Analytical Workflows To Ensure the Safety of GLP-1 Receptor Agonists

Agilent biopharma solutions Complete Analytical Workflows for GLP-1 Receptor Agonists Applications for peptide characterization, purification, and bioanalysis Introduction 03 1 Identity, Purity, and Impurity Assessment 06 1.1 Introduction 06 1.2 Molecular Weight Confirmation of a Peptide Using MS Spectral Deconvolution for OpenLab CDS and the Agilent InfinityLab LC/MSD XT System 08 1.3 An In-Depth Analysis of Semaglutide, a Glucagon-Like Peptide-1 Receptor Agonist 14 1.4 Quantification of Glucagon-Like Peptide-1 Agonist tirzepatide Using an Agilent 6495D Triple Quadrupole LC/MS System 22 2 Impurity Analysis 28 2.1 Confirmation of Peptide-Related Impurity Intact Mass Using Agilent 1290 Infinity II Bio 2D-LC and InfinityLab LC/MSD XT 28 2.2 Characterization of Forced Degradation Impurities of Glucagon‑Like Peptide-1 Agonists by LC/Q-TOF Mass Spectrometry 40 3 Sequence Confirmation 49 3.1 LC/MS Based Characterization Workflow of GLP-1 Therapeutic Peptide Liraglutide and Its Impurities 49 3.2 Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio 4 Purification Solutions 67 4.1 Introduction 67 4.2 Workflow Ordering Guide: Analysis and Purification of Synthetic Peptides by Liquid Chromatography 68 4.3 Optimizing Analysis and Purification of a Synthetic Peptide Using PLRP-S Columns 76 4.4 Efficient Purification of Synthetic Peptides at High and Low pH 84 5 Bioanalysis Studies 93 5.1 Introduction 93 5.2 Quantification of Therapeutic Peptide Exenatide in Rat Plasma 94 Introduction Peptides occupy a unique space between small molecules and biologics, offering distinct and tunable pharmacokinetic (PK) and pharmacodynamic (PD) profiles for therapeutic applications. They generally have a lower risk of triggering immune reactions compared to biologics such as monoclonal antibodies, are less expensive to produce, and penetrate tissues more effectively due to their smaller size. Although most peptides do not easily cross cell membranes, advances in engineering have improved intracellular targeting and cellular uptake. Compared to small molecules, peptides often demonstrate higher specificity and selectivity, reducing unwanted side effects. Because peptides degrade into naturally occurring amino acids, they are less likely to accumulate in tissues or cause long-term toxicity. Their size and flexibility allow them to modulate large protein surfaces and protein-protein interactions (PPIs). Manufacturing peptides is more straightforward and costeffective than producing proteins or antibodies, allowing for high purity and consistent quality. Additionally, peptides are often more stable in storage and may not require refrigeration, cutting down on logistical challenges and costs. Recent innovations in drug design, including methods to extend peptide half-life (like acylation, PEGylation, or fusion to larger proteins), alongside advances in delivery platforms, have significantly accelerated the development of next-generation peptide therapeutics. These improvements address common issues of rapid breakdown and clearance, which traditionally required frequent injections. New peptide-based therapies have also expanded into areas like targeted delivery and vaccines. Despite these advances, the increasing complexity of peptide drugs—due to varied molecular structures, conjugation strategies, and delivery formats—poses challenges for quality control. Peptides work through diverse mechanisms—acting as receptor agonists or antagonists, enzyme inhibitors, immune modulators, or disrupting intracellular signaling. They are used to treat a broad range of conditions, including metabolic and cardiovascular diseases, cancer, and infectious diseases. Manufacturing peptides is more straightforward and costeffective than producing proteins or antibodies, allowing for high purity and consistent quality. Additionally, peptides are often more stable in storage and may not require refrigeration, cutting down on logistical challenges and costs. Recent innovations in drug design, including methods to extend peptide half-life (like acylation, PEGylation, or fusion to larger proteins), alongside advances in delivery platforms, have significantly accelerated the development of next-generation peptide therapeutics. These improvements address common issues of rapid breakdown and clearance, which traditionally required frequent injections. New peptide-based therapies have also expanded into areas like targeted delivery and vaccines. Despite these advances, the increasing complexity of peptide drugs—due to varied molecular structures, conjugation strategies, and delivery formats—poses challenges for quality control. Peptides work through diverse mechanisms—acting as receptor agonists or antagonists, enzyme inhibitors, immune modulators, or disrupting intracellular signaling. They are used to treat a broad range of conditions, including metabolic and cardiovascular diseases, cancer, and infectious diseases. Figure 1. (a) Timeline of relevant milestones in the development of therapeutic peptides (only selected classes of FDA-approved drugs from 2000 onwards), (b) Classification of the FDA-approved molecules (from 1940 until October 2024) and their respective clinical use, (c) Distribution of peptidebased drugs and in-vivo diagnostics approval times from 2000 to 2024, compiled from the freely available database PepTherDia [7], with insulin-based drug data sourced from [8]. Analysis of peptide therapeutics As the complexity and diversity of peptide-based products continue to grow, the importance of robust analytical capabilities becomes increasingly evident. Sensitive and selective methods are essential for determining and monitoring critical quality attributes (CQAs), such as identity, content, purity, and impurities, to ensure the quality, safety, and efficacy of these therapeutics (Table 1). The challenge extends to the bioanalysis of these modalities, including PK and PD assessments in biological matrices. Liquid chromatography (LC) and mass spectrometry (MS) play a central role in supporting the above measurements. 5 Return to Contents Section Applications Goal Approach Identity Ensuring that the peptide has the correct structure which is, primarily, defined by the amino acid sequence and intended modifications (lipidation, amidation, PEGylation, etc.) - Comparison of retention time to reference standard by LC/UV - Molecular mass determination by LC/MS - Sequence determination by LC/MS/MS - Amino acid analysis by LC/FLD Purity and impurity Assessing the proportion of the desired peptide relative to impurities (purity) and detecting, identifying and quantifying process- (solvents, reagents, metals, host-cell materials, etc.) and product-related impurities (impurity) - Purity and product-related impurities: LC/UV, LC/MS, LC/MS/MS, 2D-LC - Process-related impurities: LC/UV, LC/ELSD, LC/RI, LC/MS, LC/MS/MS, GC/FID, GC/MS, ICPMS Content Determination of the absolute quantity of the therapeutic peptide - LC/UV, LC/MS, LC/MS/MS using external or internal calibration - Amino acid analysis by LC/FLD Bioanalysis (PK/PD) Quantification of therapeutic peptides in biological matrices (blood, plasma, serum, urine, …) - 1D or 2D-LC/MS/MS with tailored sample preparation and absolute quantification using isotopically labeled synthetic peptides or analogue peptides Table 1. Therapeutic peptide attributes studied by LC and MS. This compendium brings together practical insights and application notes focused on analytical challenges and method development for GLP-1 receptor agonists and related peptides. It is designed to support scientists involved in ensuring the quality and reliability of these innovative therapeutics as they continue to transform patient care. With content adapted from contributions by RIC Group. References 1. A.J. Pereira, L.J. de Campos, H. Xing, M. Conda-Sheridan, Peptide-based therapeutics: challenges and solutions, Medicinal Chemistry Research 33 (2024) 1275–1280. https://doi.org/10.1007/s00044-024-03269-1. 2. W. Xiao, W. Jiang, Z. Chen, Y. Huang, J. Mao, W. Zheng, Y. Hu, J. Shi, Advance in peptide-based drug development: delivery platforms, therapeutics and vaccines, Signal Transduct Target Ther 10 (2025). https://doi.org/10.1038/ s41392-024-02107-5. 3. M. Yu, M.M. Benjamin, S. Srinivasan, E.E. Morin, E.I. Shishatskaya, S.P. 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Walsh, Biopharmaceutical benchmarks 2022, Nat Biotechnol 40 (2022) 1722–1760. https://doi. org/10.1038/s41587-022-01582-x. 9. L. Wang, N. Wang, W. Zhang, X. Cheng, Z. Yan, G. Shao, X. Wang, R. Wang, C. Fu, Therapeutic peptides: current applications and future directions, Signal Transduct Target Ther 7(1) (2022) 48. https://doi.org/10.1038/s41392-022- 00904-4. 10. M. D’Hondt, N. Bracke, L. Taevernier, B. Gevaert, F. Verbeke, E. Wynendaele, B. De Spiegeleer, Related impurities in peptide medicines, J Pharm Biomed Anal 101 (2014) 2–30. https://doi.org/10.1016/j.jpba.2014.06.012. 11. FDA, 100 Years of Insulin, (2022). https://www.fda.gov/ about-fda/fda-history-exhibits/100-years-insulin (accessed July 8, 2025). 12. D. Yi, J.E. Mertz, Paul Berg and the origins of recombinant DNA, Cell 187 (2024) 1019–1023. https://doi.org/10.1016/j. cell.2024.01.007. 13. CHMP, EMA. 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Abstract Peptide biotherapeutics represent a class of pharmaceuticals that hold significant importance in modern medicine due to their unique properties and diverse therapeutic applications. Peptides are short chains of amino acids, typically comprising fewer than 50 residues, and they play crucial roles in various physiological processes within the human body. With advancements in biotechnology and pharmaceutical research, the development and use of peptide‑based therapeutics have surged, offering novel treatment options for a wide range of medical conditions. This application note presents some of the challenges when analyzing a glucagon-like peptide-1 (GLP-1) receptor agonist, semaglutide acetate, comparing different gradient conditions, temperatures, and column chemistries. Furthermore, sequence identification was achieved by LC/MS analysis using an Agilent AdvanceBio Peptide Plus column.