Study of Antibody Quality Attributes on Cell Culture Supernatant

September 23, 2015 | Applications, References |

The production of antibody-based therapeutics requires careful monitoring of quality attributes. Researchers at Institute Pasteur in collaboration with LFB Biotechnologies have used FabRICATOR to monitor important quality attributes of a therapeutic antibody, directly on the cell culture supernatant (Henninot et al. 2015).

The French researchers set up a method to improve the clone selection of antibody producing CHO-cells by identification and quantification of antibody isoforms, amino acid sequence and post-translational modifications directly on the cell supernatant. Briefly, the cell supernatant where concentrated, digested with FabRICATOR, reduced using DTT, and subsequently, the Fc, Fd and LC fragments were analyzed using LC-MS.

Using this method, Henninot et al. identified amino acid variations such as deamidation, oxidation, and pyroglutamination. Also, loss of C-terminal lysines clearly appeared in the deconvulated MS spectra. Another key quality attribute is glycosylation and in their approach, Henninot et al. could detect the various glycoforms on the Fc/2 fragment. Today’s standard method for clone selection involves large volumes of supernatant and about a week of sample treatment, whereas the method presented by Henninot et al. require only 500 μL and about 3 h of sample preparation.

Fucosylation of the N-linked glycans of IgG have impact on the antibody effector functions. Henninot et al. added the IgGZERO enzyme to cleave the glycan after the first Glc-NAc, thus leaving a Glc-NAc with or without a fucose residue attached. With this approach the fucosylation levels on the antibody could be studied and led to a better selection of clone candidates.

Taken together, Henninot et al. presents a simple and robust method to identify and quantitate important quality attributes of an antibody using FabRICATOR digestion of cell culture supernatant.