Any substance that affects the purity of the drug is called impurity. The adverse reactions of drugs in clinical use are not only related to the pharmacological activities of drugs, but also to the impurities in drugs. For example, macromolecular impurities such as polymers in antibiotics such as penicillin are the main causes of allergic reactions.
Therefore, impurity research is an important part of drug research and development.
The 2005 edition of the Pharmacopoeia of the people's Republic of China (hereinafter referred to as the "Chinese Pharmacopoeia") contains the guiding principles for the analysis of drug impurities. The former State Food and drug administration also issued the technical guiding principles for the study of chemical drug impurities in 2005, which outlines the content and ideas of impurity research.
Based on the practice of drug evaluation and the communication with some domestic and foreign pharmaceutical enterprises in impurity research practice, the author discusses personal understanding and understanding of the research ideas of chemical impurities.
1、 General principles of impurity research
The possible sources of impurities are: technological process (including unreacted reactants and reagents, intermediates, by-products and impurities mixed in reactants and reagents) and degradation process. Therefore, the general principle of impurity research is to carry out research in combination with the specific technology and product characteristics of products under research.
First of all, it is necessary to analyze the possible impurities in the product according to the specific process and product characteristics, and to have a comprehensive understanding of the source and structure of impurities in the product through the analysis of hetero mass spectrometry;
Then, on the basis of the analysis of MS, the appropriate analysis method is selected to ensure the effective detection and control of impurities;
Finally, it is necessary to determine a reasonable impurity limit based on the results of pharmacy, pharmacology and clinical research, so as to ensure the quality and safety of drugs.
2、 Analysis of hetero mass spectrometry
1. Analysis of process impurities based on synthesis route
For APIs, the impurities that may be produced in the product under research should be analyzed according to the specific synthesis process adopted.
For example, the inspection of piperazine in the quality standard of trimetazidine hydrochloride is based on the synthesis route of trimetazidine. See Figure 1.
It can be seen from Figure 1 that piperazine is one of the reactants of trimetazidine, which may remain in the final product due to incomplete reaction. It is a process impurity that needs attention in the research.
2. Analysis of possible degradation products based on product structure characteristics
The degradation products are closely related to the structural characteristics of drugs.
For example, enalapril, an antihypertensive drug, contains ethyl carboxylate group in its structure, which is easy to undergo hydrolysis reaction to generate carboxyl group, i.e. to generate enalaprila; in addition, enalapril contains one carboxyl group and one amino group in its structure, which is easy to undergo acylation reaction to generate lactam structure, i.e. to generate enalapril diketone piperazine. See Figure 2.
Therefore, the inspection of enalaprila maleate and enalapriladione piperazine under the control of relevant substances in the quality standard of enalaprila maleate contained in the second part of Chinese Pharmacopoeia (2005 Edition).
It is a kind of prediction to analyze the possible degradation products by the structural characteristics. In addition, the possible degradation products and degradation pathways can be verified by the forced destruction test.
In China, acid, alkali, oxidation, high temperature and light destructive tests are often used to verify the specificity of analytical methods, but little attention is paid to the analysis of the structure of degradation products.
Forced failure test is not necessarily limited to the above listed conditions, and a more flexible way can be adopted to better predict the possible degradation pathway and degradation products of products.
For example, for APIs, the change of products under destructive conditions in solution state can be investigated. If necessary, the forced degradation test can be carried out when the above factors are combined.
For example, the synthetic product of vitamin K, sodium menadione diphosphate, first generates menadione under certain pH value hydrolysis conditions; then, under oxidation conditions, menadione undergoes oxidation reaction to generate menadione. See Figure 3 for the reaction process.
In addition, the destructive conditions should not be too severe in the destructive test to avoid secondary degradation.
After the destructive test, the structure of potential degradation products should be confirmed in combination with the characteristics of drugs and necessary structural analysis methods, so as to facilitate the research and validation of the next analysis method and limit formulation.
3. Analysis of possible impurities in products based on comprehensive research data
The above research is to analyze and predict the possible impurities in the product, but under the actual production process and storage and packaging conditions of the product, it is not necessarily to produce all the above impurities.
Therefore, the final control of the product needs to be determined according to the actual impurities generated in the production process of the sample and the actual impurities generated in the accelerated and long-term sample retention stability research.
3、 Study on impurity analysis method
The choice of analytical method is directly related to the specificity and accuracy of impurity determination results.
Different detection methods may obtain different detection results. The fundamental principle of establishing impurity detection and analysis method is that it should be exclusive and sensitive, the main components and impurities can be separated well, and the detection limit can meet the needs of limit inspection.
The research and validation of impurity analysis methods are described in detail in the published guidelines. Here, the main problems existing in the current registration research are analyzed.
1. Study on separation method
Based on the analysis of the impurity spectrum, the establishment and verification of the method have strong pertinence.
Due to the limitations of all kinds of analysis methods, we need to pay attention to the mutual supplement and verification of different analysis methods.
For example, in the quality standard of omeprazole published in the Chinese Pharmacopoeia, HPLC method is used to determine the relevant substances at 280 nm. The analysis of the detection conditions is mainly to detect the oxidation product of omeprazole, omeprazole sulfonate, but the British Pharmacopoeia Standard specifically lists TLC method to determine the reduction product of omeprazole on this basis.
In the registration application, the most common phenomenon is that gradient elution method is used in foreign Pharmacopoeia of the same variety, while isoelution method is used in self-developed varieties without sufficient research. Gradient elution does have the disadvantages of large baseline drift and relatively cumbersome operation, but isoelution may have the disadvantages of missed detection and time-consuming.
Therefore, in the process of method change, we need to make a full comparative study of the detection ability of the method before and after the change.
In addition, it is recommended to check the peak purity of the main components in the process of analysis method specificity verification to verify whether impurities with similar structure of the main components are included in the main component chromatographic peak due to incomplete separation.
2 selection of quantitative methods
The conventional quantitative methods of impurities include impurity reference method, principal component self-control method with correction factor, principal component control method without correction factor and area normalization method.
From the analysis of current drug research registration, the principal component control method without correction factor is more commonly used.
Because the response factors of impurities and principal components may be different, the principal component control method without correction factor is not scientific.
The ideal quantitative method is the combination of the known impurity reference method and the unknown impurity principal component self-control method without correction factor.
Of course, if the research proves that the relative response factors of known impurities and principal components are in the range of 0.9 ~ 1.1, the content can be calculated by the principal component self-control method, for example, the principal component self-control method is used for the control of multiple known impurities in the quality standard of paclitaxel in the United States Pharmacopoeia.
4、 Determination of impurity limit
The general principle of impurity limit determination is to be as low as possible.
The factors influencing the determination of impurity limit are: the safety range of impurities, the actual production capacity of drugs, the degree of change during the stability period, and the variability of detection methods.
For drugs of different registration categories, due to the different amount of reference information, the way to determine the impurity limit will be different.
Determination of impurity limit of innovative drugs
The research of innovative drugs is a process of gradual cognition, during which may experience changes such as the adjustment of prescription technology, the change of miscellaneous mass spectrometry, the optimization of analytical methods, etc. the research and recognition of impurities is a process of continuous accumulation, so the determination of the limit of impurities is also phased.
From the perspective of clinical application, the main principle of determination of impurity limit is to ensure the safety of clinical samples. The determination of impurity limit is mainly based on the results obtained in the pre clinical safety research. Generally, it is required that the sample impurities used in clinical trials shall not exceed the samples used in the pre clinical safety research.
From the perspective of application for listing, the determination of impurity limit should be based on the existing basis, combined with the enlargement and optimization of production process, production data of multiple batches of products and stability information, and in line with the principle of the lowest possible limit.
Generally, from clinical application to market application, the research on impurities will be more and more sufficient, and the control of impurities will be more and more strict.
For example, in the clinical application stage, the maximum single impurity control shall not exceed 1.0%, and the total impurity control shall not exceed 3.0%; in the marketing application stage, the control process impurity A shall not exceed 0.25%, the degradation product B shall not exceed 0.25%, the other single maximum impurity shall not exceed 0.15%, and the total impurity shall not exceed 1.25%.
From clinical application to market application, the total impurity limit of the product tends to be strict, and qualitative and quantitative studies have been carried out for a single known impurity, and the limits have been formulated respectively.
In order to explain how to consider all the factors mentioned above in the determination of impurity limit, an example of review is also given.
Impurity C in a product is its degradation product, and the information available is as follows. From the analysis of the safety research results, when the impurity content is 3.57%, there is no difference between the safety results obtained and the safety results of the intact samples, indicating that the impurity C content is an acceptable safety limit when it is 3.57%.
According to the analysis of production and storage results, the measured range of impurity C in 32 batches of products is 0.04% - 0.87%, the average content is 0.38%, the standard deviation is 0.21, the average value ± 3 × standard deviation is 1.0%. The calculation limit of 1.0% is consistent with the fluctuation range of production, and it is also lower than the safety limit, so it can be used as a reasonable limit for impurity C control.
2. The determination of impurity limit of generic drugs
For imitated products, the sources of obtaining impurity safety information may be different from those of innovative drugs.
On the one hand, we can carry out the safety study of impurities by ourselves, and on the other hand, we can refer to the safety information of the listed products.
From the perspective of research cycle and research input, most of the researchers will choose the latter method, which requires a comprehensive comparative study of impurities between self-developed products and imitated products, including the types and contents of impurities. If there is no special reason, the impurity limit of the self-developed product shall not exceed the impurity limit of the imitated product.
The following is an example of impurity research of imitated injection products for specific analysis. The comparative data obtained in impurity research are listed in Table 1.
Three known impurities (a, B, d) in the imitations are higher than the listed products and 0.1% higher than the acceptance limit specified by ICH; in addition, two unknown impurities (rrt1.28 and 1.31) are also higher than 0.1%.
As for impurity a, there is no test and literature to support its safety, so we should improve the process and reduce its content so that it does not exceed the limit of the products on the market;
As for impurity B, it is a listed drug with a long history of human use. According to the literature, it can be transformed into the main drug in vivo, without safety concerns, and can accept the current level of impurities;
As for impurity D, the literature shows that its main metabolite in human body does not have safety concerns;
As for the unknown impurities in rrt1.28 and 1.31, due to the lack of test and literature to support their safety, the process should be improved to reduce them below the acceptable limit of 0.1% of the unknown impurities.
Impurity research is an important part of drug research, which runs through the whole process of drug research and development. With the recognition of the law of drug research and development, the continuous expansion of design concept and the emergence of new technologies and methods, impurity research will have new breakthroughs.