Introduction
Peptide sequencing of Semaglutide is a critical analytical process in the development and regulatory submission of GLP-1 receptor agonist-based biologics. For pharmaceutical companies advancing towards late-stage clinical trials or regulatory approval, precise peptide sequencing of Semaglutide ensures structural integrity, batch consistency, and safety compliance. At ResolveMass Laboratories, we specialize in high-resolution peptide sequencing of Semaglutide, offering comprehensive analytical support tailored for NDA, BLA, or global submissions. This case study elucidates our methodological approach and expertise in successfully executing peptide sequencing of Semaglutide for a client’s late-stage filing.
Structural Overview of Semaglutide
Semaglutide is a synthetic analog of the human glucagon-like peptide-1 (GLP-1), comprising a linear sequence of 31 amino acids. Its design incorporates specific modifications to enhance metabolic stability and prolong half-life:
- Amino Acid Sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
- Modifications:
- Substitution of alanine at position 8 with α-aminoisobutyric acid (Aib) to confer resistance against dipeptidyl peptidase-4 (DPP-4) degradation.
- Acylation of the ε-amino group of Lys20 with a C18 fatty diacid via a γ-glutamic acid spacer and two units of 8-amino-3,6-dioxaoctanoic acid (ADO), enhancing albumin binding and extending plasma half-life.
These structural features necessitate meticulous analytical techniques to confirm the integrity and consistency of Semaglutide batches.
Analytical Challenges in Peptide Sequencing of Semaglutide
The complex structure of Semaglutide presents several analytical challenges:
- Hydrophobic Modifications: The lipidation at Lys20 increases hydrophobicity, complicating chromatographic separation and mass spectrometric analysis.
- Non-Canonical Amino Acids: Incorporation of Aib and PEG-like spacers requires specialized detection and sequencing strategies.
- Sequence Confirmation: Ensuring complete sequence coverage, including modified residues, is critical for regulatory compliance.
Methodological Approach to Peptide Sequencing of Semaglutide
Our comprehensive analytical strategy encompassed the following steps:
Sample Preparation
Semaglutide samples were prepared under stringent conditions to preserve structural integrity. Denaturation and reduction steps were optimized to facilitate enzymatic digestion without compromising the lipidated moiety.
Enzymatic Digestion
A dual-enzyme approach employing Glu-C and chymotrypsin was utilized to generate overlapping peptide fragments, ensuring full sequence coverage. This strategy effectively addressed the challenges posed by hydrophobic and modified regions.
Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
High-resolution LC-MS/MS analysis was conducted using an Agilent AdvanceBio Q-TOF 6545XT system. The use of an AdvanceBio Peptide Plus column facilitated efficient separation of peptide fragments, including those with hydrophobic modifications.
Data Analysis
Advanced bioinformatics tools, including PEAKS and Mascot, were employed for de novo sequencing and database matching. Manual validation ensured accurate identification of all amino acid residues and post-translational modifications.
Nuclear Magnetic Resonance (NMR) Spectroscopy
To complement mass spectrometric data, ^1H and ^13C NMR spectroscopy were performed, providing insights into the conformational aspects and confirming the presence of specific functional groups, particularly in modified regions.
LC-MS/MS-Based Peptide Sequencing of Semaglutide: Analytical Workflow and Structural Insights
Semaglutide, a GLP-1 receptor agonist analog, is a synthetic 31-amino acid linear peptide modified to enhance metabolic stability and pharmacokinetics. The complete primary sequence, including its site-specific modifications, must be confirmed with high-resolution peptide sequencing to support CMC filings and biosimilar characterization efforts.
Amino Acid Sequence and Structural Modifications
Semaglutide consists of 31 amino acids, with substitutions and conjugations enhancing stability:
Amino Acid Sequence (N → C terminus):
rCopyEditHis–Aib–Glu–Gly–Thr–Phe–Thr–Ser–Asp–Val–Ser–Ser–Tyr–Leu–Glu–Gly–Gln–Ala–Ala–Lys–Glu–Phe–Ile–Ala–Trp–Leu–Val–Arg–Gly–Arg–Gly–OH
Key Structural Features:
- Aib (α-aminoisobutyric acid) at position 2 replaces Ala to enhance resistance to DPP-4 degradation.
- The Lys20 side-chain is acylated with a C18 fatty diacid via:
- A γ-Glu spacer,
- Two 8-amino-3,6-dioxaoctanoic acid (ADO) linkers.
These modifications render LC-MS/MS sequencing more complex due to altered hydrophobicity, ionization behavior, and fragmentation efficiency.
Analytical Workflow for LC-MS/MS Peptide Sequencing
At ResolveMass Laboratories, peptide sequencing of Semaglutide was performed through an optimized LC-MS/MS proteomic approach tailored for long-chain, modified therapeutic peptides.
1. Sample Preparation
- Protein Unfolding: Semaglutide was solubilized in 6M guanidinium chloride to fully denature the peptide.
- Reduction and Alkylation: Disulfide bonds (if present in other peptides) were reduced with DTT and alkylated with iodoacetamide (not necessary in Semaglutide but part of standard workflow).
- Desalting: Performed using C18 solid-phase extraction (SPE) cartridges to remove salts and small molecules.
2. Enzymatic Digestion
Although trypsin is the standard protease, Semaglutide’s structure required alternative enzymes due to limited cleavage sites:
- Chymotrypsin was chosen to cleave at aromatic residues (e.g., Phe, Tyr, Trp).
- Endoproteinase Glu-C provided complementary cleavage at Glu residues.
Dual digestion ensured overlapping fragments and full sequence coverage, including modified Lys20.
3. Liquid Chromatography (LC)
- A reverse-phase C18 UHPLC column (AdvanceBio Peptide Plus, 2.1×150 mm) was used.
- Gradient: 2% to 45% acetonitrile with 0.1% formic acid over 30 minutes.
- Temperature: 50°C to handle hydrophobic modifications.
The lipidated fragments were eluted later in the gradient, requiring column chemistry that tolerates strongly hydrophobic peptides.
4. Mass Spectrometry (MS/MS)
- Instrumentation: Agilent 6545XT Q-TOF or Orbitrap Exploris 480.
- Mode: Positive ion ESI, data-dependent acquisition (DDA).
- Fragmentation: Higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) were used to optimize fragment ion coverage, especially for modified residues.
Fragment ions included:
- b- and y-ions for standard amino acid identification.
- Immonium ions for residue-specific validation (e.g., for Aib, Trp).
- Neutral loss patterns used to deduce lipid tail cleavages at Lys20.
5. Data Analysis
- De Novo Sequencing: PEAKS Studio and Byonic software were employed for non-template-based sequence elucidation.
- Database Matching: Used theoretical Semaglutide sequences with specified modifications (e.g., Aib, acylated Lys).
- Manual Review: Crucial for confirming low-abundance or modified fragments, particularly in lipidated peptides.
Result: >99% sequence coverage with clear confirmation of:
- All 31 residues
- Aib presence at position 2
- Acylation at Lys20
- Absence of sequence scrambling or truncation
- Batch-specific sequence homogeneity
Role of Orthogonal Methods: NMR Confirmation
While LC-MS/MS provides residue-level sequence data, NMR spectroscopy was used for:
- Verifying the conformation of Aib and its role in α-helical propensity.
- Confirming the chemical environment of the lipid tail at Lys20.
- Supporting the stereochemistry of chiral centers (Aib, Trp, Leu).
1D and 2D (^1H, ^13C, COSY, TOCSY) NMR spectra were acquired in DMSO-d6 and H2O/D2O to study solubility and secondary structure motifs.
Significance for Regulatory Submission
Accurate LC-MS/MS peptide sequencing of Semaglutide is critical for:
- CMC filings and comparability studies (between batches or biosimilars).
- Immunogenicity risk assessment (detecting impurities or degradation).
- Pharmacokinetic bridging between reference and test products.
Results and Regulatory Compliance
The analytical workflow achieved:
- 100% sequence coverage, including modified residues.
- Accurate identification and localization of the lipidated Lys20 residue.
- Confirmation of structural integrity through complementary NMR analysis.
The comprehensive data package, including validated methods, raw and processed spectra, and detailed reports, met the stringent requirements of regulatory agencies, facilitating the client’s successful late-stage biologic filing.
Conclusion
This case study underscores ResolveMass Laboratories’ expertise in the peptide sequencing of Semaglutide, highlighting our capability to navigate complex analytical challenges and deliver data that withstands regulatory scrutiny. Our integrated approach, combining advanced mass spectrometry, enzymatic digestion strategies, and NMR spectroscopy, ensures thorough characterization of peptide therapeutics.
For detailed information on our peptide sequencing services, visit our Peptide Sequencing Service Page.
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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)
