agilent’s solutions for: glycan analysis...group/presentation title agilent restricted page 35 eic...

Agilent’s Solutions for: Glycan Analysis

Life Sciences Group

Agilent Technologies

October 2013

Biopharma Leadership Summit

1

Glycan analysis for biopharmaceuticals

• Why Analyze Glycosylation?

– Most important PTMs in

biopharmaceuticals like mAbs

– Regulatory approval

• Safety

• Efficacy

– Commercial

• Patentability

• Consistency of product

• Comparability in biosimilars

• Biobetters

August 20, 2014

Agilent Confidential

2

VVLHPNYSQVDIGLIK

Agilent solutions for mAb glycan analysis

August 20, 2014 3

mAb

Released

glycans

Digested mAb

Intact mAb

Glycosylation

site profiling

(LC-MS/MS)

Glycoform

profiling by

LC/MS

Oligo-

saccharides

Sialic acid

Monosaccharides


2-APTS label

2-AB label FLD

LIF

Digestion

GlykoPrep

15 min screening

Label-free

(non-GMP)

Routine & QC

(GMP & compliance)

Identification

(Qual/Quan)

mAb-Glyco-Chip

Agilent solutions for glycan analysis Glycoform profiling from Intact or Reduced mAb

August 20, 2014


4

Fc Region Analysis

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Deconvoluted spectrum of Fc fragment shows relative quantitation of various glycoforms

FabRICATOR

IdeS

Intact mAb Analysis

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5 x10

0

1

1.5

4 x10

0

1

2

3

4

5

6

Counts vs. Deconvoluted Mass

(amu × 10^3 )

142.0 143.0 144.0 145.0

G0

/G2

F

2G

1F

G0

F /G

1F

G1

F/G

2F

PNGaseF treatment of an IgG allows precise MW

measurements of the protein core

Agilent solutions for glycan analysis Released glycan analysis by HPLC-FLD as 2-AB

August 20, 2014


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1260 Infinity

Bio-inert HPLC/FLD

AdvanceBio

Glycan Mapping,

2.7 um +

AdvanceBio

Glycan Mapping,

1.7 um +

1290 Infinity UHPLC

Reductive Amination of Sugars

August 20, 2014


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Agilent CE/MS Setup ►MassHunter SW control

The Agilent CE/MS Advantage

Single point SW control - 7100 CE instrument

- TOF,QTOF,QQQ

- LC make-up flow

Single vendor solution: direct and competent support

Sheath Liquid Interface: robust and reliable, offering efficient control on chemistry

Fast Switch LCMS ►CEMS: quick exchange of spray-needle as connection

4x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

-

1 1

Counts vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

G0 G0F Man5 G1 G1F G2 G2F

3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

Counts vs. Acquisition Time (min)

7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5

G2

G2

F

G2

-1N

AN

A

G2

-2N

AN

A

G2

F-2

NA

NA

G2

F-1

NA

NA

CE/MS of APTS labeled glycan standards

Mixture of neutral glycan &

sialylated glycans

Mixture of neutral glycan

Galactose Mannose Fucose N-acetylglucosamine sialic acid

Page 13

Instrument type :

Agilent QTOF

CE–LIF–MS Analysis The only online combination of LIF and MS detection

The Agilent/Picometrics CE-LIF-MS Advantage

• Completely integrated CE/MS solution

• Robust and reliable sheath liquid sprayer

• LIF detection directly upfront MS detection

• Use of standard CE capillaries

Fiber optics

LIF and MS traces obtained online in a single analysis of

the glycan species enzymatically cleaved from an antibody

sample.

CE–LIF–MS systems are vital tools for the rapid analysis of complex protein moieties with high sensitivity, resolution, and reproducibility.

Standard methods at large Pharma

NP-UPLC-FLD

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mAb-Glyco Chip Kit – A Comprehensive Workflow

Solution

mAb-Glyco chip Reagent Pack

Structure Viewer

Total

workflow

solution

Glycan Database

Reporting Templates

Agilent mAb-Glyco-Chip

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N-Glycosylation analysis (mAb-Glyco-Chip)

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On-chip Deglycosylation and Analysis of Intact

Antibodies

3 sec. deglycosylation

4 x10

0

1

2

3

4

5

6

7

8

1 glycan removed 146202.21

147645.93

Counts vs. Deconvoluted Mass (amu) 145500 146000 146500 147000 147500 148000 148500 149000 149500

147806.94

147962.53

6 sec. deglycosylation

4 x10

0

1

2

3

4

5

6

Deglycosylated mab


Both glycans removed

Intact antibody prior to deglycosylation

4 x10

0

1

2

3

4

5

6

149089.58


149253.47

149415.31

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Acquire data Extract

compounds

Database search and identify

glycans

Calculate percentage and

report generation

mAb Glycan Workflow

mAb-Glyco-Chip (sample analysis)

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IgG1-A

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Conclusion: mAb-Glyco-Chip

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Method parameters

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Summary of Advantages & Disadvantages

Parameter UPLC-FLD UPLC-FLD-MS CE-LIF- (MS) mAb-Glyco chip

Advantage GMP option

Reproducibility Reproducibility Reproducibility

Robustness Robustness Robustness Robustness

Identification (MS) Identification (MS) Identification (MS)

Orthogonal

separation

speed

sensitivity

Sample

consumption

Disadvantage Time consuming Time consuming

Time consuming

Identification (RT)

Equipment cost Equipment cost Equipment cost

Trained analyst Trained analyst Trained analyst

August 20, 2014


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Analysis of Glycopeptide from mAb (Trypsin,

Pronase, Pepsin)

Group/Presentation Title

Agilent Restricted

Page 34

BPC

Sugar Oxonium Ion Helps to Identify Glycopeptide

Group/Presentation Title

Agilent Restricted

Page 35

EIC m/z 204.084 (GlcNac)

BPC Mass spectrum of the glycopeptide

TKPREEQYNSTYR

Fucose – 146 Da

Mannose – 162 Da

N-Acetylglucosamine – 203 Da

Galactose – 162 Da

Fucose – 146 Da

Mannose – 162 Da

N-Acetylglucosamine – 203 Da

Galactose – 162 Da366 528 1040

366

-

H2

O

-

2H2

O

MS/MS

Thank you !

Q&A.

August 20, 2014 36

agilent’s solutions for: glycan analysis...group/presentation title agilent restricted page 35 eic...

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