Refolding of Aggregation-Prone ScFv Antibody Fragments Assisted by Hydrophobically Modified Poly(sodium acrylate) Derivatives

Abstract

ScFv antibody fragments are a promising alternatives to full-length antibodies for both therapeutic and diagnosis applications. They can be overexpressed in bacteria, which enables easy large scale production. Since scFv are artificial constructs, they are poorly soluble and prone to aggregation, which makes them difficult to manipulate and to refold. Here, we report stabilization and refolding of scFv fragments from urea-unfolded solutions based on the use of micromolar amounts of polymers playing the role of artificial chaperons. Using fluorescence correlation spectroscopy, we determined the size and aggregation number of complexes of scFv with unmodified or hydrophobically-modified poly(sodium acrylate). The evolution of the secondary structure along the refolding procedure, in the presence or absence of 0.4 M L-arginine at scFv:polymer < 1:5 wt/wt, was determined by high-sensitivity synchrotron-radiation circular dichroism, SRCD. Measurements revealed that refolding in the presence of polymers yields native-like secondary structure, though a different folding pathway can be followed compared to refolding in the absence of polymer. This is the first report on the use of macromolecular additives to assist refolding of a multi-domain protein of therapeutic interest.3

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

“…The scFvs produced in E. coli exhibited different yields (Table 2 ), as it is commonly observed for different scFv domains. Moreover, since scFvs are naturally prone to aggregation by intermolecular cross-pairing of their VH and VL variable domains 33 – 35 , size exclusion chromatography (SEC) for each scFv was carried out in order to obtain monomeric preparations (Suppl. Fig S8).…”