A lot of the work I currently do is related to aseptic manufacturing. In fact, most of the companies I work for, have products that need to be sterile and cannot be terminally sterilized. Therefore, a filtration step is involved to sterilize the bulk drug product. To build in enough sterility assurance in the process, the bioburden before filtration needs to be determined. According to the note for guidance, the bioburden needs to be NMT 10CFU/100mL, depending on the size of the filter and the volume to be filtered.
Determination of the bioburden is done according to EP 2.6.12 or USP <61>. Both methods are harmonized. The descriptions in the monographs state more than 1 way of performing this analysis, however, as the bulk solution during manufacturing (=bulk drug product) is going to be filtrated, the filtration method will be the method of choice. In the chapter in the USP and the EP, there is also mentioned that the suitability of the method should be evaluated and it is also metioned how this should be done. In short, it will be tested whether the compound/solution you are going to filter might interfere with the ability of micro-organisms to grow.
During the testing, the bulk will be filtered over a membrane filter and after that, the filter will be washed with a solvent. The USP and EP describe several solvents that can be used. So basically the idea is that all material will be washed of the filters and the micro-organism(s) will stay on the filter. The filter will be incubated on two types of agar plates (one for the counting of bacteria (TAMC) and on for the counting of yeast and fungi (TYMC)). After incubation, the number of colonies on the filters can be counted and this way the number of CFU/volume tested can be determined. In order to determine the suitability of the method, bulk solution spiked with micro-organisms (the types are described in the EP and USP) will be filtered and the filters will be washed with solvent and the filters will be incubated. As controls, the same steps will be done with solutions containing micro-organisms, but without the bulk drug product solution.
Interpretation of results
After incubation, differences between the counts on the filters with bulk drug product and without bulk drug product cannot differ more than a factor of 2. If the difference is more than a factor of 2 for any of the micro-organisms tested, the method is not suitable and the analytical method needs to be improved, by either varying the washing solvent, the type of filter or the agar on which the plates are incubated. Eventually this should lead to a method for the determination of the prefiltration bioburden, that is suitable for all micro-organisms mentioned in the EP or USP. If the above criteria cannot be met for one of the organisms tested with any of the described methods, the method and test conditions that come closest to the criteria are used to test the product.
Validation during pharmaceutical development
Prefiltration bioburden testing is done in order to increase the sterility assurance level of the aseptic processing. This means that the test will be part of the safety characteristics of the final drug product. By saying this, validation of the bioburden testing should be done already at phase 1 of clinical development. However, if the aseptic processing part will not alter the composition of the bulk drug product solution, the validation of the bioburden testing might be delayed until a later phase into clinical development. This should of course be documented, either in a procedure, or a quality statement including a risk assessment. The reasoning for the possible delay is that when a phase 1 clinical study is being started material is usually limited, and for validation of the method at least 5 times the amount as needed for testing is necessary. Furthermore, and this is the most important reason, the sterility testing of the final drug product will be done with the same type of method as the bioburden testing, so a membrane filtration and incubation. The sterility test definitely needs to be validated, so if during this validation it is shown that the product does not interfere with bacterial or fungal growth, it can be reasoned that this will also be the case for the bioburden testing. Again, this should be well thought through and properly documented, as the method is related to safety.
Of course during the course of the development of the drug product, the bioburden method needs to be properly validated before the product goes to phase 3/commercial.