TECHNICAL NOTE

Technical Note Bulletin 4007 Rev C

Biotherapeutic

Rituximab

Cetuximab

Trastuzumab

Fluorescent Dye

N-Glycan Labeling

HILIC

Mass spectrometry

Alpha gal

UHPLC

InstantPC

Keywords

High Throughput Analysis of N-Glycans Released from Biosimilar GlycoproteinsZoltan Szabo1, Shanhua Lin1, James Thayer1, Rosa Viner2, Andreas Huhmer2, Gina Tan2, Yury Agroskin1, Dietmar Reusch3, John Yan4, Aled Jones4

1 Thermo Fisher Scientific, Sunnyvale, CA; 2 Thermo Fisher Scientific, San Jose, CA; 3 Roche Diagnostics GmbH, Penzberg, Bavaria; 4 ProZyme, Hayward, CA

SUMMARY

• Analysis of enzymatically-released N-glycans with hypdrophilic interaction liquid chromatography typically involves the addition of a fluorescent dye

• Gly-X with InstantPC N-glycan sample preparation is used to rapidly prepare InstantPC-labeled N-glycans from 4 monoclonal antibodies

• InstantPC-labeled N-Glycans are separated by hydrophilic interaction liquid chro-matography (HILIC) and detected by fluorescence and mass spectrometry (MS)

• Fluorescence and MS properties of InstantPC dye allow for robust quantitation and identification of N-glycan species

• MS1 and MS2 are utilized to characterize glycans including those containing potentially immunogenic non-human Galα1,3-Gal (alpha-Gal) epitope and N-glcy-olylneuraminic acid (NGNA)

Gly-Q

Gly-X

GlykoPrep

Glyko Enzymes

Glyko Standards

InstantPC

InstantAB

InstantQ

2-AB

APTS

PhycoLink

PhycoPro

RPE & APC Conjugates

Streptavidins

2

Figure 1: Gly-X with InstantPC N-glycan sample preparation workflow.

Figure 2: InstantPC (instant procaine) dye structure and conjugation with glycosylamine. Mass of Instant-PC-Labeled Glycan = Mass of Glycan (free reducing end) + C14N3O2H19 (261.14773 Da, monoisotopic).

Table 1: HILIC gradient details.

METHODS AND MATERIALS

Materials: 1. MabThera (rituximab), lot #B60055B01

2. Trastuzumab biosimilar (Epirus)

3. Erbitux (cetuximab), lot #10C00061B

4. mAb R (Roche)

Sample PreparationN-glycan samples were prepared using ProZyme’s Gly-X N-Glycan Rapid Release and Labeling with InstantPC Kit (Figure 1) using 40 μg material. The work-flow uses a 5-minute in-solution diges-tion with PNGase F to release N-glycans, followed by labeling with InstantPC (Figure 2) to allow for enhanced FLR and MS performance (4), and excess label is removed with a vacuum-driven cleanup plate, with a total sample preparation time of around 1 hour.

INTRODUCTION

Monoclonal antibodies (mAbs) account for around half of all marketed biopharmaceuticals, and are also the focus of investigations into fol-low-on biosimilar and biobetter molecules (1, 2). The structure of mAb N-linked glycans can potentially affect immunogenicity, pharmacokinetics and pharmacodynamics (3), making the characterization of N-glycans an essential part of the biotherapeutic development process. Analysis of N-glycans typically involve the labeling of enzymatically-released glycans with a tag to allow for fluorescence (FLR) detection; a process that often requires numerous hours or days to complete. In addition to fluorescence detection, mass spectrometry is also often utilized. Unfortunately, many of the commonly-used fluorescent tags are limited with regard to MS sensitivity. Here we present a workflow for rapid sample preparation of N-glycans released from mAbs using ProZyme’s Gly-X platform with In-stantPC label, coupled with analysis using Thermo Fisher Scientific LC-MS instrumentation and consumables.

HILIC ChromatographyN-Glycans labeled with InstantPC were separated with hydrophilic interac-tion liquid chromatography (HILIC). Injection volumes were 1 μl eluent for FLR and MS1 experiments, and 10 μl eluent diluted with appropriate organic solvents for MS2. FLR and MS1 data were interpreted with Chromeleon (Thermo Fisher Scientific).

Instrument: Dionex Ultimate 3000 UHPLC system equipped with Dionex Ultimate 3000 XRS Autosampler and fluorescence detector.

Column: Accucore-150-Amide-HILIC (150 mm x 2.1 mm), particle size: 2.6 μm (Thermo Fisher Scientific), column temperature 45 °C, gradient details listed in Table 1.

MS ConditionsAll MS experiments were performed on a Thermo Scientific Q Exactive mass spectrometer in positive mode ESI, operated at full mass scan: m/z 400-2000 at a resolution of 70,000 with automatic gain control (AGC) at 3 x106 with maximum IT: 150 ms. Data dependent MS2 was acquired for the top 5 ions at a resolution of 17,500, the AGC target was set at 1 x105, injec-tion time was 300 ms. Isolation window was set at 2 m/z. Stepped collision energy was applied at 28, 30 and 32. Xcalibur software (Thermo Fisher Sceintific) was used for data acquisition and MS2 spectra were interpreted manually and with SimGlycan 5.6 software (Premier Biosoft).

Labelwith

InstantPC 1 min, 50°C

Data AnalysisUHPLC,MS/MS

Cleanup96-well

plate15–20 min, RT

Denature40 µg

Glycoprotein3 min, 90°C

ReleaseN-glycans with

PNGase F5 min, 50°C

Tertiary amine

Glycan-NH2

ON

NNH

O

O

O

OProcaine

O

ON

GlycanNH

O

O

NH

NOH

O

O

+

Time (min)

Flow (ml/min)

% A: 100 % acetonitrile (ACN),

LC-MS grade

% B: 50 mM ammonium

formate, pH 4.40 0.4 73.0 27.0

45.0 0.4 59.0 41.045.1 0.4 45.0 55.048.9 0.4 45.0 55.049.0 0.4 73.0 27.058.0 0.4 73.0 27.0

3

RESULTS

HILIC-FLR analysis of InstantPC N-glycans with MS1• Major N-glycan species from were separated for rituximab (Figure 3), trastuzumab biosimilar (Figure 4),

cetuximab (Figure 5) and mAb R (Figure 6).

• Figures 3–6A shows the total ion chromatogram (TIC), Figures 3–6B show the FLR trace. The high FLR and MS response of InstantPC in positive mode enhanced by the tertiary amine (4) allows these two profiles to be compared, and e.g. Man5 to be identified from mass spectra (Figure 7).

• Table 2 shows reproducibility of the sample preparation for relative % area of glycan peaks for ritux-imab (n=5).

• The sample prep gives consistent results for 16–128 μg rituximab for flexibility of input amount (Figure 8).

• The low pressure of the 2.6 μm column allows use with HPLC as well as UHPLC systems.

Figure 3: Rituximab N-glycans labeled with InstantPC and separated by HILIC. (A) fluorescence detection (FLR), (B) total ion chromatogram (TIC).

5 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40-1.0e5

2.5e6

5.0e6

7.5e6

1.0e7

1.3e71.4e7

Minutes

0.0e0

5.0e6

1.0e7

1.5e7

2.0e7

2.5e7

3.0e7

G0

G0F

Man5G1[6]

G1F[6]

G1F[3]G2F

A2FG0F-N A1F

A) TIC

B) FLR

4

Figure 4: Cetuximab N-glycans labeled with InstantPC and separated by HILIC. (A) fluorescence detection (FLR),(B) total ion chromatogram (TIC).

5 6 8 10 12 14 16 18 20 22 24Minutes

26 28 30 32 34 36 38 40-1.0e5

2.5e6

5.0e6

7.5e6

1.0e7

1.3e71.4e7

-5.0e6

0.0e0

5.0e6

1.0e7

1.5e7

2.0e7

2.5e7

hybrid (hex7HexNAc4F1S1)

NA3F+3aGal

NA3FS1+2aGal(NGNA)

NA3F+aGal +F

A1F +aGal(NGNA)

G2F+2aGal

G2F+aGal +FG2F+aGal

Hex6N3F

G2F

G0F-N

G0F

Man5

G1F[6]

G1F[3]

A) TIC

B) FLR

G1F+aGal

Figure 5: Trastuzumab biosimilar N-glycans labeled with InstantPC and separated by HILIC. (A) fluorescence detection (FLR), (B) total ion chro-matogram (TIC).

5 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e60.0e0

2.0e6

4.0e6

6.0e6

8.0e6

1.0e7

1.2e7

1.4e7

1.6e7

G0

G0F

Man5 G1F[6]G1F[3]

G2FG0F-N

-1.0e5

Minutes

G0-N

A) TIC

B) FLR

5

Figure 6: mAb R N-glycans labeled with InstantPC and separated by HILIC. (A) fluorescence detection (FLR), (B) total ion chromatogram (TIC).

FucoseGalactose

Structure Key:

Mannose N-Acetylglucosamine N-Glycoylneuraminic acid (NGNA)

N-Acetylneuraminic acid (NANA)

5 6 8 10 12 14 16 18 20 22 24Minutes

26 28 30 32 34 36 38 40-1.0e5

1.0e7

2.0e7

3.0e7

4.0e74.5e7

-1.0e7

0.0e0

1.3e7

2.5e7

3.8e7

5.0e7

6.0e7

G0

G0F

Man5 G1[6]

G1F[6]

G1F[3]G2

A2A1Man6

M5N3G1F M5N3G1S1

G2F

A) TIC

B) FLR

Figure 7: Identification of Man5 from rituximab. A) TIC, B) mass spectrum. Error (ppm) = (observed mass - theoretical mass) / theoretical mass x 106.

1,492.33 1,493.75 1,495.00 1,496.25 1,497.50 1,498.75 1,500.00 1,501.25 1,502.50 1,503.75 1,505.29-1.4

12.5

25.0

37.5

50.0

62.4

1496.5938

1497.5972

Apex5 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

-1.0e5

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

Theoretical monoisotopic mass Observed Di�erence ppm

Man5 1496.5884 1496.5938 0.0054 3.63

A) TIC

B) Mass spectrum

6

HILIC separation of InstantPC N-glycans with MS2• Figures 9 and 10 show a workflow to characterize glycan species from cetuximab

and mAb R, from FLR trace to extracted ion chromatogram (XIC) to MS2 analysis in positive mode.

• Note: [3] arm position of NGNA on A1F +aGal(NGNA) is based on (5).

• Alpha-gal and NGNA-containing structures such as A1F + aGal(NGNA) in cetuximab (Figure 9) are most likely from the Fab region (6), where Erbitux produced in the mouse myeloma SP2/0 cell line is known to contain the alpha-gal N-glycan epitope.

• Hybrid-type N-glycan M5N3G1S1 is identified on mAb R (Figure 10).

Figure 8: N-Glycan relative % area (FLR) for 16–128 μg of rituximab. Error bars represent %CV (n=3).

0

10

20

30

40

50

60

G0F-N G0 G0F Man5 G1 [6] G1F[3]G1F[6] G2F A1F A2F

Rela

tive

% A

rea

16 µg32 µg64 µg128 µg

Glycan Average %CVG0F-N 0.80 5.00

G0 1.61 6.65G0F 49.45 1.60

Man5 1.33 8.10G1[6] 0.86 18.27

G1F[6] 27.51 1.06G1F[3] 11.24 5.56G2F 6.08 5.61A1F 0.49 34.83A2F 0.62 8.33

Table 2: Relative % area for N-glycans released from rituximab (n=5).

7

0 5 10 15 20 25 30 35 40 45 50Minutes

20

40

60

80

100

Rela

tive

Abun

danc

e

50015002500350045005500

Emis

sion

Cou

nts 31.12 mins

A) FLR

B) XIC

1257 1258 1259 1260 1261 1262 1263m/z

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

Rel

ativ

e Ab

unda

nce

1259.9793

1259.4788

1260.4804

1260.9816

1261.48281261.9829

C) Mass spectrum

[M+2H]2+

Accuracy: 0.84 ppmTheoretical mass: 1259.4777Observed mass: 1259.4788

A1F + aGal(NGNA)

400 800 1200 1600 2000m/z

0

10

20

30

40

50

60

70

80

90

100 528.1902

673.2362

366.13881683.6791

1845.7708

204.08761259.9685629.3107

1990.6656835.3023 1105.9299 1522.58611318.51761890.6218

483.2485290.0903 996.4090

IPC

1537.6002

Y6

Y6 Y4

Y1

Y4α

Y3

Y3

0,4X5/B6

Y3/Y4α

B3α Y1β

Y4α

Z4

Y3/Y4α

Y4/Y4α

1156.5138

B4

B5/Z4

B3B3 B4

B5

Y1

B3α

B6/Y2

1,1X0 Y1/Y1β

B4/Y5

D) MS2

Figure 9: Characterization workflow for alpha-gal and NGNA-containing N-glycan A1F +alpha Gal(NGNA) from cetuximab with MS2.

Hayward, California

Advancing GlycosciencesToll Free +1 (800) 457-9444 Phone +1 (510) 638-6900 Fax +1 (510) 638-6919 Website www.prozyme.com Technical info@prozyme.com Orders orders@prozyme.com Virtual Patent Marking www.prozyme.com/patents

References1. G. Walsh, Nat. Biotechnol. 32(10), 992–1000 (2014).

2. D.M. Ecker et al. mAbs 7(1), 9–14 (2015).

3. L. Liu, J. Pharm. Sci. 104(6), 1866–1884 (2015).

4. M. Kimzey et al. Development of an Instant Glycan Labeling Dye for High Throughput Analysis by Mass Spectrometry. Poster presented at ASMS, St. Louis MO (2015)

5. A. Wiegendt & B. Meyer, Anal Chem. 2014 86(10):4807-14 (2014)

6. D. Ayoub et al. mAbs 5(5), 699-710 (2013)

ProZyme, Gly-X and InstantPC are registered trademarks of ProZyme, Inc. in the United States and other countries. Thermo Fisher Scientific, Dionex, Accucore, Q Exactive and XCalibur are registered trademarks of Thermo Fisher Scientific in the United States and other countries. All other trademarks are the property of their respective owners.

CONCLUSIONS

1. Gly-X N-glycan sample preparation with InstantPC can be completed in around 45 minutes.

2. InstantPC N-glycan dye offers FLR and MS sensitivity.

3. HILIC separation by Accucore-150-Amide column separates major N-glycan species.

4. FLR may be used for reproducible relative quantification, MS1 spectra to assign glycan ID.

5. MS2 using the Q Exactive may be used to further characterize exotic N-glycans on the Fab region such as hybrid-type glycans and those containing potentially immunogenic epitopes such as alpha-gal and NGNA.

0 5 10 15 20 25 30 35 40 45 50Time (min)

20

40

60

80

100

Rela

tive

Abun

danc

e

100

300

500

Emis

sion

Cou

nts

23.82 mins

1075 1076 1077 1078 1079 1080 1081 1082m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive

Abun

danc

e

1076.9106

M5N3G1S1

1077.4107

1077.9112

1078.4121

1078.91391079.4152

Y4α

Y4

Y3α

Y3

Y2

C4/Z5

C6/Z2

C4/Z6

C6/Z3

Y3α/Y3

Y3α/Y4

Y3/Y4α

Y3

Y4

100 300 700 900 1100 1300 1500m/z

Rela

tive

Abun

danc

e

10

20

30

40

50

60

70

80

90

100 657.2388

1496.5846

366.1385

204.0877

292.1021 1334.5256483.2488

1172.5096528.1902 686.3378454.1582 1010.4591819.2997848.3990607.7616

IPC

B3

B1

B2

B2 B3B4

B4

Y1

Y2

852.3036

[M+2H]2+

Accuracy: -0.95 ppmTheoretical mass: 1076.9116Observed mass: 1076.9106

A) FLR

B) XIC

C) Mass spectrum

D) MS2

Figure 10: Characterization workflow for hybrid-type N-glycan M5N3G1S1 from mAb R with MS2.