The effect of the cell disruption technique on the expanded bed chromatography

Bei der Aufarbeitung intrazellulärer, biotechnologischer Produkte müssen nach dem Zellaufschluss die Zelltrümmer vom Zielprotein abgetrennt werden. Als Alternative zur Zentrifugation oder Filtration bietet sich die Expanded Bed Chromatographie an, da in diesem Verfahren die Zelltrümmerabtrennung mit einem ersten chromatographischen Reinigungsschritt verbunden wird. Die Effizienz der Expanded Bed Chromatographie hängt von den Strömungsverhältnissen in der Säule ab. Zelltrümmer erhöhen den Grad der Rückvermischung und wirken sich somit negativ auf den chromatographischen Prozess aus. In dieser Arbeit wurde der Einfluss verschiedener mechanischer Zellaufschlussmethoden (Rührwerkskugelmühle, Hochdruckhomogenisator und Ultraschallhomogenisator) auf die Strömungsverhältnisse im Expanded Bed untersucht. Es zeigte sich ein deutlicher Einfluss der Zellaufschlussmethode auf die Größe und Form der Zellbruchstücke von B. megaterium und E. coli. Diese Ergebnisse konnten mit den Resultaten der Messungen der Verweilzeitverteilung im Expanded Bed korreliert werden.To purify intracellular biotechnological products, the separation of the cell debris from the product (usually a protein) is necessary. An alternative to the centrifugation and the filtration is the expanded bed chromatography. This method combines the cell debris removal with a first chromatographic step in the purification process. The efficiency of the expanded bed chromatography process depends on the flow profile in the column. The cell debris enhances the back-mixing and therefore influences the chromatographic process negatively. In this work different mechanical cell disruption devices were tested (stirred ball mill, high pressure homogenizer and ultrasonic homogenizer). Distinct differences between the size and the shape of the cell debris from B. megaterium and E. coli deriving from different cell disruption devices could be observed. These results could be correlated with the residence time distribution in the expanded bed

Structure-Properties Correlation of Cross-Linked Penicillin G Acylase Crystals

In biocatalytic processes, the use of free enzymes is often limited due to the lack of long-term stability and reusability. To counteract this, enzymes can be crystallized and then immobilized, generating cross-linked enzyme crystals (CLECs). As mechanical stability and activity of CLECs are crucial, different penicillin G acylases (PGAs) from Gram-positive organisms have proven to be promising candidates for industrial production of new semisynthetic antibiotics, which can be crystallized and cross-linked to characterize the resulting CLECs regarding their mechanical and catalytic properties. The greatest hardness and Young’s modulus determined by indentation with an atomic force microscope were observed for CLECs of Bacillus species FJAT-PGA CLECs (26 MPa/1450 MPa), followed by BmPGA (Priestia megaterium PGA, 23 MPa/1170 MPa) and BtPGA CLECs (Bacillus thermotolerans PGA, 11 MPa/614 MPa). In addition, FJAT- and BtPGA CLECs showed up to 20-fold higher volumetric activities compared to BmPGA CLECs. Correlation to structural characteristics indicated that a high solvent content and low number of cross-linking residues might lead to reduced stability. Furthermore, activity seems to be restricted by small water channels due to severe diffusion limitations. To the best of our knowledge, we show for the first time in this study that the entire process chain for the characterization of diverse industrially relevant enzymes can be performed at the microliter scale to discover the most important relationships and limitation

The Depth-Dependent Mechanical Behavior of Anisotropic Native and Cross-Linked HheG Enzyme Crystals

Enzymes are able to catalyze various specific reactions under mild conditions and can, therefore, be applied in industrial processes. To ensure process profitability, the enzymes must be reusable while ensuring their enzymatic activity. To improve the processability and immobilization of the biocatalyst, the enzymes can be, e.g., crystallized, and the resulting crystals can be cross-linked. These mechanically stable and catalytically active particles are called CLECs (cross-linked enzyme crystals). In this study, the influence of cross-linking on the mechanical and catalytic properties of the halohydrin dehalogenase (HheG) crystals was investigated using the nanoindentation technique. Considering the viscoelastic behavior of protein crystals, a mechanical investigation was performed at different indentation rates. In addition to the hardness, for the first time, depth-dependent fractions of elastic and plastic deformation energies were determined for enzyme crystals. The results showed that the hardness of HheG enzyme crystals are indentation-rate-insensitive and decrease with increases in penetration depth. Our investigation of the fraction of plastic deformation energy indicated anisotropic crystal behavior and higher irreversible deformation for prismatic crystal faces. Due to cross-linking, the fraction of elastic energy of anisotropic crystal faces increased from 8% for basal faces to 68% for prismatic crystal faces. This study demonstrates that mechanically enhanced CLECs have good catalytic activity and are, therefore, suitable for industrial use

Process and Formulation Strategies to Improve Adhesion of Nanoparticulate Coatings on Stainless Steel

The use of ceramic nanoparticles in coatings can significantly improve their mechanical properties such as hardness, adhesion to substrate, and scratch and abrasion resistance. A successful enhancement of these properties depends strongly on the coating formulation used, and the subsequent structure formed during coating. The aim of the present work was to enhance the adhesion between nanoparticulate coatings and stainless-steel substrates. A covalent particle structure was formed and better mechanical properties were achieved by modifying alumina nanoparticles, as well as substrates, with 3-aminopropyltriethoxysilane and by using a formulation consisting of solvent, modified particles, and bisphenol-A-diglycidylether as cross-linking additive. In addition to the adhesion force needed to remove the coating from the substrate, the type of failure (adhesive or cohesive) was characterized to gain a deeper understanding of the structure formation and to identify interdependencies between process, formulation, and coating structure properties. The modification process and the formulation composition were varied to achieve a detailed conception of the relevant correlations. By relating the results to other structural properties, such as the theoretical porosity and thickness, it was possible to understand the formation of the coating structure in more detail

Quantification and modeling of macroparticle-induced mechanical stress for varying shake flask cultivation conditions

In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask

Accelerated Particle Separation in a DLD Device at Re > 1 Investigated by Means of µPIV.

A pressure resistant and optically accessible deterministic lateral displacement (DLD) device was designed and microfabricated from silicon and glass for high-throughput fractionation of particles between 3.0 and 7.0 µm comprising array segments of varying tilt angles with a post size of 5 µm. The design was supported by computational fluid dynamic (CFD) simulations using OpenFOAM software. Simulations indicated a change in the critical particle diameter for fractionation at higher Reynolds numbers. This was experimentally confirmed by microparticle image velocimetry (µPIV) in the DLD device with tracer particles of 0.86 µm. At Reynolds numbers above 8 an asymmetric flow field pattern between posts could be observed. Furthermore, the new DLD device allowed successful fractionation of 2 µm and 5 µm fluorescent polystyrene particles at Re = 0.5-25

Structure-Based Modeling of the Mechanical Behavior of Cross-Linked Enzyme Crystals

Because of their high volumetric catalytic activity, in addition to their high chemical and thermal resistances, enzymes in the form of protein crystals are an excellent choice for application as immobilized biocatalysts. However, mechanical stability is a requirement for the processability of immobilisates, in addition to the protein crystals retaining their enzymatic activity, and this is closely related to the crystal structure. In this study, the influence of protein engineering on the mechanical stability of cross-linked enzyme crystals (CLECs) was investigated using a genetically modified model protein in which additionally cysteines were introduced on the protein surface for targeted cross-linking. The results showed that the mechanical stability of crystals of the mutant proteins in the native form was decreased compared to native wild-type crystals. However, specific cross-linking of the introduced amino acid residues in the mutant proteins resulted in their increased mechanical stability compared to wild-type CLECs. In order to determine the correlation between the crystal structure and the resulting mechanical properties of CLECs to enable targeted cross-linking, a previously developed model was revised and then used for the two model proteins. This model can explain the mechanically investigated relationships, such as the anisotropic crystal behavior and the influence of a linker or mutation on the micromechanical properties and, hence, can be helpful for the tailor-made production of CLECs

Along the Process Chain to Probiotic Tablets: Evaluation of Mechanical Impacts on Microbial Viability

Today, probiotics are predominantly used in liquid or semi-solid functionalized foods, showing a rapid loss of cell viability. Due to the increasing spread of antibiotic resistance, probiotics are promising in pharmaceutical development because of their antimicrobial effects. This increases the formulation requirements, e.g., the need for an enhanced shelf life that is achieved by drying, mainly by lyophilization. For oral administration, the process chain for production of tablets containing microorganisms is of high interest and, thus, was investigated in this study. Lyophilization as an initial process step showed low cell survival of only 12.8%. However, the addition of cryoprotectants enabled survival rates up to 42.9%. Subsequently, the dried cells were gently milled. This powder was tableted directly or after mixing with excipients microcrystalline cellulose, dicalcium phosphate or lactose. Survival rates during tableting varied between 1.4% and 24.1%, depending on the formulation and the applied compaction stress. More detailed analysis of the tablet properties showed advantages of excipients in respect of cell survival and tablet mechanical strength. Maximum overall survival rate along the complete manufacturing process was >5%, enabling doses of 6 × 108 colony forming units per gram (CFU gtotal-1), including cryoprotectants and excipients

Micromechanical characterisation of protein crystals and filamentous microorganisms

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A High-Aspect-Ratio Deterministic Lateral Displacement Array for High-Throughput Fractionation

Future industrial applications of microparticle fractionation with deterministic lateral displacement (DLD) devices are hindered by exceedingly low throughput rates. To enable the necessary high-volume flows, high flow velocities as well as high aspect ratios in DLD devices have to be investigated. However, no experimental studies have yet been conducted on the fractionation of bi-disperse suspensions containing particles below 10 µm with DLD at a Reynolds number (Re) above 60. Furthermore, devices with an aspect ratio of more than 4:1, which require advanced microfabrication, are not known in the DLD literature. Therefore, we developed a suitable process with deep reactive ion etching of silicon and anodic bonding of a glass lid to create pressure-resistant arrays. With a depth of 120 µm and a gap of 23 µm between posts, a high aspect ratio of 6:1 was realized, and devices were investigated using simulations and fractionation experiments. With the two-segmented array of 3° and 7° row shifts, critical diameters of 8 µm and 12 µm were calculated for low Re conditions, but it was already known that vortices behind the posts can shift these values to lower critical diameters. Suspensions with polystyrene particles in different combinations were injected with an overall flow rate of up to 15 mL/min, corresponding to Re values of up to 90. Suspensions containing particle combinations of 2 µm with 10 µm as well as 5 µm with 10 µm were successfully fractionated, even at the highest flow rate. Under these conditions, a slight widening of the displacement position was observed, but there was no further reduction in the critical size as it was for Re = 60. With an unprecedented fractionation throughput of nearly 1 L per hour, entirely new applications are being developed for chemical, pharmaceutical, and recycling technologies