Ensuring Quality and Efficacy of GLP-1 Agonists: Analytical Challenges and Solutions in Therapeutic Peptide Production

Glucagon-like peptide-1 (GLP-1) agonists represent an exciting class of drugs that have gained significant attention in recent years for their potential to treat type 2 diabetes mellitus (T2DM) and obesity. These medications are a form of therapeutic peptide that mimics the effects of the naturally occurring GLP-1 hormone, which plays a crucial role in regulating blood sugar levels and appetite.

Functions of GLP-1 include lowering blood sugar by stimulating insulin release and reducing levels of glucagon (a hormone that tells the liver to produce glucose). GLP-1 also slows gastric emptying, contributing to a longer feeling of fullness after meals and slower digestion of food, which helps moderate blood sugar levels. Additionally, GLP-1 has neurological effects that decrease appetite, increase satiety, and reduce the desire for food^[1].

The particularly exciting thing about this class of diabetes medications is that, in contrast to many currently available diabetes drugs that address downstream effects, these medications target the root cause of diabetes.

Common GLP-1 agonists include semaglutide^[1], marketed by Novo Nordisk as a once-weekly injection for T2DM (Ozempic), a once-weekly injection for chronic weight management (Wegovy), and a once daily oral tablet^[2] for T2DM (Rybelsus). Novo’s older drug liraglutide^[1] is marketed as a once weekly injection for T2DM (Victoza) and a once weekly injection for chronic weight management (Saxenda). Eli Lilly’s GLP-1 agonist drug dulaglutide^[2] (Trulicity) is a once-weekly injection for T2DM.

GLP-1 agonist drugs can be manufactured by two methods: genetic modification of bacteria cells to secrete the drug or chemical synthesis. While use of bacteria can be an efficient way to produce very large molecule biologic drugs, this method has limitations. It is challenging to modify the genome of the bacteria to produce the desired chemical, and bacteria will only synthesize peptides from certain amino acids. This means adding customized components is not straightforward. For example, semaglutide has a fatty acid side chain that increases its half-life, allowing for once-weekly injections of Ozempic and Wegovy. For these reasons, it is often more straightforward to synthesize GLP-1 drugs, which are smaller peptides, through chemical methods. Chemical synthesis of therapeutic peptides involves adding amino acid building blocks one by one to construct the desired peptide chain. Regardless of the method used to produce a compound, the journey from discovery to clinical application of therapeutic peptides is filled with challenges, particularly in production and analytical characterization. Effective analytical techniques are pivotal in overcoming these hurdles to guarantee the quality, purity, and effectiveness of the end product.

Some considerations include:

• Purity and Impurity Detection: Ensuring high purity is critical for peptide efficacy and safety. Various impurities may be present in peptides, including missing or incorrectly inserted amino acids, incorrectly folded peptides, impurities due to oxidation or hydrolysis, isomers, or left over reagents^[1]. While some forms of impurities are safe and have no effect on your peptide or the patient who uses it, others can be toxic or have unknown effects. It is crucial to have a clear understanding of the precise composition of your peptides and the identities and amounts of any impurities present.
• Sequence Confirmation: Accurate determination of peptide sequence is essential for therapeutic specificity. Errors or sequence variants can lead to ineffective treatments or adverse effects. During synthesis of peptides, it is very easy to form variations in the sequence, thus it is vital to run testing to confirm that you have produced the correct peptide.
• Quantitative Analysis: Precise quantification of peptides is necessary throughout production stages to maintain consistency in dosage and efficacy.
• Post-Translational Modifications (PTMs): Many therapeutic peptides undergo PTMs (e.g., amidation, glycosylation) that impact their biological activity. Monitoring and characterizing these modifications are crucial for understanding peptide function.

Analytical Method Solutions:

• Purity Assessment: HPLC (high-performance liquid chromatography) coupled with ultraviolet (UV) detection, and mass spectrometry (MS) is essential for assessing therapeutic peptide purity. UV detection quantifies peptide content and identifies impurities based on absorbance at specific wavelengths and chromatographic separation. MS confirms peptide sequences and detects impurities by analyzing mass-to-charge (m/z) ratios. This combined approach ensures accurate purity assessment, aiding in method validation and regulatory compliance in peptide production.
• Impurity Identification: Impurity identification in therapeutic peptides using HPLC-MS involves chromatographic separation of peptides followed by ionization and fragmentation with MS. Mass spectra analysis reveals molecular weights and fragmentation patterns, enabling detection of impurities like oxidation products and deamidated peptides. This method provides high sensitivity and specificity, ensuring the purity and quality of therapeutic peptides for pharmaceutical use.
• Quantification: Quantifying therapeutic peptides can be achieved using HPLC with either UV detection or MS. HPLC-UV measures peptide concentrations based on absorbance at specific wavelengths, while HPLC-MS uses m/z for more precise and sensitive quantification. Both methods are vital for ensuring accurate dosage measurements in pharmaceutical applications.
• PTM Analysis: PTM analysis of therapeutic peptides using MS identifies and characterizes modifications like phosphorylation, glycosylation, and acetylation. Peptides are first separated by HPLC, then ionized and fragmented in the MS. MS accurately determines molecular weights and modification sites, essential for assessing peptide function and therapeutic potential.
• Sequence Verification: Sequence verification of therapeutic peptides using MS involves chromatographic separation followed by ionization and fragmentation. MS analyzes these fragments to confirm the peptide’s amino acid sequence by comparing experimental data with the expected pattern. This method ensures accurate identification and quality assessment of therapeutic peptides for pharmaceutical use.
• Our scientific team at Emery Pharma specializes in this type of analysis. Our general process for therapeutic peptide characterization is as follows:

1. Prepare the sample in a suitable solvent.
2. Inject the sample into an HPLC system equipped with a UV detector and MS. The system uses a gradient of aqueous and organic solvents to separate the peptide.
3. Simultaneously, monitor by UV to assess the purity and MS to confirm the peptide’s molecular weight and sequence.
4. Data from both HPLC-UV and MS are analyzed to identify the peptide, assess purity, and detect any modifications, providing comprehensive characterization essential for pharmaceutical quality control and research purposes.

As the use of therapeutic peptides such as GLP-1 agonists becomes more common, it is vital to ensure quality of products intended for use in patients. Understanding and quantifying ingredients and impurities in drugs is extremely important, and technology like MS and chromatography is ideal for the job. Our scientific team at Emery Pharma is constantly evolving to meet the latest innovative standards in safety and analysis of therapeutic peptides. For help with your therapeutic peptide projects, please inquire and speak to one of our scientists.

References:

^[1] Laurindo, L.F.; Barbalho, S.M.; Guiguer, E.L.; da Silva Soares de Souza, M.; de Souza, G.A.; Fidalgo, T.M.; Araújo, A.C.; de Souza Gonzaga, H.F.; de Bortoli Teixeira, D.; de Oliveira Silva Ullmann, T.; et al. GLP-1a: Going beyond Traditional Use. Int. J. Mol. Sci. 2022, 23, 739. https://doi.org/10.3390/ijms23020739

^[2] https://medlineplus.gov/druginfo/meds/a618008.html

^[3] https://medlineplus.gov/druginfo/meds/a619057.html

^[4] https://medlineplus.gov/druginfo/meds/a611003.html

^[5] https://medlineplus.gov/druginfo/meds/a614047.html

^[6] https://www.creative-peptides.com/blog/classification-of-impurities-in-synthetic-peptide-drugs/

Acknowledgement: Thomas Jadallah, our intern at Emery Pharma from June 2024, for his valuable contributions to this blog.