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INTRODUCTION A novel method for in-depth characterization of O-linked glycosylation of Etanercept is developed and the comparison on O-glycosylation heterogeneity between the innovator Etanercept (Enbrel) and a biosimilar is conducted in this study. Etanercept is a highly glycosylated fusion protein and a blockbuster drug. Due to its commercial success, Enbrel is an obvious target for biosimilar development. Among all the quality attributes, O-glycosylation of Etanercept plays an important role in its drug efficacy, and therefore needs to be characterized. Because of extensive O-linked glycosylation on Etanercept , characterization of O-glycosylation poses a great analytical challenge. Detailed study on O-linked glycosylation heterogeneity of Etanercept was achieved by a combination of a new sample preparation methodology and LC/MS with CID and ETD fragmentation.

CHARACTERIZATION AND COMPARISON OF O-GLYCOSYLATION BETWEEN THE INNOVATOR AND A BIOSIMILAR OF ETANERCEPT

Stephane Houel1; Ying Qing Yu1; Jonathan P. Williams2; Weibin Chen1 1Waters Corp., Milford, MA 01757; 2Waters Corp, Manchester, UK

METHODS Data acquisition:

LC: Waters ACQUTIY UPLC I-Class

Column: 2.1x150mm BEH C18 300Å, 1.7 µm

MS: Waters SynaptTM G2-S HDMS Mode: ESI positive mode Capillary Voltage: 3kV Cone: 10-25V Source Temperature: 100 °C Desolvation Temperature: 200 °C ETD settings: Reagent: 1,3-dicyanobenzene Glow discharge current: 90 A Trap wave height: 0.2-0.25 V Trap RF: 450-500 V Data Processing & Annotation: BiopharmaLynx 1.3.3 BioLynx Application Manager

Figure 1. Data were collected using a Synapt G2-S with ETD capability

Table 2: Identified glycosylation sites (in blue) from ETD spectra of Etanercept

CONCLUSION Developed a sample preparation workflow to study Core1 O-glycoproteins. Identified 5 tryptic peptides containing O-linked glycans (see table1). Assigned 12 glycosylation sites on 9 glycopeptides from Etanercept (see

table2). Highlighted differences in glycosylation patterns between the innovator and a

biosimilar. Synapt G2-S MS system with ETD can be routinely used for O-glycopeptides

sites analysis of protein therapeutics.

MaxEnt 3

T14—2 core1

T15—7 and 6 core1

Figure 4. ETD spectra of peptide T14 from Etanercept protein: raw data (A) and MaxEnt 3 deconvoluted spectrum (B). Out of 24 potential c/z ions, 23 were observed, from which the sites of the O-glycosylation modification were identified.

Figure 5. Deconvoluted ETD spectra of peptide T15 from Etanercept protein. Peptide T15 contains a total of 11 Serine or Threonine residues as potential sites for O-linked glycosylation. (A) Glycosylation sites (7 total) in a T15 peptide were identified unambiguously based on the ETD spectrum; (B-C) Modification sites (6 total) were confidently assigned from the ETD fragmentation data.

A) C) B)

A) B)

Table 1: Major O-linked glycans from the innovator of Etanercept. The number of b/y ions found and the mass error are an average of the triplicate runs.

Figure 2. sample preparation :Etanercept protein was reduced and alkylated with DTT/IAM. PNGase F (Step 1) and neuraminidase (2->3,6,8,9) (Step 2) were added to the sample and incubated overnight at 37oC. Trypsinization (Step3) was performed at 37 oC for 6h.

Figure 3. Triplicate runs of tryptic digest of the innovator Etanercept.

Replicate 1

Replicate 2

Replicate 3

or

RemovingN linked Glycans

RemovingSialic Acid

Delta Mass=

365.1322 Da

Core 1O-linked glycan

Digestion

Step 1 Step 2 Step 3

Figure 6. Extract ion chromatogram (XIC) of triplicate runs for T15 glycopeptides from A) the innovator and B) a biosimilar. C) Comparison of glycopeptides using normalized MS response. The amount of T15 containing 7 core1 structures is similar between the innovator and the biosimilar. Differences in the relative amount between the innovator and the biosimilar are observed for glycopeptides containing 6 core1 structures.

0

0.02

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0.06

0.08

0.1

0.12

0.14

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7 O‐core 6‐Ocore (45.4 min) 6‐Ocore (46.5 min)

T15

Norm

alized response

Innovator

Biosimilar

T15—7 core1 T15—6 core1

Inn

ovat

or

B)

A)

Bio

sim

ilar