mAb Quality in Cell Culture

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). In another publication, researchers at Amgen developed a technology to model and monitor the high-mannose content of the antibody in real-time using FabRICATOR digestion (Zupke et al. 2015).

Study of antibody quality attributes on cell culture supernatant

The French researchers Henninot et al. 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 GlcNAc, thus leaving a GlcNAc 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.

Henninot, A. et al., 2015. Characterization of monoclonal antibodies by a fast and easy LC- MS ToF analysis on culture supernatant. Analytical Biochemistry, pp.1–8.

More about FabRICATOR

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Real-time monitoring of antibody quality control using FabRICATOR®

The amount of high-mannose glycans at the N-linked glycan site on therapeutic antibodies have been shown to impact antibody clearance and thus the pharmacokinetics of the antibody. To follow and control the amount of high-mannose glycan in the production of an IgG1 antibody, researchers at Amgen developed a technology based on FabRICATOR for near real-time LC-MS analysis.

In the paper, Zupke et al. developed mathematical models to predict the amount of high-mannose residues on the antibody as a function of added mannose sugar. To control the high-mannose glycan in real-time, FabRICATOR was used directly on filtered cell-culture media. The antibody fragments were reduced and the 25 kDa fragments analyzed using LC-MS.

The traditional approach to study IgG glycosylation involves release of glycans using PNGaseF, labeling with a fluorescent dye and separation of the glycan structures using HILIC chromatography. Due to the time for sample preparation in this approach, it is not well suited for on-line monitoring. Instead, FabRICATOR was incubated with cell culture medium for 30 min, followed by reduction for 10 min, and then separation and analysis using LC-MS. This allowed a close to real-time response of the quality of the batch and monitoring of the high-mannose content.

Zupke and colleagues at Amgen presents an impressive study that highlights the importance of controlling critical quality attributes during the process development, that could avoid extensive product characterization.

Zupke, C. et al., 2015. Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels. Biotechnology Progress, pp.n/a–n/a.