On-line purification of monoclonal antibodies using an integrated stirred-tank reactor/expanded-bed adsorption system.

While expanded-bed adsorption (EBA) units have been used to recover proteins from whole cell cultures, the development of a more efficient, on-line process could streamline the traditional multistep process. This study implements a bench-scale on-line purification system in which whole cell cultures are loaded directly into a chromatography column to capture a monoclonal antibody (mAb) in a single step. The on-line purification system used here integrates a stirred-tank reactor (STR) and an EBA unit into a new hybrid (STR-EBA) system. To conduct this work, first, column and buffer conditions were optimized to capture immunoglobulin G from a hybridoma cell culture. A high cell removal (>95%) was achieved in part by removing the top flow distributor and mesh. Then, the 95% extent of removal was sustained for four successive cycles, each using PBS. With 20 mM phosphate buffer, however, the removal decreased from 95% to 75% stepwise. Next, the operational constraints of the EBA system were determined for the hybridoma cell culture, focusing on the effects of cell viability and density on cell removal. This study shows that the cell removal was not significantly different in the range of 80% to 0% viability. Cell density was also varied between 1 x 10(6) and 1 x 10(8) cells/mL. From 0.1 to 6 x 10(7) cells/mL, cell retention in the column was less than 5% and product recovery remained high, approximately 95%. After characterizing the working conditions of the EBA system, on-line purification was performed. With 1.1 L of culture containing 3 x 10(6) cells/mL and 100 mg/L of IgG, repeated-batch cultures were implemented. Half of the culture volume (550 mL) was directly sent to the EBA system every day, for 11 days, and the same amount of fresh medium was fed into the STR. During on-line purification, productivity was 58 mg of IgG/cycle (day) and purity was greater than 95%. Simple batch culture alone produced 17 mg of IgG/day. This result suggests that the on-line STR-EBA system can achieve higher and faster production compared with STR batch and off-line EBA purification. Overall, the STR-EBA system with repeated-batch mode was an effective and flexible system for bench-scale mAb production.

Determination of monoclonal antibody production in cell culture using novel microfluidic and traditional assays.

This study compares microfluidic technology (Protein 200 LabChip Assay kit, Agilent 2100 Bioanalyzer, referred to here as Protein 200) to the traditional approach for protein analysis, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), for the sizing and quantification of immunoglobulin G (IgG) in hybridoma cell cultures. Internal references differ between each method: purified IgG was used alone in SDS-PAGE while myosin (the upper marker) was added to each sample in Protein 200. The IgG used here were produced in cultures propagated in either a serum-free or a serum-containing medium. With serum-containing samples, there was a significant difference in the IgG concentrations (p < 0.05) between SDS-PAGE and Protein 200. The concentration determined by SDS-PAGE was significantly higher (> 30%) than by Protein 200 or by high-pressure liquid chromatography (HPLC) because the large amounts of serum albumin in the samples affect the accuracy of SDS-PAGE. Protein 200 can determine size similarly to SDS-PAGE in serum-free samples (standard error of the mean, SEM, < 1%, 95% confidence < +/-1%), unlike in serum-containing samples. The Protein 200 assay was more effective than the traditional one-dimensional SDS-PAGE in determining concentration and size of IgG in cell culture samples and it provided a miniaturized and convenient platform for rapid analysis.