Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry

Abstract

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6–9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…They checked the results with a MS detection and exposed a difference of 27.01 Da (6-9 %). The analysis of MS/MS spectra confirmed serine to asparagine substitution on the light chains [33]. The same year, disulfide isomers were separated with a high resolution.…”

“…Inter and intra-vial homogeneity was evaluated, and excellent standard deviation were calculated (0.053 for intra-vial and 0.0137 for inter-vial). Griaud et al and Miao et al reported two comprehensive studies using an array of orthogonal techniques and strategies including CE-SDS for biosimilarity assessment [33,34]. They compared the electropherograms of intact and reduced mAbs and their biosimilar candidate.…”

Unraveling the mysteries of modern size exclusion chromatography - the way to achieve confident characterization of therapeutic proteins

“…As discussed in Section 5, the significant difference of hydrophobicity between the two parent mAbs (HIC retention times of around 15 min vs. 45 min observed by running a generic linear inverse salt gradient) was likely to explain the unexpected SEC behavior [49]. Last but not least, biosimilarity evaluation also often includes SEC comparability studies for mAb [50,51], ADC [52], granulocyte colony stimulating factor (G-CSF) [53] and erythropoietin (EPO) [54] products.…”

Displacement to separate host‐cell proteins and aggregates in cation‐exchange chromatography of monoclonal antibodies

“…The heterogeneities in the mAb product further complicate its purification. Although a therapeutic protein is often considered a single entity for simplicity, it is a distribution with variations in amino acid sequence (Li et al, 2016; Parker, Pollard, Friesen, & Stanners, 1978; Santos & Tuite, 1993; Tan et al, 2012), higher‐order structure and posttranslational modifications (PTMs; Griaud et al, 2017). PTMs include glycosylation, phosphorylation, C‐terminal lysine truncation, and chemical modifications such as oxidation, deamidation, and isomerization.…”