FabRICATOR® in an Inline Electrochemical Reduction Workflow

An inline electrochemical reduction workflow for antibodies coupled with LC-MS analysis is described in this paper from the National Institute for Bioprocessing Research and Training in Dublin. The FabRICATOR enzyme (IdeS) is a key component in the subunit analysis by digesting antibodies at a single site below the hinge, generating a homogenous pool of F(ab’)2 and Fc subunits.
 

Monoclonal antibodies, mAbs, are an important class of biotherapeutics used in the diagnosis and treatment of several diseases. Due to their heterogeneity and large sizes, analytical methods to characterize them are continuously being developed and improved.
 
The analysis of mAbs is commonly divided into native, intact and subunit workflows. To evaluate mAbs at the subunit level, middle-down and middle-up techniques are commonly used. Due to the smaller size of the subunits, they are more easily separated by chromatography and high-resolution MS can efficiently be utilized. Subunits are often generated by non-biological chemical reactions or enzyme digestion, and it is important that the production of the subunits is consistent in middle-up and middle-down MS workflows. To achieve this, FabRICATOR is a perfect candidate by digesting antibodies at a single specific site below the hinge, generating a homogenous pool of F(ab’2) and Fc subunits.
 

 
Disulfide bonds are important features of mAbs, creating the 3D structure required for their bioactivity. Mispairing of disulfide bonds affects the stability and function of the mAb-based drugs, and a comprehensive characterization is crucial for mAbs carrying complex disulfide bond patterns. However, the presence of extensive disulfide bond patterns makes analysis challenging, and reduction is typically carried out using chemical reducing agents such as DTT or TCEP. To allow for an even more complete reduction, denaturing agents are often used.
 
Here, the scientists describe an inline electrochemical reduction workflow for the reduction of antibodies coupled with LC-MS analysis without the need for chemical reducing or denaturing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitates complete reduction of NISTmAb disulfide bonds. For the subunit analysis, NISTmAb was treated with FabRICATOR and completely reduced Fc/2, Fd and light chain subunits were presented after exposure to the same electrochemical conditions as for the intact NISTmAb.
 
The reduction and analysis were carried out under optimal solvent conditions, and a trapping column and switching valve were used to facilitate solvent exchange during the analysis. The level of reduction was affected by electrochemical potential, solvent organic content and temperature, and after optimization, a complete disulfide bond reduction was achieved. It is concluded that the use of an inline electrochemical cell offers a simple and fast workflow solution for LC-MS analysis of antibody subunits.

Reference

Morgan et al., 2021. Inline electrochemical reduction of NISTmAb for middle-up subunit liquid chromatography-mass spectrometry analysis. Analyst. DOI: 10.1039/d1an01184g.

FabRICATOR® – Below hinge digestion of IgG