Stability Comparison Between Microglassification and Lyophilization Using a Monoclonal Antibody.

Producing solid-state formulations of biologics remains a daunting task despite the prevalent use of lyophilization and spray drying technologies in the biopharmaceutical industry. The challenges include protein stability (temperature stresses), high capital costs, particle design/controllability, shortened processing times and manufacturing considerations (scalability, yield improvements, aseptic operation, etc.). Thus, scientists/engineers are constantly working to improve existing methodologies and exploring novel dehydration/powder-forming technologies. Microglassification™ is a dehydration technology that uses solvent extraction to rapidly dehydrate protein formulations at ambient temperatures, eliminating the temperature stress experienced by biologics in traditional lyophilization and spray drying methods. The process results in microparticles that are spherical, dense, and chemically stable. In this study, we compared the molecular stability of a monoclonal antibody formulation processed by lyophilization to the same formulation processed using Microglassification™. Both powders were placed on stability for 3 months at 40 °C and 6 months at 25 °C. Both dehydration methods showed similar chemical stability, including percent monomer, charge variants, and antigen binding. These results show that Microglassification™ is viable for the production of stable solid-state monoclonal antibody formulations.

Electrostatic spray drying for monoclonal antibody formulation.

This study explored the feasibility of electrostatic spray drying for producing a monoclonal antibody (mAb) powder formulation at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70 °C failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature and 5 kV charge enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40 °C. Low-temperature (70 °C inlet temperature) drying with an electrostatic charge (5 kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130 °C inlet temperature) without charge. This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at lower inlet temperature than traditional spray drying with better physical stability.

A Review on Mixing-Induced Protein Particle Formation: The Puzzle of Bottom-Mounted Mixers.

Mixing is an important unit operation in monoclonal antibody manufacturing. The goal is to achieve homogeneity without compromising product quality. Mixing-induced protein degradation and protein subvisible particle (SvP) formation, which impacts product quality, are associated with 2 common stress modes: mechanical shear and air-liquid interfacial stress, which can generally be overcome by formulation optimization. This review addresses a unique stress mechanism that caused SvP formation when using certain bottom-mounted mixers equipped with impellers propelled by magnetic or mechanical coupling with a drive unit. During use, the coupling assembly is submerged in the protein solution allowing liquid access into the gap between the 2 bearings. Based on data from studies of bottom-mounted mixers and other small-scale mixers, grinding of the 2 bearings is a condition for inducing particulate formation. Although grinding stress is an accepted cause, identifying the responsible stress mechanism is challenging. By applying small-scale models, researchers attempted to elucidate the modes of stress, which ranged from common stress (mechanical shear; interfacial stress/adsorption; cavitation) to more speculative hypotheses (nucleation from nano- and micro-particles; localized thermal stress). Recent literature was reviewed, and recommendations are offered to development scientists and process engineers regarding mixer design to reduce protein SvP formation during mixing of monoclonal antibody formulations.

Quantifying the Vial-Capping Process: Reexamination Using Micro-Computed Tomography.

A vial-capping process for lyophilization stopper configurations was previously quantified using residual seal force (RSF). A correlation between RSF and container closure integrity (CCI) was established, and component positional offsets were identified to be the primary source of variability in RSF measurements.To gain insight into the effects of stopper geometry on CCI, serum stoppers with the same rubber formulation were investigated in this study. Unlike lyophilization stoppers that passed CCI (per helium leak testing) even with RSF of 0 N owing to their excellent valve seal, serum stoppers consistently failed CCI when RSF was <15.8 N. When the plug was removed, both types of stoppers exhibited a comparable critical lower RSF limit (19-20 N), below which CCI could not be maintained. When CCI was retested at later time points (up to 6 mo), some previously failed vials passed CCI, suggesting that CCI improvement might be related to rubber relaxation (viscous flow), which can fill minor imperfections on the vial finish.To confirm component positional offsets are the primary sources of RSF variability, a novel quantification tool-micro-computed tomography (micro-CT)-was used in this study. Micro-CT provided images for quantification of positional offsets of the cap and stopper that directly correlated with RSF fluctuations. Serum stoppers and lyophilization stoppers are comparable in RSF variations, although lyophilization stoppers are more robust in CCI. The use of micro-CT provides a nondestructive and innovative tool in quantitatively analyzing component features of capped vials that would otherwise be difficult to investigate.

Vapor-Phase Hydrogen Peroxide Uptake by Silicone Tubing and Primary Packaging Components during Protein Drug Product Aseptic Filling: Impact of Pretreatment and Sterilization Process.

In the vapor-phase hydrogen peroxide (VPHP)-sanitized environment, VPHP uptake by product-contacting components could eventually lead to undesired oxidation of biological drug products. Silicone tubing and primary packaging materials are prominent examples of such product-contacting surfaces that are typically processed/sterilized prior to use. This study investigated the VPHP-uptake tendency of these components and how their respective processing/sterilization methods affect the uptake behaviors. Silicone tubing that was sterilized via autoclave or gamma irradiation exhibited different VPHP uptake patterns-decreased uptake rates post autoclaving vs. increased uptake rates post gamma irradiation. The reduced uptake tendency of autoclaved tubing is maintained for 14 days after sterilization, whereas the uptake tendency of irradiated tubing was mostly reversed to normal levels 1 month after irradiation. Empty glass vials adsorbed hydrogen peroxide via the diffusion of VPHP into the vial with high vial-to-vial variability. Vial pretreatment (i.e., depyrogenation) and surface hydrophilicity/hydrophobicity impacted the uptake tendency. Stoppers and empty syringes also adsorbed hydrogen peroxide but at a relatively low level. The uptake behavior of these components appeared to correlate with water levels at the surface (i.e., hydrophilicity). This study provides process development scientists and engineers an in-depth understanding of the VPHP uptake by critical product-contacting surfaces so that they can mitigate the impact on drug product quality.LAY ABSTRACT: This study investigated vapor-phase hydrogen peroxide (VPHP) absorption by biopharmaceutical drug products via VPHP uptake by critical product-contacting components during the aseptic manufacturing process with a focus on various pretreatments and processing of these components. Sterilization of silicone tubing by gamma irradiation or autoclaving resulted in different VPHP uptake profiles with different effect durations. Primary packaging components, such as vials, syringes, and stoppers, also showed different levels of VPHP uptake with surface hydrophilicity/hydrophobicity playing a critical role. These outcomes suggested that VPHP uptake is a complex phenomenon and should be carefully considered to minimize its impact on product quality. The approach and outcome of this study can benefit scientists and engineers who develop biological product manufacturing processes by providing an in-depth understanding of drug product process risks.

Stability Evaluation of Hydrogen Peroxide Uptake Samples from Monoclonal Antibody Drug Product Aseptically Filled in Vapor Phase Hydrogen Peroxide-Sanitized Barrier Systems: A Case Study.

During the manufacture of a monoclonal antibody drug product, which was aseptically filled within a vapor phase hydrogen peroxide-sanitized isolator, samples were taken to investigate the hydrogen peroxide uptake behaviors. Surprisingly, the samples had no detectable hydrogen peroxide (most results below the limit of detection). This finding was later attributed to hydrogen peroxide decomposition after the samples were stored frozen at -20°C for two weeks before testing. This case study highlights the criticality of storage conditions for hydrogen peroxide-containing samples and summarizes an investigation on hydrogen peroxide stability in water and in three monoclonal antibody solutions having a wide protein concentration range (30-200 mg/mL). Samples were stored at three temperatures (-70°C, -20°C, or 2-8°C) for up to 28 days to assess the impact of protein concentration and storage temperature on hydrogen peroxide decomposition rates. Hydrogen peroxide degraded slightly more rapidly with increasing protein concentration independent of storage condition. When stored at -20°C, hydrogen peroxide was least stable and degraded faster than when stored at 2-8°C. Hydrogen peroxide was most stable when the samples were stored at -70°C. Overall, this case study brings the hydrogen peroxide stability issue to the attention of process development scientists and engineers and offers a valuable lesson learned during process development.LAY ABSTRACT: The use of vapor phase hydrogen peroxide as a sanitizing agent for isolator and cleanroom decontamination has become common in recent years. Because of the potential impact of residual hydrogen peroxide on biopharmaceutical product quality, hydrogen peroxide uptake behaviors and mechanisms during the manufacturing process within these barriers need to be evaluated and understood. Samples taken from various small-scale and manufacturing-scale hydrogen peroxide uptake studies are often stored frozen before testing. This case study reports an important and interesting finding about hydrogen peroxide stability in samples collected for hydrogen peroxide uptake investigation, and it demonstrates the relationship between hydrogen peroxide stability and storage temperature, storage duration, and monoclonal antibody concentration. The approach and outcome of this study are expected to benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product process challenges and appropriate sample storage.

Quantifying the Vial Capping Process: Residual Seal Force and Container Closure Integrity.

Capping completes the closure of parenteral drug products in the final packaging container and is critical in maintaining an integral seal to ensure product quality. Residual seal force (RSF) is considered the sole quantifiable attribute for measuring seal "goodness" and potentially enables nonsubjective, consistent setting of cappers across manufacturing sites. However, the consistency and reliability of RSF measurement and data have been scarcely reported, and the relationship between RSF and container closure integrity (CCI) remains poorly understood.Here, we present a large data set generated from a commercial capper and the results from a laboratory capper of glass vials and rubber stoppers with aluminum caps. All RSF values exhibited significant variability. We evaluated four potential sources of variability: the capper, the RSF tester, the time-dependent nature of RSF, and the components. We determined that the capper, the tester, and the time-dependent nature are not main sources. Dimensional tolerances of the packaging components were the root cause for the container closure system (CCS) configurations tested in this study.This study correlated RSF with CCI (via helium leakage), although CCI is not sensitive to RSF; CCI was maintained even for loosely capped vials with no measurable RSF. This was attributed to the stopper's two sealing surfaces: the valve seal and the land seal. A methodology capable of differentiating the two seals' functions demonstrated that vials with only the valve seal always passed leakage testing for a selected CCS configuration in this study, while vials with only the land seal failed CCI at low RSF values. This observation allows proposal of a low RSF limit that is safe even when the valve seal is defective. Simplified statistical analysis of commercial capping data, with the input of sample size, allowed the relationship between RSF's low limit and an allowable failing rate to be established. Overall, despite the inherent variability of RSF, this study shows that it is a feasible parameter for capping process quantification and demonstrates the potential of RSF measurement in capper setup.LAY ABSTRACT: Pharmaceutical vials are typically closed off with an elastomeric stopper that is secured onto the vial with an aluminum crimp cap (or seal) such that the entire assembly is meant to protect the vial's contents from external contamination. Therefore, the capping process is critical for ensuring container closure integrity. Characterizing the effectiveness of a seal in a nonsubjective and quantifiable manner is challenging. In this communication, we report the evaluation of residual seal force measurements (the compression force that the crimp cap exerts on the stopper) to evaluate capping for a large set of samples generated on both an at-scale commercial capper and a benchtop laboratory capper. We propose a test methodology, based on a statistical approach, for establishing permissible lower residual force limits that would provide a high degree of confidence to the capping process. This is a useful tool for consistent capper setup and capping process quantification.

Vapor Phase Hydrogen Peroxide Decontamination or Sanitization of an Isolator for Aseptic Filling of Monoclonal Antibody Drug Product-Hydrogen Peroxide Uptake and Impact on Protein Quality.

A monoclonal antibody drug product manufacturing process was transferred to a different production site, where aseptic filling took place within an isolator that was decontaminated (sanitized) using vapor phase hydrogen peroxide (VPHP). A quality-by-design approach was applied for study design to understand the impact of VPHP uptake on drug product quality. Both small-scale and manufacturing-scale studies were performed to evaluate the sensitivity of the monoclonal antibody to hydrogen peroxide (H2O2) and characterize VPHP uptake mechanisms in the filling process. The acceptable H2O2 uptake level was determined to be 100 ng/mL for the antibody in the H2O2 spiking study; protein oxidation was observed above this threshold. The most prominent sources of VPHP uptake were identified to be the silicone tubing assembly (associated with the peristaltic pumps) and open, filled vials. Silicone tubing, an effective depot to H2O2, absorbs VPHP during different stages of the filling process and transmits H2O2 into the drug product solution during filling interruptions. A small-scale isolator model, established to simulate manufacturing-scale conditions, was a useful tool in understanding H2O2 uptake in relation to tubing dimensions and VPHP concentration in the isolator air (or atmosphere). Although the tubing assembly had absorbed a substantial amount of VPHP during the decontamination phase, the majority of H2O2 could be removed during tubing cleaning and sterilization in the subsequent isolator aeration phase, demonstrating that H2O2 in the final drug product solution is primarily taken up from residual VPHP in the isolator during filling. Picarro sensor monitoring demonstrated that the validated VPHP aeration process generates reproducible residual VPHP profiles in isolator air, allowing small-scale studies to provide relevant recommendations on tubing size and interruption time limits for commercial manufacturing. The recommended process parameters were demonstrated to be acceptable and rendered no product quality impact in six consecutive manufacturing batches in the process validation campaign. Overall, this case study provides process development scientists and engineers an in-depth understanding of the VPHP process and a science-based approach to mitigating drug product quality impact.LAY ABSTRACT: While the use of vapor phase hydrogen peroxide as a sanitizing agent for isolator and cleanroom decontamination has gained popularity in recent years, its impact on product quality during aseptic manufacturing of biopharmaceutical drug products is yet to be fully understood. With this scope in mind, this case study offers a detailed account of defining process parameters and developing their operating ranges to ensure that the impact to product quality is minimized. Both small-scale and manufacturing-scale studies were performed to assess the sensitivity of a monoclonal antibody to hydrogen peroxide, to characterize hydrogen peroxide uptake sources and mechanisms, and to eventually define process parameters and their ranges critical for minimizing product quality impact. The approach and outcome of this study is expected to benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product process challenges.

Mechanistic Investigation on Grinding-Induced Subvisible Particle Formation during Mixing and Filling of Monoclonal Antibody Formulations.

Processing equipment involving grinding of two solid surfaces has been demonstrated to induce subvisible particle formation in monoclonal antibody drug product manufacturing processes. This study elucidated potential stress types associated with grinding action to identify the stress mechanism responsible for subvisible particle formation. Several potential stress types can be associated with the grinding action, including interfacial stresses (air-liquid and liquid-solid), hydraulic/mechanical shear stress, cavitation, nucleation of stressed protein molecules, and localized thermal stress. More than one stress type can synergically affect monoclonal antibody product quality, making it challenging to determine the primary mode of stress. Our strategy was to assess and rule out some stress types through platform knowledge, rational judgments, or via small-scale models, for example, rheometer/rotator-stator homogenizer for hydraulic/mechanical shear stress, sonicator for cavitation, etc. These models may not provide direct evidence but can offer rational correlations. Cavitation, as demonstrated by sonication, proved to be quite detrimental to monoclonal antibody molecules in forming not just subvisible particles but also soluble high-molecular-weight species as well as low-molecular-weight species. This outcome was not consistent with that of grinding monoclonal antibodies between the impeller and the drive unit of a bottom-mounted mixer or between the piston and the housing of a rotary piston pump, both of which formed only subvisible particles without obvious high-molecular-weight species and low-molecular-weight species. In addition, a p-nitrophenol model suggested that cavitation in the bottom-mounted mixer is barely detectable. We attributed the grinding-induced, localized thermal effect to be the primary stress to subvisible particle formation based on a high-temperature, spray-drying model. The heat effect of spray drying also caused subvisible particles, in the absence of significant high-molecular-weight species and low-molecular-weight species, in spray-dried monoclonal antibody powders. This investigation provides a mechanistic understanding of the underlying stress mechanism leading to monoclonal antibody subvisible particle formation as the result of drug product processing involving grinding of solid surfaces.LAY ABSTRACT: Subvisible particles present in therapeutic protein formulations could adversely affect drug product safety and efficacy. We previously illustrated that grinding action of the solid surfaces in some bottom-mounted mixers and piston pump is responsible for subvisible particle formation of monoclonal antibody formulations. In this study, we delved into mechanistic understanding of the stress types associated with solid surface grinding. The approach was to employ several scale-down stress models with known stress types. Protein formulations stressed in these models were analytically characterized for subvisible particles and other degradants. Some commonly known stress types-such as air-liquid interface, mechanical stress, cavitation, nucleation, and thermal effect-were assessed in this study. The stress model yielding a degradation profile matching that of bottom-mounted mixers and piston pump warranted further assessment. Localized, thermal stress proved to be the most feasible mechanism. This study, along with previously published results, may further advance our understanding of these particular drug product manufacturing processes and benefit scientists and engineers in overcoming these development challenges.

Processing Impact on Monoclonal Antibody Drug Products: Protein Subvisible Particulate Formation Induced by Grinding Stress.

Subvisible particle formation in monoclonal antibody drug product resulting from mixing and filling operations represents a significant processing risk that can lead to filter fouling and thereby lead to process delays or failures. Several previous studies from our lab and others demonstrated the formation of subvisible particulates in mAb formulations resulting from mixing operations using some bottom-mounted mixers or stirrer bars. It was hypothesized that the stress (e.g., shear/cavitation) derived from tight clearance and/or close contact between the impeller and shaft was responsible for protein subvisible particulate generation. These studies, however, could not distinguish between the two surfaces without contact (tight clearance) or between two contacting surfaces (close contact). In the present study we expand on those findings and utilize small-scale mixing models that are able to, for the first time, distinguish between tight clearances and tight contact. In this study we evaluated different mixer types including a top-mounted mixer, several impeller-based bottom-mounted mixers, and a rotary piston pump. The impact of tight clearance/close contact on subvisible particle formation in at-scale mixing platforms was demonstrated in the gap between the impeller and drive unit as well as between the piston and the housing of the pump. Furthermore, small-scale mixing models based on different designs of magnetic stir bars that mimic the tight clearance/close contact of the manufacturing-scale mixers also induced subvisible particles in mAb formulations. Additional small-scale models that feature tight clearance but no close contact (grinding) suggested that it is the repeated grinding/contacting of the moving parts and not the presence of tight clearance in the processing equipment that is the root cause of protein subvisible particulate formation. When multiple mAbs, Fabs (fragment antigen binding), or non-antibody related proteins were mixed in the small-scale mixing model, for molecules investigated, it was observed that mAbs and Fabs appear to be more susceptible to particle formation than non-antibody-related proteins. In the grinding zone, mAb/Fab molecules aggregated into insoluble particles with neither detectable soluble aggregates nor fragmented species. This investigation represents a step closer to the understanding of the underlying stress mechanism leading to mAb subvisible particulate formation as the result of drug product processing.LAY ABSTRACT: Mixing and fill finish are important unit operations in drug product manufacturing for compounding (dilution, pooling, homogenization, etc.) and filling into primary packaging containers (vials, pre-filled syringes, etc.), respectively. The current trend in adopting bottom-mounted mixers as well as low fill-volume filling systems has raised concerns about their impact on drug product quality and process performance. However, investigations into the effects of their use for biopharmaceutical products, particularly monoclonal antibody formulations, are rarely published. The purpose of this study is three-fold: (1) to revisit the impact of bottom-mounted mixer design on monoclonal antibody subvisible particle formation; (2) to identify the root cause for subvisible particle formation; and (3) to fully utilize available particle analysis tools to demonstrate the correlation between particle count in the solution and filter fouling during sterile filtration. The outcomes of this study will benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product process challenges.

Filling of High-Concentration Monoclonal Antibody Formulations: Investigating Underlying Mechanisms That Affect Precision of Low-Volume Fill by Peristaltic Pump.

Filling of high-concentration/viscosity monoclonal antibody formulations into vials or syringes by peristaltic pumps is an industrial standard. Control of the peristaltic pump on fill weight/volume accuracy/precision over time, however, has not been fully disclosed in the literature. This study systematically evaluated the impact of a broad range of system/pump parameters, from tubing setup to pump parameter settings to the filling nozzle, on filling precision using a bench-top system with fill weight readings from a high-precision balance. A low fill volume of 0.3 mL was targeted to fill liquids of various viscosities (including a high-concentration monoclonal antibody formulation). Fill weight precision was reported via percent of fill weight data points (at least 100 consecutive points) falling within 3% of the target fill weight (e.g., within 0.009 g for a 0.3 g target fill weight). Experimental results suggested that the 3% precision target is challenging for filling high-viscosity liquids due to run-to-run and day-to-day variability. More importantly, none of the system/pump parameters seemed to directly correlate with fill weight precision. Photograph analysis revealed liquid suck-back height variations during fill, which correlated well with fill weight variability. Suck-back height variation was attributed to two possible root causes: (1) inconsistent liquid stream separation point at the end of fill and (2) pressure-induced variations upon suck-back. Liquid stream break-up was influenced by liquid properties as well as liquid/nozzle interactions, and pressure variations might be associated with tubing and overall mechanism of the peristaltic pump. A custom nozzle tip design featuring a hydrophobic tip and a pressure-resistance barrier enabled consistent suck-back heights for each fill and approximately 90% of fill weight data within 3% precision for a high-concentration monoclonal antibody formulation. LAY ABSTRACT: Vial and syringe filling by peristaltic pump is considered a well-established manufacturing process and has been implemented by numerous contract manufacturing organizations and biopharmaceutical companies. However, its technical details and associated critical process parameters on fill weight precision are rarely published. Such information on high-concentration/viscosity formulation filling is particularly lacking. This study aimed to identify critical filling parameters that dictate a tight control on fill weight precision. The findings of this study indicate that mitigating suck-back height variation is the key to achieving improved fill weight precision. Liquid properties, the influence of liquid/nozzle interactions, and pressure variations during suck-back are inherent to fill weight variations. Optimizing fill weight precision by manipulating pump system parameters is not a root-cause solution. The outcomes of this study will benefit scientists and engineers who develop pre-filled syringe/vial products by providing a better understanding of high-concentration formulation filling principles and challenges.

Filling of High-Concentration Monoclonal Antibody Formulations into Pre-filled Syringes: Investigating Formulation-Nozzle Interactions To Minimize Nozzle Clogging.

UNLABELLED: Syringe filling of high-concentration/viscosity monoclonal antibody formulations is a complex process that is not fully understood. This study, which builds on a previous investigation that used a bench-top syringe filling unit to examine formulation drying at the filling nozzle tip and subsequent nozzle clogging, further explores the impact of formulation-nozzle material interactions on formulation drying and nozzle clogging. Syringe-filling nozzles made of glass, stainless steel, or plastic (polypropylene, silicone, and Teflon®), which represent a full range of materials with hydrophilic and hydrophobic properties as quantified by contact angle measurements, were used to fill liquids of different viscosity, including a high-concentration monoclonal antibody formulation. Compared with hydrophilic nozzles, hydrophobic nozzles offered two unique features that discouraged formulation drying and nozzle clogging: (1) the liquid formulation is more likely to be withdrawn into the hydrophobic nozzle under the same suck-back conditions, and (2) the residual liquid film left on the nozzle wall when using high suck-back settings settles to form a liquid plug away from the hydrophobic nozzle tip. Making the tip of the nozzle hydrophobic (silicone-coating on glass and Teflon-coating stainless steel) could achieve the same suck-back performance as plastic nozzles. This study demonstrated that using hydrophobic nozzles are most effective in reducing the risk of nozzle clogging by drying of high-concentration monoclonal antibody formulation during extended nozzle idle time in a large-scale filling facility and environment. LAY ABSTRACT: Syringe filling is a well-established manufacturing process and has been implemented by numerous contract manufacturing organizations and biopharmaceutical companies. However, its technical details and associated critical process parameters are rarely published. Information on high-concentration/viscosity formulation filling is particularly lacking. This study is the continuation of a previous investigation with a focus on understanding the impact of nozzle material on the suck-back function of liquid formulations. The findings identified the most critical parameter-nozzle material hydrophobicity-in alleviating formulation drying at the nozzle tip and eventually limiting the occurrence of nozzle clogging during the filling process. The outcomes of this study will benefit scientists and engineers who develop pre-filled syringe products by providing a better understanding of high-concentration formulation filling principles and challenges.

Mixing monoclonal antibody formulations using bottom-mounted mixers: impact of mechanism and design on drug product quality.

UNLABELLED: Using bottom-mounted mixers, particularly those that are magnetically driven, is becoming increasingly common during the mixing process in pharmaceutical and biotechnology manufacturing because of their associated low risk of contamination, ease of use, and ability to accommodate low minimum mixing volumes. Despite these benefits, the impact of bottom-mounted mixers on biologic drug product is not yet fully understood and is scarcely reported. This study evaluated four bottom-mounted mixers to assess their impact on monoclonal antibody formulations. Changes in product quality (size variants, particles, and turbidity) and impact on process performance (sterile filtration) were evaluated after mixing. The results suggested that mixers that are designed to function with no contact between the impeller and the drive unit are the most favorable and gentle to monoclonal antibody molecules. Designs with contact or a narrow clearance tended to shear and grind the protein and resulted in high particle count in the liquid, which would subsequently foul a filter membrane during sterile filtration using a 0.22 µm pore size filter. Despite particle formation, increases in turbidity of the protein solution and protein aggregation/fragmentation were not detected. Further particle analysis indicated particles in the range of 0.2-2 µm are responsible for filter fouling. A small-scale screening model was developed using two types of magnetic stir bars mimicking the presence or absence of contact between the impeller and drive unit in the bottom-mounted mixers. The model is capable of differentiating the sensitivity of monoclonal antibody formulations to bottom-mounted mixers with a small sample size. This study fills an important gap in understanding a critical bioprocess unit operation. LAY ABSTRACT: Mixing is an important unit operation in drug product manufacturing for compounding (dilution, pooling, homogenization, etc.). The current trend in adopting disposable bottom-mounted mixers has raised concerns about their impact on drug product quality and process performance. However, investigations into the effects of their use for biopharmaceutical products, particularly monoclonal antibody formulations, are rarely published. The purpose of this study is three-fold: (1) to understand the impact of bottom-mounted disposable mixer design on drug product quality and process performance, (2) to identify the mixing mechanism that is most gentle to protein particle formation, (3) to apply the learning to practical mixing operations using bottom-mounted mixers. The outcomes of this study will benefit scientists and engineers who develop biologic product manufacturing process by providing a better understanding of mixing principles and challenges.

Manufacturing of High-Concentration Monoclonal Antibody Formulations via Spray Drying-the Road to Manufacturing Scale.

UNLABELLED: Spray-dried monoclonal antibody (mAb) powders may offer applications more versatile than the freeze-dried cake, including preparing high-concentration formulations for subcutaneous administration. Published studies on this topic, however, are generally scarce. This study evaluates a pilot-scale spray dryer against a laboratory-scale dryer to spray-dry multiple mAbs in consideration of scale-up, impact on mAb stability, and feasibility of a high-concentration preparation. Under similar conditions, both dryers produced powders of similar properties-for example, water content, particle size and morphology, and mAb stability profile-despite a 4-fold faster output by the pilot-scale unit. All formulations containing arginine salt or a combination of arginine salt and trehalose were able to be spray-dried with high powder collection efficiency (>95%), but yield was adversely affected in formulations with high trehalose content due to powder sticking to the drying chamber. Spray-drying production output was dictated by the size of the dryer operated at an optimal liquid feed rate. Spray-dried powders could be reconstituted to high-viscosity liquids, >300 cP, substantially beyond what an ultrafiltration process can achieve. The molar ratio of trehalose to mAb needed to be reduced to 50:1 in consideration of isotonicity of the formulation with mAb concentration at 250 mg/mL. Even with this low level of sugar protection, long-term stability of spray-dried formulations remained superior to their liquid counterparts based on size variant and potency data. This study offers a commercially viable spray-drying process for biological bulk storage and an option for high-concentration mAb manufacturing. LAY ABSTRACT: This study evaluates a pilot-scale spray dryer against a laboratory-scale dryer to spray-dry multiple monoclonal antibodies (mAbs) from the perspective of scale-up, impact on mAb stability, and feasibility of a high-concentration preparation. The data demonstrated that there is no process limitation in solution viscosity when high-concentration mAb formulations are prepared from spray-dried powder reconstitution compared with concentration via the conventional ultrafiltration process. This study offers a commercially viable spray-drying process for biological bulk storage and a high-concentration mAb manufacturing option for subcutaneous administration. The outcomes of this study will benefit scientists and engineers who develop high-concentration mAb products by providing a viable manufacturing alternative.

Filling of high-concentration monoclonal antibody formulations into pre-filled syringes: filling parameter investigation and optimization.

Syringe filling, especially the filling of high-concentration/viscosity monoclonal antibody formulations, is a complex process that has not been widely published in literature. This study sought to increase the body of knowledge for syringe filling by analyzing and optimizing the filling process from the perspective of a fluid's physical properties (e.g., viscosity, concentration, surface tension). A bench-top filling unit, comprising a peristaltic pump unit and a filling nozzle integrated with a linear actuator, was utilized; glass nozzles were employed to visualize liquid flow inside the nozzle with a high-speed camera. The desired outcome of process optimization was to establish a clean filling cycle (e.g., absence of splashes, bubbles, and foaming during filling and absence of dripping from the fill nozzle post-fill) and minimize the risk of nozzle clogging during nozzle idle time due to formulation drying at or near the nozzle tip. The key process variables were determined to be nozzle size, airflow around the nozzle tip, pump suck-back (SB)/reversing, fluid viscosity, and protein concentration, while pump velocity, acceleration, and fluid/nozzle interphase properties were determined to be relatively weak parameters. The SB parameter played an especially critical role in nozzle clogging. This study shows that an appropriate combination of optimal SB setting, nozzle size, and airflow conditions could effectively extend nozzle idle time in a large-scale filling facility and environment. LAY ABSTRACT: Syringe filling can be considered a well-established manufacturing process and has been implemented by numerous contract manufacturing organizations and biopharmaceutical companies. However, its technical details and associated critical process parameters are rarely published. The information on high-concentration/viscosity formulation filling is particularly lacking. The purpose of this study is three-fold: (1) to reveal design details of a bench-top syringe filling unit; (2) to identify and optimize critical process parameters; (3) to apply the learning to practical filling operation. The outcomes of this study will benefit scientists and engineers who develop pre-filled syringe products by providing a better understanding of HC formulation filling principles and challenges.

Investigating high-concentration monoclonal antibody powder suspension in nonaqueous suspension vehicles for subcutaneous injection.

Developing high-concentration monoclonal antibody (mAb) liquid formulations for subcutaneous (s.c.) administration is challenging because increased viscosity makes injection difficult. To overcome this obstacle, we investigated a nonaqueous powder suspension approach. Three IgG1 mAbs were spray dried and suspended at different concentrations in Miglyol® 840, benzyl benzoate, or ethyl lactate. Suspensions were characterized for viscosity, particle size, and syringeability; physical stability was visually inspected. Suspensions generally outperformed liquid solutions for injectability despite higher viscosity at the same mAb concentrations. Powder formulations and properties had little effect on viscosity or injectability. Ethyl lactate suspensions had lowest viscosity (<20 cP) and lowest syringe injection glide force (<15 N) at mAb concentrations as high as 333 mg/mL (500 mg powder/mL). Inverse gas chromatography analysis indicated that the vehicle was the most important factor impacting suspension performance. Ethyl lactate rendered greater heat of sorption (suggesting strong particle-suspension vehicle interaction may reduce particle-particle self-association, leading to low suspension viscosity and glide force) but lacked the physical suspension stability exhibited by the other vehicles. Specific mixtures of ethyl lactate and Miglyol® 840 improved overall performance in high mAb concentration suspensions. This study demonstrated the viability of high mAb concentration (>300 mg/mL) in suspension formulations for s.c. administration.

Syringe siliconization process investigation and optimization.

The interior barrel of the prefilled syringe is often lubricated/siliconized by the syringe supplier or at the syringe filling site. Syringe siliconization is a complex process demanding automation with a high degree of precision; this information is often deemed "know-how" and is rarely published. The purpose of this study is to give a detailed account of developing and optimizing a bench-top siliconization unit with nozzle diving capabilities. This unit comprises a liquid dispense pump unit and a nozzle integrated with a Robo-cylinder linear actuator. The amount of coated silicone was determined by weighing the syringe before and after siliconization, and silicone distribution was visually inspected by glass powder coating or characterized by glide force testing. Nozzle spray range, nozzle retraction speed, silicone-coated amount, and air-to-nozzle pressure were found to be the key parameters affecting silicone distribution uniformity. Distribution uniformity is particularly sensitive to low-target silicone amount where the lack of silicone coating on the barrel near the needle side often caused the syringes to fail the glide force test or stall when using an autoinjector. In this bench-top unit we identified optimum coating conditions for a low silicone dose, which were also applicable to a pilot-scale siliconization system. The pilot unit outperformed the bench-top unit in a tighter control (standard deviation) in coated silicone amount due to the elimination of tubing flex. Tubing flex caused random nozzle mis-sprays and was prominent in the bench-top unit, while the inherent design of the pilot system substantially limited tubing flux. In summary, this bench-top coating unit demonstrated successful siliconization of the 1 mL long syringe with ∼0.2 mg of silicone oil using a spraying cycle also applicable to larger-scale siliconization. LAY ABSTRACT: Syringe siliconization can be considered a well-established manufacturing process and has been implemented by numerous syringe providers. However, its technical details and associated critical process parameters are rarely published. The purpose of this study is three-fold: (1) to reveal design details of a bench-top siliconization unit, (2) to identify critical process parameters and determine their optimum range to provide consistent and even silicone coating, and (3) to demonstrate the applicability of the optimum process condition derived from the bench-top unit to a pilot siliconization unit. The outcomes of this study will benefit scientists and engineers developing pre-filled syringe products by helping them to better understanding silicone spray coating principles and their relationship to siliconization processes in a large-scale manufacturing setting.

Erythropoietin-coated ZP-microneedle transdermal system: preclinical formulation, stability, and delivery.

PURPOSE: To evaluate the feasibility of coating formulated recombinant human erythropoietin alfa (EPO) on a titanium microneedle transdermal delivery system, ZP-EPO, and assess preclinical patch delivery performance. METHODS: Formulation rheology and surface activity were assessed by viscometry and contact angle measurement. EPO liquid formulation was coated onto titanium microneedles by dip-coating and drying. Stability of coated EPO was assessed by SEC-HPLC, CZE and potency assay. Preclinical in vivo delivery and pharmacokinetic studies were conducted in rats with EPO-coated microneedle patches and compared to subcutaneous EPO injection. RESULTS: Studies demonstrated successful EPO formulation development and coating on microneedle arrays. ZP-EPO patch was stable at 25°C for at least 3 months with no significant change in % aggregates, isoforms, or potency. Preclinical studies in rats showed the ZP-EPO microneedle patches, coated with 750 IU to 22,000 IU, delivered with high efficiency (75-90%) with a linear dose response. PK profile was similar to subcutaneous injection of commercial EPO. CONCLUSIONS: Results suggest transdermal microneedle patch delivery of EPO is feasible and may offer an efficient, dose-adjustable, patient-friendly alternative to current intravenous or subcutaneous routes of administration.

Parathyroid hormone (1-34)-coated microneedle patch system: clinical pharmacokinetics and pharmacodynamics for treatment of osteoporosis.

OBJECTIVES: To evaluate the clinical PK/PD of PTH(1-34) delivered by a novel transdermal drug-coated microneedle patch system (ZP-PTH) for the treatment of osteoporosis. METHODS: Phase 1 PK studies evaluated the effect of site of administration, patch wear time and dose in normal volunteers, ages 40-85 yrs. Phase 2 was conducted in post-menopausal women with osteoporosis to determine the patch dose response compared to placebo patch and FORTEO® injection. RESULTS: Phase 1 ZP-PTH patch delivery demonstrated a rapid PTH plasma pulse profile with T(max) 3 times shorter and apparent T(1/2) 2 times shorter than FORTEO®. In Phase 2, ZP-PTH 20, 30 and 40 µg doses showed a proportional increase in plasma PTH AUC. Inter-subject and intra-subject AUC variability was similar for all patch doses and comparable to injection. All patch doses produced a significant increase in spine bone mineral density. Unexpectedly, ZP-PTH also produced an early increase in hip bone mineral density, an effect not observed with the injection. CONCLUSIONS: These studies suggest that this novel ZP-PTH patch system can deliver a consistent and therapeutically relevant PTH PK profile. Based on encouraging Phase 2 safety and efficacy data, the program is advancing into a pivotal Phase 3 clinical study.

Investigating Liquid Leak from Pre-Filled Syringes upon Needle Shield Removal: Effect of Air Bubble Pressure.

This study is to investigate the effect of headspace air pressure in pre-filled syringes on liquid leak (dripping) from the syringe needle upon needle shield removal. Drip tests to measure drip quantity were performed on syringes manually filled with 0.5 or 1.0 mL of various aqueous solutions. Parameters assessed included temperature (filling and test), bulk storage conditions (tank pressure and the type of the pressurized gas), solution composition (pure water, 0.9% sodium chloride, and a monoclonal antibody formulation), and testing procedures. A headspace pressure analyzer was used to verify the drip test method. Results suggested that leakage is indeed caused by headspace pressure increase, and the temperature effect (ideal gas expansion) is a major, but not the only, factor. The dissolved gases in the liquid bulk prior to or during filling may contribute to leakage, as these gases could be released into the headspace due to solubility changes (in response to test temperature and pressure conditions) and cause pressure increase. Needle shield removal procedures were found to cause dripping, but liquid composition played little role. Overall, paying attention to the processing history (pressure and temperature) of the liquid bulk is the key to minimize leakage. The headspace pressure could be reduced by decreasing liquid bulk storage pressure, filling at a higher temperature, or employing lower solubility gas (e.g., helium) for bulk transfer and storage. Leakage could also be mitigated by simply holding the syringe needle pointing upward during needle shield removal. LAY ABSTRACT: Substantial advances in pre-filled syringe technology development, particularly in syringe filling accuracy, have been made. However, there are factors, as subtle as how the needle shield (or tip cap) is removed, that may affect dosing accuracy. We recently found that upon removal of the tip cap from a syringe held vertically with needle pointed downwards, a small amount of solution, up to 3-4% of the 1 mL filled volume or higher for filled volume of <1 mL, leaked out from the needle. This paper identified the root causes of this problem and offered solutions from the perspectives of the syringe fill process and the end user procedure. The readers will benefit from this paper by understanding how each process step prior to and during syringe filling may affect delivery performance of the pre-filled syringe device.