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

Abstract

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 knobinto-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.

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

“…Ostrowski et al. [173] very recently applied this technology as a powerful approach for assessing product quality and provided charge variant analysis with the direct identification of all charge isoforms of the BsAb. This system provided the orthogonal separation required to resolve and identify acidic PTMs including difficult‐to‐detect deamidation and glycation events at the intact protein level.…”

“…The conventional CIEF was successfully miniaturized into a microfluidic CIEF system, which provides comparable resolution and precision and ultrafast charge heterogeneity assessment [171]. Commercially available microchip iCIEF-MS is a promising new combination and show great potential for applications such as early developmental screening and monitoring of specific protein quality attributes [172][173][174].…”

“…In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Ostrowski et al [173] very recently applied this technology as a powerful approach for assessing product quality and provided charge variant analysis with the direct identification of all charge isoforms of the BsAb. This system provided the orthogonal separation required to resolve and identify acidic PTMs including difficult-to-detect deamidation and glycation events at the intact protein level.…”

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…CIEF and iCIEF have matured into techniques that are routinely applied in analytical laboratories, especially in the biopharmaceutical industry. Exemplary applications include, but are not limited to, monoclonal antibodies [192,[209][210][211][212][213][214][215], bispecific antibodies [216][217][218], ADCs [219,220], PEGylated proteins [221,222], glycoproteins, in particular erythropoietin [213,223,224], and fusion proteins [213,225]. Because this subsection is part of the former "proteins" section, which was extended to all biopharmaceuticals, it should not go unmentioned that the range of applications has been expanded beyond proteins and includes oligosaccharides [226], LNPs and related mRNA vaccines [227][228][229][230], virus-like particles [231,232], poliovirus [230,233], adeno-associated virus [230,[234][235][236][237], papillomavirus [238], and bacteria and yeast cells [230].…”

“…It allows unam-biguous peak identification and provides additional structural information about the analyzed protein species. Different approaches have been described, for example, online CIEF-MS [218,275,282,285], microchip CIEF-MS [207,217,286], CIEF-CZE-MS [287,288], and online iCIEF-MS [289][290][291][292]. In the case of direct online CIEF-MS, the catholyte and anolyte are additionally replaced with volatile compounds, for example, ammonia (ammonium hydroxide) and formic acid, and glycerol is used as dynamic coating and viscosity enhancing agent.…”