Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling.

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.

Don't shake it! Mechanical stress testing of mRNA-lipid nanoparticles.

Shaking stress studies are typically performed during formulation development to test the liability of a drug product towards interfacial stress occurring during transport, especially if a liquid formulation is desired. We evaluated various shaking procedures using a polyA-surrogate solution and verified our findings by eGFP-LNP cell-expression experiments. Shaking on an orbital shaker in vertical and horizontal orientations at increasing speeds from 300 to 600 rpm resulted in decreasing levels of encapsulated nucleic acid content, larger LNP sizes, and decreasing PDI. We report that vertical and horizontal shaking of both polyA- and eGFP-LNPs led to white deposits on the inner glass vial surface, depending on time, rpm, and temperature. Increasing the fill volume/smaller headspace (0.3 versus 0.9 mL fill) did not mitigate this phenomenon in the studied configuration, and the use of hydrophobic primary packaging even accelerated the formation of white deposits. In contrast, we demonstrated that a lyophilized polyA-LNP dosage form was less susceptible to shaking and maintained cake integrity and product properties. Multiple vortexing steps resulted in an increase in LNP size, PDI, and a decrease in encapsulated polyA content. We conclude that shaking experiments of nucleic acid-loaded LNPs in their final configuration at intended transport conditions need to be considered during technical development.

Filling Unit Operation for Biological Drug Products: Challenges and Considerations.

One of the key unit operations during the aseptic fill-finish process of parenteral products, such as biologics, is the filling process of the formulated, sterile filtered drug substance into primary packaging containers. The applied filling technology as well as the process performance majorly impacts final drug product quality. The present review provides an overview of commonly used filling technologies during fill-finish operations of biologics including positive displacement pump systems such as radial peristaltic pump, rotary piston pump, rolling diaphragm pump, or innovative systems such as the linear peristaltic pump, as well as time-over-pressure filling technology. The article describes the operating principle of each pump system and reviews advantages and drawbacks. We highlight specific considerations for individual systems, such as the risk of protein particle formation and particle shedding from wear and tear of tubing, and discuss current literature about general challenges associated with the filling process, such as hydrogen peroxide uptake, adsorption phenomena to tubing material, and needle clogging. We suggest process development and process characterization studies to assess the impact of the filling process on product quality, and lastly provide an outlook about the use of disposable equipment during filling operations related to sustainability considerations.

Liquid crystal phase formation and non-Newtonian behavior of oligonucleotide formulations.

Viscosity behavior of liquid oligonucleotide therapeutics and its dependence on formulation properties has been poorly studied to date. We observed a high increase in viscosity and solidification of therapeutic oligonucleotide formulations with increasing oligonucleotide concentration creating challenges during drug product manufacturing. In this study, we characterized the viscosity behavior of three different single strand DNA oligonucleotides based on oligonucleotide concentration and formulation composition. We subsequently studied the underlying mechanism for increased viscosity at higher oligonucleotide concentrations by dynamic light scattering (DLS), 1H nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and polarized light microscopy. Viscosity was highly dependent on formulation composition, oligonucleotide sequence, and concentration, and especially dependent on the presence and combination of different individual ions, such as the presence of sodium chloride in the formulation. In samples with elevated viscosity, the viscosity behavior was characterized by non-Newtonian, shear-thinning flow behavior. We further studied these samples by DLS and 1H NMR, which revealed the presence of supra-molecular assemblies, and further characterization by polarized light and DSC characterized these assemblies as liquid crystals in the formulation. The present study links the macroscopic viscosity behavior of oligonucleotide formulations to the formation of supra-molecular assemblies and to the presence of liquid crystals, and highlights the importance of formulation composition selection for these therapeutics.

Metal-Induced Fatty Acid Particle Formation Resulting from Hydrolytic Polysorbate Degradation.

The occurrence of visible particles over the shelf-life of biopharmaceuticals is considered a potential safety risk for parenteral administration. In many cases, particle formation resulted from the accumulation of fatty acids released by the enzymatic hydrolysis of the polysorbate surfactant by co-purified host cell proteins. However, particle formation can occur before the accumulated fatty acids exceed their expected solubility limit. This early onset of particle formation is driven by nucleation phenomena e.g. the presence of metal cations that promote the formation and growth of fatty acid particles. To further characterize and understand this phenomenon, we assessed the potential of different metal cations to induce fatty acid particle formation using a dynamic light scattering assay. We demonstrated that the presence of trace amounts of multivalent cations, in particular trivalent cations such as aluminum and iron, may act as nucleation seed in the process of particle formation. Finally, we developed a mitigation strategy for metal-induced fatty acid particles that deploys a chelator to reduce the risk of particle formation in biopharmaceutical formulations.

Mechanistic Study of Drying Phenomena of Highly Concentrated Protein Therapeutics-Drying Kinetics and Protein Aggregation.

The fill-finish process of highly concentrated protein formulations poses several technical challenges and, in particular, the filling process is critical to ensure filling accuracy. As highly concentrated formulations comprise a significant nonvolatile fraction, drying of drug product at the filling nozzle may occur during line interruptions. In many cases, this is a result of dripping at the filling nozzle and is dependent on environmental factors. The dried product may be picked up by the units after filling interruption and, although in small quantities, the effect of drying on the quality of the drug product is currently unclear. We investigated the drying phenomenon of a highly concentrated monoclonal antibody formulation (120 mg/mL) and studied the drying kinetics and associated aggregation propensity. In this regard, we established a robust method simulating the drying process dependent on environmental conditions such as relative humidity and air flow. We revealed that the drying kinetics were characterized by an initial fast evaporation phase, which was shorter for lower relative humidity and air flow, followed by a plateau phase. Protein aggregation particularly increased during the plateau phase and was positively correlated with relative humidity. Drying kinetics and aggregate formation were modeled using Hill's equation. We highlight that drying phenomena are relevant for small-volume drug products (magnitude of 100-200 µL), in particular for dosing accuracy, but less critical for larger fill volumes in the milliliter range. Especially for the latter, they might be negligible if the dried product can fully dissolve in the first units after filling interruption and in case of consistent drug product quality because of adequate formulation choice. Ultimately, we summarize technical options to avoid drying phenomena of highly concentrated protein formulations and emphasize the importance of adequate pump parameter setting.

Intraocular pressure and injection forces during intravitreal injection into enucleated porcine eyes.

Injection of biological molecules into the intravitreous humor is of increasing interest for the treatment of posterior segment eye diseases such as age-related degenerative macular degeneration. The injection volume is limited by an increase in intraocular pressure (IOP) and 50-100 µL are typically used for most intravitreally (IVT) applied commercial products. Direct measurement of IOP is difficult and has not been studied dependent on solution properties and injection rates. We used an instrumental set-up to study IOP ex vivo using healthy enucleated porcine eyes. IOP was determined as a function of injection volume for viscosities between 1 and 100 mPas, injection rates of 0.1, 1, and 1.5 mL/min, and needle length and diameter (27/30G and 0.5/0.75″) using Dextran solutions. IOP increased exponentially for injection volumes larger than 100 µL. We did not observe differences in IOP dependent on viscosity, injection rate, and needle diameter. However, variability increased significantly for injection volumes larger than 100 µL and, unexpectedly, declined with higher viscosities. We demonstrate that the exponential increase in IOP is not reflected by injection force measurements for typical configurations that are used for IVT application. The present findings may guide injection volumes for intravitreal injection and inform injection force considerations during technical drug product development.

Opportunities in an Evolving Pharmaceutical Development Landscape: Product Differentiation of Biopharmaceutical Drug Products.

The current perspective reviews the biopharmaceutical market until end of 2020 and highlights the transforming biopharmaceutical landscape during the recent decade. In particular, the rise of biosimilars and the development of new therapeutic modalities through recent advancement in molecular biology research sustainably change the product scenery. The present manuscript describes opportunities for pharmaceutical technical development, highlighting concepts such as product differentiation to succeed in a competitive product landscape. Product differentiation offers the opportunity for numerous life-cycle options and market exclusivity through incremental improvements in standard of care treatment. In particular, different formulation options and formulation-device combinations are described, focusing on systemic delivery of monoclonal antibody products and patient-centered development. The concept of product differentiation is exemplified in a case study about HER2+ breast cancer therapy, underlining pharmaceutical technical solutions and major improvements for the patient.

Glass Leachables as a Nucleation Factor for Free Fatty Acid Particle Formation in Biopharmaceutical Formulations.

Surfactants are essential components in protein formulations protecting them against interfacial stress. One of the current industry-wide challenges is enzymatic degradation of parenteral surfactants such as polysorbate 20 (PS20) and polysorbate 80, which leads to the accumulation of free fatty acids (FFAs) potentially forming visible particles over the drug product shelf-life. While the concentration of FFAs can be quantified, the time point of particle formation remains unpredictable. In this work, we studied the influence of glass leachables as nucleation factors for FFA particle formation. We demonstrate the feasibility of nucleation of FFA particles in the presence of inorganic salts like NaAlO2 and CaCl2 simulating relevant glass leachables. We further demonstrate FFA particle formation depending on relevant aluminum concentrations. FFA particle formation was subsequently confirmed with lauric/myristic acid in the presence of different quantities and compositions of glass leachables obtained by several sterilization cycles using different types of glass vials. We further verified the formation of particles in aged protein formulation containing degraded PS20 through the spiking of glass leachables. Particles were characterized as a complex of glass leachables, such as aluminum and FFAs. Based on our findings, we propose a likely pathway for FFA particle formation that considers specific nucleation factors.

Correction to: Tissue Resistance during Large-Volume Injections in Subcutaneous Tissue of Minipigs.

Table 2 of the article 'Tissue Resistance during Large-Volume Injections in Subcutaneous Tissue of Minipigs' encountered formatting errors in the (original) pdf version with misplacement of text and columns.

Tissue Resistance during Large-Volume Injections in Subcutaneous Tissue of Minipigs.

PURPOSE: Injection devices for administration of biopharmaceuticals enable subcutaneous self-administration by patients. To meet patient specific capabilities, injection forces need to be characterized. We address the open question of whether tissue resistance significantly contributes to overall injection forces, especially for large injection volumes. METHODS: Subcutaneous tissue resistance was systematically quantified for injection volumes up to 11 mL depending on viscosity (1-20 mPa·s) and injection rates (0.025-0.2 mL/s) using Göttingen Minipigs as the animal model. The contribution of an artificially applied external force at the injection site simulating autoinjector needle cover depression was tested between 2.5-7.5 N. RESULTS: Tissue resistance reached average values of ~120 mbar for injection volumes up to 11 mL independent of viscosity and injection rate, and maximum values of 300 mbar were determined. Artificially applied external forces led to higher values, independent of the absolute applied force - maximum values of 1 bar were obtained when injecting 4.5 mL of the 20 mPa·s solution at an injection rate of 0.1 mL/s with the application of an artificial 5 N force, corresponding to ~450 mbar. All conditions yield defined injection sites suggesting tissue resistance is defined by mechanical properties of the subcutaneous tissue. CONCLUSIONS: We set our results in relation to overall injection forces, concluding that maximum values in tissue resistance may cause challenges during subcutaneous injection when using injection devices. Graphical abstract.

Near-Infrared Spectroscopy to Determine Residual Moisture in Freeze-Dried Products: Model Generation by Statistical Design of Experiments.

Moisture content (MC) is a critical quality attribute of lyophilized biopharmaceuticals and can be determined by near-infrared (NIR) spectroscopy as nondestructive alternative to Karl-Fischer titration. In this study, we create NIR models to determine MC in mAb lyophilisates by use of statistical design of experiments (DoE) and multivariate data analysis. We varied the composition of the formulation as well as lyophilization parameters covering a large range of representative conditions, which is commonly referred to as "robustness testing" according to quality-by-design concepts. We applied principles of chemometrics with partial least squares and principal component analysis. The NIR model excluded samples with complete collapse and MC > 6%. The 2 main components in the principal component analysis were MC (91%) and protein:sugar ratio (6%). The third component amounted to only 3% and remained unspecified but may include variations in process parameters and cake structure. In contrast to traditional approaches for NIR model creation, the DoE-based model can be used to monitor MC during drug product development work including scale-up, and transfer without the need to update the NIR model if protein:sugar ratio and MC stays within the tested limits and cake structure remains macroscopically intact. The use of the DoE approach and multivariate data analysis ensures product consistency and improves understanding of the manufacturing process.

Excipients for Room Temperature Stable Freeze-Dried Monoclonal Antibody Formulations.

Sucrose is a common cryoprotectant and lyoprotectant to stabilize labile biopharmaceuticals during freeze-drying and storage. Sucrose-based formulations require low primary drying temperatures to avoid collapse and monoclonal antibody (mAb) containing products need to be stored refrigerated. The objective of this study is to investigate different excipients enabling storage at room temperature and aggressive, shorter lyophilization cycles. We studied combinations of 2-hydroxypropyl-beta-cyclodextrin (CD), recombinant human albumin, polyvinylpyrroldione (PVP), dextran 40 kDa (Dex), and sucrose (Suc) using 2 mAbs. Samples were characterized for collapse temperature (Tc), glass transition temperature of the liquid (Tg') and freeze-dried formulation (Tg), cake appearance, residual moisture, and reconstitution time. Freeze-dried formulations were stored at 5°C, 25°C, and 40°C for up to 9 months and mAb stability was analyzed for color, turbidity, visible and sub-visible particles, and monomer content. Formulations with CD/Suc or CD/PVP/Suc were superior to pure Suc formulations for long-term storage at 40°C. When using aggressive freeze-drying cycles, these formulations were characterized by pharmaceutically elegant cakes, short reconstitution times, higher Tg', Tc, and Tg. We conclude that the addition of CD allows for shorter freeze-drying cycles with improved cake appearance and enables storage at room temperature, which might reduce costs of goods substantially.

Impact of dextran on thermal properties, product quality attributes, and monoclonal antibody stability in freeze-dried formulations.

Freeze-drying is commonly used to improve stability of liquid formulations of labile biopharmaceuticals. Lyo- and cryoprotectants such as sucrose are traditionally utilized as excipients, but have low glass transition (Tg') and collapse temperatures (Tc). Consequently, these formulations require low primary drying temperatures making the lyophilization cycle time-consuming and costly. We investigated different dextrans (1, 40, 150, and 500 kDa) and mixtures of dextran with sucrose as alternative excipients. The influence of dextran on thermal properties, cake appearance, and other quality attributes in the solid state was studied using bovine serum albumin as model protein. Especially at higher weight ratios of dextran to sucrose, dextrans of medium to high molecular weight (MW) of 40-500 kDa showed up to 20 °C higher Tc compared to sucrose, which was reflected in elegant lyophilisates. However, this resulted in slower reconstitution times. Addition of dextran led to lower residual moisture levels and higher Tg values compared to sucrose. We confirmed the thermal properties for two monoclonal antibodies (mAb) at two weight ratios of sucrose and dextran with different MW, and tested for stability at 40 °C for 14 days. While no loss in relative potency of the antibodies was observed after storage, size exclusion chromatography and isoelectric focusing revealed a strong increase in high molecular weight species (HMWs) and acidic species, which were dependent on the MW of the dextrans. With further characterization of selected formulations (dextran 1 kDa) by boronate affinity chromatography and mass spectrometry analysis, we demonstrated that HMWs were a result of glycation by free terminal glucose of the dextran. This chemical modification was strongly reduced when adding sucrose, which protects the protein possibly by shielding its surface. Our results demonstrate that formulation scientists need to use dextrans as excipients in freeze-dried mAb formulations with caution. A binary mixture of sucrose and dextran in adequate ratio however might potentially be superior to pure sucrose formulations allowing for faster freeze-drying cycles resulting in elegant lyophilisates and good protein stability.

Be Aggressive! Amorphous Excipients Enabling Single-Step Freeze-Drying of Monoclonal Antibody Formulations.

Short freeze-drying cycles for biopharmaceuticals are desirable. Formulations containing an amorphous disaccharide, such as sucrose, are prone to collapse upon aggressive primary drying at higher shelf temperature. We used 2-hydroxypropyl-betacyclodextrin (HPBCD) in combination with sucrose and polyvinylpyrrolidone (PVP) to develop an aggressive lyophilization cycle for low concentration monoclonal antibody (mAb) formulations. Glass transition temperature and collapse temperature of the formulations were determined, and increasingly aggressive cycle parameters were applied. Using a shelf temperature of +30 °C during primary drying, the concept of combining sublimation and desorption of water in a single drying step was investigated. Cake appearance was evaluated visually and by micro-computed tomography. Lyophilisates were further analyzed for reconstitution time, specific surface area, residual moisture, and glass transition temperature. We demonstrated the applicability of single-step freeze-drying, shortening the total cycle time by 50% and providing elegant lyophilisates for pure HPBCD and HPBCD/sucrose formulations. HPBCD/PVP/sucrose showed minor dents, while good mAb stability at 10 mg/mL was obtained for HPBCD/sucrose and HPBCD/PVP/sucrose when stored at 40 °C for 3 months. We conclude that HPBCD-based formulations in combination with sucrose are highly attractive, enabling aggressive, single-step freeze-drying of low concentration mAb formulations, while maintaining elegant lyophilisates and ensuring protein stability at the same time.

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS).

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.

Imaging Techniques to Characterize Cake Appearance of Freeze-Dried Products.

Pharmaceutically elegant lyophilisates are highly desirable implying a stable and robust freeze-drying process. To ensure homogenous and intact cake appearance after process scale-up and transfer, characterization of lyophilisates during formulation and cycle development is required. The present study investigates different imaging techniques to characterize lyophilisates on different levels. Cake appearance of freeze-dried bovine serum albumin formulations with different dextran/sucrose ratios was studied by visual inspection, three-dimensional laser scanning, polydimethylsiloxane embedding, scanning electron microscopy, and microcomputed tomography (µ-CT). The set of techniques allowed a holistic evaluation of external cake appearance and internal structure providing complementary information at macroscopic and microscopic scale. In comparison to state of the art technologies like visual inspection or scanning electron microscopy, three-dimensional laser scanning and µ-CT provided quantitative information allowing comparison of visual cake appearance. In particular µ-CT enables a global, qualitative, and quantitative characterization of external and internal cake structure with a single measurement detecting heterogeneities of lyophilisates. We even demonstrated the use of noninvasive µ-CT for qualitative imaging of internal cake structure through the glass vial. Providing meaningful characterization of the entire lyophilisate, µ-CT can serve as a powerful tool during development of freeze-drying cycles, process scale-up, and transfer.

Impact of Vial Washing and Depyrogenation on Surface Properties and Delamination Risk of Glass Vials.

PURPOSE: The proper understanding of glass delamination is important to glass manufacturers, pharmaceutical companies, and health authorities to mitigate the occurrence of glass flakes from the vial when in contact with specific drug product solutions. The surface of glass vials is altered during glass cane- and vial forming processes and is exposed to different stress conditions during drug product processing before coming in contact with the drug product solution. In this study, the impact of vial washing and depyrogenation including an evaluation of various residual water volumes on surface properties of glass vials was investigated for a defined set of vials. METHODS: 3D laser scanning microscopy was established as a new method for topographic analysis of curved surfaces of glass vials operating in high-throughput mode. A subset of vials was subsequently exposed to delamination stress testing and both the stressed solution and inner vial surface were analyzed by a panel of conventional and advanced analytical techniques including 3D laser scanning microscopy. RESULTS: The data showed that vial washing and depyrogenation strongly influenced surface properties, in particular those of uncoated vials. Surface characteristics such as pits increased depending on the process conditions, which especially applies to Expansion 33 vials. Even low residual water volumes of 50 µL after vial washing were sufficient to change the surface properties of the glass and weaken the surface in those positions prone to glass delamination. An increase in pits was related to a greater risk for glass delamination. CONCLUSIONS: Vial processing conditions need to be assessed when aiming at minimizing the glass delamination risk during parenteral product storage.

Characterization of surface properties of glass vials used as primary packaging material for parenterals.

The appropriate selection of adequate primary packaging, such as the glass vial, rubber stopper, and crimp cap for parenteral products is of high importance to ensure product stability, microbiological quality (integrity) during storage as well as patient safety. A number of issues can arise when inadequate vial material is chosen, and sole compliance to hydrolytic class I is sometimes not sufficient when choosing a glass vial. Using an appropriate pre-treatment, such as surface modification or coating of the inner vial surface after the vial forming process the glass container quality is often improved and interactions of the formulation with the surface of glass may be minimized. This study aimed to characterize the inner surface of different type I glass vials (Exp33, Exp51, Siliconized, TopLyo™ and Type I plus®) at the nanoscale level. All vials were investigated topographically by colorimetric staining and Scanning Electron Microscopy (SEM). Glass composition of the surface was studied by Time-of-Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) and X-ray Photoelectron Spectroscopy (XPS), and hydrophobicity/hydrophilicity of the inner surface was assessed by dye tests and surface energy measurements. All containers were studied unprocessed, as received from the vendor, i.e. in unwashed and non-depyrogenized condition. Clear differences were found between the different vial types studied. Especially glass vials without further surface modifications, like Exp33 and Exp51 vials, showed significant (I) vial-to-vial variations within one vial lot as well as (II) variations along the vertical axis of a single vial when studying topography and chemical composition. In addition, differences and heterogeneity in surface energy were found within a given tranche (circumferential direction) of Exp51 as well as Type I plus® vials. Most consistent quality was achieved with TopLyo™ vials. The present comprehensive characterization of surface properties of the different vial types may serve as basis to further guide the selection of adequate primary packaging based on the desired quality target product profile and to support studies of glass surface interactions with formulations. The proposed analytical method panel can be used for characterization of future glass vials either before delivery to the manufacturer or drug product manufacturing.

Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations.

Solid state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) has been used to assess protein conformation and matrix interactions in lyophilized solids. ssHDX-MS metrics have been previously correlated to the formation of aggregates of lyophilized myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal antibody (mAb) formulations and correlated to their long-term stability. After exposing lyophilized samples to D2O(g), the amount of deuterium incorporated at various time points was determined by mass spectrometry for four different lyophilized mAb formulations. Hydrogen-deuterium exchange data were then correlated with mAb aggregation and chemical degradation, which was obtained in stability studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized mAb formulations determined at the initial time point prior to storage in the dry state was directly and strongly correlated with the extent of aggregation and chemical degradation during storage. Other measures of physical and chemical properties of the solids were weakly or poorly correlated with stability. The data demonstrate, for the first time, that ssHDX-MS results are highly correlated with the stability of lyophilized mAb formulations. The findings thus suggest that ssHDX-MS can be used as an early read-out of differences in long-term stability between formulations helping to accelerate formulation screening and selection.