Investigation of Dispersion Kinetics of Particulate Lubricants and their Effect on the Mechanical Strength of MCC Tablets.

INTRODUCTION: Tablets are commonly produced by internally adding particulate lubricants, which are known to possibly lower the mechanical strength of tablets. This reduction is caused by the coverage of matrix forming components by lubricant particles, resulting in decreased interparticulate interactions. The known incompatibilities with some active compounds of the predominantly used lubricant, magnesium stearate, call for the in-depth characterization of alternative lubricants. PURPOSE: Investigation of the dispersion behavior of five commonly applied pharmaceutical lubricants by mathematically modeling the dispersion kinetics for short and extended mixing times. METHODS: The dispersion behavior of five different pharmaceutical lubricants were examined by systematically varying lubricant concentration and mixing time of binary formulations and evaluating the kinetic of tensile strength reduction by theoretically estimating the surface coverage based on particle sizes. RESULTS: For short mixing times, a unifying relationship between compactibility reduction and theoretical surface coverage was identified. Subsequently, for extended mixing times, distinct differences in the shear strength and dispersion kinetics of the investigated lubricants were found. CONCLUSIONS: The lubricant particle size controls the tensile strength reduction if short mixing times are applied. For extended mixing times, the investigated lubricants can be divided into two groups in terms of dispersion kinetics. Possible underlying reasons are discussed in detail in order to enhance the general understanding of lubricant dispersions in tablet formulations.

Temperature influence on the compression and breakage behaviour of yeast cells.

Industrial biotechnology uses microbial cells to produce a wide range of products. While the genetic and molecular properties of these organisms are well understood, less is known about their mechanical properties. Previous work has established a test procedure for single yeast cells using a nanoindentation instrument equipped with a flat-punch probe, which allows single cells (Saccharomyces cerevisiae) to be compressed between two parallel surfaces. The resulting force-displacement curves clearly showed the bursting of the cells and were used to determine characteristics such as burst force and burst energy. Other studies have investigated the influence of growth conditions and measurement conditions on the mechanical characteristics. The recent study examined the mechanical characteristics according to the temperature during compression. Temperature from 0°C to 25°C has no significant effect on the micromechanical properties. Increasing the temperature up to 35°C causes a reduction in the strength of the cells. At even higher temperatures, up to 50°C, the burst force and burst energy increase significantly. A deformation geometry model was used to calculate the cell wall tensile strength as a function of temperature. The results of these studies may facilitate the identification of efficient conditions for cell disruption and product recovery in downstream biotechnological processes.

Macroparticle-enhanced cultivation of Lentzea aerocolonigenes: Variation of mechanical stress and combination with lecithin supplementation for a significantly increased rebeccamycin production.

The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 and 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration, and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle-induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. In addition, the macroparticle-enhanced cultivation of L. aerocolonigenes was combined with soy lecithin addition to further increase the rebeccamycin concentration. With 100 g L-1 glass beads in a diameter of 969 µm and 5 g L-1 soy lecithin a concentration of 388 mg L-1 rebeccamycin was reached after 10 days of cultivation, which corresponds to the highest rebeccamycin concentrations achieved in shake flask cultivations of L. aerocolonigenes stated in literature so far.

Process engineering with planetary ball mills.

Planetary ball mills are well known and used for particle size reduction on laboratory and pilot scales for decades while during the last few years the application of planetary ball mills has extended to mechanochemical approaches. Processes inside planetary ball mills are complex and strongly depend on the processed material and synthesis and, thus, the optimum milling conditions have to be assessed for each individual system. The present review focuses on the insight into several parameters like properties of grinding balls, the filling ratio or revolution speed. It gives examples of the aspects of grinding and illustrates some general guidelines to follow for modelling processes in planetary ball mills in terms of refinement, synthesis' yield and contamination from wear. The amount of energy transferred from the milling tools to the powder is significant and hardly measurable for processes in planetary ball mills. Thus numerical simulations based on a discrete-element-method are used to describe the energy transfer to give an adequate description of the process by correlation with experiments. The simulations illustrate the effect of the geometry of planetary ball mills on the energy entry. In addition the imaging of motion patterns inside a planetary ball mill from simulations and video recordings is shown.

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.

Effect of particle size on the dispersion behavior of magnesium stearate blended with microcrystalline cellulose.

The majority of tablets manufactured contain lubricants to reduce friction during ejection. However, especially for plastically deforming materials, e.g., microcrystalline cellulose (MCC), the internal addition of lubricants is known to reduce tablet tensile strength. This reduction is caused by the surface coverage by lubricant particles, the extent of which depends on both process and formulation parameters. Previously published models to predict the lubrication effect on mechanical strength do not account for changes in the excipient particle size. In this study, the impact of both lubricant concentration and mixing time on the tensile strength of tablets consisting of three different grades of MCC and four grades of magnesium stearate (MgSt) was evaluated. By taking into account the particle size of the applied excipients, a unifying relationship between the theoretically estimated surface coverage and compactibility reduction was identified. Evaluating the dispersion kinetics of MgSt as a function of time reveals a substantial impact of the initial surface coverage on the dispersion rate, while the minimal tensile strength was found to be comparable for the majority of formulations. In summary, the presented work extends the knowledge of lubricant dispersion and facilitates the reduction of necessary experiments during the development of new tablet formulations.

The Effect of Particle Shape on the Compaction of Realistic Non-Spherical Particles-A Multi-Contact DEM Study.

The purpose of this study was to investigate the deformation behavior of non-spherical particles during high-load compaction using the multi-contact discrete element method (MC-DEM). To account for non-spherical particles, the bonded multi-sphere method (BMS), which incorporates intragranular bonds between particles, and the conventional multi-sphere (CMS), where overlaps between particles are allowed to form a rigid body, were used. Several test cases were performed to justify the conclusions of this study. The bonded multi-sphere method was first employed to study the compression of a single rubber sphere. This method's ability to naturally handle large elastic deformations is demonstrated by its agreement with experimental data. This result was validated further through detailed finite element simulations (multiple particle finite element method (MPFEM)). Furthermore, the conventional multi-sphere (CMS) approach, in which overlaps between particles are allowed to form a rigid body, was used for the same objective, and revealed the limitations of this method in successfully capturing the compression behavior of a single rubber sphere. Finally, the uniaxial compaction of a microcrystalline cellulose-grade material, Avicel® PH 200 (FMC BioPolymer, Philadelphia, PA, USA), subjected to high confining conditions was studied using the BMS method. A series of simulation results was obtained with realistic non-spherical particles and compared with the experimental data. For a system composed of non-spherical particles, the multi-contact DEM showed very good agreement with experimental data.

Influence of compression kinetics during tableting of fluidized bed-granulated microorganisms on microbiological and physical-mechanical tablet properties.

As tablets are convenient to administer to patients, ensure safe dosing and allow cost-effective production on a large scale, they are the favored dosage form for numerous active pharmaceutical ingredients but also for the administration of viable probiotic microorganisms. Granules with viable yeast cells (Saccharomyces cerevisiae) formed by fluidized bed granulation with dicalcium phosphate (DCP), lactose (LAC) or microcrystalline cellulose (MCC) as carrier materials were tableted using a compaction simulator. Besides the compression stress, the compression speed was systematically studied by varying consolidation time and dwell time. The microbial survival as well as physical properties of the tablets, e.g., porosity and tensile strength, were determined. Higher compression stresses result in lower porosities. While on the one hand this has a detrimental effect on microbial survival (due to increased pressure and shear stress during particle rearrangement / densification), on the other hand it results in higher tensile strengths. At the same compression stress, a prolonged dwell time resulted in lower porosity and thus in lower survival rates but higher tensile strength. Against that, consolidation time showed no significant influence on the considered tablet quality attributes. Since changes of the tensile strength related survival rate were negligible (due to opposite but balancing dependence on porosity), high production speeds could be used for tableting of these granules without additional loss of viability, as long as tablets with the same tensile strength are produced.

Process and formulation parameters influencing the survival of Saccharomyces cerevisiae during spray drying and tableting.

Probiotic microorganisms provide health benefits to the patient when administered in a viable form and in sufficient doses. To ensure this, dry dosage forms are preferred, with tablets in particular being favored due to several advantages. However, the microorganisms must first be dried as gently as possible. Here, the model organism Saccharomyces cerevisiae was dried by spray drying. Various additives were tested for their ability to improve yeast cell survival during drying. In addition, the influence of various process parameters such as inlet temperature, outlet temperature, spray rate, spray pressure and nozzle diameter was investigated. It was possible to dry the yeast cells in such a way that a substantial proportion of living microorganisms was recovered after reconstitution. Systematic variation of formulation and process parameters showed that the use of protective additives is essential and that the outlet temperature determines the survival rate. The subsequent compression of the spray-dried yeast reduced viability and survival could hardly be improved by the addition of excipients, but the tabletability of spray-dried yeast protectant particles was quite good. For the first time, loss of viability during compaction of spray-dried microorganisms was correlated with the specific densification, allowing a deeper understanding of the mechanism of cell inactivation during tableting.

Effect of Process Parameters, Protectants and Carrier Materials on the Survival of Yeast Cells during Fluidized Bed Granulation for Tableting.

The administration of living microorganisms is of special interest, with regard to probiotic microorganisms providing health benefits to the patient. Effective dosage forms require the preservation of microbial viability until administration. Storage stability can be improved by drying, and the tablet is an especially attractive final solid dosage form due to its ease of administration and its good patient compliance. In this study, drying of the yeast Saccharomyces cerevisiae via fluidized bed spray granulation is investigated, as the probiotic Saccharomyces boulardii is a variety of it. Fluidized bed granulation enables faster drying than lyophilization on the one hand and lower temperatures than spray drying on the other hand, which are the two predominantly used techniques for life-sustaining drying of microorganisms. Yeast cell suspensions enriched with protective additives were sprayed onto the carrier particles of common tableting excipients, namely, dicalcium phosphate (DCP), lactose (LAC) and microcrystalline cellulose (MCC). Different protectants, such as mono-, di-, oligo- and polysaccharides, but also skimmed milk powder and one alditol, were tested; as they themselves, or chemically similar molecules, are known from other drying technologies to stabilize biological structures such as cell membranes, and thus, improve survival during dehydration. With the combined use of trehalose and skimmed milk powder, survival rates were 300 times higher than without the use of protective additives. In addition to these formulation aspects, the influence of process parameters such as inlet temperature and spray rate were considered. The granulated products were characterized regarding their particle size distribution, moisture content and the viability of the yeast cells. It has been shown that thermal stress on the microorganisms is especially critical, which can be reduced, for example, by reducing the inlet temperature or increasing the spray rate; however, formulation parameters such as cell concentration also influenced survival. The results were used to specify the influencing factors on the survival of microorganisms during fluidized bed granulation and to derive their linkages. Granules based on the three different carrier materials were tableted and the survival of the microorganisms was evaluated and linked to the tablet tensile strength achieved. Using LAC enabled the highest survival of the microorganisms throughout the considered process chain.

Tableting of fluidized bed granules containing living microorganisms.

Tablets are the favored dosage form for numerous active pharmaceutical ingredients, among others because they are easy to take, ensure safe dosing and allow cost-effective production on a large scale. This dosage form is also frequently chosen for the administration of viable probiotic microorganisms. Saccharomyces cerevisiae cells granulated in a fluidized bed process, with dicalcium phosphate (DCP), lactose (LAC) and microcrystalline cellulose (MCC) as carrier materials, were tableted using a compaction simulator, varying the compression stress. The tablets were analyzed regarding physical properties, e.g., porosity and tensile strength, as well as microbial survival. Carrier material and compression stress showed a significant influence on survival rate and physical tablet properties. The dependencies were related to material specific deformation characteristics and linked to mechanistic approaches to explain the different sensitivities.

Highly protein-loaded melt extrudates produced by small-scale ram and twin-screw extrusion - evaluation of extrusion process design on protein stability by experimental and numerical approaches.

Understanding of generation, extent and location of thermomechanical stress in small-scale (< 3 g) ram and twin-screw melt-extrusion is crucial for mechanistic correlations to the stability of protein particles (lysozyme and BSA) in PEG-matrices. The aim of the study was to apply and correlate experimental and numerical approaches (1D and 3D) for the evaluation of extrusion process design on protein stability. The simulation of thermomechanical stress during extrusion raised the expectation of protein degradation and protein particle grinding during extrusion, especially when TSE was used. This was confirmed by experimental data on protein stability. Ram extrusion had the lowest impact on protein unfolding temperatures, whereas TSE showed significantly reduced unfolding temperatures, especially in combination with kneading elements containing screws. In TSE, the mechanical stress in the screws always exceeded the shear stress in the die, while mechanical stress within ram extrusion was generated in the die, only. As both extruder designs revealed homogeneously distributed protein particles over the cross section of the extrudates for all protein-loads (20-60%), the dispersive power of TSE revealed not to be decisive. Consequently, the ram extruder would be favored for the production of stable protein-loaded extrudates in small scale.

Enhanced multi-component model to consider the lubricant effect on compressibility and compactibility.

Modeling of structural and mechanical tablet properties consisting of multiple components, based on a minimum of experimental data is of high interest, in order to minimize time- and cost-intensive experimental trials in the development of new tablet formulations. The majority of commonly available models use the compressibility and compactibility of constituent components and establish mixing rules between those components, in order to predict the tablet properties of formulations containing multiple components. However, their applicability is limited to single materials, which form intact tablets (e.g. lactose, cellulose) and therefore, they cannot be applied for lubricants. Lubricants are required in the majority of industrial tablet formulations and usually influence the mechanical strength of tablets. This study combines the multi-component compaction model of Reynolds et al. (2017) with a recently published lubrication model (Puckhaber et al. 2020) to describe the impact of multiple components on a formulation consisting of two diluents and a lubricant. By that, this model combination displays a meaningful extension of existing compaction models and allows the systematic prediction of properties of lubricated multi-component tablets.

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.

Morphology engineering for novel antibiotics: Effect of glass microparticles and soy lecithin on rebeccamycin production and cellular morphology of filamentous actinomycete Lentzea aerocolonigenes.

Lentzea aerocolonigenes, as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10 g·L-1) with a median diameter of 7.9 µm, for instance, induced an up to fourfold increase in product synthesis accompanied by overall enhanced viability of biomass. Furthermore, structural elucidations showed that biopellets isolated from MPEC tend to have lower hyphal density than unsupplemented control pellets. In this context, oxygen microprofiling was conducted to better understand how internal structural changes interwind with oxygen supply into the pellets. Here, the resulting oxygen profiles are of a contradictive trend of steeper oxygen consumption with increasing glass microparticle supplementation. Eventually, MPEC was combined with another promising cultivation strategy, the supplementation of soy lecithin (7.5 g·L-1), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213 mg·L-1 after 10 days of cultivation, the highest value published so far for microparticle-supplemented shake flask cultivations of L. aerocolonigenes.

Amyloid-like aggregation of recombinant ß-lactoglobulin at pH 3.5 and 7.0: Is disulfide bond removal the key to fibrillation?

Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified ß-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant ß-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.

The role of initial spore adhesion in pellet and biofilm formation in Aspergillus niger.

Fungi grow on a great variety of organic and inorganic materials. Colony establishment and growth on solid surfaces require adhesion of spores and hyphae to the substrate, while cell-to-cell interactions among spores and/or hyphae are a prerequisite for the development of three-dimensional mycelial structures such as pellets or biofilms. Surface adherence has been described as a two-step process, comprised of the initial attachment of ungerminated conidia followed by further adhesion of the forming germ tubes and growing hyphae. In the present study, we analyzed the contribution of adhesion of ungerminated spores to pellet and biofilm formation in Aspergillus niger. Mutants deficient in melanin biosynthesis were constructed by the deletion of the alb1 gene, encoding a polyketide synthase essential for pigment biosynthesis. Δalb1 conidia have an altered surface structure and changed physicochemical surface properties. Spore aggregation in liquid culture as well as spore surface attachment differ between the wild type and the mutant in a pH-dependent manner. In liquid culture further pellet formation is unaffected by altered spore-spore interactions, indicating that germ tube and hyphal adherence can compensate for deficiencies in the initial step of spore attachment. In contrast, under conditions promoting adhesion of Δalb1 conidia to polymer surfaces the mutant forms more stable biofilms than the wild type, suggesting that initial spore adhesion supports sessile growth.

Colloidal aggregates tested via nanoindentation and quasi-simultaneous 3D imaging.

The mechanical properties of aggregated colloids depend on the mutual interplay of inter-particle potentials, contact forces, aggregate structure and material properties of the bare particles. Owing to this variety of influences, experimental results from macroscopic mechanical testings were mostly compared to time-consuming, microscopic simulations rather than to analytical theories. The aim of the present paper was to relate both macroscopic and microscopic mechanical data with each other and simple analytical models. We investigated dense amorphous aggregates made from monodisperse poly-methyl methacrylate (PMMA) particles (diameter: 1.6 µm via nanoindentation in combination with confocal microscopy. The resulting macroscopic information was complemented by the three-dimensional aggregate structure as well as the microscopic strain field and strain tensor. The measured strain field and tensor were in reasonable agreement with the predictions from analytical continuum theories. Consequently, the measured force-depth curves could be analyzed within a theoretical framework that had been frequently used for nanoindentation of atomic matter such as metals, ceramics and polymers. The extracted values for hardness and effective Young's modulus represented average values characteristic of the aggregate. On the basis of of these parameters we discuss the influence of the strength of particle bonds by introducing polystyrene (PS) between the particles.

A compactibility-based lubricant dispersion model describing the effect of formulation and paddle speed.

In rotary tablet presses, the powder flow into the dies is typically facilitated by paddle feeder. For internally lubricated formulations, the shear forces exerted by the paddle rotation can result in a considerable decrease in tablet strength due to the dispersion of lubricant agglomerates. Available models to describe the lubricant dispersion in paddle feeder allow only a limited quantitative description and transferability of the process. This study introduces an empirical dispersion kinetic which is able to precisely describe the reduction of compactibility due to the shear stresses inside the paddle feeder, even for inhomogeneously flowing material. Additionally, by blending different grades of magnesium stearate at three levels of lubricant concentration with two different grades of microcrystalline cellulose, the impact of bulk properties on the lubrication dispersion in the feed frame was investigated. It was shown, that for a given formulation, the kinetics of compactibility reduction are comparable for different magnesium stearate concentrations. Additionally, the bulk properties of the applied magnesium stearate grade critically affect the dispersion kinetics as well as the maximum compactibility reduction inside the feed frame. In summary, the developed model represents a meaningful extension of the currently available process models for pharmaceutical tablet lubrication.

Influence of the drug deformation behaviour on the predictability of compressibility and compactibility of binary mixtures.

Various studies investigate the predictability of the compressibility and compactibility of tablet formulations based on the behaviour of the pure materials. However, these studies are limited to a few materials so far probably because of the complexity of the powder compaction process. One approach preventing the excessive increase in complexity is the extension of the investigations from pure materials to binary powder mixtures. The focus of this study is on the predictability of the compressibility and compactibility of binary mixtures consisting of an active pharmaceutical ingredient (API) and the excipient microcrystalline cellulose. Three APIs with markedly different deformation behaviour were used. The API concentration and type are systematically varied. For all three material combinations it is found that the in-die compressibility of the binary mixtures can be precisely predicted based on the characteristic compression parameters of the raw materials using the extended in-die compression function in combination with a volume-based linear mixing rule. Since the tablet porosity (out-of-die) also follows a linear mixing rule, the predictability can be further extended using the method of Katz et al. In contrast, the influence of the API concentration on compactibility or rather on tablet tensile strength is non-linear and strongly dependent on the deformation behaviour of the API, making the predictability more difficult. Neither the approach of Reynolds et al. nor this of Kuentz and Leuenberger are able to predict the compactibility when clear deviations from a linear mixing rule appear.