ANALYTICAL SOLUTION FOR N-LINKED GLYCANS OF … · Glycosylation is a post-translational...
1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS Glycosylation is a post-translational modification of proteins. The sugar chains on monoclonal antibodies, IgGs, can mediate biological activity, and are therefore associated with the safety and efficacy attributes of many biopharmaceuticals. The relative amounts of the individual glycan structures must be monitored at all stages of research and development. We have developed a glycan analysis solution for these applications. N-linked glycans are released from IgGs and are labeled with 2-aminobenzamide (2-AB). The derivatized oligosaccharides are separated by HydrophILic Interaction Chromatography (HILIC) using a 1.7 micron particle UPLC column. A fluorescence detector with a low volume flow cell is specific for the 2-AB label, and enhances sensitivity beyond the predicted improvement from narrow peaks. The instrument used in this study has a low dwell volume and the capability of operating at high pressure (up to 15,000 psi), thus it provides the operating characteristics to realize the resolution, sensitivity and speed benefits of the 1.7 micron particle packing material. Fluorescent labelling is an important element of this sensitive assay. To obtain maximum sensitivity, excess reagent needs to be removed prior to UPLC. We have used a HILIC mode SPE separation to remove excess 2-aminobenzamide (2-AB), and optimized the MassPREP™ HILIC μElution Plate protocol for the IgG glycans shown below. ANALYTICAL SOLUTION FOR N-LINKED GLYCANS OF GLYCOPROTEINS: RESOLUTION AND QUANTITATION Authors: Beth L. Gillece-Castro, Thomas E. Wheat, Kim Van Tran, and Kenneth J. Fountain Affiliations: Waters Corp., Milford, MA METHODS RESULTS AND DISCUSSION Figure 8. Sialylated, acidic, oligosaccharides can be strongly retained on the Glycan Separation Technology column. High ionic strength, 100 mM or 250 mM ammonium formate at pH 4.5 yielded elution of mono to tetra-sialo gly- cans after neutral oligosaccharides (data not shown). Increasing the ionic strength of an alternative formic acid buffer to the 250mM range, 1%, also provides excellent retention of sialylated glycans for quantitation. For assays requirement LC/MS both mobile phases provide molecular weight confirmation of monosaccharide. INTRODUCTION Five injections overlayed Time 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 EU 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 EU 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 EU 0.047 0.057 0.054 0.047 0.064 0.063 0.000 0.001 0.080 0.078 100 mM 10 mM 1 mM G0F Man5 A2F Time 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 EU 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 EU 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 EU 0.047 0.057 0.054 0.047 0.064 0.063 0.000 0.001 0.080 0.078 100 mM 10 mM 1 mM G0F Man5 A2F Figure 5. UPLC-FLR chromatograms of 2-AB labeled oligosaccharides, G0F, Man5, and A2F recovered from a MassPREP™ HILIC μElution plate with varying concentrations of ammonium Tris-citrate. Chromatograms shown from the HILIC mode amide-based separation with 1.7 μm partcles. Figure 7. Reproducibility of the fluorescence area percent from a mixture of 2-AB labeled standards after purification by SPE. The values are the percent of the total for these glycans. Six replicate SPE purifications were averaged and the standard deviation is shown. These glycans are typical of N-linked human IgG oligosaccharide structures. Glycan Distribution: Variability after SPE 0 5 10 15 20 25 30 35 40 45 G0F Man5 G0F+GN G2 A2F Glycan Percent of Total FLR Figure 6. Recovery of 2-AB labeled glycans from a MassPREP™ HILIC μElution plate with varying concentrations of ammonium Tris-citrate. Citrate Recovery Comparison 0 20 40 60 80 100 120 140 100 10 1 AmmoniumCitrate in Eluent (mM) SPE Recovery (Percent) G0F Man5 A2F Figure 1. Improvements in separation were realized by the introduction of the Gly- can Separation Technology column with 1.7 μm particles. Gains were made in- cluding higher resolution, shorter analysis time, and selectivity specific to the separation of G0F and Man5. Time 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 EU x 10e4 0.000 5000.000 10000.000 15000.000 G0F Man6 G2F Man5 G1F 2 x 150 mm 3 μm 100.0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 EU x 10e4 0.000 2500.000 5000.000 7500.000 10000.000 12500.000 15000.000 35.0 Man5 Man6 G1F G2F G0F 2.1 x 150 mm 1.7 μm EU EU Time 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 EU x 10e4 0.000 5000.000 10000.000 15000.000 G0F Man6 G2F Man5 G1F Time 2 x 150 mm 3 μm 100.0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 EU x 10e4 0.000 2500.000 5000.000 7500.000 10000.000 12500.000 15000.000 35.0 Man5 Man6 G1F G2F G0F 2.1 x 150 mm 1.7 μm EU EU Figure 2. The separation of labeled glycans released from human IgG is the most common method for the structural characterization of the glycoprotein biopharmaceuticals. This example includes both neutral and charged oligosac- charides in a range of chain lengths as well as positional and branching iso- mers. Note that Man5 and Man6 are present in this example, although not normal components of human IgG glycans. Time 10.00 15.00 20.00 25.00 30.00 35.00 EU x 10e4 0.000 5000.000 10000.000 15000.000 20000.000 25000.000 1 G0 2 G0F 3 Man5 4 G0FGN 5 G1 6 G1Fa 7 G1Fb 8 G1FGN 9 Man6 10 G2 11 G2F 12 G1F+SA 13 G2F+SA 1 4 5 6 7 8 9 10 11 2 3 12 13 ACQUITY UPLC BEH Glycan, 1.7μm, 2.1 x 150 mm Time 10.00 15.00 20.00 25.00 30.00 35.00 EU x 10e4 0.000 5000.000 10000.000 15000.000 20000.000 25000.000 1 G0 2 G0F 3 Man5 4 G0FGN 5 G1 6 G1Fa 7 G1Fb 8 G1FGN 9 Man6 10 G2 11 G2F 12 G1F+SA 13 G2F+SA 1 4 5 6 7 8 9 10 11 2 3 12 13 ACQUITY UPLC BEH Glycan, 1.7μm, 2.1 x 150 mm HILIC mode chromatography utilizes aqueous/acetonitrile gradients from low to high water content. Oligosaccharide separations in HILIC mode (sometimes referred to as normal phase or NP) take advantage of the selectivity provided by the hydrophilic amide stationary phase. The sub-2 micron particle size improves resolution and decreases run time. Separation of glycans from a murine IgG1, shown in Figure 1, demonstrate these improvements. The more complicated mixture of oligosaccharides from human IgGs, shown in Figure 2, contains sialylated, complex, and bisecting GlcNAc structures. High mannose structures were also present in this mixture. * Contains up to 25% Acetonitrile MassPrep TM HILIC μElution Plate Condition 200 μL Milli-Q Water Equilibrate 2 x 200 μL 90% Acetonitrile Slowly Load Sample constituted in 90% Acetonitrile Volume: 150 μL Wash 2 x 200 μL 90% Acetonitrile Elute 2 x 50 μL 10 mM Ammonium Tris-citrate* Inject 2-AB labeled glycans directly * Contains up to 25% Acetonitrile MassPrep TM HILIC μElution Plate MassPrep TM HILIC μElution Plate Chromatographic Separation Conditions Instrument: ACQUITY UPLC pumps, injector and FLR. Column: ACQUITY UPLC BEH Glycan Separation Technology 1.7μm, 2.1 x 150 mm Eluent A: 100 mM Ammonium Formate, pH 4.5 Eluent B: Acetonitrile Temperature: 60 °C Fluorescence: λex = 330 nm, λem = 420 nm Sample amount: 15 pmol UPLC Gradient: Time Flow rate (min) (mL/min) % A % B Init 0.5 25 75 46.5 0.5 40 60 48 0.25* 100 0 49 0.25* 100 0 50 0.5 25 75 63 0.5 25 75 *Flow rate lowered during aqueous regeneration Labeling Glycans with 2-AB Released, dry glycans were derivatized with the fluorescent tag 2-aminobenzamide. The 2-AB reagent and sodium cyanoborohydride were dissolved in DMSO/Acetic acid. 1 After heating for two hours, the labeled glycans and excess reagent were diluted 10x with acetonitrile. The acetonitrile solution can then be loaded onto the HILIC μElution Plate. Up to 750 μL or 100 μg can be loaded into each of the 96 wells. Figure 3. Reproducibility over 20 injections was demonstrated with the Prozyme Human IgG glycans (included Man5 and Man 6). The chromatograms for injections numbered 2, 7, 10, 15, and 20 were overlayed Time 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 EU x 10e4 0.000 2500.000 5000.000 7500.000 10000.000 12500.000 15000.000 17500.000 20000.000 22500.000 25000.000 27500.000 ACQUITY UPLC BEH Glycan, 1.7μm, 2.1 x 150 mm G0 G2F+SA G2F G1Fb G1Fa G1 G0F+GN Man5 G0F G2 G1F+GN Man6 G1F+SA Time 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 EU x 10e4 0.000 2500.000 5000.000 7500.000 10000.000 12500.000 15000.000 17500.000 20000.000 22500.000 25000.000 27500.000 ACQUITY UPLC BEH Glycan, 1.7μm, 2.1 x 150 mm G0 G2F+SA G2F G1Fb G1Fa G1 G0F+GN Man5 G0F G2 G1F+GN Man6 G1F+SA Reproducibility of the resolution by Glycan Separation Technology Column and the ACQUITY UPLC Binary Solvent Manager is shown in Figure 3. The 2-AB labeled human IgG glycans were injected over more than 24 hours. The overlay of five fluorescence chromatograms demonstrates the reproducibility of the glycan separation profile. Using the neutral oligosaccharide 2-AB maltoheptaose, linearity of response was shown to be excellent over three orders of magnitude in the exponential plot, Figure 4. Purification of the 2-AB maltoheptaose by HILIC microelution was also linear. To extend this microelution method, recovery studies were performed on a mixture of three 2-AB labeled glycans; one high mannose, one complex and one sialylated. See Figures 5 and 6. The glycans were loaded onto the microelution plate in the presence of excess 2-AB labeling reagent. Further SPE studies with a more complex mixture (Figure 7) demonstrate reproducibility. The area for each of six glycans is reported as a percent relative to the total of area counts. The variance due to SPE recovery is decreased in relative percent. R 2 = 1 1 10 100 1000 10000 100000 1000000 1 10 100 1000 Concentration (ng/uL) A r e a FLR Area Expon. (FLR Area) Figure 4. Fluorescence Detection was shown to have a linear re- sponse over three orders of magnitude for 2-AB labeled maltohep- taose, including HILIC mode SPE and UPLC-FLR. References 1. Bigge J. C.; Patel T. P.; Bruce J. A.; Goulding P. N.; Charles S. M.; Parekh R. B. Anal. Biochem. 1995, 230, 229–238. • Glycan Separation Technology UPLC columns provide excellent resolution of mouse and human IgG oligosaccharides. • High sensitivity and linearity over 3 orders of magnitude result from the low dispersion instrument and fluorescence flow cell. • The relatively short analysis time increases the throughput of glycan chromatography. • 10 mM ammonium citrate is required for elution of the sialylated biantennary structures found on IgGs. • SPE purification by HILIC mode μElution of labeled glycans yields consistent patterns of glycoform distributions. • The combination of SPE sample preparation, the low dispersion instrument, and superior column technology provide a robust complete solution for the quantitative requirements for biopharmaceuticals. Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 EU x 10e4 0.000 10000.000 20000.000 30000.000 40000.000 50000.000 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 EU x 10e4 0.000 10000.000 20000.000 30000.000 40000.000 50000.000 1% Formic Acid 250 mM Ammonium Formate Acidic Glycans Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 EU x 10e4 0.000 10000.000 20000.000 30000.000 40000.000 50000.000 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 EU x 10e4 0.000 10000.000 20000.000 30000.000 40000.000 50000.000 1% Formic Acid 250 mM Ammonium Formate 1% Formic Acid 250 mM Ammonium Formate Acidic Glycans Mass spectrometric confirmation of glycan MWs is often required. Since high ionic strength is required to retain sialylated glycans, 1% formic acid was compared to 250 mM ammonium formate. CONCLUSIONS
Transcript of ANALYTICAL SOLUTION FOR N-LINKED GLYCANS OF … · Glycosylation is a post-translational...
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS
Glycosylation is a post-translational modification of proteins. The sugar chains on monoclonal antibodies, IgGs, can mediate biological activity, and are therefore associated with the safety and e f f i c a c y a t t r i b u t e s o f m a n y biopharmaceuticals. The relative amounts of the individual glycan structures must be monitored at all stages of research and development.
We have developed a glycan analysis solution for these applications. N-linked glycans are released from IgGs and are labeled with 2-aminobenzamide (2-AB). The derivatized oligosaccharides are separated by HydrophILic Interaction Chromatography (HILIC) using a 1.7 micron particle UPLC column. A fluorescence detector with a low volume flow cell is specific for the 2-AB label, and enhances sensitivity beyond the predicted improvement from narrow peaks. The instrument used in this study has a low dwell volume and the capability of operating at high pressure (up to 15,000 psi), thus it provides the operating characteristics to realize the resolution, sensitivity and speed benefits of the 1.7 micron particle packing material. Fluorescent labelling is an important element of this sensitive assay. To obtain maximum sensitivity, excess reagent needs to be removed prior to UPLC. We have used a HILIC mode SPE separation to remove excess 2-aminobenzamide (2-AB), and optimized the MassPREP™ HILIC µElution Plate protocol for the IgG glycans shown below.
ANALYTICAL SOLUTION FOR N-LINKED GLYCANS OF GLYCOPROTEINS: RESOLUTION AND QUANTITATION
Authors: Beth L. Gillece-Castro, Thomas E. Wheat, Kim Van Tran, and Kenneth J. Fountain Affiliations: Waters Corp., Milford, MA
METHODS RESULTS AND DISCUSSION
Figure 8. Sialylated, acidic, oligosaccharides can be strongly retained on the Glycan Separation Technology column. High ionic strength, 100 mM or 250 mM ammonium formate at pH 4.5 yielded elution of mono to tetra-sialo gly-cans after neutral oligosaccharides (data not shown). Increasing the ionic strength of an alternative formic acid buffer to the 250mM range, 1%, also provides excellent retention of sialylated glycans for quantitation. For assays requirement LC/MS both mobile phases provide molecular weight confirmation of monosaccharide.
INTRODUCTION
Five injections overlayed
Time10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
EU
10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
EU
10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
EU
0.0470.0570.054
0.0470.0640.063
0.0000.001
0.0800.078
100 mM
10 mM
1 mM
G0F Man5 A2F
Time10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
EU
10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
EU
10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50
EU
0.0470.0570.054
0.0470.0640.063
0.0000.001
0.0800.078
100 mM
10 mM
1 mM
G0F Man5 A2F
Figure 5. UPLC-FLR chromatograms of 2-AB labeled oligosaccharides, G0F, Man5, and A2F recovered from a MassPREP™ HILIC µElution plate with varying concentrations of ammonium Tris-citrate. Chromatograms shown from the HILIC mode amide-based separation with 1.7 µm partcles.
Figure 7. Reproducibility of the fluorescence area percent from a mixture of 2-AB labeled standards after purification by SPE. The values are the percent of the total for these glycans. Six replicate SPE purifications were averaged and the standard deviation is shown. These glycans are typical of N-linked human IgG oligosaccharide structures.
Glycan Distribution: Variability after SPE
0
5
10
15
20
25
30
35
40
45
G0F Man5 G0F+GN G2 A2F
Glycan
Perc
ent o
f Tot
al
FLR
Figure 6. Recovery of 2-AB labeled glycans from a MassPREP™ HILIC µElution plate with varying concentrations of ammonium Tris-citrate.
Cit rat e Recovery Comparison
0
20
40
60
80
100
120
140
100 10 1
Ammoni umCi t r at e i n E l uent ( mM )
G0F
Man5
A2F
Citrate Recovery Comparison
0
20
40
60
80
100
120
140
100 10 1
AmmoniumCitrate in Eluent (mM)
SPE
Rec
over
y (P
erce
nt)
G0FMan5A2F
Figure 1. Improvements in separation were realized by the introduction of the Gly-can Separation Technology column with 1.7 µm particles. Gains were made in-cluding higher resolution, shorter analysis time, and selectivity specific to the separation of G0F and Man5.
Time30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
EU x
10e
4
0.000
5000.000
10000.000
15000.000 G0F
Man6
G2F
Man5
G1F
Time
2 x 150 mm3 µm
100.0
T im e5.00 10.00 15.00 20.00 25.00 30.00 35.00
EU x
10e
4
0 .000
2500.000
5000.000
7500.000
10000.000
12500.000
15000.000
35.0
Man5
Man6
G1F
G2F
G0F2.1 x 150 mm
1.7 µm
EU
EU
Time30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
EU x
10e
4
0.000
5000.000
10000.000
15000.000 G0F
Man6
G2F
Man5
G1F
Time
2 x 150 mm3 µm
100.0
T im e5.00 10.00 15.00 20.00 25.00 30.00 35.00
EU x
10e
4
0 .000
2500.000
5000.000
7500.000
10000.000
12500.000
15000.000
35.0
Man5
Man6
G1F
G2F
G0F2.1 x 150 mm
1.7 µm
EU
EU
Figure 2. The separation of labeled glycans released from human IgG is the most common method for the structural characterization of the glycoprotein biopharmaceuticals. This example includes both neutral and charged oligosac-charides in a range of chain lengths as well as positional and branching iso-mers. Note that Man5 and Man6 are present in this example, although not normal components of human IgG glycans.
Time10.00 15.00 20.00 25.00 30.00 35.00
EU
x 1
0e4
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
1 G02 G0F3 Man54 G0FGN5 G16 G1Fa7 G1Fb8 G1FGN9 Man610 G211 G2F12 G1F+SA13 G2F+SA
1
4
5
6
78
9
10
11
2
3
12
13
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
Time10.00 15.00 20.00 25.00 30.00 35.00
EU
x 1
0e4
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
1 G02 G0F3 Man54 G0FGN5 G16 G1Fa7 G1Fb8 G1FGN9 Man610 G211 G2F12 G1F+SA13 G2F+SA
1
4
5
6
78
9
10
11
2
3
12
13
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
HILIC mode chromatography utilizes aqueous/acetonitrile gradients from low to high water content. Oligosaccharide separations in HILIC mode (sometimes referred to as normal phase or NP) take advantage of the selectivity provided by the hydrophilic amide stationary phase. The sub-2 micron particle size improves resolution and decreases run time. Separation of glycans from a murine IgG1, shown in Figure 1, demonstrate these improvements. The more complicated mixture of oligosaccharides from human IgGs, shown in Figure 2, contains sialylated, complex, and bisecting GlcNAc structures. High mannose structures were also present in this mixture.
Condition
200 µL Milli-Q Water
Equilibrate
2 x 200 µL 90% Acetonitrile
Slowly Load Sample
constituted in 90% Acetonitrile
Volume: 150 µL
Wash
2 x 200 µL 90% Acetonitrile
Elute
2 x 50 µL
10 mM Ammonium Tris-citrate*
Inject 2-AB labeled glycans directly
* Contains up to 25% Acetonitrile
MassPrepTM
HILIC µElution Plate
Condition
200 µL Milli-Q Water
Equilibrate
2 x 200 µL 90% Acetonitrile
Slowly Load Sample
constituted in 90% Acetonitrile
Volume: 150 µL
Wash
2 x 200 µL 90% Acetonitrile
Elute
2 x 50 µL
10 mM Ammonium Tris-citrate*
Inject 2-AB labeled glycans directly
* Contains up to 25% Acetonitrile
MassPrepTM
HILIC µElution PlateMassPrepTM
HILIC µElution Plate
Chromatographic Separation Conditions
Instrument: ACQUITY UPLC pumps, injector and FLR.
Column: ACQUITY UPLC BEH Glycan Separation Technology1.7µm, 2.1 x 150 mm
Eluent A: 100 mM Ammonium Formate, pH 4.5Eluent B: AcetonitrileTemperature: 60 °CFluorescence: λex = 330 nm, λem = 420 nmSample amount: 15 pmolUPLC Gradient: Time Flow rate(min) (mL/min) % A % BInit 0.5 25 75 46.5 0.5 40 60 48 0.25* 100 0 49 0.25* 100 050 0.5 25 7563 0.5 25 75
*Flow rate lowered during aqueous regeneration
Labeling Glycans with 2-AB
Released, dry glycans were derivatized with the fluorescent tag 2-aminobenzamide. The 2-AB reagent and sodium cyanoborohydride were dissolved in DMSO/Acetic acid.1 After heating for two hours, the labeled glycans and excess reagent were diluted 10x with acetonitrile. The acetonitrile solution can then be loaded onto the HILIC µElution Plate. Up to 750 µL or 100 µg can be loaded into each of the 96 wells.
Figure 3. Reproducibility over 20 injections was demonstrated with the Prozyme Human IgG glycans (included Man5 and Man 6). The chromatograms for injections numbered 2, 7, 10, 15, and 20 were overlayed
Time10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00
EU x
10e
4
0.000
2500.000
5000.000
7500.000
10000.000
12500.000
15000.000
17500.000
20000.000
22500.000
25000.000
27500.000
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
G0
G2
F+S
A
G2
F
G1
FbG
1Fa
G1
G0
F+G
NM
an
5G
0F
G2
G1
F+G
NM
an6
G1
F+S
A
Time10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00
EU x
10e
4
0.000
2500.000
5000.000
7500.000
10000.000
12500.000
15000.000
17500.000
20000.000
22500.000
25000.000
27500.000
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
G0
G2
F+S
A
G2
F
G1
FbG
1Fa
G1
G0
F+G
NM
an
5G
0F
G2
G1
F+G
NM
an6
G1
F+S
A
Reproducibility of the resolution by Glycan Separation Technology Column and the ACQUITY UPLC Binary Solvent Manager is shown in Figure 3. The 2-AB labeled human IgG glycans were injected over more than 24 hours. The overlay of five fluorescence chromatograms demonstrates the reproducibility of the glycan separation profile. Using the neutral oligosaccharide 2-AB maltoheptaose, linearity of response was shown to be excellent over three orders of magnitude in the exponential plot, Figure 4. Purification of the 2-AB maltoheptaose by HILIC microelution was also linear. To extend this microelution method, recovery studies were performed on a mixture of three 2-AB labeled glycans; one high mannose, one complex and one sialylated. See Figures 5 and 6. The glycans were loaded onto the microelution plate in the presence of excess 2-AB labeling reagent. Further SPE studies with a more complex mixture (Figure 7) demonstrate reproducibility. The area for each of six glycans is reported as a percent relative to the total of area counts. The variance due to SPE recovery is decreased in relative percent.
R2 = 1
1
10
100
1000
10000
100000
1000000
1 10 100 1000
Concentration (ng/uL)
A r
e a
FLR AreaExpon. (FLR Area)
Figure 4. Fluorescence Detection was shown to have a linear re-sponse over three orders of magnitude for 2-AB labeled maltohep-taose, including HILIC mode SPE and UPLC-FLR.
References
1. Bigge J. C.; Patel T. P.; Bruce J. A.; Goulding P. N.; Charles S. M.; Parekh R. B. Anal. Biochem. 1995, 230, 229–238.
• Glycan Separation Technology UPLC columns provide excellent resolution of mouse and human IgG oligosaccharides.
• High sensitivity and linearity over 3 orders of
magnitude result from the low dispersion instrument and fluorescence flow cell.
• The relatively short analysis time increases the
throughput of glycan chromatography. • 10 mM ammonium citrate is required for elution of
the sialylated biantennary structures found on IgGs. • SPE purification by HILIC mode µElution of labeled
glycans yields consistent patterns of glycoform distributions.
• The combination of SPE sample preparation, the low
dispersion instrument, and superior column technology provide a robust complete solution for t h e q u a n t i t a t i v e r e q u i r e m e n t s f o r biopharmaceuticals.
Time10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
EU x
10e
4
0.000
10000.000
20000.000
30000.000
40000.000
50000.000
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
EU x
10e
4
0.000
10000.000
20000.000
30000.000
40000.000
50000.000
1% Formic Acid
250 mMAmmonium
Formate
Acidic Glycans
Time10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
EU x
10e
4
0.000
10000.000
20000.000
30000.000
40000.000
50000.000
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
EU x
10e
4
0.000
10000.000
20000.000
30000.000
40000.000
50000.000
1% Formic Acid
250 mMAmmonium
Formate
1% Formic Acid
250 mMAmmonium
Formate
Acidic Glycans
Mass spectrometric confirmation of glycan MWs is often required. Since high ionic strength is required to retain sialylated glycans, 1% formic acid was compared to 250 mM ammonium formate.
CONCLUSIONS
