Determination of filtration and consolidation properties of protein crystal suspensions using analytical photocentrifuges with low volume samples

Abstract

Proteins and in particular pharmaceutical proteins like monoclonal antibodies are expensive products. Protein crystallization is an interesting alternative process step to the common purification and formulation of such proteins. The solid-liquid separation of proteins however, is a challenging task because the proteins are sensitive to mechanical stress, which could lead to crystal breakage and often are only available in small amounts for research and development. In this study a newly developed filtration cell for the LUMiSizer R centrifuge is used to analyze the filtration behavior with low volume samples. Isometric and needle shaped lysozyme crystals serve as model protein crystals. The needle shaped crystals showed about twice as high compressibility and much higher cake resistance than the isometric lysozyme crystals. After the filtration at maximum pressure in the centrifuge the mean particle diameter decreased compared to the unstressed median diameter. With the filtration cell the cake height and filter cake compression can be monitored in situ and the filter cake resistance and solids volume fraction can be calculated with one experiment using 200 to 300 µl sample.

Crystal Contact Engineering Enables Efficient Capture and Purification of an Oxidoreductase by Technical Crystallization

“…All mutants crystallized significantly faster than the WT, both in purified form on the static µL-scale and in non-purified form LbADH variants, which might have a direct impact on separation processes on an industrial scale, for example, during filtration of the crystal suspension. [33] After crystallization from dialyzed cell lysate, the crystal dissolution and recrystallization were successfully conducted. Starting from an initial HCP concentration of 50%, the final HCP content was below 0.5%.…”

Calculation of the Flux Density Function for Protein Crystals from Small Scale Settling and Filtration Experiments

“…From this equation the integral height specific resistance of the filter cake is obtained. For the detailed working principle of the cuvette and data analysis please refer to Radel et al [19] and Loginov et al [30]. In centrifugal filtration the pressure on the filter medium does not remain constant, but decreases with descending fluid height.…”

“…For permeation experiments in this study, Pall (Germany) Supor® membranes with a nominal pore size of 0.2 µm and a resistance of 2.1 × 10 10 -3.6 × 10 10 m −1 are used [19].…”

“…Needle [19] Rod-like ADH rod-like Figure 5: Left: mass specific filtration resistance for three lysozyme crystal shapes and rod-like alcohol dehydrogenase crystals; right: height specific filtration resistances for three lysozyme crystal shapes and rod-like alcohol dehydrogenase crystals; The data for needle shaped crystals was published in Radel et al [19]; solid lines are model calculations according to Tiller, Kwon [15] Using the resistance of a sediment or filter cake, values for flux density function above the gel point are calculated with…”

“…Investigating filtration properties of protein crystals on an early stage of process development therefore requires a much smaller laboratory setup. Radel et al [19] introduced a downscaled filtration setup for an optical laboratory centrifuge which is based on the setup published by Loginov et al [20] for investigating filtration properties at a small scale.…”