Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry

Abstract

Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of nonspecific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for in vitro characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAbdependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our in vitro data support the notion that HSA in the blood may lead to non-specific interactions with mAbs in vivo, with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.

Suppressing Nonspecific Binding in Biolayer Interferometry Experiments for Weak Ligand–Analyte Interactions

“… 4 − 9 One well-established application of BLI is the characterization of tight binding interactions, such as antibody–antigen interactions. 10 − 12 Notable examples include the identification of therapeutic antibodies against SARS-CoV-2 11 and characterization of the binding between the receptor binding domain (RBD) of SARS-CoV-2 and human ACE2. 7 , 13 , 14 …”

Probing Interactions of Therapeutic Antibodies with Serum via Second Virial Coefficient Measurements

“…Wright, et al (24) proposed a preclinical AUC method to measure weak interactions between mAbs and serum components such as HSA and IgG through determination of second virial coefficients and sedimentation interaction parameters. Kim, et al (43) used CG-MALS to probe cross-term interactions between mAbs and HSA, but also used BLI to explore the functional consequence of this nonideality on antigen binding. While these studies relied on HSA alone as a model system to determine cross-term nonideality of mAbs in serum, our results caution that such an approach may fail to qualitatively or quantitatively capture the true effects of serum.…”

Discovery Strategies to Maximize the Clinical Potential of T-Cell Engaging Antibodies for the Treatment of Solid Tumors

“…In addition, antibody libraries in non-Fab modalities are very useful for subsequent bispecific formatting with scFv and VHH components, to avoid any loss of binding or stability attributes during Fab to scFv or Fab to VHH engineering. There is an expanding set of biophysical techniques [ 55 , 56 , 57 ] and in silico tools [ 58 , 59 , 60 ] for developability assessment and engineering designs, many of which can be used in a high-throughput manner. Early manufacturability and biophysical evaluation during lead identification would advance more stable binders for bispecific TcE designs.…”