The instability of peptides is one of the main problems in their formulation research, and there are many reasons for this. But there are not many main reasons for instability of a certain peptide. A detailed study of the effects of external conditions (such as pH, temperature, light, oxygen concentration, etc.) on peptide stability can help design rational formulation. Although the mechanism by which additives stabilize peptides is not yet fully understood, the use of additives is still one of the main means to improve the stability of peptide formulations. The application of analytical methods such as CD and DSC can help quickly screen for suitable additives.
Reasons for peptide instability:
Deamidation reaction: In the deacetylation reaction, Asn/Gln residues are hydrolyzed to form Asp/Glu. The non enzymatic deamidation reaction is carried out. The amide groups in the Asn Gly structure are more easily hydrolyzed, and the amide groups located on the surface of the molecule are also more easily hydrolyzed than those inside the molecule.
There are two main reasons why peptide solutions are prone to oxidation: one is the contamination of peroxides in the solution, and the other is the spontaneous oxidation of peptides. Among all amino acid residues, Met, Cys, His, Trp, Tyr, etc. are the most easily oxidized. Oxygen partial pressure, temperature, and buffer solution also have an impact on oxidation.
Hydrolysis: Peptide bonds in peptides are easily hydrolyzed and broken. Peptide bonds formed by Asp are more easily broken than other peptide bonds, especially Asp Pro and Asp Gly peptide bonds.
Formation of incorrect disulfide bonds: Exchange between disulfide bonds or between disulfide bonds and thiol groups can form incorrect disulfide bonds, leading to changes in tertiary structure and loss of activity.
Racemization: Except for Gly, the alpha carbon atoms of all amino acid residues are chiral and easily undergo racemization reactions under alkaline catalysis. Among them, Asp residues are most prone to racemization reactions.
β - elimination: β - elimination refers to the elimination of functional groups on the β - carbon atom in amino acid residues. Cys, Ser, Thr, Phe, Tyr and other residues can be degraded through β - elimination. β - elimination is prone to occur at alkaline pH, and temperature and metal ions also have an impact on it.
Denaturation, adsorption, aggregation, or precipitation are generally related to the destruction of tertiary and secondary structures. In the denatured state, peptides are often more prone to chemical reactions and their activity is difficult to recover. In the process of peptide denaturation, intermediates are first formed. The solubility of intermediates is usually low, making them easy to aggregate and form aggregates, which in turn form visible precipitates to the naked eye.
The surface adsorption of proteins is another headache problem encountered during their storage and use, such as riL-2 adsorbing on the surface of the pipeline during Qu perfusion, causing activity loss.