Evaluation of a PAT-based in-line control system for a continuous spin freeze-drying process.

Continuous spin freeze-drying provides a range of opportunities regarding the implementation of several in-line process analytical technologies (PAT) to control and optimize the freeze-drying process at the individual vial level. In this work, two methods were developed to (1) control the freezing phase by separately controlling the cooling and freezing rate and (2) control the drying phase by controlling the vial temperature (and hence the product temperature) to setpoint values and monitoring the residual moisture content. During the freezing phase, the vial temperature closely followed the decreasing setpoint temperature during the cooling phases, and the crystallization phase was reproducibly controlled by regulating the freezing rate. During both primary and secondary drying, vial temperature could be maintained on the setpoint temperature which resulted in an elegant cake structure after every run. By being able to accurately control the freezing rate and the vial temperature, a homogeneous drying time (SD = 0.07-0.09 h) between replicates was obtained. Applying a higher freezing rate significantly increased primary drying time. On the other hand, fast freezing rates increased the desorption rate. Finally, the residual moisture of the freeze-dried formulation could be monitored in-line with a high accuracy providing insight on the required length of the secondary drying phase.

Development and Application of a Mechanistic Cooling and Freezing Model of the Spin Freezing Step within the Framework of Continuous Freeze-Drying.

During the spin freezing step of a recently developed continuous spin freeze-drying technology, glass vials are rapidly spun along their longitudinal axis. The aqueous drug formulation subsequently spreads over the inner vial wall, while a cold gas flow is used for cooling and freezing the product. In this work, a mechanistic model was developed describing the energy transfer during each phase of spin freezing in order to predict the vial and product temperature change over time. The uncertainty in the model input parameters was included via uncertainty analysis, while global sensitivity analysis was used to assign the uncertainty in the model output to the different sources of uncertainty in the model input. The model was verified, and the prediction interval corresponded to the vial temperature profiles obtained from experimental data, within the limits of the uncertainty interval. The uncertainty in the model prediction was mainly explained (>96% of uncertainty) by the uncertainty in the heat transfer coefficient, the gas temperature measurement, and the equilibrium temperature. The developed model was also applied in order to set and control a desired vial temperature profile during spin freezing. Applying this model in-line to a continuous freeze-drying process may alleviate some of the disadvantages related to batch freeze-drying, where control over the freezing step is generally poor.

Spin Freezing and Its Impact on Pore Size, Tortuosity and Solid State.

Spin freeze-drying, as a part of a continuous freeze-drying technology, is associated with a much higher drying rate and a higher level of process control in comparison with batch freeze-drying. However, the impact of the spin freezing rate on the dried product layer characteristics is not well understood at present. This research focuses on the relation between spin-freezing and pore size, pore shape, dried product mass transfer resistance and solid state of the dried product layer. This was thoroughly investigated via high-resolution X-ray micro-computed tomography (µCT), scanning electron microscopy (SEM), thermal imaging and solid state X-ray diffraction (XRD). It was concluded that slow spin-freezing rates resulted in the formation of highly tortuous structures with a high dried-product mass-transfer resistance, while fast spin-freezing rates resulted in lamellar structures with a low tortuosity and low dried-product mass-transfer resistance.

A NIR-Based Study of Desorption Kinetics during Continuous Spin Freeze-Drying.

The pharmaceutical industry is progressing toward the development of more continuous manufacturing techniques. At the same time, the industry is striving toward more process understanding and improved process control, which requires the implementation of process analytical technology tools (PAT). For the purpose of drying biopharmaceuticals, a continuous spin freeze-drying technology for unit doses was developed, which is based on creating thin layers of product by spinning the solution during the freezing step. Drying is performed under vacuum using infrared heaters to provide energy for the sublimation process. This approach reduces drying times by more than 90% compared to conventional batch freeze-drying. In this work, a new methodology is presented using near-infrared (NIR) spectroscopy to study the desorption kinetics during the secondary drying step of the continuous spin freeze-drying process. An inline PLS-based NIR calibration model to predict the residual moisture content of a standard formulation (i.e., 10% sucrose) was constructed and validated. This model was then used to evaluate the effect of different process parameters on the desorption rate. Product temperature, which was controlled by a PID feedback mechanism of the IR heaters, had the highest positive impact on the drying rate during secondary drying. Using a higher cooling rate during spin freezing was found to significantly increase the desorption rate as well. A higher filling volume had a smaller negative effect on the drying rate while the chamber pressure during drying was found to have no significant effect in the range between 10 and 30 Pa.

Lyophilization of NOTA-sdAbs: First step towards a cold diagnostic kit for 68Ga-labeling.

Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. This study investigates the possibility to lyophilize an anti-HER2 and an anti-MMR single-domain antibody fragment (sdAb)-based precursor as a first step in the development of a diagnostic kit for PET imaging. METHODS: NOTA-sdAb precursors have been lyophilized with the following formulation: 100 µg NOTA-sdAb in 0.1 M NaOAc (NaOAc), 5% (w/v%) mannitol-sucrose mix at a 2:1 ratio and 0.1 mg/mL polysorbate 80. During development of the formulation and drying cycle, factors such as cake appearance, glass transition temperature and residual moisture were analyzed to ensure qualitative and stable lyophilized samples. Stability studies of lyophilized precursor were conducted up to 18 months after storage at 2-8 °C by evaluating the precursor integrity, aggregation, functionality and 68Ga-labeling efficiency. A comparative biodistribution study (lyophilized vs non-lyophilized precursor) was conducted in wild type mice (n = 3) and in tumor bearing mice (n = 6). RESULTS: The lyophilized NOTA-anti-HER2 precursor shows consistent stability data in vitro for up to 12 months at 2-8 °C in three separate batches, with results indicating stability even for up to T18m. No aggregation, degradation or activity loss was observed. Radiochemical purity after 68Ga-labeling is consistent over a period of 12 months (RCP ≥ 95% at T12m). In vivo biodistribution analyses show a typical [68Ga]Ga-NOTA-anti-HER2 sdAb distribution profile and a comparable tumor uptake for the lyophilized compound vs non-lyophilized (5.5% vs 5.7 %IA/g, respectively). In vitro results of lyophilized NOTA-anti-MMR precursor indicates stability for up to 18 months, while in vivo data show a comparable tumor uptake (2.5% vs 2.8 %IA/g, respectively) and no significant difference in kidney retention (49.4% vs 47.5 %IA/g, respectively). CONCLUSION: A formulation and specific freeze-drying cycle were successfully developed to lyophilize NOTA-sdAb precursors for long-term storage at 2-8 °C. In vivo data show no negative impact of the lyophilization process on the in vivo behavior or functionality of the lyophilized precursor. These results highlight the potential to develop a kit for the preparation of 68Ga-sdAb-based radiopharmaceuticals.

Dual chamber cartridges in a continuous pharmaceutical freeze-drying concept: Determination of the optimal dynamic infrared heater temperature during primary drying.

The applicability of DCCs in a continuous freeze-drying concept based on spin freezing and infrared heating was evaluated. Maximum applicable filling volume was evaluated. Secondly the mechanistic model for the determination of the optimal dynamic infrared heater temperature during primary drying of regular vials during continuous freeze-drying was adapted and validated for DCCs. Finally, since spin frozen DCCs may be more prone to choked flow due to the small neck opening and the large product surface area, it was evaluated if the choked flow constraints in the model could be increased to improve the efficiency of the drying process. The experiments revealed that the maximum allowable filling volume for spin freezing at the current experimental setup was 0.8 ml which is 80% of the maximum filling volume. Applying the mechanistic model for the determination of the optimal dynamic infrared heater temperature during primary drying of the studied DCCs and experimentally verifying this determined infrared heater temperature trajectory resulted in an elegant freeze-dried product without visual signs of collapse. The experimentally determined primary drying time agreed with the one calculated based on the mechanistic model. Choked flow did not occur during the continuous freeze-drying of DCCs containing 3% sucrose or 3% mannitol.

The generation and use of recombinant extracellular vesicles as biological reference material.

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

Thermal Imaging as a Noncontact Inline Process Analytical Tool for Product Temperature Monitoring during Continuous Freeze-Drying of Unit Doses.

Freeze-drying is a well-established technique to improve the stability of biopharmaceuticals which are unstable in aqueous solution. To obtain an elegant dried product appearance, the temperature at the moving sublimation interface Ti should be kept below the critical product temperature Ti,crit during primary drying. The static temperature sensors applied in batch freeze-drying provide unreliable Ti data due to their invasive character. In addition, these sensors are incompatible with the continuous freeze-drying concept based on spinning of the vials during freezing, leading to a thin product layer spread over the entire inner vial wall. During continuous freeze-drying, the sublimation front moves from the inner side of the vial toward the glass wall, offering the unique opportunity to monitor Ti via noncontact inline thermal imaging. Via Fourier's law of thermal conduction, the temperature gradient over the vial wall and ice layer was quantified, which allowed the exact measurement of Ti during the entire primary drying step. On the basis of the obtained thermal images, the infrared (IR) energy transfer was computed via the Stefan-Boltzmann law and the dried product mass transfer resistance ( Rp) profile was obtained. This procedure allows the determination of the optimal dynamic IR heater temperature profile for the continuous freeze-drying of any product. In addition, the end point of primary drying was detected via thermal imaging and confirmed by inline near-infrared (NIR) spectroscopy. Both applications show that thermal imaging is a suitable and promising process analytical tool for noninvasive temperature measurements during continuous freeze-drying, with the potential for inline process monitoring and control.

Vacuum Induced Surface Freezing as an effective method for improved inter- and intra-vial product homogeneity.

The stochastic nature of nucleation makes it difficult to control batch homogeneity in conventional freezing, and this lack of control is in contrast with the current emphasis on Quality by Design. Among the techniques which have been developed to overcome this problem, Vacuum Induced Surface Freezing is probably the most promising for application in manufacturing, because it does not require additional equipment and can be scaled-up more easily than other proposed approaches. In this work, we summarize the impact of Vacuum Induced Surface Freezing on product morphology, and the efficiency of the subsequent drying steps as well. We will show that this controlled freezing approach is extremely beneficial for both the efficiency of the freeze-drying process, and the quality and homogeneity of the final product. The hope is that this work could contribute to the commercial implementation of controlled nucleation technology, overcoming the final resistance to its widespread use. It is our opinion that this would be a substantial improvement, beneficial for both the pharmaceutical industry and the end users.

Potential of Near-Infrared Chemical Imaging as Process Analytical Technology Tool for Continuous Freeze-Drying.

Near-infrared chemical imaging (NIR-CI) is an emerging tool for process monitoring because it combines the chemical selectivity of vibrational spectroscopy with spatial information. Whereas traditional near-infrared spectroscopy is an attractive technique for water content determination and solid-state investigation of lyophilized products, chemical imaging opens up possibilities for assessing the homogeneity of these critical quality attributes (CQAs) throughout the entire product. In this contribution, we aim to evaluate NIR-CI as a process analytical technology (PAT) tool for at-line inspection of continuously freeze-dried pharmaceutical unit doses based on spin freezing. The chemical images of freeze-dried mannitol samples were resolved via multivariate curve resolution, allowing us to visualize the distribution of mannitol solid forms throughout the entire cake. Second, a mannitol-sucrose formulation was lyophilized with variable drying times for inducing changes in water content. Analyzing the corresponding chemical images via principal component analysis, vial-to-vial variations as well as within-vial inhomogeneity in water content could be detected. Furthermore, a partial least-squares regression model was constructed for quantifying the water content in each pixel of the chemical images. It was hence concluded that NIR-CI is inherently a most promising PAT tool for continuously monitoring freeze-dried samples. Although some practicalities are still to be solved, this analytical technique could be applied in-line for CQA evaluation and for detecting the drying end point.

Developing a framework to model the primary drying step of a continuous freeze-drying process based on infrared radiation.

The continuous freeze-drying concept based on spinning the vials during freezing and on non-contact energy transfer via infrared (IR) radiation during drying, improves process efficiency and product quality (uniformity) compared to conventional batch freeze-drying. Automated control of this process requires the fundamental mechanistic modelling of each individual process step. Therefore, a framework is presented for the modelling and control of the continuous primary drying step based on non-contact IR radiation. The IR radiation emitted by the radiator filaments passes through various materials before finally reaching the spin frozen vial. The energy transfer was computed by combining physical laws with Monte Carlo simulations and was verified with experimental data. The influence of the transmission properties of various materials on the emitted IR radiation profile was evaluated. These results assist in the selection of proper materials which could serve as IR window in the continuous freeze-drying prototype. The modelling framework presented in this paper fits the model-based design approach used for the development of this prototype and shows the potential benefits of this design strategy by establishing the desired engineering parameters and by enabling the engineer to assess mechanical tolerances and material options.

Polymorphism of Indomethacin in Semicrystalline Dispersions: Formation, Transformation, and Segregation.

The crystallization of metastable crystal polymorphs in polymer matrices has been extensively reported in literature as a possible approach to enhance the solubility of poorly water-soluble drug compounds, yet no clarification of the mechanism of the polymorph formation has been proposed. The current work aims to elucidate the polymorphism behavior of the model compound indomethacin as well as the mechanism of polymorph selection of drugs in semicrystalline systems. Indomethacin crystallized as either the α- or τ-form, a new metastable form, or a mixture of the two polymorphs in dispersions containing different drug loadings in polyethylene glycol, poloxamer, or Gelucire as the result of the variation in the mobility of drug molecules. As a general rule, low molecular mobility of the amorphous drug favors the crystallization into thermodynamically stable forms whereas metastable crystalline polymorphs are preferred when the molecular mobility of the drug is sufficiently high. This rule provides insight into the polymorph selection of numerous active pharmaceutical ingredients in semicrystalline dispersions and can be used as a guide for polymorphic screening from melt crystallization by tuning the mobility of drug molecules. In addition, the drug crystallized faster while the polymer crystallized slower as the drug-loading increased with the maxima of drug crystallization rate in 70% indomethacin dispersion. Increasing the drug content in solid dispersions reduced the τ to α polymorphic transition rate, except for when the more stable form was initially dominant. The segregation of τ and α polymorphs as well as the polymorphic transformation during storage led to the inherent inhomogeneity of the semicrystalline dispersions. This study highlights and expands our understanding about the complex crystallization behavior of semicrystalline systems and is crucial for preparation of solid dispersions with reproducible and consistent physicochemical properties and pharmaceutical performance.

The relevance of shear, sedimentation and diffusion during spin freezing, as potential first step of a continuous freeze-drying process for unit doses.

Recently, a continuous freeze-drying process for the production of unit doses was presented and evaluated. In this concept, the freezing step is modified compared to traditional batch freeze-drying, as glass vials filled with a liquid formulation, are rotated around their longitudinal axis while cooled and frozen with a cold, sterile and inert gas (i.e. spin freezing). Finally, a thin frozen product layer spread over the entire vial wall is achieved. The aim of this paper is twofold: firstly, the relation between the rotation velocity and the relative difference between top and bottom of the frozen product layer thickness was determined for different vial types. Secondly, the impact of shear and centrifugal forces generated during spinning was examined, to find out whether they might cause pharmaceutical instability and sedimentation, respectively. Mechanistic and experimental evaluation showed that shear has no effect on proteins. Calculations showed that the sedimentation and diffusion velocity is too low to cause inhomogeneity in the product layer. In addition, Global Sensitivity Analysis (GSA) and Uncertainty Analysis (UA) were performed in order to account for the uncertainty of the used mechanistic model.

Global Sensitivity Analysis as Good Modelling Practices tool for the identification of the most influential process parameters of the primary drying step during freeze-drying.

Pharmaceutical batch freeze-drying is commonly used to improve the stability of biological therapeutics. The primary drying step is regulated by the dynamic settings of the adaptable process variables, shelf temperature Ts and chamber pressure Pc. Mechanistic modelling of the primary drying step leads to the optimal dynamic combination of these adaptable process variables in function of time. According to Good Modelling Practices, a Global Sensitivity Analysis (GSA) is essential for appropriate model building. In this study, both a regression-based and variance-based GSA were conducted on a validated mechanistic primary drying model to estimate the impact of several model input parameters on two output variables, the product temperature at the sublimation front Ti and the sublimation rate msub. Ts was identified as most influential parameter on both Ti and msub, followed by Pc and the dried product mass transfer resistance αRp for Ti and msub, respectively. The GSA findings were experimentally validated for msub via a Design of Experiments (DoE) approach. The results indicated that GSA is a very useful tool for the evaluation of the impact of different process variables on the model outcome, leading to essential process knowledge, without the need for time-consuming experiments (e.g., DoE).

Formulation Optimization of Freeze-Dried Long-Circulating Liposomes and In-Line Monitoring of the Freeze-Drying Process Using an NIR Spectroscopy Tool.

The effect of lyoprotectant type and concentration on the stability of freeze-dried prednisolone sodium phosphate-loaded long-circulating liposomes was investigated. Trehalose at a 5:1 carbohydrate to lipid molar ratio proved to be superior in maintaining the structural integrity and the permeability properties of the liposome bilayers, assuring the desired characteristics of the final product: a cake with a porous structure and easy to reconstitute, a similar size to the liposomes before freeze-drying, a high percent of encapsulated drug, and a low residual moisture content. Further on, the study demonstrated the possibility of near-infrared spectroscopy to provide valuable insights for detecting critical changes in acyl chain packing of the liposome bilayer. By visualizing the spectra after principal component analysis, one can predict if any harm has occurred to liposome integrity during the process. Moreover, near-infrared spectroscopy enabled us to determine the end points of primary and secondary drying without disturbing the normal freeze-drying procedure, which allowed us to gain a better understanding of the process and to improve process efficiency by optimizing the primary and secondary drying time.

Quantitative risk assessment via uncertainty analysis in combination with error propagation for the determination of the dynamic Design Space of the primary drying step during freeze-drying.

Traditional pharmaceutical freeze-drying is an inefficient batch process often applied to improve the stability of biopharmaceutical drug products. The freeze-drying process is regulated by the (dynamic) settings of the adaptable process parameters shelf temperature Ts and chamber pressure Pc. Mechanistic modelling of the primary drying step allows the computation of the optimal combination of Ts and Pc in function of the primary drying time. In this study, an uncertainty analysis was performed on the mechanistic primary drying model to construct the dynamic Design Space for the primary drying step of a freeze-drying process, allowing to quantitatively estimate and control the risk of cake collapse (i.e., the Risk of Failure (RoF)). The propagation of the error on the estimation of the thickness of the dried layer Ldried as function of primary drying time was included in the uncertainty analysis. The constructed dynamic Design Space and the predicted primary drying endpoint were experimentally verified for different RoF acceptance levels (1%, 25%, 50% and 99% RoF), defined as the chance of macroscopic cake collapse in one or more vials. An acceptable cake structure was only obtained for the verification runs with a preset RoF of 1% and 25%. The run with the nominal values for the input variables (RoF of 50%) led to collapse in a significant number of vials. For each RoF acceptance level, the experimentally determined primary drying endpoint was situated below the computed endpoint, with a certainty of 99%, ensuring sublimation was finished before secondary drying was started. The uncertainty on the model input parameters demonstrates the need of the uncertainty analysis for the determination of the dynamic Design Space to quantitatively estimate the risk of batch rejection due to cake collapse.

Modelling the primary drying step for the determination of the optimal dynamic heating pad temperature in a continuous pharmaceutical freeze-drying process for unit doses.

In the pharmaceutical industry, traditional freeze-drying of unit doses is a batch-wise process associated with many disadvantages. To overcome these disadvantages and to guarantee a uniform product quality and high process efficiency, a continuous freeze-drying process is developed and evaluated. The main differences between the proposed continuous freeze-drying process and traditional freeze-drying can be found firstly in the freezing step during which the vials are rotated around their longitudinal axis (spin freezing), and secondly in the drying step during which the energy for sublimation and desorption is provided through the vial wall by conduction via an electrical heating pad. To obtain a more efficient drying process, the energy transfer has to be optimised without exceeding the product and process limits (e.g. cake collapse, choked flow). Therefore, a mechanistic model describing primary drying during continuous lyophilisation of unit doses based on conduction via heating pads was developed allowing the prediction of the optimal dynamic power input and temperature output of the electric heating pads. The model was verified by experimentally testing the optimal dynamic primary drying conditions calculated for a model formulation. The primary drying endpoint of the model formulation was determined via in-line NIR spectroscopy. This endpoint was then compared with the predicted model based endpoint. The mean ratio between the experimental and model based predicted drying time for six verification runs was 1.05±0.07, indicating a good accordance between the model and the experimental data.

Mechanistic modelling of infrared mediated energy transfer during the primary drying step of a continuous freeze-drying process.

Conventional pharmaceutical freeze-drying is an inefficient and expensive batch-wise process, associated with several disadvantages leading to an uncontrolled end product variability. The proposed continuous alternative, based on spinning the vials during freezing and on optimal energy supply during drying, strongly increases process efficiency and improves product quality (uniformity). The heat transfer during continuous drying of the spin frozen vials is provided via non-contact infrared (IR) radiation. The energy transfer to the spin frozen vials should be optimised to maximise the drying efficiency while avoiding cake collapse. Therefore, a mechanistic model was developed which allows computing the optimal, dynamic IR heater temperature in function of the primary drying progress and which, hence, also allows predicting the primary drying endpoint based on the applied dynamic IR heater temperature. The model was validated by drying spin frozen vials containing the model formulation (3.9mL in 10R vials) according to the computed IR heater temperature profile. In total, 6 validation experiments were conducted. The primary drying endpoint was experimentally determined via in-line near-infrared (NIR) spectroscopy and compared with the endpoint predicted by the model (50min). The mean ratio of the experimental drying time to the predicted value was 0.91, indicating a good agreement between the model predictions and the experimental data. The end product had an elegant product appearance (visual inspection) and an acceptable residual moisture content (Karl Fischer).

Noncontact Infrared-Mediated Heat Transfer During Continuous Freeze-Drying of Unit Doses.

Recently, an innovative continuous freeze-drying concept for unit doses was proposed, based on spinning the vials during freezing. An efficient heat transfer during drying is essential to continuously process these spin frozen vials. Therefore, the applicability of noncontact infrared (IR) radiation was examined. The impact of several process and formulation variables on the mass of sublimed ice after 15 min of primary drying (i.e., sublimation rate) and the total drying time was examined. Two experimental designs were performed in which electrical power to the IR heaters, distance between the IR heaters and the spin frozen vial, chamber pressure, product layer thickness, and 5 model formulations were included as factors. A near-infrared spectroscopy method was developed to determine the end point of primary and secondary drying. The sublimation rate was mainly influenced by the electrical power to the IR heaters and the distance between the IR heaters and the vial. The layer thickness had the largest effect on total drying time. The chamber pressure and the 5 model formulations had no significant impact on sublimation rate and total drying time, respectively. This study shows that IR radiation is suitable to provide the energy during the continuous processing of spin frozen vials.

Impact of vacuum-induced surface freezing on inter- and intra-vial heterogeneity.

This paper aimed to study the impact of freezing on both within-batch (inter-vial) and within-product (intra-vial) heterogeneity. This analysis has been carried out using two freezing protocols, the conventional shelf-ramped method and the Vacuum Induced Surface Freezing, and placebo formulations containing both crystallizing (mannitol) and amorphous (lactose and sucrose) excipients. The freezing conditions (i.e., the temperature of freezing, the temperature and time of the equilibration phase, and the filling volume) were found to have a dramatic impact on both the within-batch and the within-product homogeneity. Overall, we observed that the control of freezing can effectively minimize the variability in product characteristics, and moisture content, within the same batch. In addition to more uniform production, the control of freezing was found to be fundamental to achieve a more uniform product than that produced by the shelf-ramped freezing method. The influence of the freezing protocol on the crystallization process of mannitol was also investigated, showing that the temperature of freezing plays a key role in the formation of the mannitol polymorphs.