High-throughput analysis system of interaction kinetics for data-driven antibody design

Abstract

Surface plasmon resonance (SPR) is widely used for antigen–antibody interaction kinetics analysis. However, it has not been used in the screening phase because of the low throughput of measurement and analysis. Herein, we proposed a high-throughput SPR analysis system named “BreviA” using the Brevibacillus expression system. Brevibacillus was transformed using a plasmid library containing various antibody sequences, and single colonies were cultured in 96-well plates. Sequence analysis was performed using bacterial cells, and recombinant antibodies secreted in the supernatant were immobilized on a sensor chip to analyze their interactions with antigens using high-throughput SPR. Using this system, the process from the transformation to 384 interaction analyses can be performed within a week. This system utility was tested using an interspecies specificity design of an anti-human programmed cell death protein 1 (PD-1) antibody. A plasmid library containing alanine and tyrosine mutants of all complementarity-determining region residues was generated. A high-throughput SPR analysis was performed against human and mouse PD-1, showing that the mutation in the specific region enhanced the affinity for mouse PD-1. Furthermore, deep mutational scanning of the region revealed two mutants with > 100-fold increased affinity for mouse PD-1, demonstrating the potential efficacy of antibody design using data-driven approach.

High‐throughput system for the thermostability analysis of proteins

“…After incubation, the culture supernatants of clone #1 were subjected to ammonium sulfate (AS) precipitation for subsequent purification using plate‐scale immobilized metal affinity chromatography (IMAC) (Figure 2 ). AS precipitation effectively removes low‐molecular‐weight components derived from culture medium (Matsunaga et al, 2023 ). However, the purity of the final sample was not enough for DSF measurements after treatment with 60% (v/v) saturated AS and IMAC, yielding initial high fluorescence (Figure 3a ).…”

“…Furthermore, the loss of scFv activity due to mutations was not considered in this study. Recently, our twin system BreviA (Matsunaga et al, 2023 ) enabled the high‐throughput interaction kinetic analysis of Fab. Combinatory analyses will facilitate the simultaneous optimization of interaction kinetics and thermal stability, deciphering the quantitative effects of mutations on different physicochemical parameters.…”

Measuring Interactions Between Proteins and Small Molecules or Nucleic Acids

“…This technique has evolved exponentially in the past few years with some machines able to measure nearly 400 interactions at a time, putting SPR at the forefront of techniques for high‐throughput experiments. This technique is often employed to characterize drug candidates because of its robust and reliable reporting of kinetic data (Matsunaga et al., 2023).…”