Template-assisted protein crystallization in microfluidics

Template-assisted protein crystallization in microfluidics
D.M, Powell*1 C.J.J. Gerard1, M.L. Briuglia1, S. Morais2, D. A. Lamprou3, J.B. Salmon2, J.H. ter Horst1
1EPSRC Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallization (CMAC), SIPBS, University of Strathclyde, Glasgow, UK;
2Laboratory of the Future – CNRS, Bordeaux, France;
3School of Pharmacy, Queen's University Belfast, Belfast, UK *[email protected]
Protein crystallization is a useful tool for 3D-structure determination through X-ray Diffraction and analysis of the structure-activity relationship. In addition, crystallization can be used as a purification technique in the manufacturing of modern protein based drugs. However, the crystallization of protein presents a great challenge due to, among others, the complexity of the intermolecular interactions between such large macromolecules often leading to a narrow window of conditions for crystallization (McPherson 2004).
Nano-template particles have been used in crystallization for induction of heterogeneous nucleation for polymorph control and promotion of nucleation rate (Artusio and Pisano 2018; Caridi et al. 2014). However, applying similar techniques to protein crystallization requires significant quantities of material. This work proposes a specially designed microfluidic tool to assess the effect of template particles on protein crystal nucleation behaviour through induction time distribution determination.
Microfluidic device
The chip (Figure 1) is composed of 78 10nL channels with dead-ends and covered by a 30-35μm thick polydimethylsiloxane (PDMS) membrane. In order to compare the effect of template particles on nucleation to nucleation without templates, the chip consists of two independent sections of 39 channels each. The permeability of PDMS to water allows only water to escape out of the crystallizing solution through the chip walls producing supersaturation in a manner resembling the hanging drop protein crystallisation method. This pervaporation process is controlled by the salt
concentration of the solution on the permeate side of the membrane on top of the chip.
As water is removed from the solution, the concentration of the components increases.
Templates
No Templates
Figure 1: Image of the microfluidic device used. Designed with two series of 39
identical channels that can be filled independently. Each channel is approximately
10nL.
Results
Induction times of template-assisted Lysozyme nucleation experiments show a
reduction compared to those in absence of template particles. Depending on the kind
of template, we measured reduced induction time of up to 50%. In addition, the use of
effective CPG template particles produced a higher fraction of crystals of the stable
tetragonal phase (Figure 2)
Crystallization of monoclonal antibodies (mAb) presents a greater challenge than the
smaller protein Lysozyme due to its large size and the resulting increase in inter- and
intramolecular forces. As such, they are especially sensitive to extremes of
temperature and pH. Crystals of the mAb Anti-CD20 of a size between 2 and 12μm
were formed, apparently coinciding with a liquid-liquid demixing. Probably the liquidliquid
phase split is the reason that no significant changes in induction time distribution
or nucleation rate were measured in the presence of template particles. Future work
looks to apply these techniques to other protein systems, such as Ovalbumin.
Figure 2. Lysozyme crystallization experiment a) without template particles. 32 crystals
formed, 0.82 crystals/droplet b) with CPG template, 64 crystals formed, 1.64
crystals/droplet.
We have demonstrated that template particles can be used in protein crystallization to
induce heterogeneous nucleation in the case of lysozyme and that the specific
microfluidic device used is a viable tool for further study of protein crystallization
behaviour.
Acknowledgements
The authors would like to thank the European Union’s FET-OPEN Horizon 2020
research and innovation program for funding this work within the AMECRYS project
(www.amecrys-project.eu/) under grant agreement no. 712965. We thank Dr. Jerry Heng
and Dr. Chen Wenqian from Imperial College London for providing some of the
a
b
nanotemplates. We also thank the Centre for Process Innovation and FUJIFILM Diosynth Biotechnologies for providing anti-CD20 material.
References
Artusio, F., and R. Pisano. 2018. 'Surface-induced crystallization of pharmaceuticals and biopharmaceuticals: A review', Int J Pharm, 547: 190-208.
Caridi, Antonella, Samir A. Kulkarni, Gianluca Di Profio, Efrem Curcio, and Joop H. ter Horst. 2014. 'Template-Induced Nucleation of Isonicotinamide Polymorphs', Crystal Growth & Design, 14: 1135-41.
McPherson, A. 2004. 'Introduction to protein crystallization', Methods, 34: 254-65.