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A STREAMLINED ANALYTICAL PLATFORM FOR GLYCAN COMPARABILITY ASSESSMENT FOR INNOVATOR AND BIOSIMILAR INFLIXIMAB William R. Alley, Jr.1, Mark Hilliard2, Jennifer Byrne2, Ying Qing Yu1, Pauline Rudd2, Min Du1 1. Waters Corporation, Milford, MA, USA 2. National Institute for Bioprocessing Research & Training, Dublin, Ireland

CONCLUSIONS We have developed a streamlined platform for

glycan analysis, from sample preparation to data collection and analysis.

Using this platform, we compared the glycan profiles of innovator and biosimilar infliximab.

Overall, the glycan profiles were very similar, especially for the abundant structures.

Some differences were observed for some lower-abundance glycans, including a complex glycan terminated with an N-glycolylneuraminic acid (elevated in the biosimilar sample) and a hybrid glycan possessing α-linked galactose units, which was observed only on the innovator samples.

Our results are in general agreement with a previous study showing glycosylation patterns between the two drugs are very similar.3

References 1. Lauber, M.A.; Yu, Y.-Q.; Brousmiche, D. W.; Hua, Z.; Koza,

S. M.; Magnelli, P.; Guthrie, E.; Taron, C. H.; Fountain, K. J. Rapid Preparation for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection, Anal. Chem., 2015, 87, 5401-5409.

2. GlycoWorks™ RapiFluor™-MS N-glycan Kit Care and Use Manual (PN: 715004793).

3. Jung, S.K.; Lee, K.H.; Jeon, J.W.; Lee, J.W.; Kwon, B.O.; Kim, Y.J.; Bae, J.S.; Kim, D.I.; Lee, S.Y.; Chang, S.J. Physicochemical characterization of Remsima, MAbs, 2014, 6, 1163-1177.

INTRODUCTION

The analysis of protein glycosylation is a rapidly developing area in many different disciplines of scientific research, including the biopharmaceutical industry where glycosylation is one of the critical quality attributes. The chemical and biochemical properties of glycans can influence the safety and effectiveness of therapeutic proteins, such as monoclonal antibodies (mAbs). Further, glycosylation patterns are important to monitor and control during the development of biosimilar products and the glycosylation patterns of these mAbs should resemble that of the innovator drug to a high degree.

To this end, we have developed a UPLC/FLR/MS analytical platform for labeled glycans. The sample preparation, including glycan labeling using a novel reagent, has been simplified and can be completed in less than 1 hr. This new tagging molecule, commercially available as RapiFluor-MSTM

(RFMS) from Waters Corp., significantly improves both the fluorescence and mass-spectral sensitivities.1 The enhanced signal allows lower-abundance glycans to be detected and confidently identified using MS and MS/MS data. The identities of the glycans are revealed through library searching of the calibrated retention time in glucose unit (GU) values and further confirmed using the MS data at sub 2-ppm mass accuracies for most glycans.

We have applied this analytical approach to analyze innovator and biosimilar infliximab mAbs and we pay particular attention to the possible immunogenic glycans, including N-glycolylneuraminic (NeuGc) acid and α-linked galactose-galactose pairs (α-Gal). In general, we have found that the major glycan structures on both mAbs are very similar and there appears to be some slight differences in some lesser abundant glycans, based on lower % relative fluorescence peak areas.

LC Conditions LC System: ACQUITY UPLC H-Class Bio LC Column: ACQUITY BEH Glycan Amide, 2.1 x 150 mm, 130 Å pore size, 1.7 μm particle size Column Temp.: 60o C Sample Temp.: 4o C Fluorescence Detection: Ex 265 nm/Em 425 nm Gradient Conditions Mobile Phase A: 50 mM ammonium formate, pH = 4.5 Mobile Phase B: Acetonitrile

MS Conditions MS System: XEVO G2-XS QToF Analyzer Mode: ESI+, sensitivity mode, ~30k resolution Capillary Voltage: 3.0 kV Cone Voltage: 80 V Source Temp.: 120o C Desolvation Temp.: 250o C m/z Range 500-2000 Scan Rate: 2 Hz Instrument Control: UNIFI 1.8

Time (min) Flow Rate (mL/min)

%A %B

0.0 0.400 25.0 75.0

35.0 0.400 46.0 54.0

36.5 0.200 100.0 0.0

39.5 0.200 100.0 0.0

43.1 0.200 25.0 75.0

47.6 0.400 25.0 75.0

55.0 0.400 25.0 75.0

Component name StructureExpected

GU? GU GUStd Dev Expectedm/z ? m/z Mass Confirmed

F(6)A2G(4)1Sg1 8.1627 0.0011 0.0500 1122.4384 0.0005 True

M4A1 8.1557 0.0081 0.0500 1122.9486 0.5107 False

F(6)A2[3]BG(4)1S(6)1 8.2239 0.0601 0.0500 1215.9806 93.5427 False

FA2G2F1 8.2365 0.0727 0.1000 1122.9486 0.5107 False

F(6)A3G(4)3 8.2393 0.0755 0.1000 1232.4857 110.0478 False

F(6)A2G(4)2Ga(3)1 8.2457 0.0819 0.1000 1130.9460 8.5081 False

F(6)A2G(4)2Ga1 iso 8.2995 0.1357 0.0500 1130.9460 8.5081 False

Comparing α-linked Galactose levels

Symbols: GlcNAc; Fu- Mannose; Galactose;

N-glycolylneuraminic acid; N-acetylneuraminic acid

10 min 5 min 10 min 1 2 3

Automated LC-MS data collection

Data processing

Review/reporting

General workflow Glycan workflow HILIC/FLR/MS data collection

Calibrate RTs, assign GUs,

apply Lockmass calibration

Search scientific library

Creating the RFMS Glycan Library

EXPERIMENTAL WORKFLOW

INSTRUMENT CONDITIONS

Quickly Identifying Differentially Expressed Glycans: The Difference Plot

Comparing Glycan Classes in UNIFI

Figure 2. FLR trace of an RFMS dextran ladder used for retention time calibration.

Figure 3. Conversion of retention times to Glucose Units (GU).

Figure 4. Exoglycosidase digestion procedure for structural characteriza-tion of a glycans and GU value as-signment.

Figure 5. Example of an entry in the Waters glycan library.

Separation Calibration

UNIFI allows users to perform a library search for structural identification. Searches are

based on GU values, which allow for more direct comparisons between instruments. GU values are determined through re-tention time calibration of an RFMS-tagged dextran ladder.

BPI Traces Reflects FLR Trace

Figure 6. Comparison of the BPI and FLR traces. The FLR trace has been annotated with the glycans that were mass confirmed in this study.

In this study, we identi-fied and quantitated, us-ing the FLR trace, 25 mass-confirmed glycans. These glycans are shown in the FLR trace of Figure 6. This figure also shows that the BPI and FLR traces are very similar.

Table 1. Comparison of different glycan classes. This table, gen-erated by UNIFI, shows that in terms of the overall glycan classes, both mAbs are very similar in their global glycan profiles.

Differences in abundance levels can be visualized by a differ-ence plot, as shown in Figure 7. This plot shows that the more highly abundant glycans are quite similar in their abun-dance levels but there are some differences in the lesser abundant glycans.

Figure 7. Difference plot comparing the biosimilar (red trace) and an innovator sample (blue trace). The green trace shows the difference in abundance level between the two samples.

Comparing N-glycolylneuraminic Acid Levels N-glycolylneuraminic acid is a non-human carbohydrate whose presence in the human body can induce a severe immunogen-ic response. It is therefore important to monitor and minimize levels of this sugar on biotheraputics.

Figure 8. This figure shows the annotated FLR trace from UNIFI and the extracted ion chromatogram for a glycan terminated with N-glycolylneuraminic acid. The XIC shows two peaks, indicating that positional isomeric species of this glycan regarding the sialic acid were present. The inset of the bottom panel shows the mass spec-trum for this glycan.

Figure 9. This figure shows the results of the library search within a specified window of GUs. In this study, a window of ± 0.10 was searched. After identifying possible glycans within this window, UNIFI examines the MS data to confirm the assignment. Within this GU window, only one glycan could be mass confirmed and was measured at sub-2 ppm mass accuracy.

Library Search Results

Summary Plot

Figure 10. A summary plot. A particular glycan can be compared across multiple samples using a summary plot of MS intensities. This plot shows that the complex glycan terminated with an N-glycolylneuraminic acid is likely elevated in the biosimilar product when compared to multiple batches of the innovator drug.

Biosimilar Innovator Difference

Figure 11. An overlay of FLR traces. To further confirm that the complex glycan terminated with N-glycolylneuraminic acid was ele-vated in its abundance levels, the FLR traces for the biosimilar and innovator samples were overlaid. This analysis also indicates that the glycan of interest is elevated in the biosimilar product.

Biosimilar

Innovator

α-Linked galactose-galactose pairs are also non-human species whose presence can induce an immunogenic re-sponse. It is also important to monitor levels of this type of linkage.

Figure 13. This figure shows the annotated FLR trace from UNIFI and the extracted ion chromatogram for a glycan containing α-linked galac-tose units. The XIC show two peaks, however the first peak is corre-sponds to another glycan whose m/z value fell within the window used to extract the ion chromatogram for the glycan of interest.

Library Search Results

Figure 14. This figure shows the results of the library search within a window of GUs. Within this window, only one glycan could be mass con-firmed and was determined to be a hybrid glycan possessing α-linked ga-lactoses.

Summary Plot

Figure 15. The summary plot. This plot shows that this particular glycan with α-linked galactose units was only present in the inno-vator samples; it was not observed in the biosimilar sample. Re-peated analyses confirmed these results.

Glycan StructureExpected 

GU? GU

GU Std D

ev

Expected 

m/z? m/z Mass Confirmed

A2G(4)2S(6)1 8.38 0.06 0.03 1122.44 85.03 FALSE

M5A1G(4)1Ga(3)

18.44 0.01 0.10 1037.40 ‐0.0002 TRUE

F(6)A2G(4)1Sg1 is

o8.35 0.09 0.05 1122.44 85.03 FALSE

F(6)A3G(4)3 8.54 0.10 0.05 1232.49 195.08 FALSE

A2G(4)2S(6)1 8.32 0.12 0.05 1122.44 85.03 FALSE

F(6)A2G(4)2Ga1 i

so8.30 0.15 0.05 1130.95 93.54 FALSE

Biosimilar

Innovator

Figure 16. The stacked FLR traces for this particular glycan also indicate that it is only present in the in-novator drug.

Figure 12. This figure shows triplicate injections (a) and triplicate sample preparations (b) of the different drugs. In each case, the same results were achieved. For these measurements, RSD values of less than 2% were achieved.

a b

DATA COLLECTION WORK-FLOW IN UNIFI 1.8

Innovator batches

Innovator batches

Biosimilar

Injection-to-injection reproducibility

Preparation-to-preparation reproducibility

Instrument Components: ACQUITY UPLC H-Class Bio System ACQUITY UPLC FTN Sample Manager ACQUITY UPLC Column Manager ACQUITY UPLC FLR Detector Xevo G2-XS QToF MS

Denature 15-μg aliquots of mAb with 1% Rapigest at 60o C for ~5 minutes

Release N-linked glycans using Rapid PNGase F for ~5 minutes at 55o C

Label glycans (released and stabilized as glyco-sylamines) with RapiFluor-MS™

Purify samples using HILIC μ-elution plate. Di-lute aqueous eluent with DMF and acetonitrile.

UPLC-MS analysis

Biosimilar

Biosimilar

Biosimilar

Biosimilar

Biosimilar

Biosimilar

Biosimilar

Biosimilar

Biosimilar Biosimilar

Sample Names Innovator Biosimilar Samples Sample 8BM Biosimilar 080M 9CM 08A12 08B03 8LM

GlycoWorks™ RapiFluor™-MS N-glycan Kit used in this study

Figure 1. Outline of the sample preparation work-flow used in this study.

RESULTS