Orthogonal Middle-up Approaches for the Characterization of the

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Orthogonal Middle-up Approaches for the Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High Resolution Mass Spectrometry Valentina D'Atri, Lucie Nováková, Szabolcs Fekete, Dwight R Stoll, Matthew Allen Lauber, Alain Beck, and Davy Guillarme Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03584 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Analytical Chemistry

Orthogonal Middle-up Approaches for the Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High Resolution Mass Spectrometry. Valentina D’Atri,*,†,¶ Lucie Nováková,¥, Beck,‡ and Davy Guillarme.†

¶

Szabolcs Fekete,† Dwight Stoll, ǂ Matthew Lauber,§ Alain

†

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU – rue Michel Servet 1, 1211 Geneva, Switzerland. ¥ Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, Czech Republic. ǂ

Department of Chemistry, Gustavus Adolphus College, Saint Peter, Minnesota 56082, United States.

§

Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696, United States. Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, Saint-Julien-en-Genevois, France.

‡

ABSTRACT: Etanercept is a recombinant Fc-fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O- and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc-fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high resolution mass spectrometry (HRMS) by using a QTOF/MS instrument to assess glycosylation of etanercept at the middle-up level of analysis (fragments of circa 25-30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e. glycosidase, sialidase and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.

Fusion proteins are a class of molecules comprised of two or more different protein domains integrated into one molecule via genetic fusion of their encoding sequences. Therapeutic fusion proteins involve several types including Fc-fusions, albumin, transferrin, immunotoxins, cytokine, antibody-enzyme fusions, and others.1 Although most of the recombinant therapeutic proteins approved by the FDA up to now are monoclonal antibodies (mAbs), about 13 Fc-fusion proteins are currently available for clinical treatment2 and some others are in clinical trials, or in preclinical development. Among Fc-fusion proteins, two of them, etanercept (Enbrel) and abatacept (Orencia), have already achieved blockbuster status with sales over US $1 billion per year. Moreover, etanercept is considered to be one of the most successful blockbuster drugs in the biopharmaceutical market.3 Fc-fusion proteins are composed of an effector structure, a peptide or a protein, that is directly attached to the Fc domain of an antibody. This domain is common for Fc-fusion proteins and is responsible for a long half-life and effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). The targeting domain is a variable part and can be formed by the extracellular domain of a receptor, a cytokine receptor, or a peptide mimetic. Modifications of these domains are also important. Glycosylation of therapeutic proteins has a strong impact on their therapeutic effect by improving pharmacokinetics and selectivity of

binding to receptors. It is the key parameter determining immunogenicity and solubility, as well as stability. Glycosylation is therefore considered to be a critical quality attribute (CQA) of glycoprotein products. The glycosylation profile is generally heterogeneous, resulting in batch-to-batch variability, and needs to be evaluated as part of a quality control strategy.4,5 The complete characterization of complex and diverse glycosylation profiles requires a combination of advanced analytical methods, usually a combination of LC and/or CE with high resolution mass spectrometry (HRMS).6–8 Etanercept is a recombinant dimeric Fc-fusion protein consisting of two different monomers, held together by 3 disulfide bonds, and having an overall molecular mass of approximately 100 kDa (Figure 1a). Each monomer contains a crystallizable fragment (Fc) of an IgG1 antibody fused to a tumor necrosis factor-α receptor (TNFR). In addition, a large number of intrachains disulfide bonds are present on each monomer, namely 11 in the TNFR domain and 2 in the Fc/2 region. A canonical Fc N-glycosylation site is present. Moreover, two additional N-glycosylation sites and 13 O-glycosylation sites are present in each TNFR domain (Section S1 and Table S1). Etanercept exhibits remarkably complicated microheterogeneity and a glycan-related mass shift of circa 30 kDa.9 The importance of a detailed glycosylation study has been emphasized in previous reports, which focused on physicochemical characterization of the etanercept originator,4,9–17 its

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biosimilars5,14,18–20 and intended copies.21 Typical protocols to describe the glycosylation profile in these papers involve release of free N- and O-glycans in the first step using PNGase F and β-elimination, respectively. Their subsequent characterization is carried out using LC-MS or LC-FLR methods. Moreover, glycosylation sites can be elucidated using peptide maps resulting from trypsin and/or other protease digestions. Other contributions to the characterization of etanercept have focused on middle-up analysis under RPLC conditions and glycan profile characterization using MALDI-TOF/MS.14 Native mass spectrometry with orbitrap technology was also found applicable for glycoform characterization as used in combination with a variety of enzymatic digestion procedures. 9 Recently, HILIC has been demonstrated to be a powerful technique for the separation of hydrophilic variants of therapeutic proteins from the class of mAbs22–24 and antibody-drug conjugates (ADCs)25 at the middle-up level of analysis, thanks to the introduction of novel wide-pore HILIC stationary phases.22 Indeed, a comparison of HILIC chromatographic profiles allows identification of the most abundant glycans and reveals the differences between individual batches or between an originator

and a biosimilar. The use of HILIC to study intact and subunitdigested proteins is a relatively recent application of separation technology, and it represents a cutting-edge opportunity for the characterization of biopharmaceutical proteins.23,25–27 The enormous potential of this chromatographic technique derives from its orthogonality to RPLC and its ability to resolve protein glyco-variants that would otherwise co-elute.28 Moreover, the technique is easily coupled to MS (without performing sample collection or pre-treatments) such that peak identification can be streamlined. However, some diligence must be exercised in order to obtain an efficient glycoprotein separation. In this regard, wide-pore HILIC stationary phases functionalized with amide groups should preferentially be used versus unbonded phases.27 In addition, use of acidic conditions and an ion-pairing reagent should be acknowledged as being critical to improving peak shape, selectivity, and resolution, since they are known to neutralize possible ionic interactions between the glycoprotein and the stationary phase. For this reason, the use of 0.1% trifluoroacetic acid (TFA) modified mobile phases was reported as an acceptable compromise for LC-MS analysis.23

Figure 1. (a) Structural features of etanercept fusion protein. Two identical chains are held together by three inter-chain disulfide bonds (in black). The tumor necrosis factor-α receptor (TNFR, in grey) acts as a TNF-α inhibitor. The crystallizable fragment (Fc, in light blue) is responsible for the interaction with cell surface receptors. N-glycans and O-glycans are represented as blue epsilons and violet sticks, respectively. IdeS enzyme and DTT cleavage sites are shown as dotted grey lines. (b) Schematic representation of some of the most common N- and O-glycoforms that might be found in etanercept along with their corresponding shorthand names and compositions. Sialidase, Oglycosidase and PNGase F cleavage sites are indicated for representative (c) O-glycans and (d) N-glycans.

Attention must also be paid to minimize breakthrough and strong solvent effects as manifest as unretained protein eluting with the void volume and/or the presence of distorted peaks throughout the chromatogram. Indeed, aqueous solutions have the strongest eluotropic strength in HILIC separations. As a result, sample diluents and high injection volumes can present challenges. To circumvent this issue, injection volumes should be minimized. In addition, ACN-rich mobile phase conditions can be used at the beginning of the gradient run to facilitate the sample loading. In particular, this latter optimization allows the sample to be mixed into higher ACN content mobile phase before reaching the column. The aim of this work was to develop a HILIC-HRMS method for the characterization of the Fc-fusion protein etanercept at a

middle-up level of analysis. To facilitate spectra deconvolution, a combination of different digestion procedures was systematically employed, including a combination of different glycosidase, sialidase, and protease enzymatic treatments.

MATERIALS AND METHODS Reagents and materials. Acetonitrile LC-MS grade was purchased from Honeywell Research Chemicals (Labicom, Czech Republic). Trifluoroacetic acid LC-MS grade (TFA, ≥ 99.7 %) was purchased from Merck (Darmstadt, Germany). Hydrochloric acid 35% analytical grade was purchased from LachNer (Neratovice, Czech Republic). DL-dithiotreitol (DTT, ≥


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Analytical Chemistry 99.0 %) and tris(hydroxymethly)aminomethane (TRIS, ≥ 99.9 %) were purchased from Sigma-Aldrich (Prague, Czech Republic). Ultrapure water was prepared by Milli Q reverse osmosis system (Millipore, Bedford, MA, USA) immediately prior to use. Immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS, FabRICATOR), O-glycosidase enzyme (OglyZOR), and sialidase enzyme (SialEXO) were purchased from Genovis Inc. (Lund, Sweden). N-glycosidase enzyme Rapid Peptide-N-Glycosidase F (PNGase F) was purchased from New England Biolabs (NA, USA) in a kit with a reaction buffer. Etanercept sample was obtained as European Union pharmaceutical-grade drug product from its respective manufacturer. Sample preparation. Etanercept stock solution (50 mg/mL) was diluted with water to achieve a concentration of 0.5 mg/mL. A variety of digestion procedures were applied for obtaining a complete characterization of the Fc-fusion protein. Digestion and reduction. 10 mM TRIS-HCl buffer pH 7.0 was added to 0.5 mg/mL etanercept, and the solution was incubated with 100 units of IdeS enzyme at 37 °C for 45 min. The digest was then subjected to reduction with 100 mM DTT by incubation at 45 °C for 30 min. This procedure was further used under the same conditions as a part of each of the following protocols. N-deglycosylation. The first step in this protocol was digestion of etanercept with IdeS. N-deglycosylation was carried out with 1 µL Rapid PNGase F enzyme using its manufacturer-provided companion buffer. The sample was incubated at 50 °C for 10 min. Reduction with DTT was carried-out thereafter. O-deglycosylation. 20 mM TRIS buffer at pH 7.0 was added to etanercept followed by addition of two enzymes for desialylation and deglycosylation, first 40 units of SialEXO and secondly 40 units of OglyZOR. The sample was incubated at 37 °C for 4h. In the next step, lyophilized IdeS enzyme was added. Lastly, the sample was reduced with DTT. Desialylation. 20 mM TRIS buffer at pH 7.0 was added to etanercept followed by addition of 40 units of SialEXO enzyme and a 4 h incubation at 37 °C. In the next step, lyophilized IdeS enzyme was added for digestion and the sample was thereafter reduced using DTT. O-deglycosylation and N-deglycosylation. 20 mM TRIS buffer at pH 7.0 was added to etanercept followed by addition of two enzymes for desialylation and deglycosylation, first 40 units of SialEXO and secondly 40 units of OglyZOR. The sample was incubated at 37 °C for 4h. In the next step, lyophilized IdeS was added and incubated for digestion. Subsequently, the digested sample was N-deglycosylated with 1 µL Rapid PNGase F and then reduced with DTT in the last step. Desialylation and N-deglycosylation. 20 mM TRIS buffer at pH 7.0 was added to etanercept followed by addition of SialEXO enzyme. The sample was subjected to a 37 °C incubation for 4h. In the next step, lyophilized IdeS enzyme was added for digestion followed first by N-deglycosylation with Rapid PNGase F and then reduction with DTT. Instrumentation and columns. A UHPLC system (ACQUITY UPLC I-Class, Waters, Milford, USA) coupled to a Q-TOF mass spectrometer (Synapt G2-Si, Waters, Milford, MA) was used for UHPLC-HRMS analyses. The mass spectrometer was operated in ESI positive ion mode to acquire continuum data in the range of 500 - 3000 m/z with a scan time of 1.0 s. Leucine-enkephalin was used as a lock mass reference

and for internal calibration. Sodium iodide was used for external calibration. Capillary voltage was set at 3.0 kV, cone voltage at 120 V, source offset at 80 V, source temperature at 150 °C, desolvation temperature at 500 °C, desolvation gas flow at 1000 L/h, and nebulizer gas at 6.0 bar. MassLynx 4.1 was used for data acquisition and analysis. Deconvolution was performed by MaxEnt1. For this study, separations were performed with a 2.1 x 150 mm column comprised of an amide-bonded, widepore hybrid particle stationary phase (ACQUITY UPLC Glycoprotein BEH Amide 300 Å 1.7 µm, Waters, Milford, MA). Chromatographic conditions. The separation was carried out in HILIC mode with gradient elution afforded by 0.1% TFA in acetonitrile and 0.1% TFA in water mobile phases and a flow-rate of 0.2 mL/min. The gradient program started at 15:85 water/ACN + 0.1% TFA, which was quickly changed to 28:72 in 0.2 min, followed by a shallow gradient to reach 37:63 in 8.5 min and subsequently 44:56 in 8.7 min. An isocratic step at 44:56 was held until 15 min. This analytical gradient was followed by a wash step and a column re-equilibration at an elevated flow-rate of 0.45 ml/min to yield a total analysis time of 27.5 min. For the entirety of the run, the column was maintained at 45 °C. 85% ACN was used as the LC wash solvent and 40% ACN was used as LC purge solvent. A 1-µL portion of sample was injected.

RESULTS AND DISCUSSION Sample preparation procedures to streamline etanercept glycan profiling. Subunit analysis offers a chance to untangle the heterogeneity inherent to the overall etanercept glyco-proteoforms. In fact, by cleaving the protein into two main subunits, namely the TNFR and Fc/2 subunits, it might be possible to streamline data analysis and to make gains in both chromatographic and MS resolution. For this purpose, IdeS digestion and DTT reduction were systematically applied. First, IdeS was used to cleave etanercept under the hinge region to yield two Fc/2 fragments and the dimeric TNFR subunit. Then, DTT was added to the sample to reduce the disulfide bonds and to release the monomeric form of the TNFR (Figure 1a). Reactions for producing the two main subunits of etanercept were also paired with treatments involving the use of orthogonal glycan-specific enzymes. To obtain the selective removal of glycans, PNGase F was used to specifically remove the N-glycans, while the combination of sialidase (SialEXO) and O-glycosidase (OglyZOR) was used to remove the O-glycans (Figure 1c-d). In addition, subunits resulting from various combinations of these enzymes were also investigated. It should be noted that the Oglycosidase reaction requires a sialidase pre-treatment in order to be effective. Accordingly, when O-deglycosylation was performed, N-glycans were also desialylated. Figure 2 shows the total ion chromatograms (TIC) obtained from HILIC-HRMS analyses of etanercept subunits. With the aim of obtaining orthogonal and complementary glycan information, six samples were selected. Specifically, IdeS digested and DTT reduced etanercept (Figure 2a) was the most complex and heterogeneous sample, since no deglycosylation was performed. Application of HILIC-HRMS for the characterization of etanercept at the subunit level. It has recently been reported that native MS with orbitrap technology can be successfully



used for glycoform characterization of etanercept, in combination with a variety of enzymatic digestion procedures 9. However, not every biopharmaceutical laboratory is equipped with such an expensive MS technology providing resolution in the range 70’000-100’000. Therefore, our objective was to develop an alternative analytical strategy involving the use of a chromatographic dimension (HILIC) in combination with QTOF/MS technology offering lower resolution (Rs of 10’000-40’000) and various enzymatic digestions. The role of the chromatography will be to help in deconvoluting MS signal and to help compensate for the lower resolution of QTOF/MS vs. orbitrapMS detection. During initial work, preliminary studies were performed to optimize HILIC mobile phase conditions. The lowest flow rate offering acceptable retention time variability on our instrument was selected (F = 0.2 mL/min). It is worth mentioning that all the analyses were performed in triplicate and retention times were reproducible to within an RSD of 0.02 minutes. In addition, the average mass accuracy was seen to be 0.8 Da across all injections (See Tables S2-S7 for detailed retention times and mass assignments). The HILIC gradient was conceived to avoid breakthrough effects. Therefore, a fast initial ramp (0.2 min) from 85 to 72% ACN was used at the beginning of the method. Then, a linear shallow gradient (slope of around 1%/min) was adopted to achieve the separation of the glycovariants related to the Fc/2 subunits of etanercept (vide infra), while an isocratic step at 56% ACN was found to be effective for the resolution of the TNFR subunits and allowed the elution of all the peaks (see Materials and Methods for method details). We also attempted to use gradient conditions to elute the TNFR subunits as sharper peaks, however this resulted in broader peaks and lower sensitivity in comparison to isocratic mode. This counterintuitive behaviour was confirmed in another study dealing with etanercept29 and can be explained by a possible precipitation/solubility issue of TNFR subunits under gradient conditions. These generic HILIC conditions were applied for the six different samples for which various enzymatic digestions were applied (Figure 2). Figure 2a shows the HILIC chromatogram obtained for the IdeS digested and DTT reduced etanercept sample. As predicted, since no deglycosylation was performed, this was the most complex and heterogeneous sample. During the first half of the chromatogram, various N-glycoforms of the Fc/2 fragment were successfully separated and identified by HRMS (mass range between 23 and 25 kDa). In the region between 11 and 16 minutes, a very broad peak was observed, due to overwhelmingly heterogeneous glycosylation, and found to correspond to different combinations of N- and O-glycoforms of the TNFR domain. In this part of the chromatogram, peaks were not baseline resolved. However, this broad peak was interrogated by HRMS (mass range between 30 and 34 kDa) in the form of 20 s time segments (vide infra). Figure 2b reports the analysis of the same sample (IdeS digested and DTT reduced etanercept) as the one in Figure 2a, but after applying a desialylation procedure. First of all, it is important to notice that some peaks related to the sialidase enzyme eluted between 6 and 7 minutes (black arrow on the chromatogram). However, they can easily be assigned by performing a blank injection of the enzyme and using MS detection. In this chromatogram, the peaks corresponding to Fc/2 fragment glycoforms were strictly identical to the ones reported in Figure 2a,

as the N-glycans on these fragments do not seem to contain sialic acids. However, the peak corresponding to the TNFR domains was much sharper since the heterogeneity on the remaining N- and O-glycans was significantly reduced (no more glycovariants related to the presence of sialic acids). In addition, its elution time was reduced, due to the decreased number of polar/acidic glycans.

Figure 2. Middle-up HILIC-HRMS analysis of etanercept. Total ion chromatograms of (a) IdeS digested and DTT reduced samples that were additionally (b) desialylated, (c) desialylated and O-deglycosylated, (d) desialylated and N-deglycosylated, (e) N-deglycosylated, and (f) fully deglycosylated. TNFR is represented in grey and the Fc/2 subunit in light blue. N-glycans and O-glycans are shown as blue epsilons and violet sticks, respectively. When desialylated, these glycans are shown in yellow. Arrows denote the presence of sialidase enzyme in the sample. See Tables S2-S7 for detailed retention times and mass assignments.

In Figure 2c, which corresponds to the desialylated and Odeglycosylated etanercept subunits, the variability on TNFR domain was further reduced, leading to a narrow peak that eluted


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Analytical Chemistry at around 11 minutes. Surprisingly, no separation of N-glycans was observed on the TNFR domains, although MS detection confirmed the presence of several N-glycoforms. This behaviour can be attributed to the presence of numerous inter-chain disulphide bridges (11) which modify the secondary structure of the TNFR part, thus limiting the accessibility of N-glycans to solvent as well as their potential hydrophilic interaction with the stationary phase. Figure 2d shows the HILIC chromatogram of the desialylated and N-deglycosylated etanercept subunits. Due to the absence of polar N-glycans, the retention of Fc/2 subunits was drastically reduced (to retention times of around 4-5 min). Regarding the TNFR domain, retention remained comparable for the desialylated O-deglycosylated TNFR sample (Figure 2c) and the desialylated N-deglycosylated TNFR sample (Figure 2d), while the peaks were quite sharp in both cases. With the desialylated N-deglycosylated TNFR sample, only the O-glycans remain a source of possible heterogeneity, but they do not contain any more sialic acid. In absence of sialic acids, O-glycans are even smaller in size (only 2 sugar units, see figure 1c) and highly comparable, so their effect on retention and peak shape is minor under HILIC conditions. Figure 2e highlights the chromatographic behaviour of the Ndeglycosylated etanercept subunits. As expected, the Fc/2 subunits behave in a same way as they did in Figure 2d. However, more variability and slightly higher retention was observed on the TNFR domains, compared to Figure 2d, due to the presence of the sialic acids on the O-glycans, and associated heterogeneity. This result also confirms that a large part of the variability observed in Figure 2a on the TNFR domains was related to the N-glycans rather than the O-glycans. Last but not least, Figure 2f shows the desialylated, O-deglycosylated and N-deglycosylated etanercept subunits. When removing all the N-glycans and O-glycans, retention in HILIC was drastically reduced, due to the lower polarity of both the Fc/2 and TNFR domains. Even in absence of glycans, a few peaks were still observed for the Fc/2 subunits and TNFR domains, which may correspond to other types of post-translational modifications, as reported in Table S7 (truncated amino acid sequences). Thanks to the appropriate subunits separation achieved by HILIC and the complementary information obtained by applying various digestion procedures, the MS peaks were more easily deconvoluted and structural information on the etanercept variants were gathered, with a QTOF/MS devices of limited resolution at the middle-up level of analysis, as reported in the next sections. Glycan characterization of the etanercept Fc/2 subunit. As previously discussed, peaks corresponding to different Fc/2 glyco-variants were well separated, which facilitated straightforward MS deconvolution and identification (Figure 3). First, the IdeS digested and DTT reduced sample (Figure 2a) was investigated to disclose the unmodified Fc/2 N-glycans distribution (Figure 3a). The set of peaks related to the Fc/2 subunits consisted of two main peaks and four minor peaks. By hyphenating HILIC with HRMS, the main peaks were identified as corresponding to fragments bearing the most abundant glycan species G0F and G1F. Less abundant species were also detected, such as afucosylated G0, M5, and G0-N, and fucosylated

G0F-N and G2F. No sialylated species were observed, in agreement with a previously reported analysis of released N-glycans.29 In addition, Fc/2 lysine variants were identified even if they were not separated under these HILIC conditions. Specifically, clipped lysine variants of the Fc/2 subunits were detected as major species, while Fc/2 subunits bearing a C-terminal lysine residue were found as minor variants (see Tables S2 for detailed mass assignment). This Fc/2 N-glycan profile obtained from an IdeS digested and DTT reduced etanercept sample was then compared to those of samples additionally treated with sialidase (Figure 2b) or N-glycosidase (Figure 2e). In the case of desialylated Fc/2 subunits (see Figure 3b and Table S3 for detailed assignments), no changes in the N-glycans pattern were detected, in accordance with the fact that no sialylated species have previously been observed in this subunit. Meanwhile, the N-deglycosylated Fc/2 subunit profile showed itself to contain only two main peaks corresponding to lysine variants (Figure 3c and Table S6 for detailed mass assignment).

Figure 3. Deconvoluted HRMS spectra of the main peaks related to the Fc/2 subunits of etanercept. N-glycosylated Fc/2 subunits eluting at 7.81 (ai), 8.20 (aii) and 8.69 (aiii) min; desialylated Fc/2 subunits eluting at 7.79 (bi), 8.18 (bii) and 8.67 (biii) min; and Ndeglycosylated Fc/2 subunits eluting at 4.46 (ci) and 4.71 (cii) min. Peaks assignments are illustrated by using shorthand glycan nomenclature alongside lysine clipping indicated as –K. See Tables S2, S3 and S6 for detailed mass assignments.

Glycan characterization of the TNFR subunit of Etanercept. Unlike the well-defined separation of the Fc/2 glyco-proteoforms, the glycosylation of the TNFR domains proved to be extremely challenging to resolve and baseline separation of each glyco-proteoforms could not be achieved. Nevertheless, orthogonal information were obtained by deconvoluting mass spectra segmented from this chromatographic region (Figure 4). The analysis of other enzymatically processed samples was even more informative. First, the analysis of the IdeS digested,



DTT reduced and fully deglycosylated etanercept sample (Figure 2f) enabled the confirmation of the subunit sequence identity, through the corresponding deconvolution of mass spectra from the peak related to the TNFR subunit (Figure 4a). Then, the TNFR glycosylation pattern was investigated step-by-step through the results obtained by the orthogonal middle-up approaches. By analysing the IdeS digested, DTT reduced, desialylated and O-deglycosylated etanercept subunits (Figure 2c), it was possible to identify the N-glycans occupying the TNFR domain, which were found to include G2F/G2 and G2F/G2F (Figure 4b). It should be noted that the sialidase pre-treatment required for effective O-deglycosylation unavoidably causes the simultane-

ous desialylation of N-glycans and thus biases their proper identification. Nevertheless, no other N-glycans were identified at this level of the analysis, and it is therefore reasonable to assume the presence of sialylated G2F and G2 N-glycans species. Similarly, the analysis of the IdeS digested, DTT reduced, desialylated and N-deglycosylated sample (Figure 2d) allowed the identification of the O-glycans occupying the TNFR subunit (Figure 4c). By removing the N-glycans and sialic acid residues, only core 1 O-glycans Galβ1-3GalNAc (C1) were left to remain on the TNFR subunit. The number of attached O-glycan cores could thereby be measured, and it was seen that the receptor was modified with 8 C1 up to 11 C1 O-glycans.

Figure 4. Deconvoluted HRMS spectra of the peaks related to the TNFR subunit of IdeS digested and DTT reduced samples additionally treated with (a) sialidase, PNGase F and O-glycosidase, (b) sialidase and O-glycosidase, (c) sialidase and PNGase F, (d) sialidase and (e) PNGase F. See Tables S2-S6 for detailed mass assignments.

Accordingly, the same distribution of 8 C1 to 11 C1 was found in combination with the G2F/G2 and G2F/G2F N-glycans in the deconvoluted spectra of the TNFR domain (Figure 4d) resulting from etanercept treated with IdeS, DTT and sialidase (Figure 2b). TNFR carrying G2F/G2 and 8 C1 O-glycans and TNFR carrying G2F/G2 and G2F/G2F N-glycans and 9 C1 to 11 C1 O-glycans were clearly identified by HRMS (Figure 4d). In addition, by separately deconvoluting the mass spectra from the two chromatographic peaks corresponding to the

TNFR subunit (Figure S2), it was possible to identify the first chromatographic peaks as corresponding to species carrying G2F/G2 and G2F/G2F N-glycans combined with 9 C1 O-glycans. Furthermore, the second chromatographic peak could be assigned to the same N-glycans combined with 10 C1 to 11 C1 O-glycans. The pre-peak shoulder in the separation could even be detected as being a G2F/G2 N-glycan composition combined with 8 C1 O-glycans (Figure 2b). So, although LC resolution was not sufficient to afford baseline separations, partitioning of


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Analytical Chemistry the protein subunits was still achieved as driven by the increasing number of glycan species decorating the TNFR. Next, the IdeS digested, DTT reduced and N-deglycosylated sample was analyzed (Figure 2e) and the unadulterated O-glycan profile of the TNFR subunit was investigated (Figure 4e). In accordance with the results obtained from the desialylated and N-deglycosylated sample (Figure 4c), an overall distribution of 8 C1 to 11 C1 O-glycans was found. Then, different combinations of mono- or di-sialylated C1 O-glycans (C1S1 or C1S2, respectively) were identified. Specifically, TNFR subunits bearing 8 C1 O-glycans were additionally sialylated with 10 to 11 Neu5Ac residues, while those with 9 C1 O-glycans contained 9 to 14 Neu5Ac residues. TNFR species with 10 C1 O-glycans were found to contain 9 to 16 Neu5Ac, and those with 11 C1 Oglycans contained 12 to 15 Neu5Ac residues (Figure 4e, see Table S6 for detailed mass assignments). By merging the information obtained by the above mentioned middle-up approaches, a list of theoretical TNFR glycoforms was generated along with their corresponding masses (Table S8). With this, it was considered that G2F/G2 and G2F/G2F would be the main N-glycan species and that 8 C1 to 11 C1 O-glycan cores could be sialylated with up 2 Neu5Ac residues. In this regard, and by assuming the virtual desialylation of the N-glycan moieties, only

1 hypothetical isomer among 5 possible combinations is taken into account (see Table S9 for comprehensive examples), without losing information on overall sialylation. In this context, IdeS digested and DTT reduced etanercept (Figure 2a) was investigated to elucidate the holistic glycosylation pattern of the TNFR subunit (Figure 5). To access this datarich chromatographic profile (between 12 and 14 min; Figure 2a), mass spectra were individually summed and deconvoluted in 20 sec segments (Figure 5). Six different glycoform scenarios were thus made tractable. Not surprisingly, the first two portions of the peak (Figure 5a-b) were mainly populated with TNFR subunits bearing 8 C1 to 9 C1 O-glycans. Meanwhile, the intermediate and final two portions of the peak (Figure 5cf) were found to correspond to TNFR subunits bearing either 9 C1 to 10 C1 or 10 C1 to 11 C1 O-glycans. Overall, the extent of the N-/O-glycans sialylation was found to range between 10 and 18 Neu5Ac (see Table S2 for detailed retention times and mass assignments). Therefore, as is consistent with the separation mechanism of HILIC, higher retention times corresponded to subunits bearing larger glycans moieties, as also highlighted by extracted ion chromatograms of selected glyco-proteoforms (Figure S3).

Figure 5. Deconvoluted HRMS spectra of the peak related to the TNFR subunit resulting from IdeS digested and DTT reduced etanercept. The peak region between 12 and 14 min was interrogated in the form of 20 sec segments (highlighted in yellow in side panels) and the summed mass spectra were independently deconvoluted (a-f). Peak assignments have been made by assuming desialylation of the N-glycans and reporting the overall number of core 1 type O-glycans and Neu5Ac residues (denoted as C1 and S, respectively). See Table S2 for detailed retention times and mass assignments.



CONCLUSIONS

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The goal of this study was to develop a HILIC-HRMS method employing a QTOF/MS instrument capable of providing detailed characterization of an Fc-Fusion protein, namely etanercept. For this purpose, a combination of simple and complementary enzymatic treatments were employed, including glycosidase, sialidase, and proteolytic digestions. With the combination of enzymatic digestion and HILIC-HRMS, it was possible to assess: i) the main PTMs (mostly C-terminal lysine clipping), ii) the subunit-specific distribution of glycans, and (iii) the overall N-/O-glycan composition and sialylation profiles of each subunit. This work clearly demonstrates the analytical potential of HILIC-HRMS to assess the complicated glycosylation characteristic of Fc-fusion proteins that could be evaluated as alternative to native MS performed with orbitrap technology. Obviously, this procedure can be applied to other types of Fc-fusion proteins, making it of broader appeal and benefit to the overall biopharmaceutical industry. In contrast to other assay approaches, this methodology provides a global overview of glycosylation patterns, which is a wealth of information useful for structure-function studies and of potential value for product comparability measurements and possibly even future manufacturing control strategies.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Description of the inherent structural heterogeneity of etanercept. Schematic representation of the N- and O-glycoforms commonly identified in etanercept. Etanercept amino acid sequence. Retention times and mass assignments of etanercept subunits upon orthogonal middle-up HILIC-HRMS analysis. Theoretical masses for TNFR subunits resulting from selected N- and O-glycan combinations. Example of possible TNFR isomers arising from different N- and O-glycans combinations (PDF).

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AUTHOR INFORMATION

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Corresponding Author * E-mail: [email protected] (22)

Author Contributions Valentina D’Atri and Lucie Nováková contributed equally to this work.

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Notes

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¶

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Davy Guillarme wishes to thank the Swiss National Science Foundation for support through a fellowship to Szabolcs Fekete (31003A_159494). Lucie Nováková wishes to thank the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15-003/0000465) co-funded by ERDF. Dwight Stoll acknowledges support from a Thought Leader Award from Agilent Technologies. Jean-Luc Veuthey from the University of Geneva is acknowledged for his useful comments and discussions.

REFERENCES (1)

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Page 8 of 9 2015, 33 (1), 155–164. Purple Book: Lists of Licensed Biological Products with Reference Product Exclusivity and Biosimilarity or Interchangeability Evaluations https://bit.ly/2oEPDqH (accessed Jul 19, 2018). Therapeutic Fc-Fusion Proteins; Chamow, S. M., Ryll, T., Lowman, H. B., Farson, D., Eds.; Wiley-Blackwell, 2014. Liu, L.; Gomathinayagam, S.; Hamuro, L.; Prueksaritanont, T.; Wang, W.; Stadheim, T. A.; Hamilton, S. R. Pharm. Res. 2013, 30 (3), 803–812. Borza, B.; Szigeti, M.; Szekrenyes, A.; Hajba, L.; Guttman, A. J. Pharm. Biomed. Anal. 2018, 153, 182–185. Parr, M. K.; Montacir, O.; Montacir, H. J. Pharm. Biomed. Anal. 2016, 130, 366–389. Gaunitz, S.; Nagy, G.; Pohl, N. L. B.; Novotny, M. V. Anal. Chem. 2017, 89 (1), 389–413. Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianférani, S. Anal. Chem. 2013, 85 (2), 715– 736. Wohlschlager, T.; Scheffler, K.; Forstenlehner, I. C.; Skala, W.; Senn, S.; Damoc, E.; Holzmann, J.; Huber, C. G. Nat. Commun. 2018, 9 (1), 1713. DiPaola, M. J. Bioanal. Biomed. 2013, 05 (05), 180–186. Houel, S.; Hilliard, M.; Yu, Y. Q.; McLoughlin, N.; Martin, S. M.; Rudd, P. M.; Williams, J. P.; Chen, W. Anal. Chem. 2014, 86 (1), 576–584. Hassett, B.; Singh, E.; Mahgoub, E.; O’Brien, J.; Vicik, S. M.; Fitzpatrick, B. MAbs 2018, 10 (1), 159–165. Schiestl, M.; Stangler, T.; Torella, C.; Čepeljnik, T.; Toll, H.; Grau, R. Nat. Biotechnol. 2011, 29 (4), 310–312. Montacir, O.; Montacir, H.; Springer, A.; Hinderlich, S.; Mahboudi, F.; Saadati, A.; Parr, M. K. Protein J. 2018, 37 (2), 164–179. Lamanna, W. C.; Mayer, R. E.; Rupprechter, A.; Fuchs, M.; Higel, F.; Fritsch, C.; Vogelsang, C.; Seidl, A.; Toll, H.; Schiestl, M.; Holzmann, J. Sci. Rep. 2017, 7 (1), 1–8. Regl, C.; Wohlschlager, T.; Holzmann, J.; Huber, C. G. Anal. Chem. 2017, 89 (16), 8391–8398. Strohl, W. R. Protein Cell 2018, 9 (1), 86–120. Cho, I. H.; Lee, N.; Song, D.; Jung, S. Y.; Bou-Assaf, G.; Sosic, Z.; Zhang, W.; Lyubarskaya, Y. MAbs 2016, 8 (6), 1136–1155. Deeks, E. D. BioDrugs 2017, 31 (6), 555–558. Matsuno, H.; Tomomitsu, M.; Hagino, A.; Shin, S.; Lee, J.; Song, Y. W. Ann. Rheum. Dis. 2018, 77 (4), 488–494. Hassett, B.; Scheinberg, M.; Castañeda-Hernández, G.; Li, M.; Rao, U. R. K.; Singh, E.; Mahgoub, E.; Coindreau, J.; O’Brien, J.; Vicik, S. M.; Fitzpatrick, B. MAbs 2018, 10 (1), 166–176. Periat, A.; Fekete, S.; Cusumano, A.; Veuthey, J.-L.; Beck, A.; Lauber, M.; Guillarme, D. J. Chromatogr. A 2016, 1448, 81–92. D’Atri, V.; Fekete, S.; Beck, A.; Lauber, M.; Guillarme, D. Anal. Chem. 2017, 89 (3), 2086–2092. Bobály, B.; D’Atri, V.; Beck, A.; Guillarme, D.; Fekete, S. J. Pharm. Biomed. Anal. 2017, 145, 24–32. D’Atri, V.; Fekete, S.; Stoll, D.; Lauber, M.; Beck, A.; Guillarme, D. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2018, 1080. Domínguez-Vega, E.; Tengattini, S.; Peintner, C.; van Angeren, J.; Temporini, C.; Haselberg, R.; Massolini, G.; Somsen, G. W. Talanta 2018, 184 (January), 375–381. Periat, A.; Kohler, I.; Bugey, A.; Bieri, S.; Versace, F.; Staub, C.; Guillarme, D. J. Chromatogr. A 2014, 1356, 211–220. Stoll, D. R.; Harmes, D. C.; Staples, G. O.; Potter, O. G.; Dammann, C. T.; Guillarme, D.; Beck, A. Anal. Chem. 2018, 90 (9), 5923–5929. Largy, E.; Cantais, F.; Van Vyncht, G.; Beck, A.; Delobel, A. J. Chromatogr. A 2017, 1498, 128–146.

Yu, K.; Liu, C.; Kim, B.-G.; Lee, D.-Y. Biotechnol. Adv.


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Analytical Chemistry

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Orthogonal Middle-up Approaches for the Characterization of the

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