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.

Recommended Best Practices for Lyophilization Validation 2021 Part II: Process Qualification and Continued Process Verification.

This work describes the lyophilization process validation and consists of two parts. Part one (Part I: Process Design and Modeling) focuses on the process design and is described in the previous paper, while the current paper is devoted to process qualification and continued process verification. The goal of the study is to show the cutting edge of lyophilization validation based on the integrated community-based opinion and the industrial perspective. This study presents best practices for batch size determination and includes the effect of batch size on drying time, process parameters selection strategies, and batch size overage to compensate for losses during production. It also includes sampling strategies to demonstrate batch uniformity as well as the use of statistical models to ensure adequate sampling. Based on the LyoHUB member organizations survey, the best practices in determining the number of PPQ runs are developed including the bracketing approach with minimum and maximum loads. Standard practice around CQA and CPP selection is outlined and shows the advantages of using control charts and run charts for process trending and quality control. The case studies demonstrating the validation strategy for monoclonal antibody and the impact of the loading process on the lyophilization cycle and product quality as well as the special case of lyophilization for dual-chamber cartridge system are chosen to illustrate the process validation. The standard practices in the validation of the lyophilization process, special lyophilization processes, and their impact on the validation strategy are discussed.

Recommended Best Practices for Lyophilization Validation-2021 Part I: Process Design and Modeling.

This work describes lyophilization process validation and consists of two parts. Part I focuses on the process design and is described in the current paper, while part II is devoted to process qualification and continued process verification. The intent of these articles is to provide readers with recent updates on lyophilization validation in the light of community-based combined opinion on the process and reflect the industrial prospective. In this paper, the design space approach for process design is described in details, and examples from practice are provided. The approach shows the relationship between the process inputs; it is based on first principles and gives a thorough scientific understanding of process and product. The lyophilization process modeling and scale-up are also presented showing the impact of facility, equipment, and vial heat transfer coefficient. The case studies demonstrating the effect of batch sizes, fill volume, and dose strength to show the importance of modeling as well as the effect of controlled nucleation on product resistance are discussed.

Foslevodopa/Foscarbidopa: A New Subcutaneous Treatment for Parkinson's Disease.

OBJECTIVE: The aim was to demonstrate that continuous s.c. infusion of a soluble levodopa (LD)/carbidopa (CD) phosphate prodrug combination effectively delivers stable LD exposure via a minimally invasive and convenient mode and has the potential to treat Parkinson's disease (PD) patients who are not well controlled on oral medication. METHODS: Foslevodopa and foscarbidopa were prepared and the equilibrium solubility and chemical stability examined in aqueous media with different values of pH. Solutions of foslevodopa/foscarbidopa (ratios ranging from 4:1 to 20:1) were prepared by dissolving pH-adjusted lyophilized materials in water and infused s.c. in healthy volunteers for ≤72 hours. Frequent blood samples were collected to measure LD and CD exposure, and safety was monitored throughout the study. RESULTS: Foslevodopa/foscarbidopa (ABBV-951) demonstrates high water solubility and excellent chemical stability near physiological pH, enabling continuous s.c. infusion therapy. After s.c. infusion, a stable LD pharmacokinetic (PK) profile was maintained for ≤72 hours, and the infusion was well tolerated. INTERPRETATION: Preparation of foslevodopa and foscarbidopa enables preclinical and clinical PK, safety, and tolerability studies in support of their advancement for the treatment of PD. In phase 1 clinical trials, foslevodopa/foscarbidopa demonstrates consistent and stable LD plasma exposure, supporting further studies of this treatment as a potentially transformational option for those suffering from PD. ANN NEUROL 2021;90:52-61.

Effect of Linker-Drug Properties and Conjugation Site on the Physical Stability of ADCs.

The physical stability of antibody drug conjugates is dictated by the properties of the antibody, linker-drug, and conjugation site. Two linker-drugs were chosen that are different in terms of hydrophobicity and polar surface area to evaluate the effect of linker-drug properties on antibody-drug conjugate (ADC) behavior. Site-specific and non-site-specific conjugation was used to investigate the role of conjugation site in conformational and colloidal stability. Finally, 2 antibodies were selected to determine if the observed results were antibody-specific. The conformational stability is affected, with the highest degree of destabilization observed when conjugation results in the removal of interchain disulfide bonds. Although conformational destabilization occurred in the domain in which conjugation occurred and domains distinct from the conjugation site, no correlation could be drawn between linker-drug properties and conformational stability. Evaluation of aggregation by size exclusion HPLC confirmed a relationship between linker-drug hydrophobicity and aggregation propensity under thermal stress in all ADCs tested. The extent of aggregation was far greater in the conjugates generated with a more hydrophobic antibody, illustrating that the properties of both the antibody and linker-drug contribute to aggregation. These studies emphasize that the distinct properties of the molecule as a whole warrant a case-by-case evaluation of each ADC.

Sensitivity Study to Assess the Robustness of Primary Drying Process in Pharmaceutical Lyophilization.

The objective of this work is to apply a sensitivity study to assess the robustness of the primary drying step of pharmaceutical lyophilization with respect to deviations in process parameters. The sensitivity study can provide valuable information regarding the effect of process input parameters on the product quality that can aid in designing robust lyophilization processes. In this study, the output response is related to its inputs using Smolyak sparse grid generalized polynomial chaos method, and the sensitivity was calculated using elementary effects method. Sensitivity of chamber pressure and shelf temperature on product temperature of 2 sucrose-based and one mannitol-based formulation was studied, and the results were analyzed in terms of risk of adverse effects due to process deviations on the product quality. The study revealed that the sensitivity varies among formulations, and preliminary information regarding the possible impact of process deviations can be obtained from the process cycle diagram. The product temperature showed greater sensitivity toward the change in the shelf temperature than toward change in the chamber pressure for the greater part of the primary drying stage. An aggressive process-deviation scenario at the late stage of primary drying was also studied for different formulations, and the results were consistent with the sensitivity study.

Leveraging Lyophilization Modeling for Reliable Development, Scale-up and Technology Transfer.

Modeling of the lyophilization process, based on the steady-state heat and mass transfer, is a useful tool in understanding and optimizing of the process, developing an operating design space following the quality-by-design principle, and justifying occasional process deviations during routine manufacturing. The steady-state model relies on two critical parameters, namely, the vial heat transfer coefficient, Kv, and the cake resistance, Rp. The classical gravimetric method used to measure Kv is tedious, time- and resource-consuming, and can be challenging and costly for commercial scale dryers. This study proposes a new approach to extract both Kv and Rp directly from an experimental run (e.g., temperature and Pirani profiles). The new methodology is demonstrated using 5% w/v mannitol model system. The values of Kv obtained using this method are comparable to those measured using the classic gravimetric method. Application of the proposed approach to process scale-up and technology transfer is illustrated using a case study. The new approach makes the steady-state model a simple and reliable tool for model parameterization, thus maximizes its capability and is particularly beneficial for transfer products from lab/pilot to commercial manufacturing.

Predictive models of lyophilization process for development, scale-up/tech transfer and manufacturing.

Scale-up and technology transfer of lyophilization processes remains a challenge that requires thorough characterization of the laboratory and larger scale lyophilizers. In this study, computational fluid dynamics (CFD) was employed to develop computer-based models of both laboratory and manufacturing scale lyophilizers in order to understand the differences in equipment performance arising from distinct designs. CFD coupled with steady state heat and mass transfer modeling of the vial were then utilized to study and predict independent variables such as shelf temperature and chamber pressure, and response variables such as product resistance, product temperature and primary drying time for a given formulation. The models were then verified experimentally for the different lyophilizers. Additionally, the models were applied to create and evaluate a design space for a lyophilized product in order to provide justification for the flexibility to operate within a certain range of process parameters without the need for validation.

Rational design of lyophilized high concentration protein formulations-mitigating the challenge of slow reconstitution with multidisciplinary strategies.

An increasing number of protein therapies require chronic administration at high doses (>200 mg) by subcutaneous (sc) injection. Due to the injection volume limitation (<1.5 mL) associated with sc administration, high protein concentration formulations at or exceeding 100 mg/mL are required to achieve the dose. Development of a high concentration protein formulation can be challenging due to increased aggregation at higher concentration and/or chemical instability, which necessitates the development of lyophilized formulation for high protein concentration drug products. Unique challenges, such as long reconstitution time for a lyophilized high protein concentration drug product, can limit practical usage and commercial marketability of the product. In this paper, a systematic approach is presented to develop a lyophilized high concentration protein formulation. The focus is on achieving reasonable reconstitution times with multidisciplinary strategies. Many strategies have been shown to provide nominal improvement in reconstitution times, such as adding wetting agents in the diluents, incorporating high annealing steps in the lyophilization cycle and reconstituting under vacuum. The reconstitution strategy of reduced diluent volume, however, has enabled significant decrease in reconstitution time (4-7-fold) of lyophilized high protein concentration formulations.

The effect of dryer load on freeze drying process design.

Freeze-drying using a partial load is a common occurrence during the early manufacturing stages when insufficient amounts of active pharmaceutical ingredient (API) are available. In such cases, the immediate production needs are met by performing lyophilization with less than a full freeze dryer load. However, it is not obvious at what fractional load significant deviations from full load behavior begin. The objective of this research was to systematically study the effects of variation in product load on freeze drying behavior in laboratory, pilot and clinical scale freeze-dryers. Experiments were conducted with 5% mannitol (high heat and mass flux) and 5% sucrose (low heat and mass flux) at different product loads (100%, 50%, 10%, and 2%). Product temperature was measured in edge as well as center vials with thermocouples. Specific surface area (SSA) was measured by BET gas adsorption analysis and residual moisture was measured by Karl Fischer. In the lab scale freeze-dryer, the molar flux of inert gas was determined by direct flow measurement using a flowmeter and the molar flux of water vapor was determined by manometric temperature measurement (MTM) and tunable diode laser absorption spectroscopy (TDLAS) techniques. Comparative pressure measurement (capacitance manometer vs. Pirani) was used to determine primary drying time. For both 5% mannitol and 5% sucrose, primary drying time decreases and product temperature increases as the load on the shelves decreases. No systematic variation was observed in residual moisture and vapor composition as load decreased. Further, SSA data suggests that there are no significant freezing differences under different load conditions. Independent of dryer scale, among all the effects, variation in radiation heat transfer from the chamber walls to the product seems to be the dominant effect resulting in shorter primary drying time as the load on the shelf decreases (i.e., the fraction of edge vials increases).

Impact of Uncontrolled vs Controlled Rate Freeze-Thaw Technologies on Process Performance and Product Quality.

Most biomolecules, owing to their marginal stability in liquid state, susceptibility to microbial growth, and tendency to foam upon storage/shipment in the liquid state, often require an alternate method of long-term storage. Cryopreservation is preferred, as it addresses most of these issues associated with liquid storage. However, the stability of the protein in the frozen state depends on the methodology of freezing/thawing and physico-chemical characteristics of the protein. A systematic study was undertaken to understand and evaluate the impact of freezing/thawing method on the process performance and product quality attributes using two freezing methods-conventional freezing in walk-in freezers and thawing in cold rooms using carboys as an uncontrolled rate method, and Celsius/CryoFin™ technologies as a controlled rate method. To assess the impact of freeze-thaw cycles on product quality, two types of proteins, a fusion protein and a peptibody (peptide fused to the Fc portion of the antibody), were used, employing appropriate stability-indicating assays. The results demonstrate superior process performance by the controlled rate freeze-thaw technology, both in terms of process times and cryoconcentration, compared to uncontrolled rate freeze thaw technology. Product impact studies indicate that the peptibody is sensitive to the method of freeze-thaw while the fusion protein is not and those that are sensitive to uncontrolled rate freeze-thaw processes can be effectively protected by controlled rate freeze-thaw technologies such as Celsius.

Development of freeze-dried biosynthetic Factor VIII: I. A case study in the optimization of formulation.

The purpose of this paper was to identify an optimal formulation, free of any human or animal derived protein, which stabilizes biosynthetic Factor VIII (rAHF) during freeze drying and storage. Factor VIII activity in samples stored at temperatures between 25 degrees C and 60 degrees C was determined using the one-stage activated partial thromboplastin time assay. Various formulations containing different combinations of a stabilizer and a bulking agent were screened for acceptable freeze-drying behavior, elegance of the resulting product, and stability during processing and storage. Degradation of freeze-dried rAHF followed the 'square root of time' kinetics. Stability of rAHF was found to increase with increasing protein concentration, indicating a self-protection effect. The addition of the antioxidant, glutathione was also shown to enhance storage stability. Given the constraint of high residual levels of NaCl from purification, the lead formulation employed mannitol as a bulking agent and trehalose as the general stabilizer. This formulation allowed an elegant product to be produced which more than met the stability requirements. However, it was also shown that elimination of residual NaCl allowed a much shorter freeze-drying cycle to produce an elegant product with greatly enhanced stability.

Mechanistic studies of glass vial breakage for frozen formulations. I. Vial breakage caused by crystallizable excipient mannitol.

The process of freeze-thaw not only subjects bioproducts to potentially destabilizing stress, but also imposes challenges to retain container integrity. Shipment and storage of frozen products in glass vials and thawing of the vials prior to use at clinics is a common situation. Vial integrity failure during freeze-thaw results in product loss and safety issues. Formulations of biomolecules often include crystallizable excipients, which can cause glass vial breakage during freeze-thaw operations. In this study, mannitol formulations served as models for mechanistic investigation of root causes for vial breakage. Several parameters and their impacts on vial breakage were investigated, including mannitol concentration (5% and 15%), different freeze-thaw conditions (fast, slow, and staging), fill configurations (varying fill volume/vial size ratio), and vial tray materials (plastic, stainless steel, corrugated cardboard, aluminum, and polyurethane foam). The results in this study were subjected to a statistical proportion test. The data showed that large fill volumes strongly correlated with higher percentage of vial cracks. Furthermore, the 15% mannitol was found to cause more breakage than 5% mannitol, especially with fast temperature gradient. Significantly more thawing vial breakage occurred in the fast compared to slow freeze-thaw with all types of vial trays. The freezing breakage was substantially lower than the thawing breakage using the fast temperature gradient, and the trend was reversed with the slow temperature gradient. An intermediate hold at -30 degrees C prior to further decrease in temperature proved to be a practical approach to minimize mannitol-induced vial breakage. Thermal mechanical analysis (TMA) and strain gage techniques were employed to gain mechanistic insights, and it was found that the primary causes for mannitol-induced vial breakage were partial crystallization during freezing and "secondary" crystallization of non-crystallized fraction during thawing. The strain on the vial's axial direction was significantly higher than the hoop direction, typically resulting in bottom lens of the vial coming off. Without a -30 degrees C hold, rapid volume expansions due to initial crystallization and secondary crystallization of mannitol were observed in TMA profiles, and these expansions were more apparent in 15% mannitol compared to 5% mannitol. With the introduction of a -30 degrees C hold step, abrupt expansions diminished in TMA profiles, suggesting that most of the mannitol crystallization occurred concurrently with ice solidification during the -30 degrees C holding step and, thus, secondary crystallization during thawing was minimal and the sudden expansion event was eliminated. Therefore, vial breakage during both freezing and thawing was reduced.

Mechanistic studies of glass vial breakage for frozen formulations. II. Vial breakage caused by amorphous protein formulations.

In an accompanying article we have described parameters that influence vial breakage in freeze-thaw operations when using crystalizable mannitol formulations, and further provided a practical approach to minimize the breakage in manufacturing settings. Using two diagnostic tools-thermal mechanical analysis (TMA) and strain gage, we investigated the mechanism of mannitol vial breakage and concluded that the breakage is related to sudden volume expansions in the frozen plug due to crystallization events. Glass vial breakage has also been observed with a number of frozen protein formulations consisting of only amorphous ingredients. Therefore, in this study, we applied the methodologies and learnings from the prior investigation to further explore the mechanism of vial breakage during freeze-thaw of amorphous protein products. It was found that temperature is a critical factor, as breakage typically occurred when the products were frozen to -70 degrees C, while freezing only to -30 degrees C resulted in negligible breakage. When freezing to -70 degrees C, increased protein concentration and higher fill volume induced more vial breakage, and the breakage occurred mostly during freezing. In contrast to the previous findings for crystallizable formulations, an intermediate staging step at -30 degrees C did not reduce breakage for amorphous protein formulations, and even slightly increased the breakage rate. The TMA profiles revealed substantially higher thermal contraction of frozen protein formulations when freezing below -30 degrees C, as compared to glass. Such thermal contraction of frozen protein formulations caused inward deformation of glass and subsequent rapid movement of glass when the frozen plug separates from the vial. Increasing protein concentration caused more significant inward glass deformation, and therefore a higher level of potential energy was released during the separation between the glass and frozen formulation, causing higher breakage rates. The thermal expansion during thawing generated moderate positive strain on glass and explained the thaw breakage occasionally observed. The mechanism of vial breakage during freeze-thaw of amorphous protein formulations is different compared to crystallizable formulations, and accordingly requires different approaches to reduce vial breakage in manufacturing. Storing and shipping at no lower than -30 degrees C effectively prevents breakage of amorphous protein solutions. If lower temperature such as -70 degrees C is unavoidable, the risk of breakage can be reduced by lowering fill volume.