Archives

Overview of GLP-1 peptide drugs:

1. Liraglutide: A daily injectable GLP-1 analog engineered to resist DPP-4 degradation. Its fatty acid modification enhances albumin binding, prolonging its half-life and improving therapeutic outcomes.
2. Semaglutide: Offered in both injectable and oral forms, Semaglutide boasts greater potency and a much longer half-life (~170 hours) than Liraglutide, making it a favored choice for weight management.
3. Tirzepatide: This dual agonist of GLP-1 and GIP delivers superior glycemic control and weight reduction, setting it apart as the latest innovation in this drug class.

Degradation Pathways of GLP-1 peptide drugs:

1. Hydrolysis: Peptide bonds degrade in aqueous conditions, producing inactive fragments. Factors like pH, temperature, and ionic content influence hydrolysis rates.
2. Oxidation: Residues such as tryptophan, tyrosine, and histidine are prone to oxidation, forming impurities that may elicit immune responses. Environmental factors like air, light, and pro-oxidant excipients exacerbate this process.
3. Thermal Degradation: Heat accelerates hydrolysis and oxidation, leading to peptide aggregation and truncated sequences that undermine drug performance.
4. Photodegradation: Exposure to light triggers structural damage to peptides. Proper storage minimizes this risk.
5. Deamidation: Asparagine and glutamine residues undergo deamidation, creating acidic variants that alter stability and pharmacokinetics.

Impurity Formation in GLP-1 peptide drugs

Daicel Pharma Standards Contribution

Regulatory Considerations for Peptide Drug Impurities

1. A generic synthetic peptide should not contain impurities at levels exceeding those found in the RLD.
2. If a new impurity is detected in the generic peptide but absent in the RLD, its level should be controlled at no more than 0.5%. Additionally, a qualification study is required to assess potential immunogenicity risks. If no significant immunogenicity risk is observed, the impurity can be controlled at ≤0.5% by testing each batch.
3. Any unknown impurity that is not present in the RLD must be controlled at ≤0.1% in every batch.

Certification of Peptide Drug Impurities: Qualitative vs Quantitative aspects

• Qualitative Characterization identifies the structural composition of impurities. The qualitative characterization of GLP-1 peptide impurities involves HRMS and ¹H NMR. Since GLP-1 peptide impurities generally contain 30–40 amino acids, HRMS analysis is required to confirm their amino acid sequences and distinguish closely related variants. Amino acid composition analysis further establishes the number and types of amino acids present in the impurity, confirming its overall sequence characteristics. Elemental analysis, particularly nitrogen determination, provides additional insight into the total peptide content within the impurity sample.
• Quantitative Characterization determines impurity concentration, requiring potency evaluation along with structural confirmation. To determine peptide impurity potency, all impurity sources including organic, inorganic, counter ions and residual solvent content must be considered. The general potency calculation formula for peptide impurities is:

Key Analytical Parameters for Certifying Peptide Drug Impurities

Conclusion

Cyclic Peptide Drugs and Their Importance

Cyclic peptides are a class of molecules that have garnered significant attention in drug development due to their unique properties, including high stability, target specificity, enhanced potency, and cell permeability. Cyclic peptides are polypeptide chains taking cyclic ring structure consisting of 5-14 amino acids with a molecular weight of about 500 to 2000 Da. The ring structure can be formed by linking one end of the peptide and the other with an amide bond, or other chemically stable bonds such as lactone, ether, thioether, disulfide, and so on.

Peptide sequence cyclization helps to bind more efficiently with their respective receptors. The cyclic structure of peptides provides a large surface area for interaction with the targeted site. Cyclic peptide drugs are impervious to enzymatic hydrolysis as they don't have free amino and carboxyl ends, thus improving their stability. Usually, cyclic peptides show better biological activity compared to their linear counterparts due to the conformational structure, therefore allowing the enhanced binding toward target molecules or the selectivity by the receptor.

Some cyclic peptide drugs include Daptomycin, Telavancin, Dalbavancin, Lanreotide, Octreotide, Linaclotide, Plecanatide, Romidepsin, Vasopressin, Oxytocin, and Calcitonin. Many cyclic peptide drugs are from natural sources like cyclosporin A, Bactenecin, Lactocyclicin, and more. However, bio-engineered, synthetic cyclic peptide drugs are becoming common now.

How Do You Synthesize Cyclic Peptides?

The synthesis of Cyclic peptides involves Solid-Phase Peptide Synthesis (SPPS). In this method, peptide synthesis occurs linearly, immobilized on a resin bead with a disulfide linker or other chemically stable bonds such as lactone, ether, and thioether. After the linear peptide formation, possible protecting groups are cleaved, and the peptides are released by adding a base. Further, deprotonation of the N-terminal thiol group strikes the disulfide linker between the peptide and resin in intramolecular cyclization, giving the desired cyclic peptide.

Overall, synthesizing cyclic peptides requires careful control of protecting groups, coupling reagents, and reaction conditions to ensure high yields and purity.

Techniques for Synthesis of Cyclic Peptides

The preparation of cyclic peptide compounds uses various techniques: • combinatorial chemistry • de novo synthesis. • chemoselective ligation-mediated cyclization

Depending on the cyclization, the methods to synthesize cyclic peptides are head-to-tail, side-chain-to-side-chain, head-to-side-chain, and side-chain-to-tail. Cyclization of side chain amino acids occurs with disulfide bridge formation between cysteine. Further, backbone-to-backbone cyclization is by amide bond formation between N-terminal and C-terminal amino acid residues.

Moreover, there are other cyclic peptides like bicyclic/ tricyclic peptides and stapled cyclic peptides. Bicyclic peptides are effective enzyme inhibitors and stapled peptides facilitate peptide cell penetration and use cross-linkers to improve physiochemical properties.

Impurities in Cyclic Peptide Drugs

Impurities may arise from the synthesis of cyclic peptides. The API-related impurities are truncations, functional group modifications, insertion or deletion of amino acids, oxidations or reduction of functional groups, aggregates, and incomplete deprotection. Chromatographic media used in purification, and solvents also may contribute to impurity generation in cyclic peptide drugs. Degradation products occur due to changes in the drug product stored for a long time or exposure to light, temperature, water, or reaction to excipients. To ensure the safety and efficacy of cyclic peptide drugs, it is important to control and minimize impurities during the synthesis and purification processes.

General peptide impurities like N-Ac-impurity, de-amidation at Gln, Asn residues, deamidation at C-terminus, trisulfide impurity, Cyanoalanice @ Asn residue, des-Gly and endo-Gly of the terminus amino acids are common in cyclic peptides too. Degradation and other truncated peptides are also a major class of impurities in these cyclic peptides.

Apart from the above-described type of impurities, other impurities like Parallel and Anti-parallel dimers are often formed in cyclic peptides.

• Seq: Cys(1)-Tyr-Phe-Gln-Asn-Cys(6)-Pro-Arg-Gly-CONH2 (sulfur linkage between cysteines)
• Parallel dimers: 1,1’, 6,6’
• Anti-parallel dimers: 1,6’,1’,6

• Seq: H-Asn-Asp-Glu-Cys(4)-Glu-Leu-Cys(7)-Val-Asn-Val-Ala-Cys(12)-Thr-Gly-Cys(15)-Leu-OH(4-12), (7-15)-bis(disulfide)
• Parallel dimers: 4,4’, 7,7’, 12,12’, 15,15’;
• Anti-parallel dimers:  4,7’,4’,7, 12,15’, 12’,15; or 4,12’,4’,12,7,15’,7’,15 or 4,15’,4’,15, 7,12’,7’,12

• Seq: H-Cys(1)-Cys(2)-Glu-Tyr-Cys(5)-Cys(6)-Asn-Pro-Ala-Cys(10)-Thr-Gly-Cys(13)-Tyr-OH (3 disulfide bridges between 1-6, 2-10 and 5-13 of cysteines)

Conclusion