Introduction

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4.3 Case Study II: Combined Solid Layer Melt Crystallization and

85 pumps. More expensive solutions are necessary to reduce the pressure even further, and in the presence of salt and protein, the vapour pressure will be lower than for pure water due to the ebullioscopic effect.

Two obvious alternatives are to freeze-out the solvent, which is the subject of this section, and membrane-based separation operations (Curcio 2003, Di Profio 2003, Curcio 2005, Di Profio 2005a, Di Profio 2005b, Curcio 2006, Gugliuzza 2009, Di Profio 2010), which are not discussed here. Specifically, the combination of solid layer melt crystallization with solution crystallization is addressed here. Two melt crystallization technologies are commonly used for industrial separations, one is solid layer melt crystallization and the other is suspension crystallization. While both employ cooled surfaces to reduce the temperature of a melt to below the temperature of solidification of the material to be purified, a major distinction between the two is that the solid layer method allows the product to grow on the heat transfer surface, whereas in the suspension crystallization the product crystals are generated in the melt. This leads to significant differences in the way the processes are operated and the quality of the product obtained (see, for example, (Ulrich 2003, Ulrich 2004)). Solid layer crystallization has the advantage that, in the simplest implementation, no moving parts are required in the equipment. In addition, solid-liquid separation is comparatively straightforward, the surplus and depleted liquor is simply drained from the crystallizer by gravitational action, after which the product can be recovered by melting and draining. For organic solids in particular, growth of the solid crystalline layer on the walls of the heat exchanger leads to a progressively increasing heat transfer resistance and due to their poor heat transfer coefficients and the increase in solid layer thickness with time. In practice, the reduction in heat transfer due to the increasing layer thickness is compensated by applying a decreasing temperature to the cooled surface to compensate for the decrease in heat transfer and to ensure effective heat dissipation.

Since the temperature gradient across the solid-liquid interface largely determines the growth mechanism of the solid layer, and this temperature gradient must be smaller than the gradient of the solidus in the vicinity of the interfacial temperature in order to ensure smooth surface growth and minimisation of inclusions. Once the temperature gradients at the solid-liquid interface exceed the gradient of the solidus, the change in composition of the liquid layer in front of the solid layer due to solute rejection may lead to constitutional supercooling, where the solution is supercooled despite possessing a higher temperature than the solid surface (Tiller 1953). Constitutional supercooling leads to unstable growth of the solid surface, often observed as dendritic growth, which itself leads to increased liquid inclusion in the solid layer and a concomitant reduction in its purity. Clearly, the temperature gradient across the interface is an important control parameter for a solid layer melt crystallization process. In practice, smooth interface growth is rarely achieved and fast growth rates more than compensate for increased impurity levels, which can be mitigated by post-crystallization treatments such as washing and sweating. In fact, sweating, that is partial melting of the crystal surface, is an attractive post-crystallization purification step due to the behaviour of impurity inclusions during crystallization. As a result of the temperature gradient, impurity

86 rich liquid inclusions tend to migrate towards the warmer surface of the growing crystal (Burton 1953, Wilcox 1968, Scholz 1993, Henning 1997), equivalent to the behaviour of impurities in zone refining. As a consequence, impurity concentrations are expected to decrease from the surface layer towards the cooling surface and in fact this is generally observed. Partial melting of the outermost layer of the crystal is therefore an effective post-crystallization purification step.

Suspension crystallization does not suffer from the heat transfer issues common in solid layer crystallization, providing the surfaces of the heat exchanger exposed to the melt are regularly scraped clean, a commonly implemented technical solution. Since suspension crystallization is not considered in the following, no detailed discussion is provided here. The interested reader may refer to the following literature for more information (Ulrich 2003, Ulrich 2004).

The rational of the process discussed below is to create sufficient supersaturation for nucleation and growth of protein crystals in solution by removing the solvent by solid layer melt crystallization and at the same time minimising the amount of precipitating agent employed, hence the coupling of solid layer melt and solution crystallization.

For the purpose of removing the solvent and concentrating an aqueous protein solution including a suitable precipitant, solid layer melt crystallization is the method of choice, since the solvent removed adheres to the surface of the heat exchanger and does not form a suspension with the liquid phase. Providing the heat exchanger is not a fixed part of the equipment, it can be removed from contact with the solution, together with the frozen layer of water, allowing easy analysis of the crystalline solvent layer. Once a sufficient amount of solvent has been removed from the solution to generate a sufficiently large degree of supersaturation with regard to the protein to be crystallized, nucleation can be allowed to occur or the solution may be seeded for controlled growth of crystals. A number of prerequisites have to be fulfilled with regard to the physical properties of the system and the process conditions imposed thereon for this procedure to be successful:

 The solvent to be frozen out (in this case water), must not form a solid solution or a compound containing a significant amount of one or more of the solution components under the conditions selected.

 None of the components solidify at a temperature higher than that of the solidification temperature of the solvent under the given thermodynamic conditions.

 The layer growth process has to be controlled carefully to avoid constitutional undercooling of the liquid in close proximity to the solid interface, in order to avoid liquid inclusions due to unstable, dendritic growth of the solid layer.

 The protein crystals must not form by epitaxy on the solid ice layer, as this would necessitate a mechanical removal of the product from the solidified solvent.

The first prerequisite has to be investigated on a case to case basis, preferably by studying the phase diagram of the system. In the case of crystallization of lysozyme chloride from an aqueous NaCl solution, there is no known risk of forming a solid phase containing water and the precipitant until temperatures lower than ca. -20 °C, where sodium chloride solidifies as a

87 dihydrate together with ice, or for salt concentrations greater than 30 %(wt/wt), where again, at temperatures below 0 °C sodium chloride dihydrate is formed (Korolev 1939). The incorporation of protein into the solid water layer was subject to investigation in the study discussed below.

The second point is fulfilled, as long as all solution components that are solid at the temperature of operation of the process are present at concentrations below their respective saturation concentrations. At the salt concentrations employed, this is the case for sodium chloride, the buffer concentrations are also sufficiently low so as not to become supersaturated and merely the lysozyme concentration is such, that crystallization should occur, and this, indeed, is desired. More importantly, the crystallization must not occur epitaxially on the ice layer (point 4) as this would lead to the need for additional, mechanical separation of the crystals, providing they have not been encased by the propagating ice layer.

Point 3 is the most important prerequisite and a key point of investigation in the case study presented.

One major advantage of the combination of melt and solution crystallization is that the process is subject to low temperatures at all times, reducing the risk of protein degradation due to extended exposure to high temperatures. Furthermore, raw materials can be limited to absolute minimum amount necessary, as the concentration change with respect to the protein and the dissolved precipitating agent can easily be calculated as a function of the water removed. As a consequence the amount of saline waste-water produced can be reduced. In order to demonstrate the suitability of the process, the fate of the protein generated in the process has to be investigated, considering the quality parameters already introduced in the study on urease.

4.3.2 Application of Combined Solid Layer Melt and Solution Crystallization to the