(270e) High Throughput Continuous Crystallization of Lysozyme in an Oscillatory Flow Baffled Crystallizer with Real Time Process Monitoring

Oliva, J., Purdue University
Nagy, Z. K., Purdue University

High throughput continuous
crystallization of lysozyme in an oscillatory flow baffled crystallizer with
real time process monitoring

are increasingly used for medicinal therapies and now make up roughly 20% of
the pharmaceutical market share. While most (small molecule) pharmaceutical
products are purified with at least one crystallization step,
biopharmaceuticals (specifically proteins) are very difficult to crystallize
due to extraordinarily slow growth kinetics1.
Moreover, large protein chains require subsequent folding operations in order
to reach a desired activity level. Until now, oscillatory flow strategies have
primarily been used in batch mode to improve the process kinetics of
biopharmaceuticals through enhanced micromixing and heat transfer2.
Known to have high surface area to volume ratios, oscillatory flow baffled
reactors (OFBR) generate turbulence by superimposing piston driven oscillatory
flow onto the net flow generated by a pump. This combination approach allows
for adequate solid suspension with greatly improved micromixing compared to the
traditional stirred tank approach3.  Herein, lysozyme was chosen as
a model compound for both crystallization and folding applications, as its
kinetic parameters are relatively well studied in literature4.

Using the
commercially available DN6 (Alconbury West Ltd) with the new Blaze probe (Blaze
900, Blaze Metrics), the oscillatory system was able to significantly reduce
the time required to crystallize lysozyme to several sizes (varying between
50-200µm). The Blaze Metrics probe allowed for real time monitoring of the
system, both in terms of steady/transient state analysis as well as monitoring
the critical quality attributes of the crystals (size and shape). These dynamic
measurements allow for real time feedback control strategies to be implemented
on this new commercially available platform.

In a separate
application, the DN6 was also used to continuously fold the newly purified
lysozyme. Proper folding of proteins is essential for their activity, and, for
enzymes, activity can be measured using a colorimetric assay utilizing
fluorescently tagged substrates. To ensure the activity of lysozyme was not
affected by the crystallization process, renatured lysozyme was examined for
its ability to degrade fluorescein-labeled Micrococcus lysodeikticus
cell walls and compared to pre-crystallization lysozyme. It was found that the
recrystallization step had little to no impact on the lysozyme’s activity. Continuous
oscillatory flow systems with proper monitoring offer advanced approaches to
generating quality crystalline proteins for high throughput applications.


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of Biochemistry and Biophysics
, 602, 3–11.

2.      Abbott,
M. S. R., Harvey, A. P., Perez, G. V., & Theodorou, M. K. (2013).
Biological processing in oscillatory baffled reactors: operation, advantages
and potential. Interface Focus, 3(1), 20120036.

3.      McDonough,
J. R., Phan, A. N., & Harvey, A. P. (2015). Rapid process development using
oscillatory baffled mesoreactors - A state-of-the-art review. Chemical
Engineering Journal
, 265(1), 110–121.

4.      Liu,
Y., Wang, X., & Ching, C. B. (2010). Toward further understanding of
lysozyme crystallization: Phase diagram, protein-protein interaction,
nucleation kinetics, and growth kinetics. Crystal Growth and Design, 10(2),