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.

Impact of Vial Capping on Residual Seal Force and Container Closure Integrity.

UNLABELLED: The vial capping process is a critical unit operation during drug product manufacturing, as it could possibly generate cosmetic defects or even affect container closure integrity. Yet there is significant variability in capping equipment and processes, and their relation to potential defects or container closure integrity has not been thoroughly studied. In this study we applied several methods-residual seal force tester, a self-developed system of a piezo force sensor measurement, and computed tomography-to characterize different container closure system combinations that had been sealed using different capping process parameter settings. Additionally, container closure integrity of these samples was measured using helium leakage (physical container closure integrity) and compared to characterization data. The different capping equipment settings lead to residual seal force values from 7 to 115 N. High residual seal force values were achieved with high capping pre-compression force and a short distance between the capping plate and plunge. The choice of container closure system influenced the obtained residual seal force values. The residual seal force tester and piezoelectric measurements showed similar trends. All vials passed physical container closure integrity testing, and no stopper rupture was seen with any of the settings applied, suggesting that container closure integrity was warranted for the studied container closure system with the chosen capping setting ranges. LAY ABSTRACT: The vial capping process is a critical unit operation during drug product manufacturing, as it could possibly generate cosmetic defects or even affect container closure integrity. Yet there is significant variability in capping equipment and processes, and their relation to potential defects or container closure integrity has not been thoroughly studied. In this study we applied several methods-residual seal force tester, a self-developed system of a piezo force sensor measurement, and computed tomography-to characterize different container closure system combinations that had been sealed using different capping process parameter settings. The residual seal force tester can analyze a variety of different container closure systems independent of the capping equipment. An adequate and safe residual seal force range for each container closure system configuration can be established with the residual seal force tester and additional methods like computed tomography scans and leak testing. In the residual seal force range studied, the physical container closure integrity of the container closure system was warranted.

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.

The Pharmaceutical Capping Process-Correlation between Residual Seal Force, Torque Moment, and Flip-off Removal Force.

The majority of parenteral drug products are manufactured in glass vials with an elastomeric rubber stopper and a crimp cap. The vial sealing process is a critical process step during fill-and-finish operations, as it defines the seal quality of the final product. Different critical capping process parameters can affect rubber stopper defects, rubber stopper compression, container closure integrity, and also crimp cap quality. A sufficiently high force to remove the flip-off button prior to usage is required to ensure quality of the drug product unit by the flip-off button during storage, transportation, and until opening and use. Therefore, the final product is 100% visually inspected for lose or defective crimp caps, which is subjective as well as time- and labor-intensive. In this study, we sealed several container closure system configurations with different capping equipment settings (with corresponding residual seal force values) to investigate the torque moment required to turn the crimp cap. A correlation between torque moment and residual seal force has been established. The torque moment was found to be influenced by several parameters, including diameter of the vial head, type of rubber stopper (serum or lyophilized) and type of crimp cap (West(®) or Datwyler(®)). In addition, we measured the force required to remove the flip-off button of a sealed container closure system. The capping process had no influence on measured forces; however, it was possible to detect partially crimped vials. In conclusion, a controlled capping process with a defined target residual seal force range leads to a tight crimp cap on a sealed container closure system and can ensure product quality. LAY ABSTRACT: The majority of parenteral drug products are manufactured in a glass vials with an elastomeric rubber stopper and a crimp cap. The vial sealing process is a critical process step during fill-and-finish operations, as it defines the seal quality of the final product. An adequate force to remove the flip-off button prior to usage is required to ensure product quality during storage and transportation until use. In addition, the complete crimp cap needs to be fixed in a tight position on the vial. In this study, we investigated the torque moment required to turn the crimp cap and the force required to remove the flip-off button of container closure system sealed with different capping equipment process parameters (having different residual seal force values).

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.

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

Structure of artificial cytoskeleton containing liposomes in aqueous solution studied by static and dynamic light scattering.

The structure of three types of liposomes (egg yolk phosphatidylcholine (EPC) without modification and EPC vesicles containing cross-linked N-isopropylacrylamide (NIPAM) networks of low and a high concentration inside the vesicles) were analyzed by static and dynamic light scattering. Upon polymerization the network was assumed to become attached to the membrane by reactive anchoring monomers. For the sample of high poly(NIPAM) content the polymer network was assumed to fill the whole space in the vesicles. The issue of the present study was to examine hard and hollow sphere behavior of the liposomes with networks of high and low poly(NIPAM) content. The theoretical scattering curves differ markedly for uniform hard and uniform hollow spheres by the presence of specific peaks. However, polydispersity washed out the peaks and led to smoothed asymptotes with fractal dimensions of df = 2 for hollow and df = 4 for hard spheres. The experimental data could efficiently be fitted with weakly polydisperse hollow spheres. No clear conclusion could be drawn from the angular dependence alone for the liposome of high poly(NIPAM) content. The two wavelengths from the HeNe and Ar lasers proved to be too long for the studied liposomes of about 100 nm in radius. However, evidence for hollow sphere behavior was found for fractionated liposomes from the ratio rho = Rg/Rh = 1.04 +/- 0.02 (theory rho = 1.00 for hollow spheres). Finally, from the molar mass and the sphere radius, an apparent density was determined. The analysis gave the expected density for the pure EPC lecithin vesicles and a poly(NIPAM) network density of 0.244 g/mL. For the liposome of low poly(NIPAM) content the network appeared to be attached to the inner surface of the lecithin shell to form a layer of about 18 nm thickness.

Mimicking a cytoskeleton by coupling poly(N-isopropylacrylamide) to the inner leaflet of liposomal membranes: effects of photopolymerization on vesicle shape and polymer architecture.

Networks of N-isopropylacrylamide (NIPAM) copolymers, coupled to spherical phospholipid bilayers, are suitable as a model for the study of the interaction between the cytoskeleton and cellular membranes, as well as for promising new drug delivery systems with triggerable drug release properties and improved stability. In this article, we describe a simple preparation technique for liposomes from egg phosphatidyl choline (EPC) encapsulating a cross-linked NIPAMminus signTEGDM copolymer skeleton (tetraethylene glycol dimethacrylate, TEGDM) which is coupled only to the inner monolayer by a novel membrane anchor monomer. Polymerization in the lipid vesicles was initiated at the inner membrane surface by the radical initiator 2,2-diethoxy-acetophenone (DEAP) permeating through the membrane from the outside. The effects of photopolymerization and polymer formation on vesicle shape and membrane integrity were studied by transmission electron microscopy (TEM), cryo-TEM, and atomic force microscopy (AFM). Upon UV irradiation, approximately 100% of the vesicles contained a polymer gel and only occasional changes in the spherical shape of the liposomes were observed. The architecture of the polymer network inside the liposomal compartment was determined by the conditions of the photopolymerization. Composite structures of polymer hollow spheres or solid spheres, respectively, tethered to spherical membrane vesicles were produced. The increased stability of the polymer-tethered lipid bilayers against solubilization by sodium cholate, compared to pure EPC vesicles, was determined by radiolabeling the lipid membrane.