Application of a Quantitative LC–MS Multiattribute Method for Monitoring Site-Specific Glycan Heterogeneity on a Monoclonal Antibody Containing Two N-Linked Glycosylation Sites

Abstract

A significant challenge of traditional glycan mapping techniques is that they do not provide site-specific glycosylation information. Therefore, for proteins containing multiple glycosylation sites, the individual glycan species present at a particular site cannot be differentiated from those species present at the other glycosylation sites on the molecule. Quantification of glycoform has previously been demonstrated using a multiattribute method (MAM), which can quantify multiple post-translational modifications including deamidation, oxidation, glycosylation variants, and fragmentation ( Rogers, R. S.; Nightlinger, N. S.; Livingston, B.; Campbell, P.; Bailey, R.; Balland, A. MAbs 2015 , 7 , 881 - 890 ; ref 1). In this paper we describe the application of an MAM based method for site specific quantification of N-linked glycan heterogeneity present on an IgG1 mAb molecule containing two distinct N-linked glycosylation sites: one present on the heavy chain (HC) variable region (Fab) and the other present on the conserved HC constant region (Fc). MAM is a peptide mapping method utilizing mass spectrometry to detect and quantify specific peptides of interest. The ionization properties of the glycopeptides with different classes of glycan structural variants, including high mannose, sialylated, and terminal galactosylated species were studied in detail. Our results demonstrate that MAM quantification of individual glycan species from both the Fab and Fc N-Linked glycosylation sites is consistent with quantification using the traditional hydrophilic interaction liquid chromatography (HILIC) analysis of enzymatically released and fluorescently labeled glycans. Furthermore, no significant impact from the glycoform on the ionization properties of the glycopeptide is observed. Our work demonstrates that the MAM method is a suitable approach for providing quantitative, site-specific glycan information for profiling of N-linked glycans on immunoglobulins.

A View on the Importance of “Multi-Attribute Method” for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy

“…Ion exchange chromatography is used to show the charge distribution on a molecule based on surface charge at a specific pH with readout of main peak, acidic peaks, and basic peaks, but does not give any site specificity for where a modification is located in the amino acid sequence. The released N-glycan HILIC assay gives the overall distribution of the combined N-linked glycans on a molecule, but if a molecule has more than one asparagine site that is subject to glycosylation, the glycan distribution shown by the HILIC glycan assay does not represent the glycan profile at either specific asparagine, neither does the HILIC glycan map provide details around the percent occupancy at the potential glycosylation sites (12). Historically, in the absence of MAM, these advanced methods, prior to the characterization with orthogonal assays, have served as surrogate measures, often capable of only assessing global rather than site-specific chemical modification at the amino acid level.…”

“…Similarly, MAM can facilitate the monitoring of host cell proteins (HCPs) with immunogenicity or safety risks, and for any HCPs at unacceptable levels, MAM can aid the optimization of purification processes for greater clearance (32). In addition to being an excellent formulation screening tool, MAM is now widely used to monitor specific oxidation, deamidation, isomerization, and/or succinimide formation events that can impact product potency and establish appropriate product expiries to mitigate their impact (8,10,(12)(13)(14)32,33).…”

Development of an LC-MS/MS peptide mapping protocol for the NISTmAb

“…Thus, peptide mapping is also a valuable tool for evaluating the stability of reference standards. When coupled with mass spectrometry, changes in the landscape of the map can be pinpointed to a particular attribute, such as increased oxidation of a certain methionine residue [ 2 – 5 ] appearance of a new sequence variant [ 6 – 9 ] or changes in glycan composition [ 10 – 12 ]. These principles also apply to the use of peptide mapping techniques for the purposes of assessing biosimilarity to an originator drug product [ 13 – 19 ].…”

Characterization of Biopharmaceuticals Focusing on Antibody Therapeutics

“…Peptide mapping approach is one of those method capable for the site-specific identification and quantitation of various PTMs. Recently multi-attribute method (MAM) has been developed as MS-based method that is able to identify and quantify several attribute at once [48,87,88]. The conventional methods such as hydrophilic interaction chromatography (HILIC) for oligosaccharide analysis, cation-exchange (CEX) chromatography, and capillary electrophoresis sodium dodecyl sulfate (CE-SDS) can be replaced by MAM approach ( Table 1).…”