Freeze-Induced Phase Transition and Local Pressure in a Phospholipid/Water System: Novel Insights Were Obtained from a Time/Temperature Resolved Synchrotron X-ray Diffraction Study.

Water-to-ice transformation results in a 10% increase in volume, which can have a significant impact on biopharmaceuticals during freeze-thaw cycles due to the mechanical stresses imparted by the growing ice crystals. Whether these stresses would contribute to the destabilization of biopharmaceuticals depends on both the magnitude of the stress and sensitivity of a particular system to pressure and sheer stresses. To address the gap of the "magnitude" question, a phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), is evaluated as a probe to detect and quantify the freeze-induced pressure. DPPC can form several phases under elevated pressure, and therefore, the detection of a high-pressure DPPC phase during freezing would be indicative of a freeze-induced pressure increase. In this study, the phase behavior of DPPC/water suspensions, which also contain the ice nucleation agent silver iodide, is monitored by synchrotron small/wide-angle X-ray scattering during the freeze-thaw transition. Cooling the suspensions leads to heterogeneous ice nucleation at approximately -7 °C, followed by a phase transition of DPPC between -11 and -40 °C. In this temperature range, the initial gel phase of DPPC, Lß', gradually converts to a second phase, tentatively identified as a high-pressure Gel III phase. The Lß'-to-Gel III phase transition continues during an isothermal hold at -40 °C; a second (homogeneous) ice nucleation event of water confined in the interlamellar space is detected by differential scanning calorimetry (DSC) at the same temperature. The extent of the phase transition depends on the DPPC concentration, with a lower DPPC concentration (and therefore a higher ice fraction), resulting in a higher degree of Lß'-to-Gel III conversion. By comparing the data from this study with the literature data on the pressure/temperature Lß'/Gel III phase boundary and the lamellar lattice constant of the Lß' phase, the freeze-induced pressure is estimated to be approximately 0.2-2.6 kbar. The study introduces DPPC as a probe to detect a pressure increase during freezing, therefore addressing the gap between a theoretical possibility of protein destabilization by freeze-induced pressure and the current lack of methods to detect freeze-induced pressure. In addition, the observation of a freeze-induced phase transition in a phospholipid can improve the mechanistic understanding of factors that could disrupt the structure of lipid-based biopharmaceuticals, such as liposomes and mRNA vaccines, during freezing and thawing.

Cryoconcentration and 3D Temperature Profiles During Freezing of mAb Solutions in Large-Scale PET Bottles and a Novel Scale-Down Device.

PURPOSE: Small-scale models that simulate large-scale freezing of bulk drug substance of biopharmaceuticals are highly needed to define freezing and formulation parameters based on process understanding. We evaluated a novel scale-down device (SDD), which is based on a specially designed insulation cover, with respect to changes in concentration after freezing, referred to as cryoconcentration, and 3D temperature profiles. Furthermore, the effect of the initial monoclonal antibody (mAb) concentration on cryoconcentration was addressed. METHODS: 2 L and 125 mL bottles were utilized. Temperatures were mapped using type T thermocouples. Frozen blocks were cut and mAb and histidine concentrations were analysed by HPLC. In addition, concentration- and temperature-dependent viscosities were measured. RESULTS: 3D freezing profiles in the SDD were comparable to large-scale bottles. The SDD accurately predicted cryoconcentration of both mAb and histidine of large-scale freezing. Concentric changes in concentration were evident as well as an unforeseen diluted core at the last point to freeze. At low initial mAb concentration cryoconcentration was substantial, while high initial mAb concentration suppressed cryoconcentration almost completely. CONCLUSION: The novel SDD gives detailed insights into large-scale freezing of mAb solutions using only a fraction of the simulated volume. It is a promising material- and cost-saving tool to understand large-scale freezing processes.

Interfacial Stress and Container Failure During Freezing of Bulk Protein Solutions Can Be Prevented by Local Heating.

Bottles and carboys are used for frozen storage and transport of biopharmaceutical formulations under a wide range of conditions. The quality of freezing and thawing in these systems has been questioned due to the formation of heterogeneous ice structures and deformation of containers. This work shows that during freezing of bulk protein solutions, the liquid at the air-liquid interface freezes first, forming an ice crust and enclosing the liquid phase. As the enclosed liquid freezes, internal pressure rises, pushing the liquid phase through the porous ice crust towards the air interface, leading to interfacial stress and protein aggregation. The aggregation of bovine serum albumin was more intense in the foam-like ice mound that was formed at the top, where bubbles were entrapped. This was characterized experimentally with the assistance of magnetic resonance imaging (MRI). An isothermal cover is proposed to prevent the early freezing of the liquid at the air interface, attenuating substantially interfacial stress to proteins and releasing hydrostatic pressure, preserving the shape and integrity of the containers.

Improving Heat Transfer at the Bottom of Vials for Consistent Freeze Drying with Unidirectional Structured Ice.

The quality of lyophilized products is dependent of the ice structure formed during the freezing step. Herein, we evaluate the importance of the air gap at the bottom of lyophilization vials for consistent nucleation, ice structure, and cake appearance. The bottom of lyophilization vials was modified by attaching a rectified aluminum disc with an adhesive material. Freezing was studied for normal and converted vials, with different volumes of solution, varying initial solution temperature (from 5°C to 20°C) and shelf temperature (from -20°C to -40°C). The impact of the air gap on the overall heat transfer was interpreted with the assistance of a computational fluid dynamics model. Converted vials caused nucleation at the bottom and decreased the nucleation time up to one order of magnitude. The formation of ice crystals unidirectionally structured from bottom to top lead to a honeycomb-structured cake after lyophilization of a solution with 4% mannitol. The primary drying time was reduced by approximately 35%. Converted vials that were frozen radially instead of bottom-up showed similar improvements compared with normal vials but very poor cake quality. Overall, the curvature of the bottom of glass vials presents a considerable threat to consistency by delaying nucleation and causing radial ice growth. Rectifying the vials bottom with an adhesive material revealed to be a relatively simple alternative to overcome this inconsistency.

Bipolar Membranes for Direct Borohydride Fuel Cells-A Review.

Direct liquid fuel cells (DLFCs) operate directly on liquid fuel instead of hydrogen, as in proton-exchange membrane fuel cells. DLFCs have the advantages of higher energy densities and fewer issues with the transportation and storage of their fuels compared with compressed hydrogen and are adapted to mobile applications. Among DLFCs, the direct borohydride-hydrogen peroxide fuel cell (DBPFC) is one of the most promising liquid fuel cell technologies. DBPFCs are fed sodium borohydride (NaBH4) as the fuel and hydrogen peroxide (H2O2) as the oxidant. Introducing H2O2 as the oxidant brings further advantages to DBPFC regarding higher theoretical cell voltage (3.01 V) than typical direct borohydride fuel cells operating on oxygen (1.64 V). The present review examines different membrane types for use in borohydride fuel cells, particularly emphasizing the importance of using bipolar membranes (BPMs). The combination of a cation-exchange membrane (CEM) and anion-exchange membrane (AEM) in the structure of BPMs makes them ideal for DBPFCs. BPMs maintain the required pH gradient between the alkaline NaBH4 anolyte and the acidic H2O2 catholyte, efficiently preventing the crossover of the involved species. This review highlights the vast potential application of BPMs and the need for ongoing research and development in DBPFCs. This will allow for fully realizing the significance of BPMs and their potential application, as there is still not enough published research in the field.

Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies.

Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to -25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1-2 weeks) and using standard characterization techniques (SEC-HPLC and DLS).

Computational fluid dynamic simulations of temperature, cryoconcentration, and stress time during large-scale freezing and thawing of monoclonal antibody solutions.

PURPOSE: Large-scale freezing and thawing experiments of monoclonal antibody (mAb) solutions are time and material consuming. Computational Fluid Dynamic (CFD) modeling of temperature, solute composition as well as the stress time, defined as the time between start of freezing and reaching Tg' at any point in the container, could be a promising approach to ease and speed up process development. METHODS: Temperature profiles at six positions were recorded during freezing and thawing of a 2L rectangular bottle and compared to CFD simulations via OpenFOAM. Furthermore, cryoconcentration upon freezing and concentration gradients upon thawing of a mAb solution were predicted and the stress time calculated. RESULTS: Temperature profiles during freezing were accurately matched by the CFD simulation. Thawing time was only 45 min to 60 min longer in the model. The macroscopic cryoconcentration of the mAb was also matched by the simulation; only a highly concentrated region in the top and a diluted core in the geometrical centre of the 2 L bottle were not well reflected in the simulation. The concentration gradient after thawing obtained by simulation as well agreed with the experimental result. In addition, CFD simulations allowed to extract the global temperature distribution, the formation of ice, and thus the distribution of stress in the freezing liquid. CONCLUSION: CFD simulations via OpenFOAM are a promising tool to describe large-scale freezing and thawing of mAb solutions and can help to generate a deeper understanding and to improve testing of the robustness of the processes.

Evaluation of Two Novel Scale-Down Devices for Testing Monoclonal Antibody Aggregation During Large-Scale Freezing.

There is a need for representative small volume devices that reflect monoclonal antibody (mAb) aggregation during freezing and thawing (FT) in large containers. We characterised two novel devices that aim to mimic the stress in rectangular 2 L bottles. The first scale-down device (SDD) consists of a 125 mL bottle surrounded by a 3D printed cover that manipulates heat exchange. The second device, a micro scale-down device (mSDD), adapts cooling and heating of 10 mL vials to extend stress time. MAb aggregation upon repeated FT was evaluated considering formation of higher molecular weight species, subvisible particles, and the increase in hydrodynamic radius, polydispersity index, and optical density at 350 nm. Three different mAb solutions were processed. Both an unshielded 125 mL bottle and the SDD can be used to predict aggregation during FT in 2 L bottles. In specific cases the unshielded 125 mL bottle underestimates whereas the SDD slightly overestimates soluble aggregate formation. The mSDD increases aggregation compared to 10 mL vials but is less representative than the SDD. Ultimately, both SDDs enable characterisation of protein sensitivity to large-scale FT with two orders of magnitude less volume and are superior to simply using smaller bottles.

Characterization of the Aeration and Hydrodynamics in Vertical-Wheel™ Bioreactors.

In this work, the oxygen transport and hydrodynamic flow of the PBS Vertical-Wheel MINI™ 0.1 bioreactor were characterized using experimental data and computational fluid dynamics simulations. Data acquired from spectroscopy-based oxygenation measurements was compared with data obtained from 3D simulations with a rigid-lid approximation and LES-WALE turbulence modeling, using the open-source software OpenFOAM-8. The mass transfer coefficients were determined for a range of stirring speeds between 10 and 100 rpm and for working volumes between 60 and 100 mL. Additionally, boundary condition, mesh refinement, and temperature variation studies were performed. Lastly, cell size, energy dissipation rate, and shear stress fields were calculated to determine optimal hydrodynamic conditions for culture. The experimental results demonstrate that the kL can be predicted using Sh=1.68Re0.551Sc13G1.18, with a mean absolute error of 2.08%. Using the simulations and a correction factor of 0.473, the expression can be correlated to provide equally valid results. To directly obtain them from simulations, a partial slip boundary condition can be tuned, ensuring better near-surface velocity profiles or, alternatively, by deeply refining the mesh. Temperature variation studies support the use of this correlation for temperatures up to 37 °C by using a Schmidt exponent of 1/3. Finally, the flow was characterized as transitional with diverse mixing mechanisms that ensure homogeneity and suspension quality, and the results obtained are in agreement with previous studies that employed RANS models. Overall, this work provides new data regarding oxygen mass transfer and hydrodynamics in the Vertical-Wheel bioreactor, as well as new insights for air-water mass transfer modeling in systems with low interface deformation, and a computational model that can be used for further studies.

Mannitol Crystallization at Sub-Zero Temperatures: Time/Temperature-Resolved Synchrotron X-ray Diffraction Study and the Phase Diagram.

Mannitol, a common pharmaceutical ingredient, exhibits complex polymorphism even in simple binary mannitol/water mixtures, with four crystalline forms observed. In this investigation, time/temperature-resolved synchrotron X-ray diffraction measurements are performed during freezing and thawing of mannitol/water mixtures. Mannitol crystallization depends strongly on the cooling rate and is initiated during cooling, if the cooling rate is lower than the critical cooling rate; otherwise, mannitol remains amorphous during freezing and crystallizes during subsequent heating above -30 °C. A temperature-composition phase diagram is constructed, reflecting eutectic and peritectic points and lower-temperature equilibria involving mannitol hemihydrate, hexagonal ice, and ß-mannitol. Comparison of the experimental data with the phase diagram reveals that the mannitol crystallization behavior does not follow the equilibrium but appears to obey the Ostwald crystallization rule. Novel insights on equilibrium and kinetics phase relationships in mannitol/water systems could lead to improved formulations and manufacturing processes for pharmaceuticals and biopharmaceuticals.

A New Perspective on Scale-Down Strategies for Freezing of Biopharmaceutics by Means of Computational Fluid Dynamics.

Common approaches to scale-down freeze-thaw systems are based on matching time-temperature profiles at corresponding points; however, little is known about the differences in anisotropy between the 2 scales. In this work, computational fluid dynamics modeling was used to investigate these differences. The modeling of the convective flow of the liquid phase within ice porous structure and volume expansion caused by freezing enabled accurate prediction of the local temperature and composition, for evaluation of potential stresses on protein stability, such as cryoconcentration and time in the nonideal environment. Overall, the small height of the scale-down containers enhances cryoconcentration. The time under stress was consistent in both scales, except when the walls of the container could deform. In general, the common approach of matching the time-temperature profile at the center of the containers was more effective as a worst-case scenario than a scale-down model. This work shows that instead of considering a single matching time-temperature location; one should aim for a more general perspective by measuring many locations. Container geometries and heat transfer rates should be designed to match stresses related to protein integrity for equivalent mass fractions between both scales, which can be achieved with the assistance of computational fluid dynamics models.

Stability of Protein Formulations at Subzero Temperatures by Isochoric Cooling.

Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP ß-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.

Controlled freeze-thawing test to determine the degree of deionization required for tartaric stabilization of wines by electrodialysis.

A controlled freeze-thawing test for wines is proposed to predict the deionization degree required for tartaric stabilization by electrodialysis. In this test, wine samples are frozen and then thawed in controlled conditions. The required deionization degree is estimated based on the difference of specific conductivity of the wines before and after the freeze-thawing cycle. The effect of freezing holding time and thawing time on the predicted deionization degree was investigated, using different types of red, rose and white table wines. For the recommended operating conditions, the freeze-thawing test gives reproducible results, which are between 5 and 9% higher than the corresponding values obtained by the mini-contact test at -4 °C during 4 h. The passive version of freeze-thawing test seems to be an expedite and reliable method that can yield in 24 h an estimate of the deionization degree of wines for tartaric stabilization by electrodialysis.

Measuring and modeling hemoglobin aggregation below the freezing temperature.

Freezing of protein solutions is required for many applications such as storage, transport, or lyophilization; however, freezing has inherent risks for protein integrity. It is difficult to study protein stability below the freezing temperature because phase separation constrains solute concentration in solution. In this work, we developed an isochoric method to study protein aggregation in solutions at -5, -10, -15, and -20 °C. Lowering the temperature below the freezing point in a fixed volume prevents the aqueous solution from freezing, as pressure rises until equilibrium (P,T) is reached. Aggregation rates of bovine hemoglobin (BHb) increased at lower temperature (-20 °C) and higher BHb concentration. However, the addition of sucrose substantially decreased the aggregation rate and prevented aggregation when the concentration reached 300 g/L. The unfolding thermodynamics of BHb was studied using fluorescence, and the fraction of unfolded protein as a function of temperature was determined. A mathematical model was applied to describe BHb aggregation below the freezing temperature. This model was able to predict the aggregation curves for various storage temperatures and initial concentrations of BHb. The aggregation mechanism was revealed to be mediated by an unfolded state, followed by a fast growth of aggregates that readily precipitate. The aggregation kinetics increased for lower temperature because of the higher fraction of unfolded BHb closer to the cold denaturation temperature. Overall, the results obtained herein suggest that the isochoric method could provide a relatively simple approach to obtain fundamental thermodynamic information about the protein and the aggregation mechanism, thus providing a new approach to developing accelerated formulation studies below the freezing temperature.

The importance of heat flow direction for reproducible and homogeneous freezing of bulk protein solutions.

Freezing is an important operation in biotherapeutics industry. However, water crystallization in solution, containing electrolytes, sugars and proteins, is difficult to control and usually leads to substantial spatial solute heterogeneity. Herein, we address the influence of the geometry of freezing direction (axial or radial) on the heterogeneity of the frozen matrix, in terms of local concentration of solutes and thermal history. Solutions of hemoglobin were frozen radially and axially using small-scale and pilot-scale freezing systems. Concentration of hemoglobin, sucrose and pH values were measured by ice-core sampling and temperature profiles were measured at several locations. The results showed that natural convection is the major source for the cryoconcentration heterogeneity of solutes over the geometry of the container. A significant improvement in this spatial heterogeneity was observed when the freezing geometry was nonconvective, i.e., the freezing front progression was unidirectional from bottom to top. Using this geometry, less than 10% variation in solutes concentration was obtained throughout the frozen solutions. This result was reproducible, even when the volume was increased by two orders of magnitude (from 30 mL to 3 L). The temperature profiles obtained for the nonconvective freezing geometry were predicted using a relatively simple computational fluid dynamics model. The reproducible solutes distribution, predictable temperature profiles, and scalability demonstrate that the bottom to top freezing geometry enables an extended control over the freezing process. This geometry has therefore shown the potential to contribute to a better understanding and control of the risks inherent to frozen storage.

Surface Characterization of Asymmetric Bi-Soft Segment Poly(ester urethane urea) Membranes for Blood-Oxygenation Medical Devices.

Asymmetric bi-soft segment poly(ester urethane urea) (PEUU) membranes containing polycaprolactone (PCL) as a second soft segment are synthesized with PCL-diol ranging from 0% to 15% (w/w). Bulk and surface characteristics of the PEUU membranes were investigated by scanning electron microscopy (SEM), static water contact angles, and surface streaming potentials and were correlated to hemocompatibility properties, namely, hemolysis and thrombosis degrees. SEM analysis reveals PEUU membranes with asymmetric cross-sections and top dense surfaces with distinct morphologies. The increase in PCL-diol content yields PEUU membranes with blood-contacting surfaces that are smoother, more hydrophilic, and with higher maximum zeta potentials. The results obtained in this work give no evidence of a correlation between hydrophilicity/zeta potentials and the hemolysis/thrombosis degree of blood-contacting surfaces of the PEUU membranes. In contrast, other hemocompatibility aspects reveal that the more hydrophilic membranes are associated with lower platelet deposition and inhibition of extreme states of platelet activation.