Challenges of nanoparticle albumin bound (nab™) technology: Comparative study of Abraxane® with a newly developed albumin-stabilized itraconazole nanosuspension.

Nanoparticle albumin bound™ (nab™) technology is an established delivery platform for development of albumin stabilized nanoparticles as drug delivery systems for poorly water-soluble drugs. By using albumin for particle stabilization, nab™ technology does not require solubilizers or emulsifiers for the formulation of poorly water-soluble drugs for intravenous use. Despite the great potential, however, to date only two products based on nab™ technology have been approved by the Food and Drug Administration: Abraxane® (nab™ paclitaxel) and Fyarro® (nab™ rapamycin). In this study, the commercially available product Abraxane® was characterized in comparison to an albumin stabilized nanosuspension for the poorly water-soluble drug itraconazole. The aim of this study was to identify critical product parameters of the nanosuspensions depending on the manufacturing process in order to assess the transferability of nab™ technology to other drugs. The colloidal properties, stabilizing protein composition and particle disintegration behavior were analyzed. In addition, studies were carried out on the impact of the key process step, the high-pressure homogenization, using a design of experiments (DoE) approach. A nanosuspension comprising spherical, stable drug nanoparticles stabilized by a large fraction of dissolved albumin around the nanoparticles were identified. During the manufacturing process, the drug core was coated with a layer of albumin, which was cross-linked to a certain level. The Abraxane® and itraconazole suspensions differed in the analyzed protein fraction, with stronger cross-linking at the particle surface for Abraxane®. Both active pharmaceutical ingredients were present in the amorphous state as nanoparticles. In vitro disintegration studies performed to mimic a strong dilution during intravenous application showed the disintegration of the nanoparticles. All in all, the analysis underlined the transferability of the nab™ technology to selected other poorly water-soluble drugs with the great advantage of eliminating solubilizers and emulsifiers for intravenous applications.

Best Practices and Guidelines (2022) for Scale-up and Technology Transfer in Freeze Drying Based on Case Studies. Part 2: Past Practices, Current Best Practices, and Recommendations.

Scale-up and transfer of lyophilization processes remain very challenging tasks considering the technical challenges and the high cost of the process itself. The challenges in scale-up and transfer were discussed in the first part of this paper and include vial breakage during freezing at commercial scale, cake resistance differences between scales, impact of differences in refrigeration capacities, and geometry on the performance of dryers. The second part of this work discusses successful and unsuccessful practices in scale-up and transfer based on the experience of the authors. Regulatory aspects of scale-up and transfer of lyophilization processes were also outlined including a topic on the equivalency of dryers. Based on an analysis of challenges and a summary of best practices, recommendations on scale-up and transfer of lyophilization processes are given including projections on future directions in this area of the freeze drying field. Recommendations on the choice of residual vacuum in the vials were also provided for a wide range of vial capacities.

Best Practices and Guidelines (2022) for Scale-Up and Tech Transfer in Freeze-Drying Based on Case Studies. Part 1: Challenges during Scale Up and Transfer.

The freeze-drying process scale-up and transfer remain a complicated and non-uniform practice. We summarized inefficient and good practices in these papers and provided some practical advice. It was demonstrated that using the same process set points/times in laboratory and commercial scale dryers may lead to loss of product quality (collapse or vial breakage). The emerging modeling approach demonstrated practical advantages. However, the upfront generation of some input parameters (vial heat transfer coefficient, minimum controllable pressure, and maximum sublimation rate) is essential for model utilization. While the primary drying step can be transferred with a high degree of confidence (e.g., using modeling), and secondary drying is usually fairly straightforward, predicting potential changes in product behavior during freezing remains challenging.

Lyophilizer Leak Rate Testing - An Industry Survey and Best Practice Recommendation.

The vacuum integrity of freeze dryers is critical for attaining adequate process control and maintaining confidence in sterility assurance which is key for the manufacture of sterile pharmaceutical products. Although discussions on the topic have been published, there is no industry standard established that is based on empirical data or that has a justifiable scientific rationale. This article provides a review of the scientific literature in the public domain and most importantly, a perspective from 14 Pharmaceutical companies on the leak rate specifications commonly used in industry. Using this information we recommend a best practice for the lyophilizer leak rate test which includes detailing necessary preparation activities following Steam-In-Place (SIP) sterilization, defining a period of stabilization to eliminate pressure and temperature fluctuations and details of the test conditions and the test period. We conclude that for routine manufacturing practice the operational leak rate should not exceed 20 µbar L/s and we provide additional guidance for large volume and older lyophilisation equipment.

Comparison of Submicron Particle Counting Methods with a Heat Stressed Monoclonal Antibody: Effect of Electrolytes and Implications on Sample Preparation.

Within this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody (mAb) solutions. These were compared with microfluidic resistive pulse sensing (MRPS), resonant mass measurement (RMM), and nanoparticle tracking analysis (NTA). For TRPS and MRPS measurements, an adjustment of ionic strength was required to achieve suitable measurement conditions. The addition of electrolytes is potentially critical for protein formulations and therefore the effect of salt concentration and pH on submicron particle levels was further investigated. Heat stress caused a sharp increase in particle levels between 250-900 nm, observable by all four techniques. Due to reduced colloidal stability, indicated by increased attractive forces and reduced aggregation onset temperatures in the presence of sodium chloride, protein aggregation was observed in heat stressed mAb only after the addition of sodium chloride. Achieving adequate ionic strength by replacing sodium chloride with other electrolytes similarly resulted in reduced colloidal stability and protein aggregation. It is recommended that protein samples prone for aggregation in the presence of high ionic strength should not be analyzed by RPS measurements after the addition of electrolytes. However, protein samples containing already required ionic strength can be analyzed by any of the four techniques.

Application of Tunable Resistive Pulse Sensing for the Quantification of Submicron Particles in Pharmaceutical Monoclonal Antibody Preparations.

Tunable resistive pulse sensing (TRPS, qNano Gold, IZON Ltd.) was investigated as a method to quantify submicron particles (SMPs) between 0.1 and 1 µm in solutions of biopharmaceuticals. To reduce sample dilution, a spiking-in approach was used to add the appropriate amount of electrolytes required for the measurement. For correct particle quantification, an electrolyte concentration of at least 50 mM sodium chloride was needed. Intra- and inter-nanopore variability were below 5% for size and below 10% for concentration measurements when analyzing polystyrene standard beads. Submicron particle counts in a stir stressed IgG1 monoclonal antibody formulation resulted in a non-symmetrical, almost bell-shaped size distribution with a maximum at 250 nm when using a NP300 nanopore (IZON Ltd.). It was shown that particle counts are heavily underestimated below 250 nm, and therefore it is recommended to quantify particle counts by TRPS in samples with heterogeneous particle size distributions (e.g., biopharmaceuticals) only starting from the maximum of the histogram towards the upper limit of detection.

Application of process analytical technology for monitoring freeze-drying of an amorphous protein formulation: use of complementary tools for real-time product temperature measurements and endpoint detection.

Process analytical technology (PAT) and quality by design have gained importance in all areas of pharmaceutical development and manufacturing. One important method for monitoring of critical product attributes and process optimization in laboratory scale freeze-drying is manometric temperature measurement (MTM). A drawback of this innovative technology is that problems are encountered when processing high-concentrated amorphous materials, particularly protein formulations. In this study, a model solution of bovine serum albumin and sucrose was lyophilized at both conservative and aggressive primary drying conditions. Different temperature sensors were employed to monitor product temperatures. The residual moisture content at primary drying endpoints as indicated by temperature sensors and batch PAT methods was quantified from extracted sample vials. The data from temperature probes were then used to recalculate critical product parameters, and the results were compared with MTM data. The drying endpoints indicated by the temperature sensors were not suitable for endpoint indication, in contrast to the batch methods endpoints. The accuracy of MTM Pice data was found to be influenced by water reabsorption. Recalculation of Rp and Pice values based on data from temperature sensors and weighed vials was possible. Overall, extensive information about critical product parameters could be obtained using data from complementary PAT tools.

Robustness testing in pharmaceutical freeze-drying: inter-relation of process conditions and product quality attributes studied for a vaccine formulation.

CONTEXT: The recent US Food and Drug Administration (FDA) legislation has introduced the evaluation of the Design Space of critical process parameters in manufacturing processes. In freeze-drying, a "formulation" is expected to be robust when minor deviations of the product temperature do not negatively affect the final product quality attributes. OBJECTIVE: To evaluate "formulation" robustness by investigating the effect of elevated product temperature on product quality using a bacterial vaccine solution. MATERIALS AND METHODS: The vaccine solution was characterized by freeze-dry microscopy to determine the critical formulation temperature. A conservative cycle was developed using the SMART™ mode of a Lyostar II freeze dryer. Product temperature was elevated to imitate intermediate and aggressive cycle conditions. The final product was analyzed using X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), Karl Fischer, and modulated differential scanning calorimetry (MDSC), and the life cell count (LCC) during accelerated stability testing. RESULTS: The cakes processed at intermediate and aggressive conditions displayed larger pores with microcollapse of walls and stronger loss in LCC than the conservatively processed product, especially during stability testing. For all process conditions, a loss of the majority of cells was observed during storage. CONCLUSION: For freeze-drying of life bacterial vaccine solutions, the product temperature profile during primary drying appeared to be inter-related to product quality attributes.

Optimization of the secondary drying step in freeze drying using TDLAS technology.

The secondary drying phase in freeze drying is mostly developed on a trial-and-error basis due to the lack of appropriate noninvasive process analyzers. This study describes for the first time the application of Tunable Diode Laser Absorption Spectroscopy, a spectroscopic and noninvasive sensor for monitoring secondary drying in laboratory-scale freeze drying with the overall purpose of targeting intermediate moisture contents in the product. Bovine serum albumin/sucrose mixtures were used as a model system to imitate high concentrated antibody formulations. First, the rate of water desorption during secondary drying at constant product temperatures (-22 °C, -10 °C, and 0 °C) was investigated for three different shelf temperatures. Residual moisture contents of sampled vials were determined by Karl Fischer titration. An equilibration step was implemented to ensure homogeneous distribution of moisture (within 1%) in all vials. The residual moisture revealed a linear relationship to the water desorption rate for different temperatures, allowing the evaluation of an anchor point from noninvasive flow rate measurements without removal of samples from the freeze dryer. The accuracy of mass flow integration from this anchor point was found to be about 0.5%. In a second step, the concept was successfully tested in a confirmation experiment. Here, good agreement was found for the initial moisture content (anchor point) and the subsequent monitoring and targeting of intermediate moisture contents. The present approach for monitoring secondary drying indicated great potential to find wider application in sterile operations on production scale in pharmaceutical freeze drying.

Non-invasive product temperature determination during primary drying using tunable diode laser absorption spectroscopy.

The goal of this work was to demonstrate the application of Tunable Diode Laser Absorption Spectroscopy (TDLAS) as a non-invasive method to determine the average product temperature of the batch during primary drying. The TDLAS sensor continuously measures the water vapor concentration and the vapor flow velocity in the spool connecting the freeze-dryer chamber and condenser. Vapor concentration and velocity data were then used to determine the average sublimation rate (g/s) which was subsequently integrated to evaluate the amount of water removed from the product. Position dependent vial heat transfer coefficients (K(v)) were evaluated using the TDLAS sensor data for 20 mL vials during sublimation tests with pure water. TDLAS K(v) data showed good agreement to K(v) data obtained by the traditional gravimetric procedure. K(v) for edge vials was found to be about 20-30% higher than that of center vials. A weighted K(v) was then used to predict a representative average product temperature from TDLAS data in partial and full load freeze drying runs with 5%, 7.5%, or 10% (w/w) sucrose, mannitol, and glycine solutions. TDLAS product temperatures for all freeze-drying runs were within 1-2 degrees C of "center vial" steady state thermocouple data.

Evaluation of a new wireless Temperature Remote Interrogation System (TEMPRIS) to measure product temperature during freeze drying.

The purpose of this research was to evaluate a new wireless and battery-free sensor technology for invasive product temperature measurement during freeze-drying. Product temperature is the most critical process parameter in a freeze-drying process, in particular during primary drying. The product temperature over time profile and a precise detection of the endpoint of ice sublimation is crucial for comparison of freeze-drying cycles. Traditionally, thermocouples are used in laboratory scale units whereas resistance thermal detectors are applied in production scale freeze-dryers to evaluate temperature profiles. However, both techniques show demerits with regard to temperature comparability and biased measurements relative to vials without sensors. A new generation of wireless temperature sensors (Temperature Remote Interrogation System, TEMPRIS) were used in this study to investigate for the first time their value when applied to freeze-drying processes. Measurement accuracy, capability of accurate endpoint detection and effect of positioning were delineated by using product runs with sucrose, mannitol and trehalose. Data were compared to measurements with 36-gauge thermocouples as well as to non-invasive temperature measurement from Manometric Temperature Measurements. The results show that the TEMPRIS temperature profiles were in excellent agreement to thermocouple data when sensors were placed center bottom in a vial. In addition, TEMPRIS sensors revealed more reliable temperature profiles and endpoint indications relative to thermocouple data when vials in edge position were monitored. The results of this study suggest that TEMPRIS may become a valuable tool for cycle development, scale-up and routine manufacturing in the future.