An accelerated surface-mediated stress assay of antibody instability for developability studies

Abstract

High physical stability is required for the development of monoclonal antibodies (mAbs) into successful therapeutic products. Developability assays are used to predict physical stability issues such as high viscosity and poor conformational stability, but protein aggregation remains a challenging property to predict. Among different types of stresses, air–water and solid–liquid interfaces are well known to potentially trigger protein instability and induce aggregation. Yet, in contrast to the increasing number of developability assays to evaluate bulk properties, there is still a lack of experimental methods to evaluate antibody stability against interfaces. Here, we investigate the potential of a hydrophobic nanoparticle surface-mediated stress assay to assess the stability of mAbs during the early stages of development. We evaluate this surface-mediated accelerated stability assay on a rationally designed library of 14 variants of a humanized IgG4, featuring a broad span of solubility values and other developability properties. The assay could identify variants characterized by high instability against agitation in the presence of air–water interfaces. Remarkably, for the set of investigated molecules, we observe strong correlations between the extent of aggregation induced by the surface-mediated stress assay and other developability properties of the molecules, such as aggregation upon storage at 45°C, self-association (evaluated by affinity-capture self-interaction nanoparticle spectroscopy) and nonspecific interactions (estimated by cross-interaction chromatography, stand-up monolayer chromatography (SMAC), SMAC*). This highly controlled surface-mediated stress assay has the potential to complement and increase the ability of the current set of screening techniques to assess protein aggregation and developability potential of mAbs during the early stages of drug development. Abbreviations :AC-SINS: Affinity-Capture Self-Interaction Nanoparticle Spectroscopy; AMS: Ammonium sulfate precipitation; ANS: 1-anilinonaphtalene-8-sulfonate; CIC: Cross-interaction chromatography; DLS: Dynamic light scattering; HIC: Hydrophobic interaction chromatography; HNSSA: Hydrophobic nanoparticles surface-stress assay; mAb: Monoclonal antibody; NP: Nanoparticle; SEC: Size exclusion chromatography; SMAC: Stand-up monolayer chromatography; WT: Wild type

Plate Reader-Based Analytical Method for the Size Distribution of Submicron-Sized Protein Aggregates Using Three-Dimensional Homodyne Light Detection

“…The surface-mediated stress assay and hydrophobic nanoparticle surface-mediated stress assay could be used to screen for any aggregation risk in therapeutic proteins. 34,35 Agitation, dropping and friability tests are typically recommended to assess aggregate formation risks in therapeutic proteins that use vials, syringes, or IV bags during formulation development [36][37][38][39] ; however, these tests are challenging to perform in the early stages of development as they require large sample amounts. 3D-HLD automatically measured the particle concentrations of aggregates in 30 formulations of two therapeutic proteins (Fig.…”

Screening for protein–protein interactions with asymmetrical flow field-flow fractionation

“…They have been expressed in mammalian cell lines and been purified as a minimum with both a Protein A purification and a size-exclusion chromatography step yielding a purity above 95 % as measured with SE-HPLC (data not shown). mAb 2 has previously been characterized in detail, and is denoted as WT in references 25,26 and as mAb-C in reference. 27 Theoretical pI is 6.9, 8.1, 8.4 and 8.4 for mAb 1-4 respectively.…”

Surface interaction patches link non-specific binding and phase separation of antibodies

“…Design, characterisation and expression of the HzATNP antibodies have been performed as described in detail previously 30,37 . After expression, all antibody samples were stored in aliquots at roughly 5-10 mg/mL in either 20mM HEPES buffer pH = 7.4, 150mM NaCl, with 0.01 % (v/v ) Tween (HS buffer) or 2mM HEPES buffer pH = 7.4, 15mM NaCl, with 0.01 % (v/v ) Tween (LS buffer) at -80 • C. Protein stocks were thawed on ice and shock frozen using liquid nitrogen, up to a maximum of three freeze-thaw cycles.…”

“…This library represents an ideal model system for conceptual investigation as it has been characterized in detail to show distinct changes in the protein biophysical properties between the individual variants. 37 We assess the impact of the changes between variants on the non-specific binding to single-stranded DNA, a common non-specificity ligand which is a valuable indicator for the overall propensity of non-specific interactions also in vivo 27,33,38 . By establishing microfluidic diffusional sizing (MDS) 39,40 (Figure 1d) as a tool for probing antibody non-specificity, we identify that non-specific binding of HzATNP antibodies to DNA at physiological salt conditions is driven by a patch of amino-acid residues that engage in hydrogen bonding.…”