Towards a universal disulphide stabilised single chain Fv format: importance of interchain disulphide bond location and vL-vH orientation

Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. Five of these disulphide bond locations were introduced into one scFv in order to compare their relative effects on expression, thermal stability, percent monomer formation and retention of antigen binding. The disulphide bond position vH44-vL100 was observed to enable the most favourable balance of biophysical properties. The vH44-vL100 disulphide bond was introduced into five additional scFv in both vL-vH and vH-vL orientations in order to investigate its general applicability. Data are presented to show the relative influence of scFv sequence, v-region organisation and interchain disulphide bond on expression yield, thermal stability and percent monomer. Introduction of the vH44-vL100 disulphide bond typically resulted in no or little increase in thermal stability and no change in percent monomer but did confer the benefit of permanently fixing monomer:dimer ratios during purification and analysis.