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.

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 sugars on the molecular motion of freeze-dried protein formulations reflected by NMR relaxation times.

PURPOSE: To relate NMR relaxation times to instability-related molecular motions of freeze-dried protein formulations and to examine the effect of sugars on these motions. METHODS: Rotating-frame spin-lattice relaxation time (T(1ρ)) was determined for both protein and sugar carbons in freeze-dried lysozyme-sugar (trehalose, sucrose and isomaltose) formulations using solid-state (13)C NMR. RESULTS: The temperature dependence of T(1ρ) for the lysozyme carbonyl carbons in lysozyme with and without sugars was describable with a model that includes two different types of molecular motion with different correlation times (τ(c)) for the carbon with each τ(c) showing Arrhenius temperature dependence. Both relaxation modes have much smaller relaxation time constant (τ(c)) and temperature coefficient (Ea) than structural relaxation and may be classified as ß-relaxation and γ-relaxation. The τ(c) and Ea for γ-relaxation were not affected by sugars, but those for ß-relaxation were increased by sucrose, changed little by trehalose, and decreased by isomaltose, suggesting that the ß-mobility of the lysozyme carbonyl carbons is decreased by sucrose and increased by isomaltose. CONCLUSION: T(1ρ) determined for the lysozyme carbonyl carbons can reflect the effect of sugars on molecular mobility in lysozyme. However, interpretation of relaxation time data is complex and may demand data over an extended temperature range.

Aqueous solubility of crystalline and amorphous drugs: Challenges in measurement.

Measurement of drug solubility is one of the key elements of active pharmaceutical ingredient (API) characterization during the drug discovery and development process. This report is a critical review of experimental methods reported in the literature for the measurement of aqueous solubility of amorphous, partially crystalline and crystalline organic compounds. A summary of high-throughput automated methods used in early drug discovery research is also provided in this report. This review summarizes the challenges that are encountered during solubility measurement and the complexities that are often overlooked. Even though there is an advantage in using the amorphous form of a drug due to its higher solubility, measurement of its solubility with useful accuracy is still a practical problem. Therefore, this review provides recommendations of preferred methods and precautions in using these methods to determine the aqueous solubility of amorphous and crystalline new molecular entities, with emphasis on the physico-chemical characterization of the solid state of the test substance.

Quality by design in formulation and process development for a freeze-dried, small molecule parenteral product: a case study.

A case study has been developed to illustrate one way of incorporating a Quality by Design approach into formulation and process development for a small molecule, freeze-dried parenteral product. Sodium ethacrynate was chosen as the model compound. Principal degradation products of sodium ethacrynate result from hydrolysis of the unsaturated ketone in aqueous solution, and dimer formation from a Diels-Alder condensation in the freeze-dried solid state. When the drug crystallizes in a frozen solution, the eutectic melting temperature is above -5°C. Crystallization in the frozen system is affected by pH in the range of pH 6-8 and buffer concentration in the range of 5-50 mM, where higher pH and lower buffer concentration favor crystallization. Physical state of the drug is critical to solid state stability, given the relative instability of amorphous drug. Stability was shown to vary considerably over the ranges of pH and buffer concentration examined, and vial-to-vial variability in degree of crystallinity is a potential concern. The formulation design space was constructed in terms of pH and drug concentration, and assuming a constant 5 mM concentration of buffer. The process design space is constructed to take into account limitations on the process imposed by the product and by equipment capability.

Emerging freeze-drying process development and scale-up issues.

Although several guidelines do exist for freeze-drying process development and scale-up, there are still a number of issues that require additional attention. The objective of this review article is to discuss some emerging process development and scale-up issue with emphasis on effect of load condition and freeze-drying in novel container systems such as syringes, Lyoguard trays, ampoules, and 96-well plates. Understanding the heat and mass transfer under different load conditions and for freeze-drying in these novel container systems will help in developing a robust freeze-drying process which is also easier to scale-up. Further research and development needs in these emerging areas have also been addressed.

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.

Solubility advantage of amorphous pharmaceuticals: II. Application of quantitative thermodynamic relationships for prediction of solubility enhancement in structurally diverse insoluble pharmaceuticals.

PURPOSE: To quantitatively assess the solubility advantage of amorphous forms of nine insoluble drugs with a wide range of physico-chemical properties utilizing a previously reported thermodynamic approach. METHODS: Thermal properties of amorphous and crystalline forms of drugs were measured using modulated differential calorimetry. Equilibrium moisture sorption uptake by amorphous drugs was measured by a gravimetric moisture sorption analyzer, and ionization constants were determined from the pH-solubility profiles. Solubilities of crystalline and amorphous forms of drugs were measured in de-ionized water at 25°C. Polarized microscopy was used to provide qualitative information about the crystallization of amorphous drug in solution during solubility measurement. RESULT: For three out the nine compounds, the estimated solubility based on thermodynamic considerations was within two-fold of the experimental measurement. For one compound, estimated solubility enhancement was lower than experimental value, likely due to extensive ionization in solution and hence its sensitivity to error in pKa measurement. For the remaining five compounds, estimated solubility was about 4- to 53-fold higher than experimental results. In all cases where the theoretical solubility estimates were significantly higher, it was observed that the amorphous drug crystallized rapidly during the experimental determination of solubility, thus preventing an accurate experimental assessment of solubility advantage. CONCLUSION: It has been demonstrated that the theoretical approach does provide an accurate estimate of the maximum solubility enhancement by an amorphous drug relative to its crystalline form for structurally diverse insoluble drugs when recrystallization during dissolution is minimal.

Determination of end point of primary drying in freeze-drying process control.

Freeze-drying is a relatively expensive process requiring long processing time, and hence one of the key objectives during freeze-drying process development is to minimize the primary drying time, which is the longest of the three steps in freeze-drying. However, increasing the shelf temperature into secondary drying before all of the ice is removed from the product will likely cause collapse or eutectic melt. Thus, from product quality as well as process economics standpoint, it is very critical to detect the end of primary drying. Experiments were conducted with 5% mannitol and 5% sucrose as model systems. The apparent end point of primary drying was determined by comparative pressure measurement (i.e., Pirani vs. MKS Baratron), dew point, Lyotrack (gas plasma spectroscopy), water concentration from tunable diode laser absorption spectroscopy, condenser pressure, pressure rise test (manometric temperature measurement or variations of this method), and product thermocouples. Vials were pulled out from the drying chamber using a sample thief during late primary and early secondary drying to determine percent residual moisture either gravimetrically or by Karl Fischer, and the cake structure was determined visually for melt-back, collapse, and retention of cake structure at the apparent end point of primary drying (i.e., onset, midpoint, and offset). By far, the Pirani is the best choice of the methods tested for evaluation of the end point of primary drying. Also, it is a batch technique, which is cheap, steam sterilizable, and easy to install without requiring any modification to the existing dryer.

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.

Reduced pressure ice fog technique for controlled ice nucleation during freeze-drying.

A method to achieve controlled ice nucleation during the freeze-drying process using an ice fog technique was demonstrated in an earlier report. However, the time required for nucleation was about 5 min, even though only one shelf was used, which resulted in Ostwald ripening (annealing) in some of the vials that nucleated earlier than the others. As a result, the ice structure was not optimally uniform in all the vials. The objective of the present study is to introduce a simple variation of the ice fog method whereby a reduced pressure in the chamber is utilized to allow more rapid and uniform freezing which is also potentially easier to scale up. Experiments were conducted on a lab scale freeze dryer with sucrose as model compound at different concentration, product load, and fill volume. Product resistance during primary drying was measured using manometric temperature measurement. Specific surface area of the freeze-dried cake was also determined. No difference was observed either in average product resistance or specific surface area for the different experimental conditions studied, indicating that with use of the reduced pressure ice fog technique, the solutions nucleated at very nearly the same temperature (-10 degrees C). The striking feature of the "Reduced Pressure Ice Fog Technique" is the rapid ice nucleation (less than a minute) under conditions where the earlier procedure required about 5 min; hence, effects of variable Ostwald ripening were not an issue.

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.

Role of thermodynamic, molecular, and kinetic factors in crystallization from the amorphous state.

Though there is an advantage in using the higher solubility amorphous state in cases where low solubility limits absorption, physical instability poses a significant barrier limiting its use in solid oral dosage forms. Unlike chemical instability, where useful accelerated stability testing protocols are common, no methodology has been established to predict physical instability. Therefore, an understanding of the factors affecting crystallization from the amorphous state is not only important from a scientific perspective but also has practical applications. Crystallization from the amorphous matrix has been linked to the molecular mobility in the amorphous matrix and recent research has focused on developing the link between these two fundamental properties of glass forming materials. Although researchers have been actively working in this area for some time, there is no current review describing the present state of understanding of crystallization from the amorphous state. The purpose of this review therefore is to examine the roles of different factors such as molecular mobility, thermodynamic factors, and the implication of different processing condition, in crystallization from the amorphous state. We believe an increased understanding of the relative contributions of molecular mobility and processing conditions are vital to increased usage of the amorphous state in solid oral dosage forms.

Study of the individual contributions of ice formation and freeze-concentration on isothermal stability of lactate dehydrogenase during freezing.

The objective of this study was to determine the individual contributions of ice formation, solute concentration, temperature, and time, to irreversible protein denaturation during freezing. A temperature-step approach was used to study isothermal degradation of frozen lactate dehydrogenase (LDH). The freeze-concentrate composition was determined using differential scanning calorimetry to enable preparation of solutions, without ice, of the same concentration as the freeze-concentrate, and thereby determine the role of the freeze-concentrate composition on LDH degradation. Both stabilizers employed in the study, hydroxyethyl starch and sucrose, conferred cryoprotection on LDH. While LDH stability was lower at 1.50-3.25% w/v sucrose than in the absence of sucrose, cryoprotection was restored at higher sucrose concentrations. pH shift during freezing, degree of supercooling, and excipient impurities were ruled out as causes for unusual LDH stability behavior at lower sucrose concentrations. Specific surface area measurements of the freeze-dried cakes showed that the ice surface area increased with an increase in sucrose concentration. No LDH degradation occurred in concentrated solutions, without ice, at the same composition as the freeze-concentrate in frozen systems where massive degradation was documented. Thus, ice formation is the critical destabilizing factor during freezing of LDH in sucrose:citrate buffer systems.

Prediction of onset of crystallization from experimental relaxation times. II. Comparison between predicted and experimental onset times.

UNLABELLED: Given a good correlation between onsets of crystallization and mobility above T(g), one might be able to predict crystallization onsets at a temperature of interest far below T(g) from this correlation and measurement of mobility at a temperature below T(g). Such predictions require that: (a) correlation between crystallization onset and mobility is the same above and below T(g), and (b) techniques used to measure mobility above and below T(g) measure the same kind of mobility [(b) demonstrated previously using dielectric and calorimetric techniques]. The objective of present work is to determine whether crystallization onset times couple with relaxation times determined above T(g), and if so to verify predictions made below T(g) (from data above T(g)) with experimental data. Model compounds were indomethacin, ketoconazole, flopropione, nifedipine, and felodipine. Onsets of crystallization measured above T(g) were coupled with dielectric mobility for indomethacin, felodipine, and flopropione. Prediction of crystallization onset times for temperatures below T(g) matched well with experimental data for indomethacin (25 degrees C, 35 degrees C: Predicted 473, 95 h; EXPERIMENTAL: 624 +/- 158, 139 +/- 49 h) and flopropione (35 degrees C, 40 degrees C; Predicted 115, 58 h; EXPERIMENTAL: 96 +/- 30, 59 +/- 10 h). The data suggests that coupling between crystallization onsets and molecular mobility at temperatures above T(g) may be exploited to develop stability testing protocol for crystallization from amorphous state.

The impact of drying method and formulation on the physical properties and stability of methionyl human growth hormone in the amorphous solid state.

The objective of this work was to investigate the impact of drying method and formulation on the physical stability (aggregation) and selected important physical properties of dried methionyl human growth hormone (Met-hGH) formulations. Solutions of Met-hGH with different stabilizers were dried by different methods (freeze drying, spray drying, and film drying), with and without surfactant. Properties of the dried powders included powder morphology, specific surface area (SSA), protein surface coverage, thermal analysis, and protein secondary structure. Storage stability of Met-hGH in different formulations was also studied at 50 degrees C and at 60 degrees C for 3 months. The dried powders displayed different morphologies, depending mainly on the method of drying and on the presence or absence of surfactant. Film dried powders had the lowest SSA (approximately 0.03 m(2)/g) and the lowest total protein surface accumulation (approximately 0.003%). Surfactant caused a reduction in the SSA of both spray dried and freeze dried powders. Spray dried powders showed greater protein surface coverage and SSA relative to the same formulations dried by other means. Greater in-process perturbations of protein secondary structure were observed with polymer excipients. Formulation impacted physical stability. In general, low molecular weight stabilizers provided better stability. For example, the aggregation rate at 50 degrees C of Met-hGH in a freeze dried trehalose-based formulation was approximately four times smaller than the corresponding Ficoll-70-based formulation. Drying method also influenced physical stability. In general, the film dried preparations studied showed superior stability to preparations dried by other methods, especially those formulations employing low molecular weight stabilizers.

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.