HIGH-PERFORMANCE GEL-PERMEATION CHROMATOGRAPHIC ANALYSIS OF IMMUNOGLOBULIN-M PRODUCED BY HYBRIDOMA CELL-CULTURE

A high-performance gel permeation chromatographic (HPGPC) method, using TSK-Gel SW(XL) 3000 and TSK-Gel SW(XL) 4000 columns installed in series, was developed for the analysis of immunoglobulin M (IgM) produced by hybridoma cell culture. The detection of this protein was achieved using ultraviolet absorption at 225 nm, yielding a detection limit of ca. 0.3-mu-g ml-1. IgM-containing samples obtained from different culture systems (T-flask, roller bottle, spinner flask, bioreactor operated in batch, fed-batch or perfusion modes) and media (Dulbecco's Modified Eagle Medium or Protein-Free Hybridoma Medium) were evaluated by this chromatographic technique and also by conventional enzyme-linked immunosorbent assay (ELISA). Concentrations of IgM in culture samples determined by both techniques were always in the same range (+/- 10%). The main advantages of HPGPC over ELISA included improved reproducibility (relative average deviation of 1-3% compared with 10-20% for ELISA), high linearity range between the signal and the concentration [at least three decades (0.3-500-mu-g ml-1) compared with one decade for ELISA (25-200 ng ml-1)] and ease of operation.