ELISA Measurement of Interferons

BioTechniques. Oct, 2002  |  Pubmed ID: 12395933

Purification of Antibody Heteropolymers Using Hydrophobic Interaction Chromatography

Journal of Chromatography. A. Jan, 2008  |  Pubmed ID: 17981289

A heteropolymer (HP) is a unique dual antibody conjugate composed of specific, chemically cross-linked monoclonal antibodies (mAbs). In this study we have demonstrated that HPs can be purified using hydrophobic interaction chromatography (HIC). Two propyl HIC resins; [PolyPropyl A and EMD Fractogel Propyl (S)] were evaluated in this study. Phosphate buffers, pH 6.5 containing ammonium sulfate or sodium sulfate were used to bind the HP to the column. A descending sulfate gradient or step gradient was used to elute the bound HP species from the column. The HP reaction mixture typically contains multiple conjugated HP species, as well as unreacted monomer mAbs. Conjugated HP product was successfully separated from unreacted antibody monomers with both propyl resins using buffers with ammonium sulfate. There was no monomer separation from HP using buffers with sodium sulfate. The purification processes, presented in this study allows the non-cross-linked antibodies to pass through the column without being bound to the resin, while the cross-linked antibodies (the HP product) bound to the column were subsequently eluted by decreasing the ammonium sulfate concentration in the running buffer. HP product was efficiently separated from free mAbs using Propyl HIC resins at both analytical and preparative scales.

MRNA Stability and Antibody Production in CHO Cells: Improvement Through Gene Optimization

Biotechnology Journal. Apr, 2010  |  Pubmed ID: 20222103

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.