Genetic heterogeneity in the leader and P1-coding regions of foot-and-mouth disease virus serotypes A and O in Africa.

Genetic information regarding the leader (L) and complete capsid-coding (P1) region of FMD serotype A and O viruses prevalent on the African continent is lacking. Here, we present the complete L-P1 sequences for eight serotype A and nine serotype O viruses recovered from FMDV outbreaks in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions revealed that the African isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus. However, similar analysis of the L region revealed random groupings of isolates from serotypes O and A. Comparisons between the phylogenetic trees derived from the structural coding regions and the L region pointed to a possibility of genetic recombination. The intertypic nucleotide and amino acid variation of all the isolates in this study supported results from previous studies where the externally located 1D was the most variable whilst the internally located 1A was the most conserved, which likely reflects the selective pressures on these proteins. Amino acids identified previously as important for FMDV structure and functioning were found to be highly conserved. The information gained from this study will contribute to the construction of structurally designed FMDV vaccines in Africa.

The effect of material properties and process parameters on die filling at varying throughputs: A pls-model-based analysis.

When tablets are manufactured on a rotary tablet press and the throughput is increased, it leads to changes in powder dynamics during die filling due to formulation characteristics and changing powder flow in the feed frame. This may result, a.o. in increased tablet weight variability, poorer content uniformity, capping and lamination. This research focuses on explaining the die filling performance depending on material properties and process settings, including throughput for small and large tablets. It was concluded that throughput had a negative impact on die filling variability, which is related to reduced residence time and lower fill fraction of feed frame and dies. Furthermore, the die filling mechanism was inherently different for large tablets in comparison to small tablets. Higher die filling consistency was observed for dense, less porous, less compressible and better flowing powders. As a result of this work, a model was developed to predict impact of formulation properties and process settings on die filling variability and its dependency on changes in throughput. This model will benefit formulation development at an early stage when active ingredient availability may be challenging as it will avoid the need to conduct experiments at high throughputs.

Identification of continuous twin-screw melt granulation mechanisms for different screw configurations, process settings and formulation.

Twin-screw melt granulation (TSMG) is a promising continuous manufacturing technology for the processing of high drug load formulations and to formulate heat- and moisture-sensitive active pharmaceutical ingredients (APIs). This study evaluates the influence of process parameters for TSMG, mainly focusing on the effect of the screw configuration combined with screw speed, throughput and barrel temperature, to elucidate the melt granulation mechanisms. For the kneading zone, the stagger angle was varied between 30°, 60° and 90°, and investigated for both the forward and the reversed direction. In addition to the process parameters, the influence of the formulation differing in their API-binder miscibility was evaluated. As responses, the granule (size, friability and porosity) and process properties such as torque were evaluated, indicating that the screw configuration is the most influential factor. Nucleation, consolidation and breakage are the granulation mechanisms for the forward and the neutral configuration, while consolidation and densification with shear elongation are identified for the reversed configuration. The formulations differ mainly in the forward and neutral configuration since the immiscible formulation shows a bimodal granule size distribution with a larger fraction of fines and weaker granules is obtained. For the reversed configuration, similar granulation mechanisms are seen for both formulations.

Elucidation of granulation mechanisms along the length of the barrel in continuous twin-screw melt granulation.

In the pharmaceutical industry, innovative continuous manufacturing technologies such as twin-screw melt granulation (TSMG) are gaining more and more interest to process challenging formulations. To enable the implementation of TSMG, more elucidation of the process is required and this study provides a better understanding of the granule formation along the length of the barrel. By sampling at four different zones, the influence of screw configuration, process parameters and formulation is investigated for the granule properties next to the residence time distribution. It showed that conveying elements initiate the granulation by providing a limited heat transfer into the powder bed. In the kneading zones, the consolidation stage takes place, shear elongation combined with breakage and layering is occurring for the reversed configurations and densification with breakage and layering for the forward and neutral configurations. Due to the material build-up in the reversed configurations, these granules are larger, stronger, more elongated and less porous due to the higher degree of shear and densification. This configuration also shows a significantly longer residence time compared to the forward configuration. Hence, the higher level of shear and the longer period of time enables more melting of the binder resulting in successful granulation.

The application of Near-Infrared Spatially Resolved Spectroscopy in scope of achieving continuous real-time quality monitoring and control of tablets with challenging dimensions.

In scope of achieving real-time release of tablets, quality attributes need to be monitored and controlled through Process Analytical Technology tools such as near-infrared spectroscopy (NIRS). The authors evaluated the suitability of NIR-Spatially Resolved Spectroscopy (NIR-SRS) for continuous real-time monitoring and control of content uniformity, hardness and homogeneity of tablets with challenging dimensions. A novel user-friendly research and development inspection unit was used as standalone equipment for the analysis of small oblong tablets with deep-cut break lines. A total of 66 tablets varying in hardness and Active Pharmaceutical Ingredient (API) content were inspected, with each tablet being analysed five times and measurements repeated on three different days. Partial Least Squares (PLS) models were developed to assess content uniformity and hardness, of which the former showed higher accuracy. The authors attempted to visualize tablet homogeneity through NIR-SRS spectra by regressing all spectra obtained during a single measurement using a content uniformity PLS model. The NIR-SRS probe demonstrated its potential towards real-time release testing through its ability to quickly monitor content uniformity, hardness and visualize homogeneity, even for tablets with challenging dimensions.

Visualization of the granule temperature using thermal imaging to improve understanding of the granulation mechanism in continuous twin-screw melt granulation.

The aim of this study is to increase process understanding of the granulation mechanism in twin-screw melt granulation by evaluating the influence of different screw configurations on granule formation and granule temperature via thermal imaging. The study used a Design of Experiments (DoE) to process a miscible and immiscible formulation (85% API/binder w/w) using a twin-screw extruder with varying screw configurations. The barrel temperature (°C), screw speed (rpm), throughput (kg/h), and kneading zone (direction and stagger angle) were varied. Granule and process properties were evaluated for samples collected at four different locations along the length of the granulation barrel to visualize the granule formation, and granule temperature was monitored by an infrared camera to measure heat transfer on the granules. The resulting temperature was linked to the granule properties and the granule formation along the length of the barrel. The most influencing factors on the granule temperature are the direction of the kneading zone and the set barrel temperature. It was observed that granule formation mainly occurred in the zones that apply more kneading on the granules. The highest temperature increase was observed when the smallest stagger angle in reverse configuration was used, and could be linked to better granule quality attributes.

Elucidation of the powder flow pattern in a twin-screw LIW-feeder for various refill regimes.

The importance of residence time distribution modeling is acknowledged as a tool for enabling material tracking and control within a continuous manufacturing line in order to safeguard both product quality and production efficiency. One of the first unit-operations into a continuous direct compression line (i.e. CDC-line) worthwhile doing extensive RTD-analysis upon are the LIW-feeders since they dose the ingredients in a controlled way following the label claim and hence can directly influence critical quality attributes like content uniformity. An NIR measurement method was developed determining the RTD of selected powders at specific feeder settings. Step-change experiments using sodium saccharin as a tracer were conducted. In order to gain and in depth understanding of the material flow, spatial samples throughout the hopper were taken at predefined timepoints during the step change experiments. This revealed the presence of a bypass trajectory along the edges of the agitator, while in the center of the agitator an inner mixing volume in which the tracer concentration lags behind seemed to be present. Finally, a model based on a plug flow and continuous stirred tank reactor was evaluated. The fitted model was not able to capture this complex flow behavior and shows the need for an extended compartmental model distinguishing between a bypass trajectory formed by the agitator and an inner mixing volume.

Preparation and evaluation of sustained-release matrix tablets based on metoprolol and an acrylic carrier using injection moulding.

Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading.

The effect of binder types on the breakage and drying behavior of granules in a semi-continuous fluid bed dryer after twin screw wet granulation.

Current study investigated the effect of different binder types on the granule drying process and the granule breakage behavior in a semi-continuous fluid bed dryer integrated in the C25 ConsiGma-system. The studied binders (i.e. hydroxypropyl pea starch, hydroxypropyl methylcellulose E15, polyvinylpyrrolidone K12, and starch octenyl succinate CO 01) required different liquid amounts to produce similar granule quality. These different liquid requirements were translated into different drying conditions for each binder to result in sufficiently dry granules at the end of a drying cycle. By comparing the size distribution of the granules before entering and after exiting the fluid bed dryer, granule breakage could be evaluated. No effect of the binder type on the granule breakage during drying was observed. However, differences in granule breakage were observed for the binders when processed with the horizontal set-up of the C25 system, as granule breakage during pneumatic transport depended on the binder type. Only one binder (hydroxypropyl pea starch) allowed to avoid granule breakage during the entire process. Furthermore, this research showed that the drying process was mainly steered by the liquid requirements for granulation, and that these liquid requirements depended on the binder used.

Continuous direct compression: Development of an empirical predictive model and challenges regarding PAT implementation.

In this study, an empirical predictive model was developed based on the quantitative relationships between blend properties, critical quality attributes (CQA) and critical process parameters (CPP) related to blending and tableting. The blend uniformity and API concentration in the tablets were used to elucidate challenges related to the processability as well as the implementation of PAT tools. Thirty divergent ternary blends were evaluated on a continuous direct compression line (ConsiGma™ CDC-50). The trials showed a significant impact of the impeller configuration and impeller speed on the blending performance, whereas a limited impact of blend properties was observed. In contrast, blend properties played a significant role during compression, where changes in blend composition significantly altered the tablet quality. The observed correlations allowed to develop an empirical predictive model for the selection of process configurations based on the blend properties, reducing the number of trial runs needed to optimize a process and thus reducing development time and costs of new drug products. Furthermore, the trials elucidated several challenges related to blend properties that had a significant impact on PAT implementation and performance of the CDC-platform, highlighting the importance of further process development and optimization in order to solve the remaining challenges.

In-depth analysis of the long-term processability of materials during continuous feeding.

This study determined the feasibility of long-term continuous powder feeding and its effect on the overall process performance. Additionally, quantitative relationships were established between material properties, process settings and screw feeding responses during gravimetric feeding. Twelve divergent raw materials were processed over longer periods using a GEA Compact Feeder integrated in a continuous direct compression line (ConsiGma™ CDC-50). The resulting gravimetric feeding responses were combined with the material properties and process settings into an overall PLS model. The model elucidated the impact of the material descriptors for density; powder flow; particle size; compressibility; permeability and wall friction angle on the feeding process. Furthermore, long-term processing of the materials exhibited challenges related to the processability and refill consistency where a significant impact of the compressibility and cohesive/adhesive properties of the materials was observed. Overall, this approach provided insights into the feasibility of long-term continuous feeding which is not possible through 'short-term' feeding trials. Additionally, throughout this study, the need for material-specific adjustments of the feeding and refill equipment was highlighted.

Predicting antigenic sites on the foot-and-mouth disease virus capsid of the South African Territories types using virus neutralization data.

Foot-and-mouth disease virus (FMDV) outer capsid proteins 1B, 1C and 1D contribute to the virus serotype distribution and antigenic variants that exist within each of the seven serotypes. This study presents phylogenetic, genetic and antigenic analyses of South African Territories (SAT) serotypes prevalent in sub-Saharan Africa. Here, we show that the high levels of genetic diversity in the P1-coding region within the SAT serotypes are reflected in the antigenic properties of these viruses and therefore have implications for the selection of vaccine strains that would provide the best vaccine match against emerging viruses. Interestingly, although SAT1 and SAT2 viruses displayed similar genetic variation within each serotype (32 % variable amino acids), antigenic disparity, as measured by r(1)-values, was less pronounced for SAT1 viruses compared with SAT2 viruses within our dataset, emphasizing the high antigenic variation within the SAT2 serotype. Furthermore, we combined amino acid variation and the r(1)-values with crystallographic structural data and were able to predict areas on the surface of the FMD virion as antigenically relevant. These sites were mostly consistent with antigenic sites previously determined for types A, O and C using mAbs and escape mutant studies. Our methodology offers a quick alternative to determine antigenic relevant sites for FMDV field strains.

Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes.

Specific recognition of phosphoinositides is crucial for protein sorting and membrane trafficking. Protein transport to the yeast vacuole depends on the Vam7 t-SNARE and its phox homology (PX) domain. Here, we show that the PX domain of Vam7 targets to vacuoles in vivo in a manner dependent on phosphatidylinositol 3-phosphate generation. A novel phosphatidylinositol-3-phosphate-binding motif and an exposed loop that interacts with the lipid bilayer are identified by nuclear magnetic resonance spectroscopy. Conservation of key structural and binding site residues across the diverse PX family indicates a shared fold and phosphoinositide recognition function.

Development and evaluation of injection-molded sustained-release tablets containing ethylcellulose and polyethylene oxide.

PURPOSE: It was the aim of the present study to develop sustained-release matrix tablets by means of injection molding of ethylcellulose (EC) and polyethylene oxide (PEO) mixtures and to evaluate the influence of process temperature, matrix composition, and viscosity grade of EC and PEO on processability and drug release. METHODS: Formulations consisting of metoprolol tartrate (MPT, concentration: 30%), EC plasticized by dibutyl sebacate, and PEO were extruded and consequently injection molded into tablets. The influence of process temperature (120°C and 140°C), matrix composition, viscosity grade of EC (4, 10, 20, 45, and 100 mPa·s) and PEO (7 × 10(6), 1 × 10(6), and 1 × 10(5) Mw) on processability and drug release was determined. RESULTS: Formulations consisting of 70% EC and 30% MPT showed incomplete drug release, whereas drug release was too fast for formulations without EC. Higher PEO concentrations increased drug release. Formulations containing 30% metoprolol, EC, and different concentrations of PEO showed first-order release rates with limited burst release. Drug release from direct compressed tablets showed faster drug release rates compared to injection-molded formulations. There was no clear relationship between the molecular weight of EC and drug release. The melting endotherm (113.9°C) of MPT observed in the differential scanning calorimeter thermogram of the tablets indicated that a solid dispersion was formed which was confirmed by X-ray diffractogram. X-ray tomography demonstrated a difference in pore structure between tablets processed at 120°C and 140°C. CONCLUSION: It was concluded that injection molding can be applied successfully to develop sustained-release PEO/EC matrix tablets.

Evaluation of torque as an in-process control for granule size during twin-screw wet granulation.

The potential of torque as in-process control (IPC) to monitor granule size in twin-screw wet granulation (TSG) was investigated. An experimental set-up allowing the collection of granules at four different locations (i.e., in the wetting zone, after the first and second kneading zone and at the end of the granulator) of the granulator screws was used to determine the change in granule size, granule temperature and the contribution of each compartment to the overall torque for varying screw speed, mass feed rate and liquid-to-solid ratio. The only observed correlation was between the granule size and torque increase after the first kneading zone because the torque increase was an indication of the degree in granule growth which was consistently observed with all applied granulation process parameters. No correlation was observed in the other locations as changes of torque were accompanied to either granule breakage and/or growth. Moreover, torque increase was correlated to higher granule temperature, suggesting that energy put into the granulator was partly used to heat up the material being processed and explains additionally the lack of correlation between granule size and torque. Therefore, this study showed that torque could not be used as IPC to monitor granule size during TSG.

Determination of a quantitative relationship between material properties, process settings and screw feeding behavior via multivariate data-analysis.

In this study, a quantitative relationship between material properties, process settings and screw feeding responses of a high-throughput feeder was established via multivariate models (PLS). Thirteen divergent powders were selected and characterized for 44 material property descriptors. During volumetric feeder trials, the maximum feed capacity (FCCmax), the relative standard deviation on the maximum feed capacity (RSDFCmax), the short term variability (STRSD) and feed capacity decay (FCdecay) were determined. The gravimetric feeder trials generated values for the mass flow rate variability (RSDLC), short term variability (STRSD) and refill responses (Vrefill and RSDrefill). The developed PLS models elucidated that the material properties and process settings were clearly correlated to the feeding behavior. The extended volumetric feeder trials pointed out that there was a significant influence of the chosen screw type and screw speed on the feeding process. Furthermore, the process could be optimized by reducing the feeding variability through the application of optimized mass flow filters, high frequency vibrations, independent agitator control and optimized top-up systems. Overall, the models could allow the prediction of the feeding performance for a wide range of materials based on the characterization of a subset of material properties greatly reducing the number of required feeding experiments.

TPLS as predictive platform for twin-screw wet granulation process and formulation development.

In recent years, the interest in continuous manufacturing techniques, such as twin-screw wet granulation, has increased. However, the understanding of the influence of the combination of raw material properties and process settings upon the granule quality attributes is still limited. In this study, a T-shaped partial least squares (TPLS) model was developed to link raw material properties, the ratios in which these raw materials were combined and the applied process parameters for the twin-screw wet granulation process with the granule quality attributes. In addition, the predictive ability of the TPLS model was used to find a suitable combination of formulation composition and twin-screw granulation process settings for a new API leading to desired granule quality attributes. Overall, this study helped to better understand the link between raw material properties, formulation composition and process settings on granule quality attributes. Moreover, as TPLS can provide a reasonable starting point for formulation and process development for new APIs, it can reduce the experimental development efforts and consequently the consumption of expensive (and often limited available) new API.

Delta-mannitol to enable continuous twin-screw granulation of a highly dosed, poorly compactable formulation.

In current study, it was investigated if the moisture-mediated polymorphic transition from δ- to ß-mannitol during twin screw granulation (TSG) also took place in high drug loaded formulations and if the specific granule morphology associated with the polymorphic transition could enable tableting of granules comprising 75% paracetamol, a poorly compactable drug. Experiments were performed on an integrated continuous manufacturing line, including a twin screw granulator, fluid bed dryer, mill and tablet press. The polymorphic transition of δ- to ß-mannitol was observed during twin screw granulation and granules exhibited the needle-shaped morphology, typical of this transition. TSG at low liquid-to-solid (L/S) ratios and use of polyvinylpyrrolidone or hydroxypropylmethylcellulose as binders inhibited the polymorphic transition, whereas screw speed, drying time, drying temperature and airflow did not affect the solid state of mannitol in the granules. Without binder and despite the high paracetamol drug load in the formulation, limited breakage and attrition was observed during drying and milling. In contrast to granules manufactured from a formulation containing paracetamol/ß-mannitol which could not be tableted due to extensive capping, granules prepared from a paracetamol/δ-mannitol formulation showed good tabletability. In conclusion, δ-mannitol is a promising TSG excipient, especially for high drug-loaded formulations with poor tabletability.

Viscosity of API/fatty acid suspensions: Pitfalls during analysis.

This article describes how to obtain reliable data during rheological analysis of active pharmaceutical ingredient/fatty acid suspensions. These materials are specifically used for prilling, an innovative pharmaceutical technique for the production of a multiparticulate dosage form. Nevertheless, presented guidelines are applicable for a wide range of pharmaceutical suspensions. Reliable rheological results can only be obtained when being aware of artefacts, such as a non-continuous medium, sedimentation, apparent wall slip and protrusion flow. To comply with the continuum hypothesis at high phase volumes (≥25% w/w), the required gap-to-particle-size ratio may be larger than the generally accepted 10:1 ratio. Reproducible flow curves that are not disturbed by sedimentation during sample analysis can be obtained faster by varying the shear rate stepwise from high to low values. While apparent wall slip (at low shear rates) can be prevented via serrated instead of smooth plates, protrusion flow (at high shear rates) during measurements with serrated plates results in non-reliable data. Therefore, in general, high viscous suspensions with yield stress can be analysed with serrated plates, while low viscous suspensions without yield stress should be analysed with geometries having smooth surfaces. By following these guidelines, accurate rheological properties of pharmaceutical suspensions can be obtained.

Influence of binder attributes on binder effectiveness in a continuous twin screw wet granulation process via wet and dry binder addition.

The effect of a wide variety of binders on the quality of granules produced via continuous twin screw wet granulation was studied. Anhydrous dicalcium phosphate was used as poorly soluble filler and was granulated applying dry or wet addition of binders. Furthermore, dry and wet binder characteristics were determined and linked to the binder effectiveness. PVA 4-88 and starch octenyl succinate exhibited the lowest granule friability at low liquid-to-solid ratios, i.e. the highest binder effectiveness, which was attributed to fast binder activation based on the fast wetting kinetics of the binder, to efficient wetting of DCP particles, and to good spreading in the powder bed. The performance of wettability measurements in an early formulation development stage is therefore considered highly important. Additionally, an increased stickiness of the binder surface caused by high binder viscosity and slow dissolution kinetics also positively influenced the binder effectiveness. In conclusion, this study revealed which binder attributes have a critical impact on the granulation process of dicalcium phosphate. Additionally, dry binder addition proved successful for creation of high quality granules.