Peptide Knowledge Center

What is Peptide Impurities


Peptide is a compound formed by multiple amino acids connected by amide bond, which can be prepared by gene recombination expression, biological extraction, chemical synthesis and other methods. The chemical synthesis of peptide drugs has its own characteristics in the preparation method, structure confirmation, quality research and so on, which can not be simply classified as small molecule chemical drugs or biological products.

Article category: Biomedical, Peptide Synthesis, Organic Synthesis, Peptide R&D

Article keywords: Peptide impurities, peptide impurity synthesis, What is peptide impurities?

According to the characteristics of impurities in the chemical synthesis of peptide drugs, they can be divided into peptide-related impurities and non-peptide impurities.

1. What is Peptide-related Impurities?

Peptide related impurities (or related substances) are related to the target molecular structure. It is one of the important indexes to reflect the chemical purity of peptide. Peptide-related impurities can be caused by the introduction of starting materials, side reactions in the production process, or degradation during long-term storage. 

Omizzur Biotech lists some common categories and sources of peptide-related impurities in the table below ,only as a common example, and can not represent the whole situation, it should be combined with product characteristics assessment and analysis, hope to help you. 

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Categories and sources of peptide-related impurities:




Missing peptide

Missing one more than one amino acid

Synthesis or degradation

Insertional peptide

Missing one more than one amino acid

Starting material or synthesis


There is one more amino acid that is different (from the target sequence)

Starting material or synthesis

Heterotropic peptide

There is one more than one differentially isomerized amino acid

Starting material, synthetic or degraded

Asp/Asn related impurities

A sequence containing aspartate/succinimide

Loss of water or ammonia from the side chain of Asp or Asn respectively and cyclization to the main chain

Sequences containing β-Asp

Water unwinding ring of aspartate/succinimide intermediates during synthesis or storage

Asp stereoisomer (diastereoisomer)

The asparagine/succinimide intermediates are differentlyisomerized and then opened during synthesis or storage

Chain break product

Complete hydrolysis of asparagine/succinimide intermediates during synthesis or storage

Pyroglutamic acid

A sequence containing n-terminal glutamine or glutamate



The end group has reactivity or changes of disulfide bond to trigger peptide polymerization

Synthesis or degradation

Sulfur-containing impurity

Sulfhydryl oxidation disulfide bond oxidation or reduction

Synthesis or degradation


Glutamine, asparagine or C-terminal deamidation

Synthesis or degradation

Acetylation of amino functional groups

Synthesis or degradation

Because the structure and properties of some peptide-related impurities are similar to those of the principal components, it may not be completely separated by a single principle analysis method, and a variety of detection methods with different principles are recommended for the detection of related substances. 

In addition to the common high performance liquid chromatography, ion exchange chromatography and capillary electrophoresis can also be used for the detection of relevant substances. Polymers can be examined by molecular exclusion chromatography, polyacrylamide gel electrophoresis and so on.

The chromatographic behavior of heterotropic peptide impurities is very close to that of principal components, so it is difficult to identify and separate them. Deuterium reagents can be used to hydrolyze peptides (such as deuterium chloride (DCI)/ deuterium water (D2O)), Marfey reagent (FDAA), phthalaldehyde (OPA) or trifluoroacetic anhydride (TFAA) are used to derivate the hydrolyzed amino acids, and the content of each chiral isomer of amino acids is determined by corresponding detection methods. The amino acids prone to racemization in the peptide chain were identified, and the corresponding isomer impurity reference products were prepared for targeted study.


The problem of specificity should be emphasized during the verification of the relevant substance inspection methodology. The separation degree of process impurities and degradation impurities with similar structure to the target peptide can be investigated, or the main peak-peak purity can be tested by crude peptide and forced degradation test samples. 

Since peptides are generally end-absorbed, peak purity cannot be effectively reflected by DAD detector, so the main peak purity can be studied by LMS and other methods. In conjunction with the specific impurities identified in the relevant substance item, the corresponding impurity reference is used for comprehensive methodological validation.

Peptide Impurities Images:

What is Peptide Impurities

2: Non-peptide impurities

Non-peptide impurities are impurities introduced in the process that are not related to the target molecular structure, including reaction reagents and solvents, metal catalysts, shrinkage agents and other residues. The study of such impurities can be carried out according to the relevant guidelines for small molecule chemicals. In the synthesis process of peptide drugs, different types of shrinking agents and protective groups are needed, and such substances may generate small molecular by-products containing warning structures in the reaction process. Reasonable control strategies should be developed. For example, targeted removal efficacy studies can be conducted with representative compounds or compounds generated near the final product process step to demonstrate that the separation and purification methods used can effectively remove the corresponding impurities.

Tip: The influence of the choice of peptide chemical synthesis process on impurities

According to the different reaction carrier, the synthesis process of peptide apis mainly includes liquid phase synthesis and solid phase synthesis. According to the sequence of peptide bond formation, the synthesis process of peptide raw materials mainly includes linear synthesis and fragment condensation synthesis. The length of the target peptide chain and the characteristics of amino acids should be considered comprehensively when selecting the synthesis process. 

It should be noted that some impurities similar to the structure of the target peptide chain will be produced during peptide synthesis, such as missing peptides, inserted peptides, misjunction peptides and differential peptides, etc. The production and accumulation of such impurities will increase the difficulty of the finished peptide purification, and the production of such peptide-related impurities should be avoided or reduced when the synthesis process is selected.


Taking solid phase synthesis technology as an example, attention should be paid to amino acid protection/deprotection strategy, selection basis of shrinkage mixture, amino acid coupling, cleavage, purification, peptide chain modification and enrichment lyophilization during process development. For the selection of peptide purification methods, factors such as the length of peptide chain, polarity, charge and purity of crude product should be comprehensively considered, and the quality of samples before and after purification should be compared to prove that the proposed purification method can effectively achieve purification effect. 

The reasonableness of freeze-drying conditions should be fully considered in the development of freeze-drying process,detailed technological parameters should be provided, and the quality of samples before and after freeze-drying should be compared. If extreme pH values or organic solvent conditions are present in processes such as filtration, necessary membrane compatibility studies should also be considered.