Monoclonal antibodies (mAbs) are used extensively for analytical, diagnostic and therapeutic applications. The purification of mAbs from cell culture supernatants typically consists of protein A, G or L affinity chromatography, often in association with other conventional chromatographic techniques such as ion exchange and gel filtration. We report the application of Gradiflow (TM) preparative electrophoresis technology, for the separation of mouse and mouse/human chimeric mAbs from cell culture supernatants in their native state. The one-step purification of murine mAb HuLym3 shows that mAbs can be purified from hybridoma cell culture supernatants to high purity, and is thus an alternative to other purification methods based on conventional and affinity chromatography for the production of mAbs for analytical and diagnostic applications. A mouse/human IgG1 chimeric mAb produced by Chinese hamster ovary cells was also purified from cell culture supernatant, and the purity achieved suggests that Gradiflow (TM) electrophoresis could replace affinity chromatography in the downstream processing of mAbs for therapeutic use. Gradiflow (TM) electrophoresis technology is scaleable and thus is applicable to industrial-scale purification of mAbs. (c) 2005 Society of Chemical Industry.