Peptide sequencing of GLP1 receptor agonists is a crucial analytical requirement in the development and validation of GLP1-based therapeutics. At ResolveMass Laboratories Inc., we’ve built a specialized platform for the accurate peptide sequencing of GLP1 analogs using LC-MS/MS and complementary structural tools. In this blog, we present a detailed case study showcasing our successful peptide sequencing of GLP1 receptor agonists for a late-stage pharmaceutical client. The project not only confirms amino acid integrity but also supports regulatory submission through validated characterization workflows.
Introduction to GLP1 Receptor Agonists
GLP1 (Glucagon-like peptide-1) receptor agonists are synthetic peptides modeled on the native GLP1 hormone. These peptides play a key role in the treatment of type 2 diabetes and obesity. Due to their structural complexity, accurate characterization through peptide sequencing of GLP1 analogs is essential for lot release, comparability studies, and regulatory filings.
Our peptide sequencing of GLP1 agonists focused on confirming amino acid sequence, identifying modifications (e.g., acylation, Aib substitutions), and evaluating degradation or truncation products. High-resolution LC-MS/MS and orthogonal methods like NMR spectroscopy were integrated into the workflow.
Why Peptide Sequencing of GLP1 Matters in Drug Development
- Verification of Primary Structure: The peptide sequencing of GLP1 ensures fidelity to the therapeutic design and biosimilar standards.
- Detection of Sequence Variants: Helps identify impurities, truncations, and synthesis errors.
- Batch Comparability: Supports bridging studies between pilot and commercial-scale batches.
- Regulatory Compliance: Provides required analytical proof for CMC documentation in the U.S., Canada, and Europe.
LC-MS/MS-Based Peptide Sequencing of GLP1 Analog
Our sequencing strategy for GLP1 analogs was developed specifically for peptides containing non-natural residues like Aib, and lipid-modified Lys.
Sample Preparation
- Solubilization in denaturing buffers
- Reduction and alkylation (when disulfide bonds are involved)
- Desalting using C18 cartridges
Enzymatic Digestion
GLP1 analogs often have limited tryptic cleavage sites. We used a dual enzyme strategy:
- Chymotrypsin for aromatic and large aliphatic residues
- Glu-C for cleaving at glutamate residues
This combination ensured full coverage for peptide sequencing of GLP1 agonists.
Chromatographic Separation
We employed reverse-phase UHPLC with:
- C18 column (150 mm × 2.1 mm)
- Gradient: 2% to 45% acetonitrile with 0.1% formic acid
Hydrophobic lipid chains required extended gradients for proper elution.
Mass Spectrometry Conditions
- Q-TOF or Orbitrap MS with nano-ESI interface
- Data-dependent acquisition (DDA)
- Fragmentation: CID and HCD to cover a wide ion range
Fragment Analysis and De Novo Sequencing
We used PEAKS Studio and Byonic software to process:
- b/y-ion series
- Neutral loss and immonium ions
- Modified residue annotation (e.g., fatty acid chain at Lys)
Confirmation of Sequence
A representative GLP1 analog:
His–Aib–Glu–Gly–Thr–Phe–Thr–Ser–Asp–Val–Ser–Ser–Tyr–Leu–Glu–Gly–Gln–Ala–Ala–Lys(C18)–Glu–Phe–Ile–Ala–Trp–Leu–Val–Arg–Gly–Arg–Gly- Aib (non-natural) confirmed by distinct immonium ions
- Lipidation confirmed by MS/MS fragment and mass shift at Lys20
- Sequence Coverage >99%
Orthogonal Characterization by NMR
To support peptide sequencing of GLP1 results:
- 1D/2D NMR used to confirm secondary structure
- Verified conjugation chemistry on modified Lys residues
Regulatory Impact and Analytical Value
- Full-length sequence confirmation submitted for BLA filing
- Used for impurity profiling and product release criteria
- Batch-to-batch consistency validated using this method
Why Choose ResolveMass for Peptide Sequencing of GLP1
- Regulatory-grade LC-MS/MS platform
- Proven experience with lipidated and modified peptides
- Scientific staff with expertise in peptide chemistry and mass spectrometry
- High-throughput capability with fast turnaround
Explore more about our services:
- Peptide Sequencing Services
- Advanced Characterization Techniques
- Method Development & Validation
- Modified Peptide Analysis
- Regulatory Support Services
Conclusion
Peptide sequencing of GLP1 receptor agonists is central to product integrity, compliance, and innovation in therapeutic peptides. At ResolveMass Laboratories Inc., our advanced analytical workflows are specifically designed to provide full structural elucidation and sequence confirmation of GLP1 analogs, enabling confident regulatory submissions and research outcomes.
If your project requires custom peptide sequencing of GLP1 receptor agonists, our expert team is ready to assist with industry-leading precision and speed.
ResolveMass Laboratories Inc.: Comprehensive Scientific Expertise You Can Rely On
ResolveMass Laboratories Inc. is a trusted Canadian contract research organization offering a wide spectrum of specialized services spanning polymer synthesis, advanced analytical testing, and custom organic synthesis. With over a decade of experience supporting pharmaceutical, biotech, and industrial clients, we bring scientific precision and regulatory insight to every project. Our core capabilities include Polymer Synthesis and Characterization, Peptide Characterization, Organic Synthesis, Nitrosamine Testing and Analysis, PFAS Testing, and Extractable & Leachable Studies, as well as a broad suite of analytical techniques such as HPLC, GC-MS, MALDI-TOF, NMR, and FTIR.
Our multidisciplinary team includes chemists, analytical scientists, and regulatory experts with advanced academic and industry backgrounds. We excel at developing customized, high-quality solutions—whether you need innovative polymer designs, impurity profiling, or confirmatory testing that meets global regulatory standards.
Clients across North America choose ResolveMass Laboratories for our deep technical knowledge, commitment to quality, and ability to deliver reproducible, reliable data that drives confident decision-making. When precision, innovation, and trust matter—ResolveMass is your partner of choice.
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📞 Request a quote for method development
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References:
Z.K. Shihabi, CAPILLARY ELECTROPHORESIS | Clinical Applications, Editor(s): Paul Worsfold, Alan Townshend, Colin Poole, Encyclopedia of Analytical Science (Second Edition), Elsevier, 2005, Pages 381-392, ISBN 9780123693976, https://doi.org/10.1016/B0-12-369397-7/00750-0. (https://www.sciencedirect.com/science/article/pii/B0123693977007500)
