21. What is the test method of microbial contamination level and heat resistance (D value) before sterilization?

Answer: The level of microbial contamination usually uses membrane filtration to retain microorganisms, and then transfer the membrane to the surface of a solid medium, cultivate and count the microorganisms. Attention should be paid to the volume of filtration and the number of retained microorganisms to ensure a sufficient detection rate (a sufficient amount of filtration) and countability (too many retained microorganisms cannot be counted).

The heat resistance test performed on each batch of products is not a D value test, but a so-called boiling test-a qualitative test. Put the filter membrane that trapped microorganisms into a test tube containing the same product liquid, boil it in a water bath for 15 minutes or more, and perform a sterility test on the liquid. If it is a heat-resistant bacteria, the D value needs to be further measured. More than 99% of the inspected products are non-heat-resistant bacteria.

The measurement of D value is quite complicated, please refer to the "Guidelines for Pharmaceutical Production Verification" (Blue Book, edited by the State Drug Administration), Chapter 3, Section 1, for a detailed introduction.

22. How to calculate the amount of inoculation based on the D value?

Answer: Calculation of spore inoculation amount:

Ni=10Do(lgNo+6)/Di

where Ni is the number of heat-resistant spores inoculated as a biological indicator

No is the limit of contaminating microorganisms in the intended product before sterilization

Do is the maximum allowable value of D for contaminating microorganisms

Di is the D value of the biological indicator heat-resistant spores in the product

23. For products that are terminally sterilized by the survival probability method, if the microbial limit is tested for each batch before sterilization, and the microbial limit detection time is 72 hours, and the actual production cycle of continuous production is much shorter than 72 hours, the test result is only Is it a reference to the sterility assurance level of the product after sterilization?

Answer: Obviously, the result of the microbiological content inspection before sterilization lags far behind the production process, and its purpose is not to be used for intermediate control of the current batch of products.

The inspection has two main meanings: first, it is used to evaluate the sterility assurance level of the batch of products; second, after long-term accumulation of multiple batches of pre-sterilization microbial content data, it can be The microbial contamination status of each process step is evaluated as a whole, so as to indicate whether the production system is effectively controlling the microbial contamination at a good level, whether it needs to be improved, and so on.

24. What is the method of microbial species and quantity research? What equipment is needed? If the residual probability method is adopted, is it necessary to measure the microbial level during the production process? If it is introduced, how much cost will it increase? As a large infusion manufacturer, use the residual probability Method, is it necessary to establish a special microbiology laboratory to test the microbial contamination level of the liquid medicine before sterilization?

Answer: The degree of microbial contamination-that is, the quantity inspection can be carried out in accordance with the microbial limit inspection method included in the Pharmacopoeia; the type of microorganisms can be identified from the following aspects: 1) Observe the colony morphology by naked eyes; 2) Microscopically inspect the morphology and Mobility; 3) General biochemical test: Gram stain or 3% KOH test; 4) Biochemical identification (ie API test) to identify species.

When the residual probability method is used, the level of microbial contamination before the product sterilization should be tested, including the boiling test of the contaminated bacteria (such as 100°C, 15 minutes) and the microbial count.

Microbiology laboratory is essential for injection manufacturers. Its basic functions should include microbial limit inspection of raw materials, microbial contamination inspection before product sterilization, product sterility inspection, bacterial endotoxin inspection, and production environment dynamic inspection (air Particles, planktonic bacteria and sedimentation bacteria count). Conditional laboratories can also carry out the following tasks: microbiological identification test (it is recommended that the central laboratory of the enterprise group carry out API identification), the calibration of biological indicators (that is, the determination of the D value, it is recommended that the central laboratory of the enterprise group carry out this work) .

25. Is sterilization process verification necessary for the overkill method? What is the difference between the product development process research of the residual probability method and the verification in actual production?

Answer: Of course, verification is required. Article 83 of the EU CGMP Annex: All sterilization processes should be verified.

The residual probability method is the design of the sterilization process, which itself needs to be verified and confirmed.

26. Has the overkill method determined that the microbial challenge test is not required?

Answer: The connotation of the over-killing method is that the microorganisms in the product are reduced by 12 logarithms. The microbial challenge test is to prove that the residual probability of microorganisms is not greater than 10-6, so the over-killing method does not need to carry out the microbial challenge test.

27. Does the terminally sterilized product require separate equipment verification for each type applied for registration? Is it possible to perform equipment verification only once, and other varieties can be used in common? Can the equipment verification materials not be attached to the variety verification materials, but only for archiving for future reference?

Answer: For terminally sterilized products, it is not necessary for each type to be registered for a separate equipment verification. If the registered variety uses the same or lower sterilization conditions, only equipment verification under higher temperature sterilization conditions can be performed; The equipment verification data for the sterilization conditions of the variety should also be attached to the verification data of the variety.

28. Can the sterilization process of non-solution dosage forms, semi-solid or powder injections calculate a value similar to the F0 value? Can SAL be calculated? What is the specific requirement?

Answer: The F0 value can not be calculated for the sterilization process of non-solution dosage form, semi-solid or powder injection, but SAL can be calculated. For details, please refer to the decision tree of EU sterilization process selection.

29. Regarding the medium filling test, whether it is for the production line verification or for the declared product (each product must be filled for three batches of verification), please ask: (1) Can the production line verification replace the filling test of the declared product? (Similar varieties)? (2) Which medium is used, are both thioglycolate and modified Martin required? (3) Does the GMP inspection require three batches of filling tests for different packaging specifications?

Answer: (1) The simulated filling test of culture medium should be carried out separately according to different products, packaging specifications, and packaging forms. For example, if the product is a powder injection, the medium simulation filling test usually fills the liquid medium first, and then the sterile powder (such as PEG sterile powder) that simulates the product; if the sterile injection solution or freeze-dried For powder injection, you only need to fill the liquid culture medium; if the packaging specifications are 2ml, 5ml, 10ml, even the same product needs to be separately tested for the media simulation filling of their respective specifications; if the packaging is There are vials or other forms (such as pre-filled syringes, ampoules), and different production lines require separate medium simulation filling tests.

In the specific work, considering that the medium simulation filling test is actually a systematic verification of the entire production line, including production equipment, environment and personnel operations, etc., if the medium simulation filling test is carried out, the worst condition is used, that is, the bottle The largest, the slowest filling speed, the largest number of personnel, and the time is longer than the normal production time. It is also not necessary to carry out the simulation filling test of each product and each specification of the medium; if it is not the worst condition, it should be based on the product, Different packaging specifications and packaging formats shall be carried out separately.

(2) Generally, a broad-spectrum medium is used, which can promote the growth of Gram-positive, negative, yeast and mold, such as soybean tryptone medium. Anaerobic medium is only used under special circumstances.

(3) Only in the first verification, each specification needs to successfully carry out 3 consecutive medium simulation filling tests, and then 2 times a year, each time a batch of medium simulation filling tests can be carried out.