Recently, I've been asked if I know why the ASTM F838 bacterial challenge test sets a maximum pressure of 30 psi (= 2.07 bar). Should this limit not be exceeded as no retention capability of the filter upwards can be provided? The answer to this question is a clear no. Filter membranes are still bacteria-retaining even at much higher pressures. For example, Millipore Durapore PVDF 0.22 µm membranes are possesing a water-wetted bubble point of 3.45 bar, which correlates with bacterial retention (see also our blog article "How is the filter’s bacterial retention capability connected with its integrity?"). So this is not the reason.
Let's start with an example. During validation of an analytical method the linearity should be evaluated. Therefore 5 concentrations with 3 replicates each are examined (in order to evaluate linearity and accuracy in one approach). At the examination level corresponding to 120% test concentration, one value is considerably higher than the other two ones. However, even taking this value into account, the acceptance criterion can be met. But the question is what to do. Is it necessary to repeat the entire 120% concentration with all three replicates, are we allowed to exclude the conspicuous one and calculate the mean by using just the two good ones, or do we have to repeat the whole linearity experiment?
For pharmaceutical companies that manufacture their products under aseptic conditions, sterile filtration is the last step before filling. The validation of the manufacturing process also includes the step of sterile filtration. Accordingly, it must be demonstrated by filter validation that the filter is able to retain bacteria and thus ensure the sterility of the product. This evidence is provided by a bacterial challenge test according to ASTM F838-15a. But, before this bacterial challenge test can be performed, it must be checked by a viability test that the product solution has no negative effect on the bacterium used in the bacterial challenge test. That sounds complicated? It shouldn’t - just imagine what would happen if your product solution is killing the test bacteria... You wouldn’t know if you get a sterile filtrate in the bacterial challenge test due to the fact that the filter did a good job or due to the fact that your product solution already killed the bacteria.
A request via our homepage prompted me to focus more intensively on the terms "dilution integrity", "dilution linearity" and "parallelism". They all belong to the area of bioanalytical methods’ validation, i.e. those methods in which a biological sample such as human or animal blood or urine is examined. Such analyzis are needed during the development of drugs in the context of preclinical and clinical studies. The analytical methods to be used, such as LC- or GC-MS or ligand binding assays (LBAs), have to be validated before use. In contrast to the validation of analytical methods for already authorized drugs, there is currently no harmonized guideline on the requirements for the validation of bioanalytical methods. Thus, the different authorities have issued their own guidelines, see also our blog article. Accordingly, confusion can easily occur. In this blog article we would like to explain the 3 terms in more detail and differentiate between them.
Sterile filters are used in pharmaceutical production, in particular in aseptic manufacturing of parenterals. As part of filter validation, evidence must be provided that the filter used for sterile filtration is able to retain bacteria and potential undesirable components such as particles or fibers, thereby ensuring the sterility of the product. The corresponding test is the bacterial challenge test according to ASTM F838-15a. Therefore, the filter is flown with a defined number of particularly small bacteria (Brevundimonas diminuta) in solution and subsequently, the filtrate is examined for bacterial growth. A sterile filtrate shows that the filter is able to retain > 107 CFU/cm2 filter surface area and is therefore suitable for its intended use.
As announced in summer this year and now in more detail in November, ICH's guideline on validating analytical methods will be revised. The content of this guideline comes from two previously separate guidelines merged in 2005, which were published more than 20 years ago. The decision to revise the validation guideline ICH Q2(R1) was made at the ICH meeting in June, together with the decision to create a new analytical procedure development guideline (ICH Q14). Currently, it is unknown whether these will be two separate documents, or whether a combined guideline will result as a possible combination should be examined by the Expert Working Group.
In our last blog post we had a look on the first approach to infer accuracy from linearity in method validations by applying normalization. This can be used to determine accuracy in case no alternative methods or impurities for spiking are available. Apart from normalization, another possibility to solve this problem exists, which will be highlighted today.
We stick to our example of the last time: a SE-HPLC method is used for purity determination. The impurities are given as the sum of minor peaks, while the main peak is our desired product. The approach to infer accuracy from linearity is allowed by the ICH Q2(1) validation guideline, when specificity (we suppose this again) and precision is given.
In today's blog, we’d like to address a quite common problem during method validations and by using a practical example showing possibilities to solve it.
A SE-HPLC method used as a purity test of a drug shall be validated. It's known that impurities (e.g. aggregates and / or fragments of the product) may arise in low concentrations. In the chromatogram, you’ll see the main peak corresponding to the intact product and a few minor peaks corresponding to impurities, which will be reported as sum.
Let's have a look at the validation parameter "accuracy". Unfortunately, neither impurities are separately available for spiking experiments nor independent alternative methods are existing. In this case, the validation guideline ICH Q2(R1) offers the following possibility for the evaluation of accuracy: "Accuracy may be inferred once precision, linearity and specificity have been established" (4.1.1 c / 4.1.2 c).