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.

Computational Screening for mAb Colloidal Stability with Coarse-Grained, Molecular-Scale Simulations.

Monoclonal antibodies (mAbs) are an important modality of protein therapeutics with broad applications for numerous diseases. However, colloidal instabilities occurring at high protein concentrations can limit the ability to develop stable, high-concentration liquid dosage forms that are required for patient-centric, device-mediated products. Therefore, it is advantageous to identify colloidally stable mAbs early in the discovery process to ensure that they are selected for development.

Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies.

Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage.

AF4 and PEG Precipitation as Predictive Assays for Antibody Self-Association.

Monoclonal antibodies (mAbs) are often formulated as high-protein-concentration solutions, which in some cases can exhibit physical stability issues such as high viscosity and opalescence. To ensure that mAb-based drugs can meet their manufacturing, stability, and delivery requirements, it is advantageous to screen for and select mAbs during discovery that are not prone to such behaviors. It has been recently shown that both these macroscopic properties can be predicted to a certain extent from the diffusion interaction parameter (), which is a measure of self-association under dilute conditions.

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.

Machine Learning Feature Selection for Predicting High Concentration Therapeutic Antibody Aggregation.

Protein aggregation can hinder the development, safety and efficacy of therapeutic antibody-based drugs. Developing a predictive model that evaluates aggregation behaviors during early stage development is therefore desirable. Machine learning is a widely used tool to train models that predict data with different attributes.

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.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity.

Carbohydrate-mediated polyethylene glycol conjugation of TSH improves its pharmacological properties.

Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance.

Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products.

Detection of high-molecular-weight amyloid serum protein complexes using biological on-line tracer sedimentation.

The systemic amyloidoses are a rare but deadly class of protein folding disorders with significant unmet diagnostic and therapeutic needs. The current model for symptomatic amyloid progression includes a causative role for soluble toxic aggregates as well as for the fibrillar tissue deposits. Although much research is focused on elucidating the potential mechanism of aggregate toxicity, evidence to support their existence in vivo has been limited.

Fluorescence-detected sedimentation in dilute and highly concentrated solutions.

Analytical ultracentrifugation (AUC) is a powerful, first-principles method for characterizing macromolecules in solution. The recent development of fluorescence-detected sedimentation for the AUC (AU-FDS) has extended the sensitivity and selectivity of the instrument which, in turn, has enabled the study of both higher affinity interactions and the sedimentation of one component in complex, concentrated solutions. While still in its infancy, AU-FDS is becoming more widespread as shown by the increasing number of literature reports citing its use.

The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal. Direct analysis by fluorescence-detected analytical ultracentrifugation.

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined.

Expression, purification, and in vitro cysteine-10 modification of native sequence recombinant human transthyretin.

Transthyretin (TTR) is a serum protein that is also a prominent component of deposits in two different types of systemic amyloid disease, senile systemic and familial TTR amyloidoses. Studies of recombinant TTR (rTTR) have provided many insights into the relationship between protein structure and amyloidogenicity. Yet, there is no existing recombinant system that results in high yield production of a protein that is identical in primary structure to human TTR.

Detailed structural analysis of amyloidogenic wild-type transthyretin using a novel purification strategy and mass spectrometry.

Wild-type transthyretin (TTR), normally a soluble plasma-circulating protein, can be amyloidogenic, i.e., form tissue-deposited fibrillar material in the extracellular matrix of various organs throughout the body.

Valence and anion binding of bovine ribonuclease A between pH 6 and 8.

Several studies have shown that divalent anion binding to ribonuclease A (RNase A) contributes to RNase A folding and stability. However, there are conflicting reports about whether chloride binds to or stabilizes RNase A. Two broad-zone experimental approaches, membrane-confined electrophoresis and analytical ultracentrifugation, were used to examine the electrostatic and electrohydrodynamic characteristics of aqueous solutions of bovine RNase A in the presence of 100 mM KCl and 10 mM Bis-Tris propane over a pH range of 6.