By Thibaut Monet-Dedreuil, Technical Specialist, RSSL
During a stability study for an Active Pharmaceutical Ingredient (API) or drug product, a sample may be found to contain impurities. These impurities might occur as degradation products, as intermediates/process impurities originating from the synthetic route, as an unwanted by-product of side-reactions, or due to contamination of the original sample.
Whilst not unexpected given the purpose of a stability study is to assess degradation in both real time and under stressed/accelerated conditions, these impurities may well be unknowns. It is important, therefore, that when found above the defined limits (ICH Q3A(R2) and ICH Q3B(R2)), they are identified, so that the implications for product safety can be understood. To this end, the impurities need to be isolated in sufficient quantity and purity for identification.
The technique best suited to this procedure is preparative LC, using low or high pressure columns. It essentially involves loading of a preparative-scale LC column with repeated doses of the sample and collecting fractions at known time intervals in order to isolate, and subsequently concentrate sufficient impurity for identification purposes. Highly sophisticated techniques, such as Nuclear Magnetic Resonance (NMR) spectrometry, Fourier Transform Infra Red (FTIR) spectroscopy and Mass Spectrometry (MS) can then be employed on the isolate to help identify the chemical impurity.
There are some limitations to the technique, not least of which is the fact that the impurity itself may be unstable under the conditions of isolation. Hence the process of isolating and concentrating the impurity is not always as straightforward as the explanation above suggests. It is critical to choose the right column stationary phase and solvents, and to control conditions carefully, so that the impurity is isolated and stable long enough for the purposes of identification.
Preparative chromatography is also suitable for isolating reference standard-quality material, again by repeatedly loading the sample onto the column and collecting fractions within a narrow band of the sample peak, where the target molecule should be at its purest. Depending on the sample, a few grams of reference standard can be produced in this manner. Again, the identification techniques of NMR, FT-IR and MS can be used to aid in the characterisation of this and subsequently establish it as a Primary Reference Standard.
Small and large molecules
The approach is appropriate for small and large molecules, depending on the nature of the columns used, meaning it has applications for both traditional pharma and biopharma customers.
Impurity identification and sample purification are both essential aspects of pharmaceutical development, and these services are an important part of the support that RSSL can offer customers in this area, whether that be for existing compounds or New Chemical/Biological Entities.