Impurity Monitoring of GLP-1 Therapeutic Peptide RetatrutideUsing a 2D-LC/TOF System

Significance of the topic

Retatrutide is a next‑generation multivalent GLP‑1/GIP/glucagon peptide therapeutic with a complex sequence and a dense impurity profile. Reliable detection and structural characterization of low‑level sequence variants, truncations, and synthesis byproducts are critical for development and QA/QC of peptide drugs. Conventional one‑dimensional reversed‑phase LC methods often fail to resolve closely related forms, while mass spectrometry (MS) preferred mobile phases can compromise chromatographic resolution. The study demonstrates a practical 2D‑LC/TOF approach that reconciles chromatographic performance with MS‑friendly conditions to support robust impurity monitoring of Retatrutide.

Objectives and study overview

The main goal was to evaluate an Agilent multiple heart‑cutting two‑dimensional LC platform coupled to an Agilent 6230C accurate‑mass TOF for targeted impurity isolation and identification of Retatrutide. Specifically, the workflow tests whether a TFA‑based first‑dimension separation (offering superior peptide resolution) can be combined with an online solvent exchange and FA‑based second dimension to enable sensitive, accurate‑mass MS analysis of selected peaks without TFA‑related ion suppression.

Methodology

• Sample: Retatrutide reconstituted at 2 mg/mL; injections of 2 µL (approximately 4 µg on‑column reported).
• Strategy: 1st dimension used TFA (0.1%) as ion‑pairing modifier for optimal UV separation; multiple heart‑cutting ASM valves transferred selected fractions into 40 µL loops for fast solvent exchange and 2nd‑dimension analysis using FA (0.1%) to enable MS detection.
• 1st‑dimension LC: Agilent Altura Peptide Plus column, 2.7 µm, 2.1 × 150 mm; mobile phases water/ACN both with 0.1% TFA; flow 0.4 mL/min; UV detection at 220 nm. Temperature: 80 °C on both columns.
• 2nd‑dimension LC: Agilent Altura Peptide Plus column, 2.7 µm, 2.1 × 50 mm; mobile phases water/ACN with 0.1% formic acid; fast gradient to perform chromatographic desalting and separation prior to MS.
• MS: Agilent 6230C Accurate Mass TOF with AJS ion source; acquisition MS only, m/z 100–3200. Typical source settings: gas temp 325 °C, drying gas 8 L/min, sheath gas 11 L/min, nebulizer 30 psi, capillary 3500 V.
• Software: MassHunter Acquisition v12.2 for instrument control and data collection.

Instrumentation

• Agilent 1290 Infinity III 2D‑LC system comprised of Bio Multisampler with thermostat, Multicolumn Thermostat, Diode Array Detector (Max‑Light cell), Bio Flexible Pump, Bio High‑speed Pump, Valve Drive(s) with ASM 2D‑LC valve and multiple heart‑cutting valves equipped with 40 µL bio loops.
• Agilent 6230C Accurate Mass TOF LC/MS System for accurate mass detection and deconvolution of intact peptide masses.

Main results and discussion

• TFA versus FA (1D RP‑LC): Under identical reversed‑phase conditions, 0.1% TFA provided substantially improved chromatographic resolution for Retatrutide compared with 0.1% formic acid. Using TFA, three impurity peaks were baseline separated from the main product; with FA one impurity (P2) co‑eluted with the main peak, illustrating the tradeoff between chromatographic selectivity (TFA) and MS compatibility (FA).
• 2D‑LC concept validation: Multiple heart‑cutting allowed targeted collection of the main peptide and the three impurity peaks from the TFA‑run and their rapid transfer into an FA‑based second dimension for MS analysis. This approach preserved the chromatographic benefits of TFA while avoiding ion suppression in the MS.
• MS characterization: Accurate mass deconvolution of the main product and three impurities yielded monoisotopic masses and mass offsets consistent with specific sequence changes or modifications. Key results summarized qualitatively:
  • Main product (M): deconvoluted monoisotopic mass ~4728.49 Da; ~94.6% UV area.
  • P1 (cut at 19.16 min; ~0.9% area): mass ~4528.36 Da, −200.13 Da vs main — consistent with deletion of one serine plus one leucine/isoleucine (combined mass loss ≈200 Da).
  • P2 (19.51 min; ~1.6% area): mass ~4615.42 Da, −113.07 Da — consistent with deletion of a single leucine or isoleucine (~113 Da).
  • P3 (20.12 min; ~2.9% area): mass ~4891.57 Da, +163.08 Da — consistent with an insertion or modification equivalent to a tyrosine residue (+163 Da) or an adduct producing a similar mass shift.
• Analytical performance: The integrated workflow enabled detection and accurate mass assignment of low‑abundance impurities down to single‑percent UV area levels, demonstrating both selectivity and sensitivity suitable for development and QA/QC contexts.

Benefits and practical applications

• Combines best chromatographic separation (TFA‑mediated) with MS‑friendly analysis (FA in 2nd dimension), addressing TFA ion suppression without sacrificing resolution.
• Targeted multiple heart‑cutting minimizes sample loss and allows focused MS characterization of peaks of interest, improving throughput compared with full 2D mapping.
• Enables confident intact‑mass based impurity annotation for peptide therapeutics, supporting release testing, stability studies, and synthetic process troubleshooting.
• Workflow is compatible with regulated QA/QC environments where TFA‑enhanced separations are standard but MS data are required for structural confirmation.

Future trends and potential applications

• Integration of MS/MS (tandem MS) in the second dimension or offline fraction collection to yield sequence‑level confirmation of impurities identified by intact mass shifts.
• Higher throughput implementations using automated heart‑cut scheduling, larger loop arrays, or faster second‑dimension gradients to screen more samples or more cuts per run.
• Application extension to other complex peptides, oligonucleotides, and biologics where strong ion‑pairing agents improve chromatography but impede direct MS analysis.
• Coupling this approach with advanced data analysis tools (automated deconvolution, database matching, and AI‑assisted annotation) to accelerate impurity identification and reduce manual interpretation time.
• Exploration of alternative chromatographic modifiers and ion‑pairing strategies that balance chromatographic selectivity with MS compatibility, potentially reducing reliance on TFA.

Conclusions

The reported multiple heart‑cutting 2D‑LC/TOF workflow enables retention of TFA‑driven chromatographic separation quality while providing MS‑friendly conditions for accurate mass detection. For Retatrutide, the method resolved and characterized three low‑level impurities with plausible structural explanations (truncations and a likely insertion), illustrating the approach's utility for peptide impurity monitoring in development and quality environments. The platform supports targeted, sensitive intact‑mass profiling and can be extended with MS/MS and automated data analysis for deeper structural confirmation.

References

1. Jastreboff AM et al. Triple–Hormone‑Receptor Agonist Retatrutide for Obesity. New England Journal of Medicine. 2023;389:514–526.
2. Agilent Technologies. Confirmation of Peptide‑Related Impurity Intact Mass Using 2D‑LC. Publication number 5994‑7654EN. 2024.