Automated Tomographic Assessment of Structural Defects of Freeze-Dried Pharmaceuticals.

The topology and surface characteristics of lyophilisates significantly impact the stability and reconstitutability of freeze-dried pharmaceuticals. Consequently, visual quality control of the product is imperative. However, this procedure is not only time-consuming and labor-intensive but also expensive and prone to errors. In this paper, we present an approach for fully automated, non-destructive inspection of freeze-dried pharmaceuticals, leveraging robotics, computed tomography, and machine learning.

Molded Vial Manufacturing and Its Impact on Heat Transfer during Freeze-Drying: Vial Geometry Considerations.

Recent advances in molded vial manufacturing enabled manufacturers to use a new manufacturing technique to achieve superior homogeneity of the vial wall thickness. This study evaluated the influence of the different manufacturing techniques of molded vials and glass compositions on vial heat transfer in freeze-drying. Additionally, the influence of using empty vials as thermal shielding on thermal characteristics of edge and center vials was investigated. The vial heat transfer coefficient Kv was determined gravimetrically for multiple vial systems. The results showed superior heat transfer characteristics of the novel manufacturing technique as well as differences in heat transfer for the different glass compositions. Empty vials on the outside of the array did not influence center vial Kv values compared to a full array. The direct contact area and vial bottom curvature and their correlation to heat transfer parameters were analyzed across multiple vial systems. A new approach based on light microscopy to describe the vial bottom curvature more accurately was described. The presented results for the contact area allowed for an approximation of the pressure-independent heat transfer parameter KC. The results for the vial bottom curvature showed a great correlation to the pressure-dependent heat transfer parameter KD. Overall, the results highlighted how a thorough geometrical characterization of vials with known heat transfer characteristics could be used to predict thermal characteristics of new vial systems as an alternative to a time-consuming gravimetric Kv determination. Primary drying times were simulated to show the influence of Kv on drying performance.

Evaluation of Packaging Materials in Freeze-Drying: Use of Polymer Caps and Nested Vials and Their Impact on Process and Product Attributes.

Current trends in the pharmaceutical industry led to a demand for more flexible manufacturing processes with smaller batch sizes. Prepackaged nested vials that can be processed as a unit were introduced into the market to fulfill this need. However, vial nests provide a different thermal environment for the vials compared to a hexagonal packaging array and could therefore influence product temperature profiles, primary drying times, and product quality attributes. Polymer caps with the possibility of vial closure inside the freeze-drying chamber were developed to remove the risks and need of a crimping process. A general concern with the use of such caps is the possibility of an increase in resistance to water vapor flow out of the vial. This case study investigated the effect of the LyoSeal® and PLASCAP® polymer caps and EZ-fill® nests on the freeze-drying process. Amorphous and partially crystalline model formulations were freeze-dried. Process data and product quality attributes were compared for regularly stoppered vials and vials with polymer caps as well as vials in a hexagonal packaging array and nested vials. The results indicated no increased resistance or impeded water vapor flow by the polymer caps. Differences in the macro- and microscopic appearances of products and a trend towards lower product temperatures were observed for the investigated nest type compared to a regular hexagonal packaging array. Consequently, the polymer caps could be used as an alternative to regular stoppers without affecting freeze-drying process data or product quality attributes, while the different thermal environment of nested vials should be considered.

Cycle Development in a Mini-Freeze Dryer: Evaluation of Manometric Temperature Measurement in Small-Scale Equipment.

The objective of this research was to assess the applicability of manometric temperature measurement (MTM) and SMART™ for cycle development and monitoring of critical product and process parameters in a mini-freeze dryer using a small set of seven vials. Freeze drying cycles were developed using SMART™ which automatically defines and adapts process parameters based on input data and MTM feedback information. The freeze drying behavior and product characteristics of an amorphous model system were studied at varying wall temperature control settings of the cylindrical wall surrounding the shelf in the mini-freeze dryer. Calculated product temperature profiles were similar for all different wall temperature settings during the MTM-SMART™ runs and in good agreement with the temperatures measured by thermocouples. Product resistance profiles showed uniformity in all of the runs conducted in the mini-freeze dryer, but absolute values were slightly lower compared to values determined by MTM in a LyoStar™ pilot-scale freeze dryer. The resulting cakes exhibited comparable residual moisture content and optical appearance to the products obtained in the larger freeze dryer. An increase in intra-vial heterogeneity was found for the pore morphology in the cycle with deactivated wall temperature control in the mini-freeze dryer. SMART™ cycle design and product attributes were reproducible and a minimum load of seven 10R vials was identified for more accurate MTM values. MTM-SMART™ runs suggested, that in case of the wall temperature following the product temperature of the center vial, product temperatures differ only slightly from those in the LyoStar™ freeze dryer.

Impact of Payload Hydrophobicity on the Stability of Antibody-Drug Conjugates.

In silico screening of toxin payloads typically employed in ADCs revealed a wide range of hydrophobicities and sizes as measured by log P and topological polar surface area (tPSA) values. These descriptors were used to identify three nontoxic surrogate payloads that encompass the range of hydrophobicity defined by the ADC toxin training set. The uniform drug to antibody ratio (DAR) ADCs were prepared for each surrogate payload by conjugation to the interchain cysteine residues of a model IgG1 subtype mAb. Linkage of these surrogate payloads to a common mAb with a matched DAR value allowed for preliminary analytical interrogation of the influence of payload hydrophobicity on global structure, self-association, and aggregation properties. The results of differential scanning fluorimetry and dynamic light scattering experiments clearly revealed a direct correlation between the destabilization of the native mAb structure and the increasing payload hydrophobicity. Also, self-association/aggregation propensity examined by self-interaction biolayer interferometry or size exclusion HPLC was consistent with increased conversion of the monomeric mAb to higher order aggregated species, with the degree of conversion directly proportional to the payload hydrophobicity. In summary, these findings prove that the payload-dependent structure destabilization and enhanced propensity to self-associate/aggregate driven by the increasing payload hydrophobicity contribute to reduced ADC stability and more complex behavior when assessing exposure and safety/efficacy relationships.

Matrix Metalloproteinase Responsive Delivery of Myostatin Inhibitors.

PURPOSE: The inhibition of myostatin - a member of the transforming growth factor (TGF-ß) family - drives regeneration of functional skeletal muscle tissue. We developed a bioresponsive drug delivery system (DDS) linking release of a myostatin inhibitor (MI) to inflammatory flares of myositis to provide self-regulated MI concentration gradients within tissues of need. METHODS: A protease cleavable linker (PCL) - responding to MMP upregulation - is attached to the MI and site-specifically immobilized on microparticle surfaces. RESULTS: The PCL disintegrated in a matrix metalloproteinase (MMP) 1, 8, and particularly MMP-9 concentration dependent manner, with MMP-9 being an effective surrogate biomarker correlating with the activity of myositis. The bioactivity of particle-surface bound as well as released MI was confirmed by luciferase suppression in stably transfected HEK293 cells responding to myostatin induced SMAD phosphorylation. CONCLUSIONS: We developed a MMP-responsive DDS for MI delivery responding to inflammatory flare of a diseased muscle matching the kinetics of MMP-9 upregulation, with MMP-9 kinetics matching (patho-) physiological myostatin levels. ᅟ: Graphical Abstract Schematic illustration of the matrix metalloproteinase responsive delivery system responding to inflammatory flares of muscle disease. The protease cleavable linker readily disintegrates upon entry into the diseased tissue, therby releasing the mystatin inhibitor.

Freeze drying of orally disintegrating tablets containing taste masked naproxen sodium granules in blisters.

Orally disintegrating tablets (ODTs) were freeze dried in blisters using the Lyostar® II SMART™ Freeze Dryer Technology. ODT formulations either without non-water soluble particles (placebo) or containing large fractions (717 mg) of taste-masked naproxen sodium (NaS) granules were freeze dried. The process data revealed differences between ODTs with and without embedded granules in the pressure rise curves as well as in the shelf (inlet) temperature adjustments during freeze-drying. Pressure rise curves of the placebo ODTs from eight hours process time showed no distinct temperature-dominated part, and the last optimization step of the shelf temperature to achieve -24.4 °C might be prone to errors. The final shelf temperature of ODTs containing granules was -23.3 °C. The detection of primary drying endpoints using SMART™ Technology or comparative pressure measurements was reliable for both ODT formulations, whereas the application of thermocouples resulted in premature endpoint indication. Product resistance of ODTs containing granules was generally elevated in comparison to ODTs without granules, but increased only slightly over the course of the drying process. In summary, the developed freeze-drying cycle was found applicable for production of elegant ODTs with incorporated taste masked NaS granules.

Investigation of the stabilizing effects of hydroxyethyl cellulose on LDH during freeze drying and freeze thawing cycles.

The objective of this study was to investigate both the cryoprotective and lyoprotective effects of the polymer hydroxyethyl cellulose (HEC) on the model protein lactate dehydrogenase (LDH) during freeze thawing and freeze drying cycles. The effect of annealing on both protein stability and the physical state of HEC was evaluated. HEC was used as a sole excipient in the protein formulations, and its stabilizing was compared to that of other excipients which are commonly used in freeze dried protein formulations. Furthermore, other quality aspects of the freeze dried samples containing solely HEC were investigated, such as, reconstitution time and product elegance. Protein stability was evaluated functionally by measuring the activity recovery of the model protein LDH. The physical state of HEC after freeze drying was investigated and compared to this of other studied solutes using differential scanning calorimetry and X-ray powder diffractometry. HEC showed superior cryoprotective effects on LDH during freeze thawing, and considerable lyoprotective effects during the freeze drying process. Annealing had limited influence on the stabilizing effect of HEC. The extensive reconstitution times of the HEC lyophilisates could be greatly improved by incorporation of the surfactant Tween 80 into the formulations prior to freeze drying.

Taste masking of naproxen sodium granules by fluid-bed coating.

The taste of oral dosage forms is an important argument regarding patient's compliance and acceptability. For this reason, it is often necessary to mask an undesirable and unpleasant taste of an active pharmaceutical ingredient. The purpose of this study was to mask the taste of naproxen sodium by a new fluid-bed coating approach. Different compositions of coating suspensions were used to coat naproxen sodium granules. It was found that products with the addition of a plasticizer were not stable at 40 °C and tended to agglomerate. Subsequently, formulations without plasticizer were used and the ratio between water and Eudragit® E was varied. Increasing the fraction of water in the suspension from 3% to 14% reduced the effective release of naproxen sodium. An optimum ratio between naproxen sodium granules and Eudragit® E was found to be 1:1.576, where less naproxen sodium was released than the threshold bitter value and an appropriate taste masking for more than 5 min was guaranteed. Investigation of the particle size distribution revealed a d(10) of 138.35 ± 21.52 µm, a d(50 )= 256.40 ± 11.27 µm and a d(90 )= 500.85 ± 69.08 µm, which guarantees an acceptable mouthfeel for patients.

"Negative" Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) tend to be less stable than their parent antibodies, which is often attributed to the hydrophobic nature of their drug payloads. This study investigated how the payload charge affects ADC stability by comparing two interchain cysteine ADCs that had matched drug-to-antibody ratios and identical linkers but differently charged auristatin payloads, vcMMAE (neutral) and vcMMAF (negative). Both ADCs exhibited higher aggregation than their parent antibody under shaking stress and thermal stress conditions. However, conjugation with vcMMAF increased the aggregation rates to a greater extent than conjugation with uncharged but more hydrophobic vcMMAE. Consistent with the payload logD values, ADC-vcMMAE showed the greatest increase in hydrophobicity but minor changes in charge compared with the parent antibody, as indicated by hydrophobic interaction chromatography and capillary electrophoresis data. In contrast, ADC-vcMMAF showed a decrease in net charge and isoelectric point along with an increase in charge heterogeneity. This charge alteration likely contributed to a reduced electrostatic repulsion and increased surface activity in ADC-vcMMAF, thus affecting its aggregation propensity. These findings suggest that not only the hydrophobicity of the payload, but also its charge should be considered as a critical factor affecting the stability of ADCs.

Evaluating the Potential of Cyclodextrins in Reducing Aggregation of Antibody-Drug Conjugates with Different Payloads.

Cyclodextrins (CDs) are versatile agents used to solubilize small drugs and stabilize proteins. This dual functionality may be particularly beneficial for antibody-drug conjugates (ADCs), as CDs may "mask" the hydrophobicity of the drug payloads. In this study, we explored the effect of CDs on the physical stability of ADCs composed of the same antibody but with different payloads (maytansinoid, auristatin, and fluorophore payloads). The aggregation of ADCs was evaluated under shaking stress conditions and elevated temperatures using size-exclusion chromatography, turbidity, and backgrounded membrane imaging. Our results showed that hydroxypropyl-(HP)-CDs effectively stabilized all ADCs during shaking stress, with increasing stabilization in the order of HPαCD < HPγCD < HPßCD at concentrations of 7.5 mM and (near) complete stabilization at 75 mM. Native CDs without surface activity also stabilized certain ADCs, although less effectively than HP-CDs under agitation stress. During quiescent incubation, the HP-CD effects were small for most ADCs. However, for an ADC with a fluorophore payload that rapidly aggregated after conjugation, HPγCD substantially reduced aggregate levels, in line with fluorescence data supporting CD-ADC interactions. In contrast, sulfobutylether-ß-CD (SBEßCD) increased the aggregation rates in all ADCs under all stress conditions. In conclusion, this study highlights the potential of appropriate CD formulations to improve the physical stability of ADCs.

Agitation-Induced Aggregation of Lysine- And Interchain Cysteine-Linked Antibody-Drug Conjugates.

Drug conjugation to an antibody can affect its stability, which depends on factors such as the conjugation technique used, drug-linker properties, and stress encountered. This study focused on the effects of agitation stress on the physical stability of two lysine (ADC-K) and two interchain cysteine (ADC-C) conjugates of an IgG1 monoclonal antibody (mAb) linked to either ∼4 MMAE or DM1 payloads. During agitation, all antibody-drug conjugates (ADCs) exhibited higher aggregation than the mAb, which was dependent on the conjugation technique (aggregation of ADC-Ks > ADC-Cs) and drug-linker (aggregation of ADCs with MMAE > ADCs with DM1). The aggregation propensities correlated well with higher self-interaction, hydrophobicity, and surface activity of ADCs relative to the mAb. The intermediate reduced mAb (mAb-SH) showed even higher aggregation than the final product ADC-Cs. However, blocking mAb-SH's free thiols with N-ethylmaleimide (NEM) strongly reduced its aggregation, suggesting that free thiols should be minimized in cysteine ADCs. Further, this study demonstrates that a low-volume surface tension method can be used for estimating agitation-induced aggregation of ADCs in early development phases. Identifying liabilities to agitation stress and their relationship to biophysical properties may help optimize ADC stability.

Features in Backgrounds of Microscopy Images Introduce Biases in Machine Learning Analyses.

Subvisible particles may be encountered throughout the processing of therapeutic protein formulations. Flow imaging microscopy (FIM) and backgrounded membrane imaging (BMI) are techniques commonly used to record digital images of these particles, which may be analyzed to provide particle size distributions, concentrations, and identities. Although both techniques record digital images of particles within a sample, FIM analyzes particles suspended in flowing liquids, whereas BMI records images of dry particles after collection by filtration onto a membrane. This study compared the performance of convolutional neural networks (CNNs) in classifying images of subvisible particles recorded by both imaging techniques. Initially, CNNs trained on BMI images appeared to provide higher classification accuracies than those trained on FIM images. However, attribution analyses showed that classification predictions from CNNs trained on BMI images relied on features contributed by the membrane background, whereas predictions from CNNs trained on FIM features were based largely on features of the particles. Segmenting images to minimize the contributions from image backgrounds reduced the apparent accuracy of CNNs trained on BMI images but caused minimal reduction in the accuracy of CNNs trained on FIM images. Thus, the seemingly superior classification accuracy of CNNs trained on BMI images compared to FIM images was an artifact caused by subtle features in the backgrounds of BMI images. Our findings emphasize the importance of examining machine learning algorithms for image analysis with attribution methods to ensure the robustness of trained models and to mitigate potential influence of artifacts within training data sets.

Comparison of product drying performance in molded and serum tubing vials using gentamicin sulfate as a model system.

In a previous study, heat transfer coefficients of different 10 mL tubing and molded vials were determined gravimetrically via sublimation tests with pure water. Contrary to "conventional wisdom", only small differences in K(v) values between tubing and molded vials were found in the pressure range relevant for pharmaceutical freeze-drying. In order to investigate the impact of these relatively small differences on the primary drying time of an actual product, freeze-drying experiments with 5% gentamicin sulfate solution as a model system were performed at 68, 100 and 200 mTorr. The primary drying times of the API in recently developed molded (EasyLyo™), tubing (TopLyo™) and polymer vials (TopPac™) were compared. At 68 and 100 mTorr the primary drying time of the drug in the glass vials only differed by 3% to 4%, while the polymer vial took around 9% longer. At 200 mTorr, the API in the EasyLyo™ vials dried approximately 15% faster compared to the other vial types. The present study suggest that molded vials that have been modified in design to have better heat transfer properties can achieve drying times comparable to tubing vials.

Miniaturized Forced Degradation of Therapeutic Proteins and ADCs by Agitation-Induced Aggregation Using Orbital Shaking of Microplates.

Microplate-based formulation screening is a powerful approach to identify stabilizing excipients for therapeutic proteins while reducing material requirements. However, this approach is sometimes not representative of studies conducted in relevant container closures. The present study aimed to identify critical parameters for a microplate-based orbital shaking method to screen biotherapeutic formulations by agitation-induced aggregation. For this purpose, an in-depth methodological study was conducted using different shakers, microplates, and plate seals. Aggregation was monitored by size exclusion chromatography, turbidity, and backgrounded membrane imaging. Both shaker quality and liquid-seal contact had substantial impacts on aggregation during shaking and resulted in non-uniform sample treatment when parameters were not suitably selected. The well volume to fill volume ratio (Vwell/Vfill) was identified as an useful parameter for achieving comparable aggregation levels between different microplate formats. An optimized method (2400 rpm [ac 95 m/s2], Vfill 60-100 µL [Vwell/Vfill 6-3.6], 24 h, RT, heat-sealed) allowed for uniform sample treatment independent of surface tension and good agreement with vial shaking results. This study provides valuable guidance for miniaturization of shaking stress studies in biopharmaceutical drug development, facilitating method transfer and comparability between laboratories.

Robustness testing in pharmaceutical freeze-drying: inter-relation of process conditions and product quality attributes studied for a vaccine formulation.

CONTEXT: The recent US Food and Drug Administration (FDA) legislation has introduced the evaluation of the Design Space of critical process parameters in manufacturing processes. In freeze-drying, a "formulation" is expected to be robust when minor deviations of the product temperature do not negatively affect the final product quality attributes. OBJECTIVE: To evaluate "formulation" robustness by investigating the effect of elevated product temperature on product quality using a bacterial vaccine solution. MATERIALS AND METHODS: The vaccine solution was characterized by freeze-dry microscopy to determine the critical formulation temperature. A conservative cycle was developed using the SMART™ mode of a Lyostar II freeze dryer. Product temperature was elevated to imitate intermediate and aggressive cycle conditions. The final product was analyzed using X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), Karl Fischer, and modulated differential scanning calorimetry (MDSC), and the life cell count (LCC) during accelerated stability testing. RESULTS: The cakes processed at intermediate and aggressive conditions displayed larger pores with microcollapse of walls and stronger loss in LCC than the conservatively processed product, especially during stability testing. For all process conditions, a loss of the majority of cells was observed during storage. CONCLUSION: For freeze-drying of life bacterial vaccine solutions, the product temperature profile during primary drying appeared to be inter-related to product quality attributes.

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.

Stability of freeze-dried products subjected to microcomputed tomography radiation doses.

OBJECTIVES: Microcomputed tomography (µCT) is a powerful analytical tool for non-invasive structural analysis. The stability of drug substances and formulations subjected to X-ray radiation may be a concern in the industry. This study examines the effect of X-ray radiation on the stability of freeze-dried pharmaceuticals. The investigation is a proof of concept study for the safety of µCT X-ray radiation doses during the non-destructive investigation of freeze-dried products. METHODS: Different formulations of clotrimazole, insulin and l-lactate dehydrogenase were freeze-dried and the products exposed to a defined dose of radiation by µCT. Conservative freeze-drying conditions were used. Irradiated and normal samples were analysed for their stability directly after freeze-drying and after stability testing. KEY FINDINGS: The stability of model compounds was well maintained during freeze-drying. Some degradation of all compounds occurred during accelerated stability testing. The results showed no differences between the irradiated and normal state directly after freeze-drying and accelerated stability testing. CONCLUSIONS: No evidence of a detrimental effect of 100 Gy X-ray exposure on a model small molecule, peptide and protein compound was found while useful structural information could be obtained. Consequently, the technology may be useful as a non-destructive tool for product inspections if the formulation proves stable.

Design of Vacuum-Induced Freezing Protocols for High Fill Volume Formulations in Freeze-Drying: A Strategic Approach.

This case study proposes a development strategy for the SynchroFreeze vacuum-induced surface freezing technology for challenging high fill volume model systems. Critical steps during the development of a nucleation protocol are discussed as an example approach for implementing vacuum-induced surface freezing for high fill volume products. Slow pressure ramps and hold steps at adequate pressures have been found to be crucial for avoiding defects caused by either excessive outgassing or incomplete degassing. The evaporative mass loss during the SynchroFreeze procedure is characterized and thermal gradients during nucleation for several model systems with concentrations in the 50-400 mg/mL range are analyzed. The technology results in a measurable mass loss that may be relevant for low fill volume formulations. Thermal data show a pronounced temperature gradient throughout the entire product solution during nucleation by vacuum-induced surface freezing. The formulation composition, concentration, and shelf temperature have been shown to influence this temperature gradient. Reliable nucleation was achieved for sucrose formulations with concentrations up to 200 mg/mL at shelf temperatures minimally below the equilibrium freezing point.

Investigation of Two Different Pressure-Based Controlled Ice Nucleation Techniques in Freeze-Drying: The Integral Role of Shelf Temperature After Nucleation in Process Performance and Product Quality.

The purpose of this study was to investigate the impact of shelf temperature modifications during application of controlled ice nucleation techniques on process data and critical product quality attributes for a challenging, high-concentration and high-fill volume amorphous model system. Different freezing programs were applied and compared for the mechanistically different depressurization and vacuum-induced surface freezing techniques. Critical process data, such as product temperature and drying time, were analyzed. The final products were characterized with a focus on product morphology, residual moisture, reconstitution time and stability. The shelf temperature directly after primary nucleation showed a major influence on process performance and product quality attributes, with an isothermal hold step at an intermediate temperature leading to optimal results in terms of homogeneity and reduction of product temperatures and drying time for the model system used. The different controlled ice nucleation techniques led to significantly different results in terms of product morphology and process data, showing that the two mechanistically different controlled nucleation techniques are not interchangeable.