Microfluidic capillary electrophoresis - mass spectrometry for rapid charge-variant and glycoform assessment of monoclonal antibody biosimilar candidates.

ABSTRACT

Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development.