(125b) Continuous Crystallisation Oscillatory Flow Platform for Protein Purification and Bio-Separation

Continuous Crystallisation
Oscillatory Flow Platform for Protein Purification and Bio-Separation

Xiaoyu Li ¹,
Huaiyu Yang¹, Wenqian Chen ¹ and Jerry Y. Y. Heng ¹
*

Department of Chemical
Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ,
UK

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

Abstract

Progress of advanced biotechnology has enhanced the
development of commercial biopharmaceutical products and the advances in
biotechnology have led to over 246 approved macromolecular drugs and vaccines with
cumulative revenues reaching $140 billion [1]. A cost effective and reliable
downstream route becomes a major industrial challenge in therapeutic protein
manufacture due to the expanded market remands. 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 alternative compared to conventional purification techniques
such as chromatography [3-5]. Moreover, crystalline proteins have a higher
purity and stability which can benefit formulation, storage, and drug delivery
steps [3, 6].

The aim of this work is to develop a continuous biocrystallisation platform for cost-effective downstream
manufacturing of therapeutic proteins. Additionally, selective crystallisation
of a protein and simultaneous crystallisation of proteins from binary protein
mixtures were investigated. Mixtures were prepared using model proteins:
lysozyme and thaumatin. >10³ of 2-4 µl hanging drops containing
mixture of protein and precipitant solutions were set-up. The crystallisation
processes were monitored under 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.
Selective crystallisation of each protein from mixtures was achieved by
manipulating crystallisation conditions such as types of precipitants,
precipitant concentration and pH of the solution. Diagrams were produced to
show the operation windows of selective crystallisation of target protein from
the mixtures. This work demonstrates our proof of concept that crystallisation
possesses the potential of separating a target protein from a complex mixture
environment.

Further, protein crystallisation from mixture is conducted
at a laboratory scale of a few µl to hundreds of µl. For future scale-up study
of the biocrystallisation process, we designed and
built a continuous oscillatory flow crystallisation platform at the ml scale,
combining two syringe pumps for feeding protein and precipitation solutions,
respectively, and an extra syringe pump to provide the oscillatory flow [7].
Crystallisation using this platform was conducted with lysozyme as model
protein. Lysozyme concentration ranging from 30 to 100 mg/mL, oscillatory
conditions with amplitude (x₀) from 10 to 25 mm and frequency (f)
from 0.05 to 0.25 Hz were investigated in a batch oscillatory flow
crystallisation platform and then successfully transferred to a continuous
oscillatory flow crystallisation platform. The steady state of continuous
crystallisation was reached at resident time 200 minutes, and the final product
crystal shape and size were consistent during the continuous process. This
study demonstrates a robust method to develop continuous oscillatory flow
crystallisation based on batch crystallisation of protein, which can contribute
to the future development of downstream bio-separation processes.

Conclusions

We demonstrate that the selective crystallisation for bioseparation of proteins is viable, as demonstrated for
the case of lysozyme-thaumatin mixtures. Further, the process for protein
crystallisation is successfully transferred from the -µl scale to a -ml scale
and finally a continuous oscillatory crystalliser. The findings in this study
can be translated to purification of other proteins with high pharmaceutical
values using crystallisation as a simpler and cost-effective alternative to
conventional multi-step chromatography to benefit the society.

References

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