High-throughput profiling of antibody self-association in multiple formulation conditions by PEG stabilized self-interaction nanoparticle spectroscopy

Abstract

Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) is an assay developed to monitor the propensity of antibody self-association, hence assessing its colloidal stability. It has been widely used by pharmaceutical companies to screen antibodies at the early discovery stages, aiming to flag potential issues with high concentration formulation. However, the original assay format is not suitable for certain formulation conditions, in particular histidine buffer. In addition, the previous data extrapolation method is suboptimal and cumbersome for processing large amounts of data (100s of molecules) in a high-throughput fashion. To address these limitations, we developed an assay workflow with two major improvements: 1) use of a stabilizing reagent to enable screening of a broader range of formulation conditions beyond phosphate-buffered saline, pH 7.4; and 2) inclusion of a novel algorithm and robust data processing schema that empowers streamlined data analysis. The optimized assay format expands the screening applicability to a wider range of formulation conditions critical for downstream development. Such capability is enhanced by a custom data management workflow for optimal data extraction, analysis, and automation. Our protocol and the R/Shiny application for analysis are publicly available and open-source to benefit the broader scientific community.

A framework for the biophysical screening of antibody mutations targeting solvent-accessible hydrophobic and electrostatic patches for enhanced viscosity profiles

“…We used Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS) to assess self-association propensity in the anti-IL-8 mAb panel. [45] Goat anti-human Fc and whole goat IgG antibodies (Jackson ImmunoResearch, PA, USA) were prepared in 20 mM acetate buffer (pH 4.3) and diluted to achieve final concentrations of 320 µg anti-Fc IgG and 80 µg goat whole IgG, then mixed with 20 nM colloidal gold nanoparticle suspension (Ted Pella Inc., CA, USA, concentration 7.0×10 11 particles /mL). After incubation and centrifugation, anti-IL-8 mAb test samples were prepared at 50 μg/mL in phosphate-buffered saline (Gibco, Thermo Fisher Scientific, MA, USA).…”

Assessing developability early in the discovery process for novel biologics

“… 50 AC-SINS is most commonly performed in a solution mimicking physiological conditions (pH 7.4, phosphate-buffered saline), 3 and its measurements have most commonly been linked to pharmacokinetic properties, 33 , 58 although it has also been used for formulation applications. 59 CS-SINS is performed in a common formulation condition (pH 6, 10 mM histidine) and has been reported to identify antibodies with low viscosity and opalescence in concentrated antibody formulations. 50 …”

High concentration formulation developability approaches and considerations

“…A recently developed method in this area, termed PEG-stabilized SINS (PS-SINS), uses polyethylene glycol (PEG) functionalization to extend the applicability of the method to a wider pH range. 38 A complementary method, which uses poly-L-lysine (PLL) functionalization (termed charge stabilized SINS, CS-SINS) to achieve a similar extension in applicability, was used to analyze the behavior of a panel of 56 antibodies composed of molecules with IgG1, IgG2, and IgG4 frameworks. 39 The authors showed that the behavior of IgG1 and IgG2 mAbs in the CS-SINS assay correlated very well with their high concentration viscosity and opalescence properties.…”

“…While high-throughput predictive assays with impressive correlations to unwanted high concentration solution behavior of proteins have been developed, it is currently impossible for a single assay to predict which liability will ultimately be manifested. 38 , 39 , 41 , 49 Furthermore, most assays have been developed for standard IgG formats. Due to the complexity of the underlying molecular mechanism of self-association, the limitation of these assays becomes relevant for alternative molecule formats (e.g., Fabs, DutaFabs, fusion proteins, Contorsbodies).…”