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Study on the stability of in vitro diagnostic reagents-summary of key points

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Stability research is an important content that runs through the entire reagent research and development stage and supports the marketing and post-marketing research of reagents, and is the basis for setting the validity period of the product. Since stability is the ability of a product to maintain stable components, properties and performance, in the process of IVD product registration, stability studies are also required to be submitted as an independent part of the registration materials. Next, based on relevant standards, I will elaborate on my basic understanding of product stability research. I welcome all colleagues to correct me.

1. Standards and introduction

1. Related reference standards and introduction:

1. Announcement on the issuance of technical guidelines for the stability of biological products

2. YYT 1579-2018 In Vitro Diagnostic Medical Devices In Vitro Diagnostic Reagent Stability Evaluation

3. ISO-23640-2011- Evaluation of the stability of in vitro diagnostic reagents

4. CLSI EP25-A stability evaluation

5. EN13640-2002+ stability test of in vitro diagnostic reagents

Because most IVD products contain biologically active ingredients, this article refers to the relevant requirements of the technical guidelines for the stability of biological products.

The principles and principles of ISO2640, EN13640 and EP25 are essentially the same in terms of content, but IS and EN are general rules, and EP25 pays more attention to details, and is more practical and instructive.

2. Scope of application

It is used to evaluate the stability of reagents, calibrators, quality control products, diluents, buffers, and kits. It is also suitable for sample collection devices that contain samples for preservation or reaction-initiating substances, but not for instruments, devices, equipment, and systems. , Sample container and sample.

3. Stability test

Stability metrics:

· Physical, biological, chemical, microbial (color, pH, particle size, sediment, growth, purity, etc.)

· Drift of analyte, detection limit, precision, recovery rate, bias of interference, etc.

Statistical confidence:

· Refer to EP05 precision, 06 linearity, 07 interference, 17 detection limit

· Appropriate choice of confidence level to identify the measured change, generally 95% confidence interval is used as

The basis of quantitative IVD evaluation, a more conservative choice of 97% or 99% confidence interval.

Acceptable standards:

· Sources and products related to input requirements, especially intended use methods and established quality objectives, historical data of similar products, and typical performance of existing products.

· Multi-level calibration quality control products, their stability can be expressed separately for each level, or the minimum stability can be marked as a whole

Note: The reference materials used in the test process should be standardized materials. It is not recommended to use randomly selected clinical samples to avoid random errors in the evaluation results and systematic deviations.

4. Stability study

Stability research mainly includes real-time stability and accelerated stability. The real-time stability includes long-term (storage period) stability, transportation stability and use stability.

4.1 Real-time stability

EP-25 proposes that real-time data is continuously monitored, and only if two consecutive points exceed the standard can it be considered out of control. After the original out of control is investigated and eliminated, the validity period is determined.

Test requirements:

· Put the product under the storage condition and start to set the zero point

· Randomly select from the stored samples and test them on each planned test day according to the stability test schedule. The samples are tested randomly.

· Check the test results of each sample to find potential outliers.

· If a sample or all-day test is unqualified due to abnormal values, quality control procedures or operational errors, re-test after finding out the actual cause and correcting it.

Reagents and equipment requirements

· Reagent volume: The product used for stability research should be a product with a scale of pilot scale or above under the condition of complying with its product production process regulations. Generally, it should be ensured that the products used for testing and the reagents, calibrators, quality control products and other samples used in the test should have a 20% margin to ensure that there are sufficient materials when testing problems occur in the research or when repeated testing is required.

· Calibrators & quality control products: The calibrators and quality control products used in the experiment should be guaranteed to be the same batch. The strains used in the microbial test should come from the same generation (first generation) passage bacterial liquid of the same engineering strain.

· Instruments and equipment: Ensure that the equipment is in the correct quality control and maintained and calibrated on time. The same type of instrument is used for each inspection and the parameters remain unchanged.

Time interval requirements:

· The time point of the test should be based on the physical and chemical properties of the reagent product, and the time interval of the stability study should be appropriately selected. For products that are sensitive to environmental factors, the inspection reagent point should be appropriately increased. The time interval covers at least the entire target shelf life. The number of time points should be selected appropriately to identify de-trends from data changes. It is recommended to extend beyond the required interval to ensure that the stability requirement interval can cover all test data without extrapolation. For example, a reagent with a shelf life of 12 months requires 13 months or 14 months.

· It is proposed in the guiding principles of the technical guidelines for the stability of biological products that the general principle for setting the time point for long-term stability research is “test once every three months in the first year, and once every six months in the second year. Starting from the third year, it can be tested once a year. If the validity period (storage period) is one year or less, the long-term stability study should be tested once a month for the first three months, and once every three months thereafter. “In practical applications In the process, the company can refer to the requirements of this guideline, or flexibly set the detection time point in the research plan according to the product’s own characteristics, but the design must be reasonable to meet the ultimate goal of the plan.

data analysis:

· Linear model: plot the X-axis (mean to be measured) and y-axis (time) of the curve. Most products should change very little (≤10%) during the test period. If the graph is non-linear, a conversion formula or a non-linear model can be adopted.

· Regression: p≥0.05 (the slope of the regression is not statistically significant), and the stability interval takes the maximum value of the test time point. P<0.05, the stability interval is the time when the acceptable standard of drift and the one-sided 95% confidence interval of linear regression intersect. (PS. The calculation can be processed by software or calculated by formula, please refer to EP-25 for formula)

Note: The stability interval should be the minimum value of the stability interval of all reagent samples.

4.1.1 Long-term (shelf life) stability

Reagents should be stored under the storage conditions claimed by the manufacturer, and humidity, temperature, etc. should be studied. In the process of long-term stability research, it is found that the performance of the product is degraded or does not meet the standard. It is recommended to identify the different components of the product to clarify the cause of the performance degradation, and focus on the observation period when re-taking stability research after improvement. Change trends and evaluate the results of improvements. For specific examples, please refer to Appendix A of EP25.

According to current experience, long-term stability research data can continue until the product registration data issuance phase is completed, which means that the stability research during registration inspection and clinical trials may not be completed.

Note: The capacity of the equipment or conditions used for the intended storage of the product should be specified

Example: The refrigerator used to store reagents in the laboratory generally claims that the temperature is controlled at 2 ℃-8 ℃, then the evaluation of the shelf life should not include below 2 ℃ or above 8 ℃.

4.1.2 Transportation stability

The manufacturer shall verify that the specified transportation conditions do not affect the expiration date of the IVD reagent.

It should be completed on the basis of the planned cold chain storage and transportation conditions, and full consideration should be given to the transportation route, means of transportation, distance, transit times, time, conditions (temperature, humidity, vibration, etc.), packaging (inner packaging, insulation measures, etc.) Situation, temperature monitor situation (number of temperature monitors, location, etc.). Simultaneously, the simulation should be carried out under the worst conditions, during which the longest distance can be set, the temperature is extremely cold or extremely hot, and the cold chain is dropped during the transit (considering the environmental temperature after the break, the number of breaks, the total time of break, etc.) The transportation stability study confirms the transportation stability of the product under the specified storage conditions and the impact of transportation on the product stability under the short-term departure from the specified storage conditions.

4.1.3 Stability in use

The stability of use should be able to reflect the daily use conditions, and the IVD reagent can maintain product performance indicators during this period.

Such as on-machine stability, reconstitution stability, bottle opening stability, etc.

(1) On-machine stability: usually refers to the effective time that the reagent can maintain its stability after entering the reagent compartment (reagent tank) of the instrument. In-machine stability research should also include such related stability research work as the time interval between PCR reagents added from the reaction solution to the machine, and so on.

(2) Reconstitution stability: usually refers to the effective time that the freeze-dried powder can be stabilized after reconstitution. (Note: The performance of the freeze-dried calibrator after re-dissolution should at least meet the accuracy requirements; the performance of the freeze-dried quality control product should be at least Meet the requirements for the validity of the quality control product assignment).

(3) Bottle-opening stability: refers to the time that the reagents that cannot be used up in one time can be stored stably. The reagents used in the open bottle stability study should be stored under the specified conditions. For example, when we conduct open bottle stability studies on PCR reagents, the remaining reagents need to be stored at -20°C each time they are used. Open bottle stability studies are usually not carried out for products packaged in single servings.

4.2 Accelerated stability

The product should be stored under the storage conditions specified by the manufacturer directly until the accelerated stability test begins. After the test begins, the product should be placed under the specified tightened conditions (including elevated temperature, elevated humidity, light and vibration, etc.). At the specified time point, the sample should be taken for testing or returned to storage conditions to predict the shelf life. Analyze the acceleration stability data (such as Arrhenius curve, nonlinear model) according to the plan, and establish the expiration date based on the acceptance criteria. For specific examples, please refer to Appendix B of EP25.

At present, some companies believe that the reagents placed at 37°C for 3 days is equivalent to 4°C for half a year, and 37°C for 7 days is equivalent to 4°C for 1 year. Some companies believe that after half a year of accelerated stability studies, the product shelf life stability can be set as In 2 years, these claims lacked scientific basis. Due to the complex structure and composition of in vitro diagnostic reagents, there may be multiple degradation pathways at the same time, and the degradation, polymerization, denaturation, inactivation, and interaction with packaging materials under different conditions are different. Therefore, in general, accelerate Stability research can only be used for general product performance research and is not recommended for deriving product expiration date.

5. Stability test plan

The test plan should include: personnel responsibilities, clear reagent identification, use conditions (such as changes in environmental factors, including the worst case), evaluation goals and objectives, reagent sample information (such as: batch, quantity, container, Source identification, concentration), potential impact of key components (PS. Key components are for stability), storage conditions, simulated transportation, time point interval for testing, testing after each time point (e.g., tested Procedure and degree), the number of tests (ps. This depends on the precision of the test method, such as taking into account the variability of reagents and instruments), the duration of stability evaluation, and the description of data analysis (eg: refer to statistical methods for data Judgment: acceptance or rejection), acceptable standards and interpretation of data, etc.

6. stability report

The stability report includes at least the test plan, the sample batches involved, test results, data analysis, pass/fail standards, and stability conclusions. This report is also part of the IVD reagent technical file.


1. Wang Jun Evaluation of the stability of in vitro diagnostic reagents, Beijing Medical Device Quality Supervision and Inspection Center of the State Food and Drug Administration, December 2013

2. Zhang Yan Wang Zhiguo Stability Evaluation of In Vitro Diagnostic Reagents Beijing Hospital of the Ministry of Health Clinical Laboratory Center

3. Zhang Li Research on the stability of in vitro diagnostic reagents [J]. Chinese Journal of New Drugs, 2006, 15 (22): 1899-1900

4. The stability study of diana_zhang, published on the Internet in August 2015

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