Enhanced CE-MS analysis of bispecific and LALA antibodies using SmartEnzymes

September 3, 2020 | Applications, References |


Gstöttner et al. used SmartEnzymes for their CE-MS analysis of highly homologous bispecifics, one of them containing LALA mutations. 


Bispecific antibodies (BsAb) offer an attractive alternative to traditional mAbs since they can bind to two different epitopes simultaneously. Because they are composed of four different peptide chains, rather than two, they are subject to additional macro- and microheterogeneity which requires proper analysis and monitoring. Bottom-up approaches together with mass spectrometry (MS) have been employed to address such heterogeneity but such methods are unable to handle minor mass shifts and does not provide domain localization information.


In a new article by Gstöttner and colleagues at Leiden University Medical Center together with Roche Penzberg, sheathless capillary electrophoresis (CE) coupled to MS was used to probe two highly homologous BsAb. They looked at both intact BsAbs and their subunits generated using SmartEnzymes. FabRICATOR was used for below hinge digestion in one of the BsAb while the second BsAb proved to be more challenging due to a LALA mutations and required above hinge digestion using FabULOUS, highlighting the versatility of the SmartEnzymes family.


Gstöttner et al., CE-MS analysis of highly homologous bispecifics.


The nature of the sheathless CE approach meant that the authors could collect and compare both the intact and subunit data using the same instrument setup. From that, marco- and microheterogeneity could be assessed. Differences between the two BsAb could be detected such as incomplete assemblies and free chains, along with PTMs such as glycation. Combining the observations, the authors conclude that the different engineering processes for each BsAbs results in varying heterogeneity. They also underscore the wide applicability of sheathless CE. Finally, they reveal the importance of SmartEnzymes and subunit analysis.


Link to Paper: Gstöttner et al. Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS. Analytica Chimica Acta 1134 (2020) p. 18-27