Some characteristics and purification of anti‐(human ovarian carcinoma)×anti‐(human CD3) single‐chain bispecific antibody

Abstract

To obtain high-quality AhOC x AhCD3 [anti-(human ovarian carcinoma)xanti-(human CD3)] with high biological activity economically and easily, some characteristics and purification of AhOC x AhCD3, a single chain bispecific antibody, were investigated. During the present study, some important properties of AhOC x AhCD3, such as molecular mass, pI, solubility, stability, hydrophobic ability, molecular status and bioactivity were primarily studied. The molecular mass of AhOC x AhCD3 was determined to be approx. 58.0 kDa by SDS/PAGE (theoretical molecular mass: 56972.5 Da, calculated on the basis of the primary structure) and the pI was approx. 7.5+/-0.4 by IEF (isoelectric focusing; theoretical pI: 8.5, predicted by the ProtParam software tool) respectively. Experiments showed that the solubility of AhOC x AhCD3 increased with the pH increase and that the AhOC x AhCD3 was easily degraded into several fragments during the storage time of samples and the mixtures of both fragment B and fragment C retained normal bioactivity. Particularly, the soluble aggregate/polymer status of AhOC x AhCD3 with bioactivity was verified by non-reducing SDS/PAGE. After optimization of purification process, AhOC x AhCD3 with electrophoretic homogeneity was successfully obtained and the yield was approx. 20%. Some important properties of AhOC x AhCD3 were first observed and a procedure for soluble AhOC x AhCD3 purification in scale-up was first established. Some characteristics of AhOC x AhCD3, especially the stability of AhOC x AhCD3, are critical for the development of this protein pharmaceutical and useful for reference to other proteins.