Bioburden method

Continue testing as described in bioburden method, steps 7 through 19, using aseptic technique. 

Enumerate the colonies on all plates according to bioburden method, steps 21 and 22. Record all data. 

Follow steps 1 through 3 in bioburden method for sampling and preparation of vials. 

Follow steps 2 through 5 in the section about stopper bioburden method for heat shocking, cooling, shaking, and sonicating samples (one container containing 20 small vials 20 half-filled vials). 

Continue testing as described in spore bioburden method steps 6 through 8. 

Microbial limits should be based on USP WFI limits for endotoxin and the detection limits of the bioburden method. Consideration should be given to where the manufacturing equipment resides, for example, class 10,000 for compounding tanks and class 100 for surge tanks, when scientifically sound microbial limits are determined. 

The LAL and bioburden methods must be developed to isolate and quantitate bacteria and endotoxins. Rinse samples are usually tested for bacterial endotoxin (LAL) and swab and rinse samples are tested for bioburden. Isolated microorganisms should be identified to an appropriate level, whenever possible. 

It is necessary to determine the bioburden and make cycle verification studies when ethylene oxide sterilization is used, as it is for other sterilization methods. The manufacturer of hospital sterilization equipment provides cycle recommendations based on the expected bioburden and the consideration of an appropriate safety factor. In ethylene oxide sterilization, it is necessary to determine if residues of the stefilant are absorbed by the sterilized article, and to examine the possible formation of other potentially toxic materials as a result of reaction with ethylene oxide. 

For sterile products, particular attention should be paid to the choice of an appropriate method of sterilization. Wherever possible a terminal sterilization process should be applied to the product in its final container-closure system, as suggested in the Ph Eur. The preferred options include steam sterilization, dry heat sterilization, and irradiation using the Ph Eur listed conditions (saturated steam at 121°C for 15 minutes dry heat at 160°C for 120 minutes irradiation with an absorbed dose of not less than 25 kGy). Where these cannot be used, the application must include justification for the alternative procedure adopted on the understanding that the highest achievable sterility assurance level should be achieved in conjunction with the lowest practicable level of presterilization bioburden. There is guidance in the form of decision trees as to the preferred options for sterilization method to be applied ... 

Justifications for the use of nonstandard (i.e., nonpreferred or nonpharmacopeial) methods of sterilization may include the heat instability of the active ingredient or an essential excipient. The choice of a method based on filtration through a microbial retentive filter and/or aseptic assembly should be justified, and the appropriate in process controls (including bioburden controls on active ingredients, excipients, bulk solutions, process time constraints etc) discussed in detail in the application. Commercial considerations should not form part of the argument for the application of a nonstandard sterilization process. The highest possible sterility assurance level should be achieved. 

Where terminal processing is not possible, the justification for alternative sterilization methods will be included in the EPAR, or at least a statement to the effect that sterile filtration/aseptic processing will be used. Presterilization bioburden issues that arose during the assessment will be included in the EPAR. 

Bioburden testing is performed on components prior to steam sterilization as verification that the sterilization parameters are sufficient for sterilization of components. The method for determining the bioburden level of components is included in the manufacturing site standard test method. For product component bioburden summary, refer to (provide reference attachment number). 

Growth promotion shall be performed per standard test method (provide number), where USP growth promotion test organisms and representative panel of microorganisms normally found environmentally or in the product bioburden shall be used in growth testing the medium-filled containers. 

The purpose is to evaluate the bioburden, spore bioburden, and endotoxin present on rubber stoppers and unprocessed glass vials. The similarity of the contents and procedure description shall be considered coincidental due to the similarity of generic methods. 

E. coli is recommended. The microbiological assessment of preservatives is required when preservatives are used in a pharmaceutical product to control microbial bioburden. The test microorganisms and methods for evaluating the efficacy of the preservative in pharmaceutical products are described in the general chapter on preservative effectiveness tests [26]. 

Assay methods for monitoring any degradation products may be used to justify the time limits for bulk storage. This time would include the period from when the product is formulated to the end of the filling operation. Because most lyophilized formulations do not contain a biological preservative, microbiological quality before sterilization by filtration must also be monitored. The unfiltered solution bioburden would include microorganisms and endotoxin levels. 

The predominant method for terminal sterilization is moist heat, and a substantial percentage of sterile products are processed in this manner. (Estimates range from 5 to 15% of all sterile products are terminally sterilized.) The sterilization often requires the attainment of a balance between sterility assurance and degradation of the material s essential properties [42],The overkill sterilization method is preferred for heat-resistant materials, and may be usable for terminal sterilization where the formulation can tolerate substantial heat input. The bioburden/biological indicator approach uses less heat input but requires increased control over the titer and resistance of the bioburden organisms present. 

The principles behind the sterilization processes are described in Chapter 20. The choice of method is determined largely by the ability of the formulation and container to withstand the physical stresses applied during the sterilization process. All products intended for sterilization should be manufactured under clean conditions and therefore will be of low microbial content (bioburden) prior to sterilization. Under these conditions, the sterilization process will not be overtaxed and will generally be within the safety limits needed to provide the required level of sterility assurance (Chapter 20). The next section emphasizes parenteral products, but the practices described apply to many other types of sterile product. 

The term bioburden refers to the amount of microbial flora that can be detected on an item or surface or in a solution. The microbial recovery method used depends on the type of material being evaluated. Aerobic bioburden counts in parenteral solutions are obtained by conducting the total aerobic count and total yeast and molds count as specified in the USP microbial limits test (<61>) or a equivalent compendial test. Alternatively, a modified membrane filtration technique can be used to allow filtration of larger volumes of solution to assess bioburden recoveries where sample results are expected to contain a negligible number of microbial flora from the overall solution. 

Microbial limit tests, as described in USP 24-NF 19 (<61>) provide the information necessary to conduct bioburden counts by quantitative estimations of viable aerobic microorganisms in pharmaceutical articles, raw materials, and finished dosage forms. Consult the EP or JP for any differences in these methods from those of the USP. Note that USP (<61>) is currently under revision and that the next version will be fully harmonized between the USP, EP, and JP. The microbial limit test comprises the total aerobic bacterial (microbial) count (TAG), the total combined yeasts and molds count (TYMC), and tests for indicator organisms. Bioburden tests are also referred to as total viable counts. This test estimates the total number of viable (culturable) aerobic microorganisms in various items including parenteral UBPS. The total viable count is obtained by adding the counts from the TAC and the TYMC. 

The TAC can be conducted using a number of microbiological methods. These are the pour-plate, membrane filtration, and multiple tube methods. The TYMC is conducted by using either the pour-plate or membrane filtration method. The TAC for bioburden is performed by adding 10 g, 10 mL, or 10 units in SCD broth or lactose broth to make 100 mL. Aliquots of this sample preparation are transferred into four standard size (15 x 100 mm) Petri dishes. Into two of the plates 15-20 mL of molten SCD agar is poured, and into the other two the same volume of Sabouraud dextrose agar (SAB) agar is poured. 

---