Improving purity and apparent thermal stability of single-chain Fv antibodies by heat treatment in a soluble E. coli cytoplasmic expression system.

Single-chain variable fragment (scFv) antibodies outperform conventional IgG antibodies due to their lower molecular weight, reduced immunogenicity, rapid clearance and efficient bacterial expression. However, it remains difficult to achieve correct folding of scFv containing two intramolecular disulfide bonds in the reducing Escherichia coli cytoplasm. We previously achieved correct folding of a trastuzumab-derived scFv by co-expressing the disulfide isomerase DsbC and the yeast sulfhydryl oxidase Erv1p in the cytoplasm of the E. coli SHuffle T7 strain. Here, we applied this system to produce the immune checkpoint inhibitor atezolizumab-derived scFv (denoted as atz-scFv). The atz-scFv was purified through a two-step chromatography process; however, insufficient disulfide bond formation led to contamination by proteins lacking a disulfide bonds. To address this, we developed and optimized a heat-treatment method in which atz-scFv was incubated at elevated temperatures to selectively precipitate protein species lacking a disulfide bond. The resulting atz-scFv exhibited increased apparent thermal stability and retained antigen-binding affinity compared to the untreated sample. This heat-treatment method offers a cost-effective alternative to traditional purification, eliminating the need for affinity chromatography while enhancing scFv stability and purity. The present results broaden scFv purification methods.