(493g) Characterization of Sedimentation and Filtration of Mechanically Labile Protein Crystals on a Small Scale

Current research investigates the bulk crystallization for purification or formulation of proteins. Compared to amorph solids, crystalline proteins offer many advantages e.g. extended shelf life or different drug release properties^1,2. One approach to use these advantages is to integrate a crystallization step in the downstream processing for protein purification and formulation^3,4. Enabling protein crystallization and handling protein crystals is often still a challenging task and subject of current research. Nowotny et al.⁵ and Hermann et al.⁶ identified crucial amino acids in the protein alcohol dehydrogenase in a rational crystal engineering approach using a molecular dynamics model to improve the crystallization behavior of the protein. Grob et al.⁷ showed the applicability of technical crystallization in downstream processing. Protein crystals often have quite different mechanical properties compared to typical inorganic or organic crystals. Cornehl et al.⁸ and Radel et al.⁹ investigated crystal breakage in solid liquid separation. For protein crystals breakage occurs already at pressures below 1 bar. Therefore Kubiack et al.^10,11 used cross-linking of protein crystals to increase mechanical stability and to enhance the micromechanical properties.

All the research described so far is typically conducted on a small scale with low sample volumes. For the process development of typical solid liquid separation steps, like filtration, the required amount is often much larger and quickly amounts to several liters. To enable the characterization of filtration and to study how filtration affects the protein crystals early in process development, we have developed a small-scale filtration device operating with low volumes. Each filtration experiment requires approximately 300 µL sample volume. The filtration process itself takes place in the centrifugal field using an analytical photocentrifuge. Thus, the filtration progress can be monitored in situ and relevant properties like cake resistance, cake height and solids volume fraction are either directly available or can be easily calculated. This allows to dramatically reduce the required amount of sample. With our filtration device we investigated the polymorphic lysozyme protein crystals (isometric, rod-like and needle shaped) and the rod-like crystals of the enzyme alcohol dehydrogenase from Lactobacillus brevis. Furthermore, we analyzed settling behavior for those crystals to calculate the flux density function. Combined with the compressive yield stress those process functions are useful for simulations of settling or filtration behavior. Additionally, we analyzed crystal breakage under shear stress with a modified ring shear tester. This in house developed device allows to shear fluid saturated particulate networks. Shear forces often involuntarily occur in technical applications e.g., in pumps or outlets.

With micro computer tomography (µCT) imaging we characterized the homogeneity and crystal size distributions in freeze dried filter cakes. µCT imaging is a powerful tool to investigate the structure of objects destruction free. Applied to filter cakes, this means it allows to characterize the structure and particles with spatial resolution.

We found that isometric lysozyme crystals have the best filtration properties (low resistance and high solids volume fraction). Rod-like lysozyme crystals have a slightly higher resistance. Both particle systems show incompressible filtration behavior. Rod-like alcohol dehydrogenase crystals have a higher filtration resistance which rises with increasing pressure. This indicates a compressible filter cake. The needle shaped lysozyme crystals show the most compressible filtration behavior. The cake resistance increases several magnitudes, and the cake height drops significantly. This strongly indicates particle breakage under normal stress. Interestingly, µCT imaging reveals a homogenous particle size distribution over the cake height for isometric and rod-like lysozyme crystals.

The isometric particles, which show no breakage at normal stress up to 1.5 bar, break at even low shear forces. A drastic shift towards smaller particles is observed when comparing unstressed, normal stressed and shear stressed crystals.

With the three-dimensional structure of filter cakes from µCT imaging we conducted computational fluid dynamics simulations (CFD) and validated them with experiments. This helps to better understand the impact of cake inhomogeneities on the filtration resistance.

In the presentation the detailed experimental filtration setup, the analysis of µCT images and conclusions of filtration and breakage behavior will be discussed. Computer simulations of the flow through particulate networks measured with µCT will also be the subject of the talk.

1. Jen A, Merkle HP. Diamonds in the rough: Protein crystals from a formulation perspective. Pharm. Res. 2001;18(11):1483–1488.
2. Basu SK, Govardhan CP, Jung CW, Margolin AL. Protein crystals for the delivery of biopharmaceuticals. Expert Opin. Biol. Ther. 2004;4(3):301–317.
3. Hekmat D. Large-scale crystallization of proteins for purification and formulation. Bioprocess. Biosyst. Eng. 2015;38(7):1209–1231.
4. Hubbuch J, Kind M, Nirschl H. Preparative Protein Crystallization. Chem. Eng. Technol. 2019;42(11):2275–2281.
5. Nowotny P, Hermann J, Li J, Krautenbacher A, Klöpfer K, Hekmat D, Weuster-Botz D. Rational Crystal Contact Engineering of Lactobacillus brevis Alcohol Dehydrogenase to Promote Technical Protein Crystallization. Crystal Growth & Design. 2019.
6. Hermann J, Nowotny P, Schrader TE, Biggel P, Hekmat D, Weuster-Botz D. Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways. Acta Crystallographica Section F: Structural Biology Communications. 2018;74(12):754–764.
7. Grob P, Huber M, Walla B, Hermann J, Janowski R, Niessing D, Hekmat D, Weuster-Botz D. Crystal Contact Engineering Enables Efficient Capture and Purification of an Oxidoreductase by Technical Crystallization. Biotechnol. J. 2020:e2000010.
8. Cornehl B, Overbeck A, Schwab A, Büser J-P, Kwade A, Nirschl H. Breakage of lysozyme crystals due to compressive stresses during cake filtration. Chem. Eng. Sci. 2014;111:324–334.
9. Radel B, Funck M, Nguyen TH, Nirschl H. Determination of filtration and consolidation properties of protein crystal suspensions using analytical photocentrifuges with low volume samples. Chemical Engineering Science. 2019;196:72–81.
10. Kubiak M, Solarczek J, Kampen I, Schallmey A, Kwade A, Schilde C. Micromechanics of Anisotropic Cross-Linked Enzyme Crystals. Crystal Growth & Design. 2018;18(10):5885–5895.
11. Kubiak M, Storm K-F, Kampen I, Schilde C. Relationship between Cross-Linking Reaction Time and Anisotropic Mechanical Behavior of Enzyme Crystals. Crystal Growth & Design. 2019;19(8):4453–4464.