A Simple and Cost-Effective Technique to Monitor the Sublimation Flow During Primary Drying of Freeze-Drying Using Shelf Inlet/Outlet Temperature Difference or Chamber to Condenser Pressure Drop.

As lyophilization continues to be a critical step in the manufacturing of sensitive biopharmaceuticals, challenges often arise during the scale up to commercial scale or the transfer from one manufacturing site to another. While data from the small-scale development of the lyophilization cycle is abundant it is typically much more difficult to extract important information from commercial scale cycles, due to the lack of process analytical technologies available on the commercial line. There is often a reluctance to include wireless temperature or pressure probes during GMP operations due to the additional contamination risk, and retrofitting equipment such as the TDLAS can be prohibitively expensive. Further, as products become more advanced, the cost of consuming the product or even the availability of material may limit the opportunities to run commercial scale trials. This paper presents two novel methods to garner critical cycle information to allow for the evaluation of cycle performance without the need for expensive analytical equipment, costly revalidation and line downtime. Critically, this can be achieved using commonly available temperature and capacitance probes on existing commercial scale equipment. The first method is a calorimetric method, based on quantifying the differences in heat transfer liquid temperature between the shelf inlet and shelf outlet. This change in temperature results from the on-going sublimation, an endo-thermic reaction occurring during lyophilization. The second method uses the differential pressure between the chamber and condenser resulting from the vapor flow from vial to condenser during primary drying. As stated by the authors both methods align well and provide valuable cycle characterization data.

Recommended Best Practices in Freeze Dryer Equipment Performance Qualification: 2022.

Best practices for performing freeze dryer equipment qualification are recommended, focusing on identifying methods to quantify shelf thermal uniformity (also known as "shelf surface uniformity"), equipment capability, and performance metrics of the freeze dryer essential to the pharmaceutical Quality by Design paradigm. Specific guidelines for performing shelf temperature mapping, freeze dryer equipment limit testing (the capability curve), and condenser performance metrics have been provided. Concerning shelf temperature mapping and equipment capability measurements, the importance of paying attention to the test setup and the use of appropriate testing tools are stressed. In all the guidelines provided, much attention has been paid to identifying the balance between obtaining useful process knowledge, logistical challenges associated with testing in the production environment vs that at laboratory scale, and the frequency of the testing necessary to obtain such useful information. Furthermore, merits and demerits of thermal conditions maintained on the cooled surfaces of the freeze dryer condenser have been discussed identifying the specific influence of the condenser surface temperature on the process conditions using experimental data to support the guidelines. Finally, guidelines for systematic leak rate testing criteria for a freeze dryer are presented. These specific procedural recommendations are based on calculations, measurements, and experience to provide useful process and equipment knowledge.

Lyophilization of Molecular Biology Reactions: A Review.

Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.

Wireless sensor networks for pharmaceutical lyophilization: Quantification of local gas pressure and temperature in primary drying.

Wireless sensor networks have become prolific in a wide range of industrial processes and offer several key advantages over their wired counterparts in terms of positioning flexibility, modularity, interconnectivity, and data routing. We demonstrate their utility in pharmaceutical lyophilization by developing a series of wireless devices to measure spatial variations in gas pressure and temperature during primary drying. The influence of shelf temperature, chamber pressure, excipient concentration, and dryer configuration are explored for various representative cycles using a laboratory-scale pharmaceutical lyophilizer. Pressure and temperature variations across the shelf for these cases are shown to vary up to 1.2 Pa and 10 °C, respectively. Experimental measurements are supported by computational fluid dynamics simulations to reveal the mechanisms driving the vapor flow. The measurements and simulation data are then combined to estimate the shelf-wise sublimation rate in the inverse sense to within a deviation of 3% based on comparison with gravimetric data. We then apply the sublimation rate profile to obtain the vial heat transfer coefficient and product mass transfer resistance for a 5% w/v mannitol formulation. Finally, these parameters are applied to a one-dimensional quasi-steady heat transfer model to predict the evolution of the product temperature over the course of primary drying. Thermocouple measurements of product temperature are compared directly to the simulated data and demonstrate accuracy comparable to existing published one-dimensional models.

Physicochemical and Quality Properties of Dried Courgette Slices: Impact of Vacuum Impregnation and Drying Methods.

The aim of this study was to determine the effects that the type of impregnating solution and drying method (freeze drying (FD) and vacuum drying (VD) at 45 °C and convective drying (CD) at 50, 60, and 70 °C) had on the physicochemical and quality properties of courgettes. Courgette slices were vacuum-impregnated (6 kPa) in freshly squeezed onion, kale, and onion and kale (50:50) juices with 3% NaCl solution (N). The application of vacuum impregnation (VI) with impregnating solutions from freshly squeezed onions and kale had a beneficial effect on the bioactive values of courgette. The highest contents of quercetin (41.84 µg/g d.m.) and carotenoids (276.04 µg/g d.m.) were found in courgette impregnated with onion juice after freeze drying. The highest values of lutein and zeaxanthin (216.42 µg/g d.m.) were recorded for courgette impregnated with kale juice and convective dried. By analysing the kinetics of convective drying, the best matching of the logistic model was found. Increasing the drying process temperature from 50 to 70 °C reduced the drying time from 15% to 36%, depending on the type of impregnating solution used. Water activity < 0.6 was recorded for courgette dried by freezing, vacuum, and convection at 60 and 70 °C. Conclusions: The vacuum impregnation process and the impregnation solutions from freshly squeezed vegetables can be used to develop new snacks with high levels of bioactive compounds. The FD method is the most appropriate considering both the bioactive compounds content and the obtained colour and water activity.

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.

Micro Freeze-Dryer and Infrared-Based PAT: Novel Tools for Primary Drying Design Space Determination of Freeze-Drying Processes.

PURPOSE: Present (i) an infrared (IR)-based Process Analytical Technology (PAT) installed in a lab-scale freeze-dryer and (ii) a micro freeze-dryer (MicroFD®) as effective tools for freeze-drying design space calculation of the primary drying stage. METHODS: The case studies investigated are the freeze-drying of a crystalline (5% mannitol) and of an amorphous (5% sucrose) solution processed in 6R vials. The heat (Kv) and the mass (Rp) transfer coefficients were estimated: tests at 8, 13 and 26 Pa were carried out to assess the chamber pressure effect on Kv. The design space of the primary drying stage was calculated using these parameters and a well-established model-based approach. The results obtained using the proposed tools were compared to the ones in case Kv and Rp were estimated in a lab-scale unit through gravimetric tests and a thermocouple-based method, respectively. RESULTS: The IR-based method allows a non-gravimetric estimation of the Kv values while with the micro freeze-dryer gravimetric tests require a very small number of vials. In both cases, the obtained values of Kv and Rp, as well as the resulting design spaces, were all in very good agreement with those obtained in a lab-scale unit through the gravimetric tests (Kv) and the thermocouple-based method (Rp). CONCLUSIONS: The proposed tools can be effectively used for design space calculation in substitution of other well-spread methods. Their advantages are mainly the less laborious Kv estimation process and, as far as the MicroFD® is concerned, the possibility of saving time and formulation material when evaluating Rp.

Equipment Capability Measurement of Laboratory Freeze-Dryers: a Comparison of Two Methods.

The objective of this investigation was to evaluate two methods for measuring the maximum sublimation rate that a freeze-dryer will support-the minimum controllable pressure method and the choke point method. Both methods gave equivalent results, but the minimum controllable pressure method is preferred, since it is easier, faster, and less subjective. The ratio of chamber pressure to condenser pressure corresponding to the onset of choked flow was considerably higher in this investigation (up to about 20:1) than in previously published reports. This ratio was not affected by the location of the pressure gauge on the condenser; that is, on the foreline of the vacuum pump versus on the body of the condenser itself. The total water loss due to sublimation as measured by tunable diode laser absorption spectroscopy was consistently within 5% of gravimetrically determined weight loss, regardless of whether the measurement took place during choked versus non-choked process conditions.

Process optimization and transfer of freeze-drying in nested vial systems.

Scale-up and transfer of freeze-drying processes is a crucial challenge in biopharma industry. With the success of small batch processing lines utilizing rack vial holding systems, further detailed knowledge about freeze-drying cycles and their scale-up for vials in a rack is required. Therefore, product temperature (TP) profiles as well as Kv values of vials nested in a Polyetheretherketon (PEEK) rack were compared to those of vials placed in a commonly used stainless steel tray. Additionally, both setups were challenged with varying fill volume and partially versus fully loaded rack. Additionally, a process developed for rack was compared to a tray freeze-drying cycle. Freeze-drying in vials placed in the rack is markedly faster for center vials and more homogeneous compared to vials in bulk tray setting, as indicated by TP and Kv values. Due to the more homogeneous drying the rack is more flexible regarding variation of the fill volume. The key point for the transfer of a freeze-drying cycle from rack to tray is to consider the higher sublimation rates in the rack by adapting chamber pressure or shelf temperature for the tray. Furthermore, transfer from one rack per shelf in a laboratory freeze-dryer to pilot scale with four racks per shelf was successful. Thus, understanding of the process in rack and tray setup was enhanced to ensure efficient scale-up and transfer of freeze-drying processes.

Freeze-Drying of Proteins.

Freeze-drying has become one of the most important processes for the preservation of biological products. This chapter provides protocols for freeze-drying of proteins and discusses the importance of formulation, cycle development, and validation. Specific formulations for stabilization of proteins are presented as well as advice on common problems with freeze-drying of proteins.

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.

High pressure-assisted vacuum-freeze drying: A novel, efficient way to accelerate moisture migration in shrimp processing.

High pressure processing (HPP), as nonthermal processing technology, has the potential to increase the drying rate due to its improvement of heat and mass exchange in different processes. In this study, the moisture migration in shrimps during HPP-vacuum-freeze drying (HPP-VFD) processes has been monitored by using low-field nuclear magnetic resonance and magnetic resonance image (MRI) in comparison with hot air-drying and VFD. Based on the T2 relaxation spectra, three water fractions corresponding to bound water (hydrogen-bonded water), immobile water (water trapped by organization structure or cell member), and free water were observed. For group B, with increasing drying time (4 to 22 hr), the transverse relaxation times of T21 , T22 , and T23 were significantly decreased (76.79%, 57.78%, and 40.9%) (P < 0.05). The content of immobile water (A22 ) and free water (A23 ) decreased (81.55% and 89.07%), whereas the bound water (A21 ) increased (7.26%). In comparison with group B, the T21 , T22 , and T23 of group C showed greater decrease (83.12%, 87.12%, and 89.57% for group C) so that HPP pretreatment could shorten the relaxation time. MRI analysis further proved that HPP-VFD drying has improved drying efficiency, and moisture migration was from the exterior to the interior part with increasing drying time. SEM analysis demonstrated that no significant damage of muscle fibers with narrower gaps was observed for groups B and C. Overall, HPP, as a pretreatment technology, could accelerate the moisture migration and improve the drying efficiency of VFD process for shrimp. PRACTICAL APPLICATION: High pressure processing (HPP) is now well known as a nonthermal processing technology and becoming increasingly acknowledged. However, there is limited information about its application in shrimp-drying process and the moisture dynamic of shrimp subjected to high pressure processing-assisted vacuum-freeze drying. This study could provide valuable information regarding the moisture status and migration in HPP-VFD shrimp monitored by LF-NMR and MRI methods. The results showed that HPP processing at 550 MPa for 10 min can be used as an interesting method for drying pretreatment, increasing its drying rate and consequently reducing its process time, and it demonstrated that the methods used in this study had good correlation coefficient with physicochemical properties of shrimp, which may be real-time and nondestructive monitoring methods for shrimp-drying process.

Vacuum lyophilization preservation and rejuvenation performance of anammox bacteria.

The storage of anaerobic ammonia oxidizing bacteria (anammox) plays an important role in the application of anammox. Glycerol, sodium alginate and DMSO were used as the cryoprotectant, and vacuum lyophilization was used to prepare the anammox bacteria powder. Simultaneously, the control experiment was set up with the same protectant and preservation time. Bacteria powders were preserved using vacuum lyophilization and preserved at 4 °C for 60 days. During the 54 days of rejuvenation, the reactors that were inoculated with bacteria powder preserved by different methods showed significant difference. The results show that the anammox bacteria powder with 3 wt% DMSO as the cryoprotectant and without the substrate solution presented the best rejuvenation effect. The average specific anammox activity was 115.84 mg-N·(g VSS·d)-1 with an activity recovery rate of 89%, and its stoichiometric ratio (Rs and Rp) was 1.33 and 0.21, which were very close to the theoretical values. The vacuum lyophilization method for the long-term preservation of anammox bacteria was effective.

Cryogenic Sample Processing with Liquid Nitrogen for Effective and Efficient Monitoring of Pesticide Residues in Foods and Feeds.

With the monitoring of hundreds of pesticides in food and feed, the comminution step is equally crucial as any other to achieve valid results. However, sample processing is often underestimated in its importance and practical difficulty to produce consistent test portions for analysis. The scientific literature is rife with descriptions of microextraction methods, but ironically, sample comminution is often ignored or dismissed as being prosaic, despite it being the foundation upon which the viability of such techniques relies. Cryogenic sample processing using dry ice (-78 °C) is generally accepted in practice, but studies have not shown it to yield representative test portions of <1 g. Remarkably, liquid nitrogen has rarely been used as a cryogenic agent in pesticide residue analysis, presumably as a result of access, cost, and safety concerns. However, real-world implementation of blending unfrozen bulk food portions with liquid nitrogen (-196 °C) using common food processing devices has demonstrated this approach to be safe, simple, fast, and cost-effective and yield high-quality results for various commodities, including increased stability of labile or volatile analytes. For example, analysis of dithiocarbamates as carbon disulfide has shown a significant increase of thiram recoveries (up to 95%) using liquid nitrogen during sample comminution. This perspective is intended to allay concerns among working laboratories about the practical use of liquid nitrogen for improved sample processing in the routine monitoring of pesticide residues in foods and feeds, which also gives promise for feasible test sample size reduction in high-throughput miniaturized methods.

Freeze-Dryer Equipment Capability Limit: Comparison of Computational Modeling With Experiments at Laboratory Scale.

The equipment capability curve is one of the bounding elements of the freeze-drying design space, and understanding it is critical to process design, transfer, and scale-up. The second bounding element of the design space is the product temperature limit beyond which the product collapses. The high cost associated with freeze-drying any product renders it crucial to operate using the most efficient cycle within the limits of the equipment and the product. In this work, we present a computational model to generate the equipment capability curve for 2 laboratory scale freeze-dryers and compare the results to experimentally generated equipment capability curves. The average deviations of the modeling results from the experiments for the 2 lyophilizers modeled are -4.8% and -7.2%. In addition, we investigate the effect of various numerical and geometric parameters on the simulated equipment capability. Among the numerical parameters, the chamber wall thermal boundary conditions exert the largest influence with a maximum value of 12.3%. Among the geometric parameters, the inclusion of the isolation valve reduces the equipment capability by 23.7%. Larger isolation valves, required for controlled nucleation technology, choke the flow in the duct at lower sublimation rates, thereby lowering the equipment capability limit.

Lyophilization of Synthetic Gene Carriers.

Lyophilization, also known as freeze drying, is a widely used method for stabilization, improvement of long-term storage stability, and simplification of handling of drugs and/or carrier systems. Lyophilization is time-consuming and energy-consuming, and hence optimized processes are required to avoid time loss and higher costs without compromising product stability. Beginning from the last decade, nonviral, synthetic carriers for gene delivery have been of increasing interest. However, these systems suffer from poor physical stability in aqueous solution or suspension. Hence, to ensure long-term storage stability lyophilization of the gene carrier systems is favored. This chapter gives an overview of the basic steps and troubleshooting for successful lyophilization of synthetic gene carriers. Furthermore, the required excipients and their mechanism of action are summarized.

Insights into the development of grapefruit nutraceutical powder by spray drying: physical characterization, chemical composition and 3D intestinal permeability.

BACKGROUND: The development of functional and nutraceutical foods comes from a greater awareness of the relationship between food and health by consumers. In recent years, the idea of purifying and encapsulating bioactive compounds through techniques such as spray drying has been well received by the food industry. The development and characterization of a grapefruit (Citrus paradisi) nutraceutical powder obtained by spray drying is of great interest owing to the different bioactive compounds and the potential health effects. RESULTS: The grapefruit powder was characterized by a low water amount (1.5 g water per 100 g powder) and a high porosity (75%). The color parameters were L* = 80.0 ± 1.8, hab * = 61.7 ± 0.4 and Cab * = 11.4 ± 0.6. The IC50 values determined for the freeze-dried oxalic acid extract (FDOA) and the freeze-dried methanol-water extract (FDMW) were 0.48 and 0.72 mg mL-1 respectively, while the total phenolic content (TPC) ranged between 1274 and 1294 mg gallic acid equivalent (GAE) per 100 g dry basis (d.b.). Regarding total flavonoid content (TFC), FDOA presented the highest amount (6592 mg quercetin equivalent (QE) per 100 g d.b.). For both extracts, the cell viability in Caco-2 and HT29-MTX was above 90% at 100 µg mL-1 . The bioavailability of the bioactive compounds was analyzed through a 3D intestinal model. Delphenidin-3-glucoside and hesperitin-7-O-glucoside presented a permeation higher than 50%, followed by hesperidin which was close to 30%. CONCLUSION: This work allows to establish that the formulation of grapefruit powder has great potential as a nutraceutical food, with spray drying being a good alternative technique in the food industry. © 2019 Society of Chemical Industry.

Representative Scale-Down Lyophilization Cycle Development Using a Seven-Vial Freeze-Dryer (MicroFD®).

We have implemented the use of a small-scale, 7-vial Micro Freeze Dryer (MicroFD®; Millrock Technology, Inc.) that has the capability to accurately control heat transfer during lyophilization. We demonstrate the ability to fine-tune the MicroFD® vial heat transfer coefficient (Kv) to match the Kv of vials in a LyoStar III laboratory-scale unit. When the MicroFD® is run under conditions that match the Kv of the LyoStar III, the resulting lyophilization performance between scales results in equivalent product temperature profiles and critical quality attributes for the same drying process. The proposed workflow demonstrates how exploitation of Kv control in the MicroFD® enables cycle development of at-scale lyophilization processes using only 7 product vials. By changing the MicroFD®Kv, laboratory and, potentially, manufacturing cycles may be simulated using only 7 product vials for tremendous active pharmaceutical ingredient savings, as long as at-scale heat transfer coefficients are well characterized.

Using two-fluid nozzle for spray freeze drying to produce porous powder formulation of naked siRNA for inhalation.

Spray freeze drying is an attractive technology to produce powder formulation for inhalation. It can be used to generate large porous particles which tend to aerosolize efficiently and do not aggregate readily. It also avoids material to be exposed to elevated temperature. In this study, we reported the use of two-fluid nozzle to produce spray freeze dried powder of small interfering RNA (siRNA). The effect of atomization gas flow rate and liquid feed rate were inspected initially using herring sperm DNA (hsDNA) as nucleic acid model. The atomization gas flow rate was found to have a major impact on the aerosol properties. The higher the atomization gas flow rate, the smaller the particle size, the higher the fine particle fraction (FPF). In contrast, the liquid feed rate had very minor effect. Subsequently, spray freeze dried siRNA powder was produced at various atomization gas flow rates. The particles produced were highly porous as examined with the scanning electron microscopy, and the structural integrity of the siRNA was demonstrated with gel electrophoresis. The gene-silencing effect of the siRNA was also successfully preserved in vitro. The best performing siRNA formulation was prepared at the highest atomization gas flow rate investigated with a moderate FPF of 30%. However, this was significantly lower than that obtained with the corresponding hsDNA counterparts (FPF ∼57%). A direct comparison between the hsDNA and siRNA formulations revealed that the former exhibited a lower density, hence a smaller aerodynamic diameter despite similar geometric size.

Use of computational fluid dynamics for improving freeze-dryers design and process understanding. Part 1: Modelling the lyophilisation chamber.

This manuscript shows how computational models, mainly based on Computational Fluid Dynamics (CFD), can be used to simulate different parts of an industrial freeze-drying equipment and to properly design them; in particular, the freeze-dryer chamber and the duct connecting the chamber with the condenser, with the valves and vanes eventually present are analysed in this work. In Part 1, it will be shown how CFD can be employed to improve specific designs, to perform geometry optimization, to evaluate different design choices and how it is useful to evaluate the effect on product drying and batch variance. Such an approach allows an in-depth process understanding and assessment of the critical aspects of lyophilisation. This can be done by running either steady-state or transient simulations with imposed sublimation rates or with multi-scale approaches. This methodology will be demonstrated on freeze-drying equipment of different sizes, investigating the influence of the equipment geometry and shelf inter-distance. The effect of valve type (butterfly and mushroom) and shape on duct conductance and critical flow conditions will be instead investigated in Part 2.