(110b) Analyzing Mechanical Labile Protein Crystals with a Special Filtration Cuvette

BACKGROUND: Recent progresses in the biotechnology industries have led to much higher product yields after the fermentation. In the case of proteins the purification of such high titers have become a challenging task. The common method is to use several chromatography steps to separate the protein with high purity. However the high concentration of protein in the mother liquor requires large amounts of toxic and explosive organic solvents and large chromatography columns. Hence another approach like selective crystallization to separate the protein from the mother liquor is becoming increasingly interesting for the pharmaceutical and biotechnology industry. Crystallized proteins not only offer advantages in the downstream processing but also have other positive properties like extended shelf life, easier product handling and in the case of therapeutic proteins different drug release properties.

After the crystallization the next step is often a solid-liquid separation. In the case of protein crystals this separation is a challenging task because in contrast to common organic or inorganic crystals protein crystals are much more fragile to mechanical stress. Even low pressure differences may lead to crystal breakage or crystal abrasion. Since therapeutic or enzymatic proteins are often expensive products a method to analyze the mechanical stability and the influence of crystal breakage on the cake filtration with low volumes and hence low product quantities is required.

METHODS: To analyze the influence of crystal breakage on the cake filtration a special filtration cuvette has been constructed which allows the filtration of volumes less than one milliliter. This 3D-printed cuvette can be used in an optical, analytical centrifuge by LUM GmbH (Berlin, Germany) to perform and monitor the filtration by means of light transmission at the same time. As a model substance hen egg-white lysozyme crystals were investigated. The particle size distribution can be measured by automatic analysis of microscope images or by analysis of sedimentation velocity. By comparing the particle size distributions before and after the filtration the crystal breakage can be quantified at different centrifugal accelerations. Due to the light transmission unit in the centrifuge the filter cake and the flux through the filter can be observed during the filtration. Different crystal shapes and sizes of lysozyme were investigated using this method.

OBJECTIVE: The objective was to analyze the mechanical stability of different shapes of lysozyme crystals during the filtration in the centrifugal field. Therefore the crystals were stressed at different accelerations in the Lumisizer® using a special 3D-printed filtration cuvette. The experimental setup and the results of experiments with different crystal shapes of lysozyme will be presented in detail.

RESULTS: Due to crystal breakage during the filtration the porosity of the filter cake decreases leading to a lower flux through the filter cake. Especially aggregates may break leading to a higher fines content and reducing the coarse content. Hence the unstressed crystal size distribution of aggregated lysozyme shows a larger coarse content and a larger median particle diameter than the crystals which were stressed at for instance 2000 or 4000rpm. Experiments with isometric and rod-like crystals have also been performed to compare the amount of mechanical stress and the reduction in the crystal size distribution. Using the filtration cuvette the filtration progess, the filter cake consolidation and the flux through the filter can be compared between the different particle systems stressed at different centrifugal forces.