can put like pins in my lifetime when certain drugs came out. I think GLP-1s is one of those moments. The same way statins effectively eliminated the massive STEMI heart attack as a cause of death in younger adults—I think GLP-1s are going to have a global impact in different ways. The direct acute impact on diabetes is going to be very palatable in a decade’s time.”
GLP-1 patents are beginning to expire. Semaglutide’s U.S. patent exclusivity began lapsing in March 2026, with multiple generics developers—including Mylan and Natco Pharma—having secured first-to-file positions. Sean flagged this transition as something he has been tracking closely since his earlier work on the genericization of liraglutide, and he described the current moment as a maturation of the same cycle: “Now we’re seeing the next phases of genericization with semaglutide, which is starting to go generic in some regions as early as next year.”
As Kelly noted: “For biosimilars and generics, the burden of proof is rigorous structural and functional comparability. You have to use that characterization information to demonstrate equivalency.” High-resolution analytical platforms are not merely development tools in this context—they are the primary mechanism by which biosimilar developers build and document their regulatory case.
The regulatory landscape is actively evolving to support this. In October 2025, the FDA released draft guidance suggesting that comparative analytical assessments (CAA) may in many cases be sufficient to prove biosimilar comparability, potentially reducing or eliminating the need for expensive and time-consuming comparative efficacy studies. A review of more than 600 PubMed biosimilar studies found that no biosimilar with proven analytical similarity has ever failed a comparative efficacy study—underscoring the predictive power of rigorous analytical characterization.
For analytical chemists, this regulatory direction elevates their role considerably. Getting the analytics right—from method development and validation through global method transfer—is the path to market for the next generation of GLP-1 products.
The sensitivity and resolution demanded by GLP-1 characterization begin well before injection onto the column. Sample preparation—including concentration, cleanup, and matrix removal—can make or break downstream performance. Both guests’ discussions highlighted how easy it is to introduce the very artifacts that analytical methods are designed to detect.
For laboratories performing nitrogen blowdown concentration ahead of LC-MS workflows, this places a premium on temperature-controlled, precisely regulated evaporation. Uncontrolled heat can drive oxidation of methionine residues, accelerate deamidation of asparagine and glutamine residues, or degrade labile fatty acid conjugates. Sean’s practical advice about temperature control—“controlling the temperature of your mobile phase and your column for these methods is a great way to add robustness”—applies equally upstream at the concentration stage.
As the commercial volume of GLP-1 therapeutics grows and biosimilar developers enter the market with their own analytical burdens, every element of the analytical chain will face greater scrutiny. Instrumentation that delivers consistent, documented, and automatable evaporation conditions is a natural component of the integrated analytical ecosystem that both Sean and Kelly described.
GLP-1 therapeutics are not just a commercial phenomenon—they are a stress test for modern analytical science. Their structural complexity demands the most capable column chemistries and detection technologies available. Their global manufacturing scale requires workflow robustness and method transferability across sites and development phases. And their approaching patent cliff is turning biosimilar comparability analytics into a primary regulatory challenge.
Both Sean and Kelly’s perspectives converge on a shared principle: analytical rigor upstream—whether in column selection, mobile phase optimization, temperature control, or sample concentration—is what makes the downstream data defensible. As Kelly summarized: “Analytical science is central to delivering the promise of making the world healthier, cleaner, and safer—not just by measuring more, but by measuring the right things earlier and measuring with confidence.”