Freeze-drying in protective bags: Characterization of heat and mass transfer.

During lyophilisation of highly potent Active Pharmaceutical Ingredients (APIs) potential contamination of the freeze-drier is an important safety issue. Since the stoppers are in semistoppered position during the lyophilization process, API may contaminate the chamber and cross-contamination may occur as well. In this study two protective bags, which enclose each tray and their influence on heat and mass transfer during freeze-drying were investigated. Sublimation tests were performed using either purified water or solutions containing trehalose as well as hydroxypropyl-ß-cyclodextrin (HPbCD) as bulking agents. During sublimation tests with purified water both bags clearly influenced heat and mass transfer compared to unpacked reference vials. The bag, which was originally designed to be used for steam sterilization, had a massive impact on drying characteristics. The bag membrane becomes the rate limiting factor, generating a separate compartment within the bag. In this compartment vapor pressure is much higher compared to the chamber pressure during primary drying, leading to altered drying conditions. However, drying was still possible. The other bag, which was specifically designed for lyophilization, also had an impact on drying behavior which could be assigned to the foil between shelf and bottom of the vials. This was detectable as differences in Kv values. Membrane resistance, however, becomes negligible when 10% (w/w) trehalose or HPbCD solutions were dried using the later bag as containment. The data reported in this work demonstrate the relevance and value of sublimation tests to understand the lyophilization process, especially when new components are implemented. The data should be considered, when freeze-drying shall be performed using such bags.

Development of cryopelletization and formulation measures to improve stability of Echis carinatus venum protein for use in diagnostic rotational thromboelastometry.

A cryopellet formulation of the diagnostic protein ecarin has been developed that is suitable for use to monitor blood coagulation via in vitro thromboelastometry. The coagulation activity of the ecarin was measured by thromelastometry using whole blood. In aqueous solution the ecarin is inactivated rapidly in a temperature dependent way that deviates from the Arrhenius equation. When kept at room temperature the ecarin activity falls to 90% of its initial value after just 39 min. The stability kinetics of ecarin during freezing and lyophilization are strongly dependent on the presence of a stabilizer comprising a collagen peptide-fraction. If this is removed by ultra-filtration the ecarin becomes highly unstable and cannot be fully stabilized even by addition of trehalose. The storage stability of ecarin in the finished pellets is excellent at temperatures below 50 °C, but deteriorates above the glass transition temperature of the pellet formulation. The cryopelletization of ecarin offer therefore a stable formulation for use in thromboelastometry that is superior to an aqueous solution and has much better handling than a regular lyophilizate in a diagnostic device.

The influence of excipients commonly used in freeze drying on whole blood coagulation dynamics assessed by rotational thromboelastometry.

BACKGROUND: Lyophilized reagents are used on a daily basis in coagulation diagnostics. They often contain a number of excipients in addition to the active compound. Some of these excipients may, however, influence coagulation dynamics. METHODS: Besides from plasmatic coagulation bulking agents may influence platelet properties. We therefore studied the influence of a variety of bulking agents (glycine, mannitol, sucrose and trehalose) as well as a surfactant (Tween® 80) on whole blood coagulation using thromboelastometry (ROTEM®) and platelet function analysis (ROTEM® platelet). RESULTS: Both disaccharides as well as Tween® 80 did not influence whole blood coagulation in the concentration range investigated. The addition of glycine and mannitol solutions to the ROTEM® measurement leads to an impaired clot formation as well as overall clot strength while clotting initiation remained barely influenced. Hypertonic glycine and mannitol solutions exhibit different clot formation impairment when correlated to their osmolar concentration and compared to equally osmolar NaCl-solutions. The effect of glycine was assigned to fibrin formation impairment identified with the FIBTEM assay. Platelet function analysis revealed that hypertonic glycine solutions do not alter platelet function but hypertonic mannitol and NaCl solutions do. CONCLUSIONS: While the influence observed for glycine may be due to fibrinogen precipitation, the mechanism of mannitol appears to be more complex as platelet function as well as fibrin-based clot formation are influenced. This study therefore demonstrates the necessity to check for coagulation impairment due to compounds contained in lyophilized reagents.

Production and characterization of rapidly dissolving cryopellets.

The procedure described in this study provides a platform technology for rapidly dissolving, single-dosed cryopellets. The different steps during cryopellet production were investigated, covering droplet generation, droplet freezing in liquid nitrogen (LN2) as well as cryopellet properties. With the setup developed, uniform droplets between 4 and 14 µL were produced. The freezing behavior was similar to approaches reported in the literature. A weight loss reported for droplets frozen in LN2 could not be confirmed. Mechanical stability as observed with texture analysis as well as dissolution time increased with increasing solid content. All cryopellets immediately disintegrated when in contact with the dissolution medium. The dissolution times of amorphous sucrose and trehalose-based cryopellets at different solid content levels were comparable. Crystalline mannitol cryopellets showed in general a higher dissolution time. The formation of δ-mannitol potentially makes the cryopellets suitable as an intermediate product for tableting.

Xenon washout during in-vitro extracorporeal circulation using different oxygenators.

BACKGROUND: Xenon anesthesia is known to have no adverse influence on myocardial contractility and cardiocirculatory function even in cardiac compromised patients. To make use of this advantages for cardiac surgery patients undergoing extracorporeal circulation (ECC) it must be known if oxygenators are diffusible for xenon in order to avoid losses of the very expensive noble gas. METHODS: Xenon saturated blood was recirculated in an in-vitro ECC. In 8 experiments four common oxygenators were investigated using continuous mass spectrometry at the exhaust port to measure xenon concentrations in the exspired gas. RESULTS: Xenon concentrations at the exhaust port of the oxygenator increased during filling the oxygenator with blood. Peak level was detected within one minute after onset of ECC. No xenon could be measured two minutes after onset of ECC. CONCLUSIONS: Using common oxygenators xenon is eliminated during ECC and lost into the atmosphere. To maintain anesthesia during ECC continuous xenon application would be necessary to compensate these losses. Due to its high price it would be too expensive to continue xenon anesthesia during ECC. Therefore it is not practicable to use the today's oxygenators and ECC equipment in xenon anesthesia.