Hyphenated Mass Spectrometry (X-MS) Techniques to Study Glycosylation Profiles

Different techniques such as hydrophobic interaction liquid chromatography (HILIC) [72], reversed-phase liquid chromatography (RPLC) [73, 74], size- exclusion chromatography (SEC) [75] and ion-exchange chromatography (IEC), as well as capillary electrophoresis (CE) have been applied to separate mAbs, their derivatives, and released glycans. The hydrophobic nature of antibodies is well suited to hydrophobic stationary phases used for RPLC, allowing selective desorption of individual subunits of peptides with the introduction of increased organic proportions within the mobile phase. Glycan moieties are hydrophilic, however, and therefore are not retained well using RPLC conditions [76]. Typical released glycan analyses utilize HILIC methods that offer better retention.

Diepold et al. [77] combined incubation at elevated temperatures and proteolytic peptide mapping, followed by quantitative LC-MS to simultaneously induce, identify, and quantify Asp isomerization and Asn deamidation. mAbs were first denatured with acidic conditions, reduced using DTT, and then transferred into a trypsin-based digestion buffer before being separated using RPLC with MS detection. Both isomerization and deamidation have been reported to impact the in vivo biological activity and the in vitro stability of any mAb product, and therefore characterization of these events is critical to the efficacy and safety of the therapeutic.

In 2008, Damen et al. executed the first LC-MS-based quantification of tras- tuzumab [78]. An advantage of using LC-MS over the typical enzyme-linked immunosorbent assay (ELISA) method is the detection of structural changes that do not just affect the binding properties. LC-MS also provides insight into the degradation patterns of a given mAb and helps identify the PTMs. The results were reported to be in good agreement with the results obtained via UV spectrophotometry and HPLC-UV analyses, and although they had lower sensitivity over ELISA methods, they gained specificity with LC-MS.

In 2011, Gilar et al. applied a HILIC-MS method to characterize the glycosylation sites of a mAb therapeutic [79]. Following tryptic digestion of trastuzumab, RPLC isolated the glycopeptides according to their hydrophobic- ity, and HILIC was then used to resolve glycoforms. In some HILIC-based analyses, quantification and identification can be performed without MS using UV detection; however, some minor glycoforms do not give sufficient UV responses. For therapeutic mAbs where glycosylation can be highly influential upon efficacy and safety, quantification and identification of these glycans are critical, which is where MS can play an important role.

For released glycan and glycopeptide analysis, HILIC is highly selective [80]. Retention within the silica-based HILIC column is dominated by hydrogen bonding; however, with ionic stationary phases, retention can also be governed by a combination of ionic and dipole-dipole interactions too. With the use of an HILIC column, polar analytes are retained and eluted with mobile phases of higher organic content [81], enabling good LC separation and improved ESI efficiency. Released M-glycan analysis typically involves the deglycosylation of the mAb with a peptide M-glycosidase, such as PNGase F [82]. This enzyme cleaves the glycan as a glycosylamine, converting asparagine to aspartic acid in the process [83]. MS and in particular LC-MS can be employed for the determination of the mass of antibody postdeglycosylation, which will benefit from increased signal intensity for each charge state as the ion current is no longer shared among the different glycoforms [84]. Analyzing the released glycans can be performed by MS with or without derivatization. 2-Aminobenzamide (2-AB) labeling is commonly used to enable fluorescence detection, but sample preparation and purification can be laborious. There are alternative options, with recent variations including a basic functional group for improved ESI in an HILIC-FLR-MS experiment [85]. Figure 10.4 describes a series of possible approaches for analyzing mAbs that ultimately enable the characterization of the oligosaccharides present.

The microheterogeneity of glycans and the presence of multiple isomers have been investigated with CE coupled to MS (CE-MS), as together this technique can offer high separation efficiencies alongside high mass resolution and mass accuracy [76]. Ma and Nashabeh were the first to demonstrate the use of CE for the analysis and monitoring of mAb N-linked glycans throughout a manufacturing process [87]. Gennaro et al. coupled this with MS to offer an online CE-LIF-MS method capable of identifying minor peaks, unidentified with previous methods [88]. The accurate mass measurements were capable of identifying CE peaks corresponding to important ADCC, regulating afuco- sylated glycan moieties along with other typical glycans observed for mAb therapeutics.

IEC and SEC methods are often used to monitor the quality and stability of mAb products during all stages of manufacture and handling. It is often the minor peaks present in IEC and SEC separations however that are critical to understanding any changes or degradations. Alvarez et al. therefore developed a two-dimensional LC strategy combining SEC with RP trap cartridges and an MS system [89]. The traps served to collect, concentrate, and desalt IEC or SEC fractions of interest to enable quantitative analysis and separation of poorly resolved and low-level peaks. The incorporation of a disulfide bond DTT reduction step also enabled chain-specific information to be obtained. This 2D LC-MS format made it possible to resolve and identify coeluting fractions while saving time and conserving sample.

A workflow demonstrating the typical strategies for mAb and glycan characterization

Figure 10.4 A workflow demonstrating the typical strategies for mAb and glycan characterization. Starting with an intact glycosylated mAb (a), reduction of the disulfide bonds separates the two heavy chains and releases light chain (b). Addition of PNGase F cleaves the glycans completely (c), which can be analyzed directly using MS [72, 74], or the free glycan can be fluorescently labeled (d) to enable fluorescence [72]. Alternatively (a) can be digested using IdeS, which cleaves specifically below the hinge to yield Fab and glycosylated Fc subunits (e). Endoglycosidase digestions alongside IdeS can be used to simplify Fc [86] (f) or HC [53] (f) glycan variants into groups with and without core fucose.

< Prev   CONTENTS   Source   Next >