LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

Abstract

The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

Rapid Analysis of Biotherapeutics Using Protein A Chromatography Coupled to Orbitrap Mass Spectrometry

“…The average mass accuracy across three runs for the annotated glycoforms was within 30 ppm. The charge envelope and deconvoluted spectra obtained using the ProA-MS method correspond well to those previously reported for bevacizumab. , …”

“…The charge envelope and deconvoluted spectra obtained using the ProA-MS method correspond well to those previously reported for bevacizumab. 14 , 25 …”

“…The charge envelope and deconvoluted spectra obtained using the ProA-MS method correspond well to those previously reported for bevacizumab. 14,25 Method precision was tested by investigating the relative standard deviation of the retention time and relative peak area acquired using six replicate injections of bevacizumab based on UV detection. The relative standard deviation for the retention time was <0.001%, indicating extremely high retention time precision across all runs, while the relative standard deviation for the peak area was 0.321%, also indicating high levels of precision.…”

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

“…Deamidation has been localized mainly to CDRs [ 15 , 16 , 17 , 18 , 26 , 28 , 37 , 44 , 73 , 74 ] and the “PENNY” peptide in the Fc region [ 9 , 16 , 27 , 31 , 32 , 33 , 36 , 37 , 39 , 75 , 76 , 77 ]. To a much lesser degree, deamidation has been detected in other sites [ 9 , 13 , 31 , 36 , 38 , 40 , 75 , 78 ]. Asn deamidation in CDRs has been demonstrated, with almost no exception, to cause decreased antigen binding [ 15 , 16 , 17 , 18 , 26 , 28 , 37 , 44 , 74 , 79 ] with no [ 16 ] or minimal [ 28 ] effect on conformation.…”

Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants

“…Typical acidic variants are species with deamidation, glycation, the sialylated glycan, trisulfide, etc. Basic variants include C-terminal unprocessed lysine, C-terminal amidation, isomerization of aspartate residues, and so on [ 8 , 9 , 10 ]. Those protein charge variants can be measured and characterized by charge indicating analytical assays, imaged capillary isoelectric focusing (icIEF), ion-exchange chromatography (IEC), or liquid chromatography/mass spectrometry (LC/MS).…”