automation of gly-x n-glycanfigure 1: hamilton nimbus workstation and gly-x n-glycan sample prep...
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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
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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.