Rapid multi‐attribute characterization of intact bispecific antibodies by a microfluidic chip‐based integrated icIEF‐MS technology

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip‐based integrated imaged capillary isoelectric focusing‐mass spectrometry (icIEF‐MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob‐into‐hole bispecific antibody. Upfront, on‐chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult‐to‐detect deamidation and glycation events at the intact protein level. In addition, on‐chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on‐chip electrospray tip and directly identified by in‐line icIEF‐MS using a connected quadrupole time‐of‐flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C‐terminal lysine, N‐terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi‐attribute characterization within a 15‐min run time.