Experiences with the use of the Microbatch Under Oil Method of Crystallisation

1. Experiences with the use of the Microbatch Under Oil Method of Crystallisation Nafeesa Noordeen & Sandra Cowan-Jacob, Novartis Pharma AG, Basel, Switzerland. Introduction: The Microbatch under oil method of crystallisation is a good way of screening many crystallisation conditions using a very small amount of protein. We have screened a variety of different proteins using the Oryx robot from Douglas Instruments (microbatch method) and the Cyberlab C200 robot from Gilson (hanging drop method of vapour diffusion). Our aim was to determine the relative success rate of microbatch versus vapour diffusion, the size of the drops that could be used for microbatch and the best way of storing these experiments. We have also started investigating using standard 96 and 384 well trays for storing of stock solutions and screening experiments. Comparison of Microbatch and Vapour diffusion for screening. Vapour diffusion (VD) experiments were done with drop ratios of 1+1 or 2+2 mL. Microbatch (MB) experiments were generally 0.5+0.5 mL or less. All screens are commercially available (i.e. Hampton Research, Jena Bioscience, Emerald biostructure). Both methods are equally successful overall in finding initial crystallisation conditions. However, different proteins can be more successful in one or the other methods. Hence, use both methods whenever possible. Comparison of different drop volumes suitable for Microbatch crystallisation: MB drop volumes as small as 0.2 + 0.2 mL are equally successful compared to larger volumes Only 6 out of a total of 10 conditions are common between MB 1+1 mL and the smaller drop volumes. There are a similar number of common conditions with VD for any of the MB volumes. For this particular protein, MB gives more unique conditions than VD and all of the VD conditions were also found using MB. * versus VD 1ml+1ml, **VD versus all MB Common conditions between MB 1ml+1ml and MB 0.5ml+0.5ml (or 0.3ml, 0.2ml): 6 Drying out of drops- Time verses Drop volume: Trays were stored at room temperature in a cupboard. This investigation gives us a general idea of how long an experiment will last. The smallest volumes dry out very quickly. This may prevent discovery of new crystallisation conditions. We have tested various methods of storage for prolonging the lifetime of the experiments with 0.2ml+0.2ml and 0.3ml+0.3ml drop volumes. Adding a reservoir of water to a particular box increases the lifetime of the experiments by approx. 25 days. As expected, polypropylene boxes are more effective than polystyrene. Methods: The oil-dropping-on method is used in preference to dispensing drops under oil. We find that the former method gives more centred drops when dispensing very small drop volumes. 15mL of paraffin oil was dispensed on top of each drop and then the whole tray was covered with about 4 ml of Al’s oil (50:50 Paraffin/Silicon) to let some evaporation occur. • Conclusions: • Microbatch and Vapour diffusion are equally successful in screening for initial crystallisation conditions. • It is possible to use small volumes (0.2ml+0.2ml) for MB screening as it gives a similar number of crystallisation conditions as larger MB volumes. • Storage in polypropylene boxes is most effective in prolonging the lifetime of small volume experiments (but only to a month ). Adding a reservoir of water to any type of storage box increases the lifetime by about 25 - 30 days. • The ability of the robot to perform 96 well experiments means that the method is moreautomated and requires less operator time per 48 wells.