Articles tagged ”LC/MS”

Biosimilar Comparability Assesment using FabRICATOR

March 31, 2017 | References |

Biosimilars are gaining in popularity as patents of innovator drugs are expiring. The analytical strategies to characterize and assess the similarity between the innovator product and the biosimilar often involve liquid chromatography and mass spectrometry (LC/MS). In a recent paper from the Freie Universität Berlin, Montacir et al. studied Rituximab and follow-on molecules using FabRICATOR digestion, reduction, and middle-down LC/MS. Using this approach the researchers found differences in the level of c-terminal lysine clipping of the Fc/2 fragment, a sequence error in the Fd fragment (as previously reported by Beck et al 2014), and a pyro-glutamic acid formation in the light chain.

The comparability assessment of biosimilars and innovator drugs using middle-down LC/MS with FabRICATOR digestion have also been published by the FDA a couple of years ago (Wang et al. 2013). Wang et al. argues that the FabRICATOR middle-down approach is very suitable for rapid fingerprinting of complex molecules, due to the high robustness and specificity of the enzyme.



Montacir, O. et al., 2017. Comparability study of Rituximab originator and follow-on biopharmaceutical. Journal of Pharmaceutical and Biomedical Analysis. E-published ahead of print.

Wang, B. et al., 2013. Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry. The Analyst, 138(10), pp.3058–3065.


Assessment of Oxidation using FabRICATOR and LC/MS

Oxidation of methionine residues in the Fc region of a therapeutic antibody may affect the binding of the antibody to Protein A and FcRn leading to difficulties in purification or increased clearence in vivo. For the variable regions of the antibody, oxidation may affect antigen binding or lead to increased immunogenicity. For these reasons, the propensity of an IgG molecule to become oxidized is a critical quality attribute to consider early in the selection of therapeutic antibody candidates. The team at Adimab have developed an high-throughput assay based on FabRICATOR digestion and LC/MS analysis to evaluate the oxidation levels of 121 clinical stage antibodies. The antibodies were digested with FabRICATOR for 30 min at 37°C and reduced with DTT to obtain Fc, Fd and LC, prior to analysis on LC/MS, an approach called middle-down. The scientists correlated the observed oxidations with a model of predicted solvent-exposed methionine residues. They authors discovered oxidation at antibodies experimentally that were not predicted in the model,  probably due to inaccurate crystal structures or differences in expression host.


Taken together, the paper by Yang et al demonstrates the robustness of the oxidation assay based on FabRICATOR digestion and subunit analysis. The 121 antibodies analyzed in the paper indicated this method applicable to early clone selection for evaluation of antibody oxidation at the subunit level.