(466e) Electric-Field-Assisted Protein Crystallization in Continuous Flow

The
competitive environment in global market for biopharmaceuticals calls for
shorter development times, improved manufacturing flexibility, reduction of
batch-to-batch variability and reduction of the footprint of production
facilities. A key method to potentially achieve these improvements is to switch
from traditional batch-wise manufacturing to continuous manufacturing¹,
which offers several potential advantages such as a reduction in the number of
required processing steps, simplified scale-up, and the ability to implement
more effective process control strategies.² The separation of a
protein from a mother liquor with impurities is a typical biopharmaceutical
process. Compared to traditional packed-bed chromatography, crystallization is
an important alternative, yielding high purities in a single processing step
that can operate over a range of scales and throughputs. Unfortunately, the
design and control of crystallization processes involving large biomolecules
such as proteins remains notoriously difficult.

The
transition of batch-wise to continuous manufacturing offers opportunities to
develop new concepts for continuous protein crystallization.³
Usually, the kinetics of protein crystallization are optimized by manipulating
relevant global variables such as concentration, temperature, and solvent
composition. However, studies on the use of local variables to manipulate
protein crystallization, such as electric fields, have also been reported⁴.
All studies of the mechanism of electric-field-assisted crystallization have
been conducted batch-wise and mainly with the objective of producing high-quality
material for crystallography or biomedical analysis. The use of low-voltage AC
electric fields for protein nucleation in a continuous flow setting for
manufacturing has, to the best of our knowledge, not been addressed. Consequently,
insights into the mechanism and guidelines for design of continuous
electric-field-assisted protein crystallization are lacking.

The
objective of this work is to investigate different electrode structures that
could be implemented in a continuous flow device to control protein nucleation
both spatially and dynamically. The long-term objective is to develop
guidelines for the optimal design of a cell with microelectrodes to enable
electric-field-assisted protein crystallization in continuous flow. To
investigate the interaction of different types of electrodes and field
properties on protein crystallization, microelectrode chambers (MEC) have been
fabricated and tested. ITO coated glasses are used as electrodes and treated
with standard lithographic techniques to obtain specific patterns (Figure 1).
Two different types of electrodes have been tested, 1) parallel plate
electrodes (Figure 1a-h) in which a potential difference is created in vertical
direction, and 2) coplanar electrodes⁵ in which a potential
difference in horizontal direction is created (Figure 1i-j). Lysozyme with a
fluorescent label is used as the model protein in this study. In a typical
experiment, supersaturated lysozyme solution is mixed with precipitant and
injected into the MECs and observed under a microscope. Results demonstrate
that under the applied electric field, the induction time for nucleation is
reduced with an average induction time of around 20 hours for some electrode
patterns. Furthermore, most of the protein crystals are found on the ITO
electrode, which is probably due to the effect of local electric field minima.
In addition, the number and quality of crystals are improved when using
electric fields, especially compared with a reference case using non-conductive
glass chambers.

In
conclusion, electric fields can induce faster protein crystallization with
higher crystal quantity and better quality. These findings can be used as
guidelines for fabricating MECs for continuous protein crystallization by
aligning the electrodes in flow channels. The advantage of the tested coplanar
electrodes over parallel electrodes is that the height of the flow channels can
be varied independently to optimize hydrodynamics whereas the height of
channels with parallel electrodes also influences the electric field properties.

Figure
1. Various electrode structures fabricated with lithographic methods to
construct MECs: (a-h) parallel electrode; (i, j) coplanar electrodes.

References

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