Protein crystals have many important applications in many fields, including pharmaceutics. Being more stable than other formulations, and having a high degree of purity and bioavailability, they are especially promising in the area of drug delivery. In this contribution, the development of a continuously operated tubular crystallizer for the production of protein crystals has been described. Using the model enzyme lysozyme, we successfully generated product particles ranging between 15 and 40 μm in size. At the reactor inlet, a protein solution was mixed with a crystallization agent solution to create high supersaturations required for nucleation. Along the tube, supersaturation was controlled using water baths that divided the crystallizer into a nucleation zone and a growth zone. Low flow rates minimized the effect of shear forces that may impede crystal growth. Simultaneously, a slug flow was implemented to ensure crystal transport through the reactor and to reduce the residence time distribution.

This paper describes a simple model-free (i.e., empirical) control strategy for crystal size tuning in a continuously operated tubular crystallizer. The crystallizer is designed for a seeded cooling crystallization process and acetylsalicylic acid crystallization from an ethanol solution was used as model system. Using a crystal size distribution (CSD) analyzer and minor initial studies, we developed a feedback controller that accurately tuned the mean crystal size within the range of 90–140 μm. In addition, we created a cleaning concept for long-term runs based on a consistency study, which demonstrated that the CSD of the products remained robust when process settings were kept constant. Sufficiently small and uniform seed crystals were generated via ultrasound irradiation.

Size, shape, and polymorphic form are the critical attributes of crystalline particles and represent the major focus of today’s crystallization process design. This work demonstrates how crystal properties can be tuned efficiently in solution via a tubular crystallizer that facilitates rapid temperature cycling. Controlled crystal growth, dissolution, and secondary nucleation allow a precise control of the crystal size and shape distribution, as well as polymorphic composition. Tubular crystallizers utilizing segmented flow such as the one presented in our work can provide plug flow characteristics, fast heating and cooling, allowing for rapid changes of the supersaturation. This makes them superior for crystal engineering over common crystallizers. Characterization of particle transport, however, revealed that careful selection of process parameters, such as tubing diameter, flow rates, solvents, etc., is crucial to achieve the full benefits of such reactors.

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Human nasal respiratory cells lose cilia in submerged cultures. This study compares the effect of extracellular matrix (ECM) molecules of the basal lamina on ciliogenesis in submerged cell cultures to ECM-free suspension cultures. Respiratory mucosa of nasal turbinates was the routine source for the cultures of nasal epithelial cells. For the submersion cultures, enzymatically isolated cells were seeded either on a layer of lethally irradiated ((60)Co, 60 Gy) murine 3T3-feeder fibroblasts or on an ECM-coated culture flask. For suspension cultures, the flasks were rotated for 3 days after cell seeding. In ECM-coated flasks, epithelial cell attachment and confluence was promoted and always much better than in cultures on a feeder layer. Respiratory cells lost cilia during the first 5 weeks in submerged cultures. Genesis of new, actively beating cilia was seen after 5-6 weeks when plastic culture dishes were coated with ECM molecules. Cells grown on uncoated plastic dishes together with 3T3-fibroblasts showed no ciliogenesis. Spheroids of epithelial cells in suspension cultures lost cilia during the 1st week and developed new cilia after 1-2 weeks in vitro. Our results suggest that ECM molecules are not the only signal for ciliary differentiation of respiratory cells in vitro, because suspension cultures are ECM free. However, the presence of ECM molecules in submerged cell cultures promotes the attachment and early confluence of seeded epithelial cells with a high density of cuboidal epithelial cells. The specific cellular shape and intense intercellular contact of these cuboidal cells may be among the most important signals inducing terminal differentiation and ciliogenesis.

Besides size and polymorphic form, crystal shape takes a central role in engineering advanced solid materials for the pharmaceutical and chemical industries. This work demonstrates how multiple cycles of growth and dissolution can manipulate the habit of an acetylsalicylic acid crystal population. Considerable changes of the crystal habit could be achieved within minutes due to rapid cycling, i.e., up to 25 cycles within <10 min. The required fast heating and cooling rates were facilitated using a tubular reactor design allowing for superior temperature control. The face-specific interactions between solvent and the crystals’ surface result in face-specific growth and dissolution rates and hence alterations of the final shape of the crystals in solution. Accurate quantification of the crystal shapes was essential for this work, but is everything except simple. A commercial size and shape analyzer had to be adapted to achieve the required accuracy. Online size, and most important shape, analysis was achieved using an automated microscope equipped with a flow-through cell, in combination with a dedicated image analysis routine for particle tracking and shape analysis. Due to the implementation of this analyzer, capable of obtaining statistics on the crystals’ shape while still in solution (no sampling and manipulation required), the dynamic behavior of the size shape distribution could be studied. This enabled a detailed analysis of the solvent’s effect on the change in crystal habit.

Between 1995 and 1998, 11 patients with disabling Menière's disease were treated at our institution with a continuous gentamicin infusion into the middle ear via a microcatheter. The patients had frequent attacks of vertigo and vomiting (functional levels 3-5). Hearing threshold on the affected side was significantly worse than on the healthy side (stage 4+5). Gentamicin was applied by a high-precision insulin pump with a flow rate of 40 mg per day directly in front of the round window. Application was stopped as soon as signs of vestibular affection appeared. A good overall control of vertiginous spells was achieved in 8 patients. Eight patients experienced complete hearing loss on the affected side, 1 experienced a slight worsening, and 1 had no hearing change. There was no correlation between the cumulative gentamicin dosage and the hearing loss. Our findings show that in terms of hearing loss and hospitalization time the continuous gentamicin application is inferior to other applications presented in the literature.

The pathogenetic mechanisms of acute cochleo-vestibular lesions are still unknown, but viral infections and vascular phenomena with impairment of microvascular perfusion are thought to play a major role. Between 1 July, 1986 and 28 February 1998, 1501 patients were treated with an infusion protocol using cortisone, dextrane 40 and pentoxifylline. Group 1 contained 1001 patients with sudden hearing loss, group 2a 107 patients with isolated tinnitus and group 2b 393 patients with labyrinthine disorders (among which were 81 patients with cochleovestibular dysfunction). The records were evaluated retrospectively. In group 1 complete hearing recovery occurred in 44.8%, partially in 40.4%, no change in 12.1% and worsened in 2.6%. In group 2a with isolated tinnitus 17.9% had a complete recovery, 43.9% partial recovery, 35.5% no change and 2.8% worsened symptoms. In group 2b vertigo disappeared in 56.8%, had partial recovery in 21.0% and did not change in 7.4%. In the 1501 patients treated, no significant side-effects were found to the medical interventions used. From these results we conclude that the infusion protocol is safe and effective in the treatment of cochleo-vestibular disorders.