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Chemo-Enzymatic Synthesis of 13C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry Begoña Echeverria,† Juan Etxebarria,† Nerea Ruiz,† Á lvaro Hernandez,† Javier Calvo,‡ Markus Haberger,§ Dietmar Reusch,§ and Niels-Christian Reichardt*,†,∥ †

Glycotechnology Group, CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebastian, Spain Mass Spectrometry Platform, CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebastian, Spain § Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, 82377 Penzberg, Germany ∥ CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Paseo Miramon 182, 20009 San Sebastian, Spain ‡

S Supporting Information *

ABSTRACT: Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of 13C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was 13C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

M

are sensitive and provide relative quantification of glycans via uniform labeling,10 they are more time-consuming, expensive, and in general more prone to error due to additional sample preparation steps. More importantly, current methods only provide relative but no absolute quantification of individual glycans, e.g., for diagnostic applications or quantification of immunogenic glycan levels. While the relative quantification of glycans is thought to be sufficient to track changes in glycosylation between samples in many biopharmaceutical applications, a clinical use of glycans as disease markers that goes beyond glycan ratios would require methods that measure absolute concentrations of individual glycans. Isotopic dilution mass spectrometry has been the golden standard for absolute metabolite quantification in newborn screening,11 quantification of immunosuppressor levels,12 in screening for illicit drugs,13 and in various approaches for quantitative proteomics.14 An application of isotopic dilution to glycan analysis, however, has been hampered largely by a lack of heavy isotope labeled glycan standards.

ost eukaryotic proteins, both membrane bound and soluble, and the large majority of commercial recombinant therapeutic proteins are modified with N-glycans that can have a major impact on protein solubility, structure, immunogenicity, circulatory half-life, and consequently drug efficacy.1−3 Changes in protein glycosylation are also a hallmark of many cancers, infectious and autoimmune diseases, and the growing number of congenital disorders of glycosylation (CDG)4 suggesting an increasingly important role of glycans as biomarkers.5−7 Consequently, robust and quantitative methods for the analysis of glycans are not only required for mapping glycan structure to function but also highly relevant in biopharmaceutical quality control and in the development of glycans as selective and complementary disease markers. Many profiling methods require the enzymatic or chemical release of the glycans from the peptide backbone. The resulting mixture of glycans can then be chemo-selectively derivatized with a fluorescent label8 like 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), or 9-aminopyrene-1,4,6-trisulfonic acid (APTS), separated by HPLC or capillary electrophoresis and analyzed by fluorescence and/or mass spectrometry detection. Alternatively, glycans can be profiled, often after permethylation, directly by mass spectrometry although isobaric structures remain unresolved, unless diagnostic fragment ions can be produced by tandem MS.9 While the chromatographic methods © 2015 American Chemical Society

Received: August 15, 2015 Accepted: October 19, 2015 Published: October 19, 2015 11460

DOI: 10.1021/acs.analchem.5b03135 Anal. Chem. 2015, 87, 11460−11467

Article

Analytical Chemistry

silica cartridges. All aqueous solutions were prepared from nanopure water produced with a Diamond UV water purification system (Branstead International). Pooled glycan containing fractions were lyophilized on an ALPHA-2-4 LSC freeze-dryer from Christ. All organic solvents were concentrated using rotary evaporation. 1 H, 13C, and HSQC experiments spectra were acquired on a Bruker 500 MHz spectrometer. Chemical shifts were reported in ppm (δ) and referenced to the residual signal of the solvent used (MeOD 4.87 ppm; CDCl3 7.26 ppm; D2O 4.79 ppm). Splitting patterns are designated as s, singlet; d, doublet; t, triplet; m, multiplet. Coupling constants (J) are reported in Hz. High-resolution mass spectra were acquired on a Waters LCT Premier XE instrument, (Waters) equipped with a standard ESI source by direct injection. The instrument was operated with a capillary voltage of 1.0 kV and a cone voltage of 200 V. Cone and desolvation gas flow were set to 50 and 600 L/h, respectively; source and desolvation temperatures were 100 °C. MALDI-TOF mass analyses were performed on an Ultraflextreme III time-of-flight mass spectrometer equipped with a pulsed N2 laser (337 nm) and controlled by FlexControl 3.3 software (Bruker Daltonics). Procedure A: Hydrogenation Reaction. The oligosaccharide was dissolved in MeOH (10 mg/mL) and hydrogenated with a H-Cube reactor on 10% Pd/C cartridge (full H2 mode; 1 mL/ min; 50 °C). The solution of the final compound was concentrated and purified on a graphitized carbon cartridge. The fractions containing product were concentrated and freezedried. Procedure B: β-1,4-Galactosylation. (B-1) Complete Extension. Enzymatic galactosylation was carried out at a glycan concentration of 2 mg/mL. Briefly, a solution of the corresponding glycan (1 equiv), UDP-Gal (uridine 5′diphosphogalactose disodium salt) (2 equiv), bovine serum albumin BSA (0.5 mg), bovine milk β-1,4-galactosyltransferase, alkaline phosphatase, and MnCl2 (10 mM) in HEPES buffer (50 mM, pH = 7.4) was incubated at 37 °C overnight. The resulting mixture was heated at 95 °C for 5 min to precipitate the enzyme. After centrifugation the supernatant was purified on the carbon cartridge (ACN/water + 0.1% TFA). The fractions containing the compound were pooled and freezedried. (B-2) Partial Extension. Enzymatic galactosylations were carried out at a total glycan concentration of 2 mg/mL. Briefly, a solution of 10 (1 equiv) was treated with β-1,4galactosyltransferase (200 mU) and uridine diphosphate galactose (UDP-Gal, mmol, 1.25 equiv) in HEPES buffer 50 mM at pH 7.4, containing MnCl2 2 mM and BSA. After 1 h of reaction at 37 °C the proteic fraction was precipitated by heating at 95 °C for 5 min and centrifuged. The reaction crude was purified by semipreparative HPLC (reverse phase, C18 column, formic acid 0.1%/ACN). The collected fractions were evaporated and freeze-dried. Procedure C: α-2,3-Sialylation. Enzymatic sialylations were carried out in a total glycan concentration of 10 mg/mL. Briefly, a solution of oligosaccharide (1 equiv), cytidine-5′monophospho-N-acetylneuraminic acid sodium salt (CMPNeuAc) (4.0 equiv), 20 mU of α-2,3-sialyltransferase from Pasteurella multocida, and MgCl2 (100 mM) in 300 μL of Tris· HCl buffer (100 mM, pH = 8) was incubated at 37 °C for 30 min. After that, 2.0 equiv more of CMP-NeuAc and 10 mU more of α-2,3-sialyltransferase were added. The mixture was incubated at 37 °C for 30 min. During the following hour, 1.0

Mass spectrometry outperforms the current chromatographic methods in terms of speed, simplicity, resolution, and information content, but reproducibility and quantification of the assays is compromised by limitations in the dynamic range, ion suppression in complex samples, fluctuations in spectrometer performance, or variability in matrix crystallization for MALDI-TOF MS. In addition, isobaric compounds cannot be easily resolved by MALDI-TOF MS unless glycan fragmentation by MS/MS techniques is employed. In analogy to isotopic dilution methods employed routinely in targeted metabolomics or clinical biochemistry, the use of stable isotope labeled glycans as internal standards could turn a mass MALDI-TOF mass spectrometer into a quantitative glycan analysis tool.15,16 13 C-enriched glycan standards show the same physicochemical properties as the natural isotopologue and hence ionize with equal efficiency but they are readily distinguished by an increment in mass. A major part of the experimental variability is mitigated when standard and sample to be analyzed are measured together. The two major challenges for developing an isotopic dilution method for glycan analysis have been (1) finding a suitable heavy isotope enrichment strategy which does not require the introduction of an artificial tag and (2) encountering a viable strategy for the preparation of heavy isotope enriched pure compound libraries with good coverage of the natural structural heterogeneity. Tagging of natural glycans with stable isotope enriched labels, e.g., by permethylation or anomeric reductive amination has been reported before but with these reagents only the relative quantification of analytes between samples is achieved: Two samples are labeled with the light and the heavy form of the labeling reagent and mixed in a 1:1 ratio. The ratios of light and heavy isotope labeled glycan are then used to compare the relative glycan intensities between two samples. Concerning labeling of entire glycan mixtures, however, the reproducibility of the method maybe affected by the variability of the tagging procedures.17−24

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EXPERIMENTAL SECTION Materials. Chemicals were purchased from Sigma-Aldrich or Acros Organics and were used without further purification. All organic were dried over activated 4 or 3 Å molecular sieves. Thin layer chromatography was carried out using Merck aluminum sheets silica gel 60 F254 and visualized by UV irradiation (254 nm) or by staining with vanillin solution. β-NAcetylglucosaminidase from Canavalia ensiformis (Jack bean), α-2,3-sialyltransferase from Pasteurella multocida, and cytidine5′-monophospho-N-acetylneuraminic acid sodium salt were purchased from Sigma-Aldrich. Uridine 5′-diphosphogalactose disodium salt (UDP-Gal) and guanosine 5′-diphospho-β-Lfucose sodium salt (GDP-Fuc) were purchased from Carbosynth. All aqueous solutions were prepared from nanopure water produced with a Diamond UV water purification system (Branstead International). Instrumentation. Microwave irradiation was performed on Biotage Initiator monomode oven, (Biotage AB). Hydrogenation reactions were performed in continuous-flow hydrogenation reactor H-Cube from ThalesNano Nanotechnology Inc. Purifications of compounds were performed on: SampliQ high performance graphitized carbon cartridges (1 mL) from Agilent Technologies, C18 Sep-Pak Cartridges (1 mL) from Waters (Milford), flash chromatography using Merck 62 Å 230−400 mesh silica gel or on a Biotage SP4 automated flash chromatography system, (Biotage AB) employing prepacked 11461


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Analytical Chemistry Scheme 1. 13C Labeling of a Bi-Antennary N-Glycan Precursora

(a) MeONa, MeOH, NH2CH2CH2NH2, MW 120 °C; (b) (13CH313CO)2O, MeOH, 60%; (c) 10% Pd/C, H2, MeOH, 72%; in red color, incorporated 13C isotopes.

a

equiv of CMP-NeuAc was added every 30 min at 37 °C. MeOH (500 μL) was added to the resulting mixture to precipitate the enzyme. After centrifugation, the supernatant was purified by a carbon cartridge (H2O/ACN gradient in the presence of NH4HCO3 40 mM). The fractions containing product were concentrated and freeze-dried. Procedure D: α-1,6 Fucosylation. Enzymatic fucosylation reactions were carried out at a glycan concentration of 2 mg/ mL. Briefly, a solution of glycan (1 equiv), GDP-Fuc (guanosine 5′-diphospho-β-L-fucose sodium salt) (1.2 equiv), α-1,6-fucosyltransferase, and 20 mM MnCl2 in MES buffer (40 mM, pH = 6.5) was incubated at room temperature overnight. The resulting mixture was heated at 95 °C for 5 min to precipitate the enzyme. After centrifugation, the supernatant was purified on the carbon cartridge with the elution conditions (ACN/water, 1/1 + 0.1% TFA). The fractions containing product were concentrated and freeze-dried. Procedure E. N-Acetyl-glucosaminidase. Hydrolysis reactions were carried out in a glycan concentration of 2 mg/mL. Briefly, a solution of 10 (1 equiv) and N-acetyl glucosaminidase from Conavalia ensiformis (100 mU) in ammonium acetate buffer 50 mM at pH 4.5 was incubated at r.t. for 6 h. The reaction was quenched by the addition of MeOH (20 μL). The reaction crude was purified by semipreparative HPLC (reversed phase, C18 column, formic acid 0.1%/ACN). The collected fractions were evaporated and freeze-dried. NMR Quantification of Labeled N-Glycan Standards. For the NMR quantification of the labeled standards, we used maleic acid, 99.99% purity as an internal standard (standard for quantitative NMR, TraceCERT Fluka). For each labeled standard, a 10 mM stock solution in dH2O was prepared. A volume of 10−20 μL of these stock solutions were added separately to a 3 mM solution of the maleic acid standard in D2O and the resulting mixtures were used for NMR quantification. NMR-spectra were acquired with a long relaxation delay (30 s) and at a 30° pulse angle. The quantified stock solutions in dH2O were used to prepare the standard formulation used in the glycan quantification by MALDI-TOF MS. Limit of Detection and Limit of Quantification. For determining LOD and LOQ, we plotted the intensities (peak areas) of five isotopologues of the quantified labeled standard G0F obtained by MALDI-TOF MS against their corresponding quantities in picomoles. The limit of detection was determined to be 0.03 pmol at a signal-to-noise ratio of 3, while the limit of quantification was 0.14 pmol corresponding to a signal-to-noise ratio of 10 (see inset in Figure S1).

Absolute Quantification of Fc Glycans on an IgG by MALDI-TOF MS. A sample (50 μg) of IgG antibody, previously purified with A/G protein affinity beads, was desalted and spiked with a known amount of the corresponding 13 C-labeled N-glycan standards. The mixture of IgG and Nglycan standards was treated with PNGase F (5 U) in dH2O for 1−18 h at 37 °C. After complete deglycosylation, the sample was filtered with a 10 kDa size exclusion filter and the mixture of light and heavy N-glycans was collected as a dH2O solution (total volume 100 μL). The glycan sample was treated with 0.1% TFA solution to remove the amino glycosides and loaded directly into the MALDI plate. Sodium citrate tribasic was used as an additive for the removal of potassium adducts in the MALDI spectra. DHB (5 mg/mL in ACN) was used as matrix.

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RESULTS AND DISCUSSION We present here the first library of 13C-enriched N-glycan standards which has been obtained by the enzymatic derivatization of a synthetic and heavy isotope-labeled Nglycan heptasaccharide with three recombinant glycosyltransferases and a glycosidase. A mass increment of 8 Da over the natural isotopologue was obtained by acetylating with bis-13Clabeled acetic anhydride the four free intermediate glucosamine residues of a biantennary glycan precursor which had been synthesized following our previously reported strategy25 (Scheme 1, details in the Supporting Information) In addition, performing the enzymatic reactions on a partially protected UV-active intermediate allowed us to produce all positional isomers in a single reaction and separate them by preparative HPLC. As one possible example for the use of this novel library of heavy isotope glycan standards we present a method for the absolute glycan quantification by MALDI-TOF MS of a therapeutic monoclonal antibody expressed in CHO cells. We and others have employed recombinant glycosyltransferases in the past to prepare structurally varied libraries of N-glycans as ligands for glycan arrays.25−29 In these efforts, however, we had been limited to the synthesis of fully extended compounds as the separation of intermediates and only partially processed product mixtures is complicated by the lack of UVabsorbing chromophores in the molecule. Recently, the Boons group had reported a strategy for the synthesis of complex asymmetrically substituted multiantennary N-glycans,30 technical limitations related to reaction monitoring and purification of intermediates make this approach however less suitable for the rapid generation of compound libraries.31 During the preparation of this manuscript, the Wang group published the synthesis of a collection of N-glycans employing a similar strategy.32 11462


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Analytical Chemistry Scheme 2. Chemoenzymatic Synthesis of a Library of 13C-Enriched Bi-Antennary Glycansa

(A) Enzymatic derivatization of semi-protected scaffold 2, (B) product mixture after incubation of 2 with β-1,4 galactosyltransferase, (C) product mixture after incubation of 2 with N-acetyl-glucosaminidase, (D) preparative chromatographic fractionation of product mixture 4−6, (E) preparative chromatographic fractionation of product mixture 2, 11−13. Reagents and conditions: (a) β-1,4-GalT, UDP-galactose, alkaline phosphatase, MnCl2, HEPES; (b) 10% Pd/C, H2; (c) GDP-fucose, α-1,6-FucT, MnCl2, MES; (d) β-1,4-N-acetyl-glucosaminidase, NH4OAc buffer, r.t.; (e) CMP-NeuAc, α-2,3-SialylT, MgCl2, Tris·HCl. a

Partially protected glycan 11 was incubated with a bacterial α2,3 sialyltransferase (SialT) from Pasteurella multocida to provided the sialylated glycan 14 which was purified and hydrogenated to arrive at the fully deprotected compound 17. This glycan was fucosylated with our recombinant FucT to produce 18 and further galactosylated on the 3-arm to arrive at 19 in pure form. On the other hand, unprotected biantennary glycan 3 was enzymatically elongated to arrive at the core-fucosylated glycan 20. Bis-galactosylation with bovine GalT produced the standard 21. This was further treated with the SialT to give rise to the core-fucosylated bis-sialylated glycan 22. Finally, the hydrogenolysis of compound 11 provided glycan 23 which was treated with recombinant FucT to produce the core-fucosylated standard 24. Purity and identity of all isotopically enriched standards was confirmed by NMR and MALDI-TOF MS (see Supporting Information). With only a small number of enzymatic transformations and the deconvolution of reaction mixtures by PADS, we have prepared a library of 16 pure and stable isotope labeled N-glycans as internal standards for the absolute glycan quantification by mass spectrometry (Schemes

Chemo-Enzymatic Synthesis of Glycan Standards. We were also keen to avoid the use of temporary fluorescent tags for the purification of glycan mixtures as this ads several additional steps to the overall process including an often lowyielding tagging step31,33 and therefore investigated the enzymatic derivatization of the partially protected glycan 2, which is easily monitored thanks to five UV-absorbing benzylic ethers present in the molecule (Scheme 1), a process we have termed Protecting Group Aided Detection and Separation (PADS). Scheme 2D shows the HPLC profile for the reaction of β-1,4-N-acetylglucosaminidase on benzylated substrate 2, producing structures that either lack a single (4, 5) or both GlcNAc residues (6). Removal of the benzylether functions through traceless hydrogenation furnished unprotected standards 7−9. Only 7 was a substrate for the recombinant α-1,6 fucosyltransferase (FucT)34 giving rise to glycan standard 10. After careful adjustment of the reaction conditions, the enzymatic extension of 2 with bovine β-1,4-galactosyltransferase (GalT) produced nearly equimolar amounts of the benzylated glycans 11−13. Again hydrogenation produced cleanly the deprotected isotope-labeled glycans 15 and 16. 11463


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Figure 1. MALDI-TOF MS spectra of IgG1 glycan profile in the presence of internal standards (marked with an asterisk).

Table 1. Absolute and Relative Quantification of 8 Glycans Present in Monoclonal IgG1 Sample day 1 (n = 10) M5 MGnF G0 G1 G2 G0F GIF G2F

day 2(n = 10)

day 3(n = 10)

interday(n = 3)

relative quantiation (I.S.)

nmol/mL

S.D.

% CV

nmol/mL

S.D.

% CV

nmol/mL

S.D.

% CV

nmol/mL

S.D.

% CV

%

S.D.

% CV

3.6 4.2 13.5 13.4 2.5 111.8 152.0 36.1

0.2 0.2 0.8 0.5 0.4 1.9 3.6 1.7

5.3 3.9 6.0 3.5 13.8 1.7 2.4 4.6

3.6 4.4 16.0 13.4 2.7 114.3 154.5 35.8

0.2 0.3 0.4 0.5 0.4 2.1 3.3 1.7

6.6 6.5 2.3 3.5 13.4 1.8 2.2 4.8

3.6 4.3 15.2 13.7 3.0 111.9 153.3 34.6

0.3 0.3 1.2 0.5 0.4 2.8 1.9 1.8

9.6 6.2 7.9 3.4 13.1 2.5 1.3 5.1

3.6 4.3 14.9 13.5 2.7 112.6 153.3 35.5

0.0 0.1 1.3 0.2 0.2 1.4 1.3 0.8

0.7 2.8 8.7 1.4 8.1 1.3 0.8 2.3

1.1 1.3 4.4 3.9 0.8 33.1 45.0 10.4

0.0 0.0 0.3 0.1 0.1 0.1 0.2 0.3

1.7 1.6 7.6 2.2 8.0 0.4 0.4 2.6

cleavage with the peptide N-glycosidase F (PNGase F) to account for any loss of glycans in the sample preparation process. After release from the protein, the glycans were isolated by ultracentrifugation and analyzed directly by MALDI-TOF MS (Figure 1). Sodium citrate was added to the sample to ensure homogeneous adduct formation. Figure 1 shows the IgG1 glycosylation profile in the presence of the 13C-enriched glycan standards and a close-up for two individual analyte−standard pairs. The significant mass increment of 8 Da over the natural compound results in a clear separation of the isotopic profiles of analyte and standard which is important for an unambiguous glycan quantification that may utilize any or all detectable isotopologues as internal standards. Sample glycan concentrations were determined by (1) analyzing the isotopic profile for every standard to determine between which isotopologues the stringent linearity criteria is fulfilled and thus avoid the use of peaks with inappropriate signal intensity, (2) selecting the analyte isotopologue with

1 and 2). This collection of heavy-isotope labeled standards 3, 7−10, 15−24, 23−25 (Schemes 1 and 2, see the Supporting Information for synthesis of 25) covers all major Fc-glycans of antibodies produced in Chinese hamster ovary (CHO) cells which are being extensively employed for the expression of therapeutic mAbs.35 For their application as internal standards, we prepared stock solutions for every glycan and determined their concentration by quantitative NMR with a certified internal NMR standard. A preformulated solution containing the eight standards 3, 10, 15, 16, 20, 21, 24, and 25 (Scheme 2) selected to cover all major mAb glycan structures was prepared by pooling aliquots of the individual stock solutions. Absolute Glycan Quantification by Mass Spectrometry. With the labeled and quantified standard solutions in hand, we optimized the sample preparation and the MALDI-TOF MS acquisition method on a therapeutic IgG1 reference sample. An aliquot of the solution containing the eight standards was spiked to the purified IgG sample prior to the enzymatic glycan 11464


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Figure 2. Glycan profile during 14-day fermentation (a) relative and (b) absolute glycan quantification over time After glycan cleavage on IgG1 at 4 different concentrations with PNGase F the released glycans were transferred to a second 96-well plate by centrifugation, dried, and resuspended with matrix solution for quantification by MALDI-TOF MS. Table 2 shows the excellent reproducibility between samples prepared in different wells.

highest intensity that falls within the intensity covered by the range of linearity, (3) integrating signal peak areas of the selected analyte, (4) calculating the analyte glycan concentration from the linear regression function (for a detailed explanation of the exact quantification algorithm see the Supporting Information). To automate the quantification of glycans, particularly for the batch analysis of samples from processed mass spectrometry acquisition data, a dedicated software application based on the same algorithm was used. Table 1 shows values for both relative and absolute quantification of IgG 1 glycans and the method precision for 10 sample acquisitions on 3 days. Excellent run-to-run and sample-to-sample reproducibility with coefficients of variation of 0.5−5% and below 15% for glycans present below 1% were obtained in all experiments. Employing a dilution series on a second batch of IgG1, we determined the limit of quantification (LOQ) and limit of detection (LOD) to 0.1−0.4 pmol and 0.014−0.16 pmol, respectively, depending on the analyte (Figure S1, Supporting Information). Glycan analysis by quantitative MALDI-TOF MS outperformed other profiling methods in analysis time,

simplicity, quantification and structural information content at a precision comparable to 2-AB labeling making this method particularly appealing for high-throughput applications in process development.36 As the glycosylation profile of a biopharmaceutical drug depends both on the selected clone producing the protein as well as conditions of fermentation, protein glycosylation is an increasingly important critical quality attribute in process development and manufacturing that requires careful monitoring.37,38 To study the effect of fermentation time and protein titer on the glycosylation profile, we analyzed 14 samples taken daily from a 14 day CHO cell culture batch expressing a recombinant IgG1 (Figure 2). Figure 2 shows that the glycan profile is clearly dominated by the two glycans G0F and G1F with changing ratios throughout the fermentation process. All other glycans including low-abundance structures Man5, MGnF, and G2 could be quantified in most samples at concentrations as low as 12 pmol/mL. Figure 2A shows the absolute quantification of glycans during the fermentation process which is well aligned with the mAb titer. Rapid access to quantitative data for immunogenic or drug efficacy altering 11465


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Analytical Chemistry Table 2. Absolute Glycan Quantification for a Therapeutic mAb at 4 Different Concentrations 90 μg mAb (3 samples) glycan

50 μg mAb (3 samples)



pmol

S.D

% CV

Rel. %

pmol

S.D

% CV

Rel. %

pmol

S.D

% CV

Rel. %

Man5 MGnF G0 G0F G1 G1F G2 G2F

18.9 20.3 50.5 508.8 40.3 543.9 14.9 98.6

1.3 2.9 8.4 42.9 2.8 24.1 3.7 10.4

6.9 14.2 16.6 8.4 7.0 4.4 24.9 10.5

1.5 1.6 3.9 39.3 3.1 42.0 1.1 7.6

9.3 8.2 30.0 284.9 25.0 301.3

0.1

1.3

6.0

25.7 2.7 5.8 2.6

4.7 3.1 11.4 128.7 10.1 132.9

0.3

7.7 7.7 1.4 7.9

1.3 1.1 4.2 39.8 3.5 42.1

2.5 5.0 1.9 2.7

21.6 3.9 18.7 2.0

1.5 1.0 3.6 40.7 3.2 42.0

6.1 61.6 7.2 64.5

0.2 1.5 1.5 1.5

2.7 2.4 20.9 2.3

3.9 39.9 4.7 41.7

57.7

5.1

8.8

8.1

25.2

3.7

14.7

8.0

14.4

1.9

13.0

9.3

total glycan

1296.0

716.4

316.1

pmol

S.D

% CV

0.7

Rel. % 0.5

154.6

standards that cover the most relevant variations in antennae, monosaccharide composition, core substitutions, and charge of the mammalian N-glycome. In this line, efforts toward the synthesis of labeled larger multiantennary glycans are well under way. In conclusion, preformulated solutions of 13C labeled glycans are finally accessible for the absolute and rapid quantification of glycans in biopharmaceutical drug development and for the development of economic and quantitative mass spectrometry based glycan biomarker assays. As these standards are compatible with protocols for the derivatization of free reducing oligosaccharides their use as internal standards will also improve the precision and accuracy of LC−MS methods and allow the quantification of isomers. Finally, as most mammalian glycans including O-glycans and glycolipids contain at least a single hexosamine residue, we anticipate that this method will be extendable to other glycan classes.

glycans by MALDI-TOF MS could help in the formulation of drugs and the decision on batch acceptance or rejection. The absolute quantification of glycans by isotopic dilution also increases confidence in the relative glycoprofiling results. The control and optimization of glycan profiles is increasingly monitored during clone selection and process development and hence high-throughput methods for profiling protein glycosylation with high reproducibility are required to handle the large sample numbers. For this purpose, we evaluated a 96-well format for sample preparation and mass spectrometry acquisition using ultrafiltration plates. Our initial trials on IgG1 showed an important loss of material of up to 40% due to unspecific binding of the protein to the filtration device making it inaccessible to the PNGase F for glycan cleavage, which was readily quantified by our method but would have gone unrecognized by a relative quantification method (Figure S2, Supporting Information). Passivation of the plates with a BSA solution prior to their use, however, avoided unspecific binding and permitted glycan recovery of close to 100% (Table 2). While a conventional relative glycan profiling method would have missed the loss of product in the sample preparation step, this example highlights the value of a rapid and absolute glycan quantification method to quantify material recovery in all process steps including sample preparation for analytical purposes. Employing our quantitative MALDI profiling method, 96 samples can be analyzed in less than 3 h compared to several days of analysis time employing a conventional 2-AB labeling method.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03135. Procedures for the chemical synthesis of synthetic precursor glycans, explanatory figure for LOD and LOQ determination, and algorithms employed for the quantification of nonlabeled glycans via isotopic dilution (PDF)

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CONCLUSIONS By employing 13C-enriched glycans as internal standards we have turned qualitative glycan profiling by MALDI-TOF into a rapid and robust method for glycan identification and absolute quantification. Stable-isotope enriched standards for all major biantennary structures were prepared by chemo-enzymatic derivatization of a single synthetic biantennary glycan precursor. The enzymatic extension of intermediates, partially protected by benzylether protecting groups, permitted the facile fractionation of even asymmetrically substituted structures by preparative HPLC. For the relative glycan quantification from absolute values the quantification of all or nearly all analytes present in a given mixture is required. For simple profiles this can be achieved as we have shown here by quantifying every glycan with its corresponding heavy isotopologue internal standard. For more complex mixtures where this would not be a practical approach, individual glycans will be quantified by cross-quantification with a reduced number of reference

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Manuel Martin-Lomas for many helpful discussions and for proofreading the manuscript. Funding is acknowledged from The Spanish Ministry of Economy and Competiveness (MINECO, CTQ2011-27874 grant) and the Government of the Basque Country, (Etortek Grant 2014-15)

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REFERENCES

(1) Dalziel, M.; Crispin, M.; Scanlan, C. N.; Zitzmann, N.; Dwek, R. A. Science 2014, 343, 1235681. (2) Solá, R. J.; Griebenow, K. BioDrugs 2010, 24, 9−21. (3) Cummings, R. D.; Pierce, J. M. Chem. Biol. 2014, 21, 1−15.

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