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On the purification of 100’s potential API candidates by reversed-phase preparative chromatography
UCB is a global biopharmaceutical company dedicated to the research, development and commercialization of innovative medicines with a focus on the fields of central nervous system disorders and immunology disorders.
At UCB Biopharma in Braine-l’Alleud (Belgium), one of the two research centers, day after day, numerous intermediates and potential Active Pharmaceutical Ingredient (API) candidates are sent to the Preparative Chromatography Team for purification.
These crude mixtures are rarely optimized, as shown on Picture 1, and can be purified in Liquid or SFC mode. Liquid purifications are realized in Reverse Phase (RP) or Normal Phase (NP), generally depending on the amount to purify, on the compound’s polarity, and taking into account chemist’s constraints. Usually, big quantities (> 3-4 g) are separated in NP on silica (~1 kg in a 8 cm ID column) and smaller quantities (< 2 g) in RP on C18 (~500 g in a 8 cm ID column).
The method used for these RP purifications is relatively standard using an adapted gradient Acetonitrile/Water with Ammonia as buffer. This parameter is very important for the choice of the stationary phase. Indeed, a robust phase maintaining its efficacy at a pH around 10 is needed.
For a long time, several phases from different providers have been continuously tested and compared for the lifetime, the pressure behavior, and above all the selectivity and the loading capacity.
During the last 3 years, around 1500 injections from 100 mg to 2 g were completed on Kromasil Classic, Eternity and EternityXT (C18, 10 µm). EternityXT, which we are still using today, was particularly appreciated because of its great lifetime allowing us to reduce the frequency of packing. Indeed, previously, some phases needed a new packing after some months (between 1 and 4), but with EternityXT a lifetime around 8 months can be reached.
It could also be interesting to note that the workflow is so robust that a change of the phase is generally not desired. Indeed, each new crude mixture is systematically analyzed on an analytical column and thanks to the retention times of the target and the impurities, the adapted gradient to use is tuned for successful purification. But when the stationary phase in the prep column is continuously changing, it is very difficult for the operators to complete their work. Fortunately, it is not the case with the Kromasil EternityXT which stabilizes very quickly. Further, we have been able to work successfully with this phase and only repack with fresh EternityXT material after 18 months of usage, with a minimum of 2 injections per day and 250 days per year.
