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Development and Verification of Analysis Methods for Disposable Systems

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The dissolution/precipitate evaluation is an important part of the process-specific verification of filters and disposable systems in the pharmaceutical field to examine the applicability of equipment (including filters and disposable systems) in specific processes. This article describes the development and validation of the dissolution/precipitate analysis method for the verification test of the dissolution/precipitate, and introduces the dissolution/precipitate test, which provides a reference for the users of filters and disposable systems in the pharmaceutical field.

Analytical Method Development

In order to ensure the reliability of the analytical method, to accurately qualitatively and quantitatively dissolve/precipitate, the analytical method should be developed and verified in advance, and certain acceptance standards should be established. Suppliers of pharmaceutical production equipment should establish sensitive chromatographic methods to better identify polymers that may be introduced into the pharmaceutical production process line, additives in polymers, and polymer-related degradation products. Usually disposable system suppliers are better able to understand, control and identify the polymeric materials used in the production of disposable systems. The following takes LC/MS and GC/MS as examples to briefly introduce the development and verification of analytical methods.


Specificity refers to the method used to accurately determine the characteristics of the substance to be tested when other ingredients may exist. It is difficult for suppliers of single-use systems to fully control all raw material information for the production of single-use systems. For example, resin manufacturers do not disclose formulas or semi-finished materials used in single-use systems. However, some suppliers have better established the retention time and standard library of compounds that are derived from disposable system materials and may appear in pharmaceuticals. When analyzing the eluate/precipitate, the specific standard in the standard library and the tested substance are injected and analyzed under the same conditions to identify the tested substance, and a high level of confidence can be obtained to verify its specificity. If only the published compound data or the information provided by the library is compared with the retention time of the tested substance or the mass spectrum fragment, the tested substance cannot be accurately identified.


Sensitivity is usually characterized by Limit of Detection (LOD) and Limit of Qualification (LOQ). The limit of detection (LOD) refers to the lowest amount of the test substance in the sample that can be detected, and the limit of quantification (LOQ) refers to the lowest amount of the test substance in the sample that can be accurately quantified.

The signal to noise (S/N) method is often used to determine sensitivity. Compare the signal measured by the known concentration standard with the signal measured by the blank sample, and set the signal-to-noise ratio to 3:1 (S/N = 3:1) and 10:1 (S/N = 10:1) respectively The corresponding concentrations are determined as LOD and LOQ. Different disposable system components have different eluates/precipitates. It is advisable to select representative compounds as standard products and obtain their LOD and LOQ values ​​to characterize sensitivity. When analyzing dissolution/precipitate, for a specific method, it is not appropriate to report its general LOD and LOQ values ​​(for example, less than 0.1ppm), because the compound type and matrix are different, the response factor and ionization efficiency will occur Therefore, the specific LOD and LOQ values ​​of such compounds should be reported.


Accuracy is how close the measurement result is to the true value. It is generally expressed as a percentage (%) between the actual measured value and the formulated true concentration value, such as 85%. The standard is directly added to the matrix to calculate the percentage to examine the accuracy of the method, and the result should meet certain acceptance criteria.

When analyzing the eluate, generally select compounds that are easily or frequently migrated out of the disposable system and are representative in the corresponding analysis method as the standard to examine the accuracy; when analysing the eluate, the accuracy is identified by the leaching test The compound is measured as a standard product. Regardless of the analysis of eluates or precipitates, it is often necessary to pre-treat the sample before analysis. This is because some solvents (such as water) are not suitable for direct injection of gas chromatography, and some pharmaceutical components (such as proteins or surfactants) can contaminate or clog the chromatographic column. Although the preprocessing is time-consuming, it can obtain representative analysis data and avoid damage to the analysis instrument. Common pretreatment methods include liquid-liquid extraction, solid phase extraction, protein sedimentation and other methods. The recovery rate of the added standard is checked to confirm whether the pretreatment method is appropriate and to help evaluate the amount of eluates and precipitates. Due to the relatively simple components of the model solvent, it is easier to determine the pretreatment method for the eluate samples. This is conducive to determining the pretreatment method of the precipitate sample in the future, and the dissolution data provides supporting evidence, which can more accurately identify the compounds with lower content in the precipitate, avoiding missed chemical substances or causing false negative results, which will affect the subsequent safety assessment The objectivity.

Comparing the spectra of the product control and the precipitate (Figure 1 and Figure 2), it is easy to identify the partial analyte, such as the absorption peaks at RT=0.52min, 2.27min and 3.24min. In the spectrum of the product control, there is a strong absorption peak at RT=1.65-2.00 min. It is impossible to determine whether there is a precipitate at this place. Comparing the dissolution spectrum (Figure 3), there is an absorption peak at RT=1.89min, which is a potential precipitate. The precipitate is further subjected to ion scanning analysis (Figure 4) to confirm that the substance is a precipitate. Then qualitatively and quantitatively analyze the substance to obtain accurate analysis results.

It can be seen that the eluate data is helpful to discover potential eluates, can identify the types of eluates to the greatest extent, and avoid missed or false negatives of eluates. At the same time, in rare cases, the precipitate extract may also contain trace reaction products produced by the reaction of the migrated chemical substance and the drug component. Using the eluate spectrum as a reference, possible additional reaction products can be identified, thereby obtaining more accurate eluate data, making subsequent safety assessments more objective and true, and reducing risks.

Precision refers to the degree of closeness between the results obtained from multiple sampling and determination of the same sample under specified test conditions. It is generally expressed by deviation, standard deviation or relative standard deviation.

Under the same conditions, the precision of the results measured by the same analyst is called repeatability. In the same laboratory, among the three factors of analysis time, analyst, and analysis equipment (even all factors that may cause deviation of test results, such as reagents, temperature, sampling method, etc.), changing any of these factors will cause the difference between the measurement results. Precision is called intermediate precision. The precision between measurement results between different laboratories and different analysts is called reproducibility.

Before sample analysis, the applicability of the system must be investigated, such as detection limit, quantification limit, precision, accuracy, etc., to ensure that the instrument is operating normally, in good condition, and the results are stable and reliable. As one of the inspection items of system applicability, before each analysis of the dissolution/precipitate samples, the standard solution is used for multiple consecutive injections, and the relative standard deviation is determined according to the retention time and peak area, and during the sample running process Use a standard solution of known concentration to investigate the deviation of the response value to confirm the validity of the analysis result.

Linearity and Range

Linearity refers to the degree to which the test result is directly proportional to the concentration of the tested substance within a certain range. Range refers to the range of high and low concentration limits used by the test method to achieve a certain degree of precision, accuracy and linearity. Generally, the standard curve should contain at least 5 concentration points, and the sample concentration should be within the range of the standard curve. The lowest concentration point is the limit of quantification. Generally, if a related class of substances is used, semi-quantitative analysis is an acceptable method. The advantage is that less resources can be consumed in a shorter time, and the approximate concentration of the compound can be obtained. In addition, when standard products cannot be obtained through commercial channels, semi-quantitative analysis is a reasonable choice. If you need more accurate results to evaluate high-risk processes, you need to add standards to the matrix to obtain standard curves with different concentrations in the linear range for quantitative analysis.

Due to the different response factors (that is, the ratio of peak area to concentration) of different compounds in the instrument, semi-quantitative analysis cannot determine the exact concentration of the compound. As shown in Figure 5, the three absorption peaks A, B, and C in the liquid chromatogram represent three different compounds. Standards of A, B, and C were used for quantitative analysis and another compound D was used for semi-quantitative analysis. The two quantitative results are shown in Table 1 and Table 2. In most cases, quantitative analysis is more accurate than semi-quantitative analysis. From the results, the semi-quantitative analysis results of the three compounds are respectively 23%, 67% and 66% higher than the actual concentrations, while the deviation of the quantitative analysis results is only 1%. It can be seen that the results of quantitative analysis are much more accurate than semi-quantitative analysis. When choosing a quantitative (fully quantitative or semi-quantitative) method, you should be cautious and strike a balance between seeking more accurate results and the resources (such as time and cost) required to invest.


Durability refers to the degree to which the measurement results will not be affected when there are small changes in the measurement conditions.

In order to make the method usable for daily inspection, the durability of the analytical method should be studied during the development phase of the analytical method. If there are harsh test conditions, it should be noted in the method. For GC/MS and LC/MS, the typical variable factors are: the stability of the solution to be tested, the composition and pH value of the mobile phase, the stationary phase, the same type of chromatographic column of different brands or batch numbers, column temperature, flow rate, Inlet and detector temperature, etc.

Dissolution/precipitate verification

The dissolution/precipitate verification is to examine the applicability of filters and disposable systems in a specific process to prove that their application will not affect the quality and efficacy of the drug, and will not bring safety risks. The verification process is mainly divided into the following six steps:

(1) Determine the disposable system that directly contacts the drug;

(2) Understand its material composition, contact methods with drugs, contact conditions and production processes;

(3) Carry out dissolution test on the disposable system, predict potential precipitates based on the dissolution, and conduct risk assessment;

(4) Carry out the precipitate test on the disposable system with the drug product to obtain the precipitate data;

(5) Carry out safety assessment based on the precipitate data;

(6) Summarize the impact of disposable systems on drugs and draw conclusions on applicability.

During the dissolution test, the fluid should be kept flowing as much as possible during the direct contact between the filter or the disposable system and the test fluid, so as to obtain the dissolution extract. Perform qualitative and quantitative analysis on the obtained leachable extract, so as to obtain the type and amount of leachables (that is, potential leachables). Before performing some analysis, it may be necessary to pretreat the extract. As mentioned above, the recovery rate of the standard product should be investigated during the pretreatment to verify the pretreatment method and ensure accuracy.

After obtaining the dissolution data, calculate the daily intake of the dissolution according to the production batch of the drug and the daily intake of the clinical drug, and then compare it with the data such as Permitted Daily Exposure (PDE) to confirm the filter Or whether the leachate produced by the disposable system under the process conditions poses a safety risk to the drug product.

Because the precipitates are usually a subset of the eluates, that is, there are more types of compounds in the eluates than the corresponding precipitates, and the amount of elution is larger than the amount of precipitation, so it is generally considered that the eluate represents the worst condition of the precipitate. If the dissolution does not exceed the safety threshold, the disposable system can be considered suitable for the specific process conditions; if the safety threshold is exceeded, the precipitate test can be considered, combining the production batch of the drug and the daily intake of the drug, using the precipitate The data is then evaluated for safety.


The use of disposable systems for the production of biopharmaceutical products has aroused widespread interest and enthusiasm in the entire pharmaceutical industry, and has good application prospects. As people pay more and more attention to health and drug quality, the risks that may be brought about by the application of disposable systems have attracted great attention from regulatory agencies, standards associations, industry alliances, suppliers and end users. Discussion and research, and constantly promulgating relevant regulations, technical documents and solutions to promote the development of the entire industry. Therefore, the dissolution/precipitate verification of the disposable system not only meets the requirements of laws and regulations, but also conforms to the development trend of the industry. Technically, you can refer to the industry-recommended solutions and analysis methods to comprehensively evaluate the applicability of the disposable system. At the same time, with the continuous development of analytical technology, the level of analysis of trace substances has gradually improved, and the analysis of eluates/precipitates will become more and more accurate. With the emphasis on laws and regulations, the improvement of methods and standards, and the improvement of technology, disposable systems will definitely have a more profound impact on the pharmaceutical industry.

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