pH and excipient profiles during formulation of highly concentrated biotherapeutics using bufferless media.

Several models have been developed to describe the shifts in pH and excipient concentrations seen during diafiltration of monoclonal antibody (mAb) products accounting for both Donnan equilibrium and electroneutrality constraints. However, these models have assumed that the mAb charge is either constant or only a function of pH, assumptions that will not be valid when formulating highly concentrated mAbs using bufferless or low-buffered media due to the change in local H concentration at the protein surface. The objective of this study was to incorporate the effects of both pH and ionic strength on the mAb charge, through the use of a charge regulation model based on the amino acid sequence of the mAb, into an appropriate mass balance model to describe the pH and excipient profiles during diafiltration.

Biophysical and Sequence-Based Methods for Identifying Monovalent and Bivalent Antibodies with High Colloidal Stability.

In vitro antibody discovery and/or affinity maturation are often performed using antibody fragments (Fabs), but most monovalent Fabs are reformatted as bivalent IgGs (monoclonal antibodies, mAbs) for therapeutic applications. One problem related to reformatting antibodies is that the bivalency of mAbs can lead to increased antibody self-association and poor biophysical properties (e.g.

Facile Preparation of Stable Antibody-Gold Conjugates and Application to Affinity-Capture Self-Interaction Nanoparticle Spectroscopy.

Protein-nanoparticle conjugates are widely used for conventional applications such as immunohistochemistry and biomolecular detection as well as emerging applications such as therapeutics and advanced materials. Nevertheless, it remains challenging to reproducibly prepare stable protein-nanoparticle conjugates with highly similar optical properties. Here we report an improved physisorption method for reproducibly preparing stable antibody-gold conjugates at acidic pH using polyclonal antibodies from a wide range of species (human, goat, rabbit, mouse, and rat).

Measurements of Monoclonal Antibody Self-Association Are Correlated with Complex Biophysical Properties.

Successful development of monoclonal antibodies (mAbs) for therapeutic applications requires identification of mAbs with favorable biophysical properties (high solubility and low viscosity) in addition to potent bioactivities. Nevertheless, mAbs can also display complex, nonconventional biophysical properties that impede their development such as formation of soluble aggregates and subvisible particles as well as nonspecific interactions with various types of surfaces such as nonadsorptive chromatography columns. Here we have investigated the potential of using antibody self-interaction measurements obtained via affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) at dilute concentrations (0.

Discovery of highly soluble antibodies prior to purification using affinity-capture self-interaction nanoparticle spectroscopy.

Self-association of monoclonal antibodies (mAbs) at high concentrations can result in developability challenges such as poor solubility, aggregation, opalescence and high viscosity. There is a significant unmet need for methods that can evaluate self-association propensities of concentrated mAbs at the earliest stages in antibody discovery to avoid downstream issues. We have previously developed a method (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) that is capable of detecting weak antibody self-interactions using unusually dilute mAb solutions (tens of µg/ml).

High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum.

Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations.

Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions.

A limitation of using mAbs as therapeutic molecules is their propensity to associate with themselves and/or with other molecules via nonaffinity (colloidal) interactions. This can lead to a variety of problems ranging from low solubility and high viscosity to off-target binding and fast antibody clearance. Measuring such colloidal interactions is challenging given that they are weak and potentially involve diverse target molecules.

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy.

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties.