Automated Workflow for Monoclonal Antibody N-linked Glycan Analysis with AssayMAPBravo and InstantPC LabelingDye

Shuai Wu1; Zach Van Den Heuvel1; Steve Murphy1; Justin Hyche2; Aled Jones2

1Agilent Technologies Inc., Santa Clara, CA 2ProZyme, Hayward, CA

ASMS 2018MP-061

Poster Reprint

2

Introduction Experimental

N-glycan sample preparation1

Within AssayMAP N-Glycan Sample Prep application, a reagent calculator automatically calculates the volume of all the reagents needed. The robot dispenses the reagents from a master reagent plate to prepare all the reagent plates for the following steps. 6 samples including ProZyme control human IgG, Agilent CHO mAb, Herceptin, NISTmAb, rituximab and SILuMAb were normalized to 1 µg/µL. 50 µL mAbsamples (4 replicates for each mAb, n=4) were denatured using the denature reagent, immobilized in parallel onto 24 RX cartridges. A blocking step was used afterwards to block all the active binding sites on the RX cartridges. N-Glycanase was aspirated on the cartridge at 45 °C and mAbs were incubated for 30 minutes. At the end of the reaction, released N-glycans were eluted into the InstantPC dye and labeled. InstantPC-labeled N-glycans were cleaned up using the AssayMAP CU cartridges and eluted with a 50 µL final elution volume (Figure 1).

N-glycan analysisLC/Q-TOF was set up according to Table 1 and 2. The FLD was set to λEx=285 nm and λEm=345 nm, with PMT gain = 10. Data analysis was performed with MassHunter BioConfirm using an internal N-glycan database. 1 µL sample injection represents N-glycan released from 1 µg mAb.

Glycosylation plays an important role in maintaining antibody stability and efficacy. Therefore, glycans are carefully monitored throughout the antibody development process and manufacturing. High-throughput glycan sample preparation is essential during the clone selection and cell culture optimization process. We have developed an automated workflow using the AssayMAP Bravo and ProZyme GlykoPrep InstantPC kit to release, label and purify N-glycans. Using a cartridge based platform, the method fully integrates the newly developed InstantPC dye and the automated liquid handler to streamline N-glycan sample preparation. The InstantPC-labeled N-glycans generate enhanced signals in both fluorescence detection (FLD) and mass spectrometry (MS) measurements.

Experimental

Figure 1. Automated workflow of glycan releasing,labeling, purification using the AssayMAP Bravo andGlykoPrep InstantPC kit.

Table 1. UHPLC HILIC-FLD condition2

Table 2. Q-TOF Parameters

3

1x10

0

0.5

1

1.5

2

2.5

3

FLD1 - A:Ex=285, Em=345 Control_glycan-r001.d

6.99.7

18.714.410.5

7.9 19.2 21.615.914.9 20.717.65.9 12.611.4

1x10

0

0.5

1

1.5

2

2.5

3

FLD1 - A:Ex=285, Em=345 CHO mAb_glycan-r001.d

6.9

9.7

10.4 14.45.9 7.7

1x10

0

0.5

1

1.5

2

2.5

3

FLD1 - A:Ex=285, Em=345 Herceptin_glycan-r001.d

6.9

9.7

10.45.9 7.74.6 14.48.5

Response Units vs. Acquisition Time (min)4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 231x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

FLD1 - A:Ex=285, Em=345 NISTmAb_glycan-r001.d

6.9

9.7

10.414.35.6 8.5 17.815.87.7 20.44.6 12.6 21.7

1x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

FLD1 - A:Ex=285, Em=345 rituximab_glycan-r001.d

6.9

9.7

10.414.4

7.85.9 19.516.98.5

1x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

FLD1 - A:Ex=285, Em=345 SILuMAb_glycan-r001.d

6.9

9.7

5.9 10.414.48.4 16.9 19.5

Response Units vs. Acquisition Time (min)4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

6x10

0

1

2

3

4

5

+ESI EIC(650.0000-749.2578, 749.2878-1450.0000) Scan Frag=120.0V Control_glycan-r001.d

* 7.0

9.8

14.5* 18.710.5

8.019.316.0 21.717.6 20.712.65.9 11.49.0

6x10

0

1

2

3

4

5

+ESI EIC(650.0000-749.2578, 749.2878-1450.0000) Scan Frag=120.0V CHO mAb_glycan-r001.d

6.9

9.7

10.4 14.45.9 * 7.88.94.4 12.5 17.4

6x10

0

1

2

3

4

5

+ESI EIC(650.0000-749.2578, 749.2878-1450.0000) Scan Frag=120.0V Herceptin_glycan-r001.d

6.9

* 9.7

10.45.9 7.7 14.44.7 8.5 12.79.2 16.911.3

Counts vs. Acquisition Time (min)4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 236x10

0

0.5

1

1.5

2

2.5

3

3.5

4

+ESI EIC(650.0000-749.2591, 749.2891-1450.0000) Scan Frag=120.0V NISTmAb_glycan-r001.d

6.9

9.7

10.414.45.6 8.57.7 15.8 17.812.6 20.54.7

6x10

0

0.5

1

1.5

2

2.5

3

3.5

4

+ESI EIC(650.0000-749.2591, 749.2891-1450.0000) Scan Frag=120.0V rituximab_glycan-r001.d

6.9

9.7

10.414.4

7.85.9 8.5 17.0 19.5

6x10

0

0.5

1

1.5

2

2.5

3

3.5

4

+ESI EIC(650.0000-749.2591, 749.2891-1450.0000) Scan Frag=120.0V SILuMAb_glycan-r001.d

6.9

9.7

10.45.9

14.48.4 16.9

Counts vs. Acquisition Time (min)4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Results and Discussion

G0

G0F

G0FB

G1F[3]

G1FB[6]G2

G2F

G2FB G2S1

G2FS1

G2FS1B G2S2 G2FS2 G2FS2B

G0F-N Man5

G0-N

G0

G0F

G0FB

G1F[6]

G1F[3]G1FB[6]

G2

G2FG2FB

G2S1

G2FS1

G2FS1BG2FS2

G2FS2B

G0F-N Man5

G0-N

G1F[6]

G2F+aGalG2F+aGal

G1F-N

G1FS1

G1

G1F-N

G1FS1

Figure 2. LC/Q-TOF analysis of InstantPC-labeled N-glycans from all samples. A. ProZyme Control human IgG glycan; B. CHO mAb glycan; C. Herceptin glycan; D. NISTmAb glycan; E. rituximab glycan; F. SILuMAb glycan.

A

B

C

D

E

F

Figure 3. LC/FLD analysis of InstantPC-labeled N-glycans from all samples. A. ProZyme Control human IgG glycan; B. CHO mAb glycan; C. Herceptin glycan; D. NISTmAbglycan; E. rituximab glycan; F. SILuMAb glycan.

A

B

C

D

E

F

Qualitative Analysis of InstantPC-labeled N-glycans The ProZyme control human IgG contains 17 well characterized N-glycans. Based on retention time and accurate mass, these glycans were quickly identified with high mass accuracy (< 2 ppm) in the 5 other mAbs (not labeled in Figure B to F). In addition, 5 other common N-glycans (labeled in B, C, D) were identified by accurate mass and monosaccharide composition. FLD results showed the same retention time and similar relative peak abundance for these glycans.

G2S2G1

This information is subject to change without notice.

© Agilent Technologies, Inc. 2018

For Research Use Only. Not for use in diagnostic procedures.

An automated N-glycan releasing, labeling, cleanup and elutionworkflow was implemented using the AssayMAP Bravo and GlycoPrepInstantPC Kit.

• AssayMAP enables high-throughput and highly reproducible samplepreparation.

• InstantPC enhances N-glycan signal in both MS and FLD.

• AdvanceBio Glycan Mapping column provides excellent separation forN-glycans. 6545XT AdvanceBio LC/Q-TOF provides highly sensitiveand accurate mass measurement to enable N-glycans identification.

• BioConfirm enables batch analysis and automatic plotting of N-glycancontent.

Results and Discussion

Conclusions

1GlykoPrep-plus Rapid N-Glycan Sample Preparation with InstantPCAutomated on AssayMAP Technology_Prozyme Instruction manual_GPPNG-PC2Analysis of Monoclonal Antibody N-glycans by Fluorescence Detection and Robust Mass Selective Detection and Robust Mass Selective Detection Using the Agilent LC/MSD XT, Agilent Technologies, 5991-8071EN

References

Quantitative Analysis of InstantPC-labeled N-glycansUsing an internal glycan database, BioConfirm quickly identified and integrated 10 labeled N-glycans over the 24 samples using retention time and accurate mass. Figure 4A was automatically generated by the BioConfirm software, which represented the percentage of 10 N-glycans within each mAb. Figure 4B was generated based on the peak area and relative standard deviation calculated by BioConfirm. The absolute abundance showed how much glycan was detected from 1 µg of each mAb. Fucosylated glycans were the most abundant glycans as expected. Almost all RSD% were less than 10% showing high reproducibility of the automated workflow.

A

B

Figure 4A. Relative abundance of the top 10 most abundant and common N-glycans in the control sample and 5 mAbs.4B. Absolute abundance of the 10 N-glycans in all samples. RSD% were labeled on the top of each glycan.

Table 3. A summary of 22 N-glycansidentified from all the samples