Mass spectrometry for glycosylation analysis of biopharmaceuticals

Accepted Manuscript Title: Mass spectrometry for glycosylation analysis of biopharmaceuticals Author: Viktoria Dotz, Rob Haselberg, Archana Shubhakar, Radoslaw P. Kozak, David Falck, Yoann Rombouts, Dietmar Reusch, Govert W. Somsen, Daryl L. Fernandes, Manfred Wuhrer PII: DOI: Reference:

S0165-9936(15)00199-5 http://dx.doi.org/doi: 10.1016/j.trac.2015.04.024 TRAC 14490

To appear in:

Trends in Analytical Chemistry

Please cite this article as: Viktoria Dotz, Rob Haselberg, Archana Shubhakar, Radoslaw P. Kozak, David Falck, Yoann Rombouts, Dietmar Reusch, Govert W. Somsen, Daryl L. Fernandes, Manfred Wuhrer, Mass spectrometry for glycosylation analysis of biopharmaceuticals, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi: 10.1016/j.trac.2015.04.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mass spectrometry for glycosylation analysis of biopharmaceuticals Viktoria Dotz a, Rob Haselberg a, Archana Shubhakar b, Radoslaw P. Kozak b, David Falck c, Yoann Rombouts c, d, e, Dietmar Reusch f, Govert W. Somsen a, Daryl L. Fernandes b, Manfred Wuhrer a, c, * a

Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands b Ludger Ltd., Culham Science Center, Oxfordshire, UK c Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands d Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands e CNRS, UMR 8576, Villeneuve d'Ascq, France f Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany

HIGHLIGHTS  Biopharmaceuticals are very promising for the treatment of multiple diseases  Therapeutic proteins often bear sugar chains that can influence their pharmacology  Glycosylation has to be characterized and controlled throughout drug development  Mass spectrometry is a key technology in analysis of drug glycosylation  Recent trends in mass spectrometry analysis of therapeutic glycoproteins ABSTRACT Biopharmaceuticals are drugs of biotechnological origin used as vaccines or for the treatment of non-communicable diseases, such as cancer or anemia. Due to their high efficacy and specificity, the market for novel and biosimilar biopharmaceuticals is growing immensely. This growth is accompanied by new challenges in quality control and analytical characterization during drug development and production. Glycosylation is one of the structural modifications that occur during production of many protein-based drugs and can have significant effects on their pharmacological properties. Mass spectrometry (MS) is a promising technique for high-quality analytical characterization of glycosylation, starting from early drug development through to final lot release. Here, we review the most recent trends in biopharmaceutical glycosylation analysis by MS with and without coupling to liquid chromatography or capillary electrophoresis, and draw comparisons with established, nonMS methods. We discuss future prospects for the emerging MS approaches for the biotech industry and biopharmaceutical research. Keywords: Biopharmaceutical Biosimilar Capillary electrophoresis coupled to mass spectrometry (CE-MS) Glycomics Glycoproteomics Glycosylation Critical Quality Attributes (GCQA) Liquid chromatography coupled to mass spectrometry (LC-MS) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) Mass spectrometry Post-translational modification Abbreviations: AA, Aminobenzoic acid; AB, Aminobenzamide; CE, Capillary electrophoresis; CGE, Capillary gel electrophoresis; CID, Collision-induced dissociation; CIEF, Capillary isoelectric focusing; CZE, Capillary zone electrophoresis; EPO, Erythropoietin; FTICR, Fourier transform-ion cyclotron resonance; HCD, Higherenergy C-trap dissociation; HILIC, Hydrophilic interaction liquid chromatography; HPAEC-PAD, High-

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performance anion-exchange chromatography with pulsed amperometric detection; HPLC, High-performance liquid chromatography; Ig, Immunoglobulin; IM-MS, Ion mobility-mass spectrometry; IT, Ion trap; mAbs, Monoclonal antibodies; MALDI-TOF, Matrix-assisted laser-desorption/ionization time-of-flight; MS, Mass spectrometry; PGC, Porous graphitized carbon; PNGase, Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase; PTM, Post-translational modification; QbD, Quality by Design; RP, Reversed phase * Corresponding author: Tel.: +31 20-5987527. E-mail address: [email protected] (M. Wuhrer)

1.

Relevance of glycosylation in biopharmaceuticals

Biopharmaceuticals or biologic drugs have been gaining increasing importance on the drug market, since these therapeutically active biomolecules often have greater efficacy and specificity than classic small-molecule drugs. They have been shown to be particularly promising in the treatment of non-communicable diseases, such as cancer or rheumatoid arthritis, but also as vaccines [1]. Since the prevalence of non-communicable diseases is expected to increase worldwide, the biopharmaceutical market – currently estimated at >US$199 billion – is also expected to grow at annual rate of ~13.5% [2]. Most biologic drugs tend to cause less adverse reactions than small molecules because they are very structurally similar to endogenous proteins, such as immunoglobulin G (IgG)based monoclonal antibodies (mAbs), cytokines or hormones. Nonetheless, clinical safety and efficacy testing are key pillars in biologic-drug development. Notably, due to the structural variability of biologic drugs as a consequence of the manufacturing process in living cells and their inherent structural complexity, quality assessment and control come at a cost. Post-translational modifications (PTMs) during biopharmaceutical production contribute greatly to the structural variety. Moreover, manipulation and characterization of PTMs is challenging, but very important, since PTMs can significantly affect the physicochemical and pharmacological properties of the therapeutic. The attachment of sugars (glycosylation) is a common PTM in biologic drugs and it can greatly affect clinical safety and efficacy profiles primarily via its influence on immunogenicity, solubility, protein folding and serum half-life [3,4]. Two main types of glycosylation occur in protein-based drugs: N- and O- linked, where glycan chains are attached to specific asparagine and serine/threonine residues, respectively (Fig. 1). The Nglycan composition of IgG (and mAbs) has been shown to regulate efficacy by affecting its receptor affinity [5]. The impact of O-glycosylation in biopharmaceuticals, such as erythropoietin (EPO) or blood-coagulation factors, is less well investigated due to many obstacles in O-glycan analytics, but O-glycosylation is also expected to play an important role in drug quality [6]. The main obstacles due to glycosylation, which impede biologic-drug manufacture, arise from the following aspects.  Complexity. Glycans are oligomers themselves, and proteins may carry many different glycans, adding to the structural complexity of the overall glycoprotein.  Microheterogeneity. Different glycosylation sites on a protein may carry different glycans. Site-specific glycosylation may contribute to functional variation and influence clinical safety and efficacy.  Variability. Cell-culture conditions partly define the glycosylation phenotype. Changes in glycosylation patterns due to inconsistency of manufacturing conditions are the major source of batch-to-batch variability. To address these challenges, the biopharmaceutical industry is employing new analytical technologies to improve the characterization of glycosylation during drug design and manufacture. Accordingly, guidelines from health agencies including US Food and Drug Administration, European Medicines Evaluation Agency, and the International Conference on Harmonization contain specifications for drug glycosylation [4]. 2 Page 2 of 14

The variability of drug glycosylation embodies potential risks to patients and exposes companies to litigation for producing unreliable therapeutics. In this context, the Quality by Design (QbD) concept offers solutions. In practice, this entails identifying, characterizing and optimizing Glycosylation Critical Quality Attributes (GCQAs) starting at the early stages of drug development through to post-approval batch release (Fig. 2) [7]. So far, the best GCQAs evaluated include:  the number of sialic acids attached to the glycan/glycoprotein, known to affect serum halflife of various biopharmaceuticals;  the distribution of the main mAb glycoforms, which indicates process consistency; and,  the presence of non-human, potentially immunogenic glycan structures, such as Nglycolylneuraminic acid and alpha1,3-galactosylation. QbD in glycoprotein-drug development is supported by a range of orthogonal methods for structural and quantitative glycosylation analysis. In practice, these are predominantly drawn from three complementary analytical platforms:  high-performance liquid chromatography (HPLC);  capillary electrophoresis (CE); and,  mass spectrometry (MS). This review points out the most recent trends and future directions in MS methods used in development and production of biologic drugs. We discuss the advantages of MS for rapid, high-throughput and high-automation glyco-analysis and the challenges of using MS for drug-glycosylation characterization.

2.

Current methods in glycosylation analysis of biopharmaceuticals

In principle, the whole range of methods available for glycan analysis, as extensively reviewed in [8], can also be applied to glycosylation analysis in biopharmaceuticals. However, glycosylation analysis in a biotech laboratory is limited to only a small choice of approaches with, preferably, high degrees of robustness, repeatability, accuracy, speed and automation providing qualitative and/or quantitative information sufficient to meet the requirements of regulatory agencies (Table 1). Previously, the main focus was on batch-to-batch consistency of glycosylation, which is implied in the quality control at a later stage of drug development, as depicted in Fig. 2. However, a more thorough evaluation of biopharmaceutical glycosylation at all stages of drug development and production becomes obligatory, since glyco-engineering is emerging. This is demonstrated by the targeted insertion of additional glycosylation sites in darbepoetin alpha or alpha 1-antitrypsin [9-11], concurrent with increasing availability of cost-effective glyco-analytical methods. Glycosylation in a protein-based biopharmaceutical can be analyzed on three different levels:  intact or reduced protein;  glycopeptides after enzymatic cleavage; and,  N-/O-glycans after enzymatic/chemical release and (optional) derivatization. Most traditional methods rely on chromatographic or electrophoretic separation combined with spectrophotometric detection. For example, in intact-protein glycoprofiling, electrophoretic methods without prior derivatization [e.g., capillary isoelectric focusing (CIEF), capillary-gel electrophoresis (CGE) and capillary-zone electrophoresis (CZE)] have become methods of choice in pharmacopeias and companies in the context of, e.g., product development, lot release and stability analyses [12,13]. Glyco-analysis at the protein level has the great advantage of reducing sample preparation to a minimum, thereby reducing costs and errors. However, more structural information can be obtained at the peptide and oligosaccharide levels. Accordingly, the most common methods traditionally applied for the analysis of lot-to-lot variability of biopharmaceutical glycosylation are based on 3 Page 3 of 14

oligosaccharide profiling [1]. For that purpose, N- and O-linked oligosaccharides first need to be released from the protein. The enzyme used for N-glycan release from mammalian proteins is peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase) F. In contrast, no enzyme is available for an efficient release of O-glycans. As a consequence, laborious chemical methods with harsh conditions, such as β-elimination or hydrazinolysis, are applied, resulting in consecutive monosaccharide loss (“peeling”) [14]. For spectrophotometric detection, released glycans need to be derivatized, commonly by attachment of a fluorophore [e.g., 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), or 1-aminopyrene-3,6,8-trisulfonate] at the reducing end [15,16]. Separation is then commonly achieved by hydrophilic interaction liquid chromatography (HILIC)-HPLC or UPLC [16,17], or by C(G)E with fluorescence detection [18,19]. As presented in a multilaboratory study, fluorescent labeling is the critical step in profiling due to possible incomplete derivatization, resulting in greater laboratory variability than MS-based Nglycomic methods [20]. In O-glycan profiling, MS-based methods were clearly preferred over spectrophotometric methods due to their advantage of not being limited to nonreductive, peeling-prone releasing techniques [21]. However, recent progress in the suppression of peeling may make the combination of chemical release and reducing-end labeling more appealing [22–25]. A label-free alternative to fluorescent detection of released glycans is high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). This technique is less tedious, but also less sensitive and less selective than LC-fluorescence approaches. According to the European Pharmacopeia, HPAEC-PAD and weak anionexchange-HPLC-fluorescence after AA-labeling, both were suitable for evaluation of lot consistency in a comparative study of released, sialylated N-glycans from 15 different EPO preparations [26]. Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF)MS was presented in the same study as a suitable rapid alternative, having the advantage of revealing additional, potentially antigenic, glycan structures. Due to its operation at very high pH and the requirement of specialized equipment and carbonate-free solvents, HPAEC-PAD provides less flexibility and ease-in-use than HPLC-fluorescence or MS-based techniques. The main drawback of the non-MS techniques presented is the inability of characterizing unknown compounds, as may be necessary after major process changes, since identification is performed only by referring to retention or migration time. Also, the unique versatility of MS-based methods offers an optimal basis for a rapidly growing, dynamic field, such as biopharmaceutical research. MALDI-MS and ESI-MS provide options for quick screening and obtaining detailed structural information via proteomic and glycomic approaches [i.e., on the (glyco)protein, (glyco)peptide or oligosaccharide level using the same instrumentation]. Extensive reviews on the analytical techniques available for characterizing protein biopharmaceuticals were recently published [27,28]. In the following, we describe recent MS approaches for the glycosylation analysis of biopharmaceuticals. 2.1.

MALDI-MS

The best established MS technique in biopharmaceutical glyco-analysis is MALDI-TOFMS [1]. It has been applied for many years in oligosaccharide profiling, particularly in early drug development. The main strengths of MALDI-TOF-MS are its outstanding speed, low consumption of sample, and high-throughput and automation potential (Table 1). In several comparative studies on N-glycans released from different proteins, including biopharmaceuticals, MALDI-TOF-MS showed results comparable to other, coupled MS techniques or non-MS methods, as listed in European Pharmacopoeia [9,20,29]. For example, batch-to-batch variability in terms of glycan branching, degree of sialylation, and Oacetylation of sialylated N-glycans was determined in recombinant EPO via negative-ion 4 Page 4 of 14

reflectron-mode MALDI-TOF-MS. Profiles obtained were in good agreement with nanoLC– ESI-MS data [9]. Notably, sialic acids are known to be prone to in-source and metastable decay, especially in positive-ion mode. Permethylation is therefore commonly performed prior to MALDI-MS of released N- or O- glycans, as it improves sensitivity, stabilizes the labile sialic-acid bonds, and enables the detection of non-sialylated and sialylated glycan species in positive-ion mode [8,20,21,29,30]. This approach was recently applied to characterize N-glycans from influenza antigen [31]. Striking differences between vaccine-manufacturing cell platforms were revealed, and are very relevant to efficient vaccine development. Permethylation has also been used to enhance robustness in (relative) quantitation by introducing isotope-labeled agents, such as 13CH3I [32]. For the same reason, reductive amination with 13[C6]AA has been applied [30]. These and other derivatization techniques for various glycomics applications were recently reviewed [33]. In addition, linkage-specific derivatization by lactone formation and ethyl esterification was recently shown to have great potential for the MALDI-TOF-MS characterization of sialylated biopharmaceuticals, enabling differentiation of 2,3-linked and 2,6-linked sialic acids [34]. MALDI-TOF-MS of released glycans has also been used for the elucidation of additional modifications, such as sulfation or phosphorylation of N-glycans in biopharmaceuticals [11]. Likewise, due to its speed and low consumption of sample, MALDI-TOF-MS is suitable for oligosaccharide sequencing via exoglycosidase digestion, providing information on the anomericity of glycosidic linkages [16]. This approach provides an attractive, sensitive alternative to traditional linkage-analysis techniques, such as methylation GC-MS or NMR. Furthermore, for native glycans, detailed structural information can be obtained via MALDIMS/MS, since sodiated adduct ions, commonly observed in MALDI, result in cross-ring fragments upon high-energy collision-induced dissociation (CID) TOF/TOF-MS [35]. MALDI-MS can be less suitable for the analysis of smaller oligosaccharides, in particular, certain O-glycan species. This is related to an overlap with background peaks in the lower mass range originating from the MALDI matrix. Direct ESI-MS of smaller oligosaccharides or MALDI-TOF-MS of glycopeptides can therefore be a better approach in certain cases. If applied on glycopeptides, MALDI-MS provides information on site occupancy [31] and possible contamination of the original protein sample. Off-line LC-MALDI-TOF-MS and -MS/MS have been applied successfully to sitespecific characterization of several O-glycan modifications in a recombinant Fc fusion protein [36]. Reliable relative quantitation of glycopeptides by preventing in-source and metastable decay using MALDI-Fourier transform ion-cyclotron resonance (FT-ICR) equipment was demonstrated for plasma IgG [37]. However, since the maintenance of FTICR instruments and their operation requires a high degree of expertise and effort (Table 1), their use has been limited to research so far. 2.2.

Direct ESI-MS and -MS/MS

Another soft ionization technique, which is well established in glycomics and (glyco)proteomics, is ESI-MS. Direct ESI-ion-trap (IT)-MSn has provided good linearity and reproducibility in rapid, automated screening of released 13[C6]AA-labeled N-glycans, with good performance on sialylated species [30]. Another ESI-IT-MS approach with direct infusion and CID MS/MS was able to detect unexpected isobaric N-glycans in mAbs [38]. A recently published method allowed fully-automated, high-throughput, relative quantitation of Fc-glycopeptides from IgG by direct infusion on an Orbitrap instrument [39]. Glycan sequence could be verified via CID in the same experiment (Fig. 3). However, with the increasing complexity of glycan samples and the presence of interfering components, direct MS methods may suffer from sensitivity losses due to ionization suppression. Coupling with a separation technique will limit ionization suppression 5 Page 5 of 14

and often allow resolving isomeric or isobaric structures, thereby greatly supporting MS characterization of glycosylated samples. 2.3. LC-ESI-MS and LC-MS/MS Robust and unambiguous glycomic and glycoproteomic analysis of various glycoproteins including biopharmaceuticals has been performed on standard LC-MS instrumentation [27,40–43]. Starting with intact or reduced protein, LC-MS is a straightforward technique for profiling glycoforms in biopharmaceuticals, as applied in biosimilar research [10,44,45], clone selection [38], batch-to-batch comparison [46,47], or as routine check for molecular weight and heterogeneity in biopharma industry. LC-MS is commonly applied in reversed-phase (RP) or size-exclusion chromatography modes with ESI-TOF-MS detection [43,48]. Advantages in intact protein MS are high reproducibility and speed due to minimal, if any, sample preparation [42,43]; also, potential impurities may be detected within the same run. Notably, if multiple glycosylation sites are present, leading to a higher degree of heterogeneity, intact protein MS and MS/MS (top-down approach) will become challenging [42]. During the past few years, glycoprofiling of biopharmaceuticals by LC-MS at the glycopeptide level has gained enormous popularity, mainly due to its unique option to obtain site-specific information and the ability to separate glycopeptides of interest, which are often hard to ionize, from the more hydrophobic, interfering non-glycosylated peptides within one run. In a classical bottom-up approach, the glycoprotein of interest is digested by a proteolytic enzyme, directly in solution in case of high-purity glycoprotein, or in gel after electrophoretic pre-purification. An example protocol for N- and O- glycan analysis in biopharmaceuticals with discussion of various critical aspects and alternative options was given by Kolarich et al. [41]. Trypsin is the protease most commonly used for proteomic and glycoproteomic bottom-up approaches. However, depending on the glycoprotein structure, other proteases or protease mixtures have been shown as more suitable, such as Lys C for Oglycosylation analysis [6,41,49]. More recently, IdeS and other enzymes for limited proteolysis generating larger peptides for a faster middle-up glycoprofiling were successfully applied for lot variability and biosimilar analysis of mAbs via LC-ESI-(Q)TOF-MS [44,50]. C18-RP materials commonly used for bottom-up proteomics may not retain certain glycopeptides due to increased analyte hydrophilicity compared to non-glycosylated peptides, requiring additional use of porous graphitize carbon (PGC) [41] or HILIC [51]. HILIC separation of glycopeptides is based on the properties of the peptide and the glycan moiety, and tolerates various substitutions, such as sulfation [52]. A recent approach demonstrated that on-line HILIC pre-enrichment of N-glycopeptides prior to C18-LC-QTOF-MS/MS resulted in efficient site-specific glycoprofiling [51]. The detection of both glycan and peptide fragmentation in the same MS/MS spectrum via elevated-energy CID allowed confirmation of both peptide and glycan sequence, and site assignment. LC-ESI-IT-MS and MS/MS have been applied to assign the site of a novel O-linked modification in a mAb and the N- and O- glycosylation sites in an Ig fusion protein, respectively, by using CID [49,53]. Though LC-ESI-IT-MS is widely used for glycopeptide analysis in biopharmaceuticals, LC-ESI-(Q)TOF-MS is gaining interest due to its higher resolution and mass accuracy [10,51,54,55]. Electron-transfer dissociation (ETD) is an attractive alternative fragmentation mode next to CID. While CID preferably results in the fragmentation of the glycan portion, ETD provides information on the amino-acid sequence, while leaving the glycan moiety intact [56]. Similarly, higher-energy C-trap dissociation (HCD) performed on an Orbitrap instrument has proved useful in characterizing both the glycan and peptide moiety, while also providing ultra-high resolution and mass-accuracy MS and MS/MS spectra [57]. A software 6 Page 6 of 14

tool for confident glycan/protein sequencing and site assignment on the basis of combined CID/HCD/ETD data has been introduced, facilitating simultaneous identification of both features [58]. These advanced fragmentation techniques are particularly useful in biopharmaceutical research. Moreover, they facilitate site-specific O-glycan analysis, which is more challenging than N-glycans due to the lack of a single consensus sequence and thus higher heterogeneity [41,56]. These approaches may also be extended for a distinction between isomeric N- or O-glycan structures by using the same instrumentation [59]. In multistage tandem MS experiments, more detailed information on both peptide and glycan moiety can be obtained. In addition, site assignment even in case of peptides with multiple glycosites is feasible. Alternatively, a data-independent, so-called MSE, peptide-mapping approach with ramping collision energies on a high-resolution and accuracy mass spectrometer has been applied to N- and O-,glycopeptides to obtain both site-specific glycan and peptide sequence information within the same run [31,45,56]. The generation of multiple fragment spectra during (glyco)peptide mapping results in vast amounts of data, particularly in dataindependent analysis, leading to a major bottleneck. Notably, inter-laboratory reproducibility has been shown to be largely affected by data analysis, including the choice and the number of glycopeptides for quantification. In addition, sample-preparation parameters, such as choice of proteolytic enzyme and digestion conditions, contribute to uncertainty, as shown for the analysis of a glycoprotein with a single N-glycosylation site [42]. However, another study comparing four different glycosylationprofiling strategies in IgG has shown RP-LC-ESI-QTOF-MS to be superior to MALDI-TOFMS glycopeptide profiling or CGE-LIF relative quantitation of released N-glycans [60]. In addition, analyzing O-glycopeptides is advantageous over released O-glycans, since degradation due to peeling during the chemical release is omitted [6]. For the LC-MS analysis of released glycans, HILIC and PGC materials are used for separation of reducing, as well as reduced, oligosaccharides [40,52,61]. The resolving power of PGC, applied in nanoLC-chip format, was demonstrated by the MS identification of nearly 400 N-glycan structures released from darbepoetin alpha, a hyperglycosylated recombinant form of EPO [9]. Another PGC-nanoLC-chip-based approach with QTOF-MS/MS was used to generate a comprehensive N-glycan library from eight different mAbs, including 70 identified structures, with 25 being fully characterized [61]. On-line PGC-LC-ESI-MS in negative-ion mode also proved highly reproducible for profiling released non-derivatized O-glycans from IgA [21]. Unfortunately, PGC lacks retention of released monoccharides and certain disaccharides, such as O-linked Tn or T antigens [40]. In contrast, HILIC-based stationary phases provide size-dependent fractionation from monoccharides up to large oligosaccharides, including neutral, charged and native next to fluorescently-labeled glycans {reviewed in [52]}. HILIC includes silicabased ion-exchange, zwitterionic and non-ionic stationary phases with new developments towards smaller particle sizes and column dimensions enabling faster analysis and higher sensitivity via UPLC-MS technology [45,62]. Most HILIC solvents are MS compatible, as they contain volatile acids, bases and buffers, and high ACN concentrations. Nevertheless, the buffers cause background noise, which can be an issue in nanoUPLC-MS, where low fmol amounts of glycans are applied [52]. LC-MS analysis of released glycans is commonly conducted on QTOF [9,61], IT [55], [40] or Orbitrap [62] instruments with the possibility of low-energy or high-energy CID MS/MS. 2.4. CE-MS CE-MS is gaining attention in the characterization of biopharmaceuticals. Due to the incompatibility of MS detection with CGE and CIEF methods, currently mainly CZE-MS 7 Page 7 of 14

methods are used for glycosylation characterization of biopharmaceuticals. Regarding detection, the same developments as described in sub-section 2.3 for LC-MS/MS can be seen in the CE-MS field, as summarized in Table 1. For the analysis of released glycans, only a few methods have been published. This has to do with the reduced separation in the absence of gel buffers. Two promising methods enabled high resolving separations by CZE-MS for APTSlabeled N-glycans of therapeutic antibodies [63,64]. As APTS is used in these studies to introduce charge, both off-line [63] and on-line [64] fluorescence detection could be performed. Recently, a CE-MS method was introduced to determine glycan heterogeneity without derivatization [65]. The separation is less efficient than separations of APTS-labeled glycans, but co-migrating glycans could be distinguished and identified due to the use of MS detection, discerning 22 and more than 70 different N-glycan structures for IgG and recombinant EPO, respectively. CZE-MS has proved very useful for characterizing site-specific microheterogeneity of biopharmaceuticals at the glycopeptide level. In contrast to LC-based separations, CZE separation is mainly based on the attached glycan rather than on the peptide moiety [66]. Both N- and O- glycosylation of EPO were studied after protein digestion, revealing more than 10 glycoforms per glycosylation site, including N-acetylneuraminic-acid and Nglycolylneuraminic-acid residues [67]. N-glycosylation of four mAbs was characterized on the peptide level using CZE-MS, revealing 10–16 different glycoforms [68]. CZE-MS has been most extensively used for the characterization of intact glycoproteins [69]. Most efficient separations of glycoproteins are obtained when using low-pH background electrolytes in combination with coatings that minimize or cancel out electroosmotic flow. Differences in charged sugar units and charged amino acids cause the most significant shift in migration time, whereas differences in neutral sugars also contribute to glycoform separation, but to a lesser extent. Overall, this leads to a complex, but also very comprehensive, profile revealing the overall glycosylation pattern (Fig. 4). For example, interferon-β-1a (1 Nglycosylation site) was shown to contain 18 glycoforms and more than 80 proteoforms, whereas EPO (3 N-glycosylation sites, 1 O-glycosylation site) presented over 70 glycoforms and more than 250 proteoforms [70]. 2.5. IM-MS One of the newer developments in glycoproteomics is the combination of ion-mobility spectrometry with MS (IM-MS). In IM, ions are separated based on size, shape and charge in the gas phase [47]. Since the mechanism of separation is very different from most chromatographic separations and the time scale of separation ranges between that of LC and MS experiments, IM-MS is well suited to complement LC-MS experiments. IM-MS has rarely been applied to glyco-analysis of biopharmaceuticals [47]. Harvey et al. acquired clean mass spectra from glycans after direct infusion of a non-purified HIV protein/PNGase F digest into an IM-MS instrument, although the sample contained interfering compounds, such as detergent [71]. Efficient separation of background ions from glycans released from sub-mg amounts of protein could be achieved based on different IM drift times as typically observed for different biomolecule classes. Conceptually, the rapid separation of glycan isomers, such as the differentiation of isomeric complex and hybrid [72] or bianntenary glycans [73], could become a distinctive feature for the application of IM-MS. However, at the moment, the drift-time resolution of commercial IM-MS instruments allows at most a partial separation of many relevant glycan isomers. The (partial) separation of precursor ions also opens new possibilities for CID-fragmentation analysis. For example, in MSE-like approaches IM-separated precursor ions can be linked to their fragment ions via their drift time [47,72]. IM can also be applied to distinguish isomeric precursor structures via separation of their isomeric fragments [74]. 8 Page 8 of 14

3. The future of drug glycosylation analysis MS techniques have become integral parts of drug-glycosylation analysis in academia and industry, due to their high resolution and unambiguous assignment of mass to composition, particularly if coupled to separation techniques, such as LC, CE and IM. The combined methods provide not only the option of mass or spectral matching, but also retention/migration-time matching and isomer differentiation. Due to the increasing success of biopharmaceuticals on the drug market in recent years, a number of novel protein-based drugs, including mucin-based cancer vaccines, Fc fusion proteins and glyco-engineered optimized agents, can be anticipated in the near future [3,6]. New developments of biopharmaceutical production on non-mammalian platforms add to this development. As a result, (glyco)analytical strategies of high efficiency with regard to method development, routine analysis time, sample amount, and expertise will be sorely needed to meet quality requirements. This may even become more rigorous in the future due to the great significance of glycosylation as a CQA of pharmaceuticals. MS will therefore play a crucial role in analytical monitoring during drug development and lot release, as it has the potential to provide all these features due to its unique versatility as both a stand-alone technique and in combination with separation techniques. Moreover, it is powerful at several levels of glycoprotein analysis (i.e. glycan, glycopeptide and intact-protein levels by using standard proteomic instrumentation). Analytical platforms based on MS are therefore very promising, since they facilitate combined analysis of PTMs, including glycosylation, and the amino-acid sequence of a biopharmaceutical, as has been demonstrated, e.g., for biosimilar studies [44]. Further developments in MS/MS techniques, as seen in the combined application of CID and ETD or HCD, will promote the co-evolution of such integrated platforms. Increased availability of high-resolution and high-accuracy instruments will further support unambiguous glycosite assignment and characterization of peptide and glycan structures within a single measurement. Furthermore, since MS analysis at the peptide and protein level has opened up the possibility of direct site-specific information, a trend towards bottom-up and, more recently, top-down or middle-down approaches is rapidly emerging. Another, very recent approach for intact-protein analysis is native MS, as exemplified by a study profiling the various glycoforms of a half antibody [75]. Native MS keeps the noncovalent interactions and fold of a protein largely intact and thus allows in-depth analysis of, e.g., stability, binding properties and conformation, which can also be useful to obtain information on glycosylation [43, 75]. Furthermore, native MS shows a more concise chargestate distribution than conventional denaturing approaches, often resulting in higher signal-tonoise ratios and reduced spectrum complexity [43]. In addition, improvements in analytical instrumentation and overall workflow should lead towards a high degree of automation, optimally including all steps from sample preparation to data analysis. In addition, in clone selection, medium development, pharmacokinetic and pharmacodynamic studies, process development and characterization, and lot release, a high sample throughput can be useful. This can be facilitated, e.g., by the use of immobilized protein A for mAb purification in 96-well plate format [39] or by exoglycosidase arrays for obtaining linkage information [56]. Still, data analysis remains a major bottleneck in high-throughput glyco-analysis, since classic proteomics and glycomics databases do not sufficiently provide combined glycoproteomic data. Efforts towards automated data extraction for targeted (relative) glycan quantification or automated glycopeptide identification and quantitation based on MS and MS/MS data were published recently [34,76,77]. The challenges of, and the available tools for, automated glycopeptide data analysis were recently reviewed [78]. Down-scaling of sample preparation and analysis also plays an important role in glycosylation analytics and has found widespread application, as demonstrated by numerous publications presenting methods using (chip-based) nanoLC [9,61]. Kits for glycan release, fluorescent labeling and 9 Page 9 of 14

purification, as well as permethylated or tagged glycan standards are commercially available, facilitating routine glycan analytics by enhancing method robustness and ease of use. However, there is still a long way to go before glycomics and glycoproteomics can reach the level of sophistication established in proteomics, particularly with regard to quantitation via MS. In proteomics, the use of internal standards and state-of-the-art MS equipment has led to established techniques for accurate, precise quantitation, with particular validity when isotopic labeling is used. The advent of commercially-available isotopically-labeled glycans and glycopeptides will allow very robust, quantitative MS glyco-analysis of biopharmaceuticals. Hydrogen/deuterium exchange (HDX)-MS is another heavy-isotope-based MS technique recently applied to glycoproteomics. The impact of sialylation on glycoprotein-ligand interactions was studied in a bottom-up approach using an ETD-capable Orbitrap instrument [79]. Similar approaches may be particularly useful in studies on drug-target interactions during discovery. In conclusion, biopharmaceutical development will benefit from the emerging glycoanalytical approaches using MS, especially in the context of QbD by their unique potential for simplifying and optimizing quality assessment and control of drugs. Acknowledgements This work was supported by the European Union's Seventh Framework Program (FP7Health-F5-2011) under Grant Agreement N° 278535 (HighGlycan). David Falck acknowledges financial support by Hoffmann La Roche. References [1] E. Higgins, Glycoconj J 27 (2010) 211. [2] Research and Markets, Biopharmaceuticals - A Global Market Overview, M2PressWIRE. 2013. [3] A.R. Costa, M.E. Rodrigues, M. Henriques, R. Oliveira, J. Azeredo, Crit Rev Biotechnol (2013). [4] P. Hossler, S.F. Khattak, Z.J. Li, Glycobiology 19 (2009) 936. [5] J.N. Arnold, M.R. Wormald, R.B. Sim, P.M. Rudd, R.A. Dwek, Annu Rev Immunol 25 (2007) 21. [6] P.W. Zhang, T.; Bardor, M.; Song, Z., Pharm Bioprocess 1 (2013) 89. [7] I.J. del Val, C. Kontoravdi, J.M. Nagy, Biotechnol Prog 26 (2010) 1505. [8] W.R. Alley, Jr., B.F. Mann, M.V. Novotny, Chem Rev 113 (2013) 2668. [9] M.J. Oh, S. Hua, B.J. Kim, H.N. Jeong, S.H. Jeong, R. Grimm, J.S. Yoo, H.J. An, Bioanalysis 5 (2013) 545. [10] A. Harazono, N. Hashii, R. Kuribayashi, S. Nakazawa, N. Kawasaki, J Pharm Biomed Anal 83 (2013) 65. [11] A. Lusch, M. Kaup, U. Marx, R. Tauber, V. Blanchard, M. Berger, Mol Pharm 10 (2013) 2616. [12] A. Staub, D. Guillarme, J. Schappler, J.L. Veuthey, S. Rudaz, J Pharm Biomed Anal 55 (2011) 810. [13] M.J. Little, D.M. Paquette, P.K. Roos, Electrophoresis 27 (2006) 2477. [14] G. Zauner, R.P. Kozak, R.A. Gardner, D.L. Fernandes, A.M. Deelder, M. Wuhrer, Biol Chem 393 (2012) 687. [15] L.R. Ruhaak, G. Zauner, C. Huhn, C. Bruggink, A.M. Deelder, M. Wuhrer, Analytical and Bioanalytical Chemistry 397 (2010) 3457. [16] K.J. Lee, S.M. Lee, J.Y. Gil, O. Kwon, J.Y. Kim, S.J. Park, H.S. Chung, D.B. Oh, Glycoconj J 30 (2013) 537. [17] Y. Du, F. Wang, K. May, W. Xu, H. Liu, J Chromatogr B Analyt Technol Biomed Life Sci 907 (2012) 87. 10 Page 10 of 14

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Captions Fig. 1. Model glycoprotein with exemplary glycan structures. Commonly found in biopharmaceuticals are N- and O- type glycosylation, in which oligosaccharides are attached to an asparagine residue and to a serine or threonine, respectively. Fig. 2. Stages of biologic-drug development involving glycosylation analysis. Therapeutic glycoproteins need to be optimized and monitored throughout all stages of product development, also with respect to their glycosylation. Glycosylation Critical Quality Attributes (GCQAs) should be determined to ensure optimal efficacy and consistent drug quality. Fig. 3. Fully automated, high-throughput ESI-Orbitrap-MS(/MS) of therapeutic IgG Fc glycopeptides. (A) Direct ESI-MS profile of the HILIC-SPE-purified tryptic IgG1 Fc glycopeptides. (B) Deconvoluted CID-MS/MS of the G1F3+ (Hexose4N-acetylglucosamine4fucose1) glycoform at m/z 933.04. For glycan symbols, see Fig. 1; for method description and m/z list, see [39]. Fig. 4. Sheathless CE-MS of recombinant human interferon-β (A) and recombinant human erythropoietin (B). (A1) and (B1) are base peak electropherograms; (A2) extracted ion electropherograms for 5 selected glycoforms including their deamidated products at the indicated m/z value (11+ charge state) with assigned glycan structures (for symbols, see Fig. 1); (B2) contour plot zoomed at the 14+ charge state of the glycoforms indicating the compositions of the 13-fold sialylated glycans. {Reprinted (adapted) with permission from [65]. ©2013, American Chemical Society}.

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Table 1. Common methods for the analysis of glycosylation in biopharmaceuticals Separation

Analysis

MS/MS

Ease of use b)

Automation

Maintenance and costs

Analyte level

Remarks

CE

UV

-

+++

+++

+

Glycoproteins

Identification limited to migration time; little structural information, low sensitivity

(nano)UPLC or CE

Fluorescence

-

+++

+++

+

Oligosaccharides, monosaccharides

Derivatization necessary; quantitation straightforward

HPAEC

PAD

-

++

++

+

Oligosaccharides, monosaccharides

Lower sensitivity than fluorescence

-

MALDITOF(/TOF)-MS

CID, LID a)

+++

+++

+

Glycopeptides, oligosaccharides

Metastable decay; neutral vs. Sialylated separately in +/– ion mode

(nano)UPLC or CE

IT

CID, ETD

++

++

++

Glycopeptides, oligosaccharides

Msn for in-depth analysis

(Q)TOF

CID, ETD

++

++

++

Glycoproteins, glycopeptides, oligosaccharides

High resolution & mass accuracy

Orbitrap

CID, ETD, HCD

++

++

++

Glycoproteins, glycopeptides, oligosaccharides

High resolution & mass accuracy

a)

CID, Collision-induced dissociation; LID, Laser-induced dissociation; ETD, Electron-transfer dissociation; HCD, Higher-energy C-trap dissociation. moderate; +++, high.

b)

+, low; ++,

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