Porosity and mechanically optimized PLGA based in situ hardening systems.

Goal of the present study was to develop and to characterize in situ-hardening, porous PLGA-based systems for their future application as bone grafting materials. Therefore, we investigated the precipitation behavior of formulations containing PLGA and a water-miscible solvent, DMSO, PEG 400, and NMP. To increase porosity, a pore forming agent (NaCMC) was added and to enhance mechanical properties of the system, an inorganic filler (α-TCP) was incorporated. The behavior upon contact with water and the influence of the prior addition of aqueous media on the morphology of the corresponding hardened implants were investigated. We proved cell-compatibility by live/dead assays for the hardened porous polymer/ceramic-composite scaffolds. The IsHS formulations can therefore be used to manufacture hardened scaffolds ex vivo by using molds with the desired shape and size. Cells were further successfully incorporated into the IsHS by precultivating the cells on the α-TCP-powder prior to their admixing to the formulation. However, cell viability could not be maintained due to toxicity of the tested solvents. But, the results demonstrate that in vivo cells should well penetrate, adhere, and proliferate in the hardened scaffolds. Consequently, we consider the in situ hardening system being an excellent candidate as a filling material for non-weight-bearing orthopedic indications, as the resulting properties of the hardened implant fulfill indication-specific needs like mechanical stability, elasticity, and porosity.

Residual transglutaminase in collagen - effects, detection, quantification, and removal.

In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4 ×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.

Optimization of a pharmaceutical freeze-dried product and its process using an experimental design approach and innovative process analyzers.

The aim of the present study was to examine the possibilities/advantages of using recently introduced in-line spectroscopic process analyzers (Raman, NIR and plasma emission spectroscopy), within well-designed experiments, for the optimization of a pharmaceutical formulation and its freeze-drying process. The formulation under investigation was a mannitol (crystalline bulking agent)-sucrose (lyo- and cryoprotector) excipient system. The effects of two formulation variables (mannitol/sucrose ratio and amount of NaCl) and three process variables (freezing rate, annealing temperature and secondary drying temperature) upon several critical process and product responses (onset and duration of ice crystallization, onset and duration of mannitol crystallization, duration of primary drying, residual moisture content and amount of mannitol hemi-hydrate in end product) were examined using a design of experiments (DOE) methodology. A 2-level fractional factorial design (2(5-1)=16 experiments+3 center points=19 experiments) was employed. All experiments were monitored in-line using Raman, NIR and plasma emission spectroscopy, which supply continuous process and product information during freeze-drying. Off-line X-ray powder diffraction analysis and Karl-Fisher titration were performed to determine the morphology and residual moisture content of the end product, respectively. In first instance, the results showed that - besides the previous described findings in De Beer et al., Anal. Chem. 81 (2009) 7639-7649 - Raman and NIR spectroscopy are able to monitor the product behavior throughout the complete annealing step during freeze-drying. The DOE approach allowed predicting the optimum combination of process and formulation parameters leading to the desired responses. Applying a mannitol/sucrose ratio of 4, without adding NaCl and processing the formulation without an annealing step, using a freezing rate of 0.9°C/min and a secondary drying temperature of 40°C resulted in efficient freeze-drying supplying end products with a residual moisture content below 2% and a mannitol hemi-hydrate content below 20%. Finally, using Monte Carlo simulations it became possible to determine how varying the factor settings around their optimum still leads to fulfilled response criteria, herewith having an idea about the probability to exceed the acceptable response limits. This multi-dimensional combination and interaction of input variables (factor ranges) leading to acceptable response criteria with an acceptable probability reflects the process design space.

Importance of using complementary process analyzers for the process monitoring, analysis, and understanding of freeze drying.

The aim of the present paper is to demonstrate the importance of using complementary process analyzers (PAT tools) for the process monitoring, analysis, and understanding of freeze drying. A mannitol solution was used as a model system. Raman spectroscopic, near-infrared (NIR) spectroscopic, plasma emission spectroscopic, and wireless temperature measurements (TEMPRIS) were simultaneously performed in-line and real-time during each freeze-drying experiment. The combination of these four process analyzers to monitor a freeze-drying process is unique. The Raman and NIR data were analyzed using principal component analysis (PCA) and multivariate curve resolution (MCR), while the plasma emission spectroscopic and wireless temperature measurement data were analyzed using univariate data analysis. It was shown that the considered process analyzers do not only complement but also mutually confirm each other with respect to process step end points, physical phenomena occurring during freeze drying (process understanding), and product characterization (solid state). Furthermore and most important, the combined use of the process analyzers helped to identify flaws in previous studies in which these process analyzers were studied individually. Process analyzers might wrongly indicate that some process steps are fulfilled. Finally, combining the studied process analyzers also showed that more information per process analyzer can be obtained than previously described. A combination of Raman and plasma emission spectroscopy seems favorable for the monitoring of nearly all critical freeze-drying process aspects.