1. Background

     peptide drugs have the advantages of high biological activity, strong specificity, weak toxic reaction, low aggregation in vivo, less interaction with other drugs, and high affinity with receptors in vivo. In recent years, polypeptide drugs have been widely used in the prevention and treatment of cancer, metabolism, cardiovascular and other diseases, and are one of the hot research and development spots of pharmaceutical enterprises at home and abroad.

　　As a special class of drugs, the structure of synthetic peptides is obviously different from that of proteins and small molecule drugs. At the same time, the amino acid sequence, potential secondary and tertiary structure, preparation process and quality control requirements of polypeptide drugs are significantly different from classical small molecule chemical drugs, with diversity and complexity, which may also be the reason that domestic and foreign guidance documents rarely involve such varieties. USP released the "Quality attributes of synthetic peptide drug substances" [1] on June 11, 2021, which has great guiding significance for the quality research strategy of synthetic peptide drugs. Therefore, this paper extracts and discusses the important content of the guiding principle.

　　2.Main content

　　Peptide drug-related impurities and their contributing factors were analyzed from the following aspects: (1) starting materials, (2) peptide characterization, (3) peptide content detection, and (4) impurities.

　　2.1Material control

　　The materials used for the synthesis of peptides include SM, solvents and reagents required for the production of peptide apis. Starting materials (SM) are raw materials, intermediates or active ingredients and peptide fragments used in the production of peptide apis. Typically, the starting material quality control items are identification and purity, including related substances and enantiomer impurities, which have a direct impact on the quality of the polypeptide drug product. Acceptable criteria for SM quality control are determined based on process development, process validation data, historical batch data, and risk assessment.

　　2.2 Peptide characterization methods

　　 The quality standard should fully reflect the key quality attributes (CQA) of the product. Summary of common detection methods for drug characterization and quality control of peptides

　　2.3 Content and peptide content

　　The content of polypeptide apis is generally determined by anhydrous and non-counter ion meters, usually by chromatographic methods. When HPLC is used, quantitative standards, such as pharmacopeia standards or homemade working reference products, are generally used.

　　The earliest traditional method for determining peptide content is the simple mass balance method, which subtracts the content of water, counter-ions, related substances and other impurities in the sample by 100%. In addition, N analysis, Kjeldahl nitrogen determination, or chromatography with a nitrogen chemiluminescence detector (NCD) can be used.

　　Amino acid quantitative analysis is also a traditional analytical method for the determination of peptide content, but due to the relatively complex operation process of this method, the determination results may be different. When using this method to calculate the peptide content, the peak area of each amino acid in the hydrolyzed sample was compared with that of the amino acid standard, which was calculated by external standard method. In addition, the polypeptide hydrolysis method should be validated to ensure good recovery, and only stable amino acids with good recovery can be selected to calculate the average peptide content.

　　Although quantitative nuclear magnetic (qNMR) generally requires an internal standard that can be completely separated from the peptide-related signal peak, it is still an alternative method for peptide content determination. The versatility of qNMR makes it suitable for the analysis of peptides without suitable UV-absorbing groups, and it is very suitable for the study of peptide aggregation.

　　The determination of peptide content containing tryptophan (Trp) or tyrosine (Tyr) can be obtained by measuring the absorbance of UV at 280nm, and the molar absorption coefficient can be calculated using theoretical values based on the peptide sequence, but other analytical methods should be used to verify the determination results. UV assay is a simple and rapid method for frequent detection, on-line real-time determination and process analysis.

　　2.4Impurity

　　The impurity spectra of polypeptide apis may vary significantly according to the different production processes used, so it is difficult to summarize the impurity spectra and control strategies uniformly. In addition, because the production process of peptide drugs is more complex for smaller molecules, the impurity research guidelines applicable to small molecules may not be applicable to peptides.

　　2.4.1 sort

　　The USP 1086 Study Guide for Impurities in apis classifies impurities into three categories: organic, inorganic, and residual solvents. However, from practical applications, polypeptide impurities can be divided into polypeptide-related impurities and non-polypeptide impurities. (1) Polypeptide-related impurities, also known as "related substances", refer to a class of impurities related to the structure of the target molecule. The classification, sources and identification methods of polypeptide-related impurities are summarized in Table 3. It should be noted that only the most common impurities in polypeptides are summarized here, and the actual research process should not be limited to this. (2) Non-peptide impurities refer to a class of impurities introduced in the production process that have nothing to do with the target molecular structure, including residual materials, reagents and additives such as catalysts and solvents, as well as reaction by-products introduced thereby, as detailed in Table 4.

　　Peptide purity is usually determined by HPLC, which should be able to separate starting materials, process-related impurities, and degradation impurities. Multiple analytical methods are recommended to detect as many impurities as possible. For peptides containing hydrophobic residues, RP-HPLC method is preferred. Instead, try using ion exchange chromatography or high performance liquid chromatography in composite mode. For hydrophilic peptides, the stationary phase (HILIC column) of hydrophilic interaction chromatography may obtain better separation, and anion or cation exchange chromatography may also obtain better results. In addition, liquid chromatography-mass spectrometry (LC-MS) is very effective for the identification of impurities.

　　Limit establishment: In order to reasonably establish acceptable limits for polypeptide-related impurities, impurities can first be further divided into specific impurities and non-specific impurities, specific impurities can be further divided into known impurities and unknown impurities, impurities confirmed by structure are classified as known impurities, quality control impurities and their limits are based on toxicological studies, clinical trials or scientific assessments based on the structure of impurities. Limits for both known and unknown impurities shall be specified in the release criteria. The ICH Q3A (R2) Guidelines for the Study of Impurities in new apis specify a limit of 0.10% or 0.05% for a single unknown impurity, but clearly state that it does not apply to peptide products; The EP sets a higher acceptance limit (0.5%) and is recognized by the EU member States' drug authorities; Other regulatory authorities such as the US FDA believe that the control limits of unknown impurities in new peptide products should be analyzed according to the characteristics of specific varieties.

　　2.4.2 Control strategy for polypeptide-related impurities

　　The control strategy of polypeptide-related impurities should be based on the characteristics of the process, and fully consider the impurities introduced by the starting material and the impurities introduced during the synthesis and purification of the peptide. A properly validated process control analysis procedure may be used to ensure the quality of the final API, especially if the impurity content is too low to be accurately determined by release methods, and other analytical methods should be developed.

　　3. Sources and common analytical methods of polypeptide-related impurities

　　Note: The detection of isomer impurities can not be directly detected by conventional amino acid chiral column. Generally, the peptide needs to be hydrolyzed and derivated, separated by hand gas/liquid chromatography column, and analyzed by GC-MS or HPLC-MS. The method needs to be verified.

　　4. Control strategy for non-polypeptide-related impurities

　　If a reagent used in the manufacturing process has a risk of residue in the API, the solvent residue should be specified in the quality standard, such as trifluoroacetic acid (TFA) used in cracking and Fmoc deprotection. Another category of potential process impurities is elemental impurities, several sources of which are described in ICH Q3D (R1), the 232-element impurity Limit, and the 233-element impurity analysis method: intentional addition of catalyst residues during synthesis, introduction into process equipment or storage containers, or introduction into starting materials. Acceptable limits for elemental impurities are determined based on the allowable daily exposure (PDE) from toxicological data, as specified in ICH Q3D (R1).

　　5. Conclusion

　   peptide drugs are different from small molecule drugs because of their complex structure, long synthetic steps, and many kinds of starting materials. Therefore, the quality control of synthetic polypeptide should be characterized by the analytical method of space separation considering different principles. (1) Identification. The analytical methods used in the quality standard focus on method specificity and choose more deterministic methods such as HPLC, MS, NMR, etc., rather than color response identification. (2) Impurity analysis. The analysis of impurity spectrum should be based on the specific analysis of the adopted process route, rather than completely copying the pharmacopoeia or import registration standards of various countries, and distinguish which are process impurities and which are degraded impurities, so as to formulate reasonable measures to optimize the process and inhibit the generation of impurities [2]. In addition, the analysis of impurities should be as much as possible to choose different principles of analysis methods, polypeptide-related substances analysis methods usually use reverse high performance liquid chromatography (RP-HPLC), which is to be separated by the polarity difference of the analyte; In addition, we should try to use ion exchange chromatography based on charge separation (IEX-HPLC), molecular exclusion chromatography based on molecular weight separation (SEC-HPLC), hydrophobic chromatography based on hydrophobicity (HIC-HPLC), chiral analysis based on isomers, etc. The impurities of polypeptide drugs can also be comprehensively characterized by capillary electrophoresis (CE) and liquid chromatograph-mass spectrometry (LC-MS), so as to avoid the limitation of using a single method that some impurities can not be detected in the main component. If there is data indicating the intestinal glucagon analogues exenatide, The purity of the product tested by RP-HPLC was 97.8%, but the purity of the product tested by SCX-HPLC was only 88.0%, and the impurity [N-His1] was as high as 9.8%[3]. In addition, small molecule drugs usually use DAD detector to determine the main peak purity to determine the specificity of the analysis method, but for polypeptide drugs, the impurities and the structure of related substances are very similar, the absorption wavelength such as 214nm or 220nm is the maximum absorption wavelength of the peptide bond of the non-target molecule characteristic absorption. Therefore, the peak purity result cannot determine whether the target peak of the peptide contains other impurities, and LC-MS is recommended to confirm the peak purity.