HPLC Separation of Tirzepatide on NanoPak-C All Carbon Microbeads

Tirzepatide, with its dual glucose-dependent insulinotropic polypeptide (GIP) / glucagon-like peptide-1 (GLP-1) activity and strong clinical data in type 2 diabetes and obesity, is rapidly becoming one of the most important peptide drugs in the metabolic disease space. As process chemists and analytical scientists scale up Tirzepatide manufacturing, they need HPLC methods that remain stable under both acidic and alkaline conditions while preserving peak shape, recovery, and retention-time precision.

We have published an application note describing an optimized HPLC method for separating Tirzepatide using NanoPak C all-carbon reversed-phase media under both acidic and alkaline conditions.

In this post, we will go beyond that application note and explain what these results mean for method development, purification, and QC workflows.

Why is the separation of Tirzepatide challenging?

Tirzepatide is a long-acting peptide drug that enhances insulin secretion by amplifying the incretin

response. This response involves gut-derived hormones, mainly GIP and GLP-1, which are released after eating and help trigger insulin secretion. Tirzepatide acts as a dual agonist at GLP-1 and GIP receptors, producing effects similar to those of the body’s own incretins.

Figure 1. Depiction of Tirzepatide structure. Image adapted from [1].

In chromatography, Tirzepatide behaves like other GLP-1-class peptides. It is large, amphiphilic, tends to aggregate, and its solubility and shape depend on pH. To separate and purify Tirzepatide effectively, you need to carefully choose:

• Stationary phase chemistry

• Mobile phase composition and pH

• Flow rate and temperature

• Gradient design and loading strategy

For GLP 1 analogs such as Semaglutide and Liraglutide, all-carbon microbeads have already demonstrated:

• C18-like efficiency, sensitivity, and loading capacity

• Robust performance from acidic to strongly basic pH

The study presented in the application note establishes Tirzepatide’s behavior on NanoPak C under acidic and alkaline conditions using a basic analytical gradient.

The results demonstrated that:

• Mobile-phase pH and sample environment significantly influence Tirzepatide retention on the all-carbon stationary phase. When the sample is in DI water at pH 2.5 (acidic), Tirzepatide comes out at 5.3 minutes. At pH 8.5 (alkaline), it elutes at 6.8 minutes.

  
  

• Tirzepatide is retained longer under alkaline conditions, consistent with reduced protonation and stronger hydrophobic interactions with the carbon surface.

Even with the change in pH, Tirzepatide still shows a sharp, symmetric peak in both cases, with clear separation and stable retention.

Why do these results matter for process development and QC?

For process development teams, being able to separate Tirzepatide at different pH levels offers several practical benefits:

• Match purification to Tirzepatide's dissolution and aggregation behavior. Tirzepatide and related GLP-1 analogs often show better solubility and reduced aggregation in mildly to strongly alkaline solutions. The ability to run chromatography at pH 8.5 on a pH-stable stationary phase is a major advantage.

  
  

• Use the same platform for both acidic and alkaline methods. NanoPak-C all-carbon allows you to develop methods under acidic conditions (for LC–UV compatibility and legacy workflows) and under alkaline conditions (for solubility-driven or MS-friendly methods) on the same column family.

  
  

• Improve robustness at scale. All carbon microbeads tolerate harsh cleaning cycles, high aqueous loading, and pH swings without the dewetting or loss of bonded phase seen with silica C18, thereby reducing column replacement costs and downtime.

For QC laboratories, the same acidic/alkaline flexibility and stable retention behavior translate directly into more robust routine testing:

• Reliable release and in-process testing. Clear separation and consistent retention times at both pH levels support robust assay methods for Tirzepatide drug substance and drug product.

• Stability and stress studies on one platform. A single NanoPak-C method can be adapted to monitor Tirzepatide under acidic and basic stress conditions, simplifying stability protocols and trending.

• Side-by-side comparison with existing C18 methods. QC teams can benchmark NanoPak-C all-carbon against current silica C18 methods while keeping similar gradients and detection setups, making method transfer and validation more straightforward.

To download our application note on the Separation of Tirzepatide on the NanoPak-C All Carbon HPLC column, including chromatograms and detailed HPLC conditions, please check here.

For inquiries or collaborations, or if you are interested in evaluating NanoPak-C all-carbon microbeads, please contact our technical team at inquiry@millennialscientific.com, call us at 855 388 2800, or fill out our online contact form at www.millennialscientific.com/contact.

References

1) S. Lindner, S. Keim, S. Haddadzadegan, O. Romero, K. Zoeller, G. Stern, I. Cesi, K. Kafedjiiski, A. Bernkop-Schnürch, Strategies to Improve the Lipophilicity of Hydrophilic Macromolecular Drugs, Advanced Healthcare Materials 15 (2025).