Analysis of N-glycans from a Monoclonal Antibody by Capillary Electrophoresis and Mass Spectrometry

Application Note

Biopharma

Abstract

Glycosylation is one of the most important post-translational modifi cations involving

the attachment of glycan moieties to proteins. Alterations in the glycosylation pat-

terns have a profound impact on the immunogenicity and overall biological activity.

Glycan characterization is of crucial importance to biopharma-based applications.

Various analytical techniques have been widely used to profi le the N-glycans and

face signifi cant challenges in accurate detection of low levels of glycan species.

This Application Note demonstrates the analysis of recombinant monoclonal anti-

body glycans (mAb) by employing capillary electrophoresis and mass spectrometry

(CE-MS). This method involves enzymatic cleavage of glycans from mAb by PNGase F

followed by fl uorescent (8-aminopyrene-1,3, 6-trisulfonic acid trisodium salt–APTS)

labeling of glycans, and analysis of the glycans by using an Agilent 7100 CE system

coupled to an Agilent 6520 Accurate Mass Q-TOF LC/MS. We identifi ed seven

glycans from a recombinant mAb and the relative percentage ratio of individual

glycan moieties reveals the presence of both the major and minor forms of glycan

modifi cations. This Application Note demonstrates faster separation of CE along with

the sensitive detection limits of MS indicating the coupling of CE-MS to be a success-

ful and promising alternative analytical solution to LC/MS for the characterization of

glycans from mAbs/or glycoproteins.

Authors

Suresh Babu C.V and Ravindra Gudihal

Agilent Technologies India Pvt. Ltd

Bangalore, India

2

Introduction

In recent years, monoclonal antibod-ies (mAb) have become the emerging potential protein drug candidates for biopharmaceutical industries. The drug discovery pipeline comprises a series of meticulously controlled and evalu-ated steps, demanding careful and critical monitoring of the therapeutic stability and effi cacy of the target compounds. Therefore, a compre-hensive characterization of mAbs at every stage is highly benefi cial prior to commercialization. Among various well studied protein post-translational modifi cations, glycosylation is known to play an important fundamental role in several biological processes, for example, protein degradation and transcription, infl uencing health and disease progression1. A reliable, robust and sensitive analytical technique is needed to identify the glycans attached to protein molecules of interest.

Recently, capillary electrophoresis (CE) has gained much attention in analyz-ing the glycoproteins, delivering high effi ciency separations in shorter run times. Enzymatically released glycans are labeled with a fl uorescent chromo-phore (8-aminopyrene-1,3, 6-trisulfonic acid trisodium salt–APTS, anionic) and subjected to CE separation with high sensitivity laser-induced fl uores-cence (LIF) or mass spectrometry (MS) detection. The APTS labeling imparts negative charge to glycans, aiding in enhanced electrophoretic separations and is also amenable to electrospray ionization (negative mode), making the

entire separations and detection pro-cess highly compatible with the fl uo-rescent labeling chemistry. Coupling CE to MS is benefi cial in identifying unknown and assigning the glycan modifi cation or mass information2.

This Application Note demonstrates the CE-QTOF MS analysis of N-glycans derived from recombinant mAb. The CE-MS method with APTS labeling helps to identify all glycan moieties attached to mAb. Further relative percentage ratios of each glycan were presented to show the major and minor forms of glycan modifi cations.

Materials and method

ChemicalsMonoclonal antibody, PNGase F and PVA coated capillaries were from Agilent Technologies, Inc. e-aminocaproic acid and 1 M sodium cyanoborohydride in tetrahydrofuran (THF) from Sigma-Aldrich (St. Louis, MO, USA). APTS was obtained from Molecular Probes, Invitrogen (Eugene, OR, USA). Glycan standards were purchased from Prozyme (Hayward, CA, USA). PhyTip Columns were from PhyNexus (San Jose, CA, USA).

Enzymatic deglycosylation, glycan extraction and APTS labelingDeglycosylation of mAb were per-formed using PNGase F. The mAb (15 mg/mL) was treated with PNGase F in 0.25 M Tris buffer (pH 7.6) overnight at 37 °C. The deglycosylated protein

was heat precipitated and centrifuged. The supernatant containing released glycans were dried and labeled with APTS by reductive amination. To the dried glycan sample, 2.5 µL of 50 mM APTS in 1.2 M citric acid and 2.5 µL of 1 M sodium cyanoborohydride in THF were added and incubated overnight at 37 °C in a water bath. The reaction mixture was diluted with 50 µL water to stop the reaction. Unbound excess dye was removed using PhyNexus normal phase polyamide resin contain-ing PhyTip which was conditioned with 95% ACN. After sample application, the column was washed with 95% ACN and fi nally N-glycans were eluted with 20% ACN.

CE-MS instrumentationThe CE-ESI-MS analysis was per-formed using the 7100 CE system with a CE-MS capillary cassette (G1603A) coupled to the 6520 Accurate-Mass Q-TOF equipped with dual electrospray source and orthogonal coaxial sheath liquid interface (G1607B)3. Separations and spray stability were optimized using the blank buffers and a standard. A sheath-liquid CE-MS interface with a low fl ow rate (5 µL/min) is maintained to preserve the high effi ciency sepa-ration of CE and to provide a stable fl ow and spray conditions essential for electrospray ionization. Q-TOF parameters were optimized automati-cally through MS tuning programs and the MS system was calibrated using an ESI tuning mixture.

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Table 1 shows the CE-MS parameters. The total compound list was extracted using the MassHunter molecular feature extractor (MFE) algorithm.

Table 1CE-MS conditions.

Capillary Electrophoresis (CE)

CE: Agilent 7100 Capillary Electrophoresis System

Sample: Released N-glycans from mAb

Injection: 40 seconds at 30 mbar

Capillary: PVA, total length 60 cm, 50 µm id

Buffer: 40 mM e-aminocaproic acid pH 4.5

Voltage: -25 kV

External pressure: 10 mbar

Temperature: 20 °C

Mass Spectrometry (MS)

MS: Agilent 6520 Accurate-Mass Q-TOF LC/MS

Ionization mode: ESI

Acquisition mode: MS (mass range 400–3,200 m/z)

Sheath liquid: Isopropanol:water (1:1 v/v) with 0.2% NH3 at 5 µL/min

Drying gas fl ow: 5 L/min

Nebulizer: 8 psi

Drying gas temperature: 250 °C

Fragmentor: 175 V

Vcap: 3,200 V

Accu time: 980.3 ms/spectrum

Acc rate: 1.02 spectra/s

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Results and discussion

Athough CE-LIF is routinely used for glycan analysis, CE-MS has an advantage in identifying the unknown migrating species present in the electrophoretic run providing molecular weight information. In this Application Note CE was coupled to Q-TOF MS using sheath-liquid interface for assignment of glycans mass informa-tion from mAb. Scheme 1 outlines the glycan profi ling steps for mAb using CE-MS. Briefl y, the released glycans from antibody were labeled with APTS, followed by CE-MS analysis. Figure 1 shows the CE-MS total compound pro-fi le for N-linked glycans released from recombinant mAb. Using a PVA-coated capillary, all the mAb glycans were migrated within a 15-minute separation time. We successfully identifi ed the uncharged N-linked glycan species G0, G0F, G1, G1F, G2, and G2F in replicate runs. In addition, mono-sialylated glycan moiety G2F+1NANA was also observed. All the peak assignments were based on accurate mass meas-urements from Q-TOF analysis.

Scheme 1Schematic overview of the glycoprofi ling of mAb using CE-MS.

×105

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

Acquisition time (min)

Cou

nts

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

G2F+1NANA

G0

G0F

G1

G1F

G2

Total compound electropherogram

G2F

Figure 1CE-MS of APTS labeled N-glycans released from mAb.

Glycosylated monoclonal antibody (mAb)

PNGase F

Protein precipitation

Released glycans

Labeled glycans

CE-MS

Fluorescent labeling with APTS and purification using normal phase polyamide resin

Non glycosylated mAb and released glycans

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Table 2 summarizes the glycans identifi ed in this study. Note that the G2 species was successfully identi-fi ed by CE-MS, but not with LC/MS analysis (data not shown) indicating the signifi cant advantage of CE-MS based analysis (complementary value of CE-MS).

Glycanabbreviation

Monoisotopicmeasured mass

Most abundant charge state measured

Glycanstructures

Monoisotopictheoretical mass

Relativepercentage

G0 1,757.4575 877.7212 (-2) 1,757.4512 20.8

G0F 1,903.5121 950.7484 (-2) 1,903.5091 34.0

G1 1,919.5071 958.7389 (-2) 1,919.5040 2.3

G1F 2,065.5649 1,031.7756 (-2) 2,065.5619 4.2

G2 2,081.5666 1,039.7751 (-2) 2,081.5568 3.1

G2F 2,227.6228 1,112.8041 (-2) 2,227.6147 1.2

G2+1NANA 2,518.7191 838.5664 (-3) 2,518.7101 34.4

Galactose Mannose Fucose N-acetylglucosamine Sialic acid

Table 2Summary of mAb N-glycans identifi ed using CE-MS.

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The mass spectrum for individu-ally resolved glycans is depicted in Figure 2. The most abundant charge states observed in the mass spectrum are indicated. Estimating the abso-lute/relative glycan quantity levels

Figure 2Mass spectrum of APTS labeled mAb N-glycans.

0

2

6877.7212

838.5678943.7623

1024.78871097.8227

972.2266924.6664852.5647 1258.83901134.25511073.2859 1171.2227

×103 [M-3H]3-

G2F+1NANA

0

0.5

1.5877.7199

838.8957 943.26131097.3168

1024.7865971.2223924.6579852.8991 1258.33801134.3397999.7881

1178.85061066.2195 1215.2396808.5550767.7263

×104

[M-2H]2-G0

0

1

31024.2866950.7421

1097.3127

878.2173838.55931178.8422

998.7005 1072.7867 1135.28081162.8106

923.6758808.5900768.2694 863.3876 1215.6788

×103[M-2H]2-

G0F

0

0.5

1.5 1024.7879958.7487

1097.3223

877.7156838.5629 1178.32551071.2325 1146.7222774.2202 1206.1727803.9364

×103 [M-2H]2-

G1

0

4

8 1097.8186

1178.84471031.7698

958.7413877.7165

×102

[M-2H]2-G2

1039.7614

×103

00.25

0.75

1.25 1097.3163

950.7420 1024.2871

1178.8440877.7149838.8955

Mass-to-charge (m/z)

Cou

nts

760 800 840 880 920 960 1000 1040 1080 1120 1160 1200 1240 1280

[M-2H]2- G2F

1112.7964

0

0.5

3

×103

[M-2H]2-1097.8227

1031.7764

877.7195 959.2501 1178.8469838.5697741.5310

G1F

is of paramount importance in the therapeutic product development pipe-line. Therapeutic products are sensi-tive to degradation and any minimal modifi cations. The percentage volume relative to total compound peaks

volume was estimated and relative quantifi cation of mAb released glycans are shown in Table 2. In the present case, the major form of the glycoform was found to be G2+1NANA.

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These results clearly indicate that CE-MS can be used effectively as an alternative analytical tool to moni-tor glycan profi les. CE-MS coupled separations avoid contaminations, due to minimal sample handling steps and the buffers used for CE separations are highly compatible with the electrospray ionization. The current approach is sensitive, accurate in glycan profi ling as compared to conventional CE-LIF.

Conclusions

This Application Note provides an Agilent CE-MS based solution for mAb glycan profi ling. The powerful data processing capabilities of Agilent MassHunter and BioConfi rm suite enable in successful and detailed iden-tifi cation/profi ling of the glycoforms of mAb. The glycan pattern was reason-ably resolved allowing separation of major and minor forms of glycans.

References

1.Li Y, Tao SC, Bova GS, Liu AY, Chan DW, Zhu H, Zhang H. Detection and verifi cation of glycosylation patterns of glycoproteins from clinical specimens using lectin microarrays and lectin-based immunosorbent assays. Anal. Chem., 83 (22), pp 8509–8516, 2011.

2.Gennaro LA, Salas-Solano O. On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies. Anal. Chem., 80 (10), pp 3838-3845, 2008.

3.Suresh Babu C.V and Ravindra Gudihal, “Glycopeptide Analysis of Antibodies by Capillary Electrophoresis and Q-TOF Mass Spectrometry”, Agilent publica-tion number 5990-7138EN, 2011.

www.agilent.com/chem/ce

© Agilent Technologies, Inc., 2012Published September 1, 20125991-1020EN