Peptide factors and protein drugs are primarily cleared from the body through degradation, excretion, and receptor-mediated endocytosis. Peptide factors with a molecular weight of less than 20 kDa are easily filtered through the glomeruli during metabolism. They are partially degraded by proteases in the renal tubules and excreted in the urine, leading to a short half-life. To maintain therapeutic efficacy, large and frequent doses are required, which can cause patient discomfort and side effects. In recent years, researchers have focused on chemical modifications, gene fusion, site-directed mutagenesis, and formulation improvements to develop long-acting peptide drugs.
1. Causes of Peptide Instability
1.1 Deamidation Reaction: In this process, Asn/Gln residues hydrolyze to form Asp/Glu. Non-enzymatic deamidation is influenced by environmental conditions and peptide structure. Higher pH and temperature promote deamidation, especially in Asn-Gly structures. Amide groups on the surface of molecules are more prone to hydrolysis than internal ones.
1.2 Oxidation: Peptide solutions oxidize for two main reasons: contamination with peroxides and spontaneous oxidation. Among amino acid residues, Met, Cys, His, Trp, and Tyr are the most susceptible to oxidation. Oxygen partial pressure, temperature, and buffer solutions also affect oxidation.
1.3 Hydrolysis: Peptide bonds are prone to hydrolytic cleavage, with Asp-containing bonds, particularly Asp-Pro and Asp-Gly, being the most vulnerable.
1.4 Formation of Incorrect Disulfide Bonds: Exchange between disulfide bonds or between disulfide bonds and thiol groups can lead to incorrect disulfide bonds, altering the tertiary structure and causing loss of activity.
1.5 Racemization: Except for Gly, the α-carbon atoms of all amino acid residues are chiral and can undergo racemization under alkaline conditions. Asp residues are the most prone to racemization.
1.6 β-Elimination: This refers to the elimination of groups from the β-carbon atom of amino acid residues. Cys, Ser, Thr, Phe, Tyr residues are susceptible to β-elimination, especially under alkaline pH conditions, with temperature and metal ions also influencing the process.
1.7 Denaturation, Adsorption, Aggregation, or Precipitation: Denaturation is generally associated with the disruption of tertiary and secondary structures. In the denatured state, peptides are more prone to chemical reactions, and their activity is difficult to recover. During denaturation, intermediates form, which often have low solubility and tend to aggregate, leading to visible precipitation. Surface adsorption of proteins is another issue during storage and use, which can lead to loss of activity, as seen with riL-2 adhering to tubing during perfusion.
2. Methods to Improve Peptide Drug Stability
2.1 Site-Directed Mutagenesis: By replacing residues that cause peptide instability or introducing residues that enhance stability through genetic engineering, peptide stability can be improved. For example, in the peptide chain of interleukin-2, Cys-125, which easily forms incorrect disulfide bonds, was replaced with Ser or Ala, resulting in twice the biological activity and a half-life extended from a few minutes to 1.2 hours.
2.2 Chemical Modifications: Selecting appropriate modification methods and controlling the degree of modification can enhance biological activity, thermal stability, resistance to protease degradation, reduce antigenicity, and prolong the half-life of peptides in the body. For example, single methoxy polyethylene glycol (mPEG) is widely used to extend the half-life of protein drugs. However, random chemical modifications can lead to product heterogeneity and reduced drug activity, so enzyme-mediated site-specific modifications are being explored.
2.3 Gene Fusion: Gene fusion techniques can extend the half-life of peptide and protein drugs by increasing molecular weight or altering receptor affinity. Human serum albumin (HSA) is a commonly used carrier protein with a molecular weight of about 66 kDa and a half-life of two weeks. HSA fusion proteins have been used to extend the half-life of growth hormone, interferon-α2, and IL-2. Other methods include fusing cytokines or antibodies to prolong drug efficacy.
2.4 Additives: Additives like sugars, polyols, gelatin, amino acids, and certain salts can improve peptide stability. They work by forcing more water molecules around the peptide, stabilizing its structure, or replacing water during freeze-drying to stabilize the natural conformation.
2.5 Freeze-Drying: Many chemical reactions, such as deamidation, β-elimination, and hydrolysis, require water. Reducing the water content through freeze-drying increases the denaturation temperature of peptides, enhancing stability.
