High-performance gel permeation chromatographic analysis of immunoglobulin M produced by hybridoma cell culture

Abstract

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 μg 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 μg ml −1) compared with one decade for ELISA (25−200 ng ml −1)] and ease of operation.