Decoding GLP-1 Peptide Analysis: Expert Insights from Phenomenex's Sean Orlowicz

In collaboration with Separation Science

The analytical chemistry world has been transformed by the explosive growth of GLP-1 receptor agonists—therapeutic peptides like semaglutide (Ozempic, Wegovy) and tirzepatide (Mounjaro) that are revolutionizing diabetes and obesity treatment. But behind every successful GLP-1 therapeutic lies a complex analytical challenge that requires sophisticated chromatography methods, specialized column technologies, and deep expertise in peptide separations.

In a recent episode of the Concentrating on Chromatography podcast, presented in collaboration with Separation Science—the premier learning platform for analytical scientists—we sat down with Sean Orlowicz, Principal Marketing Development Manager for Pharmaceuticals at Phenomenex, to explore the analytical complexities of GLP-1 analysis.

With over 22 years of experience in chromatography and column surface chemistry, Sean shared invaluable insights on method development, troubleshooting strategies, and the future of peptide analysis.

The GLP-1 Opportunity: From Innovation to Genericization

Sean's journey into GLP-1 analysis began over a decade ago when he first encountered exenatide in 2013, but his real immersion came through supporting the genericization of liraglutide (Victoza) in the early 2020s.

"This was one of the first peptides I saw going generic," Sean explained. "We were working with both synthetic peptide manufacturers on their DMF filings and drug product manufacturers on their ANDA filings. Everyone was developing methods around peptides and their related substances."

Today, the market has matured significantly. Semaglutide is beginning genericization in some regions as early as 2026, and Phenomenex supports clients across the entire value chain—from API manufacturing where synthetic peptides are purified by reverse phase chromatography at commercial scale, all the way to analytical workflows testing purity and aggregation.

The Unique Analytical Challenges of Peptide Separations

What makes GLP-1 peptides so challenging to analyze? According to Sean, peptides occupy a unique gray area between small molecules and large biologics.

"Peptides fall in the gray spaces between all of those terms," Sean noted. "Are they small molecule? They're very manageable from a small molecule chemist perspective in terms of the number of residues. But they're also very specific and targeting, kind of like biopharma therapeutics."

The Impurity Challenge

The real analytical difficulty lies in the nature of peptide impurities. Unlike traditional small molecule pharmaceuticals where impurities might have significantly different structures, peptide-related substances are remarkably similar to the parent compound.

"A lot of their impurities are a deletion of one amino acid, one oxidation at one position, or God forbid it's isomerization—the same exact molecular formula, just in a different shape," Sean explained. "When you think of a large peptide like 31 amino acid residues and it's got one less amino acid, relatively that's a very small change."

The Phenomenex technical note on semaglutide separation illustrates this challenge perfectly, identifying 13 known impurities including:

D-amino acid isomers (D-Asp⁹, D-His¹, D-Phe⁶, D-Ser⁸, D-Ser¹¹)
Truncated sequences (3-31-Semaglutide, 3-31-Linear)
Modified forms (Des side chain-Semaglutide)
Plus three unknown impurities detected at low concentrations

This complexity stands in stark contrast to typical small molecule oral tablets, which commonly have between two and six impurities called out in USP monographs.

The Two Critical Variables: Selectivity and Efficiency

Sean broke down the chromatographic separation of peptides into two essential variables that matter most: selectivity and efficiency.

Understanding Selectivity

"Selectivity is harder to wrap your head around as a chromatographer," Sean admitted. "I am a chromatography geek—I've been one for more than 20 years. My career is focused on the surface chemistry of chromatography columns, and that really affects selectivity or alpha in the resolution equation."

Traditional reverse phase separations rely on hydrophobic differences—using C18 columns to separate molecules based on Van der Waals forces and carbon-carbon interactions. But peptide impurities present a different challenge.

"The differences between these peptides and the related substances are amino acids, oxidations, or deamidations—these are not carbon-type differences. These are more ionic or polar in nature. It's these secondary or non-Van der Waals forces type separations that make the difference in the ability to separate these compounds on a chromatographic column."

This is why every manufacturer offers dozens of C18 columns in their catalogs—they're not all equal. They all have slight differences in selectivity based on those secondary interaction mechanisms.

The Aeris Peptide XB-C18 Advantage

Years ago, Phenomenex discovered that their Aeris XB chemistry had unique selectivity for peptides during peptide mapping work with protein digests. Fast forward to GLP-1 therapeutics, and they were pleased to learn empirically that the same selectivity advantage benefited synthetic peptide manufacturers.

The Aeris Peptide XB-C18 column features di-isobutyl side chains on the XB-C18 ligands, yielding a uniformly bonded stationary phase with increased ligand spacing relative to conventional C18 phases. This allows for more effective interactions with longer peptides (20+ residues).

The Efficiency Factor

"The cherry on top is my second variable in the chromatographic equation: efficiency," Sean emphasized. "Empirically in my experience, efficiency plays a huge role in these peptide separations because they have very small differences."

When separating peaks with small resolution—particularly when the main API peak is at 90-something percent and related substances are at 0.5%—peak symmetry and height become critical.

"If my peaks aren't very symmetrical and very tall, if they're not very efficient, I'm not gonna be able to accurately integrate them. I'm gonna manually draw baselines and that's gonna lead to precision problems."

This is where core-shell particle technology provides the advantage. Aeris columns are built on superficially porous particles specifically designed for efficiency. The core silica particle foundation minimizes analyte diffusion, resulting in narrower peaks and outstanding resolution.

The technology also scales from traditional HPLC systems (400-600 bar) to sub-2-micron UHPLC applications, maximizing efficiency across instrument platforms.

Method Development: Beyond Copy and Paste

One of Sean's most powerful messages to the chromatography community was about breaking free from routine method development approaches.

"I had an epiphany recently that the enemy of innovation is the existence of copy and paste," Sean shared. "Worldwide, HPLC reverse phase method development is approached relatively the same way: my favorite column and the mobile phase that's sitting on the system today."

For peptide separations, this approach won't suffice.

Mobile Phase Considerations

Sean encouraged analysts to explore unique organic modifiers beyond the standard trifluoroacetic acid (TFA) approach.

"TFA is a great ion pairing agent—it magically makes peaks seem more symmetrical—but maybe it's not the best organic modifier for all peptide separations," he noted.

The semaglutide method described in the Phenomenex technical note uses a sophisticated buffer system with ammonium dihydrogen phosphate (100mM) and perchloric acid as the pH modifier, combined with acetonitrile and methanol in the organic phase.

Column Selection Strategy

Don't just bang your head against the wall with your favorite column, Sean advised. If a particular column isn't working well, it might be because the specific peptide is very hydrophobic or missing a residue that the column's selectivity can't separate.

Phenomenex recently published work on tirzepatide where they found a different chemistry to be more beneficial than the Aeris XB-C18—demonstrating that no single column works for every peptide.

Temperature Control Matters

"A lot of people overlook the temperature control of their method as a robustness issue," Sean cautioned. "But I can tell you from lots of experience that controlling the temperature of your mobile phase and your column for these types of methods is a great way to add robustness."

Regulatory Considerations and Method Robustness

GLP-1 therapeutics face intense regulatory scrutiny for good reason—they're widespread sterile injectables affecting a massive patient population.

The Robustness Imperative

"These methods must be robust because the impurities that may affect therapeutic benefits, bioavailability, or toxicity are very small, and the chromatographic resolution needs to be there," Sean emphasized.

Methods also face the challenge of global transferability. Sean frequently receives calls requesting help troubleshooting methods being transferred from Indianapolis to China, or between various CDMOs and CROs worldwide.

"We all want to pretend that methods are completely transferrable, but for any of us that have worked in the real world of method transfer, they're not," Sean acknowledged. "The analogy I like to use is recipes. We write down the ingredients and how much, but if you give a great recipe to a bad cook, food's not gonna taste very good."

Building Robustness Upfront

Sean's advice for building robust methods includes:

Spend time during development and validation testing robustness parameters
Test three batches of columns to ensure lot-to-lot reproducibility
Use high-quality mobile phase reagents
Consider instrument aspects: gradient delay volumes, system dwell volumes, flow cell volumes
Evaluate injector types (loop injectors versus flow-through)
Test method performance across different instrument platforms

The semaglutide related substances method developed by Phenomenex demonstrates this robustness principle. The optimized 85-minute gradient successfully separated all impurities with resolution of at least 1.3, and system suitability testing showed reproducible results with RSD <5% across six replicate injections.

Even when impurity concentrations were varied from 0.2% to 2%, the resolution remained unchanged and consistently above 1.3.

Size Exclusion Chromatography: A New Frontier for Many

For many companies, GLP-1 therapeutics represent their first foray into peptides and sterile injectable products. This means learning entirely new workflows, including size exclusion chromatography (SEC) for aggregation testing.

The FDA requires drug products to be tested for aggregation—monitoring the formation of dimers and trimers in solution under shipping and storage conditions. As peptides aggregate, potency decreases significantly, and in some cases, aggregates can become therapeutically harmful.

"We've gotten phone calls in the last couple years from people who have never run size exclusion chromatography," Sean noted. "They have a lot of questions."

Phenomenex recently launched a new size exclusion column specifically aimed at GLP-1s, designed for the molecular weight range and hydrophobic characteristics of these smaller peptides. Sean mentioned that liraglutide, in particular, is "one of the most hydrophobic peptides I've ever worked on."

The Role of Mass Spectrometry in GLP-1 Analysis

Sean, who spent several years focused on mass spectrometry, shared an insightful perspective on the complementary role of MS in peptide analysis.

"There's kind of two people in a mass spectrometry lab," Sean joked. "There's the chromatographer that only looks at the mass spec and sees a detector, and then there's the mass spectrometrist that looks at the HPLC and only sees an injector. I try to be both".

High-Resolution MS in Research

High-resolution mass spectrometry plays a significant role in research and development, particularly as scientists explore GLP-1 therapeutics for new therapeutic areas beyond diabetes and obesity—including addiction and other conditions.

"We're seeing a lot of backwards movement using high-resolution mass spectrometry to understand how these specific drugs are targeting proteins and receptors that are beneficial to other therapeutic areas," Sean explained.

Triple Quad MS in Quality Control

Perhaps more interesting is the growing adoption of triple quadrupole mass spectrometry in process analytical and quality control applications, especially in emerging markets like India where MS has historically been less prevalent due to cost.

"As a chromatographer, it's very challenging for me to separate chromatographically a 31 residue peptide by the position of a lysine. That is hard work," Sean noted. "You know what a triple quad mass spec does really well? It separates them in the mass spectra".

This specificity makes MS particularly valuable for detecting oxidated products and other modifications without necessarily requiring complete chromatographic baseline separation—though ion suppression must still be considered.

Practical Troubleshooting Tips for Peptide Analysis

Drawing from decades of supporting clients globally, Sean offered several practical tips for analysts working with GLP-1 peptides:

Do Your Homework

"My first advice is just Google. Use the Google," Sean said with a smile. "Lean on vendors like Phenomenex and others that have done this before. Learn what not to do from others—learn from other people's mistakes".

While there may not be an exact solution for your unique problem online, you can find valuable insights about approaches that don't work, saving you time and resources.

Be Patient with Run Times

If you're coming from a small molecule background where 10-20 minute methods are standard, adjust your expectations.

"You're gonna have to be a little more patient with these types of methods. They're typically 45 minutes, and in some cases they're 100 minutes," Sean explained. "It sounds like we're going backwards, but we're not. We're using very high efficient particles often on UHPLC instruments at high pressures to get massive efficiencies".

The semaglutide assay method runs 45 minutes, while the related substances method extends to 85 minutes—but both deliver the resolution needed for regulatory compliance.

Leverage Available Resources

Sean noted an interesting challenge: "There's not a lot of peer-reviewed literature out there" on GLP-1 methods. Innovator companies kept their methods proprietary, and compendial monographs are still limited.

This means vendors who are actively focused on the GLP-1 market—and Phenomenex certainly is—become valuable resources for method development guidance, application notes, webinars, and technical support.

The Future of GLP-1 Therapeutics and Analysis

Looking ahead, Sean sees GLP-1 therapeutics as "a story of pharma for the next 10 years".

Transformative Impact

Sean drew a powerful analogy to another pharmaceutical revolution: statins and the near-elimination of STEMI heart attacks.

"I come from a very large Irish family, and my uncles—if they passed away in their late fifties from a large STEMI heart attack, that was essentially natural causes. That doesn't happen anymore. Less than 30,000 STEMI heart attacks globally a year now. Statins did that," Sean reflected. "I do think GLP-1s are going to have an impact globally in different ways, and the direct acute impact on diabetes is going to be something that's very palatable in a decade's time".

Analytical Innovation Continues

Phenomenex is developing new products not just for peptides, but for oligonucleotides and adeno-associated viruses (AAVs)—staying ahead of where pharma is heading.

The company recently launched its Biozen dSEC-1 SEC column specifically designed for GLP-1 molecular weight ranges and hydrophobicity challenges.

Educational Support

Phenomenex has significantly ramped up educational content, publishing numerous technical notes with real sample data—not just single standard peaks in gradients.

"We obtained 13 commercial and identified impurities that we've found through insights from our customers are chromatographically important, and we talked about their separation," Sean shared, referencing the comprehensive semaglutide work.

Preparative Purification: The Other Side of the Story

While much of the interview focused on analytical methods, Sean's background in large-scale organic synthesis gave him unique appreciation for preparative chromatography—the purification step that produces the API.

"Most synthetic peptide therapeutics manufactured today are purified by reverse phase chromatography," Sean noted. "I love that—it's seeing chromatography in front of you, not just on a screen as a Gaussian peak. You've got a fraction, you've got rotovaps going".

This large-scale purification represents an economically driven area where companies are working to optimize throughput, reduce costs, and improve yields.

Key Takeaways for Separation Scientists

Based on Sean's extensive expertise and the technical materials shared, here are the critical lessons for analysts working with GLP-1 peptides:

Method Development:

Don't rely on copy-paste approaches—peptides require thoughtful method development
Consider alternative ion pairing agents beyond TFA
Evaluate multiple column chemistries for optimal selectivity
Implement rigorous temperature control

Column Selection:

Prioritize both selectivity (for separating similar structures) and efficiency (for accurate integration
Core-shell particles offer efficiency advantages for peptide separations
Consider pore size appropriate for peptide molecular weight

Robustness Testing:

Test multiple column lots during validation
Evaluate method performance across instrument platforms
Consider gradient delay volumes and system dwell in method transfer
Build in adequate resolution (≥1.3) to ensure method specificity

Complementary Techniques:

Implement SEC for aggregation monitoring
Consider MS for enhanced specificity in impurity detection
Evaluate high-resolution MS for research applications

Resources and Support:

Leverage vendor expertise and application notes
Access educational platforms like Separation Science for learning
Attend conferences and engage with the separation science community
Don't hesitate to reach out for troubleshooting support

Conclusion

The rise of GLP-1 therapeutics represents both an enormous pharmaceutical opportunity and a significant analytical challenge. As Sean Orlowicz's insights demonstrate, successful peptide analysis requires a sophisticated understanding of chromatographic principles, careful method development, and access to specialized column technologies.

Whether you're a small molecule chemist entering the peptide world for the first time or an experienced analytical scientist optimizing existing methods, the combination of selectivity-focused column chemistry, high-efficiency particles, robust method development practices, and comprehensive validation will be essential for success.

As these therapeutics continue their genericization journey and expand into new therapeutic areas, the analytical chemistry community will play a critical role in ensuring drug quality, safety, and efficacy for millions of patients worldwide.

Additional Resources

Learn More:

Visit Phenomenex.com for GLP-1 application notes and technical support
Explore SepScience.com for chromatography education and training modules
Download the complete Phenomenex technical note on semaglutide impurity separation

Connect with Sean:

LinkedIn: Sean Orlowicz

About This Episode:

This interview was conducted in collaboration with Separation Science, the premier online learning platform for analytical scientists, providing expert content on chromatography, mass spectrometry, sample preparation, and related laboratory techniques.labxmediagroup+1

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