A new approach to quantification of mAb aggregates using peptide affinity probes

Abstract

Using mAbs as therapeutic molecules is complicated by the propensity of mAbs to aggregate at elevated concentrations, which can lead to a variety of adverse events in treatment. Here, we describe a proof-of-concept for new methodology to detect and quantify mAb aggregation. Assay development included using an aggregated mAb as bait for screening of phage display peptide library and identifying those peptides with random sequence which can recognize mAb aggregates. Once identified, the selected peptides can be used for developing quantitative methods to assess mAb aggregation. Results indicate that a peptide binding method coupled with mass spectrometric detection of bound peptide can quantify mAb aggregation and potentially be useful for monitoring aggregation propensity of therapeutic protein candidates.

The structural basis of nanobody unfolding reversibility and thermoresistance

“…Therefore, it is of considerable interest to study its determinants for avoiding risk factors or potentially restoring nanobody folding reversibility through protein engineering. However, a comparison of aggregation propensities among a set of binders as diverse as ours remains challenging 43 , 44 . Turbidity signals are qualitative due to their dependence on aggregate size and shape 44 .…”

Assessment of Extracellular Vesicles Purity Using Proteomic Standards

“…Figure 2B shows that the major component of both preparations is the same with hydrodynamic diameter near 8 nm. This is too small for EVs and most likely represents either protein(s) such as IgG 21 or lipoprotein complexes such as high-density lipoprotein, 22 which coisolate with EVs. The bigger components (not visible by number in Figure 2B) are present based on intensity of scattering and their pattern change after SEC (Figure 2C).…”

A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material

“…Firstly, N Ab at ϑ m Fc may be a fundamental property of a given antibody due to glycan variation and the locations of PAG binding domains on the structure, leading to some antibody monomers binding in orientations which favour closer packing on the surface [35]. The ϑ m Fc will also be sensitive to non-specific aggregation and clustering of IgG [29], although aggregates of the NISTmAb IgG are only observed following deliberate physical agitation for days at room temperature [36] and the antibody samples tested here are not expected to contain significant aggregates due to appropriate storage. The lack of correlation between ϑ m Fc and ESI monomeric purity may best be explained by the orientation and higher packing density of low mass antibody degradation products, which may have access to a greater number of binding sites on the PAG surface than monomeric IgG [20].…”

“…The biosensor-based technique presented here to estimate antibody monomeric purity can be performed in 15 min and may be combined with additional rapid tests for IgG aggregates as described for the NISTmAb in ref. [36].…”