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.

Evaluation of Predictors of Protein Relative Stability Obtained by Solid-State Hydrogen/Deuterium Exchange Monitored by FTIR.

PURPOSE: Hydrogen/deuterium (H/D) exchange over a range of temperatures suggests a protein structural/mobility transition in the solid state below the system glass transition temperature (Tg). The purpose of this study was to determine whether solid-state protein stability correlates with the difference between storage temperature and apparent Td where an abrupt change in mobility occurs, or alternatively, the extent of H/D exchange at a single temperature correlates directly to protein stability in lyophilized solids. METHODS: Solid-state H/D exchange was monitored by FTIR spectroscopy to study the extent of exchange and the apparent transition temperature in both pure recombinant human serum albumin (rHSA) and rHSA formulated with sucrose or trehalose. H/D exchange of freeze-dried formulations at 11% RH and temperatures from 30 to 80°C was monitored. Protein stability against aggregation at 40°C/11% RH for 6 months was assessed by size exclusion chromatography (SEC). RESULTS: Both sucrose and trehalose showed equivalent protection of protein secondary structure by FTIR. The rHSA:sucrose formulation showed superior long-term stability at 40°C by SEC over the trehalose formulation, but the apparent Td determined from H/D exchange was much higher in the trehalose formulation. Instead, the extent of H/D exchange (X∞) was lower in the sucrose formulation at the temperature of the stability studies (40°C) than found for the trehalose formulation, which was consistent with better stability in the sucrose formulation. CONCLUSIONS: While apparent Td did not correlate with protein stability for rHSA, the extent of H/D exchange, X∞, did.

Stability of Freeze-Dried Protein Formulations: Contributions of Ice Nucleation Temperature and Residence Time in the Freeze-Concentrate.

Controlling ice nucleation, at a fixed higher temperature, results in larger ice crystals, which can reduce the ice/freeze-concentrate interface area where proteins can adsorb and partially unfold. Moreover, limited work has been done to address any effects on short-term stability due to a slow ramp or long isothermal hold after the ice nucleation step. The objective was to evaluate the effect of the ice nucleation temperature and residence time in the freeze-concentrate on in-process or storage stability of representative proteins, human IgG, and recombinant human serum albumin. The results suggest a higher ice nucleation temperature can minimize aggregation of protein pharmaceuticals, which are labile at ice/aqueous interface. Apart from the ice nucleation step, the present study identified the residence time in the freeze-concentrate as the critical factor that influences protein stability post ice nucleation. At a temperature where enough mobility exists (i.e., above Tg' of the formulation), the long residence time in the freeze-concentrate can result in significant protein aggregation during the process. In addition to stability, the findings revealed that not only the ice nucleation temperature but also the thermal history of the formulation post ice nucleation defines the surface area of ice and the porous structure of the freeze-dried cake.

Applications of the Tunable Diode Laser Absorption Spectroscopy: In-Process Estimation of Primary Drying Heterogeneity and Product Temperature During Lyophilization.

The aim of this research was to evaluate the impact of variability in ice sublimation rate (dm/dt) measurement and vial heat transfer coefficient (Kv) on product temperature prediction during the primary drying phase of lyophilization. The mathematical model used for primary drying uses dm/dt and Kv as inputs to predict product temperature. A second-generation tunable diode laser absorption spectroscopy (TDLAS)-based sensor was used to measure dm/dt. In addition, a new approach to calculate drying heterogeneity in a batch during primary drying is described. The TDLAS dm/dt measurements were found to be within 5%-10% of gravimetric measurement for laboratory- and pilot-scale lyophilizers. Intersupplier variability in Kv was high for the same "type" of vials, which can lead to erroneous product temperature prediction if "one value" of vial heat transfer coefficient is used for "all vial types" from different suppliers. Studies conducted in both a laboratory- and a pilot-scale lyophilizer showed TDLAS product temperature to be within ±1°C of average thermocouple temperature during primary drying. Using TDLAS data and calculations to estimate drying heterogeneity (number of vials undergoing primary drying), good agreement was obtained between theoretical and experimental results, demonstrating usefulness of the new approach.

Freezing of Aqueous Solutions and Chemical Stability of Amorphous Pharmaceuticals: Water Clusters Hypothesis.

Molecular mobility has been traditionally invoked to explain physical and chemical stability of diverse pharmaceutical systems. Although the molecular mobility concept has been credited with creating a scientific basis for stabilization of amorphous pharmaceuticals and biopharmaceuticals, it has become increasingly clear that this approach represents only a partial description of the underlying fundamental principles. An additional mechanism is proposed herein to address 2 key questions: (1) the existence of unfrozen water (i.e., partial or complete freezing inhibition) in aqueous solutions at subzero temperatures and (2) the role of water in the chemical stability of amorphous pharmaceuticals. These apparently distant phenomena are linked via the concept of water clusters. In particular, freezing inhibition is associated with the confinement of water clusters in a solidified matrix of an amorphous solute, with nanoscaled water clusters being observed in aqueous glasses using wide-angle neutron scattering. The chemical instability is suggested to be directly related to the catalysis of proton transfer by water clusters, considering that proton transfer is the key elementary reaction in many chemical processes, including such common reactions as hydrolysis and deamidation.

Impact of Natural Variations in Freeze-Drying Parameters on Product Temperature History: Application of Quasi Steady-State Heat and Mass Transfer and Simple Statistics.

Inter- and intra-batch variability in heat and mass transfer during the drying phase of lyophilization is well recognized. Heat transfer variability between individual vials in the same batch arise from both different positions in the vial array and from variations in the bottom contour of the vials, both effects contributing roughly equally to variations in the effective heat transfer coefficient of the vials, Kv. Both effects can be measured in the laboratory, and variations in average Kv values as a function of vial position in the array for lab and production can be calculated by use of the simple steady-state heat and mass transfer theory. Typically, in the laboratory dryer, vials on the edge of the array, "edge vials," run 2-4°C warmer than "center vials," but differences between laboratory and manufacturing temperatures are modest. The variability in mass transfer can be assigned to major variations in ice nucleation temperature (both intra-batch and inter-batch), including major differences between laboratory and manufacturing. The net effect of all random variations, for each class of vial, can be evaluated by a simple statistical model-propagation of error, which then allows prediction of the distribution in product temperatures and drying times, and therefore prediction of percent of vials dry and percent of vials collapsed and proximity to the edge of failure for a given process. Good agreement between theoretical and experimentally determined maximum temperatures in primary drying and percent collapsed product demonstrates the calculations have useful accuracy.

Effect of Controlled Ice Nucleation on Stability of Lactate Dehydrogenase During Freeze-Drying.

Several controlled ice nucleation techniques have been developed to increase the efficiency of the freeze-drying process as well as to improve the quality of pharmaceutical products. Owing to the reduction in ice surface area, these techniques have the potential to reduce the degradation of proteins labile during freezing. The objective of this study was to evaluate the effect of ice nucleation temperature on the in-process stability of lactate dehydrogenase (LDH). LDH in potassium phosphate buffer was nucleated at -4°C, -8°C, and -12°C using ControLyo™ or allowed to nucleate spontaneously. Both the enzymatic activity and tetramer recovery after freeze-thawing linearly correlated with product ice nucleation temperature (n = 24). Controlled nucleation also significantly improved batch homogeneity as reflected by reduced inter-vial variation in activity and tetramer recovery. With the correlation established in the laboratory, the degradation of protein in manufacturing arising from ice nucleation temperature differences can be quantitatively predicted. The results show that controlled nucleation reduced the degradation of LDH during the freezing process, but this does not necessarily translate to vastly superior stability during the entire freeze-drying process. The capability of improving batch homogeneity provides potential advantages in scaling-up from lab to manufacturing scale.

Lyophilized Drug Product Cake Appearance: What Is Acceptable?

Cake appearance is an important attribute of freeze-dried products, which may or may not be critical with respect to product quality (i.e., safety and efficacy). Striving for "uniform and elegant" cake appearance may continue to remain an important goal during the design and development of a lyophilized drug product. However, "sometimes" a non-ideal cake appearance has no impact on product quality and is an inherent characteristic of the product (due to formulation, drug product presentation, and freeze-drying process). This commentary provides a summary of challenges related to visual appearance testing of freeze-dried products, particularly on how to judge the criticality of cake appearance. Furthermore, a harmonized nomenclature and description for variations in cake appearance from the ideal expectation of uniform and elegant is provided, including representative images. Finally, a science and risk-based approach is discussed on establishing acceptance criteria for cake appearance.

Modeling the Secondary Drying Stage of Freeze Drying: Development and Validation of an Excel-Based Model.

Although several mathematical models of primary drying have been developed over the years, with significant impact on the efficiency of process design, models of secondary drying have been confined to highly complex models. The simple-to-use Excel-based model developed here is, in essence, a series of steady state calculations of heat and mass transfer in the 2 halves of the dry layer where drying time is divided into a large number of time steps, where in each time step steady state conditions prevail. Water desorption isotherm and mass transfer coefficient data are required. We use the Excel "Solver" to estimate the parameters that define the mass transfer coefficient by minimizing the deviations in water content between calculation and a calibration drying experiment. This tool allows the user to input the parameters specific to the product, process, container, and equipment. Temporal variations in average moisture contents and product temperatures are outputs and are compared with experiment. We observe good agreement between experiments and calculations, generally well within experimental error, for sucrose at various concentrations, temperatures, and ice nucleation temperatures. We conclude that this model can serve as an important process development tool for process design and manufacturing problem-solving.

Freeze-Drying Process Development and Scale-Up: Scale-Up of Edge Vial Versus Center Vial Heat Transfer Coefficients, Kv.

This report presents calculations of the difference between the vial heat transfer coefficient of the "edge vial" and the "center vial" at all scales. The only scale-up adjustment for center vials is for the contribution of radiation from the shelf upon which the vial sits by replacing the emissivity of the laboratory dryer shelf with the emissivity of the production dryer shelf. With edge vials, scales-up adjustments are more complex. While convection is not important, heat transfer from the wall to the bands (surrounding the vial array) by radiation and directly from the band to the vials by both radiation and conduction is important; this radiation heat transfer depends on the emissivity of the vial and the bands and is nearly independent of the emissivity of the dryer walls. Differences in wall temperatures do impact the edge vial effect and scale-up, and estimates for wall temperatures are needed for both laboratory and manufacturing dryers. Auto-loading systems (no bands) may give different edge vial heat transfer coefficients than when operating with bands. Satisfactory agreement between theoretical predictions and experimental values of the edge vial effect indicate that results calculated from the theory are of useful accuracy.

Protein Internal Dynamics Associated With Pre-System Glass Transition Temperature Endothermic Events: Investigation of Insulin and Human Growth Hormone by Solid State Hydrogen/Deuterium Exchange.

Lyophilized proteins are generally stored below their glass transition temperature (Tg) to maintain long-term stability. Some proteins in the (pure) solid state showed a distinct endotherm at a temperature well below the glass transition, designated as a pre-Tg endotherm. The pre-Tg endothermic event has been linked with a transition in protein internal mobility. The aim of this study was to investigate the internal dynamics of 2 proteins, insulin and human growth hormone (hGH), both of which exhibit the pre-Tg endothermic event with onsets at 50°C-60°C. Solid state hydrogen/deuterium (H/D) exchange of both proteins was characterized by Fourier transform infrared spectroscopy over a temperature range from 30°C to 80°C. A distinct sigmoidal transition in the extent of H/D exchange had a midpoint of 56.1 ± 1.2°C for insulin and 61.7 ± 0.9°C for hGH, suggesting a transition to greater mobility in the protein molecules at these temperatures. The data support the hypothesis that the pre-Tg event is related to a transition in internal protein mobility associated with the protein dynamical temperature. Exceeding the protein dynamical temperature is expected to activate protein internal motion and therefore may have stability consequences.

Influence of Miscibility of Protein-Sugar Lyophilizates on Their Storage Stability.

For sugars to act as successful stabilizers of proteins during lyophilization and subsequent storage, they need to have several characteristics. One of them is that they need to be able to form interactions with the protein and for that miscibility is essential. To evaluate the influence of protein-sugar miscibility on protein storage stability, model protein IgG was lyophilized in the presence of various sugars of different molecular weight. By comparing solid-state nuclear magnetic resonance spectroscopy relaxation times of both protein and sugar on two different timescales, i.e., (1)H T1 and (1)H T1ρ, miscibility of the two components was established on a 2-5- and a 20-50-nm length scale, respectively, and related to protein storage stability. Smaller sugars showed better miscibility with IgG, and the tendency of IgG to aggregate during storage was lower for smaller sugars. The largest sugar performed worst and was phase separated on both length scales. Additionally, shorter protein (1)H T1 relaxation times correlated with higher aggregation rates during storage. The enzyme-linked immunosorbent assay (ELISA) assay showed overlapping effects of aggregation and chemical degradation and did not correspond as well with the miscibility. Because of the small scale at which miscibility was determined (2-5 nm) and the size of the protein domains (∼2.5 × 2.5 × 5 nm), the miscibility data give an indirect measure of interaction between protein and sugar. This reduced interaction could be the result of steric hindrance, providing a possible explanation as to why smaller sugars show better miscibility and storage stability with the protein.

Addition of Monovalent Electrolytes to Improve Storage Stability of Freeze-Dried Protein Formulations.

This study investigates the effect of low levels of electrolytes on storage stability in freeze-dried sucrose-based protein formulations. Both bovine serum albumin and recombinant human serum albumin were freeze dried with sucrose and alkali halides (LiCl, NaCl, KCl, RbCl, and CsCl) at selected low levels. All formulations were stored at 50 °C and 65 °C up to 2 months and then assayed for protein aggregation. The data demonstrate that low levels of LiCl and NaCl enhance stability. No obvious correlations with either protein secondary structure or global dynamics (structural relaxation time) were found. However, good correlations were found between stability and both free-volume hole size via positron annihilation lifetime spectroscopy (PALS) and fast dynamics by neutron scattering. Volume changes on mixing and the partial molal volume of salt were also studied in an effort to detect decreases in free volume. These data did not support the hypothesis that reduction in free volume was the primary mechanism for salt-induced stabilization. Finally, a positive effect of postlyophilization annealing on stability was demonstrated. In summary, we find that small amounts of LiCl and NaCl significantly stabilize these proteins, which is a result at variance with conventional formulation wisdom.

Addition of Amino Acids to Further Stabilize Lyophilized Sucrose-Based Protein Formulations: I. Screening of 15 Amino Acids in Two Model Proteins.

In small amounts, the low molecular weight excipients-sorbitol and glycerol-have been shown to stabilize lyophilized sucrose-based protein formulations. The purpose of this study was to explore the use of amino acids as low molecular weight excipients to similarly enhance stability. Model proteins, recombinant human serum albumin and α-chymotrypsin, were formulated with sucrose in combination with one of 15 amino acid additives. Each formulation was lyophilized at 1:1:0.3 (w/w) protein-sucrose-amino acid. Percent total soluble aggregate was measured by size-exclusion chromatography before and after storage at 50 °C for 2 months. Classical thought might suggest that the addition of the amino acids to the sucrose-protein formulations would be destabilizing because of a decrease in the system's glass transition temperature. However, significant improvement in storage stability was observed for almost all formulations at the ratio of amino acid used. Weak correlations were found between the extent of stabilization and both amino acid molar volume and side-chain charge. The addition of amino acids at a modest level generally improves storage stability, often by more than a 50% increase, for lyophilized sucrose-based protein formulations.

Stabilization of proteins in solid form.

Immunogenicity of aggregated or otherwise degraded protein delivered from depots or other biopharmaceutical products is an increasing concern, and the ability to deliver stable, active protein is of central importance. We review characterization approaches for solid protein dosage forms with respect to metrics that are intended to be predictive of protein stability against aggregation and other degradation processes. Each of these approaches is ultimately motivated by hypothetical connections between protein stability and the material property being measured. We critically evaluate correlations between these properties and stability outcomes, and use these evaluations to revise the currently standing hypotheses. Based on this we provide simple physical principles that are necessary (and possibly sufficient) for generating solid delivery vehicles with stable protein loads. Essentially, proteins should be strongly coupled (typically through H-bonds) to the bulk regions of a phase-homogeneous matrix with suppressed ß relaxation. We also provide a framework for reliable characterization of solid protein forms with respect to stability.

Optimization of a Raman microscopy technique to efficiently detect amorphous-amorphous phase separation in freeze-dried protein formulations.

A confocal Raman microscopic technique was optimized to more efficiently detect amorphous-amorphous phase separation in freeze-dried protein formulations. A Renishaw Raman inVia confocal microscope was used to collect 100-200 µm line maps (2 µm step size) of freeze-dried protein-excipient formulations. At each point across the line map, the composition was evaluated from the intensity of the nonoverlapping peaks representative of each component. Collection aperture, scan time, and line map length significantly contributed to the phase-separation analysis, whereas different sample preparation methods did not affect the analysis. Using the optimized parameters (i.e., large aperture 5 s scan time, 200 µm line map), phase separation was successfully detected in binary polymer formulations and was comparable to the previously developed Raman method. However, the previous method required 2.5 h/sample, whereas the optimized method only requires 0.5 h/sample. Phase separation was detected in the following protein-excipient formulations: lysozyme-trehalose (1:1), lysozyme-isomaltose (1:1), ß-lactoglobulin-dextran (1:1), ß-lactoglobulin-dextran (1:3), and ß-lactoglobulin-trehalose (1:1). Phase separation was not detected in lysozyme-sucrose (1:1) and ß-lactoglobulin-sucrose (1:1) formulations. The optimized method successfully detected phase separation in several protein formulations, where phase separation was previously suspected, and promised to be a useful tool for detection of phase separation in amorphous therapeutic formulations.

Lyophilization process design space.

The application of key elements of quality by design (QbD), such as risk assessment, process analytical technology, and design space, is discussed widely as it relates to freeze-drying process design and development. However, this commentary focuses on constructing the Design and Control Space, particularly for the primary drying step of the freeze-drying process. Also, practical applications and considerations of claiming a process Design Space under the QbD paradigm have been discussed.

Is the pre-Tg DSC endotherm observed with solid state proteins associated with the protein internal dynamics? Investigation of bovine serum albumin by solid state hydrogen/deuterium exchange.

DSC thermograms of solid state pure proteins often show a distinct endotherm at a temperature far below the glass transition temperature of the system (Tg). We hypothesized this endotherm represents enthalpy recovery associated with an internal mobility transition of the protein molecule. Although the existence of an internal transition has been postulated, whether this endotherm is associated with such a transition has not previously been discussed. The purpose of this study was to investigate the origin of the pre-Tg endotherm in lyophilized bovine serum albumin (BSA). Due to strong glass behavior, the system Tg was determined by extrapolating Tg data of disaccharide/BSA formulations to zero saccharide. A small pre-Tg endotherm around 40-60 °C was observed in amorphous BSA equilibrated at 11%RH. The apparent activation energy suggested the endotherm was "α-mobility"-related. A solid state hydrogen/deuterium exchange study using FTIR was conducted over a temperature range spanning the endotherm. We found a fast phase, followed by essentially a plateau level which is highly temperature dependent in the 40-60 °C range, suggesting enhanced internal protein motion as the system passes through the temperature range of the endotherm. These results suggest the pre-Tg endotherm is associated with a protein internal mobility transition.

Accurate prediction of collapse temperature using optical coherence tomography-based freeze-drying microscopy.

The objective of this study was to assess the feasibility of developing and applying a laboratory tool that can provide three-dimensional product structural information during freeze-drying and which can accurately characterize the collapse temperature (Tc ) of pharmaceutical formulations designed for freeze-drying. A single-vial freeze dryer coupled with optical coherence tomography freeze-drying microscopy (OCT-FDM) was developed to investigate the structure and Tc of formulations in pharmaceutically relevant products containers (i.e., freeze-drying in vials). OCT-FDM was used to measure the Tc and eutectic melt of three formulations in freeze-drying vials. The Tc as measured by OCT-FDM was found to be predictive of freeze-drying with a batch of vials in a conventional laboratory freeze dryer. The freeze-drying cycles developed using OCT-FDM data, as compared with traditional light transmission freeze-drying microscopy (LT-FDM), resulted in a significant reduction in primary drying time, which could result in a substantial reduction of manufacturing costs while maintaining product quality. OCT-FDM provides quantitative data to justify freeze-drying at temperatures higher than the Tc measured by LT-FDM and provides a reliable upper limit to setting a product temperature in primary drying.