Flow as a Control Strategy to Promote Biomolecule Nucleation.

The main aim of this chapter is to provide an (at least semiquantitative) explanation of biomolecular crystallization (nucleation and growth) experimental results in solution flow, in order to further assist with optimization of the process. An overview will be provided of biomolecular crystal nucleation and growth in forced solution flows, but also in the presence and absence of natural (buoyancy-driven) convective flows. The chapter focuses first on the theoretical analysis of the different mass transfer modalities, diffusion versus convection dominated (both of crystal building blocks and of impurities), leading to biomolecule-depleted and to self-purifying (i.e., impurity-depleted) zones. The same topics will then be addressed, describing the experimentally investigated influence of forced flows, artificially created through air pressure, peristaltic pumping, oscillatory mixing, stirring, shearing, and other methods, both in small-scale laboratory and in large-scale industrial settings. In addition to forced flows, work on the influence of gravity-induced naturally occurring solution convection and crystal sedimentation will be reviewed, as well as the effects of its absence when crystallization trials are carried out under microgravity environments. By developing the principle of the separation of the nucleation and growth stages of crystallization ("double-pulse technique") not only in time but also in space, the reported results have shown that at solution flow velocities larger than those of buoyancy-driven convection, most crystals nucleated heterogeneously, while bulk-nucleated crystals predominated at lower flow velocities. Lastly, fluid flow also allowed the investigation of the important role of impurities of biological origin on the kind of crystal nucleation, homogeneous or heterogeneous. In view of the diverse needs for protein crystals (structure determination by conventional X-ray crystallography or by XFEL, industrial production of pharmaceuticals), flow-utilizing strategies may be adjusted to each specific crystallization need. Experiments could also be undertaken that would optimize the flow parameters separately and successively for each stage of the crystallization process (nucleation, growth, cessation of growth). Finally, it would be extremely informative to widen the range of proteins used in crystallization experiments involving flow, besides the usual lysozyme and a handful of others that have already been studied.