2. Biosynthesis (gene recombination technology)

Gene recombination technology is to construct the gene sequence of polypeptide on the vector to form recombinant DNA expression vector, and to express, extract and purify polypeptide molecules in prokaryotic or eukaryotic cells.

This method is suitable for the preparation of target peptides with more than 50 amino acids and is easy to obtain. With the improvement of the technology of peptide production by genetic engineering, the development and clinical application of peptide drugs by genetic engineering have been accelerated.

3. Chemical synthesis (including liquid-phase and solid-phase methods)

Liquid phase method is the first developed peptide synthesis method: the process is to prepare the required amino acid or short peptide into a solution, in which the amino end of one amino acid and the carboxyl end of another amino acid or short peptide and the side chain group not involved in the reaction are protected by chemical groups, while the carboxyl end of the amino acid or short peptide involved in the reaction needs to be activated, and then coupling occurs in the solution 。

After the reaction, the raw materials and activators which were not involved in the reaction were separated and removed by various methods to obtain the purified peptide products. The synthesis of polypeptide in liquid phase is mainly divided into natural chemical connection and stoodinger connection.

Natural chemical linkage is the basic method of peptide segmented synthesis, and its limitation is that the synthesized peptide must contain Cys residues, which limits the application scope of natural chemical linkage method. The extension of natural chemical linkage method includes chemical regioselective linkage, delectable linkage and light sensitive linkage;Staudinger joining method is another basic fragment joining method, which opens up a broader way for peptide fragment joining. Orthogonal chemical joining method is the extension of Staudinger joining method, which can improve the condensation rate between fragments by simplifying the phosphine thioester auxiliary group.

SPPs (very important!) is a popular method of peptide synthesis at present. The principle is to fix the carboxyl of the first amino acid at the C-terminal of the peptide sequence on the insoluble carrier (resin + linker) through esterification, and then take the amino acid as the amino component to form peptide bond after removing the amino protection group and reacting with the excessive activated carboxyl component After that, the deprotection, condensation and washing operations are repeated to extend the length of peptide chain, so as to obtain the required length of peptide chain.

Finally, the peptide chain is cut off from the resin by acid (such as trifluoroacetic acid) and all protective groups are removed at the same time. After purification by high performance liquid phase, the required peptide can be obtained.

After years of research and development, it not only overcomes the time-consuming and cumbersome of liquid-phase synthesis, but also reduces the loss caused by operation. The biggest advantage is that all purification steps in synthesis are completed by simple washing and filtration, greatly reducing the difficulty of purification. It has the advantages of convenient and quick operation, simple operation and high yield. Its advantage also shows that it is easy to realize automation and make it become It is the preferred method for peptide synthesis.

Peptide synthesizer

The world's first real peptide synthesizer appeared in the late 1960s to the early 1970s. It uses nitrogen bubbles to stir reactants, and uses computer program control to realize limited automatic synthesisTable products Beckman 990 peptide synthesizer launched by Beckman company and Vega's 296 peptide synthesizer launched by Vega's biotechnology company; the second generation peptide synthesizer was born in the 1980s, representing PS3 peptide synthesizer launched by protein technologies company and act peptide synthesizer model 90 launched by advanced Chemtech company; the third generation peptide synthesizer The synthetic instrument was born in the 1990s. Its representative products are abi433 peptide synthesizer of Applied Biosystems in the United States and cs336 non dead angle peptide synthesizer of CS bio.

Quality control of polypeptide drugs

The quality control of peptide drugs comes from the synthesis of peptide drugs. The quality research can refer to the general research ideas of chemical drugs. Quality control mainly includes properties, identification and inspection, including general impurities, related substances, residual solvents, crystal form, particle size, clarity and color of solution, loss on drying and water, isomers, in addition, there are special inspection items for peptide drugs, such as amino acid ratio, biological activity inspection, related peptide, anti ion content, peptide diagram analysis, polymers, antihypertensive substances Inspection and other items, content and titer determination. For the synthesized peptides, the physical and chemical properties need to pay attention to the isoelectric point, specific rotation and the solubility of buffer besides the general conventional items.

Because peptide drugs are easy to be enzymolysis, short half-life and poor fat solubility, they are mostly administered by injection. The main dosage forms are freeze-dried powder injection and injection. For general injection and freeze-dried powder injection, due to the unstable and easy degradation of polypeptide substances, the quality control project focuses on the inspection of related substances. The related substances for peptide synthesis can be divided into four categories, mainly including:

1. Process impurities, such as missing peptide, breaking peptide, inserted peptide, unprotected peptide and other peptide related substances brought in during synthesis;

2. Degradation products produced by unstable factors such as deamidation of degradation impurity ~ peptide, oxidation, reduction, hydrolysis, disulfide bond mismatch, β - elimination, etc;

3. Polymer dimer and polymer;

4. Optical impurity ~ racemized, non enantiomeric impurity.

Peptide drugs are mainly injection and injection powder, which are easy to degrade and produce degradation impurities in the process of production and storage. The main detection methods of polypeptide drugs are: reversed phase high performance liquid chromatography, capillary electrophoresis, high performance liquid chromatography-mass spectrometry, high performance molecular exclusion chromatography.

reference:

1. R Ismail，I Csóka.Novel strategies in the oral delivery of antidiabetic peptide drugs-insulin，GLP1 and its analogs[J]. European Journal of Pharmaceutics & Biopharmaceutics, 2017.

2. S Eskandari，T Guerin，I Tot，et al.Recent advances in self-assembled peptides: Implications for targeted drug delivery and vaccine engineering[J].Advanced Drug Delivery Reviews，2016.

3. S Shi，PKNguyen，HJ Cabral et al. Development of peptide inhibitors of HIV transmission[J].Bioactive Materials，2016，1 (2) :109-121

4. Qu Peng, Song Li, Zhao Haodong, et al. Research progress in peptide synthesis [J]. Modern Chinese medicine, 2015

5. Tian Wenjing, Ren Xue, Liao Haiming, et al. Research progress of peptide drug quality control [J]. Journal of drug analysis, 2013

6. Nie Caihui, Xu Hanmei. Development status of polypeptide drugs [J]. Pharmaceutical progress, 2014

7. Wang kequan, Xu Hanmei. Research progress of polypeptide drugs [J]. Pharmaceutical progress, 2015

8. Part of the data comes from Thomson Reuters

   Shengnuo bio PRODUCT

Best sales manager contact

About Shengnuo

Chengdu Shengnuo Biotechnology Co., Ltd. has "Chengdu polypeptide drug engineering technology research center" in Chengdu, mainly engaged in polypeptide, polypeptide drug and beauty peptide research. Our zero defect has passed the FDA certification, and now it has become the first-class professional peptide drug and product development, technology transfer, technical service and peptide drug industry in the scale production and export of China's parks.