Preparation of Piroxicam nanosuspensions by high pressure homogenization and evaluation of improved bioavailability.

OBJECTIVE: Inflammation is a natural response of the organism, involving events responsible for releasing chemical mediators and requiring treatments of symptoms such as pain, redness, heat, swelling, and loss of tissue function. Piroxicam (PRX) is a non-steroidal anti-inflammatory drug with the effect of nonselective COX inhibitor activity; however, it shows poor bioavailability caused by the poor and slow water solubility. In this study, we developed PRX nanosuspensions with 200-500 nm in diameter to increase the bioavailability of PRX by improving its solubility. METHODS: PRX nanosuspensions were fabricated by High pressure homogenization method with PVA, SDS and Tween 80. The nanosuspensions were characterized by XRD, FTIR, DSC, and in vitro release. In vivo pharmacokinetic properties and anti-inflammatory effects were also investigated in rabbits. RESULTS: PRX nanosuspensions significantly increased the solubility (14.89 ± 0.03 mg/L for pure PRX and 16.75 ± 0.05 mg/L for PRX nanosuspensions) and dissolution rate as compared to the pure PRX (p < 0.05). Orally administered PRX nanosuspension (AUC 0-t is 49.26 ± 4.29 µg/mL × h) significantly improved the bioavailability of PRX (AUC 0-t is 28.40 ± 12.11 µg/mL × h). The anti-inflammatory effect of PRX nanosuspension was also investigated in rabbits and it was observed that PRX nanosuspension treatment significantly improved the inhibition of COX-2 and NFκB expression as compared to the PRX treatment (p < 0.05). CONCLUSIONS: The results in this study indicate that PRX nanosuspension is a promising nanomedicine for enhancing the anti-inflammatory activity of PRX and has a high potential for the treatment of inflammation.

Selective laser sintering 3D printing - an overview of the technology and pharmaceutical applications.

Food and Drug Administration (FDA) has approved a drug product (Spritam®) and many medical devices manufactured by three-dimensional printing (3DP) processes for human use. There is immense potential to print personalized medicines using 3DP. Many 3DP methods have been reported in the literature for pharmaceutical applications. However, selective laser sintering (SLS) printing has remained least explored for pharmaceutical applications. There are many advantages and challenges in adopting a SLS method for fabrication of personalized medicines. Solvent-free nature, availability of FDA approved thermoplastic polymer/excipients (currently used in hot melt-extrusion process), minimal/no post-processing step, etc. are some of the advantages of the SLS printing process. Major challenges of the technology are requirement of at least one thermoplastic component in the formulation and thermal stability of drug and excipients. This review provides an overview of the SLS printing method, excipient requirements, process monitoring, quality defects, regulatory aspects, and potential pharmaceutical applications.

Use of a structure-borne sound-based in-process sensor system to identify Weld seam irregularities during electron beam welding.

In this publication, an in-process quality assurance method for electron beam welding based on a structure-borne sound emission test for the detection of weld irregularities arising in the process is presented. For this purpose, different sheet materials, i.e., AISI 304, AZ31 and AlMg3, were welded in a butt-joint and the resulting process noises were recorded by means of two acoustic emission sensors specifically designed for structure-borne sound. During the welding experiments, typical irregularities, e.g. incidence points, pore lines and cracks, were deliberately induced. Subsequently, the recorded acoustic signals were examined with regard to defect-specific abnormalities. Various methods in the time and frequency domain as well as pre-trained machine learning models were used to analyze the acoustic emission data. The results show that the investigated irregularities can be identified and distinguished from other process emissions, eventually enabling a robust means of identification for weld seam irregularities and, thus, opening pathways towards cost-effective in-process quality control.

Metabolic modulation to improve MSC expansion and therapeutic potential for articular cartilage repair.

BACKGROUND: Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy. METHODS: In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T2) to characterize the effects of AA on improved MSC expansion and chondrogenic potential. RESULTS: Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a > 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair. CONCLUSIONS: Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion.

Real-time in situ monitoring of CRM-197 and polysaccharide conjugation reaction by fluorescence spectroscopy.

Aims: Process analytical technology (PAT) is increasingly being adopted within the pharmaceutical industry to build quality into a process. Development of PAT that provides real-time in situ analysis of critical quality attributes are highly desirable for rapid, improved process development. Conjugation of CRM-197 with pneumococcal polysaccharides to produce a desired pneumococcal conjugate vaccine is a significantly intricate process that can tremendously benefit from real-time process monitoring. Methods: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time. Results & conclusion: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time.

In-Line Rheo-Optical Investigation of the Dispersion of Organoclay in a Polymer Matrix during Twin-Screw Compounding.

The dispersion mechanisms in a clay-based polymer nanocomposite (CPNC) during twin-screw extrusion are studied by in-situ rheo-optical techniques, which relate the CPNC morphology with its viscosity. This methodology avoids the problems associated with post extrusion structural rearrangement. The polydimethylsiloxane (PDMS) matrix, which can be processed at ambient and low temperatures, is used to bypass any issues associated with thermal degradation. Local heating in the first part of the extruder allows testing of the usefulness of low matrix viscosity to enhance polymer intercalation before applying larger stresses for clay dispersion. The comparison of clay particle sizes measured in line with models for the kinetics of particle dispersion indicates that larger screw speeds promote the break-up of clay particles, whereas smaller screw speeds favor the erosion of the clay tactoids. Thus, different levels of clay dispersion are generated, which do not simply relate to a progressively better PDMS intercalation and higher clay exfoliation as screw speed is increased. Reducing the PDMS viscosity in the first mixing zone of the screw facilitates dispersion at lower screw speeds, but a complex interplay between stresses and residence times at larger screw speeds is observed. More importantly, the results underline that the use of larger stresses is inefficient per se in dispersing clay if sufficient time is not given for PDMS to intercalate the clay galleries and thus facilitate tactoid disruption or erosion.

An oxygen-uptake-rate-based estimator of the specific growth rate in Escherichia coli BL21 strains cultivation processes.

The cell cultivation process in a bioreactor is a high-value manufacturing process that requires excessive monitoring and control compatibility. The specific cell growth rate is a crucial parameter that describes the online quality of the cultivation process. Most methods and algorithms developed for online estimations of the specific growth rate controls in batch and fed-batch microbial cultivation processes rely on biomass growth models. In this paper, we present a soft sensor - a specific growth rate estimator that does not require a particular bioprocess model. The approach for online estimation of the specific growth rate is based on an online measurement of the oxygen uptake rate. The feasibility of the estimator developed in this study was determined in two ways. First, we used numerical simulations on a virtual platform, where the cell culture processes were theoretically modeled. Next, we performed experimental validation based on laboratory-scale (7, 12, 15 L) bioreactor experiments with three different Escherichia coli BL21 cell strains.

At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization.

Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.

Evaluation of the Tempo® System: Improving the Microbiological Quality Monitoring of Human Milk.

Background: Bacteriological testing of donor human milk is mostly done both before and after pasteurization to control contamination in the end-product and meet the microbiological standards. Although the plate count method represents a reliable and sensitive technique and is considered the gold standard for bacteriological testing, it is recognized for being time-consuming and requiring qualified personnel. Recently, faster testing technologies, mostly geared toward the food industry, have been developed. Among these, the bioMérieux TEMPO® system uses the most probable number method to assess microbiological content in a semi-automated fashion. Objective: The performances of the TEMPO® system in enumerating bacterial quality indicators in human milk were assessed and compared to the reference plate count method. Methods: Naturally and artificially contaminated human milk samples were used to compare the analytical performances of the TEMPO® system to the plate count technique. More specifically, bacteria belonging to the genera Bacillus, Enterobacteriaceae, Staphylococcus aureus, and total aerobic flora were screened using both methods. Bacteria isolated on agar plates containing selective media were identified by supplemental testing. Bacterial testing results and method parameters were compared using linear regression analyses and Bland-Altman approaches. Results: Naturally contaminated milk samples (n = 55) tested for total aerobic flora showed < 1 log (CFU/ml) discrepancy between the two methods in the output results for 98% of the samples. Comparative linear regression analyses demonstrate good correlations between the two methods (R 2 > 0.9). At lower levels of bacterial contamination, the TEMPO® method precision (C.V. < 8%) and accuracy (> 83%) were comparable to plate counts. Conclusions: The analytical performances of the TEMPO® system for human milk bacteriological testing are equivalent to the reference plate count method. Results from the TEMPO® system are available within a 24-h turnaround time from sample inoculation without the need for further supplemental testing, suggesting that this semi-automated method could be implemented within milk bank operations as an in-process monitoring technology to optimize end-product quality and safety.

In-Depth Evaluation of Data Collected During a Continuous Pharmaceutical Manufacturing Process: A Multivariate Statistical Process Monitoring Approach.

The present work presents an in-depth evaluation of continuously collected data during a twin-screw granulation and drying process performed on a continuous manufacturing line. During operation, the continuous line logs 49 univariate process variables, hence generating a large amount of data. Three identical 5-h continuous manufacturing runs were performed. Multivariate data analysis tools, more specifically latent variable modeling tools such as principal component analysis, were used to extract information from the generated data sets unveiling process trends and drifts. Furthermore, a statistical process monitoring strategy is presented. The approach is based on the application of multivariate statistical process monitoring to model the variables that remain around a steady state.

Automated and enhanced clone screening using a fully automated microtiter plate-based system for suspension cell culture.

Recently, we established an automated microtiter plate (MTP)-based system for suspension cell culture for high-throughput (HT) applications in biopharmaceutical process development. In the present report, the new system was evaluated regarding its potential to improve clone screening by allowing high-throughput fed-batch cultivation at an early stage. For this purpose, a fully automated procedure was compared to a mainly batch mode-based manual standard process. The new system performed daily measurements of viable cell density and product concentration for a total of 96 clones in biological duplicates that were evaluated for final clone selection. This resulted in a more than fivefold increase in sample throughput and 4 weeks of time saving compared to the reference process. The top clone characterized by the highest cell specific productivity was identified only by the new process. In contrast, this clone was lost in the expansion phase of the reference procedure. Overall, the new system identified more high-productive clones, offering more alternatives and flexibility for process development. In-process monitoring of glucose and lactate levels representing crucial secondary selection criteria further enhanced top clone identification. Clone characterization at an early stage was further extended by linking the MTP-based cell culture system to additional HT-analytic systems for N-glycosylation analysis as well as gene expression analysis by reverse transcriptase-quantitative polymerase chain reaction. These powerful tools connected to the automated MTP-based cell culture system lead to considerably advanced quality and speed of clone screening, and increase the probability of selecting the most suitable clone. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2760, 2019.

Process monitoring and evaluation of a continuous pharmaceutical twin-screw granulation and drying process using multivariate data analysis.

The present study aims at acquiring an in-depth process knowledge about a twin-screw granulation and fluid bed drying process performed on the commercially available continuous line. Batch Statistical Process Monitoring (BSPM) principles are used to describe and monitor the variables with a relevant time-related trajectory. The continuous granulator operates in a truly continuous manner and variables logged by this unit do not present time-relevant features. On the other hand, the fluid bed dryer is divided in six identical cells, which are sequentially filled and discharged, ensuring a continuous flow of material. Multiple variables logged at the dryer and subsequent product control unit, present time-relevant features. A profound analysis of these variables logged during normal operation, as well as an in-depth description of the startup period of the different units, were achieved. The BSPM concepts allows to monitor the time relevant variables of this continuous manufacturing line, to detect and diagnose deviations from normal operation and assign possible causes for the disturbances.

Process monitoring and visualization solutions for hot-melt extrusion: a review.

OBJECTIVES: Hot-melt extrusion (HME) is applied as a continuous pharmaceutical manufacturing process for the production of a variety of dosage forms and formulations. To ensure the continuity of this process, the quality of the extrudates must be assessed continuously during manufacturing. The objective of this review is to provide an overview and evaluation of the available process analytical techniques which can be applied in hot-melt extrusion. KEY FINDINGS: Pharmaceutical extruders are equipped with traditional (univariate) process monitoring tools, observing barrel and die temperatures, throughput, screw speed, torque, drive amperage, melt pressure and melt temperature. The relevance of several spectroscopic process analytical techniques for monitoring and control of pharmaceutical HME has been explored recently. Nevertheless, many other sensors visualizing HME and measuring diverse critical product and process parameters with potential use in pharmaceutical extrusion are available, and were thoroughly studied in polymer extrusion. The implementation of process analytical tools in HME serves two purposes: (1) improving process understanding by monitoring and visualizing the material behaviour and (2) monitoring and analysing critical product and process parameters for process control, allowing to maintain a desired process state and guaranteeing the quality of the end product. SUMMARY: This review is the first to provide an evaluation of the process analytical tools applied for pharmaceutical HME monitoring and control, and discusses techniques that have been used in polymer extrusion having potential for monitoring and control of pharmaceutical HME.

A novel approach to monitor coating amount by short-wavelength near-infrared spectroscopy using a tracer with a long-chain hydrocarbyl group.

Investigation into the use of near-infrared (NIR) as a Process Analytical Technology has been conducted for in-process monitoring of coating amounts for oral pharmaceutical products. However, the low specificity of NIR spectra has made it time consuming and costly to establish quantitative calibration models for commercial production. Here we revealed that long-chain hydrocarbyl group compounds containing saturated hydrocarbon chains, such as cetyl and stearyl, exhibit specific and strong absorption in the short wavelength (SW)-NIR region (800-1,100 nm) with limited interference from peaks corresponding to other components. To simplify the quantitative model, we used cetanol as a model tracer of coating amount to enhance detection sensitivity and analytical precision. The coating amount on crystalline cellulose granules was determined only from the intensity of NIR absorption at a single wavelength, which was attributed to the tracer. The results showed close agreement with quantitative analyses from gas chromatography and measurement of weight gain. In conclusion, we determined coating amount with considerable accuracy from NIR absorption at a single wavelength in the SW-NIR region using the long-chain hydrocarbyl containing compound as a tracer, thereby eliminating the need for complicated statistics.