TECHNICAL NOTE

Technical Note Bulletin 4010 Rev A

Automation

Biotherapeutic

Capillary Electrophoresis

Enbrel

Fluorescent Dye

Glycoprotein

HILIC

InstantPC

InstantQ

MabThera

N-Glycans

Keywords

Automation of Gly-X N-Glycan Sample Prep with InstantPC and InstantQ dyesLoredana Serafini*, Ted Haxo†, Emily Dale†, Adele Taylor†, Katherine M. Brendza**Gilead, Foster City, CA;†ProZyme, Inc., Hayward, CA

SUMMARY

The Gly-X™ N-glycan sample preparation protocol, including 5-minute rapid deglyco-

sylation, instant labeling and cleanup is carried out in 96-well format plates compatible

with most liquid handling workstations. Here we present methods for automation of

Gly-X N-glycan sample prep with InstantPC™ (LC/MS) and Gly-X with InstantQ™ (CE) on

the Hamilton Microlab NIMBUS liquid handler.

The method is designed to process 8–24 samples, utilizing up to 3 full columns of 8

samples. Samples were prepared in a 96-well PCR plate and reagents were prepared

per kit instructions. Samples, reagents and any appropriate dilutions of reagents were

placed on the platform deck in either vials or into troughs. All subsequent steps were

fully automated on the NIMBUS system without further user intervention. Samples

were ready for analysis by LC/MS (InstantPC dye) or CE (InstantQ dye) on the Gly-Q™

Glycan Analyzer in about 1.5 hours total time.

Gly-Q

Gly-X

GlykoPrep

Glyko Enzymes

Glyko Standards

InstantPC

InstantAB

InstantQ

2-AB

APTS

PhycoLink

PhycoPro

RPE & APC Conjugates

Streptavidins

NOTICE: ProZyme was purchased by Agilent in July 2018. Documents for products and product lots manufactured before August 2019 will contain references to ProZyme. For more information about these products and support, go to: www.agilent.com/en/contact-us.

2

Figure 1: HAMILTON NIMBUS workstation and Gly-X N-Glycan sample prep kit.

Figure 2: Automation overview: Workflow on Hamilton NIMBUS for Gly-X Rapid Release and

Labeling with InstantPC™ and with InstantQ™ kits.

METHODS AND MATERIALS

Sample Preparation KitsGly-X Rapid Release and Labeling with

InstantPC Dye Kit (GX96-IPC)

Gly-X Rapid Release and Labeling with

InstantQ Dye Kit (GX96-IQ)

MabThera® lot # H0102B03,

Enbrel® lot # 1075801

Automation StationHamilton Microlab NIMBUS liquid han-

dler with:

• 96-well head

• 2 heater blocks (set at 90°C and 50°C)

• MPE positive pressure automation

module

Deck Configuration • Load tips – 50 μL and 1000 μL, see

Figure 4

• Load Reagent Source Plate (empty

PCR plate, semi-skirted)

• Load Cleanup Plate stack on deck,

not MPE (Gly-X Cleanup Plate and

waste tray, waste tray remains on

deck and is not transferred to MPE)

• Load collection plate

• Preheat heaters to 50°C and 90°C

Reagent Preparation• Gly-X Denaturant – Dilute 1:1 with DI

water. Prepare 120 μL for 24 wells.

Place in a 0.5 mL Sarstedt screw top

vial and place in the reagent block

on deck

INTRODUCTION

Biopharmaceutical research and development of monoclonal antibodies

has grown exponentially to become the fastest growing class of therapeu-

tic agents. Recombinant monoclonal antibodies contain a conserved N-gly-

cosylation site at Asn297 in the Fc region, with differences in glycosylation

patterns observed depending on the host cell line and culture conditions

used during protein production.1 Glycan heterogeneity has been shown to

impact the physiochemical and pharmacokinetic properties of therapeu-

tic antibodies, with particular glycoforms having very specific effects on

antibody function.1,2 For example, minimizing N-glycans that contain core

fucosylation has been shown to enhance Fc receptor binding and antibody

dependent cell-mediated cytotoxicity (ADCC) activity.2 These findings have

given rise to engineering efforts to control glycoform profiles for optimum

bioefficacy and reliable batch-to-batch consistency.2,3 Various approaches

have been tested to alter glycoprofiles including directed mutations to the

protein Fc region, and modifications to the host cell line, either by molecu-

lar or chemical interference that redirect cellular glycosylation pathways.3,4

In light of these efforts, glycan analysis has become an important tool for

quality control and analytical characterization of this critical quality attribute

(CQA) during therapeutic antibody production and development.5,6 Here

we test two purified recombinant monoclonal antibodies with the goal of

developing a streamlined, walkaway N-glycan sample preparation work-

flow that can be implemented as a part of cell line screening and process

development using a standard liquid handler and off-the-shelf sample

preparation kits.

BarcodeScan

96-WellPlate

CleanupPlate Wash Elute

UHPLCor CE

MeasureAdd ElutionBuffer

AddSample

CollectGlycans

EquilibratePlate

[MPE]2 [MPE]2 [MPE]2

[MPE]2

Add WashBuffer

Repeat

Pipetting, Gripper, MPE Offline Analysis Pipetting

AddReagent(s)

TransferSample

Incubate

Repeat

PrepareProtein

3

Automation Protocol DesignThe automation protocol was developed to provide the most efficient use

of the previously installed NIMBUS system, processing 8–24 samples at a

time. Glycoprotein samples (20 μL, 2 mg/mL) were placed in the a 300 μL

semi-skirted 96-well PCR plate on the NIMBUS deck. Reagents for dena-

turation, deglycosylation and labeling were placed in vials on the platform

deck prior to starting the automation workflow (Figure 3). The automation

design begins with transferring required volumes of these stock reagents

into a 96-well Reagent Source Plate (using single tips, see Figure 5). The

number of columns for each transferred reagent matches the number of

columns of glycoprotein samples to be processed (up to 3 columns). The

automation design then moves the glycoprotein samples (using 8 chan-

nels, see Figure 5) into the wells of the source plate (containing reagents),

mixing and then returning to the mixture to the Sample Plate followed by

incubation at the specified temperature. In this manner, all incubations are

in the Sample Plate, and 8 channels can be used to transfer the samples

back and forth from Sample Plate to Reagent Source Plate and back to the

Sample Plate. Dye removal and cleanup is performed with the Gly-X Clean-

up Plate and the NIMBUS MPE positive pressure manifold.

• N-Glycanase Working Solution –

Prepare N-Glycanase working solu-

tion according to kit instructions,

then dilute 1:1 with DI water. Prepare

120 μL of 1:1 dilution for 24 wells.

Place in a 0.5 mL Sarstedt screw

top vial and place in reagent block

on deck

• InstantDye Working Solutions

• For InstantPC, prepare 1 vial of

InstantPC in 150 μL of InstantPC

Dye Solvent per the kit instruc-

tions and place on the deck

• For InstantQ, prepare 1 vial of

InstantQ in 400 μL InstantQ Dye

Solvent, and mix 1:1 with Activa-

tion Reagent, according to the

kit instruction. Place in a 0.5 mL

Sarstedt screw top vial and place

in reagent block on deck

• Cleanup reagents

• For InstantPC, prepare 100 mL of

Load/Wash buffer according to

the kit instructions and place into

two troughs on deck

• For InstantQ, place 100 mL etha-

nol into two troughs on deck

• Elution of labeled N-Glycans

• For InstantPC, add InstantPC

Eluent to trough on deck (100 μL

per sample required)

• For InstantQ, place water in

trough on deck. (100 μL per

sample required)

• Load Samples

• Place samples (20 μL each) into

columns of a semi-skirted PCR

plate and place on deck.

Figure 3: Hamilton NIMBUS deck layout. Location of plates, vials, troughs, heaters and MPE posi-

tive pressure manifold.

Heater,50°C

MPE2

PositivePressureStationCaps, Empty

CollectionPlate

Heater,90°C

Reagentsin Vials

50 μLTips

SamplePlate

Load/Wash

Elution Wash(InstantQ)

Caps, ReagentSource Plate

1000 μLTips

Cleanup Plate+ Storage Plate

Figure 4: Tip layout. A) 50 µL tips. The first two columns (red) are designated for single tips use tips

for dispensing reagents from vials into the Reagent Source Plate. The remaining columns are for

multichannel dispensing and transfers. B) 1000 µL tips. All columns are for multichannel dispensing

and transfers.

50 µL Tips 1000 µL Tips

4 3 2 14 3 2 1

123

4

Wash. Using 8 new tips per column, dispense 600 µL of Load/

Wash buffer to each column of the Cleanup Plate with samples.

Move Cleanup Plate to manifold and apply pressure gradient

(Figure 5). When complete, move the Cleanup Plate back to

original deck location. Using the same tips, repeat wash and

pressure steps 2 more times. Move the Cleanup Plate over the

top of the Collection Plate.

Elute. Using 8 new tips, transfer 100 µL of InstantPC Eluent to

each of the columns containing samples. Move the stack con-

taining Elution Plate and Cleanup Plate onto the manifold and

apply pressure gradient (Figure 5). When complete, move the

stack back to original location. Move the Cleanup Plate off the

Collection Plate back to the waste plate location. Using 8 new

tips per column, mix eluted samples. Procedure is complete.

CLEANUP AND ELUTION (InstantQ DYE)

Prime. Using 8 channels on the 96-channel head, transfer

400 µL of water to each well on the Cleanup Plate. Once all

columns to be used are loaded with water, move the Cleanup

Plate to the positive pressure manifold. Apply a step pressure

gradient to the plate (Figure 5). When complete, move the

Cleanup Plate back to original deck location. Using 8 channels

on the 96-channel head, transfer 600 μL of ethanol to each

well on the Cleanup Plate. Once all columns to be used are

loaded with ethanol, move the Cleanup Plate to the positive

pressure manifold. Apply a step pressure gradient to the plate

(Figure 5). When complete, move the Cleanup Plate back to

original deck location. Repeat ethanol wash a second time.

Load. Using 8 channels on the 96-channel head, transfer

400 µL of ethanol into the first column of the Cleanup Plate.

Using the same 8 tips, transfer 150 µL of ethanol into the first set

of samples and mix. Using the same tips, transfer the samples

to the Cleanup Plate positions with ethanol and mix. Repeat for

additional columns if appropriate. Once all samples are loaded

on plate, move the plate to the positive pressure manifold. Apply

a step pressure gradient to the plate (Figure 5). When complete,

move the Cleanup Plate back to original deck location.

Wash. With 8 new tips per column, dispense 600 µL of etha-

nol to each column of the Cleanup Plate with samples. Move

Cleanup Plate to manifold and apply pressure gradient (Figure

5). When complete, move the Cleanup Plate back to original

deck location. Using the same tips, repeat wash and pressure

steps 2 more times (total of 3 washes).

Elute. Move the Cleanup Plate over the top of the Collection

Plate. Using 8 new tips, transfer 150 µL of water to each of the

columns containing samples. Move the stack containing Elution

Plate and Cleanup Plate onto the manifold and apply pressure

gradient (Figure 5). When complete, move the stack back to

original location. Move the Cleanup Plate off the Collection

Plate back to the waste plate location. Using 8 new tips per

column, mix eluted samples. Procedure is complete.

Automation Details

DISPENSE REAGENTS INTO REAGENT SOURCE PLATE

Using single tip mode of the 96-channel head, transfer each of

the 3 reagents from tubes into sections of a semi-skirted PCR

plate. Dispense such that the number of columns in the Reagent

Source Plate for each reagent matches the number of columns of

the glycoprotein samples. For 24 samples, dispense 4 μL Gly-X

Denaturant into columns 1–3; dispense 4 μL of Working N-Gly-

canase solution into columns 4–6; for InstantDye, dispense into

columns 7–9; 5 μL for InstantPC, 20 μL for InstantQ (for 8 sam-

ples use columns 1, 4, and 7). For each transfer, pick up a larger

volume and dispense in multiple wells to shorten the overall

operation.

DENATURE SAMPLES

Using 8 channels on the 96-channel head, pick up the samples

and transfer to the corresponding Gly-X Denaturant location on

the Reagent Source Plate, mix and return to the Sample Plate.

Pick up plate and move to 90°C heater. Wait 3 minutes. Move

back to original Sample Plate location. Wait 1 minute.

DEGLYCOSYLATE

Using 8 channels of the 96-channel head, pick up the samples

and transfer to the corresponding N-Glycanase location on the

Reagent Source Plate. Mix and return samples to the Sample

Plate. Pick up plate and move to the 50°C heater. Wait 5 minute.

Move plate back to the original Sample Plate location and wait

1 minute.

LABEL

Using 8 channels on the 96-channel head, pick up the samples

and transfer to the corresponding InstantDye location on the

Reagent Source Plate, mix and return to the Sample Plate. Pick up

plate and move to 50°C heater. Wait 1 minute. Move back to original

Sample Plate location. Wait 1 minute.

CLEANUP AND ELUTE (InstantPC DYE)

Load. Using 8 channels on the 96-channel head, transfer

400 µL of Load/Wash buffer into the first column of the Cleanup

Plate. Using the same 8 tips, transfer 150 µL load/Wash buffer

into the first column of samples and mix. Using the same tips,

transfer the samples to the Cleanup Plate positions with load

wash buffer and mix. Repeat for additional columns if appro-

priate. Once all samples are loaded onto the Cleanup Plate,

move the plate to the positive pressure manifold. Apply a step

pressure gradient to the plate. (see Figure 5). When complete,

move the plate back to original deck location.

Step Pressure Duration1 1 psi 40 seconds2 3 psi 20 seconds3 6 psi 20 seconds4 10 psi 20 seconds5 55 psi 10 seconds

Figure 5: Pressure Gradient for MPE positive pressure manifold.

5

Row 1 2 3A Enbrel Water Blank MabTheraB MabThera Enbrel Water BlankC Water Blank MabThera EnbrelD Enbrel Water Blank MabTheraE MabThera Enbrel Water BlankF Water Blank MabThera EnbrelG Enbrel Water Blank MabTheraH MabThera Enbrel Water Blank

Figure 6: Sample Plate layout design. Glycoprotein samples and water blank loca-

tions for preparing N-glycan samples labeled with InstantPC and InstantQ.

Figure 7: MabThera InstantPC labeled N-Glycans. Average peak areas observed using a 15-minute UHPLC-HILIC method on an Amide column (2.1 x 100 mm, 1.7 μm). (light green) Samples Prepared with the NIMBUS automation platform; (dark green) Samples prepared with the manual method.

RESULTS

Comparison of Automated and Manual MethodMabThera (monoclonal IgG) and Enbrel (Fc fusion protein) were

placed into the sample plate in a checkerboard layout design

(Figure 6) to verify well-to-well performance. Water blanks

were included to test for sample crossover. This same test

was performed using InstantPC dye followed by analysis by

UHPLC-HILIC and with InstantQ dye followed by analysis on the

Gly-Q System. The average percent area of InstantPC-labeled

glycans prepared using the automation workflow are in good

correlation with results from samples prepare by the manual

method (Figures 7, 8). Crosstalk between samples was not

observed as demonstrated by the absence of labeled glycans

in the water blank well (Figure 9). Similar results were obtained

using InstantQ dye in the automation workflow, demonstrating

that the Gly-X kits provide robust results regardless of prepara-

tion method.

0

5

10

15

20

25

30

35

40

G0 G0F G1 G1F[6] G1F[3] G2F

Are

a %

HamiltonManual

Average % Peak Area, N=8 Stdev % CV

Glycan Automated Manual Automated Manual Automated vs Manual

G0 0.85 0.86 0.02 0.02 0.52%G0F 37.75 36.82 0.32 0.22 1.77%G1 2.16 2.27 0.11 0.05 3.27%G1F[6] 33.18 32.81 0.25 0.22 0.79%G1F[3] 11.22 11.23 0.12 0.07 0.07%G2F 10.90 11.02 0.12 0.13 0.81%A1F 1.88 1.85 0.04 0.08 1.23%A2F 1.57 1.40 0.16 0.02 7.99%

6

EU

22232425262728293031323334353637383940

Minutes0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2 7.6 8.0 8.4 8.8 9.2 9.6 10

Figure 9: Overlay of UHPLC-HILIC traces from water blanks (n=8) of automated Gly-X N-glycan sample preparation with InstantPC Dye. Peak at left is free dye peak.

Figure 8: Enbrel InstantPC labeled N-Glycans. Average peak areas observed using a 15-minute method on an Amide column (2.1 x 100 mm, 1.7 μm). (Light orange)

Samples Prepared with the NIMBUS automation platform; (dark orange) Samples prepared with the manual method.

0

5

10

15

20

25

G0 G0F

Man5

G1F[

6]

G1F[

3]

G2

G1FS

1

G2F A1 A1F A2 A2F

Are

a %

HamiltonManual

Average % Peak Area, N=8 Stdev %CV

Glycan Automated Manual Automated Manual Automated vs Manual

G0 1.06 1.10 0.01 0.00 2.6%G0F 18.76 18.77 0.16 0.05 0.0%Man5 5.44 5.32 0.02 0.03 1.6%G1F[6] 9.70 9.77 0.07 0.01 0.5%G1F[3] 4.55 4.60 0.03 0.01 0.7%G2 2.49 2.92 0.08 0.01 11.3%G1FS1 3.16 3.19 0.01 0.00 0.8%G2F 5.84 5.93 0.05 0.01 1.0%A1 14.06 13.96 0.08 0.04 0.5%A1F 20.53 20.39 0.47 0.04 0.5%A2 4.25 4.21 0.06 0.01 0.6%A2F 10.17 9.85 0.13 0.03 2.3%

7

Sign

al (R

FU)

-764

79,236

69,236

59,236

49,236

39,236

62,236

19,236

9,236

89.236

Sign

al (R

FU)

-1,103

18,897

38,897

78,897

58,897

98,897

118,897

Glucose Units (GU)3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5

A1

A2

A2F

G0

G0F

G1[6]

G1F[3]

G1F[6]

G2F

G2G1FS1

A1F

Man5

G0F-N

+

G1[3]

DP3

DP15

A1

A2

A2F

G0

G0F

G1[6]

G1F[3]

G1F[6]

G2F

G2G1FS1

A1F

Man5

G0F-N

+

G1[3]

DP3DP15

A) Hamilton Method

B) Manual Method

Glucose Units (GU)3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5

Figure 10: Overlay of InstantQ labeled N-glycans prepared from Enbrel samples (n=8) separated with the Gly-Q System. A) Sample preparation performed on the NIMBUS

automation platform; B) Sample preparation using the manual method.

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

ProZyme, Gly-X, Gly-Q, InstantPC and InstantQ are trademarks of ProZyme, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

References 1. Glycoengineering of protein-based therapeutics. Donadio-Andréi S, Iss C, El Mai N, Calabro V, Ronin C. Carbohydr Chem. 2012;38: 92–123

2. Boosting ADCC and CDC activity by Fc engineering and evaluation of antibody effector functions. Kellner C, Derer S, Valerius T, Peipp M. Methods. 2014;65(1):105-13

3. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies. Yu X, Baruah K, Harvey DJ, Vasiljevic S, Alonzi DS, Song BD, Higgins MK, Bowden TA, Scanlan CN, Crispin M. J Am Chem Soc. 2013;135(26):9723-32

4. The choice of mammalian cell host and possibilities for glycosyla-tion engineering. Butler M, Spearman M. Curr Opin Biotechnol. 2014;30:107-12

5. The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins. O’Flaherty R, Trbojević-Akmačić I, Greville G, Rudd PM, Lauc G. Expert Rev Proteomics. 2018;15(1):13-29

6. N-Glycan Analysis of Biotherapeutic Proteins. Jones A. BioPharm Intl. 2017;30(6)20-5

CONCLUSIONS

1. Adaptation of the Gly-X protocol to the Hamilton NIMBUS

liquid handler used off-the-shelf InstantPC and InstantQ kits

(GX96-IPC, GX96-IQ) and did not require modification of the

existing installed NIMBUS system.

2. Automation provides walkaway Gly-X N-glycan sample

preparation in approximately 1.5 hrs.

3. No crosstalk was detected between N-glycan samples pre-

pared on the NIMBUS system.

4. Results from manual and automated methods are compara-

ble, allowing day to day operational flexibility.

A

Average % Area StdevGlycan Hamilton Manual Hamilton ManualG0 1.16 1.17 0.03 0.02G0F 17.87 18.16 0.19 0.10G1F[6] 9.95 10.12 0.12 0.04G1F[3] 4.55 4.55 0.08 0.03Man5 + G0F-N 6.95 7.05 0.07 0.06G2 3.57 3.54 0.06 0.04G2F 6.01 5.98 0.11 0.07G1FS1 (2,3) 3.20 3.17 0.03 0.02A1 (2,3) 15.82 15.46 0.12 0.09A1[6]F (2,3) 18.87 18.64 0.12 0.09A2 (2,3) 4.15 4.17 0.10 0.02A2F(2,3) 7.90 8.00 0.16 0.07

B

Average % Area StdevGlycan Hamilton Manual Hamilton ManualG0F 36.97 37.25 0.14 0.26G2F 11.68 11.66 0.23 0.13G1F[6] 35.24 35.46 0.26 0.17G1F[3] 11.40 11.55 0.12 0.10Man5 + G0F-N 2.94 2.97 0.12 0.04A2F(2,3) 1.19 1.26 0.09 0.08

Figure 11: Comparison of Gly-X InstantQ sample preparation using NIMBUS auto-

mation with the manual method for A) Enbrel InstantQ-labeled N-glycans; B) Rituxan

InstantQ-labeled N-glycans.