Despite advances in processes and formulations, monoclonal antibody (mAb) processing still must identify and quantify free thiols in final products to minimize the risks of antibody aggregation, improper binding, denaturation, and degradation in the final product. Existing analytical methods to detect free thiols are limited by such issues as applicability, low sensitivity, localization capability, and complicated sample preparation steps that can introduce artifacts.
The solution may be a label-free method that combines hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-MS). This lets free thiols in mAb processing be localized directly and quantified at domain-specific resolution.
“Subunit HILIC-MS method is an effective alternative for rapid free thiol monitoring in therapeutic mAbs,” Shunhai Wang, PhD, a director at Regeneron, tells GEN. “It is ideally suited in candidate selection and process development, where quick turn-around is desired.” Writing in the Journal of the American Society for Mass Spectrometry, Wang and his Regeneron colleagues report this approach also shows how point mutations in the Fc sequence affected the stability of disulfide bonds.
Speed vs. specificity
After digesting a recombinant IgG4 mAb and selectively reducing its interchain disulfide bonds, the scientists used HILIC-MS/MS to analyze the remaining disulfide bonds in the Fd, LC, and Fc/2 subunits.
Fc/2 subunits’ two domains, CH2 and CH3, each contain one intradomain disulfide bond, Wang and colleagues point out. HILIC provided domain-specific separation of free thiol species at the Fc/2 level. This was confirmed by performing targeted MS2 analysis of each Fc/2 species in its elution window, using higher energy collisional dissociation (HCD) fragmentation.
When tested against a wide selection of mAbs, quantification was comparable to the conventional bottom-up approach. Additionally, targeted second-stage MS analysis of the fragmentation patterns caused by higher-energy collision-induced dissociation enabled free thiols to be localized. Consequently, they report, “direct mapping of free thiols into individual domains through the use of HCD fragmentation…eliminated the need for offline free thiol localization.” Direct mapping saves time and reduces sample handling, which lowers the risk that artifacts will be introduced.
“The current method can consistently cover four of the six intradomain disulfide bonds in therapeutic mAbs, including the highly variable and crucial VH domain, so it should already offer great utility in routine applications,” Wang says. “Further method development, particularly regarding HILIC conditions, is likely required to cover all six intradomain disulfide bonds.”
“Additionally, it will be very interesting to apply this technique to multi-specific mAbs (e.g., alternative antibody formats with more than 2 Fab domains) because of its unique capability of resolving chain-specific free thiols.” That task, he says, is challenging for a bottom-up approach.”
