On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

Abstract

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product analysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was successfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.

Specific Effects of Synthetic Oligopeptides on Cultured Animal Cells

“…The experimental error involved in the estimation of cell density, and viability was ±10%. The monoclonal antibody (MAb) concentration was determined by immunoturbidimetry (9). The experimental error associated with the estimation of MAb concentrations was ±5%.…”

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G

“…In parallel, several biosensors have been developed for measurement of hIgG, including piezoelectric (Chen et al, 2007;Li et al, 2003;Liu et al, 2003;Lu et al, 2000) and piezomagnetic biosensors (Ogi et al, 2006), surface plasmon resonance biosensors (Dong et al, 2008;Suzuki et al, 2002), and electrochemical biosensors such as amperometric biosensors (Bian et al, 2005;Dutra et al, 2000;Messina et al, 2005;Wilson and Nie, 2006), potentiometric biosensors (Li and Gao, 2008), and field-effect transistors (Cid et al, 2008;Kim et al, 2008). Furthermore, several online immuno-monitoring systems were successfully developed for the measurement of hIgG based on turbidimetric assays (Fenge et al, 1991;Middendorf et al, 1993), and fluorescence measurements (Reif et al, 1994). In addition, a rapid capillary electrophoresis technique was employed for the determination of the molecular mass of heavy and light chains of hIgG (Lausch et al, 1993).…”

Survival Factor-Like Activity of Small Peptides in Hybridoma and CHO Cells Cultures

“…The hybridoma cultures were inoculated at a density of (250 ± 50) × 10 3 cells mL −1 and incubated until the decline phase, i.e., for 7 days. The monoclonal antibody (MAb) concentration in hybridoma culture supernatants was determined by immunoturbidimetry (12). Briefly, aliquots of media were diluted by 5% poly(ethylene glycol) and incubated for 1 h with porcine anti‐mouse immunoglobulin affinity purified antibody.…”