Reasons why peptides are unstable

Deamidation reaction: In the deacylation reaction, Asn/Gln Residues are hydrolyzed to form Asp/Glu.
Proceeding of non-enzymatic deamidation reaction. exist Asn-Gly- The amide groups in the structure are easier to hydrolyze, and the amide groups located on the surface of the molecule are also easier to hydrolyze than the amide groups inside the molecule.
 

There are two main reasons why oxidized polypeptide solutions are easily oxidized.
First, there is peroxide contamination in the solution.
The second is the spontaneous oxidation of polypeptides.
Among all amino acid residues, Met, Cys and His, Trp, Tyr Etc. are most susceptible to oxidation. Oxygen partial pressure, temperature and buffer solution also have an impact on oxidation.

Hydrolysis: Peptide bonds in polypeptides are easily broken by hydrolysis. Depend on Asp The peptide bonds involved in the formation are more easily broken than other peptide bonds, especially Asp-Pro and Asp-Gly Peptide bonds.

Forming incorrect disulfide bonds: Exchange between disulfide bonds or between disulfide bonds and sulfhydryl groups can form incorrect disulfide bonds, resulting in changes in tertiary structure and loss of activity.

Racemize: remove Gly Except, all amino acid residues αCarbon atoms are all chiral and are prone to racemization reactions under base catalysis. in Asp The residues are most susceptible to racemization reactions.

β-eliminate: β-Elimination refers to the amino acid residues in βElimination of groups on carbon atoms. Cys, Ser, Thr, Phe, Tyr All residues can pass through β-Eliminate degradation.
in alkaline PH prone to happen β-Elimination, temperature and metal ions also have an impact on it.

Denaturation, adsorption, aggregation or precipitation denaturation are generally related to the destruction of tertiary structure and secondary structure. In the denatured state, peptides are often more susceptible to chemical reactions and their activity is difficult to recover. During the denaturation process of polypeptides, intermediates are first formed. Usually the solubility of intermediates is low and they are easy to aggregate and form aggregates, which in turn form visible precipitation.
 

1) site-directed mutagenesis: The stability of polypeptides can be improved by genetic engineering means to replace residues that cause polypeptide instability or to introduce residues that can increase polypeptide stability.

2) chemical modification: There are many chemical modification methods for polypeptides, and the most studied ones are PEG Grooming. PEG It is a water-soluble polymer compound that is degradable in the body and non-toxic. PEG After combining with polypeptides, it can improve the solubility of the polypeptide, adjust biocompatibility, improve thermal stability, resist degradation by proteases, reduce antigenicity, and extend half-life in the body. Choosing the appropriate modification method and controlling the degree of modification can improve or improve the biological activity of the original product.

3) additive: The stability of peptides can be improved by adding additives such as sugars, polyols, gelatin, amino acids, and certain salts. Sugars and polyols increase the stability of the peptide by forcing more water molecules around the protein at low concentrations. During the freeze-drying process, the above-mentioned substances can also replace water and form hydrogen bonds with the polypeptide to stabilize the natural conformation of the polypeptide, and can also increase the glass transition temperature of the freeze-dried product. In addition surfactants such as SDS, Tween, Pluronic, It can prevent surface adsorption, aggregation and precipitation of polypeptides.

4) Freeze-dried: A series of chemical reactions that occur in polypeptides such as deamidation, β-Elimination, hydrolysis, etc. all require the participation of water, and water can also be used as the mobile phase for other reactants. In addition, reducing the water content can increase the denaturation temperature of the polypeptide. Therefore, lyophilization improves the stability of peptides