SpeB proteolysis with imaged capillary isoelectric focusing for the characterization of domain-specific charge heterogeneities of reference and biosimilar Rituximab

Abstract

The charge variations of therapeutic monoclonal antibody reveal important information of the post-translational modifications that may potentially impact the potency and safety of pharmaceutical products, especially during the evaluation of biosimilarity of therapeutic proteins. In this work, a novel SpeB-based proteolysis strategy coupling with imaged capillary isoelectric focusing was developed for the determination of domain-specific charge heterogeneities of innovator and generic Rituximab drug products from United States, European and Indian markets. It was observed that innovator Rituximab from the United States and Europe share highly similar peak distributions and charge heterogeneities with 26.2-26.6% Fc/2, 28.9-29.3% LC and 44.4-44.5% Fd peak areas detected, respectively, while multiple basic variations of Fc/2 and less acidic LC and Fd species were found from generic Rituximab from India with 20.9% Fc/2, 32.3% LC and 46.9% Fd peak areas detected. It was also demonstrated that structural changes caused by Carboxypeptidase B treatment and deamidation study at pH extremes could be sensitively captured with the established method, with the results further indicating that the generic product's basic variations of Fc/2 were un-cleaved Lysine residues, while the lack of certain acidic peaks on LC and Fd probably was due to the lower level of deamidation. This new strategy could become a useful tool to reveal domain-specific charge heterogeneities profiles of a variety of therapeutic monoclonal antibodies in regulated environments.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

“…Two studies demonstrated that the two drugs showed no clinical differences in terms of toxicity, tumor response rates, prolonged progression-free survival, and overall survival when prescribed for diffuse large B-cell lymphoma [28,29]. However, from a molecular perspective, MabThera ® and Reditux™ exhibited a significant variability [14,[30][31][32][33]. Nupur et al [33] used a multi-analytical approach to assess the biosimilarity of the two biotherapeutics and reported an increased level of core-afucosylated and oligomannose glycans in Reditux™ compared to MabThera ® , as well as differences in charge variant patterns.…”

Proteoform-Resolved FcɤRIIIa Binding Assay for Fab Glycosylated Monoclonal Antibodies Achieved by Affinity Chromatography Mass Spectrometry of Fc Moieties

“…Bacterial enzymes are important tools for middle-up approaches, since they cleave IgG specifically within the hinge region. Robust and simple workflows for the middle-up analysis of (therapeutic) mAbs are established (Zhang et al, 2016; Moelleken et al, 2017; Sjögren et al, 2017; Bern et al, 2018; van der Burgt et al, 2019). IdeS, SpeB, and Kgp are frequently used commercial IgG hinge-specific proteases (cleavage sites and products are indicated in Figure 1 and Supplementary Figure 1, respectively).…”

Rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in diffuse large B-cell lymphoma

“…Eight biosimilars of rituximab have been approved by a regulatory health authority with preclinical and clinical information. 69 – 82 From all these biosimilars, only four were approved after conducting a comparative trial in DLBCL patients.…”