Peptide Impurity Synthesis

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As a special class of drugs, synthetic peptides are significantly different from classical small-molecule chemical drugs in their amino acid sequences, potential secondary and tertiary structures, preparation process and quality control requirements, and are diverse and complex. United States The Peptide Expert Group of the Pharmacopoeia Committee published related articles in 2014, discussing the quality control standards and corresponding analytical methods of peptide drugs. The amino acid sequence, peptide spectrum, amino acid composition, etc. in the quality control items are unique quality control items for polypeptide drugs, and are also routine inspection items for peptide drugs. Compared with classical small molecule drugs, synthetic peptide drugs have certain particularities in identification and related substance inspection items.


To study the generation of impurities in peptide synthesis, the main aspects are as follows:

1. The process lacks impurities, and the solid-phase synthesis of peptides cannot be 100% condensation in every step. It is inevitable that there will be the loss of some amino acids, and the polypeptide chain itself may break, and some structures may be modified.

2. Degrade impurities. For example, some amino acids are prone to hydrolysis, oxidation or even polymerization. Ichq3 also requires the study and control of solvent residues, elemental impurities and genotoxicity. At present, when applying for polypeptide products, in addition to the first two types of impurities, the latter solvent residues, elemental impurities and genotoxic impurities should be considered.


At present, Omizzur provides customized synthesis of impurities of a series of drug peptides to facilitate your research in impurities. Some products are as follows. If you need other products, please consult our customer service personnel:


Impurities generated during the synthesis of peptide drugs

ImpuritiesSource of impuritiesIdentification methodHPLC detection
Missing peptideProcess of synthesisLC-MS or LC-MS/MS+
Inserted peptideStarting material or synthesisLC-MS or LC-MS/MS+
Cleavage peptideProcess of synthesisLC-MS  +
EpipeptideStarting material or synthesisSynthetic epipeptide as control for HPLC+/-
Alternative (Leu /Ile)Starting material Synthetic or purified analogs / amino acid analysis-
Side chain groups are modifiedSynthesis or stabilityLC-MS or LC-MS/MS+/-
Disulfide modificationSynthesis or stabilityLC-MS or LC-MS/MS++
Gln / Asn / C-terminal deamidationPeptide degradationLC-MS or LC-MS/MS+/-
AminoacetylationPeptide degradationLC-MS or LC-MS/MS++
PolymerPurification or storage//


1. epipeptide impurity

Epipeptide impurities are peptide chains formed by amino acid residues with one or more unexpected chiral configurations in the amino acid sequence. Epipeptide impurities may form optical isomers in raw amino acids, so they need to be strictly controlled; It may also be formed by epimerization during peptide chain synthesis


2 chain end impurities

There are two kinds of chain end impurities: one is the impurities produced by N-terminal acylation or C-terminal deacylation. This kind of impurity is easy to obtain impurity reference substance in the process of synthesis or preparation, and the research is not difficult


3 side chain impurities

The side chain impurities can be divided into two categories: the impurities introduced by the side chain protective group and the impurities introduced by the amino acid side chain with reactive activity. In the process of peptide synthesis, there may be incomplete removal of side chain protective groups in the process of acid hydrolysis. At the same time, the carbon positive ions formed may be coupled with potential nucleophilic sites in the peptide chain to form other impurities


4 oxidation / reduction impurities

In the process of solid-phase synthesis, oxidation or reduction reaction may occur to specific amino acid residues. Impurities due to the oxidation of cysteine and methionine residues have been found in cabetoxin and eldoxin respectively


5 polymer impurities

During the purification and placement of peptide drugs, a certain amount of polymer impurities may be generated due to the reactivity of the end groups or the change of disulfide bonds. For example, there are as many as 6 kinds of polymer impurities found and identified in desmopressin. Polymer impurities can generally be effectively detected by molecular exclusion chromatography


Quality control items and analysis methods of peptide drugs

Inspection itemsAnnotation
StructureThe sequence is described by a 3-letter code; If the sequence is short, the ChemDraw structure can be used
HPLCThe method is the same as that of related substances, and is calculated based on the content of the reference substance
M/SShall be within ± 1.0 mass unit of theoretical value
MS-MS sequencingComplex for longer sequences
IR
Applicable to some peptides
NMRMay be difficult to solve the spectrum when the sequence is long
Amino acid analysisHydrolysis process shall be specified
Advanced structureUsed to study the secondary or tertiary structure of peptides in aqueous solution (Circular dichroism, NMR and FTIR )
Enantiomeric purityAnalysis of chiral amino acids
Residual solventFocus on the solvent used in the last step of the production process
Heavy metalMetal catalyst used in the production process; Heavy metals introduced by production equipment should also be considered
Microbial limit / bacterial endotoxinThis inspection is required for API for injection


Example: some peptide impurity research varieties:


NameImpuritiesProbability

Exenatide :

Endo-39Ser-ExenatideHigh
Endo-34Ser-Exenatide Medium
Endo-9Ser-ExenatideMedium
Endo-12Ser-ExenatideMedium
Des-4Gly-ExenatideHigh
Des-29Gly-ExenatideMedium
Des-34Gly-ExenatideMedium
……………………

Calcitonin Salmon:


Des-Thr21 Calcitonin Salmon:

High

Endo-Asn4 Calcitonin Salmon

High
Endo-Asn27 Succinamide Calcitonin SalmonMedium
Des-Pro23 Calcitonin Salmon:High

Des-Gly28 Calcitonin Salmon:

High
Des2-Ser Calcitonin SalmonHigh
Formyl Lys11 Calcitonin SalmonHigh
Formyl Lys18 Calcitonin SalmonHigh
Acetyl-Lys18Calcitonin Salmon High
……………………

Vasopressin[Asp5] vasopressinHigh
[Glu4] vasopressinMedium
[Gly9-OH] vasopressinMedium
[His2]-VasopressinMedium
[D-Cys6]-VasopressinMedium
[D-Arg8]-VasopressinMedium

[Leu7]-Vasopressin


……………………

Teriparatide [Des-Ser1]-PTH Teriparatide High
(Asp10)-PTH(1-34) Teriparatide   High
[D-Met18]TeriparatideMedium
[D-Asn33]-TeriparatideMedium
Glu6-PTH(1-34) Teriparatide Medium
PTH(1-31) Teriparatide  Medium
[Endo-Ser1]-PTH Teriparatide Medium
S-34-F[Met(0)18] Teriparatide Medium
……………………