A scaling relationship between thermodynamic and hydrodynamic interactions in protein solutions.

Weak protein interactions are associated with a broad array of biological functions and are often implicated in molecular dysfunction accompanying human disease. In addition, these interactions are a critical determinant in the effective manufacturing, stability, and administration of biotherapeutic proteins. Despite their prominence, much remains unknown about how molecular attributes influence the hydrodynamic and thermodynamic contributions to the overall interaction mechanism.

Designing Robust Monoclonal Antibody Drug Products: Pitfalls of Simplistic Approaches for Stability Prediction.

Thermal stability attributes including unfolding onset (T) and mid-point (T) are often utilized for efficient development of monoclonal antibody (mAb) products during lead selection and formulation screening workflows. An assumption of direct correlation between thermal and kinetic physical stability underpins this basic approach. While literature reports have substantiated this general approach under specific conditions, clear exceptions have been highlighted alongside.

A biomimetic chip to assess subcutaneous bioavailability of monoclonal antibodies in humans.

Subcutaneous (subQ) injection is a common route for delivering biotherapeutics, wherein pharmacokinetics is largely influenced by drug transport in a complex subQ tissue microenvironment. The selection of good drug candidates with beneficial pharmacokinetics for subQ injections is currently limited by a lack of reliable testing models. To address this limitation, we report here a cutaneous Co-lture issue-on-a-chip for njection imulation (SubCuTIS).

Ultradilute Measurements of Self-Association for the Identification of Antibodies with Favorable High-Concentration Solution Properties.

There is significant interest in formulating antibody therapeutics as concentrated liquid solutions, but early identification of developable antibodies with optimal manufacturability, stability, and delivery attributes remains challenging. Traditional methods of identifying developable mAbs with low self-association in common antibody formulations require relatively concentrated protein solutions (>1 mg/mL), and this single challenge has frustrated early-stage and large-scale identification of antibody candidates with drug-like colloidal properties. Here, we describe charge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS), an affinity-capture nanoparticle assay that measures colloidal self-interactions at ultradilute antibody concentrations (0.

Characterization of Opalescence in low Volume Monoclonal Antibody Solutions Enabled by Microscale Nephelometry.

Monoclonal antibody (mAb)-based drugs are often prone to unfavorable solution behaviors including high viscosity, opalescence, phase separation, and aggregation at the high concentrations needed to enable patient-centric subcutaneous dosage forms. Given that these can have a detrimental impact on manufacturability, stability, and delivery, approaches to identifying, monitoring, and controlling these behaviors during drug development are critical. Opalescence presents a significant challenge due to its relationship to liquid-liquid phase separation.

A single molecular descriptor to predict solution behavior of therapeutic antibodies.

Despite the therapeutic success of monoclonal antibodies (mAbs), early identification of developable mAb drug candidates with optimal manufacturability, stability, and delivery attributes remains elusive. Poor solution behavior, which manifests as high solution viscosity or opalescence, profoundly affects the developability of mAb drugs. Using a diverse dataset of 59 mAbs, including 43 approved products, and an array of molecular descriptors spanning colloidal, conformational, charge-based, hydrodynamic, and hydrophobic properties, we show that poor solution behavior is prevalent (>30%) in mAbs and is singularly predicted (>90%) by the diffusion interaction parameter ( ), a dilute-solution measure of colloidal self-interaction.

Particle shape enhances specificity of antibody-displaying nanoparticles.

Monoclonal antibodies are used in numerous therapeutic and diagnostic applications; however, their efficacy is contingent on specificity and avidity. Here, we show that presentation of antibodies on the surface of nonspherical particles enhances antibody specificity as well as avidity toward their targets. Using spherical, rod-, and disk-shaped polystyrene nano- and microparticles and trastuzumab as the targeting antibody, we studied specific and nonspecific uptake in three breast cancer cell lines: BT-474, SK-BR-3, and MDA-MB-231.

Polar solvents decrease the viscosity of high concentration IgG1 solutions through hydrophobic solvation and interaction: formulation and biocompatibility considerations.

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL.

Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter.

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions.

Effect of PEGylation on protein hydrodynamics.

We studied the effect of PEGylation on protein hydrodynamic behavior using hen egg-white lysozyme (HEWL) as a model protein. HEWL was PEGylated with a linear, 20 kDa PEG using reductive amination to produce PEG1-, PEG2-, and PEG3-HEWL. Near- and far-UV-CD spectroscopy revealed no significant effect of PEGylation on HEWL higher order structure.

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions.

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.

Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation.

The concentration-dependence of the diffusion and sedimentation coefficients (k(D) and k(s), respectively) of a protein can be used to determine the second virial coefficient (B₂), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B₂ under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k(D) and k(s) via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions.

Characterization of antibody aggregation: role of buried, unpaired cysteines in particle formation.

Proteins are susceptible to degradation upon exposure to a variety of stresses during product manufacturing, transportation and storage. In this study, we investigated the aggregation properties of a monoclonal antibody during agitation stress. Agitation exclusively led to insoluble aggregates, or particle formation.

Ion-specific modulation of protein interactions: anion-induced, reversible oligomerization of a fusion protein.

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E.

Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies.

Purpose: The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies.

Methods: Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG(2) mAbs.

Anion binding mediated precipitation of a peptibody.

Purpose: Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions.

Methods: The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.

Self-buffering antibody formulations.

Monoclonal antibodies (mAbs) often require the development of high-concentration formulations. In such cases, and when it is desirable to formulate a mAb around pH 5.0, we explored a novel approach of controlling the formulation pH by harnessing the ability of mAbs to "self-buffer.