(124d) Effect of Mixing Intensity on Lysozyme Crystallization in a Meso Oscillatory Flow Reactor

Protein crystallization as a process step requires rapid and quantitative crystallization. As most of the industrial processes, recovery yield and purity must be high and the crystallization time low, so that growth kinetics should be fast. It also requires a minimum of agitation to keep crystals in suspension throughout the process and ensure good mixing of the crystallization solution [1,2]. Crystallization operations are usually designed as stirred tank processes, where large tanks are often characterized by low mixing efficiency that may lead to excess local concentrations and, consequently, affect the characteristics of the final product [2]. Further, shear forces exerted by the impeller can be significant and thus damage protein crystals [2]. Therefore, a crystallizer with uniform mixing and minimized shear forces is desirable to overcome the aforementioned problems.

In the present work we report the effects of mixing intensity on protein crystallization under oscillatory flow mixing. For this, we studied the crystallization of a model protein, lysozyme, in a meso oscillatory flow reactor (OFR) [3]. The meso-OFR consists of a 35 cm long and 3 mm internal diameter glass jacketed tube provided with smooth periodic constrictions (SPC) with an approximate volume of 4 mL. The fluid is typically oscillated in the axial direction and this motion interacts with the constrictions forming vortices, the mixing intensity being controlled by the oscillation frequency (f) and amplitude (x₀). This provides precise control of mixing, from gentle to the most intense, and efficient heat and mass transfer. The intensity of mixing applied to the meso-OFR can be described by the oscillatory Reynolds number Re_o:

Re_o = (2Ï€fx₀ρd)/μ (1)

where d is the column diameter (m), ρ the fluid density (kg.m^−3), μ the fluid viscosity (kg.m^−1.s^−1), x₀ the oscillation amplitude (m) and f the oscillation frequency (s^-1).

Materials and Methods

The experimental set-up involves one meso-OFR placed vertically, a mixing chamber, a linear motor (LinMot, Switzerland) to ensure oscillation of the fluid, and an in-line flow cell connected to a multi-channel optic spectrometer system (ScanSpec UV−vis, Sarspec, Portugal) to monitor the turbidity of the crystallization solution by measurement of the absorbance at 400 nm.

Batch lysozyme crystallization trials were carried out at different oscillation f and x₀, at 20 ºC and pH 4.7. Both lysozyme (100 g.L^-1) and sodium chloride (6% (w/v)) solutions were prepared in sodium acetate buffer at pH 4.7 and filtered (0.22 µm). Crystallization assays were started by injecting simultaneously equal parts of both solutions in the meso-OFR through a syringe pump (NE-4000, New Era, United States of America).

The crystals obtained were characterized by optical microscopy (Standard 20, Zeiss, Germany) to evaluate their shape and size. In addition, their activity was determined by spectrophotometry through the rate of lysis of Micrococcus lysodeikticus, and crystal yield was estimated based on the initial and final concentration of lysozyme in solution measured by spectrophotometry.

Results and Discussion

Typical turbidity curves obtained during lysozyme crystallization experiments showed four phases. Initially the turbidity increases slowly (growth of clusters and nucleation). In a second phase the turbidity increases sharply (combination of nucleation and growth). Then the turbidity decreases (sedimentation and/or aggregation of the crystals) and stabilizes. As shown in previous works [4], monitoring of the turbidity of the crystallization solution can provide a mean of detection of the different stages in protein crystallization, although in these conditions it is not possible to distinguish between the nucleation and growth steps. The slopes of the turbidity curves corresponding to the nucleation and growth phase were also determined for the different agitation conditions. Preliminary results suggest an increase in the slope value with the increase of the mixing intensity. We also verified that tetragonal crystals were formed for all the operating conditions. One can also notice the decrease of crystal size with the increase of x₀ at a fixed f, as well as with the increase of f at a fixed x₀. The general trend is that mean crystal size decreases with the increase of Re_o. The results obtained are in agreement with previous studies [5], being the decrease of mean crystal size with the increase of mixing intensity often attributed to erosion and attrition due to collisions of crystals with crystallizer. Further, the protein collected remains active at the end of the experiments, since most of the samples exhibited at least 70% of activity when compared to the initial lysozyme solution. Finally, crystal yield values were higher than 50% for all the operating conditions studied.

Acknowledgments: This work was financially supported by: (i) the Project POCI-01-0145-FEDER-006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the European Regional Development Fund (ERDF), through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds, through FCT - Fundação para a CieÌ‚ncia e a Tecnologia; (ii) the Project NORTE‐01‐0145‐FEDER‐000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); iii) the Project POCI-01-0145-FEDER-016816 (PTDC/QEQ-PRS/3787/2014) funded within the scope of the Project 9471 – Reforçar a Investigação, o Desenvolvimento Tecnológico e a Inovação, and co-funded by the European Regional Development Fund (ERDF) and by FCT - Fundação para a Ciência e Tecnologia; iv) national funds through FCT-Portuguese Foundation for Science and Technology-under the projects: UID/BIO/ 04469/2013; IF exploratory project [IF/01087/2014]; post-doctoral Fellowship [SFRH/BPD/96132/2013]. A. Ferreira is an Investigator FCT (IF/01087/2014).

References:

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[2] Schmidt. S., Havekost. D., Kaiser. K., Kauling. J., Henzler. H.-J., Crystallization for the Downstream Processing of Proteins, Eng. Life Sci., 5, 3 (2005).

[3] Ferreira. A., Rocha. F., Teixeira. J.A., Vicente. A., Apparatus for mixing improvement based on oscillatory flow reactors provided with smooth periodic constrictions, (2016).

[4] Hu. H., Hale. T., Yang. X., Wilson. L.J., A spectrophotometer-based method for crystallization induction time period measurement, J. Cryst. Growth, 232, 1-4 (2001).

[5] X. Ni, A. Liao, Effects of mixing, seeding, material of baffles and final temperature on solution crystallization of l-glutamic acid in an oscillatory baffled crystallizer, Chem. Eng. J., 156, 1 (2010).