Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Capillary isoelectric focusing (cIEF) was online coupled to a Q‐TOF MS by a flow‐through microvial interface for the analysis of therapeutic mAb. Intact molecular weights obtained from the mass spectrum deconvolution of separated charge variants provided information on the structural heterogeneity of therapeutic mAbs. A sandwich cIEF–MS configuration composed of anolyte, sample, and catholyte segments sequentially injected into a neutrally coated capillary was used for the charge heterogeneity separation of four mAbs. Acetic acid and ammonium hydroxide were used in places of the non‐volatile acids and bases commonly used for IEF but are incompatible with online MS detection. Glycerol was added as the anti‐convective reagent. A chemical modifier was mixed with the cIEF effluent in the flow‐throw microvial to maintain the ESI stability and to mitigate ion suppression from the co‐eluted carrier ampholytes and glycerol. Analysis of mAb samples have shown relative populations of two basic variants originating from C‐terminal lysine process and acidic variant of deamidation. The lysine clippings, deamidation, and sialic acid modification in oligosaccharide chains were revealed in infliximab. Two lysine clipping variants and a deamidated variant were observed in adalimumab. The duplicate analyses of a reference mAb demonstrated five charge variants separated by cIEF due to some unidentified modifications, as their mass spectra shared close similarities. The mAb analyses demonstrated the feasibility of the cIEF–MS method, and they demonstrated how charge and structural variants and minor differences in therapeutic mAbs are observed with this technology. Online cIEF–MS is an information rich technology with high throughput, demonstrated by the initial data presented here.