(240g) Bio-Separation Via Selective Protein Crystallisation Facilitated By Porous Silica Templates: A Case Study Using Mesoporous Nanoparticles in Lysozyme-Thaumatin Mixture

Bio-separation via selective protein
crystallisation facilitated by porous silica templates: A case study using
mesoporous nanoparticles in lysozyme-thaumatin model binary protein mixture

Xiaoyu Li ¹,
Wenqian Chen ¹, and Jerry Y. Y. Heng¹ *

1. Department of Chemical
Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ,
United Kingdom

* Corresponding Author:
Jerry Y. Y. Heng. E-mail: jerry.heng@imperial.ac.uk

Abstract

Recent progress of advanced biotechnology has enhanced
the development of commercial biopharmaceutical products. Over 246 approved
biotechnology drugs and vaccines had been developed with cumulative revenues
reaching $140 billion [1]. To meet the growing market demand of protein-based
biopharmaceuticals, a cost effective and reliable downstream route becomes a
major industrial challenge in therapeutic protein manufacture. Crystallisation
has been used for the purification of more than 95% small-molecule
pharmaceuticals in various conventional chemical industries [2].
Crystallisation of proteins as downstream separation and isolation steps is
considered to be more efficient and cost-effective compared to conventional
purification techniques such as chromatography [3-5]. Additionally, crystalline
proteins have a higher purity and stability which can benefit formulation,
storage, and drug delivery steps [3, 6]. Even so, control of protein
crystallisation remains challenging after decades of investigation. To address
this problem, recent researches showed heterogeneous nucleants with designed
properties have the potential to facilitate selective protein crystallisation
and thus lead to better control of the crystallisation processes [7, 8].

Here, we report a demonstration of selective
crystallisation of target protein from a model binary protein mixture achieved
by addition of mesoporous silica nanoparticles (NPs) as heterogeneous
nucleants. In this work, mixtures were prepared using model proteins: lysozyme
and thaumatin. Protein mixtures with a composition range of 0 – 100 mg/mL
lysozyme and 0 – 100 mg/mL thaumatin were crystallised using Hanging-drop
vapour-diffusion (HDVD) crystallisation technique. >10³ of 2-4 µl
hanging drops containing mixture of protein and precipitant solutions were
set-up. The crystallisation processes were monitored under optical microscope
for 1-2 weeks. Protein crystals of these proteins were distinguishable from
their habits (shapes). Both model proteins can be crystallised out by adding
the same precipitant solution. Diagrams were produced to show the operation
windows of selective crystallisation of target protein from the mixtures.
Further, mesoporous silica NPs such as SBA-15 were used in the crystallisation
experiments and experiments revealed that certain silica nanoparticles were
able to promote lysozyme crystallisation while hinder thaumatin crystallisation
in the range of this study. Therefore the mesoporous NPs were able to shift the
operating window of target protein crystallisation. Furthermore, scaled-up –mL
batch crystallisation were conducted to prove the results revealed by HDVD
experiments. This work demonstrates that mesoporous silica facilitated
crystallisation possesses the potential of separating a target protein from a
complex mixture environment which can contribute to the future development of
downstream bio-separation processes.

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