(523c) Design of Continuous Enzymatic Reactive Crystallization for Beta-Lactam Antibiotic Synthesis

Design of Continuous Enzymatic Reactive
Crystallization for Beta-lactam Antibiotic Synthesis

Matthew A. McDonald¹, Andreas S. Bommarius¹, and
Ronald W. Rousseau¹

¹School of Chemical and Biomolecular Engineering,

Georgia Institute of Technology, Atlanta, GA 30332-0100, USA

                  β-lactam
antibiotics, despite a long and storied history, find continued widespread use in
our fight against infection.  In
2010 the two most common classes of β-lactam antibiotics, those derived
from penicillin and cephalosporin, accounted for nearly 60% of world antibiotic
consumption, with over 20 billion doses given of penicillin-derived antibiotics
alone [1].  Their high rate of use,
both historical and contemporary, has contributed to a growing deadly allergy
to β-lactams, particularly those derived from penicillin.  The rate of allergy is significant enough
for the FDA to recently require that all penicillin-derived antibiotics be
manufactured, processed, and packaged in facilities separate from those used
for any other pharmaceuticals [2]. 
Traditionally, β-lactam antibiotics have been made through chemical
synthesis requiring use of several protection groups and resulting in
unsustainable amounts of potentially contaminated waste [3]. 

                  An
alternative route that has received considerable interest involves using the
enzyme penicillin G acylase (PGA) to synthesize semi-synthetic
penicillin-derived antibiotics from an acyl donor (e.g. phenylglycine
methyl ester for ampicillin) and 6-aminopenicillanic acid
(6-APA) [3].  However batch
processing, even enzymatic, results in only moderate yields and thus
inefficient use of materials.  PGA
also catalyzes the hydrolysis of the acyl donor (primary hydrolysis) and the hydrolysis
of the antibiotic (secondary hydrolysis, ampicillin cleaves into phenylglycine and 6-APA), leading to unnecessary
waste. 

                  This
work focuses on the design of continuous reactive crystallization of ampicillin
catalyzed by PGA, such that the precipitated ampicillin is protected from
secondary hydrolysis and is of high purity.  The design of such a system requires
models for kinetics of reaction and crystallization, solubility of all components,
and robust controls for continuous operation.  The dependence of the kinetics and
solubility on pH promises a means of control in which yield and selectivity can
be maximized.

                  Crystallization
kinetics (nucleation and growth) were determined via online chord-length
distribution and concentration versus time data in batch crystallizers [4].  Change in pH was used to induce
supersaturation, and the effect of crystallization on pH was recorded.  Since the goal is steady state operation
of a continuous reactive crystallizer, seed crystals should be used in all
experiments, the loading of seed crystals being another important design
parameter.  Crystallization kinetics
were combined with the reaction kinetics scheme of Youshko and Svedas [5].  The reaction kinetics were
modified to include the effect of enzyme protonation state on the rates of
synthesis and primary and secondary hydrolysis.  Batch experiments across a range of pH
values were used to factor in the equilibrium of the enzyme-substrate complex
and enzyme-product complex protonation states.  The effect of pH on the rates of
ampicillin synthesis, primary hydrolysis, and secondary hydrolysis were
determined independent of each other. 
A combined model was used to determine the yield and selectivity for
ampicillin over the hydrolysis product. 
The largest obstacle to high conversion remains the accumulation and
precipitation of the hydrolysis product phenylglycine, an intolerable impurity
in the final project.

                  Model
simulations show that continuous operation can increase yield considerably over
batch processes.  Simulations also
show the substantial benefit of crystallization and reaction occurring in the
same vessel.  Different vessel
designs (i.e. stirred tank, tubular reactor) have also been investigated via
the model and show significant differences between competing designs.  A continuous stirred tank arrangement
will be implemented experimentally to validate the model.  Preliminary results also suggest
controls over important variables including crystal size distribution are
possible while maintaining high yield, selectivity, and purity.

[1] Gelbrand,
Helen, et al. "The State of the WorldÕs Antibiotics 2015." Wound Healing Southern Africa 8.2 (2015): 30-34.

[2] 21 C.F.R ¤ 211.42 2015

[3] Giordano,
Roberto C., Marcelo PA Ribeiro, and Raquel LC Giordano.
"Kinetics of β-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial
enzymatic reactor optimization."Biotechnology
advances 24.1 (2006): 27-41.

[4] Encarnación-Gómez, Luis G., Andreas S. Bommarius, and Ronald W. Rousseau. "Crystallization Kinetics of Ampicillin
Using Online Monitoring Tools and Robust Parameter Estimation." Industrial
& Engineering Chemistry Research 55.7 (2016): 2153-2162.

[5] Youshko, M. I., and V. K. Śvedas. "Kinetics of ampicillin
synthesis catalyzed by penicillin acylase from E.
coli in homogeneous and heterogeneous systems. Quantitative characterization of
nucleophile reactivity and mathematical modeling of the process." Biochemistry (Moscow) 65.12 (2000): 1367-1375.