Chinese peptide company Consideration of research and development and consistency evaluation of polypeptide generic drugs

Consideration of research and development and consistency evaluation of polypeptide generic drugs


Chinese Journal of new drugs, Vol. 29, No. 8, 2020


Hu Yuxi, Han Tianjiao, Hu Yanchen

Drug evaluation center of State Drug Administration

State Key Laboratory of natural and Biomimetic Drugs, School of pharmacy, Peking University


In this paper, combined with the relevant technical requirements of new registration classification and consistency evaluation in recent years, on the basis of “the influence of preparation process and process control on related substances of synthetic peptide drugs” and “Research on quality control and impurity spectrum of synthetic peptide drugs”, the key issues in the research and development and consistency evaluation of polypeptide generic drugs were discussed in order to help new drug researchers improve their applications Report data quality.

key word

Peptide; generic drug; conformity evaluation; research and development

With the maturity of peptide synthesis technology and the improvement of drug preparation technology, peptide drugs have become the focus of drug research and development companies at home and abroad. Among the 46 new drugs approved by FDA in 2017, 6 are polypeptide drugs, which is the highest annual approved number of polypeptide drugs, which proves that the development of peptide drugs has entered the “fast lane”.

The development of peptide drugs in China started relatively late. The accessibility of raw materials and the technical bottleneck of high-end preparations restrict the development of peptide drugs. With the key support of major new drug creation projects and the “Twelfth Five Year Plan” of biomedical industry, in recent years, the technical difficulties of peptide drugs have made major breakthroughs, and the number of domestic polypeptide drugs has gradually increased.

However, due to the differences in technical strength and the imperfection of guiding documents for the development of peptide drugs, the quality of the application materials submitted by the applicants is uneven, and some of the submitted materials have serious defects, which affect the development and registration of such products.

In view of the above situation, the author has published two papers [2-3], combining with the relevant technical guidance documents at home and abroad, discussed the synthesis process, quality standard and impurity research of polypeptide drugs.

This paper is based on the announcement of several policies on drug registration review and approval issued by the State Drug Administration (No. 230 in 2015), announcement on issues related to the quality and efficacy consistency evaluation of generic drugs (No. 100 in 2017), and technical requirements for quality and efficacy consistency evaluation of generic drugs for chemical injection (Draft for comments) 》According to the requirements of relevant policy documents, this paper reviews the problems existing in the listed peptide drugs, and discusses the problems that should be paid attention to in the consistency evaluation and generic drug application of these drugs.


Current situation of polypeptide drugs in China

In recent years, the sales of polypeptide drugs in China have maintained a relatively rapid growth trend, of which octreotide, somatostatin, triptorelin, leuprorelin and other varieties have sales of more than 100 million yuan. However, the polypeptide drugs which can be produced independently in China are of primary and low added value. At present, most of the drugs with broad market prospects are still mainly imported, such as glucagon like peptide-1 (GLP-1) analogues, etc., as shown in Table 1.

Table 1 lists the polypeptide drugs listed in Europe, the United States and China after 2000. There are many polypeptide drugs on the market in cancer, anti infection, diabetes, immunity and other treatment fields. Through comparison, it can be seen that most of the drugs in China are later than those in Europe and the United States. Some drugs with expired patents and definite curative effect have not been listed in China or only a few enterprises have imitated them.

There may be several reasons:

① Accessibility of API. At present, there are few domestic enterprises with the ability to produce high-quality polypeptide APIs commercially, and most of the APIs produced by enterprises with API production capacity are only used by our company’s preparations, and there are few high-quality APIs with pharmaceutical grade that can be purchased in the market. Therefore, the development of polypeptide drugs is limited at the source.

② The key preparation technology needs to break through. For some special peptide preparations, such as sustained-release microspheres, liposomes, inhaled preparations and so on, there are few enterprises mastering the core technology of the above preparations in China, which on the other hand restricts the marketing of such peptide drugs.

③ Attention of R & D enterprises. Because polypeptide drugs are a kind of special drugs between macromolecular protein drugs and small molecular chemical drugs, their research and development and production are very difficult. Most pharmaceutical enterprises in China have not been involved in this kind of drugs and pay less attention to them.

Therefore, the number of imitations is much lower than that of small molecular chemical drugs. For example, after the patent of parecoxib sodium expired, dozens of enterprises applied for production, while the patent of terlipressin, a polypeptide drug, had already expired. At present, only a few domestic generic pharmaceutical enterprises were allowed to produce.

Some polypeptide drugs that have been put on the market earlier in China. In recent years, China has become an ICH member country. The technical standards of drugs are in line with international standards. With the comprehensive development of drug conformity evaluation in China, polypeptide drugs that have been put on the market in the early stage may not meet the technical requirements of conformity evaluation, and the polypeptide manufacturers with production approval number will face great challenges.

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Determination of reference preparation

According to the regulatory documents issued by China’s drug regulatory agencies, the quality and efficacy of generic drugs should be consistent with those of reference preparations.

In 2017, the general office of the CPC Central Committee and the general office of the State Council issued the opinions on deepening the reform of the review and approval system and encouraging the innovation of pharmaceutical and medical devices, which clearly required that China should gradually establish an orange peel book system and clarify the reference preparations of various drugs.

Since generic drugs bridge the information of quality control, safety and effectiveness of reference preparations, applicants should attach great importance to the selection of reference preparations when developing polypeptide generic drugs.

In addition to referring to the announcement on the procedures for the selection and determination of reference preparations for chemical generic drugs (No. 25, 2019), a comprehensive investigation should be conducted on the rationality of the formulation, the standardization and scientificity of clinical trials, and the authority of the instructions when the products are marketed abroad. Taking oxytocin for injection as an example, some pharmaceutical considerations in the selection of reference preparation are shared.

Oxytocin injections listed in the United States, Europe and Japan have the possibility of obtaining the qualification of domestic reference preparations, such as app pharmaceuticals, LLC of the United States, mylan Products Ltd of Europe, and Takeda Shimin Industry Co., Ltd. of Japan.

For the imitation injection, Q1 and Q2 are consistent with the reference preparation in principle, that is, the type and dosage of excipients in the prescription are consistent; secondly, according to the requirements of “basic considerations for chemical injection (Trial Implementation)”, the types and dosage of excipients used in the injection should be reduced as far as possible on the premise of meeting the needs.

Whether alcohol is included in the prescription is the main difference among European, American and Japanese products. There is no ethanol in the products listed in Japan and the United States, and there is ethanol in the products listed in Europe.

At the same time, it is found that there is no ethanol in the products approved recently in Europe. Therefore, the role and necessity of ethanol in the prescription should be analyzed in detail, and the reference preparation should be defined in combination with the pre marketing clinical research and instructions.


Quality of polypeptide API

The quality control of polypeptide raw materials is relatively complex. In “the influence of preparation process and process control on the related substances of synthetic peptide drugs” and “Research on quality control and impurity spectrum of synthetic peptide drugs”, how to realize the quality control of polypeptide drugs by combining the quality control of protected amino acids, condensation process control, purification control and intermediate control was described.

Therefore, it is necessary to make clear that the process control of synthetic peptide drugs is more important, and it is the key point to improve the quality of application and consistency evaluation application of polypeptide generic drugs. On the basis of comprehensive research, it is possible to achieve full quality control of polypeptide drugs. It may not be able to effectively evaluate product quality only by means of detection. Here are two examples that may cause quality risk of polypeptide drugs.

For small molecule chemicals, the difference of hetero mass spectra caused by different synthetic routes is limited. Through the corresponding analysis and research, the differences of potential hetero mass spectra can be fully covered. Polypeptide drugs are different from small molecular chemical drugs in many synthetic routes.

The same peptide chain can be prepared by solid-phase synthesis, liquid-phase synthesis, solid-liquid-phase combination of fragment synthesis and other means, that is to say, there are many synthetic routes, resulting in a large difference between the process impurities and the reference preparation.

According to the current application data, the method of gradual coupling from C-terminal to N-terminal is basically used in domestic preparation, while most foreign original research enterprises use solid-phase synthesis to prepare peptide fragments, which are then coupled into the whole peptide chain.

The rationality of the synthetic route is not analyzed for the time being, but different synthetic routes may lead to great difference in impurity spectra. Taking exenatide in USP as an example, although there are as many as three analytical methods for related substances contained in the Pharmacopoeia, some applicants still found that there were undetectable process impurities in the method after development. The methods included in the Pharmacopoeia may mainly come from the original research method.

Due to the different synthetic routes adopted by the imitation enterprises, the impurity spectra produced are inconsistent.

Only using the pharmacopoeia or the original import registration standards of related substances analysis methods may not be able to fully reveal the level of impurities in generic drugs, which will lead to product quality risks.

Therefore, it is necessary to carry out detailed impurity spectrum analysis for polypeptide drugs, and comprehensively verify the detection ability of the analysis method with corresponding impurity reference substance. Only using the existing analysis methods for investigation, it is likely to cause quality control defects such as missing impurity detection.

In terms of impurity content, it should be noted that some impurities may have great influence on the stability of peptide drugs even if they meet the safety level range. For example, a polypeptide drug is included in the Pharmacopoeia of the people’s Republic of China, which requires that the maximum unknown impurity should not exceed 2.0%.

In the process of evaluation, it was found that according to the method of Pharmacopoeia, there was a single impurity peak after the main peak of reference preparation, and there were two impurity peaks after the main peak of most domestic preparations. One impurity peak had the same relative retention time with the reference preparation impurity peak, which should be the same impurity, and the extra impurity peak was new unknown impurity.

The impurity content does not exceed the control limit of the maximum unknown impurity.

Although the initial impurity level was low and met the requirements of Pharmacopoeia, the new unknown impurities increased rapidly in the process of stability study, and it was close to the control limit after 12 months, while the reference preparation still maintained an impurity peak after the main peak. The impurity may be hydrolytic impurity of amides, and a small amount of impurities in the system may have similar “chain initiation” reaction, resulting in the impurity growth level of the imitation product is much higher than that of the reference preparation.


Structure of polypeptide drugs

Structure is the basis. If there are structural differences, the activity of the generic product may be weaker than that of the reference preparation, or even not. The structure confirmation of polypeptide drugs is quite different from that of small molecular drugs, and may have high-level structures. The commonly used methods to reflect the structure of peptide drugs are as follows.

First order structure confirmation method: the molecular weight can be determined by mass spectrometry, the peptide sequence can be identified by mass spectrometry combined with enzyme digestion, or the peptide sequence can be identified by Edman degradation.

Methods to confirm the secondary structure: the secondary structure of peptide is actually the local structure formed by hydrogen bond of peptide chain. The research methods include X-ray single crystal diffraction, two-dimensional NMR, FT-IR and far ultraviolet CD.

Among them, X-ray single crystal diffraction and two-dimensional NMR are the most accurate, but the single crystal of polypeptide is not easy to cultivate, and does not represent the real situation in the solution. However, two-dimensional NMR takes a long time, is difficult to analyze, and the analysis flux is low, so these two methods are generally used less; at present, FT-IR and far ultraviolet CD are the most commonly used methods.

Third order structure confirmation method: for complex polypeptides, there may be tertiary structure. The common methods of tertiary structure research include fluorescence spectrum and near ultraviolet CD, etc. the principle is to use the micro environment change of aromatic amino acid side chain to bring the difference between fluorescence emission spectrum and near ultraviolet CD spectrum.

However, this method can only be used for qualitative comparative study, and can not be used to confirm the structure. It can only be used as a reference for the study of three-level structure.

Fourth order structure confirmation method: functional aggregation of polypeptides and proteins is a reflection of high-level structure, which contains two aspects of information: the number of aggregation monomer and the spatial conformation after aggregation. Common analytical methods include field flow separation and ultracentrifugation.

Biological activity research methods: due to the uncertainty of the high-level structure confirmation, the correctness of the high-level structure of peptide drugs can be indirectly proved by testing the biological activity. According to the statistics in Table 2, among the 11 polypeptide drugs included in USP, 4 preparations and 1 API need to be tested for biological activity. More and more peptide drugs need to increase the research and detection of biological activity.

At present, there are two ways to study the activity of peptides and proteins in vitro and in vivo. In vitro evaluation methods include, but not limited to, enzyme kinetics, binding capacity and biological activity; In the case of binding force determination, surface plasmon resonance (SPR), biofilm interference (BLI) and micro differential scanning calorimetry (nanodsc) are used to measure the binding force between molecules. If the binding force is to the receptor on the cell surface, the homogeneous time-resolved fluorescence technology is generally used;

Biological activity is generally achieved by stimulating cells with drugs and detecting the chemical substances produced by them by homogeneous time-resolved fluorescence techniques. In vivo evaluation generally uses animals or animal organs. If the mechanism is clear, the specific compound index is detected. If the mechanism is not clear, the specific therapeutic effect is observed. For example, the EAE model of mice can be used to evaluate the delayed and alleviating effect of glatiride acetate on the symptoms of multifunctional sclerosis.

For peptide drugs, the structure confirmation part is different from small molecule chemical drugs. After the structure of small molecule chemical drugs is clear in the part of raw materials, further research is not needed in the preparation development. However, due to the uncertainty of the high-level structure of peptide drugs, some of them need to be compared with the reference preparation in order to determine the consistency of a certain structure content with the reference preparation.

At the same time, because the high-level structure of polypeptide drugs is related to the environment in which they are located, it may be out of the actual use environment of drugs in the process of structure testing. Therefore, it is necessary to use a variety of complementary analysis methods to study the structural consistency of generic and reference preparations.

It needs to be clear that not all peptide drugs have high-level structure, and only a few of them have high-level structure in the synthesis. It is suggested that the product developers should first search the reference preparation information, literature, patents and other aspects to determine whether there is a high-level structure, and then select the appropriate structure confirmation means to study, so as to avoid over development.

Study on peptide preparation

Most of the polypeptide drug preparations are injections, with a small amount of oral preparations, such as desmopressin, linalopeptide, somaluptide, and a small amount of inhaled preparations such as desmopressin and salmon calcitonin.

In this paper, the potential differences between peptide drug injections and small molecular drugs are mainly discussed based on the technical requirements for quality and efficacy consistency evaluation of generic chemical injection (Draft).

5.1 prescription technology

Excipients selection: Although the pharmaceutical administration allows the injection generic drugs to be different from the reference preparations in antibacterial agents, buffers, pH regulators, antioxidants, metal ion complexing agents, etc., the selected excipients should be as consistent as possible with the reference preparations for polypeptide preparations The stability of peptide chain is poor, acid, alkali and metal ions may cause peptide chain degradation;

there are many exposed amino and carboxyl groups in the structure of peptide drugs, which may interact with excipients to form related impurities; peptide drugs may have high-level structure, and slight differences in pH value and ionic strength may cause structural changes.

Solution preparation process: some peptide drugs are sensitive to pH value, oxygen content, ionic strength, etc. in the preparation process, short-term separation from the stable environment will cause adverse effects on peptide drugs.

It is more important to grasp the details of the process. It is necessary to investigate the order of raw and auxiliary materials, mixing time, stirring speed and whether it needs to avoid light and deoxidization to determine whether it has an impact on the product quality.

For example, a polypeptide preparation was found to be stable in a relatively narrow pH range in the early process development process, and the pH control range of the proposed key process parameters was also relatively narrow. In the first process validation, it was found that the pH value after solution preparation was within the proposed control range, but the pH value of some final products was beyond the proposed control range.

The main reason is that after batch scale-up, the stirring performance of commercial production equipment is low, the dissolution time of peptide drugs is prolonged, and the pH value after drug configuration needs to reach steady state after a long time. After the detailed investigation of the mixing speed, time and other process parameters, the stable production of three consecutive batches of products was realized.

For example, for a polypeptide preparation, when the stability of three batches of process validation products was retained for 12 months, the increase rate of impurities in one batch of products was significantly greater than that of the other two batches.

After reviewing the actual batch production records, it was found that there was a pH value callback problem in the products with large changes. Since the product process is to add the API into the solution with adjusted pH value, the ionic strength of the system will increase after callback, which is the main reason for the degradation phenomenon. The applicant’s supplementary study showed that if the ionic strength in the system increased, the accelerated test could obviously find that the drug degradation rate increased.

Therefore, strict restrictions were made in the process procedures, such as the failure of pH value adjustment, it was directly treated as waste liquid and re produced.

Lyophilization process: the stability of peptide drugs is limited, and freeze-drying injection is often selected when injection route is adopted. From the application data, most of the applicants only carry out the research for the purpose of freeze-drying, ignoring the impact of freeze-drying process on stability.

In the process of freeze-drying, there may be many problems, such as buffer crystallization, pH shift, stabilizer morphology change, peptide space conformation change and so on, which can directly affect the stability of peptide drugs. For example, for an imported polypeptide preparation, the proposed freeze-drying process needs to be kept at 40 ℃ for a long time.

Since the proposed storage condition of the drug is 2-8 ℃, questions about the process are raised during the evaluation process, and the applicant is required to further provide the rationality of the freeze-drying process. The applicant provided the product stability ratio under different freeze-drying process conditions

Compared with the data, the results showed that when the temperature was higher in the second heating stage, the degradation impurities in the product during long-term storage were significantly less than that of products freeze-dried at low temperature.

This process can be considered as “annealing” in freeze-drying process, which can release drug energy and improve microstructure, so as to further stabilize peptide drugs. Professor Michael Pikal of the United States has carried out in-depth research on this aspect [6], which can guide the development of freeze-drying process for polypeptide drugs.

Special packaging form: some hypoglycemic drugs use pre filling technology in cartridge. Due to the particularity of preparation process and use method, special research methods should be established. For example, in the production process of cartridge bottles, silicification process directly affects the smoothness of the inner wall, and then affects the accuracy of dosage.

The spraying amount and uniformity of silicone oil should be controlled accordingly. Due to the particularity of the drug delivery mode, such as air bubbles in the preparation, the pressure response will be inconsistent and the dosage will be inaccurate. Therefore, multiple injections can be adopted in the filling process to reduce the introduction of gas;

This kind of medicine involves many punctures and other problems, so it is necessary to conduct a complete investigation on the sealing property of the packaging materials.

Compatibility problem: some peptide drugs are sensitive to metal impurities that may be introduced into the production equipment, and the introduction of trace impurities may cause peptide chain degradation, resulting in the degradation rate of the imitation product faster than that of the reference preparation.

In order to study the compatibility of drugs with production equipment, filters and drug packaging materials, researchers should pay more attention to the inorganic impurities such as metal ions on the basis of the research contents of small molecular chemical drugs. In addition, the dosage of peptide drugs is usually small.

In order to ensure the accuracy of drug dosage, attention should be paid to the adsorption of drugs by production equipment, filter and packaging materials. If necessary, excessive feeding can be used to solve the problem, but sufficient basis should be provided.

For example, in the preliminary development of a polypeptide preparation, the applicant found that metal ions could be introduced into the proposed production equipment, resulting in a significantly accelerated degradation rate of the product. Due to the difficulty of replacing the production equipment, the applicant proposes to use disposable solution preparation bag in the liquid preparation process, which avoids the introduction of metal ions, thus ensuring the product quality.

Exclusive solvents and syringes: some polypeptide reference preparations have exclusive solvents and injection devices. When conducting research, generic pharmaceutical enterprises need to pay attention to whether the drugs are compatible with clinical common solvents and syringes, and the necessity of exclusive solvents and syringes should be fully evaluated.

For example, terlipressin reference preparation has a special solvent sodium chloride aqueous solution. The appendix of product quality standard specifies the concentration and pH value of sodium chloride in sodium chloride aqueous solution in detail.

At present, the commonly used compatible solvent in clinical practice does not meet the requirements of the control limit of reference preparation. The domestic listed terlipressin for injection did not specify the clinical compatible solvent, so it is necessary to evaluate the necessity of exclusive solvent and the influence of compatibility on product quality and patient tolerance.

5.2 quality research

Researchers should combine the characteristics of polypeptide drugs and carry out targeted investigation in quality research. For the peptide drugs with poor stability, attention should be paid to whether the pH value, properties, re dissolution time and initial impurity level are consistent with the reference preparation.

There may be a phenomenon that the control limit of quality standard is wide, and the imitation product can meet the requirements of the proposed quality standard, but there is still a certain difference compared with the reference preparation.

The above differences may indicate that the microenvironment of polypeptide drugs is different from that of the reference preparation. In the long-term stability study, problems may occur in the inspection items such as pH value, related substances and insoluble particles.

Researchers should pay attention to the above differences, understand and find out the reasons, so as to avoid problems in the long-term stability investigation and affect the application progress.

Related substances are one of the key projects in the quality research of polypeptide drug preparations. The quality research of preparation should be combined with the investigation results of related substances of API, pay attention to the investigation of degradation impurities, and focus on polymer impurities.

Polypeptides contain disulfide bonds, exposed amino groups and carboxyl groups, which are easy to form polymers due to dehydration of disulfide bonds or amino carboxyl groups.

The researchers should make clear whether the polymer impurities are the polymer formed by covalent bond, the aggregate formed by intermolecular force or the advanced structure fixed by peptide chain. If it is a polymer formed by covalent bond, there may be a greater risk of immunogenicity. It is necessary to determine the structural consistency with the impurity in the reference preparation and ensure that its content is within the safe limit.

5.3 stability issues

The main reason is that the stability of polypeptide drugs is poor in solution state, and the compatibility stability can reflect the quality difference with reference preparation. Some polypeptide drugs need to be diluted by a large multiple if they are used according to the clinical actual concentration.

The real quality changes of the products may not be detected according to the analysis method in the proposed quality standard. If necessary, the dosage of compatible solution can be reduced, and the stability comparison with the reference preparation at the same compatibility concentration proves that the quality of the imitation product is not lower than that of the reference preparation.

It should be noted that the storage conditions of some polypeptide generic drugs on the market in China are more stringent than those of the reference preparations. For example, carbetoxine, the storage conditions of the reference preparations should not exceed 30 ℃ and should not be frozen. The storage conditions of domestic generic drugs were airtight and stored at 2-8 ℃.

The difference of storage conditions indicates that the stability of the products may be different. Further discussion may be due to the comprehensive reasons such as raw material drug, prescription, production technology, production equipment, packaging container and so on. The above problems show that the quality of generic drug is not consistent with the reference preparation, and the quality needs to be improved and improved.

The stability of the generic drug should be consistent with the reference preparation, and the storage conditions should not be more stringent than the reference preparation in principle.



Because polypeptide drugs are special preparations between small molecule chemical drugs and protein drugs, although there are some difficulties in research and development, on the basis of full identification of drug risks, the establishment of corresponding control measures can effectively guarantee the product quality.

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