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Some characteristics and purification of anti‐(human ovarian carcinoma)×anti‐(human CD3) single‐chain bispecific antibody
Author(s) -
Zhou Feng,
Wei DongZhi,
Tong WangYu,
Zhu YiQi,
Jiang YunMei
Publication year - 2007
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20060149
Subject(s) - molecular mass , solubility , isoelectric point , chemistry , isoelectric focusing , chromatography , pi , yield (engineering) , enzyme , biochemistry , organic chemistry , materials science , metallurgy
To obtain high‐quality AhOC×AhCD3 [anti‐(human ovarian carcinoma)×anti‐(human CD3)] with high biological activity economically and easily, some characteristics and purification of AhOC×AhCD3, a single chain bispecific antibody, were investigated. During the present study, some important properties of AhOC×AhCD3, such as molecular mass, pI, solubility, stability, hydrophobic ability, molecular status and bioactivity were primarily studied. The molecular mass of AhOC×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×AhCD3 increased with the pH increase and that the AhOC×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×AhCD3 with bioactivity was verified by non‐reducing SDS/PAGE. After optimization of purification process, AhOC×AhCD3 with electrophoretic homogeneity was successfully obtained and the yield was approx. 20%. Some important properties of AhOC×AhCD3 were first observed and a procedure for soluble AhOC×AhCD3 purification in scale‐up was first established. Some characteristics of AhOC×AhCD3, especially the stability of AhOC×AhCD3, are critical for the development of this protein pharmaceutical and useful for reference to other proteins.