Determining the filtration properties of different protein crystals in the centrifugal field using low volume samples

B. Radel*, T.H. Nguyen, H. Nirschl, Karlsruhe Institute of Technology (KIT), Germany

Due to advances in the microbial production of target molecules, product titers after fermentation have increased in recent years. In the case of proteins, such high titers are a challenge for downstream processing. Typically, centrifugation and several chromatography steps separate the target protein from the impurities and the mother liquor. High titers in the mother liquor require large chromatography columns and large amounts of solvents making the downstream process expensive. Additional precautions regarding toxicity and explosive properties may be required when using organic solvents. Therefore, alternative methods such as selective preparative protein crystallization are becoming more and more interesting for the pharmaceutical industry. Another positive aspect of crystalline proteins is a longer shelf life and different drug release properties. After the crystallization step, the protein crystal must be separated from the mother liquor. Dead-end cake filtration is often used for this solid-liquid separation step. Compared to conventional crystals, protein crystals have a lower mechanical stability. Even at low pressures, crystal breakage or abrasion can occur, which worsens the filtration behavior.

In current research Nowotny et al. have modified the amino acid sequence of the enzyme alcohol dehydrogenase from Lactobacillus brevis in order to improve the crystallization properties. Kubiak et al. used cross-linking of lysozyme crystals, which increased the mechanical stability. For cost reasons, the screening of such modifications is typically carried out on a small scale with only a few milliliters of sample volume. Typical filtration experiments in e.g. a nutsche filter require much larger volumes.
To characterize the filtration behavior, we have developed a 3D printed filtration cell which, in combination with an optical analytical centrifuge, enables a filtration experiment with a sample volume of approx. 300┬ÁL. Thus, the influence of protein engineering and crystal modification on filtration can be determined on a small scale. The flux density function, which describes sedimentation and filtration properties as a function of the solids volume fraction, is calculated from experimental data obtained with the filtration cell.

Lysozyme and alcohol dehydrogenase crystals serve as model protein crystals. Since lysozyme is polymorphic, i.e. depending on the crystallization conditions the crystals are either isometric, rod-like or needle shaped, the influence of crystal size and morphology on the filtration can be investigated well. Both protein crystals show...

Session: L11 - Centrifugal Cake Filtration
Day: 23 October 2019
Time: 16:45 - 18:00 h

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