Wang Liangyou, Tianma, Suzhou: control of impurities in key process of peptide synthesis

< br / >, In particular, the prediction and strategy of difficult sequences in peptide synthesis, the formation of disulfide bonds, the synthesis of cyclopeptides and the modification methods such as PEGylation, phosphorylation and glycosylation of peptides have been studied in depthThe research projects that he participated in or presided over and completed include: the key funded project of National Natural Science Foundation of China (39430120), the Youth Science Fund Project of National Natural Science Foundation of China (30300436), nihgrantca1142994, and the major new drug creation (2011zx09102-001-16), the major special subject of National Science and technologythe polypeptide imitations with large-scale production process have been developed, including thymosin, octreotide, trepressin, desmopressin, bivalirudin, etifibatide, sermorin, somatostatin, leuprorelin, de Sherry Lin, triptorelin, goserellin, Bushrui Lin, oxytocin, cabergoline, Rick, digalaric, Ghani Rick, praline Lin peptide, Lanrui peptide, tripatide, salmon calcitonin, exenatide, lilalutide, somalutide, etcAmong them, 2 peptide APIs have obtained GMP certificate in China, 4 have passed CDE technical evaluation, and 1 has obtained kdmf Certificate in South KoreaThe varieties of peptide innovation drug process development that we cooperated in included vasopressin (clinical approval in 2016), antibacterial peptide (clinical approval in 2018), etc< br / >< br / >< br / > < br / > peptide molecules are large or small, and the small ones may consist of two or three or five amino acidsIt may be composed of dozens of amino acidsInsulin, for example, has two chains, a total of 51 amino acidsThere are 20 kinds of natural amino acids< br / >In fact, polypeptide drugs only account for about 2%, and there are about 80 kinds of polypeptide drugs on the market at presentIn 2018, the sales volume of peptides in the world was 28.8 billion< br / >! < br / > the chemical synthesis of polypeptide has a history of more than 100 yearsThe first discovery of polypeptide synthesis was made by German scientists, who won the Nobel PrizeIn 1965, insulin was synthesized for the first time in China, which was quite a sensation At that time, Peking University, Shanghai Institute of organic chemistry, Shanghai Institute of Biochemistry and other units jointly tackled key problems, and gradually developed China's peptide industry from scratch to now, forming a very complete industry < br / > < br / > in the history of polypeptide chemistry, many scientists have won the Nobel Prize, including Sanger of the UK Professor Merrifield and Tam have come to China International polypeptide Conference for many times < br / > three < br / > < br / > impurity control in peptide synthesis < br / > < br / > at present, peptide synthesis mainly includes chemical synthesis and biological synthesis Chemical synthesis method, we use more solid-phase method and liquid-phase method, biological method has fermentation method, enzyme method, gene engineering recombinant method Each of these methods has its own advantages and disadvantages There may be many methods to be adopted in production For example, in solid-phase synthesis, liquid-phase synthesis may also be considered The solid phase method may also be used to make some fragments in liquid phase synthesis Therefore, there are many ways to synthesize polypeptides < br / > for the solid-phase method, the first consideration may be the selection of resin, which is the carrier of our solid-phase synthesis and is very important Nankai University in China and Lanxiao in Xi'an have done very well in this respect < br / > the most basic reaction in peptide synthesis is condensation, in which two amino acids are condensed to form peptide bonds The condensing agent used in peptide synthesis has gone through several generations and can be produced in China < br / > the main reason why solid-phase synthesis of polypeptides has been recognized is that it is very simple in operation, because the synthetic people all know that the most difficult chemical reaction is the post-processing of the reaction Solid phase synthesis is to simplify the post-treatment Since professor Merrifield invented solid-phase peptide synthesis in 1963, it has been very popular Because in the factory, it's impossible for everyone to be an organic chemistry major to do the synthesis, and they often need some operators Solid phase synthesis is very suitable for simple operators, and even automation can be achieved Although it is not a very economic method, it is still a mainstream peptide production method < br / > at present, the evaluation of chemical synthetic peptide drugs is based on chemical drugs, so in pharmaceutical research, it is basically similar to small molecule synthetic drugs The main research contents include the research on the preparation process, structure confirmation, preparation prescription, quality and stability of API It is basically the same as other chemical drugs, but the polypeptide may have some characteristics of its own < br / > For example, the main chain of foreign lilaluptide is reconstructed by biological method, but it has a chemical modification at the end Therefore, the original product of lilaluptide has a great advantage in price Now most of the imitations are chemical synthesis, which has no advantage in cost < br / > Because it has two chains and three pairs of disulfide bonds, it is difficult to synthesize If chemical synthesis is used, the cost cannot be compared with biological method, so all insulin on the market is obtained by gene recombination < br / > whether it is to make peptide generic drugs or peptide innovative original drugs, the application materials are now required to be submitted in the form of CTD The core content of < br / > CTD format is actually the main part of the DMF file Module 2 is the quality overview, in which 2.3s3.2 is the impurity research in the synthesis process It requires to list all impurities and their analysis, and effectively control the impurities Solvent residue is also an important content in the impurity research < br / > module 3 of DMF file puts forward very specific requirements for impurity research To do DMF file, a lot of efforts should be spent on impurity research and control in 3.2s3.2 < br / > at present, the concept of MS is quite popular for people who make medicine Because the synthesis process of polypeptide is complex, there are many kinds of impurities in it, so many control methods need to be developed In the past, the focus of polypeptide drug imitation was mainly to make products that met the quality standards of the original research or Pharmacopoeia But the current review thinking is different It requires you to start with the original materials and control the whole process That is to say, our previous passive mode of "starting with the end" must be changed into the active control mode of "starting with the source" < br / > Chemical synthetic peptides only focus on the identification of chemical structure, including the control of impurities, but it has no other biological impurities, such as heteroproteins or some research contents of host DNA Therefore, most of the domestic peptide imitation still prefer to use chemical synthesis method, because the biological peptide imitation products still need clinical trials < br / > before, if the original peptide drug was developed by biological method, the imitated product must also be subject to clinical trial, which is not recognized by laws and regulations This is a bit like biosimilar in the internal method of biological drugs, such as anti monoclonal antibody and other products Each product must be subject to clinical trial But in the past two years, FDA has also changed some rules It believes that polypeptides, whether biological or chemical, can be imitated if you can analyze the structure clearly For example, the original research is a biological method If you synthesize it by chemical method, and you prove that its structure and activity are the same, you can declare it as a generic drug, which greatly reduces the obstacles in previous laws and regulations < br / > < br / > for this reason, the vast majority of domestic peptide imitation products are chemically synthesized, so the evaluation should be faster For example, in the past few years, there was a product, trypatide, which was originally developed by biological method Some domestic enterprises have applied for this product Because of the clinical research, the whole research cycle is very long Therefore, the imitation of chemical synthesis method has advantages in application of R & D expenses and approval time < br / >? In fact, the most easy to think of is the lack of impurities in the process, because solid-phase synthesis, not every step is 100% condensation, inevitably there will be some amino acids missing, and the polypeptide chain itself may break, some structures may be modified These impurities are the focus of research and belong to process impurities < br / > < br / > there are also degradation 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, except for the first two kinds of impurities, the solvent residues, element impurities and gene toxic impurities should be considered when applying for peptide products < br / > < br / > and now according to the requirements of CDE review, the source and destination of impurities, especially the quality control of starting materials, have greatly improved the requirements < br / >? < br / > there are many kinds of amino acids used in peptide synthesis, for example, the commonly used double protected lysine L-lysine, which may contain D-type impurities In addition, during the preparation of this double protective amino acid, it may lose a protective group, form a dimer, introduce beta alanine, or even exchange the positions of the two protective agents, or both protective groups are lost These impurities may be brought into the product, so it is necessary to analyze these impurities when starting material control < br / > just now, just take an amino acid as an example, but in peptide synthesis, several or even more than ten kinds of protective amino acids are often used, so for the analysis department, QC needs to do a lot of work < br / > How to ensure that the condensation of each amino acid is complete in the peptide synthesis process? Ninhydrin detection is the most widely used method This reaction is very sensitive As a qualitative central control method, it is widely used at present There are other methods, such as TNBS method, Tetrachlorobenzoquinone method If you want to control