Cathepsin B Cleavage of vcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific.

Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different sites is not a critical parameter to control in manufacturing of the vc-MMAE-based ADC conjugated at reduced disulfide bonds.

Drug product Formulation and Fill/Finish Manufacturing Process Considerations for AAV-Based Genomic Medicines.

Adeno-associated viruses (AAVs) have become the delivery medium of choice for a variety of genomic medicine applications i.e., gene therapy, gene editing/regulation, and ex-vivo cell therapy. AAVs are protein-DNA complexes which have unique stability characteristics that are susceptible to various stress exposure conditions commonly seen in the drug product (DP) life cycle. This review takes a comprehensive look at AAV DP formulation and process development considerations that could impact critical quality attributes (CQAs) during manufacturing, packaging, shipping, and clinical use. Additional aspects related to AAV development reviewed herein are: (1) Different AAV serotypes with unique protein sequences and charge characteristics potentially leading to discrete stability profiles; (2) Manufacturing process challenges and optimization efforts to improve yield, recovery and purity especially during early development activities; and (3) Defining and identifying CQAs with analytical methods which are constantly evolving and present unique characterization challenges for AAV-based products.

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.

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.

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.

Isomerization of Asp-Asp motif in model peptides and a monoclonal antibody Fab fragment.

Isomerization of aspartyl (Asp or D) residues is a critical degradation route to consider for stable monoclonal antibody formulations. Among the known hotspot sequences, the DD motif is relatively understudied. To gain mechanistic insights, we used model hexapeptides, YADXFK, YADDXK, and DIDDDM, as surrogates for the hotspots in a Fab protein (YADDFK and DIDDDM), to characterize the rate-pH profile of Asp isomerization. Compared with the YADGFK peptide, isomerization of D3 (the first D in the DD pair) in YADDFK was highly pH dependent. Comparison of rate-pH profiles of YADDFK, YADNFK, and YADHFK revealed a charge effect of the n + 1 residue-isomerization rate is accelerated by the positive side chain and reduced by negative side chain at n + 1 residue. Studies on YADDFK, YADDAK, and YADDGK indicated a mutual impact of D3 and D4 on their respective isomerization rates through charge effect. Comparison of rate-pH profile of DIDDDM sequence in peptide models with that in the complementary determining region of the Fab showed a faster rate in the Fab than in peptides, presumably because of contribution from structural factors in the former.

Identification of amino acid residues responsible for the release of free drug from an antibody-drug conjugate utilizing lysine-succinimidyl ester chemistry.

Unexpected release of free drug was observed during the stability testing of an antibody-drug conjugate (ADC). The ADC was designed to use lysine-succinimidyl ester chemistry to conjugate small molecule cytotoxic drugs to the antibody. To elucidate the mechanism of the release of free drug, a succinimidyl ester analog, 7-hydroxy-4-methyl-3-coumarinylacetic acid N-succinimidyl ester, and a series of peptides were used to probe the potential side reaction of succinimidyl ester with other amino acid residues. Cysteine and tyrosine residues were found to be reactive to succinimidyl ester, and the bonds formed through these reactions were found to be labile. Combining the fluorescent property of the succinimidyl ester analog and mass spectroscopy analysis, specific cysteine and tyrosine residues of the antibody were found to be reactive to succinimidyl ester and the bonds formed through this reaction were susceptible to hydrolysis.