AFFINITY-CHROMATOGRAPHY WITH IMMUNOCHEMICAL DETECTION APPLIED TO THE ANALYSIS OF HUMAN METHIONYL GRANULOCYTE-COLONY-STIMULATING FACTOR IN SERUM

An on-line, automated, HPLC method was developed for the separation an d detection of recombinant human methionyl granulocyte colony stimulat ing factor (GCSF) and GCSF modified with poly(ethylene glycol) (PEG-GC SF) in rat serum. The automated method consists of an initial immunoaf finity chromatography step, a microbore reverse phase separation, and postcolumn immunochemical detection. Calibration curves were construct ed with a lower limit of 1.5 ng of GCSF (80 fmol) and 7.5 ng of PEG-GC SF in 120 mu L of rat serum, In vivo serum samples from rats dosed wit h PEG-GCSF were also analyzed. The development of the method is discus sed as well as its general application to the detection of metabolites of recombinant proteins in body fluids.