End-To-End Workflow Solutions for Therapeutic Peptides

Importance of the Topic

Peptide therapeutics, composed of chains of up to 40 amino acids, have rapidly advanced as a class of targeted drugs. Their high specificity for receptors and lower systemic toxicity compared to small molecules or biologics make them vital in treating metabolic disorders such as type 2 diabetes and obesity. However, challenges including metabolic instability, poor bioavailability and complex impurity profiles necessitate robust analytical workflows to ensure quality, safety and compliance throughout development and production.

Objectives and Study Overview

This resource guide outlines end-to-end analytical solutions for therapeutic peptide research, from raw material verification in the warehouse to final product QA/QC in manufacturing. It presents standardized workflows for identity confirmation, purity assessment, impurity profiling, sequence verification, preparative purification, quantitative analysis in biological matrices, elemental impurity testing and residual solvent determination.

Methodology and Instrumentation

The workflows integrate a suite of analytical techniques and instruments:

• Raw Material Identification: Handheld Raman (Vaya) for through-barrier checks; FTIR (Cary 630) and HPLC (1290 Infinity III bio LC) for lab-based verification.
• Purity Analysis: Reversed-phase LC/UV and LC/MS (single-quad MSD and TOF LC/MS) for peptide separation and quantitation.
• Amino Acid Analysis: HILIC-LC/MS with pre-column derivatization on Infinity III LC and AdvanceBio AAA columns.
• Impurity and Aggregation Profiling: 1D and 2D LC with Q-TOF or quadrupole-TOF detection for mapping sequence variants, diastereomers and aggregates.
• Sequence Confirmation and Isomer Characterization: ExD-enabled LC/Q-TOF for deep sequence coverage; ion mobility LC/Q-TOF (6560C) to resolve structural isomers by collision cross section.
• Preparative Purification: Analytical to preparative-scale reversed-phase LC systems (1260/1290 Infinity) with compatible columns for milligram to gram isolation.
• Quantitative Analysis in Biological Matrices: Automated sample prep (AssayMAP Bravo) with triple quadrupole LC/TQ for pharmacokinetic studies of GLP-1 analogs.
• Final Product QC: Multicell UV-Vis spectroscopy (Cary 3500) for rapid concentration checks and QC assays.
• Elemental Impurity Testing: ICP-MS (7850 or 7900) for trace metals per USP <232>/ICH Q3D guidelines.
• Residual Solvent Analysis: Headspace GC or GC/MS (8890 GC, 8697 HS, 5977 MSD) following USP <467> methods.

Instrumentation

Key instruments include Agilent Vaya handheld Raman spectrometer, Cary 630 FTIR, 1290 Infinity III bio LC, 6230B TOF LC/MS, 6545XT AdvanceBio LC/Q-TOF with ExD cell, 6560C ion mobility LC/Q-TOF, 1260/1290 Infinity II preparative LC systems, 6495D LC/TQ, Cary 3500 UV-Vis, 7850 ICP-MS and 8890 GC with 8697 headspace sampler.

Key Results and Discussion

• Warehouse RMID with Vaya Raman enables non-destructive, through-barrier identity checks under cGMP conditions.
• LC/UV and LC/MS workflows deliver peptide purity assessments with high resolution, sensitivity and throughput.
• AdvanceBio AAA kits and LC/MS provide accurate amino acid composition and sequence confirmation down to isomeric distinctions.
• 2D-LC/Q-TOF and IM-MS approaches resolve closely related impurities and aggregates, enhancing risk assessment.
• Preparative LC systems maintain analytical-scale robustness while scaling up isolations from milligrams to grams.
• Triple quadrupole LC/TQ workflows achieve low-ppt quantitation of peptide drugs in complex matrices for PK/PD studies.
• Validated ICP-MS and headspace GC methods ensure compliance with elemental and residual solvent limits in final drug products.

Benefits and Practical Applications

• Integrated platforms reduce method development time by reusing columns, software and sample prep protocols across applications.
• High-throughput and automated workflows support accelerated timelines from discovery to commercial supply.
• Comprehensive impurity profiling enhances safety and regulatory readiness.
• Scalable preparative solutions enable seamless transition from R&D to manufacturing purification.

Future Trends and Opportunities

Advancements are expected in multimodal separation (combining chromatography with ion mobility), AI-driven data analysis for rapid impurity identification, further miniaturization of workflows, and integration of multi-omics platforms to study peptide interactions and metabolism in biological systems.

Conclusion

This guide demonstrates a coherent suite of analytical tools and workflows for therapeutic peptides, addressing every stage from raw material verification through final QC. Leveraging Agilent’s instrumentation and software, researchers and manufacturers can achieve robust, compliant and efficient peptide drug development.

References

• Tripodi A, Coffey A. An In-Depth Analysis of Semaglutide, a Glucagon-Like Peptide-1 Receptor Agonist. Agilent Technologies application note 5994-7419EN, 2024.
• Ryu CY. Rapid Confirmation of GLP-1 Analog (Liraglutide) Using Agilent InfinityLab LC/MSD iQ. Agilent application note 5994-7415EN, 2024.
• Franklin R, Meeuwsen J. Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio LC/Q-TOF System. Agilent application note 5994-7506EN, 2024.
• Babu S. Characterization of Forced Degradation Impurities of GLP-1 Agonists by LC-Q-TOF MS. Agilent application note 5994-7794EN, 2024.
• Sanderson J. Residual Solvent Analysis in Hemp Consumer Products Using Headspace GC/MS. Agilent application note 5994-5237EN, 2022.
• Whitecotton L, McCurdy E, Jones C. Validating Performance of an Agilent ICP-MS for USP <232>/ICH Q3D Elemental Impurities. Agilent application note 5991-8335EN, 2022.
• Amino Acid Analysis – ‘How-To’ Guide. Agilent Technologies application note 5991-7694EN, 2021.