
==== Front
Cell Rep Methods
Cell Rep Methods
Cell Reports Methods
2667-2375
Elsevier

S2667-2375(24)00207-8
10.1016/j.crmeth.2024.100834
100834
Article
A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format
Ortega-Rodriguez Uriel 1
Bettinger John Q. 1
Zou Guozhang 14
Falkowski Vincent M. 1
Lehtimaki Mari 1
Matthews Alicia M. 1
Biel Thomas G. 1
Pritts Jordan D. 1
Wu Wells W. 2
Shen Rong-Fong 2
Agarabi Cyrus 1
Rao V. Ashutosh 1
Xie Hang 3
Ju Tongzhong tongzhong.ju@fda.hhs.gov
15∗
1 Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA
2 Facility for Biotechnology Resources, United States Food and Drug Administration, Silver Spring, MD 20993, USA
3 Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA
∗ Corresponding author tongzhong.ju@fda.hhs.gov
4 Present address: Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA

5 Lead contact

07 8 2024
19 8 2024
07 8 2024
4 8 1008342 10 2023
10 5 2024
16 7 2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Summary

Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.

Graphical abstract

Highlights

• Establishes a single-workflow, one-pot glycomic method

• The one-pot method simultaneously profiles N- and O-glycans from glycoproteins

• The one-pot method measures relative abundances of permethylated N- and O-glycans

• The one-pot method reports the N- to O-glycan ratios in glycoproteins

Motivation

Glycosylation is known to impact the efficacy, half-life, stability, and safety of therapeutic glycoproteins, which define glycosylation as a critical quality attribute (CQA). The glycans on therapeutic protein drugs are controlled to ensure drug quality, lot-to-lot consistency, and bioequivalence of biosimilars to their reference products. Structural analysis of glycans is still challenging, as no universal method for the release of all major types of glycans exists. Traditional methods for structural N- and O-glycomics involve different procedures for glycan release, often in separate workflows, requiring two different samples and separate manipulation and analysis. Therefore, determining the relative quantity of N- to O-glycans on the same glycoprotein is challenging. We developed a one-pot glycomic method for simultaneous analysis of N- and O-glycans from the same sample and in a single workflow to facilitate the comprehensive assessment of glycosylation during the biomanufacture and release of protein-based drug products, which represents a significant advancement in the analytical assessment of N- and O-glycosylation of glycoprotein therapeutics.

Ortega-Rodriguez et al. develop a one-pot method to more comprehensively analyze complex sugar chains (glycosylation) of glycoproteins in a single workflow. This analytical method can be leveraged as a control strategy for glycosylation of therapeutic glycoproteins and biosimilars and can also facilitate investigation of functional glycomics.

Keywords

glycomics
biotechnology
glycan analysis
mass spectrometry
N-glycans
O-glycans
protein drugs
Published: August 7, 2024
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pmcIntroduction

Glycosylation is a highly diverse post-translational modification of proteins that can impact protein folding, sorting, activity, function, and stability.1 A comprehensive understanding of glycan structure is critical for uncovering the diverse functional implications of glycosylation. Regarding therapeutic proteins, glycosylation is often identified as a critical quality attribute (CQA) because glycans can impact therapeutic efficacy, stability, pharmacodynamic (PD) and pharmacokinetic properties, safety, bioavailability, as well as immunogenicity.2,3,4 Two major glycosylation types, N-glycosylation on Asn residues and mucin-type O-glycosylation of Ser or Thr residues, commonly exist on therapeutic proteins manufactured in mammalian host cell substrates, such as Chinese hamster ovary (CHO) cells, HEK293 cells, and murine cells. While monoclonal antibody (mAb) drugs usually contain N-glycans, fusion proteins such as etanercept5 and abatacept,3 hormones such as erythropoietin (EPO), enzyme replacement therapies, and other therapeutic proteins possess both N- and O-glycans. N-glycosylation of therapeutic proteins can directly impact their quality and safety, and major gaps remain in understanding of the role of O-glycans in safety, efficacy, and quality of several therapeutic proteins.

As O-glycans on cellular glycoproteins are known to be involved in many important biological functions, including homeostasis, cell adhesion, cell signaling, leukocyte trafficking, and immunity,1,6,7,8 O-linked glycosylation on biotherapeutics remains under investigated when compared to N-linked glycans.9 A few targeted studies have reported that O-glycans can affect immunogenicity10,11,12,13 and potency.5 Specifically, an O-glycovariant of etanercept engineered exclusively with sialyl-Tn and sialyl core 1 and O-glycans possessed increased tumor necrosis factor alpha (TNFα) binding affinity and cell-based potency.5 Investigation of O-glycosylation is still quite challenging due to the lack of a consensus sequence for O-glycosylation site determination, and there is no universal enzyme that is capable of cleaving all O-glycans.14 For N-glycan characterization studies, the peptide:N-glycosidase F (PNGase F) is commonly used to release all the mammalian N-glycans from the peptide backbone, and N-glycosylation site consensus sequences (sequons), Asn-Xxx-Ser/Thr (Xxx≠Pro), are known.15 The lack of feasible and specific methods to analyze O-glycans and relatively homogeneous sialyl core 1 O-glycans on glycoproteins produced from CHO cells are largely the cause of the undefined impact of O-glycans in therapeutic proteins on drug product safety and efficacy. The most common approach for release of O-glycans is reductive β-elimination, which is time consuming and has many limitations, including requirement of harsh basic conditions, which often leads to chemical degradation of glycans (i.e., peeling reactions).16 Although some enzymatic tools for O-glycan release are available, O-glycanases or endo-α-N-acetylgalactosaminidases, which belong to the GH101 family, reported thus far have restricted specificity to only core 1 and core 3 disaccharides.17,18,19,20 As the most common O-glycans on glycoproteins are sialylated core 1, core 2, or core 3 based, the available O-glycanases and traditional β-elimination method are not practically useful for characterizing O-glycans or monitoring them to ensure drug quality.

The structural characteristics of both N- and O-glycans, e.g., sialylation and exposed β-galactose (terminal β-Gal), can be carefully characterized with a comprehensive relative N- and O-glycan ratios to support and improve drug product safety, efficacy, and quality. However, current methodologies for structural characterization of glycans often require separate workflows and analysis of N- and O-glycans in separate experiments, which is not adequate to perform quantitatively comparative analysis. In our previous etanercept O-glycovariant study, we employed an early rendition of our one-pot glycomic approach to structurally characterize N- and O-glycans from several O-glycovariants of etanercept in tandem.5 Here, we describe in detail an optimized methodology for profiling N-and O-glycans in a one-pot experimental format. We demonstrate the reproducibility and applicability to assess N- to O-glycan profiles, their ratio, and lot-to-lot comparability of complex Fc-fusion proteins and we discuss major challenges in streamlining the procedure to allow feasible and practical applications across therapeutic glycoproteins. We also discuss considerations on the applicability of the one-pot method to analysis of cellular glycoproteins from cell extracts.

Results

Qualification of quantifying permethylated glycan standards on MALDI-TOF/MS

To validate the quantitative nature of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the analysis of N- and O-glycans, we employed a series of experiments using purified glycan standards as reference material. First, N- and O-glycan standards were mixed in a priori known amounts and the glycan ratios were measured by MALDI-TOF analysis after permethylation (Figures 1A–1D). We found that individual glycan ratios measured by MALDI-TOF (Figure 1A) strongly correlate with theoretically anticipated results (r = 0.94; Figure 1E) based on the amount of each glycan standard reported on the provided labels. Nonetheless, there were minor disagreements between the measured and anticipated glycan ratios. Discrepancies between measured and anticipated results could have many sources, including sources that are intrinsic to the method (i.e., differences in ionization efficiencies between glycans) or intrinsic to the reference material (i.e., errors in the labeled amounts of each glycan standard). We therefore sought to further validate the quantitative nature of MALDI-TOF/MS compared to 2-aminobenzamide (2-AB) labeling and ultra-high-performance liquid chromatography fluorescence (UHPLC-FLR) analysis (Figure 1B), an industry standard for the quantification of glycans. Not only are the results obtained by MALDI-TOF/MS better correlated with the anticipated results (Figures 1C and 1E), especially for the less-abundant glycans (MALDI-TOF r = 0.94, 2-AB UHPLC-FLR r = 0.84; Figure 1E), but there is also a strong correlation between the results obtained by the two methods (r = 0.91). In addition, the mean precision between the two methods is not significantly different from one another (Figure 1D). Taken together, these results indicate that MALDI-TOF/MS analysis of complex mixtures performs at least on par with industry standard methods.Figure 1 Qualification of permethylated glycan standards on MALDI-TOF/MS and 2-AB UHPLC-FLR

(A) Representative MALDI-TOF spectra of permethylated glycan standards.

(B) Representative UHPLC-FLR chromatogram of 2-AB-labeled glycan standards.

(C) Comparison of glycan abundances (mean ± SD) determined by MALDI-TOF (blue) or 2-AB UHPLC-FLR (red) to theoretical ratios of mixed glycans (green).

(D) Distribution of SD of glycan standards determined by MALDI-TOF (blue) or 2-AB UHPLC-FLR (red); a t test was performed in GraphPad Prism to compare the SD values for glycan abundances.

(E) Correlation between theoretical relative abundance (x axis) and measured glycan abundances (y axis) for MALDI-TOF (red) and 2-AB UHPLC-FLR (blue). Data are represented as mean ± SD.

We note, however, that accurate quantification of true glycan ratios is a traditionally difficult task and often requires the addition of internal standards or “tune mixes.”21,22 Because the generation and validation of internal standards can often be time and resource intensive, it is commonly more useful to rely on relative quantifications between samples (i.e., the up- or downregulation of particular glycans between production runs). To make accurate measurements of relative abundances between samples, it is necessary that a method be precise and that changes in the magnitude of the readout proportionally reflect changes in the abundance of each analyte. We have demonstrated the former using mixtures of glycan standards above. To demonstrate the latter, standardized response curves for three N-glycans and one O-glycan were generated. The responses for each glycan tested demonstrated good linearity (Figure S1A). Importantly, N- and O-glycans have different isotope distributions (Figure S1B), which should be considered in calculating their abundance, as discussed in a later section. Furthermore, the standardized slopes for the four glycans tested do not significantly differ from one another (Table S1). Therefore, these results suggest that shifts in MALDI-TOF/MS signal intensity are equally proportional to shifts in glycan abundance for different glycan species. These data demonstrate that MALDI-TOF/MS is suitable for the accurate measurement of glycan up- and downregulation between samples.

Simultaneous analysis of N- and O-glycans of same glycoprotein samples by incorporating current N-glycan and O-glycan procedures

The main goal for this study was to establish a reproducible and robust method for simultaneous analysis of N- and O-glycans on protein drugs from the same aliquot of sample. To initiate and optimize the methodology, bovine fetuin was selected as the model glycoprotein. Bovine fetuin is an α2-HS-glycoprotein that bears predominantly large bi- and tri-antennary complex N-glycans at three N-glycosylation sites and a mixture of sialylated core 1 and core 2 O-glycans reportedly at six O-glycosylation sites (Table S2), which provided an overall anticipated glycan profile during method development.23,24 The established workflow for one-pot glycan profiling is depicted in Figure 2A. In brief, denatured and reduced glycoproteins were alkylated and then transferred to a 30-kDa molecular-weight cutoff filter device for filter-aided sample preparation (FASP) in preparation for de-N-glycosylation by PNGase F. O-glycopeptides were then enzymatically generated by nonspecific proteolysis with proteinase K on the filter, and finally small peptides, N-glycans, and O-glycopeptides were isolated through the filter by centrifugation. After selective removal of peptides by hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) cartridges, the enriched N-glycan/O-glycopeptide mixture underwent reduction and permethylation in preparation for analysis by MALDI-TOF/MS.Figure 2 Optimization of the one-pot method for simultaneous analysis of N- and O-glycans of glycoproteins

(A) Workflow of one-pot method.

(B) Differentiation of permethylated N- and O-glycans by unique reducing ends.

(C) One-pot N- and O-glycan profiling of a model glycoprotein fetuin.

(D) Summary of glycans identified in six reactions of fetuin plotted by % abundance (mean ± SD).

(E) N- to O-glycan ratio of fetuin (top right) and abundance of the total sialylated N- and O-glycan species of fetuin (bottom right). Data are represented as mean ± SD.

To isolate N-glycans and O-glycopeptides in tandem, a commercial FASP protein digestion kit designed for universal sample preparation for peptide analysis by MS was utilized. The FASP approach is compatible with a broad range of biological sample types, including purified proteins, whole-cell lysates, and tissue samples.25,26,27 A FASP-based approach has been described for isolation and separation of N-glycans (FANGS) and remains a current method for N-glycan profiling of cell substrates, complex glycoprotein mixtures, and experimental protein therapeutics.28,29,30

Several SPE approaches were investigated to remove native peptides and selectively enrich N-glycans and O-glycopeptides after FASP filtration. Active charcoal was selected initially based on its performance for purification of neutral and acidic N-glycans from previous studies.28,30,31 To assist in recovery of both N-glycans and O-glycopeptides, the concentration of acetonitrile (ACN) was increased from 30% to 50% can, which is frequently used for N-glycan purification.28,31,32 This change was to ensure a full recovery of O-glycopeptides while eliminating most of the non-glycosylated peptides. An increase in low-mass peptide contaminants was also observed when the organic concentration was increased, which complicated the spectra in the O-glycan mass range (Figure S2A). Alternative approaches were evaluated due to this observation, including a combination of C18 and active charcoal in which the FASP filtrate was acidified and passed through C18 cartridges to separate out native peptides or non-glycosylated peptides, followed by enrichment of N-glycans/O-glycopeptides by active charcoal. However, hydrophilic N-glycans were not retained on the C18 cartridge, and a portion of the O-glycopeptides were retained and required an additional elution step with 10%–20% ACN. Separate elution steps did not meet the conditions for avoiding separation of N-glycans and O-glycopeptides/O-glycans required to facilitate relative quantification of total glycosylation. Instead, aminopropyl (NH2) SPE cartridges were chosen based on its capabilities to efficiently remove native peptide contaminants while achieving full recovery of N-glycans and O-glycopeptides in HILIC mode.

Optimization of the mobile phases determined the use of 0.1% trifluoracetic acid (TFA) as a pairing agent to enhance recovery of acidic glycans and glycopeptides that were lost completely using a mobile phase without TFA (Figure S2B). It is possible that negatively charged acidic glycans are expelled from the stationary phase by electrostatic repulsion from negatively charged hydroxy groups within the silica. The addition of TFA may neutralize charged silica and acidic glycans by removing electrostatic repulsion. This SPE medium also has a weak anion exchange property, and TFA is necessary to elute acidic glycans.

After successful separation of N-glycans and O-glycopeptides from native peptides, the mixture was reduced with ammonia borane prior to permethylation. This step facilitates differentiation of N- from O-glycans by unique reducing-end reduced fragments obtained during fragmentation by tandem MS. A unique mass shift of 16 Da after reduction and permethylation will be present only in N-glycans by tandem mass spectra and the signature fragment of sodiated and reduced hexosamine (HexNAc) with 316.2 Da, and 490.1 Da fragment for reduced N-Acetylglucosamine (GlcNAc) with core Fucose will be present in the MS2 (Figure 2B), respectively. A chemoenzymatic approach as described by Goetz et al. in which O-glycans are released by permethylation of short O-glycopeptides was chosen to simplify the O-glycan release process while maintaining a single workflow.16 This approach is considered superior to traditional chemical O-glycan release methods because it greatly reduces undesired peeling reactions that can compromise the recovery of intact O-glycans.16

With the optimized procedures, three representative MS spectra of O- and N-glycans, each from the same fetuin sample, are shown in Figure 2C. The O-glycan species include mono-sialyl core 1, di-sialyl core 1 and di-sialyl core 2 O-glycans corresponding to the peak of m/z of 879.4, 1,240.6, and 1,689.8, respectively, with the mono-sialyl core 1 structure as the major O-glycan. The N-glycan species include mono-sialylated biantennary structure (m/z 2,447.2), di-sialylated bi-antennary structure (A2G2S2, m/z 2,808.4), di-sialylated tri-antennary structure (m/z 3,257.8), and fully tri-sialylated tri-antennary structure (A3G3S3, m/z 3,618.8), as well as tetra-sialylated tri-antennary structure (A3G3S4, m/z 3,980.0), with A2G2S2 and A3G3S3 as the dominant structures. Glycan relative abundances were calculated by their corresponding peak intensity (the sum of peak areas of isotopic masses) using AssignMALDI.33 AssignMALDI calculates the abundance of glycans by the sum of intensities across the entire isotopic range of glycan species. Such a feature is critical for analyzing N- and O-glycans in the same mixture, as both classes of glycans can differ in isotope distributions (Figures S1B).

The same N- and O-glycan structures were detected using conventional glycan methods (Figures S3A and S3B). More importantly, the calculated glycan relative quantity/abundance was similar between conventional methods and the one-pot format (Figure S3C). The major observed glycan species from both methods were also in agreement with previously reported results.16,29,34 Furthermore, each of the assigned glycan structures from the one-pot format had consistent relative abundance (%) from six repeated runs (Figure 2D). The calculated ratio of N-glycans (∼70%) to O-glycans (∼30%) was ∼7:3, based on the abundances calculated by AssignMALDI, which indicates that N-glycan occupancy is higher than O-glycans (Figure 2E, top panel). Because N-glycosites tend to be more completely occupied compared to O-glycosites, which have variable and sometimes low occupancies, an N:O-glycan ratio of 7:3 was not surprising, also due to the contamination of other N-glycoproteins in the commercial fetuin sample. To better understand this observation, we sought to map the number of O-glycosites on bovine fetuin. O-glycoproteomic analysis revealed six sites of O-glycosylation on bovine fetuin (Table S2, and Supplemental Material_O-Glycopeptide Mapping_CR-METHODS-D-23-00314.psmtsv.xlsx), consistent with what is reported in the Uniprot database but greater than what has been previously reported.23,35 It was not possible to estimate occupancies for each glycosite in our analysis due to the heterogeneity at each site. Given that bovine fetuin contains six O-glycosites and only three N-glycosites, it is feasible that the true N:O-glycan ratio on fetuin is close to the measured amount of 7:3.

Next, the total sialylation was calculated by the sum of the abundance of glycan species bearing at least one sialic acid residue divided by the summed abundance of all glycan species to reveal that 100% of all detected species are sialylated (Figure 2E, bottom panel). Taken together, these data establish an optimized method for comprehensive N- and O-glycan profiling from the same glycoprotein sample in a one-pot format.

Applicability of method to analyze N- and O-glycans of Fc-fusion proteins drugs: Etanercept, abatacept, and aflibercept

To demonstrate the capabilities of the one-pot method in routine glycan profiling of protein therapeutics, we processed three commercial Fc-fusion protein drug products (abatacept, etanercept, and aflibercept), which were manufactured in CHO cells under good manufacturing practices (GMPs). The MALDI-TOF mass spectra revealed two major sialylated core 1 O-glycan species, mono-sialyl core 1 (m/z 879.4) and di-sialyl core 1 (m/z 1,240.6) from abatacept and etanercept, but no detectable O-glycan peaks were present in aflibercept (Figure 3A). Based on high-resolution liquid chromatography tandem mass spectroscopy (HR-LC-MS/MS) of the mono-sialyl core 1 O-glycan species, the species were confirmed as a linear sialyl core 1 O-glycan, e.g., α2,3 sialyl core 1, Neu5Acα2,3Galβ1,3GalNAc-OH by the presence of two signature ions (m/z 504.24, 486.23), which are only possible by mono-substitution of the internal HexNAc residue (Figure S4, top panel). All three fusion protein drugs had mainly neutral biantennary complex N-glycans, and a minor fraction of high-mannose N-glycan, Man5 (M5) (m/z 1,595.8) only present in etanercept and aflibercept. Specifically, N-glycans commonly found on CHO-produced immunoglobulin (Ig) G1-Fc (FA2, FA2G1, FA2G2) were detected as peaks with m/z 1,851.9, 2,056.0, and 2,260.1, respectively. The sialylated complex biantennary N-glycan species, FA2G1S2 (m/z 2,417.2), A2G2S1 (m/z 2,621.3), and FA2G2S2 (m/z 2,982.4) were also identified in all three glycoprotein drugs with different relative abundance. Interestingly, A2G2S1 (m/z 2,447.1) and M5 (m/z 1,595.8) were only present in etanercept and aflibercept but were not detectable in abatacept. Trace tri- and tetra-antennary N-glycans such as FA3G1S1, FA3G2S1, FA3G3S1, FA3G2S2, FA3G3S2, FA3G3S3, and FA4G4S2 were notably identified in abatacept. The relative abundance (%) of glycan species was calculated from three preparations of each glycoprotein drug and then compared (Figure 3B). In total, O-glycans accounted for ∼7% and ∼12% of total glycosylation in abatacept and etanercept, respectively, which translates to an N- to O-glycan (N:O) ratio of 13.3:1, and ∼11:1.5, respectively (Figure 3B, top right). This is the first direct quantitative measurement of N- versus O-glycosylation of these drugs, which has not been reported in any previous studies.36,37Figure 3 Analysis of Fc-fusion glycoproteins by one-pot glycomic method and test of reproducibility with etanercept

(A) MALDI-TOF/MS spectra of N- and O-glycans of Fc-fusion proteins: abatacept, etanercept, and aflibercept (n = 3).

(B) Summary of unique glycan abundance found in abatacept, etanercept, and aflibercept followed by the N- and O-glycan ratio of each glycoprotein and total % sialylation of N- and O-glycans combined.

(C) Summary of the reproducibility of one-pot method using the data from 22 reactions of etanercept lot 1 with the abundance of glycan species identified in etanercept.

(D) N- and O-glycan ratio.

(E) Summary of abundance of total sialylated and asialylated glycan species; abundance of sialylated N- and O-glycan species is shown in the upper panel. Other glycan determinants include N-glycan fucosylation, total sialylation, and abundance of mannose receptor (MR) and asialoglycoprotein receptor (ASGPR) target glycans, summarized in the lower panel. Data are represented as mean ± SD.

Sialic acid content is likely a CQA due to its correlation with pharmacokinetics (PK) and elimination rate of glycoproteins, so asialylated N-glycan content is routinely determined and controlled.38 Unlike fetuin, which bears predominantly sialylated glycan species (Figure 2), sialylated glycan species account for ∼50%, ∼48%, and 35% of total glycosylation in abatacept, etanercept, and aflibercept, respectively (Figure 3B bottom right). This observation was expected as neutral N-glycan species frequently occupy the N-glycosylation site at the Fc within the CH2 domain and are rarely extended or sialylated due to reduced accessibility by glycosyltransferases in catalyzing this process.39 Overall, the individual N-glycosylation and O-glycosylation data were consistent with the published studies.5,36,37 Taken together, these data demonstrate that the one-pot method can reliably profile N- and O-glycans of therapeutic glycoproteins simultaneously and determine the N- and O-glycan ratios.

Reproducibility of the one-pot method

Analytical procedures for biologics must meet proper reproducibility standards as described in the International Conference on Harmonization (ICH) document Q2(R1) and the US Food and Drug Administration (FDA) guidance for industry on analytical procedures and methods validation for drugs and biologics (https://www.fda.gov/media/87801/download). Multiple runs of the one-pot glycan analyses using the same lot of etanercept (lot 1) were performed to validate the reproducibility of the method. The data from 22 individual glycan preparations of etanercept performed across multiple weeks were analyzed and combined as a summary in Figure 3C. The relative abundance of each glycan species identified in etanercept was similar to the data described in Figure 3B, which was performed in a separate experiment; however, the etanercept in this experiment was processed immediately after dispensing fresh drug product from the autoinjector, while the previous lot tested had been frozen at −80C for long-term storage. The standard deviation (SD) and coefficient of variation (CV) values for each glycan species identified are presented in Table 1. Two major glycan species, FA2 (mean, 32.8%; SD, ±5.9%) and FA2G1 (mean, 16.7%; SD, ±1.97%), which account for 49.4% of the entire glycan profile, showed the most reproducibility based on CV range of 11.8%–17.9% (Table 1). Glycan species between a range of 4.8% and 14.1% abundance, FA2G2S1 (mean, 14.1%; SD, ±4.3%), mono-sialyl core 1 (mean, 13.6%; SD, ±3.95%), A2G2S1 (mean, 9.3%; SD, ±2.8%), and FA2G2 (mean, 4.8%; SD, ±1.4%) had a CV range of 29.0%–30.1% (Table 1). Minor species below 3.1%, such as M5 (mean, 3.1%; SD, ±1.7%), di-sialyl core 1 (mean, 1.8%; SD, ±0.86%), FA2G1S1 (mean, 1.6%; SD, ±1.12%), A2G2 (mean, 1.1%; SD, ±0.6%), FA2G2S2 (mean, 0.6%; SD, ±0.5%), A2 (mean, 0.5%; SD, ±0.3%), A2G1(mean, 0.2%; SD, ±0.2%), and A2G2S2 (mean, 0.1%; SD, ±0.1%), showed the least consistency as the CV ranged from 52.9% to 175.2%. In these cases, the low intensity of these glycans contributed greatly to the variance, while the range of SD was still rather tight between 0.1% and 1.7%. Consequently, consistent with previous data (Figure 3B), total O-glycans accounted for an average of 15.4% ± 4.1% of total glycosylation and the N- to O-glycan ratio was determined as ∼8.5:1.5 N:O (Figure 3D). These data demonstrate that the one-pot method is highly reproducible in determining the relative abundances of N- and O-glycans from the same sample.Table 1 Summary of N- and O-glycans and their relative abundances identified in lot 1 of etanercept (n = 22)

Glycan structure	m/z [M + Na]+	Mean (%)	SD	CV	
Mono-sialyl core 1	879.4	13.61%	±3.95%	29.0%	
Di-sialyl core 1	1,240.6	1.80%	±0.86%	47.6%	
M5	1,595.8	3.05%	±1.68%	55.1%	
A2	1,677.8	0.45%	±0.29%	63.8%	
FA2	1,852.0	32.75%	±5.86%	17.9%	
A2G1	1,881.9	0.21%	±0.17%	85.1%	
FA2G1	2,056.1	16.66%	±1.97%	11.8%	
A2G2	2,086.1	1.10%	±0.58%	52.9%	
FA2G2	2,260.2	4.80%	±1.42%	29.5%	
FA2G1S1	2,417.2	1.57%	±1.12%	71.5%	
A2G2S1	2,447.2	9.29%	±2.79%	30.0%	
FA2G2S1	2,621.3	14.08%	±4.24%	30.1%	
A2G2S2	2,808.4	0.07%	±0.13%	175.2%	
FA2G2S2	2,982.5	0.55%	±0.49%	89.3%	

Determining the limit of quantitation (LOQ) is difficult without a calibration curve containing defined concentrations of analytes, and the lack of standards of each glycan species at different dilutions presents a challenge. The variance of glycans at ≤3.1% abundance was ≥55.1% and glycans ≤0.5% such as A2G1 and A2G2S2 had even higher CVs of 85.1% and 175.2%, respectively (Table 1). Based on these data, the estimated LOQ of the one-pot method is about 3% of relative abundance when using 50 μg of glycoprotein starting material. As the MS detection is very sensitive, our data also demonstrated that the limit of detection (LOD) of the one-pot method is about 0.1% relative abundance of glycans.

Taken together, tight SDs between ±0.1% and 5.9% were observed for all 14 glycan species identified. Specifically, major glycan species with average abundance above 10% had the lowest variance among all 22 reactions. These data support that the reproducibility of the one-pot method in profiling and quantifying glycan species and their structural characteristics has the potential to be an analytical procedure for the control of N- and O-glycans on glycoprotein drug products.

Quantification of sialylated versus neutral glycans relevant to a drug’s potential PK

Glycosylation on drug products can impact PK and mAb effector functions.2 Figure 3E summarizes the glycan sialylation, core fucosylation of N-glycans, and abundance of potential glycan determinants that can target therapeutic glycoproteins, such as etanercept, for clearance by mannose receptor (MR) and asialoglycoprotein receptor (ASGPR). Regarding sialylation of etanercept, 59% ± 8.4% of all glycans were asialylated and 41% ± 8.4% of all glycans carried at least one sialic acid residue. O-glycans accounted for 15.4% ± 4.1% of the total profile, while only 25.6% ± 8.0% of N-glycans were sialylated (Table 1).

Like most protein therapeutics manufactured in CHO cell substrates, core fucosylated N-glycan species were more abundant than their afucosylated counterparts, accounting for 83.3% ± 2.5% and 16.7% ± 2.5% of total N-glycans, respectively (Figure 3D). Core fucosylation was confirmed by the presence of protonated signature ions consistent with reduced and fucosylated HexNAc residues (m/z 468.3, m/z 450.3) by collision-induced dissociation (CID) in our HR-LC-MS/MS experiments (Figure S4, bottom panel). In this study, no evidence of fucosylated O-glycans was obtained in any Fc-fusion proteins produced from CHO cells, which mainly synthesize mono- and di-sialyl core 1 O-glycans.5

The relative abundance of glycans bearing terminal GlcNAc or mannose (M5, A2, FA2) and galactose residues (A2G1, FA2G1, A2G2, FA2G2), which are respective targets for selective uptake and clearance by MR and ASGPR, is summarized in Figure 3E (bottom right panel). MR-target glycans accounted for 36.3% ± 6.9% and ASGPR-target glycans accounted for 22.8% ± 2.6% compared to 41% ± 8.4% sialylated glycans. Although 59.1% total glycans (36.3% MR + 22.8% ASGPR) are potentially critical to the PK, these neutral N-glycan species were predominantly located in the Fc domain and less accessible to MR and ASGPR than those within the functional domain, TNFR2 (Figure S5). Specifically, four major neutral N-glycan species were identified in the Fc domain, including M5 (m/z 1,595.8), FA2 (m/z 1,851.9), FA2G1 (m/z 2,056.0), FA2G2 (m/z 2,260.2), and other minor neutral species (Figure S5, bottom panel). While three low-abundance sialylated species (A2G1S1, m/z 2,243.1; A2G2S1, m/z 2,447.2; FA2G2S1, m/z 2,621.3) were found in the Fc domain, most complex N-glycan species were localized in the TNFR2 domain (Figure S5, middle panel) with some di-sialylated glycans being exclusive to the TNFR2 domain, including A2G2S2 (m/z 2,808.4) and FA2G2S2 (m/z 2,982.5). Interestingly, the TNFR2 domain was also found to be differentially O-glycosylated (Figure S5). The two major O-glycan species mono- and di-sialyl core 1 (m/z 879.3 and m/z 1,240.6 respectively) were predominantly found in the TNFR2 domain, and only a minor peak of mono-sialyl core 1 was detected in the Fc domain, most likely found in T225 of the Fc hinge region, which was reportedly O-glycosylated in fusion proteins.37,40 These data are consistent with other studies showing that etanercept and other Fc-fusion proteins are differentially sialylated based on accessibility to exposed N-glycosylation sites on the receptor domain in contrast to the Fc-glycosylation site in which N-glycans are typically buried within the two heavy chains.37,38 Furthermore, sialylation of N-glycans within the functional domain will enhance half-life through reduced binding with ASGPR, which can be reduced by a single sialic acid residue.38,39

Lot-to-lot comparability of N- and O-glycans within three lots of etanercept

The N- and O-glycans of etanercept from three different lots in triplicate were analyzed to further demonstrate the applicability and repeatability of the one-pot glycomic method in the context of drug substance and product release testing. MALDI-TOF/MS analysis of each lot revealed 14 glycan species in agreement with our previous reproducibility data, which were derived from lot 1 (Figure S6). In all three lots, the most abundant N-glycan species identified was FA2 (m/z 1,852.0), which accounted for 30.6% ± 3.6% (lot 1), 27.1% ± 6.8% (lot 2), and 23.7% ± 4.0% (lot 3) of total glycans, respectively (Figure 4A) with a variance of 19.4% among three lots (mean, 27.2%; SD, 5.3%) (Table S3). In all three cases, the most abundant O-glycan species detected was mono-sialyl core 1 (m/z 879.4), which accounted for 15.6% ± 0.3% (lot 1), 14.1% ± 1.7% (lot 2), and 10.8% ± 2.4% (lot 3), respectively, with a variance of 19.2% among all three lots (Table S3). Kruskal-Wallis test with Dunn’s multiple comparisons was used to identify statistically significant lot-to-lot glycosylation differences. From an individual glycan standpoint, lot 1 A2G2 (p = 0.0219), and FA2G2 (p = 0.0219) were significantly different compared to lot 3 (Figure 4A). All other glycan species identified were not statistically different between lots. Calculation of the total N- and O-glycan abundance (Figure 4B, top left panel) revealed some minor differences among different lots: total O-glycan abundances were 18.3% ± 0.2% for lot 1, 15.8% ± 2.1% for lot 2; and 16.5% ± 2.97% for lot 3; total N-glycan abundances were 81.7% ± 0.2% for lot 1, 84.2% ± 2.1% for lot 2, and 83.5% ± 2.97% for lot 3. However, these minor differences were not statistically significant by Dunn’s multiple comparisons test. The overall N- to O-glycan (N:O) ratios were close to 8.5:1.5 as reported previously in lot 1 as (lot 1, 8.1:1.;8 lot 2, 8.4:1.6; and lot 3, 8.3:1.7) for all three lots. Importantly, such information is not possible to obtain with conventional approaches in which N- and O-glycans are processed separately. A one-pot format thus allows an appropriate setting for direct comparison of total N- and O-glycosylation.Figure 4 Lot-to-lot comparability with three lots of etanercept

(A) Comparison of relative abundance of glycan species detected in three lots of etanercept.

(B) Lot-to-lot comparison of etanercept in N- and O-glycan ratio, total and N- and O-glycan specific sialylation and fucosylation status of N-glycans, and abundance of MR and ASGPR target glycans. Data are represented as mean ± SD. Non-parametric analyses; Kruskal-Wallis and Mann-Whitney tests were performed at alpha level of 0.05 to study glycosylation differences between three lots; ∗p < 0.05.

In all three lots, 45.5%–49% of all glycans identified were sialylated (lot 1, 49% ± 5.6%; lot 2, 46.2% ± 7.6%; lot 3, 45.5% ± 1.8%); N-glycans accounted for the majority of sialylated species detected (lot 1, 30.7% ± 5.8%; lot 2, 30.4% ± 8.1%; lot 3, 29.0% ± 2.8%) (Figure 4B, top right panel). In all cases, asialylated N-glycan species (lot 1, 51.0% ± 5.7%; lot 2, 53.8% ± 7.6%; lot 3, 54.5% ± 1.8%) were more abundant than their sialylated counterparts, which was consistent with the results in Figure 3. While these data indicate variability of sialylated glycans between different lots of etanercept, the differences reported were not statistically significant by Dunn’s multiple comparisons test.

Regarding core fucosylated N-glycans, consistent with the earlier results of etanercept in Figure 3, the majority of N-glycans identified in all three lots were core fucosylated: lot 1, 83.7% ± 1.6%; lot 2, 81.3% ± 2.0%; lot 3: 82.5% ± 2.0%, with a variation of 3.9% between these three lots (mean, 82.5%; SD 1.2%; CV, 1.4%) (Figure 4B, bottom left panel). Afucosylated N-glycans made up a minor fraction of total glycosylation from a range of 16.3%–18.7% (lot 1, 16.3%; lot 2, 18.7%; lot 3, 17.5%) with a variation of 6.7% (mean, 17.5%; SD, 1.2%; CV, 6.7%) among these three lots. These data indicate some variability in core fucosylation of N-glycans between lots of etanercept; however, the results of the Dunn’s multiple comparisons test determined that these differences were not statistically significant (Figure 4B, bottom left panel).

Potentially critical glycan species bearing terminal GlcNAc, mannose, and galactose residues were summarized to indicate the abundance of MR- and ASGPR-target glycans in comparison to sialylated species in each lot (Figure 4B, bottom right panel). MR-target glycans bearing terminal GlcNAc or mannose (M5, A2, FA2) accounted for 28.8%–31.4% of total glycan species (lot 1, 31.4% ± 3.7%; lot 2, 28.8% ± 6.9%; lot 3, 28.8% ± 2.3%) and ASGPR-target glycans (A2G1, FA2G1, A2G2, FA2G2) made up roughly 19.6%–25.7% of the glycan species (lot 1, 19.6% ± 2.1%; lot 2, 224.9% ± 1.5%; lot 3, 25.7% ± 2.7%). There were no statistically significant differences in the glycans observed between the lots as determined by non-parametric Kruskal-Wallis with Dunn’s multiple comparisons test (Figure 4B, bottom right panel). The tight SD of relative abundances in the major glycan species within the same lots of etanercept further demonstrated the repeatability of the method. Taken together, these data demonstrate the applicability of the one-pot glycomic method for profiling and quantifying N- and O-glycan species and glycan structural attributes to assess lot-to-lot glycan comparability of therapeutic glycoprotein drugs.

Intermediate precision of the method

To evaluate the intermediate precision of the method, two analysts (analysts 1 and 2) performed the one-pot glycan method using etanercept lot 1 and analyzed the sample in triplicate. Prior to MALDI-TOF/MS analysis, each analyst took measurements of 2,5-dihydroxybenzoic acid (2,5-DHB) matrix alone to confirm that no glycan peaks were present in the matrix prior to glycan analysis (Figure 5A). Etanercept samples yielded peaks consistent with the mass of 14 glycan species described in analysis of lot 1 (Figures 3 and 4). As previously determined, the major species identified was FA2 (A1 mean, 31.0% ± 3.4%; A2 mean, 18.8% ± 3.7%) with a variance of 29.7% (mean, 24.9% ± 7.4%) between both analysts (Table S4). The major O-glycan species identified was sialyl core 1 (A1 mean, 15.7% ± 0.3%; A2 mean, 12.8% ± 2.4%), which had a variance of 15.7% (mean, 15.7% ± 2.2%) between both analysts. Regarding the N:O-glycan ratio (Figure 5B, top left), the abundance of O-glycans (A1 mean, 18.6% ± 1.1%; A2 mean, 17.1% ± 2.9%) and N-glycans (A1 mean, 81.6% ± 0.3%; A2 mean, 83.9% ± 3.4%) was consistent between both analysts, with variance of 5.7% and 2.4%, respectively. When quantifying sialylated species (Figure 4B top right), A1 determined that 48.6% ± 5.6% of glycans were sialylated compared to 59.4% ± 6.4% from A2 with a variance of 14.2% between both analysts. The differences between sialylated and asialylated glycan species were most likely attributed to a difference in recovery of FA2, the major glycan identified in etanercept by the one-pot glycan method. Differences between sialylated and asialylated glycan species were not statistically significant based on p values of 0.2 by non-parametric two-tailed Mann-Whitney test. Minor differences in N-glycan fucosylation were observed between A1 and A2. (Figure 5B, bottom left) A1 determined 83.5% ± 1.9% of N-glycans were fucosylated compared to A2, which reported 78.7% ± 1.2% (Figure 5B, bottom left). The variance between both analysts was 4.2% (mean, 81.1% ± 3.4%). Regarding afucosylated N-glycans, A1 determined 16.5% ± 1.9% N-glycans were afucosylated compared to A2, which reported 21.3% ± 1.2% This variation is mostly due to a ∼12.2% difference in recovery of the FA2 N-glycan between analysts 1 and 2. No statistically significant differences in fucosylation, sialylation, or N- to O-glycan ratio were observed based on the results of a non-parametric Mann-Whitney test. Furthermore, analyst 2 had no experience with glycan analysis prior to learning the one-pot glycomic protocol for this experiment, which demonstrates the strength of the method as a simplified approach for non-glycomic experts to learn. Overall, these data support that the one-pot method is reproducible for profiling N- and O-glycans from the same sample and effective in relative quantitation of glycans from protein therapeutics.Figure 5 Intermediate precision of one-pot glycomic method performed by two analysts

(A) MALDI-TOF/MS scans of 2,5-DHB matrix alone, followed by N- and O-glycan profiles of three preparations of etanercept by analyst 1 (A1) and analyst 2 (A2), respectively.

(B) Comparison of N- and O-glycan ratio, sialylation, and fucosylation of glycans measured in etanercept by two analysts demonstrate the intermediate precision of one-pot glycan method. Data are represented as mean ± SD. Variability between two analysts was assessed using non-parametric Mann-Whitney test ∗p < 0.05.

Applying the one-pot glycomic method for cell-line glycan profiling

To assess the applicability of the one-pot glycomic method to cellular glycan profiling, cell lysates were prepared from suspension CHO (CHO S) cells. Briefly, protein lysates were prepared by solubilizing the cell pellets in 4% sodium dodecyl sulfate (SDS) lysis buffer followed by heat denaturation and brief sonication prior to proceeding with the optimized one-pot glycomic method. The resulting mass spectra revealed the presence of 32 glycan species (Figure 6). The major N-glycans identified were high-mannose-type species (M5, 1,595.8 m/z; M6, 1,799.9 m/z, 2,004.0 m/z; M7, 2,208.1 m/z; M9, 2,412.2 m/z). Additionally, an array of complex bi-antennary (FA1, 1,606.8 m/z; FA2, 1,851.9 m/z; FA2G2, 2,260.2 m/z; FA2G2S1, 2,621.4 m/z; A2G2S2, 2,808.5 m/z; FA2G2S2, 2,982.5 m/z), tri-antennary (FA3, 2,097.1 m/z; FA3G3, 2,709.4 m/z; FA3G3S1, 3,070.6 m/z; FA3G3S2, 3,431.8 m/z; FA3G3S3, 3,793.0 m/z), and tetra-antennary complex type N-glycans (FA4G4, 3,158.7 m/z; FA4G4S1, 3,519.8 m/z; FA4G4S2, 3,880.9 m/z; FA4G4S3, 4,242.2 m/z) were also identified. A minor population of polylactosamine (PolyLacNAc) N-glycans was also observed in both asialylated (FA4G5, 3,607.8 m/z; FA4G6, 4,056.9 m/z; FA4G7, 4,506.1 m/z) and sialylated forms (FA4G5S1, 3,968.9 m/z; FA4G5S2, 4,330.2 m/z). Clearly, only mono-sialyl core 1 (879.5 m/z) and di-sialyl core 1 (1,240.6 m/z) O-glycans were identified in CHO S cell lysates. Although adaptation of the one-pot method to cellular glycan profiling was possible, challenges in reproducible recovery of O-glycans were encountered when multiple replicates of the same lysate were processed for analysis. N-glycan species on the other hand were consistently obtained with the optimized method as demonstrated in Figure S7, which summarizes the intensity of three major classes of N-glycans (high mannose, complex, paucimannose) identified in two biological replicates of CHO wild type (WT) processed in triplicate.Figure 6 Simultaneous N- and O-glycan profiling of cellular glycoproteins

N- and O-glycans from CHO S cell line were prepared with optimized one-pot method and analyzed on MALDI-TOF/MS (n = 6). The representative spectra are shown.

Discussion

The quantitative MS-based analysis of complex analytes is challenging due to the unequal ionization of different analytes.41 To minimize the ionization differences among glycans in a sample, permethylation is often applied.42 However, even after permethylation, differences in ionization efficiencies have been observed.41 As the UHPLC-FLR method qualifies quantification of glycans after labeling with fluorescence dyes, such as 2-AB, we performed a comparative experiment for quantifying glycans with standards in different aliquots of the same mixtures of 11 glycans, including one sialylated O-glycan and 10 different N-glycans. Our comparative analysis of glycan standards revealed minor discrepancies in the quantitative profiles obtained from analysis of 2-AB-labeled glycans and permethylated glycans (Figure 1). While some variations were observed in the relative abundance of a few glycan species, the overall trends were highly consistent between the two methods. Statistical analysis indicated a close agreement in glycan quantification, with a correlation coefficient between the measured means exceeding 0.9 for most glycan species. In addition, MALDI-TOF analysis was strongly correlated with the anticipated results (r = 0.94) based on known amounts of each glycan species (Figure 1E). Major differences between the two methods can likely be attributed to differences in the labeling and processing protocols. For example, the two methods required different post-labeling cleanup strategies. It is conceivable that each of these methods suffers from different biases. Overall, our results support the quantitative nature of MALDI-TOF analysis of permethylated glycans, especially in the context of relative rather than absolute quantifications, and highlight the complementary nature of MS- and fluorescence-based methods for the quantitative analysis of complex glycan mixtures.

Our report demonstrates the first development of a one-pot chemoenzymatic method for simultaneous profiling of N- and O-glycans of glycoproteins. The glycan profiles derived from the same sample facilitates quantification and characterization of the ratio of O- to N-glycans and other PK-relevant glyco-determinants in a single workflow. Prior to these findings, such information was not possible to obtain without an appropriate comparison setting, such as the traditional methods that analyze N- and O-glycans separately from different aliquots of samples. Furthermore, unique N-glycan reducing-end fragments can be used to facilitate differentiation of N-glycans from O-glycans by collision-induced dissociation or laser-induced dissociation in tandem MS. Although N-glycans are typically larger, and differ in structures from O-glycan species, unique reducing-end reduced fragments could assist in differentiation of large O-glycans with polylactosamine moieties, which could potentially overlap or be larger than some short N-glycans such as paucimannosidic observed in CHO cell lines. Permethylation of glycans for analysis by MS has been proved to have merits including improved ionization, better signal-to-noise ratio, enhanced analytical sensitivity, cleaner spectrum, and (more importantly) the quantification of glycans based on the relative abundance of all glycans identified in the analyzed samples.31 Therefore, the relative abundance of different glycan species, the ratio of total O-glycans versus N-glycans, and the sialylated species versus neutral glycans can be determined based on their individual peak areas in the MS spectra.

Chemical methods are currently employed for intact O-glycan release. Under alkaline conditions, the O-glycosidic linkage between the reducing glycan sugar and serine/threonine residues is labile and readily hydrolyzed, which is called β-elimination. The alkaline conditions can be generated by using different chemicals, from strong base NaOH to mild bases such as NH4OH, LiOH, and hydroxylamine. However, the major limitation of these methods is the peeling process in which released O-glycans are subjected to degradation from the reducing end. To address this issue, reducing reagents are often introduced to limit peeling reactions. However, these techniques still cannot eliminate the peeling, while the mild alkaline such as NH4OH cannot efficiently release all O-glycans from glycoproteins. Another issue with traditional β-elimination is the need to remove excess reactants that are incompatible with downstream MS analysis. In the one-pot method, O-glycopeptides were first generated, and then O-glycans were released though a principle of β-elimination in the presence of NaOH during solid-phase permethylation, and the resultant free O-glycans with free reducing ends are immediately methylated. Our method does not only efficiently release all O-glycans but also possesses undetectable peeling. Furthermore, potential peeling reactions will not occur on the N-glycans since the reducing ends of released N-glycans are reduced prior to the permethylation. Finally, we have demonstrated the reproducibility of techniques not only by analyzing the same lot of protein drugs processed at different times and lot-to-lot consistency but also intermediate precision of method through analysis of the protein drugs by two different scientists at different times. When assessing the performance of the one-pot method in a lot-to-lot glycan analysis, we observed tight SDs from intra-lot samples, further supporting the reproducibility of the method in quantifying lot-to-lot glycan differences. Comparative analysis of the same lot by two different analysts did result in inter-analyst variability; however, no statistical differences were observed.

Additional technical lessons learned during method development and validation include considerations for digestion steps, enrichment steps, and permethylation steps. To elaborate, O-glycans were more efficiently released from short O-glycopeptides than from intact glycoproteins. Thus, proteinase K or even pronase digestion is a necessary step to ensure the efficient release of O-glycans and reproducibility of the method. HILIC is the method of choice to selectively enrich and recover neutral and charged N-glycans and O-glycopeptides, instead of active charcoal, a well-established and widely used method for purification of oligosaccharides. Completed permethylation using solid-phase format is essential for the clean spectra, high sensitivity, and relative quantifications of glycans. C18 cleanup is optional to further purify the permethylated glycans prior to MS analysis.

Limitations of the one-pot method

At present, the major limitation of the one-pot method is its laborious nature because it takes 2 days to complete analysis and obtain glycan profiles. Reaction condition optimization using increased enzyme concentrations, rapid digesting versions of enzymes, characterized isolation/purification steps and resins/columns, and so on will shorten the procedure time. In addition, the method cannot determine (1) the specific O-glycosylation sites, (2) site-specific glycans, or (3) the occupancy of N- and O-glycosylation due to a loss of the information as glycans are liberated from the peptide backbone and the protein of interest. Currently, the glycosite information is obtained from the integrated comprehensive peptide mapping approach, which can also provide the information of site-specific glycans of major species, and occupancy of the glycosites.

We highlight the one-pot method’s reproducibility and precision rather than the absolute accuracy. While the absolute quantification of glycan profiles in a complex mixture is still a challenging task that requires orthogonal and complimentary methods and availability of all glycan standards, our analysis of some glycan standard response curves suggests that MALDI-TOF of permethylated glycans alone is sufficient for the relative quantification of glycan profiles between samples. With the one-pot method, consistent data on relevant glycan species within a sample can be obtained and relative shifts in abundance can be reliably measured across samples. The one-pot method is therefore suitable for ensuring lot-to-lot consistency and similarity of a biosimilar to its reference product in glycosylation.

Finally, the method was applied to cellular N- and O-glycomics using whole-cell lysates, but a defined O-glycan profile as compared to purified glycoproteins was not consistently obtained. The major issue is most likely due to the sample complexity as whole cellular extracts contain more proteins and other cell components, and a detergent is often needed to solubilize the membrane-bound proteins. Another consideration is protective mucin domains present in cell-membrane fractions, which are often resistant to proteolysis and reduce access for proteinase K to cleave the peptide backbone, therefore affecting the recovery of cellular O-glycans. Mucin domains contain tandem repeated sequences (TRs) of O-glycosylated threonine and serine residues that form rigid bottlebrush-like structures. These rigid domains are resistant to proteolysis,43 which could result in inefficient digestion with proteinase K, therefore leaving various extracellular and transmembrane O-glycoproteins trapped on the FASP filter unit. While the one-pot glycomic method facilitated glycan profiling of purified proteins, its performance for cellular glycan profiling leaves much room for improvement. A potential strategy to overcome the limitations of protective mucin domains is to incorporate mucin-selective proteases such as secreted protease of C1 esterase inhibitor (StcE) to the one-pot glycomic method. StcE is a bacterial protease from Escherichia coli, which can cleave mucin domains through recognition of a discrete peptide- and glycan-based motif.44,45 Enhanced proteolysis of mucin domains could facilitate recovery of O-glycans and result in reproducible cellular glycan profiling with simple workflow. Sequential treatment of proteins in lysate with PNGase F, StcE, and proteinase K or mixtures of proteases could facilitate enhanced recovery of N- and O-glycans, and potentially result in reproducible cellular glycan profiling with a single workflow. This strategy should also improve the recovery of O-glycopeptides from purified glycoproteins, including glycoprotein drugs for accurately profiling O-glycans. Despite the challenges encountered in whole-cell glycan profiling, the one-pot method could recover highly complex N-glycan species including extended polyLacNAc and tri- and tetra-antennary complex N-glycans consistently. With further optimization, and incorporation of mucin-selective proteases, the one-pot method could become a robust approach for cellular glycan profiling.

In summary, this is the first one-pot method to simultaneously profile the N- and O-glycans of purified glycoproteins, including therapeutic protein drugs, which provides more information than individually profiled N- and O-glycans of glycoproteins. The method was analyzed for repeatability, reproducibility, and applicability using different glycoprotein drug products. The method is potentially applicable for the characterization and release testing of drug substances and products to improve monitoring the drug product quality and lot-to-lot consistency and the bioequivalence of biosimilars to their reference products in glycosylation. This feasible analytical method can be leveraged as a control strategy for glycosylation of therapeutic glycoproteins and can also facilitate the investigation of functional glycomics.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Fetuin from fetal bovine serum	Millipore Sigma	F3004	
Borane-ammonia complex	Millipore Sigma	682098	
Acetonitrile, Optima™ LC/MS Grade	Fisher Chemical	A955	
Water, Optima™ LC/MS Grade	Fisher Chemical	W6500	
Trifluoroacetic Acid, Optima™ LC/MS Grade	Fisher Chemical	A116-10X1AMP	
Methanol, Optima™ LC/MS Grade	Fisher Chemical	A456	
Acetic Acid, Optima™ LC/MS Grade	Fisher Chemical	A11350	
Formic Acid, 99.0+%, Optima™ LC/MS Grade	Fisher Chemical	A1171-AMP	
ENBREL® (etanercept)	Amgen	58406-010-01	
EYLEA® (aflibercept)	Regeneron	61755-005-01	
ORENCIA® (abatacept)	Bristol-Myers Squibb	0003-2187-10	
Glycerol Free PNGase F	New England Biolabs	P0709S	
Proteinase K	Millipore Sigma	70-663-4	
IdeZ Protease	New England Biolabs	P0770S	
Iodomethane	Millipore Sigma	289566	
Sodium hydroxide, beads, 20–40 mesh	Millipore Sigma	367176	
Dimethyl sulfoxide	Millipore Sigma	276855	
Sodium dodecyl sulfate	Millipore Sigma	436143	
Dithiothreitol	Millipore Sigma	3860	
NIST N-glycan standard reference material	NIST	SRM3655	
Sialylated Core 1 O Glycan, C1S(3)1	Ludger	CO-C1(S3)1-10U	
Chloroform	Millipore	CX1058	
Trypsin-ultra	NEB	P8101S	
O-glycoprotease (IMPa)	NEB	P0761S	
C13 Iodomethane	Sigma-Aldrich	277185	
	
Critical commercial assays	
	
FASP Protein Digestion Kit	Abcam	ab270519	
Glycoworks 2-AB labeling reagent kit	Waters	186007034	
	
Deposited data	
	
Glycoproteomic LC-MS/MS spectra	PRIDE repository	PXD052172	
	
Experimental models: Cell lines	
	
Hamster: FreeStyle CHO Cells, Wild Type	ThermoFisher Scientific	Cat# R80007	
	
Software and algorithms	
	
GraphPad Prism version 10.0.3	https://www.graphpad.com/	N/A	
FlexControl Version 4.2	Bruker Daltonik GmbH	N/A	
FlexAnalysis Version 4.2	Bruker Daltonik GmbH	N/A	
Glycoworkbench 2.1	https://code.google.com/archive/p/glycoworkbench/	N/A	
Assign-MALDI	https://github.com/FDA/AssignMALDI	N/A	
Metamorpheus	https://github.com/smith-chem-wisc/MetaMorpheus	N/A	
	
Other	
	
Thermo Scientific™ HyperSep™ Aminopropyl (NH2) Cartridges 50mg	Thermo Scientific	03-251-276	
Micro SpinColumns, C18	Harvard Apparatus	74–4601	
Micro SpinColumns, Empty, 20 μm frit	Harvard Apparatus	74–4400	
MTP 384 target plate ground steel BC	Bruker Daltonik	8280784	
MALDI-TOF-Mass Spectrometer	Bruker Daltonik	ultrafleXtreme	
Bravo Streptavidin Cartridge	Agilent	G5496-60010	
Orbitrap Eclipse Tribid Mass Spectrometer	Thermo Fisher	FSN04-10000	

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tongzhong Ju (Tongzhong.Ju@fda.hhs.gov).

Materials availability

This study did not generate any new unique reagents.

Data and code availability

• LC-MS/MS data for glycoproteomic analysis is available via ProteomeXchange with identifier PRIDE: PXD052172.

• This paper does not report original code.

• Any additional information needed to re-analyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Cell lines: FreeStyle CHO S cells were cultured according to the manufacturer’s guidelines (Cat# R80007, ThermoFisher Scientific). Briefly, on day 0, CHO S cells were rapidly thawed in a 37°C water bath for approximately 1 min and then transferred into a 125 mL shaker flask (Cat# 4115-0125, VWR) containing 30 mL of complete FreeStyle CHO expression medium (Cat# 12651022, ThermoFisher Scientific). The cells were subsequently incubated at 37°C with agitation at 125 RPM in an atmosphere of 5% CO2. Maintenance of the CHO S cells involved passaging 7.5 million cells on day 3 into 30 mL of FreeStyle CHO expression medium, with subsequent passages occurring on days 5 and 7 under identical conditions.

Method details

Quantification of relative glycan abundances

The relative abundance (%) of each assigned glycan was calculated by their corresponding peak intensity (the sum of peak areas of isotopic masses) using the freely available AssignMALDI program.33 AssignMALDI is an automated pipeline that picks, assigns and calculates the abundance of glycans across multiple files. It returns results as aligned spectra and allows users to edit assignments for manual curation if needed. Importantly, AssignMALDI sums intensities across the entire isotopic range of glycan species. Such a feature is particularly important when analyzing N- and O-glycans simultaneously due to the large difference in isotope distributions between these two classes of molecules (Figure S1B).

MALDI-TOF analysis of glycan standards

N-glycan (Cat# SRM3655, NIST) and O-glycan (Cat# CO-C1(S3)1-10U, Ludger) standards were mixed at prespecified molar ratios for a total of approximately 400 pmol. Glycans were dried down in a SpeedVac and reconstituted in 200 μL of a DMSO/NaOH slurry (120 mg/mL). Following reconstitution, 50 μL of iodomethane (Cat# 289566, Millipore Sigma) was added to each sample and samples were mixed by vigorous vortexing. Samples were then incubated in a thermoblock at 23°C with shaking at 800 rpm for 1 h. Following incubation, samples were centrifuged at 5,000xg for 5 min and the supernatants were transferred to fresh collection tubes. Subsequently, 500 μL of LC-MS grade water was added to each sample and the samples were observed to turn cloudy, indicating successful permethylation. A chloroform extraction of permethylated glycans was performed by adding 500 μL of HPLC grade chloroform to each sample. Samples were mixed vigorously and centrifuged at 5,000xg for 1 min. The top aqueous layer was removed and a fresh 500 μL of LC-MS grade water was added to each sample. Samples were vortexed vigorously and centrifuged at 5,000xg for 1 min and the top aqueous layer was removed. This process was repeated for a total of five times. After the final round of chloroform extraction, the organic fraction, containing permethylated glycans, was dried down in a SpeedVac. Dried and permethylated glycans were reconstituted in 22 μ L of 50% methanol, 50% water. Reconstituted glycans were mixed with 22 μL of 2,5-dihydroxybenoic acid matrix (DHB,10 mg/mL containing 1mM NaCl in 30% ACN, 0.1% TFA) and 0.5 μL was spotted onto an MTP ground steel target plate. Glycan analysis was performed on a Bruker UltrafleXtreme in positive ion reflector mode with a mass window of 700–3500 m/z. N and O-glycans were detected as sodiated ions. The relative abundance (%) of each assigned glycan was calculated by AssignMALDI. The Z score was set to 30 and the mass tolerance set to 0.3 Da. Data was searched using a custom database of permethylated N and O-glycans.

Relative response curves for glycan standards

N-glycan and O-glycan standards were permethylated as described above. Each glycan standard was permethylated in two aliquots with either C12 or C13 labeled iodomethane (Cat# 289566 Millipore Sigma, Cat# 277185, Sigma-Aldrich). Following permethylation, glycan standards were reconstituted in 50% methanol. Standards labeled with C12 iodomethane were prepared at different concentrations by dilution with 50% methanol to generate a standard ladder. A constant amount of matching C13 labeled glycan standard was added to each sample and used as an internal quantitative standard. MALDI-TOF analysis was performed as described above. Peak area as a function of concentration was calculated using Flexanalysis. Peak areas for each sample were normalized by the intensity of C13 labeled standard in each sample. Relative response factors were calculated by measuring the slope of each standard curve after standardizing by the maximum measured response (highest concentration sample).

2-AB labeling and UPLC analysis of glycan standards

N-glycan (NIST) and O-glycan (Ludger) standards were mixed at prespecified molar ratios for a total of 800 pmol. The glycan mixture was then separated into three aliquots of either 200 pmol or 400 pmol per sample. Glycan standards were derived from the same stock as those used for MALDI-TOF analysis. Glycans were dried down in a SpeedVac and the 2-AB labeling was performed using a GlycoWorks 2-AB labeling kit (Cat# 186007034, Waters) according to the manufacturer’s instructions. Briefly, 300 μL of glacial acetic acid was added to 700 μL of DMSO. Subsequently, 800 μL of the acetic acid/DMSO mixture was added to the entire contents of the 2-AB vial (10 mg, PN 186007034-6, Waters) and vortexed vigorously to resuspend. The entire 2-AB containing solution (800 μL) was then added to the entire contents of the sodium cyanoborohydride vial (PN 186007034-5, 12 mg, Waters) and vortexed vigorously. Finally, 20 μL of the prepared labeling reagent was added to each sample and samples were incubated at 65°C for 3 h.

Glycan samples were diluted with 800 μL of Acetonitrile (ACN) with 0.1% TFA and loaded on a 50 mg HyperSep-NH2 column (Cat# 60108-424, ThermoFisher) and the flow-through was collected and reloaded a total of three times to maximize recovery of N- and O-glycans. The column resin was washed with 5 mL of 90% ACN, 0.1% TFA to remove salts and excess label and finally N- and O-glycans were eluted with 300 μL of 10% ACN, 0.1% TFA, and the column was eluted 2 times for maximized recovery. The eluent was dried down in a SpeedVac and the dried glycans were reconstituted in 50 μL of 30% ammonium formate (pH 4.4), 70% ACN and transferred to an autosampler vial.

2-AB labeled glycans were analyzed by HILIC separation on a Waters Acquity H class UPLC using a BEH Amide column (130 Å, 1.7 μm, 2.1 × 150 mm). Each injection used 10 μL of sample. The column temperature was set to 60°C and the sample temperature was set 8°C. Mobile phase A was 50mM ammonium formate (pH 4.4) and mobile phase B was ACN. Prior to injection the column was equilibrated in 20% mobile phase A, 80% mobile phase B for a total of 15 column volumes. A 65-min gradient was used to separate glycan species. The elution setup started with a 45-min linear gradient from 20% to 35% mobile phase A at a flow rate of 0.4 mL/min. From 45 to 46.5 min the gradient was ramped up to 100% mobile phase A at a flow rate of 0.2 mL/min and this condition was maintained for 3 min. From 49.5 to 53.1 min the gradient was gradually reduced to 20% mobile phase A and from 53.1 to 57.6 min the flow rate was increased to 0.4 mL/min and this condition was maintained until 65 min.

The fluorescent detector was set with excitation at 330 nm and emission at 420 nm and a data collection rate of 10 points/second. Peaks were integrated in Empower 3 (Waters) using manual definition of peak boundaries. Peaks were identified based on alignment with an IgG N-glycan standard library (Cat# 186006349, Waters).

Sample preparation for O-glycoproteomic analysis

Fifty micrograms of Bovine Fetuin (Cat# F3004, Sigma-Aldrich, St. Louis, MO) was processed using a Filter Aided Sample Prep (FASP) protein digestion kit (Cat# ab270519, Abcam). Aliquots of Bovine Fetuin were brought up to 30 μL in 100mM Tris-HCl buffer pH 8.5 containing 10 mM DTT. Samples were denatured and reduced by incubation at 95°C for 5 min. After cooling to room temperature, samples were diluted to 100 μL with a solution of 8M urea supplemented with 40mM IAA and incubated at room temperature for 20 min in the dark. Subsequently, 200 μL of 8M urea was added to the top of 30 kDa molecular weight cutoff ultrafiltration devices. The entire glycoprotein mixture was transferred to the tops of ultrafiltration devices and centrifuged at 15,000xg for 15 min. Filters were washed for a total of 3 times in 200 μL of 8M Urea, centrifuging at 15,000xg for 15 min after each wash. Filters were additionally washed for a total of three times in 200 μL of 50mM Ammonium Bicarbonate. After the final wash, 100 μL of 50mM ammonium bicarbonate containing 1500 U of glycerol-free PNGase F (Cat# P0709S, NEB) was added to each sample. Samples were incubated at 37°C for 16–18 (overnight) hours. Following treatment with PNGase F, the samples were centrifuged at 15,000xg for 15 min and the flowthrough containing released N-glycans was discarded. Filters were washed with 10% acetonitrile containing 0.1% TFA to remove any residual N-glycans. Filters were then washed with 200 μL of 50mM TEAB for a total of three times. After the final wash filters were transferred to fresh collection tubes and 100 μL of 50mM TEAB containing 1 μg of trypsin was added to each sample. Samples were incubated at 37°C for 16–18 (overnight) hours. In the morning, peptides were eluted by centrifugation at 15,000xg for 15 min and the filters were washed once with 100 μL of 0.1% TFA in order to maximize recovery of peptides.

Eluted peptides were dried down in a SpeedVac and subsequently reconstituted in 50 μL of 20mM Tris-HCl, pH 8.0. Subsequently, 5 μL of O-glycoprotease (NEB, Cat# P0761) was added to the sample and samples were digested for 16–18 h at 37°C. In order to remove O-glycoprotease from the sample, peptides were passed through a 30 kDa centrifugal filter and the flowthrough containing digested peptides was retained.

Peptides were dried down in a SpeedVac and subsequently reconstituted in 100 μL of 0.1% TFA in preparation for C18 solid-phase extraction (SPE). C18 microspin columns (Cat# 74–4601, Harvard Apparatus) were activated by washing with 200 μL of 50%/0.1% acetonitrile/TFA for a total of five times, centrifuging at 1,000xg for 2 min in-between each wash. C18 microspin columns were equilibrated by washing with 200 μL of 0.1% TFA for a total of 3 times, centrifuging at 1,000xg for 2 min in-between each wash. Columns were transferred to fresh collection tubes and peptide samples were applied to the top. C18 microspin columns were centrifuged at 1,000xg for 2 min and the flowthrough was reapplied to the column for a total of five times; in order to maximize recovery of glycopeptides. Bound peptides were washed with 200 μL of 0.1% TFA for a total of three times. C18 microspin columns were transferred to fresh collection tubes and peptides were eluted with 50 μL of 50%/0.1% acetonitrile/TFA. Eluted peptides were dried in a SpeedVac and prepared for LC-MS/MS analysis.

LC-MS/MS analysis of O-glycopeptides

(Glyco)peptides were analyzed as described previously with minor modification.46 Dried peptides were reconstituted in 200 μL of 0.1% TFA. Peptides were analyzed on an Orbitrap Eclipse Tribrid mass spectrometer and approximately 1 μg of the peptide mixture was injected per LC-MS/MS run. (Glyco)peptides were separated on an Acclaim PepMap C18 RLSC nanoviper column (2 μm, 100 Å, 75 μm i.d. x 15 cm, ThermoFisher part# 164534) over a 60-min run. Solvent A was 5%/0.1% ACN/FA and Solvent B was 0.1% FA in acetonitrile. The gradient increased from 0% to 90% B over the first 45 min at a flow rate of 0.3 μL/min. The gradient was held at 90% B from 45 min to 50 min at a flow rate of 0.3 μL/min. The gradient was then decreased from 90% B to 5% from 50 min to 55 min and held at 5% B from 55 min to 60 min. The column temperature was set to 35°C. Survey scans of peptide precursors were collected in the Orbitrap from 400 to 2000 Th with an AGC target set to standard, a maximum injection time set to auto, RF lens at 30%, and a resolution of 120,000 at 200 m/z. Monoisotopic precursor selection was enabled for peptide isotopic distributions, precursors were selected for data-dependent MS/MS scans for 12 dependent scans and dynamic exclusion was set to 30 s with a ±10 ppm window. Higher-energy collisional dissociation (HCD) “scouting” MS/MS scans were performed at 36% normalized collision energy (NCE) and an orbitrap resolution of 30,000 for a maximum injection time of 60ms, an AGC target set to standard and a scan range set to auto. Product-dependent EThCD child scans were triggered by the following oxonium ion m/z ratios (204.09, 366.14, 126.055, 138.0549, 144.0655, 168.0654, 186.076, 274.0921, 292.1027) with a low and high mass tolerance of 10 ppm and 25 ppm, respectively. EThCD scans were carried out using a supplemental activation of 25% NCE, a maximum injection time of 54 ms, and an orbitrap resolution of 30,000. AGC target was set to standard and scan range was set to auto.

Analysis of O-glycoproteomic data

All raw data was searched using O-pair search in Metamorpheus V1.0.5 (https://github.com/smith-chem-wisc/MetaMorpheus).47 Raw files were searched using a FASTA file containing Uniprot derived sequences of bovine fetuin A and common contaminants.35 Protein sequences were digested with Trypsin/IMPa were IMPa digests N-terminally to serine or threonine residues. The calibrate task was enabled with a maximum of eight missed cleavages and a precursor mass tolerance of 15 ppm and a product tolerance of 25 ppm. A carbamidomethyl modification on cysteine was set as fixed and methionine oxidation was set as a variable modification. The global PTM discovery (GPTMD) task was enabled using the default list of common biological and common artifact modifications. The O-glycosearch task was enabled using a database of 22 O-glycans. The maximum number of glycans allowed per peptide was set to six and oxonium ion filtering was enabled. The parent scan type was set to HCD and the child scan type was set to EThCD. Final results were obtained by filtering the identified sites to retain only those with a level 1 confidence (site localization supported by fragment ions). O-glycoproteomics data for Fetuin is available as a supplementary table (Supplemental Material_ O-Glycopeptide Mapping_CR-METHODS-D-23-00314.psmtsv.xlsx).

One pot N-glycan and O-glycopeptide isolation

N-glycans and O-glycopeptides were isolated from Bovine Fetuin (Cat# F3004, Sigma-Aldrich, St. Louis, MO), or Fc-fusion glycoprotein drug products (etanercept, abatacept, and aflibercept) which were commercially manufactured in CHO cells under good manufacturing practices (GMP). Glycoproteins were processed with materials from a Filter Aided Sample Prep (FASP) protein digestion kit (Cat# ab270519, Abcam). Fifty micrograms of glycoprotein in 30 μL of 100mM Tris-HCL pH 8.5 containing 10mM DTT in a 0.5 mL Eppendorf tube was denatured and reduced by incubating at 95°C for 5 min. The volume of the denatured glycoprotein solution was then adjusted to 100 μL with fresh Urea solution (8M in 100mM Tris-HCL pH 8.5), containing a final concentration of 40mM iodoacetamide (IAA) and the proteins in the solution were alkylated in the dark for 20 min. The entire glycoprotein mixture was transferred to a 30 kDa molecular weight cutoff ultrafiltration device followed by washing the tube with 200 μL of 8M Urea solution (without IAA) and transferring to the device. The ultrafiltration device was centrifuged at 15,000 x g for 15 min, and the filter was washed three times with Urea solution and 2x buffer exchanged with 50mM ammonium bicarbonate (pH 7.5). The filter unit was transferred to a new collection tube and 100 μL of 50mM ammonium bicarbonate containing 1500 U of glycerol-free PNGase F (Cat# P0709S, NEB) was added. Samples were incubated at 37°C for 16–18 (overnight) hours. One hundred microliters of 100mM Tris-HCL pH 8.5 containing 20 μg of Proteinase K (Cat# 70-663-4, Millipore Sigma) was added to the filter and the unit was incubated overnight at 50°C. Released N-glycans and short O-glycopeptides were recovered by centrifugation for 15 min at 15,000 x g, followed by washing the filter with 100 μL of 0.1% Trifluoroacetic acid (TFA) in water. For selective enrichment of N-glycans and O-glycopeptides, the filtrate was diluted with 800 μL of Acetonitrile (ACN) with 0.1% TFA and loaded on a 50 mg HyperSep-NH2 column (Cat# 60108-424, ThermoFisher) and the flow-through was collected and reloaded a total of three times to maximize recovery of N-glycans and O-glycopeptides. The column resin was washed with 3 mL of 90% ACN, 0.1% TFA to remove salts and peptides and finally N-glycans and O-glycopeptides were eluted with 300 μL of 10% ACN, 0.1% TFA, and the column was washed 2 times for maximized recovery. The eluate was dried by a SpeedVac and reduced as described.28 Excess reactants were removed by adding 200 μL of 10% Acetic acid (Cat# A11350, ThermoFisher) in methanol followed by vacuum centrifugation to dryness a total of 5 times. After a series of methanolic evaporations, the samples were reconstituted in 5 μL of LC-MS water, 65 μL of Dimethyl sulfoxide (Cat#276855, Sigma)., followed by adding 35 μL of iodomethane (Cat# 289566, Millipore Sigma) and permethylated by solid-phase sodium hydroxide as described.28,48 Permethylated N and O-glycans (released by β-elimination during permethylation as reported by Goetz et al.16). Essentially, 20–40 mesh Sodium Hydroxide (NaOH) beads (Cat#367176, Sigma) soaked in acetonitrile were packed into a 3cm depth into an empty spin column with a 20μm frit (Cat#74–4400, Harvard Apparatus). The columns were washed with a total of 6 mL of DMSO. Glycan mixtures were loaded on the NaOH columns and incubated for 20 min, at room temperature, followed by brief centrifugation at 240 x g to collect the flow through. The glycan mixtures were loaded and incubated on the NaOH columns an additional two times. The permethylation reaction was quenched by adding 200 μL of LC-MS water, and excess iodomethane was removed by pipetting air into the sample several times with a 200 μL pipette. The glycan mixture was purified directly by C18 microspin columns (Cat# 74–4601, Harvard Apparatus). Briefly the entire glycan mixture containing DMSO, and water was passed through the C18 microspin column, collected and reloaded through the column a total of three times. DMSO was removed by washing the resin bed with 500 μL of 5% ACN, 0.1% TFA. Permethylated N&O-glycans were eluted three times with 200 μL 85% ACN, 0.1% TFA, and the final eluate was dried by vacuum centrifugation.

Analysis of permethylated N-and-O-glycans by MALDI-TOF/MS

The permethylated glycans were resuspended in 10 μL of 50% methanol, and 0.5 μL was spotted to an MTP ground steel target. The glycan spot was overlayed with 0.5 μL of 2,5-dihydroxybenoic acid (DHB,10 mg/mL containing 1mM NaCl in 30% ACN, 0.1% TFA) and mixed by pipetting up and down several times and left to dry. Glycan analysis was performed on a Bruker UltrafleXtreme in positive ion reflector mode with a mass window of 600–5000 m/z. N and O-glycans were detected as sodiated ions. Glycan mass calculations were performed using Glycoworkbench 2.1.49

The relative abundance (%) of each assigned glycan was calculated by their corresponding peak intensity (the sum of peak areas of isotopic masses) using the freely available AssignMALDI program.33 AssignMALDI is an automated pipeline that picks, assigns and calculates the abundance of glycans across multiple files. It returns results as aligned spectra and allows users to edit assignments for manual curation if needed. Importantly, AssignMALDI sums intensities across the entire isotopic range of glycan species. Such a feature is particularly important when analyzing N- and O-glycans simultaneously due to the large difference in isotope distributions between these two classes of molecules (Figure S1B).

Analysis of permethylated N-and-O-glycans by NanoC18 RP LC-MS/MS

LC-MS analysis was performed to obtain high resolution tandem mass spectra to confirm signature ion fragments of reduced N-glycans for differentiation from O-glycans. Briefly, five microliters of permethylated glycans previously suspended in 50% methanol was transferred to a new microtube and dried by s SpeedVac. The dried glycans were reconstituted in 50 μL of 3% Acetonitrile, 0.1% Formic acid solution. Particulates were removed by centrifugation at 21,000 x g for 10 min, and the supernatant was transferred to autosampler vials. High resolution tandem mass spectra were obtained by either a Fusion Orbitrap MS (ThermoFisher, San Jose, CA) or a Xevo G2-XS Q-TOF Nano-LC/MS (Waters, Milford, MA) based on instrument availability.

For the glycans analyzed on the Fusion orbitrap, briefly, permethylated glycans were first loaded onto a trap cartridge (ThermoFisher Acclaim PepMap C18 nanotrap, 5 μm, 100 μm i.d. X 20 mm, 100 Å), then eluted onto a reversed phase Easy-Spray column held at 60°C (ThermoFisher PepMap, C18, 3 μm, 75 μm i.d. X 15 cm, 100 Å) using a linear 120 min gradient of ACN (30−70%) containing 0.1% formic acid at 300 nL/min flow rate. The eluted glycans were analyzed by Fusion Orbitrap. The data-dependent acquisition (DDA) mode was enabled, and each Orbitrap MS1 scan (60,000 resolution) was followed by Orbitrap MS2 scans (15,000 resolution) using top speed (acquired as many MS2 scans as possible within 3 s cycle time). Precursor ion fragmentation was performed using CID at collision energy of 35. Automatic gain control (AGC) targets were set as “standard” for both MS and MS2. The spray voltage and ion transfer tube temperature were set at 1.8 kV and 250°C respectively.

For glycans analyzed on the waters Xevo G2-XS Q-TOF, one microliter of sample was injected and trapped on a nanoEase M/Z Symmetry C18 trap column (100 Å, 5 μm, 180 μm × 20 mm) at 99:1 Water:ACN containing 0.1% FA with a flow rate of 5 μL/min for 3 min. Samples were analyzed using a waters Xevo G2-XS Q-TOF Nano-LC/MS system utilizing a nano-EASE M/Z HSS C18 T3 column (100 Å, 1.8 μm, 75 μm × 100 mm). A gradient of 2%–40% ACN for 30 min, 40–90% ACN for 31-35min, 90-2% ACN from 40 to 60min was used at a flow rate of 0.5 μL/min. Both water and ACN mobile phases contained 0.1% FA and the column temperature was maintained at 45°C. Acquisition utilized sensitivity/Positive mode scanning over the range of 100 Da–2000 Da with a scan time of 0.5 s. A collision energy ramp of 15-35V was used. Leucine-Enkephalin was utilized as a lock mass standard with 3 scans every 10 s and the mass correction was applied.

Micropurification of IgG Fc and TNFR2 fragments after IdeZ treatment for domain-specific N- and O-glycan analysis

IdeZ treatment of Protein A-captured etanercept was performed as described.50 Briefly, 50 μg of biotinylated Protein A (TCI America, P2407VIAL) was immobilized on an Agilent Bravo Streptavidin Cartridge (Agilent G5496-60010) and unreacted protein was washed out 3 times with phosphate buffered saline (PBS) pH 7.2. One hundred micrograms of etanercept were loaded on the cartridge and passed through a total of three times to maximize binding. The cartridge was washed three times with 100 μL of PBS and domain cleavage was performed by adding 50 μL of Glycobuffer 2 containing 1.6 units/μL of IdeZ (NEB, P0770S) and incubating at 37OC for 1 h. TNFR2 domain was obtained by washing of the cartridge with 100 μL of PBS into a collection plate as described.50 Acidic elution of the Fc domain was performed by washing the cartridge with citrate buffer followed by N&O-glycan analysis.

Preparation of cell lysates for FASP

A pellet containing 10 million cells was resuspended in 600 μL of lysis buffer (100mM Tris-HCL pH 8.5 with 4% SDS and 0.01M DTT), vortexed briefly and incubated at 95°C for 5 min. To reduce viscosity of the lysate, the tube was sonicated for 15 min. The lysate was then clarified by centrifugation at 16,000xg for 10 min, and the supernatant was transferred to a fresh 1.5mL tube and frozen at −80°C until ready for processing with the optimized one-pot method.

Quantification and statistical analysis

Statistical analysis of data on glycan standards was performed in GraphPad Prism version 10.2.2. Means and standard deviations for relative glycan abundances were calculated using the row statistics function. Correlations between the measured and anticipated means of relative glycan abundances measured by MALDI-TOF/MS and UHPLC-FLR were measured using a simple linear regression. Standardized response curves were measured using a simple linear regression. Within the simple linear regression module, the option for testing whether or not the slopes are significantly different was enabled with a pp-value cutoff of 0.05. GraphPad Prism compares slopes in a framework that is equivalent to an F test to compare a global model where slope is shared among the data sets with a model where each dataset gets its own slope. Due to small sample size, non-parametric analyses, Kruskal-Wallis and Mann-Whitney tests were performed. Kruskal-Wallis and Dunn’s multiple comparisons tests were performed at alpha level of 0.05 to study differences between the mean glycosylation component content between three lots. Variability of the method results between two analysts was assessed using non-parametric Mann-Whitney test.

Supplemental information

Document S1. Figures S1–S7 and Tables S1–S4

Data S1. O-glycopeptide mapping of bovine fetuin glycoproteomics reveals six occupied O-glycan sites, related to Table S2, Figure 2, and STAR Methods on analysis of O-glycoproteomic data

Document S2. Article plus supplemental information

Acknowledgments

We would like to thank David Powers (FDA) for critical review of the manuscript. This project was supported by OPQ BsUFA III Regulatory Science Pilot Program, CDER Domestic Manufacturing Initiatives, OPQ Centers of Excellence, and the CDER Regulatory Science and Research Committee at the FDA to T.J. We also thank Oak Ridge Institute for Science and Education (ORISE) for their support. This project was also partially supported by CDER Regulatory Science and Research Committee at the FDA to T.G.B., C.A., and A.R. and by the Office of Women's Health/FDA.

The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the FDA and the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.

Author contributions

U.O.-R., J.Q.B., G.Z., and T.J. designed the experiments. U.O.-R., J.Q.B., and G.Z. optimized the methodology. U.O.-R., J.Q.B., G.Z., V.M.F., T.G.B., J.D.P., and W.W.W. performed the research. M.L. and J.Q.B. performed the statistical analyses. H.X., T.G.B., C.A., A.R., and T.J. acquired the funding. U.O.-R., J.Q.B., and T.J. wrote the paper. All authors reviewed and revised the manuscript. T.J. supervised the research. U.O.-R., J.Q.B., G.Z., T.G.B., and A.M.M. contributed to new reagents/analytical tools.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.crmeth.2024.100834.
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