Improved Diffusion Interaction Parameter Measurement to Predict the Viscosity of Concentrated mAb Solutions.

During the developability assessment of therapeutic monoclonal antibody (mAb) candidates, utilization of robust high-throughput predictive assays enables rapid selection of top candidates with low risks for late-stage development. Predicting the viscosities of highly concentrated mAbs using limited materials is an important aspect of developability assessment because high viscosity can complicate manufacturability, stability, and administration. Here, we report a high-throughput assay measuring protein-protein interactions to predict mAb viscosity. The diffusion interaction parameter (kD) measures colloidal self-association in dilute solutions and has been reported to be predictive of the mAb viscosity at high concentrations. However, kD of Amgen early stage IgG1 mAb candidates measured in 10 mM acetate at pH 5.2 containing sucrose and polysorbate (denoted A52SuT) shows only weak correlation to their viscosities at 140 mg/mL in A52SuT. We hypothesize that kD measured in A52SuT reflects primarily long-range electrostatic repulsions because most of these mAb candidates carry strong net positive charges in this low ionic strength formulation with pH (5.2) well below pI values of mAb candidates. However, the viscosities of high concentration mAbs depend heavily on short-range molecular interactions. We propose an improved kD method in which salt is added to suppress charge repulsions and to allow for detection of key short-range interactions in dilute solutions. Salt types and salt concentrations were screened, and an optimal salt condition was identified. This optimized method was further validated using two test mAb sets. Overall, the method improves the Pearson R2 between kD and viscosity (6-230 cP) from 0.24 to 0.80 for a data set consisting of 37 mAbs.

Development of in silico models to predict viscosity and mouse clearance using a comprehensive analytical data set collected on 83 scaffold-consistent monoclonal antibodies.

Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.

Antibodies Adsorbed to the Air-Water Interface Form Soft Glasses.

When monoclonal antibodies are exposed to an air-water interface, they form aggregates, which negatively impacts their performance. Until now, the detection and characterization of interfacial aggregation have been difficult. Here, we exploit the mechanical response imparted by interfacial adsorption by measuring the interfacial shear rheology of a model antibody, anti-streptavidin immunoglobulin-1 (AS-IgG1), at the air-water interface. Strong viscoelastic layers of AS-IgG1 form when the protein is adsorbed from the bulk solution. Creep experiments correlate the compliance of the interfacial protein layer with the subphase solution pH and bulk concentration. These, along with oscillatory strain amplitude and frequency sweeps, show that the viscoelastic behavior of the adsorbed layers is that of a soft glass with interfacial shear moduli on the order of 10-3 Pa m. Shifting the creep compliance curves under different applied stresses forms master curves consistent with stress-time superposition of soft interfacial glasses. The interfacial rheology results are discussed in the context of the interface-mediated aggregation of AS-IgG1.

High-Pressure, Low-Temperature Induced Unfolding and Aggregation of Monoclonal Antibodies: Role of the Fc and Fab Fragments.

The effects of high pressure and low temperature on the stability of two different monoclonal antibodies (MAbs) were examined in this work. Fluorescence and small-angle neutron scattering were used to monitor the in situ effects of pressure to infer shifts in tertiary structure and characterize aggregation prone intermediates. Partial unfolding was observed for both MAbs, to different extents, under a range of pressure/temperature conditions. Fourier transform infrared spectroscopy was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubation at elevated pressures and subzero ° C temperatures was independent of the extent of tertiary unfolding and reversibility. Several combinations of pressure and temperature were also used to discern the respective contributions of the isolated Ab fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed significantly different partial unfolding profiles and reversibility. There was not a simple correlation between stability of the full MAb and either the Fc or Fab fragment stabilities across all cases, demonstrating a complex relationship to full MAb unfolding and aggregation behavior. That notwithstanding, the combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress.

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

A Rapid, Small-Volume Approach to Evaluate Protein Aggregation at Air-Water Interfaces.

Non-native protein aggregation is a common concern for biopharmaceuticals. A given protein may aggregate through a variety of mechanisms that depend on solution and physico-chemical stress conditions. A thorough evaluation of aggregation behavior for a protein under all conditions of interest is necessary to ensure drug safety and efficacy. This work introduces a rapid, small-volume approach to evaluate protein aggregation propensity upon exposure to air-water interfaces (AWI). A microtensiometer apparatus is used to aerate a small volume of a protein solution with microbubbles for short periods of time (≤10 s). Sub-visible particles that form are captured and analyzed using backgrounded membrane imaging. This allows one to capture all particles in the solution while being sample sparing. The surface-mediated aggregation of two model monoclonal antibodies (MAbs) and a globular protein (aCgn) was tested as a function of pH and temperature. Temperature had a negligible effect under the rapid interface turnover time scales with this technique. Electrostatic protein-protein interactions, mediated by pH changes, were more influential for particle formation via AWI. Nonionic surfactants substantially reduced particle formation for all MAb solutions, but not aCgn. The results are contrasted with expectations when exposing samples to much larger air-water interfacial stress.

Resolving Liquid-Liquid Phase Separation for a Peptide Fused Monoclonal Antibody by Formulation Optimization.

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (kD) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40 °C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization.

Temperature Dependence of Protein Solution Viscosity and Protein-Protein Interactions: Insights into the Origins of High-Viscosity Protein Solutions.

Protein solution viscosity (η) as a function of temperature was measured at a series of protein concentrations under a range of formulation conditions for two monoclonal antibodies (MAbs) and a globular protein (aCgn). Based on theoretical arguments, a strong temperature dependence for protein-protein interactions (PPI) indicates highly anisotropic, short-ranged attractions that could lead to higher solution viscosities. The semi-empirical Ross-Minton model was used to determine the apparent intrinsic viscosity, shape, and "crowding" factors for each protein as a function of temperature and formulation conditions. The apparent intrinsic viscosity was independent of temperature for aCgn, while a slight decrease with increasing temperature was observed for the MAbs. The temperature dependence of solution viscosity was analyzed using the Andrade-Eyring equation to determine the effective activation energy of viscous flow (Ea,η). While Ea,η values were different for each protein, they were independent of formulation conditions for a given protein. PPI were quantified via the osmotic second virial coefficient (B22) and the protein diffusion interaction parameter (kD) as a function of temperature under the same formulation conditions as the viscosity measurements. Net interactions ranged from strongly attractive to repulsive by changing formulation pH and ionic strength for each protein. Overall, larger activation energies for PPI corresponded to larger activation energies for η, and those were predictive of the highest η values at higher protein concentrations.

Kinetics and Competing Mechanisms of Antibody Aggregation via Bulk- and Surface-Mediated Pathways.

Non-native protein aggregation is a long-standing obstacle in the biopharmaceutical industry. Proteins can aggregate through different mechanisms, depending on the solution and stress conditions. Aggregation in bulk solution has been extensively studied in a mechanistic context and is known to be temperature dependent. Conversely, aggregation at interfaces has been commonly observed for liquid formulations but is less understood mechanistically. This work evaluates the combined effects of temperature and compression/dilation of air-water interfaces on aggregation rates and particle formation for anti-streptavidin immunoglobulin gamma-1. Aggregation rates are quantified via size-exclusion chromatography, dynamic light scattering, and microflow imaging as a function of temperature and extent of air-liquid interface compressions. Competition exists between bulk- and surface-mediated aggregation mechanisms. Each has a largely different temperature dependence that leads to a crossover between the dominant aggregation mechanisms as the sample temperature changes. Surface-mediated aggregation rates are pH dependent and correlate with electrostatic protein-protein interactions but do not mirror the pH dependence of bulk aggregation rates that instead follow trends for conformational stability. Mechanistic insights were informed by quiescent incubation of solutions before and after interface compressions. Detailed mechanistic conclusions require direct dynamic observation at the interface. Microbubble tensiometry is introduced as a promising tool for such measurements.

High-Throughput Analytical Light Scattering for Protein Quality Control and Characterization.

We present a review of high-throughput techniques for the characterization and quality control of proteins in the course of purification, evaluation, and formulation, based on static and dynamic light scattering. Multi-angle static light scattering (MALS) in combination with rapid, low-volume UHPLC size exclusion chromatography is effective in characterizing key biophysical properties, while dynamic light scattering (DLS) in high-throughput microwell-plate format provides large quantities of data in a short time to screen many conditions, excipients, cell lines, or candidate biotherapeutics.

How Well Do Low- and High-Concentration Protein Interactions Predict Solution Viscosities of Monoclonal Antibodies?

Protein-protein interactions (PPI) and solution viscosities were measured at low and high protein concentrations under a range of formulation conditions for 4 different monoclonal antibodies. Static light scattering was used to quantify the osmotic second virial coefficient (B22) and the zero-q limit static structure factor (Sq=0), versus protein concentration (c2) from low to high c2. Dynamic light scattering was used to measure the collective diffusion coefficient as a function of c2 and to determine the protein interaction parameter (kD). Static light scattering and dynamic light scattering were combined to determine the hydrodynamic factor (Hq=0), which accounts for changes in hydrodynamic PPI as a function of c2. The net PPI ranged from strongly repulsive to attractive interactions, via changes in buffer pH, ionic strength, and choice of monoclonal antibodies. Multiple-particle tracking microrheology and capillary viscometery were used to measure monoclonal antibodies solution viscosities under the same solution conditions. In most cases, even large and qualitative changes in PPI did not result in significant changes in protein solution viscosity. This highlights the complex nature of PPI and how they influence protein solution viscosity and raises questions as to the validity of using experimental PPI metrics such as kD or B22 as predictors of high viscosity.

Biophysical characterization and molecular simulation of electrostatically driven self-association of a single-chain antibody.

Colloidal protein-protein interactions (PPI) are often expected to impact key behaviors of proteins in solution, such as aggregation rates and mechanisms, aggregate structure, protein solubility, and solution viscosity. PPI of an anti-fluorescein single chain antibody variable fragment (scFv) were characterized experimentally at low to intermediate ionic strength using a combination of static light scattering and sedimentation equilibrium ultracentrifugation. Surprisingly, the results indicated that interactions were strongly net-attractive and electrostatics promoted self-association. Only repulsive interactions were expected based on prior work and calculations based a homology model of a related scFv crystal structure. However, the crystal structure lacks the charged, net-neutral linker sequence. PyRosetta was used to generate a set of scFv structures with different linker conformations, and coarse-grained Monte Carlo simulations were used to evaluate the effect of different linker configurations via second osmotic virial coefficient (B22 ) simulations. The results show that the configuration of the linker has a significant effect on the calculated B22 values, and can result in strong electrostatic attractions between oppositely charged residues on the protein surface. This is particularly relevant for development of non-natural antibody products, where charged linkers and other loop regions may be prevalent. The results also provide a preliminary computational framework to evaluate the effect of unstructured linkers on experimental protein-protein interaction parameters such as B22 .

High-Throughput Screening and Analysis of Charge Variants of Monoclonal Antibodies in Multiple Formulations.

Among different biopharmaceutical products, monoclonal antibodies (mAbs) show a high level of complexity, including heterogeneity due to differences in size, hydrophobicity, charge, and so forth. Such heterogeneity can be related to both cell-based production and any of the stages of purification, storage, and delivery that the mAb is subjected to. Choosing the right formulation composition providing both physical and chemical stabilities can be a very challenging process, especially when done in the limited time frame required for a typical drug development cycle. Charge variants, a common type of heterogeneity for mAbs, are easy to detect by ion exchange, specifically cation exchange chromatography (CEX). We have developed and implemented a high-throughput CEX-based approach for the rapid screening and analysis of charge modifications in multiple formulation conditions. In this work, 96 different formulations of antistreptavidin IgG1 and IgG2 molecules were automatically prepared and analyzed after incubation at high temperature. Design of experiment and statistical analysis tools have been utilized to determine the major formulation factors responsible for chemical stability of antibodies. Regression models were constructed to find the optimal formulation conditions. The methodology can be applied to different stages of preformulation and formulation development of mAbs.

Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering.

Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified. The present report compares these measures of aggregation rates across a range of solution conditions that result in different aggregation mechanisms for anti-streptavidin (AS) immunoglobulin gamma-1 (IgG1). The results illustrate how the two methods provide complementary information when deducing aggregation mechanisms, as well as cases where they provide new mechanistic details that were not possible to deduce in previous work. Criteria are presented for when the two techniques are expected to give equivalent results for quantitative rates, the potential limitations when solution non-idealities are large, as well as a comparison of the temperature dependence of AS-IgG1 aggregation rates with published data for other antibodies.

Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes.

Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δµ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.

Acetate- and Citrate-Specific Ion Effects on Unfolding and Temperature-Dependent Aggregation Rates of Anti-Streptavidin IgG1.

Controlling and predicting unwanted degradation, such as non-native aggregation, is a long-standing challenge for mAbs and other protein-based products. mAb aggregation rates are typically sensitive to temperature, pH, and the addition of excipients. Quantitatively comparing temperature-dependent aggregation rates across multiple possible formulations is a challenge in product development. A parallel temperature initial rate method is used to efficiently and accurately determine initial rates for anti-streptavidin (AS) IgG1 aggregation as a function of pH, [NaCl], and in the presence of acetate versus citrate buffer. Parallel temperature initial rates are shown to agree with results from a traditional, isothermal method and permits direct comparison of the formulations across almost 3 orders of magnitude of aggregation rates. The apparent midpoint unfolding temperatures (through differential scanning calorimetry) and the effective activation energy values (Ea) are generally higher in acetate buffer compared with citrate buffer, which is consistent with preferential accumulation of citrate ions compared with acetate ions that was speculated in previous work (Barnett et al., J Phys Chem B, 2015). Static light scattering and Kirkwood-Buff analysis show that AS-IgG1 has stronger net repulsive protein-protein interactions in acetate compared with citrate buffer, also consistent with increased values of Ea. In an extreme case, aggregation of AS-IgG1 is effectively eliminated across all practical temperatures at pH 4 in 10 mM sodium acetate but proceeds readily in citrate buffer.

Structural Changes and Aggregation Mechanisms for Anti-Streptavidin IgG1 at Elevated Concentration.

Non-native protein aggregation may occur during manufacturing and storage of protein therapeutics, and this may decrease drug efficacy or jeopardize patient safety. From a regulatory perspective, changes in higher order structure due to aggregation are of particular interest but can be difficult to monitor directly at elevated protein concentrations. The present report focuses on non-native aggregation of antistreptavidin (AS) IgG1 at 30 mg/mL under solution conditions that prior work at dilute concentrations (e.g., 1 mg/mL) indicated would result in different aggregation mechanisms. Time-dependent aggregation and structural changes were monitored in situ with dynamic light scattering, small-angle neutron scattering, and Raman scattering and ex situ with far-UV circular dichroism and second-derivative UV spectroscopy. The effects of adding 0.15 M (∼5 w/w %) sucrose were also assessed. The addition of sucrose decreased monomer loss rates but did not change protein-protein interactions, aggregation mechanism(s), or aggregate structure and morphology. Consistent with prior results, altering the pD or salt concentration had the primary effect of changing the aggregation mechanism. Overall, the results provide a comparison of aggregate structure and morphology created via different growth mechanisms using orthogonal techniques and show that the techniques agree at least qualitatively. Interestingly, AS-IgG1 aggregates created at pD 5.3 with no added salt formed the smallest aggregates but had the largest structural changes compared to other solution conditions. The observation that the larger aggregates were also those with less structural perturbation compared to folded AS-IgG1 might be expected to extend to other proteins if the same strong electrostatic repulsions that mediate aggregate growth also mediate structural changes of the constituent proteins within aggregates.

Specific-ion effects on the aggregation mechanisms and protein-protein interactions for anti-streptavidin immunoglobulin gamma-1.

Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein-protein interactions were quantified in terms of the protein-protein Kirkwood-Buff integral (G22) determined from static light scattering and in terms of the protein effective charge (Zeff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein-protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Zeff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein-protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems.

Accelerated formulation development of monoclonal antibodies (mAbs) and mAb-based modalities: review of methods and tools.

More therapeutic monoclonal antibodies and antibody-based modalities are in development today than ever before, and a faster and more accurate drug discovery process will ensure that the number of candidates coming to the biopharmaceutical pipeline will increase in the future. The process of drug product development and, specifically, formulation development is a critical bottleneck on the way from candidate selection to fully commercialized medicines. This article reviews the latest advances in methods of formulation screening, which allow not only the high-throughput selection of the most suitable formulation but also the prediction of stability properties under manufacturing and long-term storage conditions. We describe how the combination of automation technologies and high-throughput assays creates the opportunity to streamline the formulation development process starting from early preformulation screening through to commercial formulation development. The application of quality by design (QbD) concepts and modern statistical tools are also shown here to be very effective in accelerated formulation development of both typical antibodies and complex modalities derived from them.