RFMS Glycan Characterization Techniques for Biotherapeutics

Dr Mark Hilliard, NIBRT

Waters THE SCIENCE OF WHAT’S POSSIBLETM

The Complexity of Glycosylation

Glycosylation is the most common posttranslational modification. Glycosylation of proteins is a complex process leading to

‘glycoforms’ of the same protein.

Glycans are branched, therefore leading to a higher degree of structural complexity (unlike DNA and proteins ).

Sugar chains can be linked to the protein through either the nitrogen atom of asparagine residues (N-linked glycans) or via the hydroxyl group of serine and threonine residues (O-linked glycans).

Biopharmaceuticals and recombinant proteins: Quality control – the determination of correct product glycosylation is essential in order to ensure the efficacy and safety of therapeutic products.

International Conference on Harmonisation (ICH) Guideline Q6B requires carbohydrate content, structure, and glycosylation sites

present on therapeutic proteins to be characterised as extensively as possible

Motivation for Understanding Glycosylation

Classes of N-Linked Glycans

Complex Hybrid High Mannose

Asn

Asn Desialylation of intravenous immunoglobulin abrogates its anti-inflammatory properties Kaneko et al (2006). Science; 313(5787): 670-673

Loss of sialylation decreases EPO half-life from 2 h to 10 min Fukuda et al (1989). Blood; 73(1): 84-89

Asn Presence of gal-α(1,3)-gal can induce anaphylaxis (shock) and can be present on biotherapeutics Chung et al (2006). N Engl J Med; 358(11): 1109-1117

Influence on Biopharmaceutical Production

Asn

Asn

Half of the population have antibodies against β(1,2)-xylose and α(1,3)-core fucose Bardor et al (1995). Glycobiology; 13(6): 427-434

Glycosylated Therapeutic Proteins

Over 190 EMA/FDA Approved Therapeutic Proteins. Over 127 are Glycoproteins (>66%) (Walsh: Nature

Biotechnology 28, 917–924 (2010)

Glycan Release Considerations

• Different enzymes exist that facilitate the easy release of N-glycans from glycoproteins.

PNGase F / PNGase A cleavage site Endoglycosidase cleavage site: Endo F1,

Endo H, Endo M, Endo B

N X≠P

S/T

High Mannose N-glycans

Endo D cleaves paucimannose sugars

UPLC (Ultra performance liquid chromatography ): See More, Faster

70 80 90 100 110 120 Minutes

5 µm Tosoh TSKgel Amide-80 4.6 mm x 250 mm

3 h gradient

2 4 6 8 10 12 14 0 Minutes

1.7 µm Waters BEH Glycan 2.1 mm x 150 mm

30 min gradient

↑Increased resolution ↓Deceased Run times

BEH material less retentive

2AB

2AB

The elution times of glycans are expressed in glucose units (GU) by reference to a dextran ladder.

Each individual glycan structure has a GU value that is related to the

number, linkage of its monosaccharides.

% 50 m

M

Am

monium

form

ate pH

4.4

4.0

5.0

6.0

7.0

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

y=ax+bx2+cx3+dx4+ex5

How Are Glucose Unit (GU) Values Generated? Part 1

Retention Time (min)

5.0

6.0

7.0 8.0

9.0 10.0 11.0

5.41

5.77

6.25

6.71

6.

84

6.95

7.

08

7.20

7.52

7.63

7.

76

8.05

8.41

8.81

9.04

9.67

10.0

10

.2

5.89

0 16

Dextran ladder

Herceptin®N-linked glycans F(6)A2

Derived GU value

How Are Glucose Unit (GU) Values Generated? Part 2

GU Value Caveats • Whilst GU values are helpful, their validity is dependent upon:

– The column chemistry and separation conditions used for their generation, – The sugar ladder used for their annotation, – The method used to fit the regression line, – Associated R2 value.

10 20 30 40 50 60 70 80 90 100 110 120 130 140Retention Time [min]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16GU

Glc (α1→6)n

Glc (α1→4)n

Mittermayr & Guttman, Electrophoresis, 33, 2012, 1000-7

GU Increments with Sugar Addition Linkage position

2 3

4

8 6

2 3

4

8 6

2 3

4

8 6

2 3

4

8 6

Linkage position

unknown linkage

β - linkage

- linkage

Linkage type

unknown linkage

- linkage

α - linkage

Linkage type

Glc

GlcNAc

Gal

GalNAc

Fuc

NeuNAc

Man

Symbol for sugar

Xylose

NeuNAc

Man

Xylose

Exoglycosidase Digestions

• Exoglycosidases are enzymes that remove monosaccharides from glycans with a defined linkage specificity.

ABS NAN 1

ABS α2-3/6/8 NAN 1 α2-3/8

Sialidase

ABS

AMF

BKF

X1-2F

AMF

BKF

BKF α1-6/2 X1-2F α1-2 Fucosidase

AMF α1-3/4

SPG BTG

SPG BTG

BTG

SPG β1-4 BTG β1-3/4/6 Galactosidase

GUH

GUH

GUH β1-2/4/6 Hexosaminidase

RELEASED LABELLED GLYCANS

Wash

Elute Elute

Dry

Resuspend

Dry

2-AB Label

Formic Acid

Elute

Biotherapeutic denaturation with RapiGest SF

PNGase F

Exoglycosidase array of Etanercept by UPLC analysis

Search Glycobase 3.2 for preliminary identification

Overview of N-linked Glycan Release with UPLC Analysis

Identification

Glycan identity confirmed by mass spectrometry HILIC-FLR-MS/MS

FLR

MS1

Biotherapeutic Protein Core

Glycan Structures

Mr. Potato Head “Biotherapeutic”

We tend to analyse these in isolation. This can lead to a “miss” understanding of how the structure exists, even though

our data is correct

• Peptide mapping√ • Intact m/z√

• Glycan analysis √

Taking into account how all of these structures work together will deliver a true

understanding of the biotherapeutic.

• Site Specific /Site Heterogeneity PTM Analysis • Glycoform/Isoform Intact Mass Analysis

• 3 Dimensional Structure

Good analysis is not enough, how does all the data fit together – that is the real question

Biotherapeutic Protein Core • Peptide sequence • Intact mass analysis • PTM’s

Released Glycan structures • Glycan characterization • Sialylated glycans speciation • Immunogenic glycans

A classic approach is to release the glycan's and then analyse them separately

to the peptide sequence

RapiFluor-MS (RFMS) label provides over X10 FLR and X100 MS sensitivity compared to the 2AB label

A

B

MS1 analysis

MS1 analysis

Ribonuclease B 2AB

Ribonuclease B 2AA

Ribonuclease B RFMS

UPLC-HILIC-FLR analysis of Ribonuclease B glycans

2-aminobenzoic acid 2-aminobenzamide

2-aminopyridine 2-aminoacridone Aniline

There are a number of Tags available, but the standard labeling method can take days

to complete!

2AB/2AA N-Glycan sample preparation

Labeling can take 1-2 days, sometimes longer!!

RapiFluor-MS N-Glycan sample preparation

From Glycoprotein to labeled N-glycans, to GU values with m/z data in 2 HOURS!

5.29

5.45

5.77

6.52

6.

65

7.40

7.97

8.52

5.0 6.0 7.0 8.0 9.0 10.0

5.28

5.44

5.76

+ Galactosidase (ABS+BTG)

5.25

5.41

5.73

6.48

6.

61

7.37

+ Sialidase (ABS)

Dextran ladder

0.6 GU = 1 Sialic acids

1.2 GU = 2 Sialic acids

Undigested

1.1 GU = 2 x N-Acetylgalactosamine

4.39

4.76

+β-Hexosaminidase (ABS+BTG+GUH)

1.62 GU = 2 x Galactose

0.7 GU = x Galactose

Herceptin® (Trastuzumab) RFMS labeled N-linked Glycan Structural Assignments

Herceptin® (Trastuzumab) N-linked Glycan Structural Assignment using Glycobase

NIBRT GlycoBase accessed through www.glycoase.nibrt.ie or through Biopharmaceutical Platform Solution with UNIFI : Waters

https://shar.es/1juHbx

RFMS labeled N-glycans analysed by UPLC-HILIC-FLR-QDa at NIBRT

UPLC Stack

FLR detector

QDa

The NIBRT QDa was donated by Waters as part of the Enterprise Ireland (EI) NIBRT-Waters project

UPLC-HILIC-FLR-QDa analysis EU

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tens

ity

0.0

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

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1.4x107

1.6x107

1.8x107

2.0x107

2.2x107

2.4x107

Minutes10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00

FLR*

QDa

Overlay of raw FLR and QDa TIC data

Herceptin RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa

*GU values can also be determined with an

appropriate processing method and dextran ladder

FLR*

QDa

UPLC-HILIC-FLR-QDa analysis Extracted glycan m/z values from QDa data

*GU values can also be determined with

an appropriate processing method and dextran ladder

FLR with GU values

Herceptin RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa

EU

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UPLC-HILIC-FLR-QDa analysis

FLR*

Overlay of raw FLR and QDa TIC data

Enbrel® (etanercept) RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa

QDa

*GU values can also be determined with an

appropriate processing method and dextran ladder

Enbrel® (etanercept) RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa

UPLC-HILIC-FLR-QDa analysis Extracted glycan m/z values from QDa data

FLR*

QDa

FLR with GU values

Undigested

Herceptin RFMS N-linked Glycans and UPLC-QDa Exoglycosidase digestion array TIC (500-1200 m/z)

Development of a novel workflow for monitoring O-Glycans utilising the new Waters QDa system

N-linked sites= 3 • 2 in the TNF-α receptor

• 1 in the Fc region

O-linked sites=13, hinge

region. Very challenging.

TNF-α receptor

Fc region

Etanercept, Enbrel

What about O-Glycans?

S

T

S

*Typical CHO O-glycans: Core 1, Mono- and Disialyl Core 1. Found on Enbrel, FC-Fusion and EPO biotherapeutics

P

Characterization of O-glycosylation is considerably more complicated than the analysis of their N-linked counterparts:

• Generally lower carbohydrate to protein ratio, sensitivity considerations

• Multicore structures, see below • Chemical release often necessary

*

Chemical release is also possible, normally base catalyzed beta elimination type reactions. But there can be issues with chemical artifacts (peeling products).

Base: Hydrazine, hydroxide, ammonia

Reducing sugars possible (labeling) , peeling reactions problematic

Reducing agents

More stable alditols formed, subsequent labeling not possible

OH

How do we release O-Glycans?

LC-PGC-MS (Agilent /Thermo Fisher)

UPLC-HILIC-FLR-MS (MS friendly tags preferred)

LC-MS-UNIFI O-Glycan database (Waters)

UPLC Analysis of Procainamide labeled Bovine Fetuin O-Glycans (HILIC Plate clean up)

Ammonia-based β-elimination release (16hrs 65c)

Desalting via evaporation (1 day)

Procainamide labeling (or other MS friendly tag)

HILCI plate clean up vs traditional paper chorography

UPLC method optimization for QDA

N-linked Glycans

Preliminary assignments

O-linked Glycans

UPLC-HILIC- FLR

Peeling product!

UPLC-HILIC-FLR-QDa Analysis of Procainamide labeled Bovine Fetuin O-Glycans

FLR

QDa

529.03

N glycan source Peeling product

Acknowledgments

Waters THE SCIENCE OF WHAT’S POSSIBLETM