Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density

Abstract

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.

Monoclonal antibody capture and viral clearance by cation exchange chromatography

“…Ion exchange chromatography is a frequently discussed alternative mAb capture strategy (Arunakumari et al, 2007; Follman and Fahrner, 2004; Lain et al, 2009; Necina et al, 1998) that also can demonstrate viral clearance (Miesegaes et al, 2010a). Attractive aspects include lower resin costs relative to Protein A and resistance to highly concentrated alkaline cleaning buffers such as NaOH.…”

Hexamer peptide affinity resins that bind the Fc region of human immunoglobulin G

“…Human immunoglobulin G is usually isolated from pooled human plasma, produced in hybridoma cell culture as antigen‐specific monoclonal antibodies (MAbs), or produced as polyclonal antisera. The crude source of HIgG can be subjected to a combination of different processes, such as precipitation (4,5), ion‐exchange chromatography (IEC) (6), hydrophobic interaction chromatography (7) or affinity chromatography (AC) (8–10) for purification of HIgG. ImClone Systems, for instance, integrates IEC and Protein A affinity chromatography in the manufacture of Erbitux®, a monoclonal antibody (MAb) that targets the epidermal growth factor receptor on the surface of normal and tumor cells (11).…”

Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules

“…Preliminary experiments were performed using affinity and single‐mode chromatography ligands, namely protein A (affinity), carboxymethyl Sepharose (cation exchanger), and Q Sepharose (anion exchanger), whose interactions with IgG and BSA are well described in the literature, in order to demonstrate the potential and feasibility of the multiplex structure to screen different chromatographic conditions and to find optimal working windows.…”