Characterization of outcomes of amino acid modifications using a combinatorial approach to reveal physical and structural perturbations: A case study using trastuzumab biosimilar

Kamble, Ritu; Puranik, Amita; Narvekar, Aditya; Dandekar, Prajakta; Jain, Ratnesh

doi:10.1016/j.jchromb.2022.123430

Cited by 2 publications

(1 citation statement)

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“…Aggregation results in enriched level of hydrophobic variants, which partly explains the shift in retention . DTL M 255 ISR and W 420 QQGNVFSCSV M 431 HEALHNHYTQK in the Fc region of trastuzumab is situated close to the CH2–CH3 interface, potentially resulting in changed effector functions or reduced binding to protein A. Oxidation of these residues has been reported to activate the unfolding of IgG, , hence increasing the accessible surface area. The CH2–CH3 domains are also less stable and known to trigger conformational change .…”

Section: Resultsmentioning

confidence: 99%

Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants

Ewonde Ewonde,

Böttinger,

De Vos

et al. 2024

Anal. Chem.

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This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography−mass spectrometry (LC−MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N 30 ) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M 255 , W 420 , and M 431 ) was observed for peptides situated at the Fc region close to the CH2−CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

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