Chromatofocusing studies involving a monoclonal Fab'.

Abstract

Isoelectric focusing (IEF) of the Fab' derivative of murine monoclonal antibody ZCE-025 is known to detect at least six bands having isoelectric points (pI) ranging from 5.4 to 7.8. Chromatofocusing was employed to separate these bands. Electrophoresis of the starting materials under nonreducing conditions indicated all of the materials to migrate as Fab'. The electrophoresis of urine samples obtained from Balb/c and nude mice 8 hr after the i.v. injection of the various 125I bands revealed the low pI bands to migrate approximately as a 125I-Fab'. The higher pI band activity was located in lower molecular weight regions. Serum samples taken at 8 hr postinjection from the above mice revealed a series of what appeared to be high molecular weight complexes and some low molecular weight species. Biodistribution studies in comparison Balb/c mice and nude mice revealed that the low pI 125I-Fab' bands gave an organ and tumor uptake at 8 hr very similar to Fab', while the high pI 125I-Fab' bands were rapidly excreted into the urine and feces and did not concentrate in the tumor. The data suggest that the population of molecules making up the Fab' of this antibody is heterogeneous and variably stable. Theoretically, some of the entities observed could be counter productive to successful radioimmunoimaging. It is also possible that some of the labeled molecules are associating in vivo with endogenous proteins that might, in some Mabs, affect the biodistribution of the radiopharmaceutical.