Influence of mixing intensity on lysozyme crystallization in a meso oscillatory flow reactor

The influence of mixing intensity on protein crystallization has been investigated for the first time in a meso oscillatory flow reactor (meso-OFR). For this, lysozyme crystallization was studied in batch at different oscillation amplitudes and frequencies. Turbidity was monitored over time to measure induction time and follow the crystallization process. Results suggest that the crystallization mechanism may be characterized by the occurrence of primary heterogeneous nucleation followed by attrition-induced secondary nucleation. Results also evidence the non-monotonic dependence of both induction time and mean crystal size on mixing intensity. Indeed, results show that the two parameters decrease with increasing mixing intensity at Reo below 189, while for further increase of Reo both remain almost constant. Based on the literature, we suggest that such behaviour may be explained by the influence of mixing intensity on protein clusters size distribution in solution. Further, induction time and mean crystal size show a stronger dependency on the oscillation amplitude compared to the oscillation frequency, in particular at the lower amplitudes. Finally, it is shown that lysozyme keeps its activity at the end of the experiments and crystal yields are quite reasonable.This work was the result of the project: (i) POCI-01-0145FEDER-016816 (PTDC/QEQ-PRS/3787/2014) funded by the European Regional Development Fund (ERDF), through COMPETE2020, Programa Operacional Competitividade e Internacionalização (POCI), and by national funds through Fundação para a Ciência e a Tecnologia (FCT), Project 9471, Reforçar a Investigação, o Desenvolvimento, Tecnológico e a Inovação (Projeto 9471, RIDTI); (ii) POCI-01-0145-FEDER006939 (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 Ciência e a Tecnologia; (iii) 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); (iv) UID/BIO/04469/2013, IF exploratory project [IF/01087/2014], and Postdoctoral Fellowship [SFRH/BPD/96132/2013]. A.F. is an Investigador FCT.