8. The Cubic Lattice Crystallizer !LINK!
On the basisof the rising demand for protein crystals, large-scalecrystallization processes are increasingly important, particularlyas the range of biotechnologically produced proteins is expanding.However, a systematic understanding of large-scale protein crystallizationprocesses, especially with respect to scale-up, control, and optimization,is scarce. In general, scale-up of crystallization processes is notstraightforward. This is even more true for protein crystals, as theirstructure and quality are highly dependent on the process conditions.Impeller tip speed, mean power input, and agitation rate are typicallyused as criteria for scale-up in technical-scale stirred batch crystallizers.12 However, it is well-known that for all scale-upmethods, mixing times, maximum shear rates, and local dissipationrates of turbulence vary vastly between small and large-scale systems.For mechanically fragile systems (such as proteins), this is problematicas in order to reach the same macro-mixing, much higher maximum shearrates are realized in the impeller zone.
8. The Cubic Lattice Crystallizer
To obtain good heat transferand to avoid the formation of foldsand creases that may potentially obstruct free particle flow, thereactor tube was loosely coiled on metal cylinders (Ø = 21.5cm) immersed in the water baths. At the outlet of the crystallizer,samples were collected for analysis. For the microscopic examinationsamples of the suspension were directly dripped on the microscopeslides. The yield of the process was determined via spectrophotometricmeasurements. Product samples were taken by dripping the product suspensioninto Eppendorf tubes. Crystals could now be separated from the suspensionvia centrifugation (15000g, 20 C, 2 min, centrifuge:Hettich 320R, Tuttlingen, Germany), and the remaining concentrationof lysozyme in the supernatant was determined photometrically at 280nm by comparing it to a lysozyme standard (extinction coefficient A280nm = 2.48).
The yield of the process was calculated byanalyzing the concentrationof lysozyme at the inflow and outflow. Starting at 49.9 g/L, the concentrationin the supernatant dropped to 45.1 g/L after the first water bath(nucleation zone). After the second water bath which had a slightlyhigher temperature, the concentration dropped to 29.5 g/L. At theoutlet of the crystallizer, the final concentration was 15.8 g/L,which corresponds to an overall yield of the process of 68% with aresidence time of 113.4 min. Although the differences in operatingtemperatures in the three zones of the continuous crystallizer mayappear small, they are optimal to minimize nucleation in water bath2 and 3 and to simultaneously guarantee high growth rates throughoutthe reactor. This was confirmed by microscope analysis when increasednumbers of microcrystals obtained during experiments at lower temperaturein water bath 2 and 3, respectively.
Obviously, the length ofthe nucleation zone has an impact on theproduct crystals and can be adapted to meet particular requirements.The chosen value is a good compromise between the size of crystalsproduced, the crystal size distribution, and the obtained yield. Theoverall length of the crystallizer was limited by nonuniform flowpatterns potentially caused by exceeding air volumes inside the reactor.The use of bigger air slugs or smaller bubble intervals contributedto this phenomenon, coupled with higher flow rates or smaller productionrates.
In contrast to agitated systems (data not shown here), weachievedthe formation of well-defined crystals and high yields within shortresidence times in a tubular crystallizer. Running the crystallizerat a low average velocity of 1.9 mm/s ensured a laminar flow and verylow shear forces at the surface of the particles. Mixing induced bythe air bubbles proved to be sufficient to overcome sedimentationof crystals inside the reactor. In experiments without introductionof air slugs, crystal transport was ineffective, leading to a highresidence time distribution and possible accumulation of crystalsinside the crystallizer. Moreover, product analysis of these experimentsshowed increased numbers of crystals aggregates.
These findings are in line with preliminary tests using batchreactors(see Supporting Information) in our study.Without agitation, crystals of over 100 μm in size (most ofwhich were sticking to the wall) were obtained within 24 h in the10 mL batch experiments. An agitation speed of 180 rpm led to theformation of a crystalline suspension with crystals not exceeding100 μm within the same time range. Higher power input led tothe formation of mostly amorphous protein precipitate. Agitated batchcrystallizers of industrial scale impart high shear forces which aredetrimental to the fragile 3D structure of proteins and their respectivecrystals. Moreover, by interaction with surfaces (potentially enhancedby agitation), the formation of amorphous precipitate is likely tobe induced.41 This confirms the need forthe development of new high-throughput protein crystallization processes. 041b061a72