orthogonal methods for glycoanalysis: ce and uplc®

www.prozyme.com

ABSTRACTThe biopharmaceutical drug development process often requires the application of orthogonal methods to show method selectivity and specificity, confirm and identify components, and show that the appropriate level of due diligence has been performed for full product characterization. For N-glycan analysis, the predominant methods are liquid chromatography and capillary electrophoresis. The method ultimately chosen for routine analysis or product release has largely been a matter of preference, but the importance of utilizing orthogonal analyses in the decision-making process should not be overlooked. Presented here are the analyses of different analyte mixtures by both liquid chromatography (Ultra-Performance LC, UPLC®) and capillary electrophoresis (CE with laser-induced fluorescence, CE-LIF); multiple separation programs were performed for each method. By comparing these results, it becomes clear that each method has its strengths and weaknesses. These differences highlight the importance of performing orthogonal analyses for product characterization.

INTRODUCTIONBoth liquid chromatography and capillary electrophoresis are well-established platform technologies for N-glycan analysis. A comparison between the two was previously reported in 20071. With recent advances in separations technologies, a new comparison was warranted; a more in-depth comparison was performed2 demonstrating that UPLC and CE were complimentary for all biantennary N-Glycans of interest and offer truly orthogonal protocols to assess the bias of the individual methods: the coeluting/comigrating peaks in either platform will be distinguishable on the other platform. As a result of this work, ProZyme agreed to support a Multi-site N-Glycan Mapping Study3 (Mapping Study) by providing sample preparation reagents and supplies, System Certification Standards and Test Articles. The Mapping Study encompassed one method on each platform, defined by the vendors of the analytical instruments: Waters Corporation for UPLC, and Beckman Coulter, Inc.® for CE, who both provided significant materials and technical support. An international team, including over 20 independant

laboratories from biopharmaceutical companies, universities, analytical contract laboratories, instrument vendors and national authorities in the United States, Europe and Asia was formed to evaluate the precision and robustness of sample preparation and analysis of 2-aminobenzamide (2-AB)- and 8-aminopyrene-trisulfonic acid (APTS)-labeled N-glycans using UPLC and CE. All participants used the same lot of chemicals, reagents, columns/capillaries and test articles to run their assays, according to methods defined by each instrument vendor. After a predefined system suitability test to assure that systems were performing within specifications, the participants prepared a Protein Test Article that was analyzed with 5 different glycan samples, including a pre-labeled, homo-oligomer ladder standard, 3 pre-labeled N-glycan standard libraries (containing high-mannose, complex-type afucosylated and fucosylated species), and a pre-labeled N-glycan Test Article. Results are being reviewed and analyzed and will be presented/published at a future date.Questions arose among the participants during the planning for the Mapping Study regarding alternative separation programs (both UPLC and CE) to improve resolution or throughput, the trade-off between resolution and throughput, intra- and intersite method transferability, and how to approach peak identification. We have undertaken to address some of these questions here.Experiments were performed by both Ultra-Performance Liquid Chromatography with fluorescence detection of 2-AB (UPLC-FLR) and Capillary Electrophoresis with fluorescence detection of APTS (CE-LIF). In order to mimic the N-glycans typically observed on monoclonal antibodies and many Fc-fusion proteins, the Protein Test Article was prepared by mixing human IgG and Ribonuclease B (RNase B). To challenge the resolving power of each method and to emphasize the importance of employing orthogonal methods, two Glycan Test Articles were prepared by mixing N-glycan standards known to be somewhat difficult to resolve. Additionally, ProZyme’s pre-labeled Biantennary & High-Mannose Partitioned N-linked Glycan Libraries (product code GKSB-520 for 2-AB, and GKSP-520 for APTS) were used to assist in peak assignmnet; a ladder was also included to evaluate GU-values[Glyko 2-AB-(Glucose Homopolymer, product code GKSB-503, and Glyko® APTS-(Maltodextrin Ladder), product code GKSP-503]. Samples were

Orthogonal Methods for Glycoanalysis: CE and UPLC®

Zoltan Szabo; Jennie Truong; Shiva Pourkaveh; Samnang Tep; Ted Haxo; Michael Kimzey; Susan Fuller; Sybil Lockhart; Justin Hyche; Aled Jones; Jo Wegstein


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prepared by GlykoPrep workflows (product codes GP96NG-AB and GP96NG-APTS): deglycosylation was performed on GlykoPrep RX Cartridges; N-glycans were labeled with either Rapid-Reductive-Amination™ 2-AB or APTS; and post-labeling cleanup was performed on the associated GlykoPrep CU Cartridges (Figure 1).

FIgURe 1: Sample Preparation Workflow

Digest

Label

Cleanup

GS96-RX

Analysis

APTSGS96-APTS

2-ABGS96-AB

CEUPLC

C2GS96-C2

CUGS96-CU

PROTEIN TEST ARTICLE

MeTHODSUPLC Methods

Analytical SystemLC System: Waters ACQUITY UPLC® (H-class) with FLR detector, Empower™ software

Columns• Waters ACQUITY UPLC BEH Glycan 1.7 µm (Glycan Separation

Technology, GST column)

Method Column Size Waters Part Number

5-Minute Gradient 2.1 x 50 mm 186004740

10-Minute Gradient 2.1 x 100 mm 186004741

60-Minute Gradient A8,9 2.1 x 150 mm 186004742

60-Minute Gradient B 2.1 x 150 mm 186004742

• No guard column was used• Column Temperature: 60°C

Sample Preparation Recommendations• Prepare samples following protocols set forth in corresponding

manual for GlykoPrep® Rapid N-Glycan Preparation with 2-AB (ProZyme product code GP96NG-AB).

• Inject 1 µl of aqueous sample per UPLC run.• Remaining samples may be stored frozen in the dark. Use

sealing film if storing in 96-well plates.

Solvents• Solvent A: Acetonitrile• Solvent B: 100 mM Ammonium Formate pH 4.4

excitation and emission• Excitation: 360 nm• Emission: 428 nm

5-Minute gradient — Method Details

Time (min) Flow Rate (ml/min) % A % B

0 1.5 73 274 1.5 55 404.05 1.5 30 604.25 1.5 30 604.3 1 73 274.7 1.5 73 275 1.5 73 27

10-Minute gradient — Method Details


0 1 75 258 1 60 408.1 0.5 40 608.5 0.5 40 608.6 1 40 608.8 1 75 2510.0 1 75 25

60-Minute gradient A8,9 — Method Details


0 0.5 78 2238.5 0.5 55.9 44.139.5 0.25 20 8044.5 0.25 20 8046.5 0.5 78 2260 0.5 78 22

60-Minute gradient B — Method Details


0 0.75 75 2554 0.75 62.5 37.555 0.25 40 6056 0.25 40 6057 0.5 75 2558 0.75 75 2560 0.75 75 25

Ce Methods

Analytical System: CE System: Beckman Coulter® PA800 plus with LIF detector, 32-Karat™ software

Capillaries: Beckman Coulter N-CHO and eCAP (developmental)

Method Capillary Beckman Part Number

10-Minute Separation10 eCAP NA

15-Minute Separation12 N-CHO 477601



Sample Preparation Recommendations• Prepare samples following protocols set forth in corresponding

manual for GP96NG-APTS• Load each sample at 2 psi for 10 seconds• Remaining samples may be stored frozen in the dark.


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Separation Buffers: Method-specific; see individual methods

excitation and emission• Excitation: 360 nm• Emission: 428 nm

10-Minute Separation10 — Method Details

Length 60 cm (effective: 50 cm)

I.D. 30 µmSeparation Buffer 25 mM boric acid, 8 mM ammonium acetate; pH 9.0Temperature 35°CVoltage 30 kV for 10 minutesPolarity Reversed



I.D. 50 µmSeparation Buffer 40 mM, -aminocaproic acid, 40 mM acetic acid,

0.2% hydroxymethylcelluloseTemperature 20°CVoltage 30 kV for 15 minutesPolarity Reversed



I.D. 50 µmSeparation Buffer N-linked Carbohydrate Separation Gel Buffer

(Part No. 477623)Temperature 20°CVoltage 30 kV for 20 minutesPolarity Reversed



I.D. 50 µmSeparation Buffer 1:1 Mixture of N-linked Carbohydrate Separation Gel

Buffer (Part No. 477623) and eCAP ds DNA 1000 Gel Buffer (Part No. 477628)

Temperature 20°CVoltage 20 kV for 35 minutesPolarity Reversed

Samples. The Protein Test Article was prepared by mixing human IgG with RNase B, to yield an N-glycan profile with predominantly neutral biantennary fucosylated and high-mannose species. The two Glycan Test Articles were prepared by mixing a set of five and six N-glycan standards, respectively; some of the glycans were known to be difficult to resolve.

Liquid Chromatography. All analyses were performed on Waters ACQUITY UPLC® systems equipped with a fluorescence detector (ex: 360 nm/em: 428 nm); three different instruments were used by three different analysts, respectively, to analyze the Protein Test Article and Glycan Test Article No. 1 using the same instrument program. Four different instrument programs were used by an individual analyst to analyze the Protein Test Article and both Glycan Test Articles, the 60-minute (A) method provided by Waters being the one used in the Mapping Study; all four methods utilized an increasing ammonium formate gradient to separate the N-glycans over a Waters BEH GST column at 60°C (note that different column lengths were employed). See Table 1 for a summary of method parameters.

UPLC Method gradient Time minutes

Run Time minutes

Column Length mm

Column Temperature °C

5-minute gra-dient 4 5 50 60

10-minute gradient 8 10 100 60

60-minute gradient-A3,4 38.5 60 150 60

60-minute gradient-B 56 60 150 60

Ce Method Separation Voltage kV

Run Time minutes

Capillary Length cm

Capillary Temperature °C

10-minute separation5 30 10 60 35




TABLe 1: Summary of Method Parameters

Capillary electrophoresis. All analyses were performed on a single Beckman Coulter® PA800 plus Pharmaceutical Analysis System equipped with a LIF detector (ex: 488 nm/em: 520 nm); three different analysts performed analyses of the Protein Test Article and Glycan Test Article No. 1. Four different instrument programs were used by an individual analyst to analyze the Protein Test Article and both Glycan Test Articles, the 20-minute method provided by Beckman Coulter being the one used in the Mapping Study. Three of the methods utilized Beckman Coulter NCHO coated capillaries at 20°C (different separation buffers were used); the fourth method utilized a Beckman Coulter eCAP developmental capillary at 35°C. See Table 1 for a list of instrument programs.

ReSULTS & DISCUSSIONglykoPrep Sample Preparation. The quality of analysis is greatly affected by sample preparation; even the most capable instruments will show distortions caused by impurities or bias introduced during sample preparation. Important factors include the completeness of deglycosylation, labeling efficiency and/or fidelity of the labeling chemistry and effectiveness of post-labeling cleanup. All of the methods described here provide high-quality results as the GlykoPrep workflow has been optimized to yield quantitative deglycosylation, high labeling efficiency and effective removal of excess/unreacted dye, salts and artifacts without loss of glycans. In addition, the typical GlykoPrep workflow requires less than five hours from glycoprotein to purified fluorescently labeled N-Glycans.Analysis of Results. A similar semi-automated data analysis approach was utilized across the two platforms: peak tables were assembled based upon the retention and migration times of the N-glycan libraries for each separation program; the profiles were autoprocessed with the corresponding software (manual identification performed when needed due to minor shifting of peaks); all peaks with relative peak areas below 0.1% were excluded from the final profile.UPLC Results. The Protein Test Article was analyzed by 3 different analysts, on 3 different instruments, on different days; triplicate analysis was performed on each of 3 replicates yielding an n = 9 for each analyst with an overall n = 27. See


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separation programs, 3 different separation buffers and one additional capillary to investigate the trade-off between speed and resolution; different separation buffers and capillary dimensions were required as simply adjusting the separation voltage and time do not significantly impact resolution. The shorter methods tend to yield less resolved data that may be deemed appropriate for screening purposes, but more detailed information is lost. The longest method yielded the most information pertaining to peak identities and is more appropriate for characterization purposes, but at the expense of speed and throughput (Figure 7).The Glycan Test Articles were analyzed using the same four separation programs. See Figures 8 and 9; it is clear that greatest resolution is achieved with the longest method.UPLC and Ce. By comparing the results obtained by the various instrument programs across the two platforms, it is clear that neither UPLC nor CE is the complete answer. For the instrument programs chosen for use in the Mapping Study, the UPLC

Figure 2 for representative chromatograms and Table 2 for the corresponding data. The Protein Test Article was further analyzed using 3 different gradient programs to investigate the trade-off between speed and resolution. Faster methods yield reasonable data and may be deemed appropriate for screening purposes, but more detailed information is lost. Longer methods yield more accurate results pertaining to peak identities due to their greater resolution and are more appropriate for characterization purposes, but at the expense of speed and throughput (Figure 3). The Glycan Test Articles were analyzed using the same four gradient programs (Figures 4 and 5). Again, it is clear that peak resolution is greatly affected by gradient length.Ce Results. The Protein Test Article was analyzed by 3 different analysts on different days; triplicate analysis was performed on each of 3 replicates yielding an n = 9 for each analyst with an overall n = 27. See Figure 6 for representative electropherograms and Table 3 for the corresponding data. The Protein Test Article was further analyzed using 3 different

FIgURe 3: Comparison of UPLC Methods – Protein Test Article. Overlay of chromatograms for the Protein Test Article acquired by 4 different methods. Upper trace: 5-minute gradient. Second from the top: 10-minute gradient. Second from the bottom: Multi-site N-Glycan Mapping Study3 gradient. Bottom trace: 60-minute gradient-B. Increased resolution is achieved with longer gradients.

FIgURe 2: Comparison of UPLC Across 3 Analysts. Overlay of all chromatograms acquired by 3 analysts using the Multi-site N-Glycan Mapping Study gradient. Profiles have not been normalized; stable flurescence intensities highlight reproducibility of the workflow. For glycan ID's, refer to Legend.

FIgURe 4: Comparison of UPLC Methods – glycan Test Article No. 1. Overlay of chromatograms from the Glycan Test Article acquired by 4 different gradients. Upper trace: 5-minute gradient. Second from the top: 10-minute gradient. Second from the bottom: Multi-site N-Glycan Mapping Study3 gradient. Bottom trace: 60-minute gradient-B. Increased resolution is achieved with longer gradients. For glycan ID's, refer to Legend.

FIgURe 5: Comparison of UPLC Methods – glycan Test Article No. 2. Acquired by 4 different gradients. Upper trace: 5-minute gradient. Second from the top: 10-minute gradient. Second from the bottom: Multi-site N-Glycan Mapping Study3 gradient. Bottom trace: 60-minute gradient-B. Increased resolution is achieved with longer gradients. For glycan ID's, refer to Legend.


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Analyst 1 (n = 9) Analyst 2 (n = 9) Analyst 3 (n = 9) Overall (n = 27)

Peak ID Average Std Dev Average Std Dev Average Std Dev Average Std Dev %CV

1 0.13 0.01 0.12 0.01 0.14 0.01 0.13 0.01 9.82 0.77 0.03 0.74 0.03 0.82 0.01 0.78 0.04 5.13 0.13 0.01 0.12 0.02 0.13 0.01 0.13 0.01 9.74 17.87 0.40 17.96 0.15 18.48 0.12 18.10 0.37 2.05 2.26 0.05 2.29 0.04 2.49 0.03 2.35 0.11 4.86+7 4.29 0.08 4.27 0.02 4.41 0.03 4.33 0.08 1.88 0.46 0.01 0.45 0.01 0.49 0.02 0.47 0.02 4.0unknown 1 0.37 0.02 0.36 0.03 0.36 0.01 0.36 0.02 5.59 19.33 0.24 19.32 0.15 19.21 0.05 19.29 0.17 0.910 8.22 0.11 8.23 0.05 8.22 0.03 8.22 0.07 0.811 4.30 0.03 4.29 0.03 4.22 0.02 4.27 0.05 1.113 1.38 0.05 1.61 0.02 1.25 0.01 1.41 0.16 11.212 0.41 0.01 0.39 0.03 0.42 0.01 0.41 0.02 6.014 1.12 0.01 1.10 0.02 1.16 0.01 1.13 0.03 2.4unknown 2 0.29 0.02 0.27 0.04 0.29 0.01 0.28 0.02 8.715 15.81 0.05 15.71 0.09 15.35 0.06 15.62 0.21 1.3unknown 3 0.14 0.01 0.16 0.02 0.13 0.02 0.14 0.02 15.616 1.69 0.02 1.69 0.06 1.61 0.01 1.66 0.05 3.1unknown 4 0.17 0.01 0.19 0.02 0.19 0.02 0.19 0.02 8.917 0.68 0.03 0.71 0.03 0.68 0.06 0.69 0.04 6.0unknown 5 0.31 0.02 0.29 0.01 0.30 0.02 0.30 0.02 5.618 2.13 0.08 2.05 0.13 1.93 0.03 2.03 0.12 6.0unknown 6 0.13 0.02 0.11 0.01 0.25 0.04 0.16 0.07 41.419 0.86 0.04 0.81 0.02 0.84 0.01 0.84 0.03 4.0unknown 7 0.13 0.02 0.13 0.01 0.14 0.00 0.13 0.01 10.0unknown 8 0.11 0.01 0.11 0.02 0.11 0.01 0.11 0.01 9.220 2.38 0.03 2.35 0.09 2.35 0.01 2.36 0.06 2.421 9.23 0.46 9.07 0.15 8.85 0.05 9.05 0.31 3.5unknown 9 0.43 0.03 0.41 0.02 0.45 0.01 0.43 0.03 6.122 1.42 0.08 1.36 0.03 1.32 0.01 1.37 0.06 4.7unknown 10 0.12 0.01 0.13 0.02 0.13 0.01 0.12 0.02 13.823 0.97 0.02 0.98 0.06 0.97 0.03 0.97 0.04 3.824 0.23 0.03 0.25 0.03 0.24 0.01 0.24 0.03 10.925 0.14 0.02 0.13 0.03 0.15 0.01 0.14 0.02 13.226 0.97 0.10 1.01 0.04 0.99 0.03 0.99 0.07 6.727 0.84 0.10 0.88 0.04 0.90 0.02 0.87 0.07 7.6

TABLe 2: Comparison of UPLC Across 3 Analysts. Results show high reproducibility for major peaks; low-level unknowns show the highest variability. Refer to Legend for Peak IDs.

FIgURe 6: Comparison of Ce Across 3 Analysts. Overlay of all electropherograms acquired by 3 analysts using the Multi-site N-Glycan Mapping Study separation program. Profiles have not been normalized; stable fluorescence intensities highlight reproducibility of the workflow. For glycan ID's, refer to Legend.

FIgURe 7: Comparison of Ce Methods – Protein Test Article. Overlay of electropherograms from the Protein Test Article acquired by 4 different separation programs. Upper trace: 10-minute separation5. Second from the top: Multi-site N-Glycan Mapping Study3 separation. Second from the bottom: 15-minute separation. Bottom trace: 35-minute separation6. Increased resolution is achieved with longer programs.


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Analyst 1 (n = 9) Analyst 2 (n = 9) Analyst 3 (n = 9) Overall (n = 27)

Peak ID Average Std Dev Average Std Dev Average Std Dev Average Std Dev %CV

24 0.14 0.02 0.14 0.03 0.15 0.01 0.14 0.02 14.825 0.07 0.01 0.08 0.01 0.09 0.00 0.08 0.01 16.426 0.88 0.08 0.86 0.04 0.91 0.03 0.88 0.06 6.427 0.68 0.11 0.67 0.06 0.72 0.03 0.69 0.08 11.0unknown 1 0.23 0.03 0.26 0.07 0.27 0.01 0.25 0.05 18.418 1.97 0.01 1.93 0.04 1.99 0.02 1.96 0.03 1.719 0.57 0.02 0.60 0.03 0.63 0.01 0.60 0.03 5.2unknown 2 0.23 0.01 0.22 0.04 0.24 0.00 0.23 0.02 10.421+2 9.53 0.09 9.61 0.13 9.55 0.14 9.56 0.12 1.322+5 2.69 0.19 2.62 0.14 2.72 0.07 2.68 0.14 5.4unknown 3 0.38 0.09 0.32 0.09 0.31 0.03 0.34 0.08 23.2unknown 4 0.11 0.01 0.08 0.02 0.24 0.02 0.14 0.08 53.04+13+7 21.67 0.32 21.88 0.50 21.42 0.08 21.66 0.38 1.88 0.33 0.01 0.33 0.03 0.33 0.03 0.33 0.03 8.26 2.99 0.03 3.02 0.06 2.97 0.04 3.00 0.05 1.69+17 22.33 0.06 22.24 0.38 22.29 0.05 22.29 0.22 1.010+11+14 13.42 0.06 13.42 0.10 13.47 0.07 13.44 0.08 0.6unknown 5 0.04 0.01 0.04 0.01 0.05 0.01 0.05 0.01 18.420+12 2.07 0.03 2.06 0.01 2.02 0.01 2.05 0.03 1.4unknown 6 0.07 0.01 0.06 0.01 0.08 0.01 0.07 0.01 14.615+23 18.37 0.19 18.31 0.14 18.27 0.06 18.31 0.14 0.816 1.28 0.03 1.27 0.03 1.29 0.01 1.28 0.03 2.0

TABLe 3: Comparison of Ce Across 3 Analysts. Results show high reproducibility for major peaks; low-level uknowns show the highest variability. Refer to Legend for Peak IDs.

method yields better resolution of the Protein Test Article and Glycan Test Article No. 1. It is important to note however that the gradient is 60 minutes, while the CE separation program used is 20 minutes; this clearly demonstrates the trade-off between speed and resolution, even across platforms. When directly comparing Protein Test Article results from the shortest methods used by each platform (i.e., the 5-minute UPLC gradient and the 10-minute CE separation), the CE method yields better resolution of peaks; 16 peaks are resolved by UPLC and 23 peaks resolved by CE. A comparison of both 10-minute methods shows that the UPLC method is able to resolve 28 peaks, while the CE method resolves only 23. The highest resolving method for each platform yielded similar results; the UPLC method has 2 co-eluting N-glycan peaks (G1[6] and G0FB), while the CE method is able to resolve all of the known

peaks (Figure 10). The highest resolving CE method is 35 minutes, while the highest resolving UPLC method is 60 minutes. In this regard, the CE method outperforms the UPLC method. It should however be noted that the N-glycans of interest were completely eluted within 35 minutes using the UPLC method, indicating that the gradient could be truncated to decrease the total run time and improve throughput (if desired). Additionally, when comparing the peak-to-peak resolution, the highest resolving UPLC method yields greater average resolution values (UPLC = 3.28; CE = 1.71).The results for the Glycan Test Articles are similar to those obtained for the Protein Test Article. The shortest methods for both UPLC and CE are unable to fully resolve the glycan standards in the mix; the 5-minute UPLC method is only able

FIgURe 8: Comparison of Ce Methods – glycan Test Article No. 1. Overlay of electropherograms from the Glycan Test Article acquired by 4 different separation programs. Upper trace: 10-minute separation5. Second from the top: Multi-site Mapping Study3 separation. Second from the bottom: 15-minute separation7. Bottom trace: 35-minute separation6. Increased resolution is achieved with longer programs. For glycan ID's, refer to Legend.

FIgURe 9: Comparison of Ce Methods – glycan Test Article No. 2. Overlay of electropherograms from the Glycan Test Article No. 2 acquired by 4 different separation programs. Upper trace: 10-minute program5. Second from the top: Multi-site Mapping Study3 separation. Second from the bottom: 15-minute separation. Bottom trace: 35-minute separation6. Increased resolution is achieved with longer programs. For glycan ID's, refer to Legend.


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to fully resolve three of the standards in each mixture (other standards are only slightly resolved, appearing as shoulders); the 10-minute CE method is only able to fully resolve four of the five standards in Glycan Test Article No. 1 and five of the six standards in Glycan Test Article No. 2. The highest resolving method for both platforms was able to resolve all five glycans in Glycan Test Article No. 1; only the highest resolving CE method was able to resolve all six glycans in Glycan Test Article No. 2.Orthogonality. The orthogonality of the platforms is highlighted by the differences in resolution and peak selectivity; this is more clearly observed with the Glycan Test Articles. When comparing Glycan Test Article No. 1 by the two 10-minute methods, it is observed that both methods are only able to resolve four of the five peaks with two of the standards co-eluting/-migrating in each; however, the unresolved N-glycan-pairs are different between the platforms: the 10-minute UPLC method is unable to resolve A1F and Man-8, while the 10-minute CE method is unable to resolve G1F[6] and Man-8. When comparing Glycan

FIgURe 10: Comparison of Highest-Resolving UPLC and Ce Methods – Protein Test Article. Overlay of N-glycan profiles from the Protein Test Article acquired by the highest-resolving UPLC and CE methods. Upper trace: 60-minute gradient-B. Bottom trace: 35-minute separation6. Both methods yield fairly comparable results; the UPLC method has more resolvable peaks. For glycan ID's, refer to Legend.

FIgURe 11: Overlay of Partitioned Libraries with the Protein Test Article (UPLC). Acquired by the highest-resolving UPLC method. Use of glycan standards can facilitate the peak identification process for many commonly observed glycans. Red font indicates glycans not included in the N-glycan Legend.

FIgURe 12: Overlay of Partitioned Libraries with the Protein Test Article (UPLC). Acquired by the highest-resolving UPLC method. Use of glycan standards can facilitate the peak identification process for many commonly observed glycans. Red font indicates glycans not included in the N-glycan Legend.

FucoseGalactoseMannoseN-acetylglucosamineN-acetylneuraminic acid

GO 3

GOF 4

GOFB 6

G1 7+8

G1F 9+10

G1FB 11+12

G2 14

G1FS1 18

A1 19

A1F 21

A1FB 22

A2 24

A2B 25

A2F 26

A2FB 27

GO-N 1

GOF-N 2

Man5 5

Man6 13

G2F 15

G2FB 16

Man7 17

Man8 20

2 34

Man9 23

A B

FIgURe 13: Comparison of gU Values from Different Chromatographic Programs (UPLC) and electrophoretic Separations Programs (Ce). (A) Calculated GU values from 2 different UPLC methods. Differences in GU values are observed for all glycan types. Larger differences appear to be associated with sialylated species. (B) Calculated GU values from 2 different CE methods. Large differences in GU values are observed for all glycan types.

Legend – N-glycan Structures, Peak ID Numbers and Notations. Peak ID Numbers identify the associated structures in the N-Glycan Profiles.


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Test Article No. 2 by the two 10-minute methods, it is observed that both methods are only able to resolve five of the six peaks with two of the standards co-eluting/-migrating in each. The unresolved N-glycan-pairs are different between the platforms: the 10-minute UPLC method is unable to resolve G0FB and G1, while the 10-minute CE method is unable to resolve G0FB and G2. Differences in selectivity are reinforced when comparing the two highest-resolving methods: both methods resolve Glycan Test Article No.1, but Glycan Test Article No. 2 is only resolved using CE. The UPLC method shows that larger neutral glycans (i.e., high mannose; -7, -8, -9, etc.) elute in the same time frame as smaller sialylated glycans (i.e., monosialylated biantennary; with/without fucose); this can convolute data analysis and the characterization of unknown peaks, as the ability to “cluster” similar glycans is somewhat lost. The CE method shows that similarly-sized/-charged glycans migrate closely together; this can be troublesome as it may limit the ability to clearly resolve peaks; even the highest-resolving CE method used here does not fully separate G1F[6] and G2. Utilizing both methods during the characterization process should be considered to ensure the greatest coverage possible.Biantennary and High-Mannose Partitioned N-linked glycan Library and gU Values. A set of glycan standard libraries (Partitioned Libraries) and a ladder were included in these experiments; each was analyzed on both platforms with their respective instrument programs. The Partitioned Libraries provide an alternative to GU-values for the assignment of glycan peaks; see Figures 11 and 12. Using a set of known standards allows for the direct comparison (i.e., aligning) of peaks to aid in peak assignment. When a ladder is used, GU-values are calculated for peaks of interest and cross-referenced against a database; the database may provide multiple structural options and is helpful in the identification of peaks. Users of the ladder should be aware that GU-values may vary depending on the instrument and/or program used; see Figures 13 A and B. Regardless of whether glycan libraries or ladders are used, the identities of peaks should always be confirmed by other means, such as glycosidase treatments or mass spectrometry.

CONCLUSIONSglykoPrep. Sample preparation using GlykoPrep provides a rapid and simple workflow for the quantitative preparation of samples, which reproducibly yields high-quality data (evident in the reproducibility of total fluorescence observed between replicates). The inherent flexibility allows for a wide range of applications, from the in-depth characterization of a few samples to high-throughput screening for hundreds of samples. Additionally, the GlykoPrep workflow is amenable to, and unbiased for, both UPLC and CE.Speed vs. Resolution. Rapid separation methods are very attractive and useful for high-throughput screening applications, such as cell-line selection and optimization of culture conditions, as less-detailed information is needed. More in-depth characterization work requires longer methods that yield greater resolution.UPLC and Ce. Overall data quality between the two platforms is comparable. When using high-resolution methods, both platforms perform equally well. The CE method is 35 minutes, while the UPLC method is 60 minutes.

Orthogonality. The orthogonality of the platforms is clearly evident when comparing the types of N-glycans that are difficult to resolve for each. In most cases, peaks not well- or unresolved on one platform will be resolved on the other. For instance, the 10-minute UPLC method does not resolve A1F and Man-8, while the 10-minute CE method does. Conversely, the 10-minute CE method does not resolve G1F[6] and Man-8, while the 10-minute UPLC method does.Biantennary and High-Mannose Partitioned N-linked glycan Library. The Partitioned Libraries provide an effective means for the assignment of peaks that is more definitive than comparison against a ladder, as the assignment process is more direct. Glucose unit (GU) values are affected by the instrument program, which can make it difficult to cross-reference published GU-based repositories when an instrument program does not exactly match. Although the elution or migration of the N-glycans within a pre-labeled Partitioned Library are also affected by the instrument programs, the individual components are known, which allows for direct comparison regardless of changes to the instrument program.

ReFeReNCeS1. Domann et al. Separation-based Glycoprofiling Approaches using Fluorescent Labels.

Proteome Research 2007, 1, 70-76.2. Kimzey et al. N-Glycan Profiling Using Orthogonal Methods: UPLC® and CE; Poster

presented at: HPLC 2012 38th International Symposium on High Performance Liquid Phase Separations and Related Techniques, 2012 June 16-21; Anaheim, CA.

3. N-Glycan Mapping Study Using Orthogonal Methods: CE and UPLC. Workshop at CE in Biotechnology & Pharmaceutical Industries: 15th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules (CE Pharm 2013), October 6-10, 2013, Arlington, VA, USA.

4. Ahn, J. et al. UPLC-FLR Method Development of 2-AB-Labeled Glycan Separation in Hydrophillic Interaction Chromatography (HILIC). Application Note. Waters Corporation, Milford, MA, USA.

5. Szabo, Z. et al. Rapid High-Resolution Characterization of Functionally Important Monoclonal Antibody N-Glycans by Capillary Electrophoresis. Analytical Chemistry, 2011, 83, (13), 5329-5336.

6. Rampal, S. et al. Separation of Fucosylated, non-Fucosylated, and Complex Carbohydrates Associated with Monoclonal Antibodies using Capillary Electrophoresis. Chromatography Today 2011, 3, 38-40.

7. Hamm, M. et al. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection. Pharmaceuticals, 2013, 6, 393-406.

8. N-Glycan Mapping Study Using Orthogonal Methods: CE and UPLC. Workshop at CE in Biotechnology & Pharmaceutical Industries: 15th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules (CE Pharm 2013), October 6-10, 2013, Arlington, VA, USA.

9. Ahn, J. et al. UPLC-FLR Method Development of 2-AB-Labeled Glycan Separation in Hydrophillic Interaction Chromatography (HILIC). Application Note. Waters Corporation, Milford, MA, US

10. Szabo, Z. et al. Rapid High-Resolution Characterization of Functionally Important Monoclonal Antibody N-Glycans by Capillary Electrophoresis. Analytical Chemistry, 2011, 83, (13), 5329-5336.

11. Rampal, S. et al. Separation of Fucosylated, non-Fucosylated, and Complex Carbohydrates Associated with Monoclonal Antibodies using Capillary Electrophoresis. Chromatography Today 2011, 3, 38-40.

12. Hamm, M. et al. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser- Induced Fluorescence Detection. Pharmaceuticals, 2013, 6, 393-406.

AcknowledgementsWe thank Beckman Coulter, Inc. for making the developmental capillaries available to us for this work, and for the use of the BC PA 800 plus Pharmaceutical Analysis System as part of our technical collaboration.

Trademarks & TradenamesProZyme, Glyko, GlykoPrep and Rapid-Reductive-Amination are trademarks or registered trademarks of ProZyme, Inc. in the United States and other countries; Waters®, ACQUITY® UPLC® and BEH Technology™ are trademarks of Waters Corporation, Milford,MA, USA; Beckman Coulter® and Beckman Coulter, Inc.® are registered trademarks of Beckman Coulter, Inc.

orthogonal methods for glycoanalysis: ce and uplc®

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