Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development.

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development.

Shipping-Induced Aggregation in Therapeutic Antibodies: Utilization of a Scale-Down Model to Assess Degradation in Monoclonal Antibodies.

It is vital to understand the impact of transportation on monoclonal antibody (mAb) product quality during drug product development. Fully representative real-time shipment studies are resource intensive, so in this work, we studied laboratory agitation methods to mimic the effect of real-time shipment on aggregation, specifically subvisible particle formation. The agitation methods studied include a rotator, orbital shaker, vortexer, and shipping simulator vibration table. The simulator is able to predict the particle formation behavior during real-time shipment for a number of mAbs in vial and prefilled syringe configurations, with a correlation of about 90%, whereas the other methods of agitation were inconsistent. This study demonstrates that using a shipping simulator vibration table provides an opportunity for consistent and predictive development studies of shipping stress with minimal resource requirements during early- or late-stage drug product development.

Surfactant Effects on Particle Generation in Antibody Formulations in Pre-filled Syringes.

Protein aggregation and particle formation have been observed when protein solutions contact hydrophobic interfaces, and it has been suggested that this undesirable phenomenon may be initiated by interfacial adsorption and subsequent gelation of the protein. The addition of surfactants, such as polysorbate 20, to protein formulations has been proposed as a way to reduce protein adsorption at silicone oil-water interfaces and mitigate the production of aggregates and particles. In an accelerated stability study, monoclonal antibody formulations containing varying concentrations of polysorbate 20 were incubated and agitated in pre-filled glass syringes (PFS), exposing the protein to silicone oil-water interfaces at the siliconized syringe walls, air-water interfaces, and agitation stress. Following agitation in siliconized syringes that contained an air bubble, lower particle concentrations were measured in the surfactant-containing antibody formulations than in surfactant-free formulations. Polysorbate 20 reduced particle formation when added at concentrations above or below the critical micelle concentration (CMC). The ability of polysorbate 20 to decrease particle generation in PFS corresponded with its ability to inhibit gelation of the adsorbed protein layer, which was assessed by measuring the interfacial diffusion of individual antibody molecules at the silicone oil-water interface using total internal reflectance fluorescence (TIRF) microscopy with single-molecule tracking.

Effect of the siliconization method on particle generation in a monoclonal antibody formulation in pre-filled syringes.

Silicone oil is used as a lubricant in glass pre-filled syringes (PFS) but can contribute to the generation of particles within protein formulations in PFS. To mitigate the production of such particles, various silicone oil coating processes have been proposed. In this study, three siliconization methods (the "covalent" method, the "baked silicone oil" method, and the "liquid silicone oil" method) were used to coat glass syringes with silicone oil. Glide forces were determined for syringes coated by each method. Then, a monoclonal antibody formulation or a buffer solution were incubated in the coated syringes in either the presence or absence of an air bubble, and the syringes were rotated end-over-end to induce air bubble movement within the syringe. The particle concentrations were measured throughout the incubation period using flow microscopy. The coating method did not affect particle concentrations measured in buffer alone, nor did the coating method affect particle concentrations measured in antibody formulations in the absence of an air bubble. Particle concentrations were influenced by the syringe coating method in protein formulations agitated in the presence of an air bubble, with the most particles formed in syringes lubricated with liquid silicone oil. Fewer particles were produced in syringes lubricated with baked silicone oil, and the fewest particles were produced in syringes with covalently-attached silicone oil. However, the glide forces measured in syringes coated with silicone oil by each method are inversely correlated with the measured particle concentrations.

Gelation of a monoclonal antibody at the silicone oil-water interface and subsequent rupture of the interfacial gel results in aggregation and particle formation.

The formation of viscoelastic gels by a monoclonal antibody (mAb) at the silicone oil-water interface was studied using interfacial shear rheology. At a concentration of 50 µg/mL, the mAb formed gels in less than 1 h, and the gelation time decreased with increasing protein concentration. To probe the effects of mechanical rupture of the interfacial gel layers, a layer of silicone oil was overlaid on the surface of aqueous solutions of mAb, and the interface was ruptured periodically with a needle. Rupture of the interfacial gel resulted in formation of subvisible particles and substantial losses of mAb monomer, which were detected by microflow imaging and quantified by size-exclusion chromatography, respectively. Resonance mass measurement showed that levels of both protein particles and silicone oil droplets increased as the gel was repeatedly ruptured with a needle. In contrast, in samples wherein the interfacial gels were not ruptured, much lower levels of aggregates and particles were detected. Addition of nonionic surfactants (polysorbate 20 or polysorbate 80) protected against aggregation and protein particle formation, with increased protection seen with increasing surfactant levels, and with the greatest inhibition observed in samples containing polysorbate 80.

A comparative study of monoclonal antibodies. 1. Phase behavior and protein-protein interactions.

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. This work explores the phase behavior of eight monoclonal antibodies (mAbs) that exhibit liquid-liquid separation, aggregation, gelation, and crystallization. The phase behavior has been studied systematically as a function of a number of factors, including solution composition and pH, in order to explore the degree of variability among different antibodies. Comparisons of the locations of phase boundaries show consistent trends as a function of solution composition; however, changing the solution pH has different effects on each of the antibodies studied. Furthermore, the types of dense phases formed varied among the antibodies. Protein-protein interactions, as reflected by values of the osmotic second virial coefficient, are used to correlate the phase behavior. The primary findings are that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, though there is appreciable variability among the antibodies in the apparent strengths of the intrinsic protein-protein attraction manifested. However, the osmotic second virial coefficient does not provide a clear basis to predict the type of dense phase likely to result under a given set of solution conditions.

Interactions and phase behavior of a monoclonal antibody.

Protein phase behavior is implicated in numerous aspects of downstream processing either by design, as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. An improved understanding of protein phase behavior is, therefore, important for developing rational design strategies for important process steps. This work explores the phase behavior of a monoclonal antibody (mAb), IDEC-152, which exhibits liquid-liquid separation, aggregation, gelation, and crystallization. A systematic study of numerous factors, including the effects of solution composition and pH, has been conducted to explore the phase behavior of this antibody. Phenomena observed include a significant dependence of the cloud point on the cation in sulfate salts and nonmonotonic trends in pH dependence. Additionally, conditions for crystallization of this mAb are reported for the first time. Protein-protein interactions, as determined from the osmotic second virial coefficient, are used to interpret the phase behavior.

Effects of ammonium sulfate and sodium chloride concentration on PEG/protein liquid-liquid phase separation.

When added to protein solutions, poly(ethylene glycol) (PEG) creates an effective attraction between protein molecules due to depletion forces. This effect has been widely used to crystallize proteins, and PEG is among the most successful crystallization agents in current use. However, PEG is almost always used in combination with a salt at either low or relatively high concentrations. Here the effects of sodium chloride and ammonium sulfate concentration on PEG 8000/ovalbumin liquid-liquid (L-L) phase separation are investigated. At low salt the L-L phase separation occurs at decreasing protein concentration with increasing salt concentration, presumably due to repulsive electrostatic interactions between proteins. At high salt concentration, the behavior depends on the nature of the salt. Sodium chloride has little effect on the L-L phase separation, but ammonium sulfate decreases the protein concentration at which the L-L phase separation occurs. This trend is attributed to the effects of critical fluctuations on depletion forces. The implications of these results for designing solution conditions optimal for protein crystallization are discussed.

Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth.

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.