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.

Keywords: aggregation; antibodies; developability; formulation; interfaces; stability; surfaces.

Figure 1.

Surface-induced aggregation of IgG4 wild type in the hydrophobic nanoparticle surface-mediated aggregation assay. (a) Size distribution of our model IgG4 at the initial time point in absence and presence of hydrophobic nanoparticles, which trigger the formation of micron size aggregate in a time scale of few seconds. The IgG4 sample is stable in absence of hydrophobic nanoparticles. Incubation was performed at room temperature in 10 mM NaCl, 10 mM HEPES, 10 mM MES buffer at pH 6.6. (b) Bright field (top) and fluorescence (bottom) microscopy images of aggregates stained by ANS. Scale bar is 50 μm. (c) The monomer loss scales linearly with the hydrophobic surface introduced by the nanoparticles (results shown were taken after 30 min incubation), and (d) is constant after 30 min stagnant incubation at room temperature (ratio NPs:mAb 1:50).

Figure 2.

Correlation between the monomer loss induced by our HNSSA (1:50 surface ratio NPs:mAb) and an agitation stress assay performed with an air headspace in glass vials incubated at 1400 rpm for 1.5 h. The variants were formulated in both assays at 0.50 mg/mL in 20 mM HEPES, 10 mM NaCl at pH 7.6. The HNSSA and agitation stress assays were performed in duplicates. The different points correspond to the indicated variants, the color gradient from red to blue reflects the CamSol solubility score, from low to high solubility, respectively. The color coding is preserved throughout the presented work. Error bars correspond to one standard deviation.

Figure 3.

Behavior of the variant library relative to the WT in accelerated thermal stability studies (6 weeks incubation at 45°C). (a) The variant ranking in terms of soluble aggregate formation relative to the WT correlates directly to the CamSol solubility score. (b) Correlation between the variant rakings of soluble aggregates against insoluble aggregates. Soluble variants tend to form soluble aggregates, while insoluble variants tend to form insoluble aggregates.  In (a) and (b), a high rank value (>0) corresponds to high amounts of aggregates formed compared to the WT, while a low rank value (<0) corresponds to a lower amount of aggregates formed compared to the WT. The bright and big marker points correspond to the average rankings between the two replicate incubations, while small markers correspond to the individual ranking obtained for each replicate incubation. The samples were prepared as quintuples (run 1) and triplicates (run 2) and incubated in 20 mM HEPES, 10 mM NaCl at pH 7.6.

Figure 4.

Variants ranking in the HNSSA compared to the ranking in the accelerated thermal stability. The variants ranking in the HNSSA correlates (a) inversely with the soluble aggregates ranking in the accelerated thermal stress assay, (b) directly with the ranking of the amount of insoluble aggregates and (c) directly with the ranking of the total amount of aggregates formed (soluble and insoluble) evaluated by SEC. (d) Aggregation and solubility scores obtained with the monomer loss in the HNSSA (not the rankings) and in the thermal stability at 45°C. Bold numbers indicate the Pearson and Spearman rank-order correlation coefficients, while the values indicated below correspond to the respective p-values. High (>0) and low (<0) rank values correspond respectively to higher and lower amounts of aggregates formed compared to the WT. The bright and larger marker points correspond to the average rankings between two replicate incubations, while small markers correspond to the individual ranking obtained for each replicate incubation. The samples were prepared in 20 mM HEPES, 10 mM NaCl at pH 7.6 and incubated in two runs for 6 weeks at 45 °C. The HNSSA was performed in duplicates, and the nanoparticles introduced in a 1:50 NPs:mAb ratio.

Figure 5.

Developabilixty score matrix showing the Pearson’s and Spearman’s correlation rank coefficient of the HNSSA and other developability assays. Bold numbers indicate the Pearson and Spearman rank-order correlation coefficients, while the values indicated below correspond to the respective p-values.

Figure 6.

In silico predictors score matrix showing the Pearson’s and Spearman’s correlation rank coefficients between the HNSSA, the CamSol score and three other common in silico predictors. Bold numbers indicate the Pearson and Spearman rank-order correlation coefficients, while the values indicated below correspond to the respective p-values.