Charge Variants Analysis of a Bispecific Antibody Using a Fully Automated One-step Capillary Isoelectric Focusing - Mass Spectrometry Method

Abstract

Background: Bispecific antibody (BsAb) therapeutics have emerged as the next-generation immuno oncology therapy. The architecture of bsabs is inherently more complex than that of mAb therapeutics. As a result, prior knowledge of critical quality attributes (CQAs) assessment of mAbs is no longer inclusive for bsabs. Objective: The main objective of this work is to develop a fully automated one-step capillary isoelectric focusing – mass spectrometry (cIEF-MS) workflow for the charge variant analysis of a bispecific antibody molecule. Method: A number of critical factors for the method development are investigated: the performance of two commonly used ampholytes are compared; the impact of protein concentration for the cIEF-MS assay is examined; as for sample preparation, off-line and on-line desalting are compared; various combinations of Pharmalyte® 3-10 and 8-10.5 are considered. Results: In this fully automated workflow, the charge variants of this BsAb molecule are clearly separated and accurately identified. Based on six repeat injections, RSDs of the migration time of the identified charge variants are between 3 and 6%. The identified masses of each charge variant show a variation between 0.48 and 1.40 Da. The delta masses of the basic and acidic variants are from the most basic to the most acidic, -58.59, 162.26, 453.44, -907.47, 1,563.60, and 1,566.98 Da, respectively. Conclusion: Overall, the separation resolution, system sensitivity, robustness, and reproducibility of this fully automated cIEF-MS workflow, as demonstrated using this BsAb example, proves it a powerful assay for the quality assessment of recombinant protein therapeutics.

Prokaryotic cell membrane‑based protein technologies (Review)

“…Among these, SDS-PAGE is generally performed in all cases of separation of biomolecules, whereas in multilevel proteomics, the determination of the peptide, proteoform and protein complex is achieved by capillary electrophoresis utilized with MS (59). In recombinant protein therapeutics to perform quality assessment and unravel protein molecules, capillary isoelectric focusing-MS can be utilized and charge variant analysis can also be performed, demonstrated with bispecific antibody (60). NMR and MS are two techniques widely used to study and determine biological molecules.…”

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].…”

“…In an application note, the background noise of different ampholytes was compared, indicating differences between brands [253]. During CIEF-MS, Wu et al described a significantly higher background noise in the high mass range for Servalytes compared to Pharmalytes [218]. Lot-to-lot (in)consistency has been an issue and a recent patent suggest an LC-MS approach to ampholyte analysis before use in (i)CIEF [254].…”

“…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.…”

“…During CIEF–MS, Wu et al. described a significantly higher background noise in the high mass range for Servalytes compared to Pharmalytes [218]. Lot‐to‐lot (in)consistency has been an issue and a recent patent suggest an LC–MS approach to ampholyte analysis before use in (i)CIEF [254].…”