Automated Tomographic Assessment of Structural Defects of Freeze-Dried Pharmaceuticals.

The topology and surface characteristics of lyophilisates significantly impact the stability and reconstitutability of freeze-dried pharmaceuticals. Consequently, visual quality control of the product is imperative. However, this procedure is not only time-consuming and labor-intensive but also expensive and prone to errors. In this paper, we present an approach for fully automated, non-destructive inspection of freeze-dried pharmaceuticals, leveraging robotics, computed tomography, and machine learning.

Cycle Development in a Mini-Freeze Dryer: Evaluation of Manometric Temperature Measurement in Small-Scale Equipment.

The objective of this research was to assess the applicability of manometric temperature measurement (MTM) and SMART™ for cycle development and monitoring of critical product and process parameters in a mini-freeze dryer using a small set of seven vials. Freeze drying cycles were developed using SMART™ which automatically defines and adapts process parameters based on input data and MTM feedback information. The freeze drying behavior and product characteristics of an amorphous model system were studied at varying wall temperature control settings of the cylindrical wall surrounding the shelf in the mini-freeze dryer. Calculated product temperature profiles were similar for all different wall temperature settings during the MTM-SMART™ runs and in good agreement with the temperatures measured by thermocouples. Product resistance profiles showed uniformity in all of the runs conducted in the mini-freeze dryer, but absolute values were slightly lower compared to values determined by MTM in a LyoStar™ pilot-scale freeze dryer. The resulting cakes exhibited comparable residual moisture content and optical appearance to the products obtained in the larger freeze dryer. An increase in intra-vial heterogeneity was found for the pore morphology in the cycle with deactivated wall temperature control in the mini-freeze dryer. SMART™ cycle design and product attributes were reproducible and a minimum load of seven 10R vials was identified for more accurate MTM values. MTM-SMART™ runs suggested, that in case of the wall temperature following the product temperature of the center vial, product temperatures differ only slightly from those in the LyoStar™ freeze dryer.

Stability of freeze-dried products subjected to microcomputed tomography radiation doses.

OBJECTIVES: Microcomputed tomography (µCT) is a powerful analytical tool for non-invasive structural analysis. The stability of drug substances and formulations subjected to X-ray radiation may be a concern in the industry. This study examines the effect of X-ray radiation on the stability of freeze-dried pharmaceuticals. The investigation is a proof of concept study for the safety of µCT X-ray radiation doses during the non-destructive investigation of freeze-dried products. METHODS: Different formulations of clotrimazole, insulin and l-lactate dehydrogenase were freeze-dried and the products exposed to a defined dose of radiation by µCT. Conservative freeze-drying conditions were used. Irradiated and normal samples were analysed for their stability directly after freeze-drying and after stability testing. KEY FINDINGS: The stability of model compounds was well maintained during freeze-drying. Some degradation of all compounds occurred during accelerated stability testing. The results showed no differences between the irradiated and normal state directly after freeze-drying and accelerated stability testing. CONCLUSIONS: No evidence of a detrimental effect of 100 Gy X-ray exposure on a model small molecule, peptide and protein compound was found while useful structural information could be obtained. Consequently, the technology may be useful as a non-destructive tool for product inspections if the formulation proves stable.

Evaluation of Packaging Materials in Freeze-Drying: Use of Polymer Caps and Nested Vials and Their Impact on Process and Product Attributes.

Current trends in the pharmaceutical industry led to a demand for more flexible manufacturing processes with smaller batch sizes. Prepackaged nested vials that can be processed as a unit were introduced into the market to fulfill this need. However, vial nests provide a different thermal environment for the vials compared to a hexagonal packaging array and could therefore influence product temperature profiles, primary drying times, and product quality attributes. Polymer caps with the possibility of vial closure inside the freeze-drying chamber were developed to remove the risks and need of a crimping process. A general concern with the use of such caps is the possibility of an increase in resistance to water vapor flow out of the vial. This case study investigated the effect of the LyoSeal® and PLASCAP® polymer caps and EZ-fill® nests on the freeze-drying process. Amorphous and partially crystalline model formulations were freeze-dried. Process data and product quality attributes were compared for regularly stoppered vials and vials with polymer caps as well as vials in a hexagonal packaging array and nested vials. The results indicated no increased resistance or impeded water vapor flow by the polymer caps. Differences in the macro- and microscopic appearances of products and a trend towards lower product temperatures were observed for the investigated nest type compared to a regular hexagonal packaging array. Consequently, the polymer caps could be used as an alternative to regular stoppers without affecting freeze-drying process data or product quality attributes, while the different thermal environment of nested vials should be considered.

Molded Vial Manufacturing and Its Impact on Heat Transfer during Freeze-Drying: Vial Geometry Considerations.

Recent advances in molded vial manufacturing enabled manufacturers to use a new manufacturing technique to achieve superior homogeneity of the vial wall thickness. This study evaluated the influence of the different manufacturing techniques of molded vials and glass compositions on vial heat transfer in freeze-drying. Additionally, the influence of using empty vials as thermal shielding on thermal characteristics of edge and center vials was investigated. The vial heat transfer coefficient Kv was determined gravimetrically for multiple vial systems. The results showed superior heat transfer characteristics of the novel manufacturing technique as well as differences in heat transfer for the different glass compositions. Empty vials on the outside of the array did not influence center vial Kv values compared to a full array. The direct contact area and vial bottom curvature and their correlation to heat transfer parameters were analyzed across multiple vial systems. A new approach based on light microscopy to describe the vial bottom curvature more accurately was described. The presented results for the contact area allowed for an approximation of the pressure-independent heat transfer parameter KC. The results for the vial bottom curvature showed a great correlation to the pressure-dependent heat transfer parameter KD. Overall, the results highlighted how a thorough geometrical characterization of vials with known heat transfer characteristics could be used to predict thermal characteristics of new vial systems as an alternative to a time-consuming gravimetric Kv determination. Primary drying times were simulated to show the influence of Kv on drying performance.

Design of Vacuum-Induced Freezing Protocols for High Fill Volume Formulations in Freeze-Drying: A Strategic Approach.

This case study proposes a development strategy for the SynchroFreeze vacuum-induced surface freezing technology for challenging high fill volume model systems. Critical steps during the development of a nucleation protocol are discussed as an example approach for implementing vacuum-induced surface freezing for high fill volume products. Slow pressure ramps and hold steps at adequate pressures have been found to be crucial for avoiding defects caused by either excessive outgassing or incomplete degassing. The evaporative mass loss during the SynchroFreeze procedure is characterized and thermal gradients during nucleation for several model systems with concentrations in the 50-400 mg/mL range are analyzed. The technology results in a measurable mass loss that may be relevant for low fill volume formulations. Thermal data show a pronounced temperature gradient throughout the entire product solution during nucleation by vacuum-induced surface freezing. The formulation composition, concentration, and shelf temperature have been shown to influence this temperature gradient. Reliable nucleation was achieved for sucrose formulations with concentrations up to 200 mg/mL at shelf temperatures minimally below the equilibrium freezing point.

Investigation of Two Different Pressure-Based Controlled Ice Nucleation Techniques in Freeze-Drying: The Integral Role of Shelf Temperature After Nucleation in Process Performance and Product Quality.

The purpose of this study was to investigate the impact of shelf temperature modifications during application of controlled ice nucleation techniques on process data and critical product quality attributes for a challenging, high-concentration and high-fill volume amorphous model system. Different freezing programs were applied and compared for the mechanistically different depressurization and vacuum-induced surface freezing techniques. Critical process data, such as product temperature and drying time, were analyzed. The final products were characterized with a focus on product morphology, residual moisture, reconstitution time and stability. The shelf temperature directly after primary nucleation showed a major influence on process performance and product quality attributes, with an isothermal hold step at an intermediate temperature leading to optimal results in terms of homogeneity and reduction of product temperatures and drying time for the model system used. The different controlled ice nucleation techniques led to significantly different results in terms of product morphology and process data, showing that the two mechanistically different controlled nucleation techniques are not interchangeable.

Correction to: Restoration of MARCKS enhances chemosensitivity in cancer.

In the original article, the title of the article is "Restoration of MARCK enhances chemosensitivity in cancer". The authors would like to change the article title to "Restoration of MARCKS enhances chemosensitivity in cancer" by adding a letter "S" to the word MARCK.

Restoration of MARCKS enhances chemosensitivity in cancer.

PURPOSE: Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. METHODS: CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). RESULTS: We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. CONCLUSIONS: Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

Influence of drug load on dissolution behavior of tablets containing a poorly water-soluble drug: estimation of the percolation threshold.

Drug load plays an important role in the development of solid dosage forms, since it can significantly influence both processability and final product properties. The percolation threshold of the active pharmaceutical ingredient (API) corresponds to a critical concentration, above which an abrupt change in drug product characteristics can occur. The objective of this study was to identify the percolation threshold of a poorly water-soluble drug with regard to the dissolution behavior from immediate release tablets. The influence of the API particle size on the percolation threshold was also studied. Formulations with increasing drug loads were manufactured via roll compaction using constant process parameters and subsequent tableting. Drug dissolution was investigated in biorelevant medium. The percolation threshold was estimated via a model dependent and a model independent method based on the dissolution data. The intragranular concentration of mefenamic acid had a significant effect on granules and tablet characteristics, such as particle size distribution, compactibility and tablet disintegration. Increasing the intragranular drug concentration of the tablets resulted in lower dissolution rates. A percolation threshold of approximately 20% v/v could be determined for both particle sizes of the API above which an abrupt decrease of the dissolution rate occurred. However, the increasing drug load had a more pronounced effect on dissolution rate of tablets containing the micronized API, which can be attributed to the high agglomeration tendency of micronized substances during manufacturing steps, such as roll compaction and tableting. Both methods that were applied for the estimation of percolation threshold provided comparable values.