PULSE SPR: A High Throughput Method to Evaluate the Domain Stability of Antibodies

Abstract

Downstream purification of therapeutic antibodies requires candidates to be stable under various stress conditions, such as low pH. Current approaches to assess the conformational or colloidal stability of biologics may require significant amounts of material, and the testing may occur over an extended period of time. Furthermore, typical methodologies for early stability testing often do not directly address low pH stability, but focus more on conditions suitable for long-term drug product storage. Here we report a high-throughput method that measures protonation-induced unfolding of ligand binding sites for stability evaluation by surface plasmon resonance or PULSE SPR. This method, which requires only several micrograms of sample, is highly sensitive to the structural integrity of ligand binding sites and correlates well with thermal and chemical conformational stability. Combined with ligands targeting different regions of antibodies, this method allows a comprehensive assessment of antibody domain stability. By applying PULSE SPR, we found that antibodies with different complementarity-determining regions (CDRs), frameworks, formats, and interchain disulfide bonds showed different stabilities under acidic conditions. Additionally, biologics that aggregated in solution also had poor low pH stability. Taken together, PULSE SPR is a high-throughput and low sample consumption method that can be adopted to evaluate antibody domain stability and aggregation at low pH, which are two important aspects of therapeutic biologics.

A non-clinical comparative study of IL-23 antibodies in psoriasis

“…DSC and nanoDSF measurements were performed as previously described. 45 Briefly, antibody samples were prepared at a concentration of 1 mg/mL in 4.4 mM Succinate, 225 mM Sorbitol, 0.02% Polysorbate 20, pH 6.0 (risankizumab) or 3.87 mM L-histidine, 7.15 mM L-histidine monohydrochloride monohydrate, 7.9% w/v sucrose, 0.05% w/v polysorbate 80, pH 5.8 (ustekinumab, guselkumab, and tildrakizumab). The thermal ramps were performed from 20°C to 95°C with a scan rate of 1°C/min.…”

“…Binding kinetics of IL-23 antibodies for recombinant human sc or heterodimeric IL-23 were determined by SPR at 37°C using an anti-Fc capture assay approach as described. 45 Briefly, IL-23 antibodies were captured on an anti-human Fc chip. IL-23, ranging in concentration from 36 nM to 0.15 nM in a randomized 3-fold dilution series, was injected over both reference and test surface for 5 minutes at 50 µL/minute.…”

Getting Smaller by Denaturation: Acid-Induced Compaction of Antibodies

“…The nonnative conformation of IgG1 induced by this pH shift is aggregation-prone at a neutral pH, with structural origins that remain unclear. [10,[59][60][61][62][63][64] Because the acid-denatured state is unstable at a neutral pH, the IgG1 molecules transition toward native or aggregated states. It is unclear whether the molecular characteristics of the acid-denatured IgG1, such as intramolecular aggregation, slow or accelerate these reactions.…”

“…Here, we report an exceptional case where denaturation makes proteins smaller, using an immunoglobulin G1 (IgG1) monoclonal antibody. Acid-solution induces the denaturation of IgG1, including the loss of the binding function [10] and native structures. [11,12] However, the structures of (which was not certified by peer review) is the author/funder.…”

Matching pH values for antibody stabilization and crystallization suggest rationale for accelerated development of biotherapeutic drugs

“…Using DSF or DSC (differential scanning calorimetry) thermal unfolding events of antibody C H 3 domain ( T m 3) at temperatures above 80 °C have been reported (Brader et al, ; Zhou et al, ). However, the corresponding fluorescent signals are usually quite weak, if present at all.…”