Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Mechanistic Insights into the Adsorption of Monoclonal Antibodies at the Water/Vapor Interface.

Monoclonal antibodies (mAbs) are active components of therapeutic formulations that interact with the water-vapor interface during manufacturing, storage, and administration. Surface adsorption has been demonstrated to mediate antibody aggregation, which leads to a loss of therapeutic efficacy. Controlling mAb adsorption at interfaces requires a deep understanding of the microscopic processes that lead to adsorption and identification of the protein regions that drive mAb surface activity. Here, we report all-atom molecular dynamics (MD) simulations of the adsorption behavior of a full IgG1-type antibody at the water/vapor interface. We demonstrate that small local changes in the protein structure play a crucial role in promoting adsorption. Also, interfacial adsorption triggers structural changes in the antibody, potentially contributing to the further enhancement of surface activity. Moreover, we identify key amino acid sequences that determine the adsorption of antibodies at the water-air interface and outline strategies to control the surface activity of these important therapeutic proteins.

Structure and interaction of therapeutic proteins in solution: a combined simulation and experimental study.

The aggregation of therapeutic proteins in solution has attracted significant interest, driving efforts to understand the relationship between microscopic structural changes and protein-protein interactions determining aggregation processes in solution. Additionally, there is substantial interest in being able to predict aggregation based on protein structure as part of molecular developability assessments. Molecular Dynamics provides theoretical tools to complement experimental studies and to interrogate and identify the microscopic mechanisms determining aggregation. Here we perform all-atom MD simulations to study the structure and inter-protein interaction of the Fab and Fc fragments of the monoclonal antibody (mAb) COE3. We unravel the role of ion-protein interactions in building the ionic double layer and determining effective inter-protein interaction. Further, we demonstrate, using various state-of-the-art force fields (charmm, gromos, amber, opls/aa), that the protein solvation, ionic structure and protein-protein interaction depend significantly on the force field parameters. We perform SANS and Static Light Scattering experiments to assess the accuracy of the different forcefields. Comparison of the simulated and experimental results reveal significant differences in the forcefields' performance, particularly in their ability to predict the protein size in solution and inter-protein interactions quantified through the second virial coefficients. In addition, the performance of the forcefields is correlated with the protein hydration structure.

Competitive Adsorption of a Monoclonal Antibody and Nonionic Surfactant at the PDMS/Water Interface.

Interfacial adsorption of monoclonal antibodies (mAbs) can cause structural deformation and induce undesired aggregation and precipitation. Nonionic surfactants are often added to reduce interfacial adsorption of mAbs which may occur during manufacturing, storage, and/or administration. As mAbs are commonly manufactured into ready-to-use syringes coated with silicone oil to improve lubrication, it is important to understand how an mAb, nonionic surfactant, and silicone oil interact at the oil/water interface. In this work, we have coated a polydimethylsiloxane (PDMS) nanofilm onto an optically flat silicon substrate to facilitate the measurements of adsorption of a model mAb, COE-3, and a commercial nonionic surfactant, polysorbate 80 (PS-80), at the siliconized PDMS/water interface using spectroscopic ellipsometry and neutron reflection. Compared to the uncoated SiO2 surface (mimicking glass), COE-3 adsorption to the PDMS surface was substantially reduced, and the adsorbed layer was characterized by the dense but thin inner layer of 16 Å and an outer diffuse layer of 20 Å, indicating structural deformation. When PS-80 was exposed to the pre-adsorbed COE-3 surface, it removed 60 wt % of COE-3 and formed a co-adsorbed layer with a similar total thickness of 36 Å. When PS-80 was injected first or as a mixture with COE-3, it completely prevented COE-3 adsorption. These findings reveal the hydrophobic nature of the PDMS surface and confirm the inhibitory role of the nonionic surfactant in preventing COE-3 adsorption at the PDMS/water interface.

Investigating the Orientation of an Interfacially Adsorbed Monoclonal Antibody and Its Fragments Using Neutron Reflection.

Interfacial adsorption is a molecular process occurring during the production, purification, transport, and storage of antibodies, with a direct impact on their structural stability and subsequent implications on their bioactivities. While the average conformational orientation of an adsorbed protein can be readily determined, its associated structures are more complex to characterize. Neutron reflection has been used in this work to investigate the conformational orientations of the monoclonal antibody COE-3 and its Fab and Fc fragments at the oil/water and air/water interfaces. Rigid body rotation modeling was found to be suitable for globular and relatively rigid proteins such as the Fab and Fc fragments but less so for relatively flexible proteins such as full COE-3. Fab and Fc fragments adopted a 'flat-on' orientation at the air/water interface, minimizing the thickness of the protein layer, but they adopted a substantially tilted orientation at the oil/water interface with increased layer thickness. In contrast, COE-3 was found to adsorb in tilted orientations at both interfaces, with one fragment protruding into the solution. This work demonstrates that rigid-body modeling can provide additional insights into protein layers at various interfaces relevant to bioprocess engineering.

Conformational Changes and Drivers of Monoclonal Antibody Liquid-Liquid Phase Separation.

Liquid-liquid phase separation is a phenomenon within biology whereby proteins can separate into dense and more dilute phases with distinct properties. Three antibodies that undergo liquid-liquid phase separation were characterized in the protein-rich and protein-poor phases. In comparison to the protein-poor phase, the protein-rich phase demonstrates more blue-shift tryptophan emissions and red-shifted amide I absorbances. Large changes involving conformational isomerization around disulfide bonds were observed using Raman spectroscopy. Amide I and protein fluorescence differences between the phases persisted to temperatures above the critical temperature but ceased at the temperature at which aggregation occurred. In addition, large changes occurred in the structural organization of water molecules within the protein-rich phase for all three antibodies. It is hypothesized that as the proteins have the same chemical potential in both phases, the protein viscosity is higher in the protein-rich phase resulting in slowed diffusion dependent protein aggregation in this phase. For all three antibodies we performed accelerated stability studies and found that the protein-rich phase aggregated at the same rate or slower than the protein-poor phase.

Understanding the Stabilizing Effect of Histidine on mAb Aggregation: A Molecular Dynamics Study.

Histidine, a widely used buffer in monoclonal antibody (mAb) formulations, is known to reduce antibody aggregation. While experimental studies suggest a nonelectrostatic, nonstructural (relating to secondary structure preservation) origin of the phenomenon, the underlying microscopic mechanism behind the histidine action is still unknown. Understanding this mechanism will help evaluate and predict the stabilizing effect of this buffer under different experimental conditions and for different mAbs. We have used all-atom molecular dynamics simulations and contact-based free energy calculations to investigate molecular-level interactions between the histidine buffer and mAbs, which lead to the observed stability of therapeutic formulations in the presence of histidine. We reformulate the Spatial Aggregation Propensity index by including the buffer-protein interactions. The buffer adsorption on the protein surface leads to lower exposure of the hydrophobic regions to water. Our analysis indicates that the mechanism behind the stabilizing action of histidine is connected to the shielding of the solvent-exposed hydrophobic regions on the protein surface by the buffer molecules.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Recent Advances in Studying Interfacial Adsorption of Bioengineered Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of biotherapeutics; as of 2020, dozens are commercialized medicines, over a hundred are in clinical trials, and many more are in preclinical developmental stages. Therapeutic mAbs are sequence modified from the wild type IgG isoforms to varying extents and can have different intrinsic structural stability. For chronic treatments in particular, high concentration (≥ 100 mg/mL) aqueous formulations are often preferred for at-home administration with a syringe-based device. MAbs, like any globular protein, are amphiphilic and readily adsorb to interfaces, potentially causing structural deformation and even unfolding. Desorption of structurally perturbed mAbs is often hypothesized to promote aggregation, potentially leading to the formation of subvisible particles and visible precipitates. Since mAbs are exposed to numerous interfaces during biomanufacturing, storage and administration, many studies have examined mAb adsorption to different interfaces under various mitigation strategies. This review examines recent published literature focusing on adsorption of bioengineered mAbs under well-defined solution and surface conditions. The focus of this review is on understanding adsorption features driven by distinct antibody domains and on recent advances in establishing model interfaces suitable for high resolution surface measurements. Our summary highlights the need to further understand the relationship between mAb interfacial adsorption and desorption, solution aggregation, and product instability during fill-finish, transport, storage and administration.

The Impact of the Metal Interface on the Stability and Quality of a Therapeutic Fusion Protein.

Subvisible particle formation, which occurs after the sterile filtration step of the fill/finish process, is a challenge that may occur during the development of biotherapeutics with complex molecular structures. Here, we show that a stainless steel pump head from a rotary piston pump produces more protein aggregates, past the limit of the acceptable quality range for subvisible particle counts, in comparison to a ceramic pump head. The quartz crystal microbalance was used to quantify the primary layer, proteins irreversibly adsorbed at the solid-liquid interface, and the secondary diffuse gel-like layer interacting on top of the primary layer. The results showed that the mass of protein irreversibly adsorbed onto stainless steel sensors is greater than on an aluminum oxide surface (ceramic pump mimic). This suggests that the amount of adsorbed protein plays a role in surface-induced protein aggregation at the solid-liquid interface.

Comparison of Polysorbate 80 Hydrolysis and Oxidation on the Aggregation of a Monoclonal Antibody.

Polysorbates are used ubiquitously in protein therapeutic drugs to help minimize adsorption to surfaces and aggregation. It has been recognized that polysorbate can itself degrade and in turn result in loss of efficacy of therapeutic proteins. We studied the 2 main pathways of polysorbate 80 (PS80) degradation, enzymatic ester hydrolysis, and oxidation. Degraded polysorbates were quantified through mass spectrometry to identify the loss of individual components. Next Langmuir trough adsorption isotherms were used to characterize changes in the surface activity of the degraded polysorbates. PS80 degraded via hydrolysis results in slower surface adsorption rates, whereas the oxidized PS80 show increased surface activity. However, the critical micelle concentration remained unchanged. A monoclonal antibody was formulated with stock and degraded polysorbates to probe their ability to prevent aggregation. Hydrolyzed polysorbate resulted in a large increase in particle formation during shaking stress. Oxidized PS80 was still protective against aggregation for the monoclonal antibody. Monomer loss as measured by SEC was comparable in formulations without PS80 to those with esterase hydrolyzed PS80. Monomer loss for oxidized PS80 was similar to that of nondegraded PS80. Hydrolysis of PS80 resulted in free fatty acids which formed insoluble particles during mechanical agitation which stimulated protein aggregation.

Cis/Trans Isomerization of Unsaturated Fatty Acids in Polysorbate 80 During Light Exposure of a Monoclonal Antibody-Containing Formulation.

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.

Interfacial Adsorption of a Monoclonal Antibody and Its Fab and Fc Fragments at the Oil/Water Interface.

The physical stability of a monoclonal antibody (mAb) solution for injection in a prefilled syringe may in part depend on its behavior at the silicone oil/water interface. Here, the adsorption of a mAb (termed COE-3) and its fragment antigen-binding (Fab) and crystallizable (Fc) at the oil/water interface was measured using neutron reflection. A 1.4 ± 0.1 µm hexadecane oil film was formed on a sapphire block by a spin-freeze-thaw process, retaining its integrity upon contact with the protein solutions. Measurements revealed that adsorbed COE-3 and its Fab and Fc fragments retained their globular structure, forming layers that did not penetrate substantially into the oil phase. COE-3 and Fc were found to adsorb flat-on to the interface, with denser 45 and 42 Å inner layers, respectively, in contact with the oil and a more diffuse 17-21 Å outer layer caused by fragments adsorbing in a tilted manner. In contrast, Fab fragments formed a uniform 60 Å monolayer. Monolayers were formed under all conditions studied (10-200 ppm, using three isotopic contrasts), although changes in packing density across the COE-3 and Fc layers were observed. COE-3 had a higher affinity to the interface than either of its constituent fragments, while Fab had a lower interfacial affinity consistent with its higher net surface charge. This study extends the application of high-resolution neutron reflection measurements to the study of protein adsorption at the oil/water interface using an experimental setup mimicking the protein drug product in a siliconized prefilled syringe.

Protein Adsorption and Layer Formation at the Stainless Steel-Solution Interface Mediates Shear-Induced Particle Formation for an IgG1 Monoclonal Antibody.

Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates. SVP particles produced were monitored using flow microscopy or flow cytometry. Neutron reflectometry and a quartz crystal microbalance with dissipation monitoring were used to characterize adsorption and properties of NISTmAb at the stainless steel interface. Pump/shear cell experiments showed that the NISTmAb concentration and interface material had a significant effect on SVP formation, while the effects of interfacial shear rate and passage number were less important. At the higher NISTmAb concentrations, the adsorbed protein became structurally altered at the stainless steel interface. The primary adsorbed layer remained largely undisturbed during flow, suggesting that SVP formation at high NISTmAb concentration was caused by the disruption of patches and/or secondary interactions.

Effects of Protein Conformation, Apparent Solubility, and Protein-Protein Interactions on the Rates and Mechanisms of Aggregation for an IgG1Monoclonal Antibody.

Non-native protein aggregation is a key degradation pathway of immunoglobulins. In this work, the aggregation kinetics of an immunoglobulin gamma-1 monoclonal antibody (IgG1 mAb) in different solution environments was monitored over a range of incubation temperatures for up to seven months using size exclusion chromatography. Histidine and citrate buffers with/without sodium chloride were employed to modulate the mAb's conformational stability, solubility (in the presence of polyethylene glycol, PEG), and protein-protein interactions as measured by differential scanning calorimetry, PEG precipitation, and static light scattering, respectively. The effect of these parameters on the mechanism(s) of mAb aggregation during storage at different temperatures was determined using kinetic models, which were used to fit aggregation data to determine rate constants for aggregate nucleation and growth processes. This approach was used to investigate the effects of colloidal protein-protein interactions and solubility values (in PEG solutions) on the mechanisms and rates of IgG1 mAb aggregation as a function of temperature-induced structural perturbations. Aggregate nucleation and growth pathways for this IgG1 mAb were sensitive to temperature and overall conformational stability. Aggregate growth, on the other hand, was also sensitive to conditions affecting the solubility of the mAb, particularly at elevated temperatures.

Evaluating the Role of the Air-Solution Interface on the Mechanism of Subvisible Particle Formation Caused by Mechanical Agitation for an IgG1 mAb.

Mechanical agitation of monoclonal antibody (mAb) solutions often leads to protein particle formation. In this study, various formulations of an immunoglobulin G (IgG) 1 mAb were subjected to different controlled interfacial stresses using a Langmuir trough, and protein particles formed at the interface and measured in bulk solution were characterized using atomic force microscopy and flow digital imaging. Results were compared to mAb solutions agitated in glass vials and unstressed controls. At lower pH, mAb solutions exhibited larger hysteresis in their surface pressure versus area isotherms and increased number of particles in bulk solution, when subjected to interfacial stresses. mAb samples subjected to 750-1000 interfacial compression-expansion cycles in 6 h contained high particle numbers in bulk solution, and displayed similar particulation trends when agitated in vials. At compression rates of 50 cycles in 6 h, however, particle levels in mAb solutions were comparable to unstressed controls, despite protein aggregates being present at the air-solution interface. These results suggest that while the air-solution interface serves as a nucleation site for initiating protein aggregation, the number of protein particles measured in bulk mAb solutions depends on the total number of compression cycles that proteins at the air-solution interface are subjected to within a fixed time.

Calculating the mass of subvisible protein particles with improved accuracy using microflow imaging data.

Although formation of subvisible particles (1-100 µm) during manufacturing and/or storage is a major stability concern with protein therapeutics, particle numbers are often too low to permit for direct experimental measurement of their protein content (mass). The objective of this work was to develop a novel, accurate, and easy-to-implement method to calculate the mass of subvisible protein particles using particle number, size, and morphology data obtained from microflow imaging (MFI) measurements. The method was evaluated using (1) spherical and nonspherical polystyrene standards and (2) shake and stir-stressed IgG1 mAb solutions. For extensively stressed mAb samples, in which protein mass loss after particle removal could be measured experimentally, calculated results were in good agreement and showed improvements in accuracy and precision compared with other methods. Improved estimates of protein mass in particles were made possible by using morphological data to better model particle volume, and by using literature-based values for protein density and particle composition. This method improves estimations of protein particle mass when total amounts are too low to be measured experimentally and also facilitates a better understanding of protein particle formation by accounting for particle mass as well as number.

Structural characterization of IgG1 mAb aggregates and particles generated under various stress conditions.

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions.

Radar chart array analysis to visualize effects of formulation variables on IgG1 particle formation as measured by multiple analytical techniques.

This study presents a novel method to visualize protein aggregate and particle formation data to rapidly evaluate the effect of solution and stress conditions on the physical stability of an immunoglobulin G (IgG) 1 monoclonal antibody (mAb). Radar chart arrays were designed so that hundreds of microflow digital imaging (MFI) solution measurements, evaluating different mAb formulations under varying stresses, could be presented in a single figure with minimal loss of data resolution. These MFI radar charts show measured changes in subvisible particle number, size, and morphology distribution as a change in the shape of polygons. Radar charts were also created to visualize mAb aggregate and particle formation across a wide size range by combining data sets from size-exclusion chromatography, Archimedes resonant mass measurements, and MFI. We found that the environmental/mechanical stress condition (e.g., heat vs. agitation) was the most important factor in influencing the particle size and morphology distribution with this IgG1 mAb. Additionally, the presence of NaCl exhibited a pH and stress-dependent behavior resulting in promotion or inhibition mAb particle formation. This data visualization technique provides a comprehensive analysis of the aggregation tendencies of this IgG1 mAb in different formulations with varying stresses as measured by different analytical techniques.