Practical Advice on Scientific Design of Freeze-Drying Process: 2023 Update.

OBJECTIVE: The purpose of this paper is to re-visit the design of three steps in the freeze-drying process, namely freezing, primary drying, and secondary drying steps. Specifically, up-to-date recommendations for selecting freeze-drying conditions are provided based on the physical-chemical properties of formulations and engineering considerations. METHODS AND RESULTS: This paper discusses the fundamental factors to consider when selecting freezing, primary drying, and secondary drying conditions, and offers mathematical models for predicting the duration of each segment and product temperature during primary drying. Three simple heat/mass transfer primary drying (PD) models were tested, and their ability to predict product temperature and sublimation time showed good agreement. The PD models were validated based on the experimental data and utilized to tabulate the primary drying conditions for common pharmaceutical formulations, including amorphous and partially crystalline products. Examples of calculated drying cycles, including all steps, for typical amorphous and crystalline formulations are provided. CONCLUSIONS: The authors revisited advice from a seminal paper by Tang and Pikal (Pharm Res. 21(2):191-200, 2004) on selecting freeze-drying process conditions and found that the majority of recommendations are still applicable today. There have been a number of advancements, including methods to promote ice nucleation and computer modeling for all steps of freeze-drying process. The authors created a database for primary drying and provided examples of complete freeze-drying cycles design. The paper may supplement the knowledge of scientists and formulators and serve as a user-friendly tool for quickly estimating the design space.

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

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

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

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

A refined phase diagram of the tert-butanol-water system and implications on lyophilization process optimization of pharmaceuticals.

While water is the solvent of choice for the lyophilization of pharmaceuticals, tert-butyl alcohol (TBA) along with water can confer several advantages including increased solubility of hydrophobic drugs, decreased drying time, improved product stability and reconstitution characteristics. The goal of this work was to generate the phase diagram and determine the eutectic temperature and composition in the "water rich" region (0.0 to 25.0% w/w TBA) of TBA-water mixtures. Solutions of different compositions were frozen and characterized by low temperature differential scanning calorimetry and powder X-ray diffractometry (XRD). The thermal events observed during warming, and their characterization by XRD, enabled the generation of phase boundaries as well as the eutectic temperature and composition. While TBA crystallized as a dihydrate in frozen solutions, on heating, the dihydrate transformed to a heptahydrate. TBA heptahydrate and ice (22.5% w/w TBA) formed a eutectic at ∼-8 °C.

Mechanical Characterization of Vial Strain During Freezing and Thawing Operations Using Amorphous Excipients.

The purpose of this study was to investigate the mechanical stresses and strains acting on pharmaceutical glass tubing vials during freezing and thawing of model pharmaceutical formulations. Strain measurements were conducted inside of a laboratory-scale freeze-dryer using a custom wireless sensor. In both sucrose and trehalose formulations at concentrations between 5 % and 20 % w/v, the strain measurements initially increased before peaking in magnitude at temperatures close to the respective glass transition temperatures of the maximally freeze concentrated solutes, Tg'. We attribute this behavior to a shift in the mechanical properties of the frozen system from a purely elastic glass below Tg' to a viscoelastic rubber-like material above Tg'. That is, when the interstitial region becomes mechanically compliant at temperature above Tg'. The outputs were less predictable below 5 % w/v and tended to exhibit two separate peaks in strain output, one near the equilibrium melting temperature of pure ice and the other near Tg'. The peaks merged at concentrations between 4 and 5 % w/v where the largest strain magnitude was observed. The strain on primary packaging has traditionally been applied to evaluate the risk of damage or breakage due to, for example, crystallization of excipients. However, data collected during this study suggest there may be utility in formulation design or as a process analytical technology to minimize potentially destabilizing stresses and strains in the frozen formulation.

Mannitol as an Excipient for Lyophilized Injectable Formulations.

The review summarizes the current state of knowledge of mannitol as an excipient in lyophilized injectable small and large molecule formulations. When compared with glycine, the physicochemical properties of mannitol make it a desirable and preferred bulking agent. Though mannitol is a popular bulking agent in freeze-dried formulations, its use may pose certain challenges such as vial breakage or its existence as a metastable crystalline hemihydrate in the final cake, necessitating appropriate mitigation strategies. The understanding of the phase behavior of mannitol in aqueous systems, during the various stages of freeze-drying, can be critical for the optimization of freeze-drying cycle parameters in multi-component formulations. Finally, using a decision tree as a guiding tool, we demonstrate the use of orthogonal techniques for attaining a stable and cost-effective lyophilized drug product containing mannitol.

A Kinetic Model for Spray-Freezing of Pharmaceuticals.

Spray freeze-drying (SFD), which includes spray-freezing into droplets and dynamic vacuum drying, presents a promising alternative approach to manufacture dried pharmaceuticals more efficiently than conventional vial freeze-drying. Without reliable predictive models for the SFD conditions of interest, any respective process development still relies on empirical approaches. In this work, we propose an improved modeling framework to describe the fast freezing (<1 s) that sub-millimeter droplets undergo in the present SFD process. The modeled freezing rate accounts for both the kinetics of ice growth and droplet heat transfer mechanisms. Computational fluid dynamics (CFD) simulations and experiments on bulk spray-freezing are combined to refine and validate the proposed reduced-order model. While this study is limited to water-sucrose solutions, the present modeling approach can be extended to other pharmaceutical excipients. For the cooling rates of interest, model results indicate that droplets with initial sucrose concentration higher than 20% w/w will transit to a glassy state before completion of crystallization and, consequently, devitrification is expected during post spray-freezing manipulation of the bulk material. In practice, such compact model does not only allow quantification of process parameters that cannot be measured in real time but also enable the choice of optimal spraying conditions for production of free-flowing, high-quality frozen droplets that meet the target product profile.

Reconstitution of Highly Concentrated Lyophilized Proteins: Part 1 Amorphous Formulations.

Long reconstitution times before patient administration remain an undesirable quality attribute for high concentration lyophilized protein formulations. In this study, 3 approaches were developed to study reconstitution behavior of lyophilized, amorphous cakes of a highly concentrated monoclonal antibody (mAb) by exploring their wetting, disintegration, and hydration behavior. As the mAb concentration increased from 0 to 83 mg/mL, reconstitution times were longer with poorer wetting, slower hydration, and disintegration rates. Furthermore, the effect of controlling ice nucleation temperature at -5 and -10°C during freezing followed by either conservative or aggressive drying conditions on the reconstitution times was explored in formulations containing 40 and 83 mg/mL mAb. Although no effect of either of the 2 processing conditions was noted at 40 mg/mL, aggressive drying led to faster reconstitution at both the nucleation temperatures with 83 mg/mL mAb. The present study combined with literature data suggests that below a protein-to-sugar ratio of 1, reconstitution was complete within 1 min, and when the ratio was greater than 1, the reconstitution times increased nonlinearly. Disintegration and hydration were determined to be the key mechanisms contributing to the complete reconstitution of the lyophilized, amorphous cakes of the highly concentrated mAb in vials.

Investigation of Fogging Behavior in a Lyophilized Drug Product.

Vial "fogging" is a common observation in lyophilized biological products and has been reported in the pharmaceutical industry. In addition to unappealing appearance, severe fogging that reaches the shoulder or neck of the vial can potentially compromise the container closure integrity of the vials. In this study, we performed experiments to identify parameters impacting the fogging phenomena in lyophilized drug product vials. Glass vial surface properties were found to have a significant impact on vial fogging. In line with prior published research, the study demonstrates that fogging can be mitigated by using glass vials with hydrophobic surface (such as siliconized vial or TopLyo® vial) and by extending the prefreeze 5°C hold during the lyophilization cycle. Moreover, this study shows that extending the annealing at -5°C or -10°C can also significantly reduce the fogging. Increased formulation viscosity and exclusion of a surfactant can mitigate the fogging behavior of the lyophilized product. The study shows that container closure integrity as determined by headspace analysis and vacuum decay is not compromised for the "fogging" drug product vials for this model monoclonal antibody container using a worst-case model of lyophilized "neck-wet" vials.

Bulk Dynamic Spray Freeze-Drying Part 1: Modeling of Droplet Cooling and Phase Change.

In spray freeze-drying (SFD), the solution is typically dispersed into a gaseous cold environment producing frozen microparticles that are subsequently dried via sublimation. This technology can potentially manufacture bulk lyophilized drugs at higher rates compared with conventional freeze-drying in trays and vials because small frozen particles provide larger surface area available for sublimation. Although drying in SFD still has to meet the material collapse temperature requirements, the final characteristics of the respective products are mainly controlled by the spray-freezing dynamics. In this context, the main goal of this work is to present a single droplet spray-freezing model and validate it with previously published simulations and experimental data. For the investigated conditions, the droplet temperature evolutions predicted by the model agree with experiments within an error of ±10%. The proposed engineering-level modeling framework is intended to assist future development of efficient SFD processes and support scale up from laboratory to commercial scale equipment.

Bulk Dynamic Spray Freeze-Drying Part 2: Model-Based Parametric Study for Spray-Freezing Process Characterization.

Spray freeze-drying is an evolving technology that combines the benefits of spray-drying and conventional lyophilization techniques to produce drug substance and drug product as free-flowing powders. The high surface-to-volume ratio associated to the submillimeter spray-frozen particles contributes to shorter drying and reconstitution times. The formation of frozen particles is the most critical part of this dehydration technique because it defines the properties of final product. Based on a previously proposed and validated model, the current goal is to understand the role of various controllable parameters in the spray-freezing process. More specifically, given a set of spraying conditions, the model is used to predict the minimum distance required to cool and freeze the droplets below a temperature that prevents coalescence and product agglomeration. A parametric study is carried out to map the operational limit conditions of the actual spray-freezing column apparatus under consideration. For the spray freeze-drying conditions of interest, model simulations indicate that convection contributes to at least 80% of the total droplet heat transfer and, consequently, that freezing column gas temperature and droplet diameter are the most important process parameters affecting the freezing distance.

Protein/Ice Interaction: High-Resolution Synchrotron X-ray Diffraction Differentiates Pharmaceutical Proteins from Lysozyme.

Protein/ice interactions are investigated by a novel method based on measuring the characteristic features of X-ray diffraction (XRD) patterns of hexagonal ice (Ih). Aqueous solutions of four proteins and other solutes are studied using high-resolution synchrotron XRD. Two pharmaceutical proteins, recombinant human albumin and monoclonal antibody (both at 100 mg/mL), have a pronounced effect on the properties of ice crystals, reducing the size of the Ih crystalline domains and increasing the microstrain. Lysozyme (100 mg/mL) and an antifreeze protein (1 mg/mL) have much weaker impact on Ih. Neither of the proteins studied exhibit preferred interactions with specific crystalline faces of Ih. It is proposed that the pharmaceutical proteins interact with ice crystals indirectly by accumulating in the quasi-liquid layer next to ice crystallization front, rather than directly, via a sorption on ice crystals. This is the first report, to the best of our knowledge, of major difference in the protein/ice interaction between non-antifreeze proteins. Another important finding is a detection of a second (minor) population of ice crystals, which is tentatively identified as a high-pressure form of ice, possibly IceIII or IceIX. This finding highlights a potential role of mechanical stresses in freeze-induced destabilization of proteins.

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.

Post-thaw aging affects activity of lactate dehydrogenase.

Freeze-thawing is routinely used to study freezing-induced irreversible protein denaturation in the formulation characterization and development of lyophilized proteins. In most cases, the temperature profiles of the samples are not fully monitored during freeze-thawing and therefore, the sample thermal histories are largely unknown. The objective of this study was to develop experimental protocols for the study of isothermal protein degradation using a temperature-step apparatus. Freeze-thaw experiments were performed at a freezing rate of 10 degrees C/min and various thawing rates (0.5-3.3 degrees C/min) using a temperature-step apparatus. In our efforts to design validation studies, we encountered anomalies in the recovered enzyme activity data of an enzyme, lactate dehydrogenase at the end of freeze-thawing. The effect of thawing rate was studied to explain the variability in the data. In addition, post-thaw "aging" of freshly frozen and thawed samples was performed at 5 degrees C to reduce the variability in the recovered enzyme activity. Results from these experiments implicate the use of aging of dilute multimeric enzymes at the end of freeze-thawing to control the variability in enzyme assays.

Next generation drying technologies for pharmaceutical applications.

Drying is a commonly used technique for improving the product stability of biotherapeutics. Typically, drying is accomplished through freeze-drying, as evidenced by the availability of several lyophilized products on the market. There are, however, a number of drawbacks to lyophilization, including the lengthy process time required for drying, low energy efficiency, high cost of purchasing and maintaining the equipment, and sensitivity of the product to freezing and various other processing-related stresses. These limitations have led to the search for next-generation drying methods that can be applied to biotherapeutics. Several alternative drying methods are reviewed herein, with particular emphasis on methods that are commonly employed outside of the biopharmaceutical industry including spray drying, convective drying, vacuum drying, microwave drying, and combinations thereof. Although some of the technologies have already been implemented for processing biotherapeutics, others are still at an early stage of feasibility assessment. An overview of each method is presented, detailing the comparison to lyophilization, examining the advantages and disadvantages of each technology, and evaluating the potential of each to be utilized for drying biotherapeutic products.

Investigation of PEG crystallization in frozen and freeze-dried PEGylated recombinant human growth hormone-sucrose systems: implications on storage stability.

The objectives of the current study were to investigate (i) the phase behavior of a PEGylated recombinant human growth hormone (PEG-rhGH, ∼60 kDa) during freeze-drying and (ii) its storage stability. The phase transitions during freeze-thawing of an aqueous solution containing PEG-rhGH and sucrose were characterized by differential scanning calorimetry. Finally, PEG-rhGH and sucrose formulations containing low, medium, and high polyethylene glycol (PEG) to sucrose ratios were freeze-dried in dual-chamber syringes and stored at 4°C and 25°C. Chemical decomposition (methionine oxidation and deamidation) and irreversible aggregation were characterized by size-exclusion and ion-exchange chromatography, and tryptic mapping. PEG crystallization was facilitated when it was covalently linked with rhGH. When the solutions were frozen, phase separation into PEG-rich and sucrose-rich phases facilitated PEG crystallization and the freeze-dried cake contained crystalline PEG. Annealing caused PEG crystallization and when coupled with higher drying temperatures, the primary drying time decreased by up to 51%. When the freeze-dried cakes were stored at 4°C, while there was no change in the purity of the PEG-rhGH monomer, deamidation was highest in the formulations with the lowest PEG to sucrose ratio. When stored at 25°C, this composition also showed the most pronounced decrease in monomer purity, the highest level of aggregation, and deamidation. Furthermore, an increase in PEG crystallinity during storage was accompanied by a decrease in PEG-rhGH stability. Interestingly, during storage, there was no change in PEG crystallinity in formulations with medium and high PEG to sucrose ratios. Although PEG crystallization during freeze-drying did not cause protein degradation, crystallization during storage might have influenced protein stability.

Investigation of PEG crystallization in frozen PEG-sucrose-water solutions. I. Characterization of the nonequilibrium behavior during freeze-thawing.

Our objective was to characterize the nonequilibrium thermal behavior of frozen aqueous solutions containing PEG and sucrose. Aqueous solutions of (i) sucrose (10%, w/v) with different concentrations of PEG (1-20%, w/v), and (ii) PEG (10%, w/v) with different concentrations of sucrose (2-20%, w/v), were cooled to -70 degrees C at 5 degrees C/min and heated to 25 degrees C at 2 degrees C/min in a differential scanning calorimeter. Annealing was performed at temperatures ranging from -50 to -20 degrees C for 2 or 6 h. Similar experiments were also performed in the low-temperature stage of a powder X-ray diffractometer. A limited number of additional DSC experiments were performed wherein the samples were cooled to -100 degrees C. In unannealed systems with a fixed sucrose concentration (10%, w/v), the T'g decreased from -35 to -48 degrees C when PEG concentration was increased from 1% to 20% (w/v). On annealing at -25 degrees C, PEG crystallized. This was evident from the increase in T'g and the appearance of a secondary melting endotherm in the DSC. Low-temperature XRD provided direct evidence of PEG crystallization. Annealing at temperatures

Investigation of PEG crystallization in frozen PEG-sucrose-water solutions: II. Characterization of the equilibrium behavior during freeze-thawing.

Our objective was to characterize, by DSC and XRD, the equilibrium thermal behavior of frozen aqueous solutions containing polyethylene glycol (PEG) and sucrose. Aqueous solutions of (i) PEG (2.5-50% w/w), (ii) sucrose (10% w/v) with different concentrations of PEG (1-20% w/v), and (iii) PEG (2% or 10% w/v) with different concentrations of sucrose (2-20% w/v), were cooled to -70 ° C at 5 ° C/min and heated to 25 ° C at 2 ° C/min in a DSC. Annealing was performed for 2 or 6 h at temperatures, ranging from -50 to -20 ° C. Experiments under similar conditions, on select compositions, were also performed in a powder X-ray diffractometer. Two endotherms, observed during heating of a frozen PEG solution (10% w/v), were attributed to PEG-ice eutectic melting and ice melting, and were confirmed by XRD. At higher PEG concentrations (≥ 37.5% w/w), only the endotherm attributed to the PEG-ice eutectic melting was observed. Inclusion of sucrose decreased both PEG-ice melting and ice melting temperatures. In unannealed systems with a fixed sucrose concentration (10% w/v), the PEG-ice melting event was not observed at PEG concentration ≤ 5% w/v. Annealing for 2-6 h facilitated PEG crystallization. In unannealed systems with a fixed PEG concentration (10% w/v), an increase in the sucrose concentration increased the devitrification but decreased the PEG-ice melting temperature. The PEG-ice melting temperatures obtained by DSC and XRD were in good agreement. In ternary systems at a fixed PEG to sucrose ratio, the T' g as well as the PEG-ice melting temperature were unaffected by the total solute concentration. XRD confirmed the absence of a PEG-sucrose-ice ternary eutectic. When the PEG to sucrose ratio was systematically varied, the PEG-ice and ice melting temperatures decreased with an increase in the sucrose concentration. However, at a fixed PEG to sucrose ratio, the PEG-ice melting temperature, was unaffected by the total solute concentration.

Protein stability during freezing: separation of stresses and mechanisms of protein stabilization.

Although proteins are often frozen during processing or freeze-dried after formulation to improve their stability, they can undergo degradation leading to losses in biological activity during the process. During freezing, the physical environment of a protein changes dramatically leading to the development of stresses that impact protein stability. Low temperature, freeze-concentration, and ice formation are the three chief stresses resulting during cooling and freezing. Because of the increase in solute concentrations, freeze-concentration could also facilitate second order reactions, crystallization of buffer or non-buffer components, phase separation, and redistribution of solutes. An understanding of these stresses is critical to the determination of when during freezing a protein suffers degradation and therefore important in the design of stabilizer systems. With the exception of a few studies, the relative contribution of various stresses to the instability of frozen proteins has not been addressed in the freeze-drying literature. The purpose of this review is to describe the various stages of freezing and examine the consequences of the various stresses developing during freezing on protein stability and to assess their relative contribution to the destabilization process. The ongoing debate on thermodynamic versus kinetic mechanisms of stabilization in frozen environments and the current state of knowledge concerning those mechanisms are also reviewed in this publication. An understanding of the relative contributions of freezing stresses coupled with the knowledge of cryoprotection mechanisms is central to the development of more rational formulation and process design of stable lyophilized proteins.