A lab-scale
stirred tank with a cooled tubular reactor in bypass
was applied for continuous crystallization of lysozyme and a full-length
therapeutic monoclonal antibody. The stirred tank was operated as
a mixed suspension classified product removal crystallizer. Lysozyme
was crystallized by a combination of cooling crystallization and salting-out.
The antibody was crystallized by a combination of cooling crystallization
and isoelectric crystallization. It was deduced that nucleation rates
were enhanced when the protein solutions passed through the cooled
tubular bypass. It was further deduced that crystal growth rates were
enhanced in the stirred tank which was operated at a higher temperature
compared to the tubular reactor. Classified product removal was possible
by controlled sedimentation of protein crystals. The continuous crystallization
system allowed a targeted control of crystal morphology and size.
No sedimentation occurred in the tubular reactor and precipitation
was avoided at all times. High crystallization yields of more than
90% were obtained. Crystals of the monoclonal antibody were continuously
produced for the first time with a space–time yield of up to
12 g L–1 h–1.
Technical crystallization is an attractive method to purify recombinant proteins. However, it is rarely applied due to the limited crystallizability of many proteins. To overcome this limitation, single amino acid exchanges are rationally introduced to enhance intermolecular interactions at the crystal contacts of the industrially relevant biocatalyst Lactobacillus brevis alcohol dehydrogenase (LbADH). The wildtype (WT) and the best crystallizing and enzymatically active LbADH mutants K32A, D54F, Q126H, and T102E are produced with Escherichia coli and subsequently crystallized from cell lysate in stirred mL‐crystallizers. Notwithstanding the high host cell protein (HCP) concentrations in the lysate, all mutants crystallize significantly faster than the WT. Combinations of mutations result in double mutants with faster crystallization kinetics than the respective single mutants, demonstrating a synergetic effect. The almost entire depletion of the soluble LbADH fraction at crystallization equilibrium is observed, proving high yields. The HCP concentration is reduced to below 0.5% after crystal dissolution and recrystallization, and thus a 100‐fold HCP reduction is achieved after two successive crystallization steps. The combination of fast kinetics, high yields, and high target protein purity highlights the potential of crystal contact engineering to transform technical crystallization into an efficient protein capture and purification step in biotechnological downstream processes.
Although it is generally assumed when selecting a distillation packing that the wetting of a solid surface by a liquid without mass transfer will indicate its ability to wet under distillation conditions, for systems whose surface tensions change considerably with composition this postulate is shown to be invalid. The underlying reasons for this are analyzed and illustrated by considering the wetting characteristics of aqueous 1-propanol films flowing down a vertical copper surface both under equilibrium conditions and at total reflux.
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