1

Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MSYing Qing YuWaters Corporation, Milford, MA, U.S.

IN T RO DU C T IO N

Glycosylation of therapeutic protein drugs is of particular importance because it

plays a vital role in the clinical performance of these drugs. In this work, we study

the N-linked glycans from two Coagulation Factor IX biologics that are used for

Hemophilia B treatment; one is recombinant (rFIX, BeneFIX) and the other one

is derived from human plasma (pd-FIX, Mononine). Both Factor IX proteins are

heavily glycosylated (Figure 1).1 Previous findings on their glycoforms were done

primary using orthogonal HPLC separation techniques, typically via Ion Exchange

chromatography (IEX) and hydrophilic interaction (HILIC) modes, due to the complex

nature of the Factor IX glycans. For mass profiling and structure characterization,

mass spectrometry (MS) was typically used offline for LC fractions.

Waters has developed a HILIC UPLC®/FLR/QTof MS analytical platform for

fluorescent-labeled glycan characterization. Significant improvements can be

made such as peak resolution, speed, sensitivity, and the ability to identify and

quantify even the minor glycans. An ACQUITY UPLC System is directly interfaced

to a Xevo QTof MS, eliminating the need for fractionation. Comparative analysis of

rFIX and pd-FIX glycans using this platform is demonstrated.

Figure 1. Coagulation factor IX structure.

WaT e R s sO lU T IO Ns

ACQUITY UPLC® System

ACQUITY UPLC Fluorescence Detector

ACQUITY UPLC BEH Glycan Column

Xevo® QTof MS,

SimGlycan (Premier Biosoft)

k e y W O R D s

Glycans, recombinant Factor IX protein

(rFIX), plasma derived Factor IX protein

(pd-FIX), 2-aminobenamide (2AB), sialic

acid, structure elucidation

a P P l I C aT IO N B e N e F I T s

HILIC UPLC/FLR/QTof MS is a powerful glycan

characterization tool for characterization

of complex glycans samples. Conventional

HPLC method lacks the resolution power and

sensitivity. Fractionation for MS analysis

step is eliminated since the ACQUITY UPLC

System is directly interfaced into a Xevo

QTof MS. SimGlycan Software is part of the

system solution, which interprets the collision

fragmentation data, and offers a mean to

elucidate glycan structures.

2Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS

R e sU lT s a N D D Is C U s s IO N

rFIX and pd-FIX has very different glycans profiles (Figure 3, 4). Glycans released

from rFIX are mostly fucosylated bi-, tri-, and tetra-antennary complex type

glycans. Additional lactosamine units were observed in some glycans. Man5 and

a core N-glycans with an addition of fucose were also identified (Table 1). Glycans

from pd-FIX are show more heterogeneity, especially for the larger glycans.

e X P e R IM e N Ta l

Sample

Figure 2 shows the basic glycan analysis workflow.

FIX proteins are reduced and alkylated using

DTT and IAM, followed by PNGase F enzymatic

digestion overnight to release the glycans. The

glycans are extracted using HILIC SPE device and

labeled with 2-aminobenamide (2AB) dye, and the

excess dye was removed by HILIC SPE again.2

LC ConditionsLC System: ACQUITY UPLC Detection: ACQUITY UPLC FLR Column: ACQUITY UPLC BEH Glycan Column (2.1 x 150 mm)Column temp. 60 °CSample temp. 10 °CInjection volume: 5 µLFlow rate: 0.4 mL/minMobile phase A: 100 mM Ammonium Formate (~ pH 4.3)Mobile phase B: 100% Acetonitrile (Fisher Optima)

Gradient table Time (min) Flow rate %A %B Curve Initial 0.400 39.0 70.0 60.00 0.400 50.0 50.0 6 60.10 0.250 95.0 5.0 6 63.00 0.250 95.0 5.0 6 64.00 0.300 30.0 70.0 6 65.00 0.400 30.0 70.0 6 72.00 0.400 30.0 70.0 6

MS conditionsMS system: Xevo QTOF MSIonization mode: ESI positiveCapillary voltage: 3200 VCone voltage: 30 VDesolvation temp.: 350 °CDesolvation gas: 800 L/HrSource temp: 100 °CAcquisition range: 700 to 2000 m/zCollision energies: 4 VLockmass: Cesium Iodide (CSI): 1 µg/µL in 50% MeCN

Data ManagementMassLynx 4.1SimGlycan v. 2.9 (Premier Biosoft)

Figure 2. Glycan analysis workflow.

Protein reductionand alkylation

PNGase F digestion to release N-linked glycans

Fluorescent label with 2AB

HILIC UPLC / FLR / QTof MS/MS analysis

Only the complex type glycans with polylactosamine units were observed

from pd-FIX; the level of fucosylation is from 0 to 2 (Table 1). Two sulfated

bi-antennary glycans were observed, which was not reported in the literature;

these sulfated glycans were well resolved from their non-sulfated counterpart

(Figure 4). The possibility of phosphorylation was excluded by alkaline

phosphatase reaction (data not shown). In addition, MS/MS fragmentation was

used to further confirm their identification (see Figure 5).

3Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS

Peak No. Glycan rFIX (BeneFIX)

pd-FIX (Mononine)

Observed MW

Theoretical MW

1 Man3 + 1F + — 1176.456 1176.455

2 G0F - GN + — 1379.534 1379.534

3 G0F + — 1582.612 1582.674

4 Man5 + — 1354.511 1354.502

5 1A/1F - GN + — 1541.587 1541.587

6 2A/1F + — 1906.710 1906.719

7 2A/1F + GN + — 2109.760 2109.798

8 2A/1F/1S + — 2197.816 2197.814

9 3A/1F + — 2271.840 2271.850

10 2A/2S — + 2342.880 2342.851

11 2A/2S/SO3 — + 2422.814 2422.808

12 2A/1F/2S + + 2488.912 2488.909

13 3A/1F/1S + — 2562.924 2563.945

14 2A/1F/2S/SO3 — + 2568.868 2568.866

15 4A/1F + — 2636.962 2637.982

16 3A/1F/2S + — 2854.042 2854.042

17 4A/1F/1S + — 2928.066 2928.07

18 3A/3S — + 2999.112 2999.079

19 3A/1F/3S + + 3145.112 3145.137

20 4A/1F/2S + — 3219.134 3219.174

21 3A/2F/3S — + 3291.188 3291.195

22 4A/1F/3S + — 3510.224 3510.269

23 4A/4S — + 3655.277 3655.306

24 4A/1F/4S + + 3801.358 3801.365

25 4A/1F/3S +(GN+Ga) + — 3875.401 3875.402

26 4A/2F/4S — + 3947.373 3947.423

27 4A/1F/4S +(GN+Ga) + — 4166.418 4166.497

28 4A/1F/3S +2(GN+Ga) + — 4240.533 4240.533

29 4A/1F/4S +2(GN+Ga) + — 4531.617 4531.629

30 4A/2F/2S + 2(GN+Ga) — + 4677.610 4677.687

31 4A/3F/4S + 2(GN+Ga) — + 4823.694 4823.745

32 4A/1F/4S + 3(GN+Ga) + — 4896.783 4896.761

Table 1. A complete list of 2AB labeled glycans observed from rFIX and pd-FIX. Complex type glycan with various degree of sialylation along with Man5 and Man3/1F were observed. The average mass error is less than 5 ppm.

N-Acetylglucosamine (GlcNAc)

Manose

Galactose

Fucose

Sialic Acid

GN = GlcN AcGa = Gal a tose

Example of Glycan Nomenclature: 4A/2F/4S

4Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS

Time (minutes)

10 20 30 40 50

(1) ACQUITY FLR ChA Ex330,Em420 nmRange: 73630

1: TOF MS ES+BPI61

10 }

11

12

14

1819 } }}

21

23

24

26

}30, 31

Figure 4. HILIC UPLC/FLR/MS of 2AB labeled pd-FIX (Mononine) glycans. More glycans were identified, such as sulfated glycans and multiply fucosylated glycans. The possibility of phosphoryla-tion was excluded by alkaline phosphatase reaction (data not shown). In addition, MSMS fragmentation was used to further confirm their identifi-cation (see Figure 5 A-C).

The glycan profile from both rFIX and pd-FIX is very complex. HPLC-based methods lack the resolution needed

to identify and quantify various glycan forms. The ACQUITY UPLC System coupled with the ACQUITY UPLC

BEH Glycans Column alone is able to achieve baseline separation of glycans that are different in mass and

degree of sialylation; terminal sialic acid isomers are also well separated.

The accurate mass measurement from the Xevo QTof MS offers confident assignment of the glycans. MS/MS

fragmentation along with SimGlycan Software gives further confirmation on glycan structure. Figure 6 shows

that glycan 4A/1F/4S was observed from both rFIX and pd-FIX; the LC retention time is the same for the isobaric

tetra-antennary glycans. However, the CID fragmentation showed distinct difference, which resulted from the fucose

location on the glycans (Figure 5B). Figure 7 shows another example on how CID was used to differentiate isobaric

doubly fucosylated glycans from both rFIX and pd-FIX glycans, again distinct fragment ions were used to differentiate

the location of fucose. Biological influence caused by the location of fucose was documented in the literature.

Figure 3. HILIC UPLC/FLR/QTof MS of 2AB labeled rFIX (BeneFIX) glycans. Low abundant glycans in the low mass region were observed and confirmed by MS analysis. In addition, isomeric glycans (sialic acid positional isomers) were well resolved. The peak number corresponds to the glycan listed in Table 1.

Time (minutes)

10 20 30 40 50

(1) ACQUITY FLR ChA Ex330,Em420 nmRange: 1214421

1: TOF MS ES+BPI305

1 2 34 5 6 7

89

1213

1516

17

19

2022

24

25

27

2829

32

} } } } } } }

5Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS

Pd-FIX

Pd-FIX

mass200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

1: TOF MSMS 1172.40ES+3.67e3657.216

366.132

204.083292.100 528.176

454.142

1687.5751313.311

1031.364869.311

819.256 893.279 1234.4411055.335 1396.476

1525.5182343.777

2052.687

3: TOF MSMS 1212.40ES+333

657.218

366.135

204.085342.159 528.175

1687.5231313.333

1031.348819.258869.253

1212.4981396.494

1525.497 2343.7722052.6231737.618

2424.649

(M+H)+

(M+H)+- SO3

SO3

2A/2S

2A/2S/SO3

A)

B)

Figure 5. MS/MS fragmentation of sulfated glycans such as 2A/2S/SO3 were compared with its non-sulfated counterpart, 2A/2S. Facile loss of SO3 (-80 Da) was observed. The MS/MS data, after MaxEnt3 mass deconvolution, was submitted to SimGlycan software for structure assignment; and SimGlycan validated the proposed structures.

Figure 6. Differences in fucosylation were observed for rFIX and pd-FIX. Fucose from rFIX was located at the first GlcNAc residue in the core structure; while the majority of the fucosylation site for singly fucosylated pd-FIX glycans was located at the antenna. Fragment ions at m/z 488 and 803 were the diagnostic ions for probing the fucosylation sites.

mass200 400 600 800 1000 1200 1400 1600 1800

4: TOF MSMS1901.60ES+2.15e3x10 657.2424

454.1565366.1502

204.0931 1901.6008

7: TOF MSMS 1901.60ES+294

x10 657.2350

366.1260

204.0841 803.2665 1688.5410

Tag

(M+2H)2+

4A/1F/4S

4A/1F/4S

rFIX

Pd-FIX

A)

B)

6Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS

Figure 7. Some doubly fucosylated glycans were observed for pd-FIX sample. The two diagnostic ions at m/z 488 and 803 were observed, which indicated that one fucose was at the first GlcNAc residue (core structure) and the other one was located at the antenna.

3A/2F/3S

3A/1F/3S

Pd-FIX

Pd-FIX

mass200 400 600 800 1000 1200 1400 1600 1800

6: TOF MSMS 1646.50ES+183x5 657.2099

366.1381

204.0750292.0992

488.2148803.2485

1647.06621445.64511184.3798870.3103 1834.60141979.5824

5: TOF MSMS 1573.50ES+468x5 657.2099

366.1381

204.0841

292.0992

528.1824

454.1430

803.2485

707.2417 869.3013 1688.59301031.3752 1397.49001234.3911

Tag

(M+H)+

(M+H)+

A)

B)

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, ACQUITY UPLC, UPLC, and Xevo are registered trade-marks of Waters Corporation. T he Science of What’s Possible is a registered trademark of Waters Corporation. All other trademarks are the property of their respective owners.

©2011 Waters Corporation. Produced in the U.S.A.June 2011 720004019en AG-PDF

References

Yasushi Makino, Kaoru Omichi, Nahoki Kuraya, Hideyuki Ogawa, 1. Hitoshi Nishimura, Sadaaki Iwanaga and Sumihiro Hase. J. Biochem. 128, 175-180 (2000).

Ying Qing Yu, Martin Gilar, Jennifer Kaska and John C. Gebler. 2. Deglycosylation and Sample Cleanup Method for Mass Spectrometry Analysis of N-linked Glycan. Waters Application Note. 2007. 720001146EN.

CO N C lU s IO N

Blood protein glycan characterization is known to be very

challenging for scientists working in the biopharmaceutical

field, since the glycans that attach to the protein backbone are

highly heterogeneous and complex. Our solution for complex

glycan separation and characterization is the UPLC/FLR/QTof MS

analytical platform.

In addition to shortened run time enabled by ACQUITY UPLC

technology, HILIC UPLC also offers significant improvement in peak

resolution compares to conventional HPLC method; for example,

positional sialic acid isomer separation is achieved, also the

separation of sulfated and sialyated glycans were observed.

MS/MS fragmentation and database search using SimGlycan

Software helped the glycan structure elucidation, adding more

confidence to glycan structure assignment.

The ACQUITY UPLC/FLR/Xevo QTof MS analytical platform

along with SimGlycan Software is a powerful and versatile tool

for complex glycan characterization. Glycan profiling, mass

confirmation, and structure elucidation can all be done in a single

LC/MS system. High quality data are generated with shorter

analysis and data interpretation time. This is a valuable tool for

researchers working with glycoprotein drugs.