characterization of biologics using agilent tof for the reactor isolation from the cap-pump flow...

Characterization of

Biologics Using

Agilent TOF

Systems

Dawn Stickle, Ph.D.

December 2013

Center of Excellence

Agilent Technologies

Wilmington, DE

Agilent’s Tools and Technologies for

BioPharmaceutical Analyses

Agilent’s broad technology portfolio includes:

• Liquid chromatography

• LC/MS

• Capillary electrophoresis

• Ion analysis (CE and HPLC)

• Isoelectric focusing

• Evaporative light scattering

• Gas chromatography

• GC/MS

• qPCR

• LC column technology (reversed phase, ion exchange and size exclusion)

• Protein standards and protein processing kits

• Lab-on-a-Chip

Agilent LC/MSD TOF

Two Stage Ion

Mirror

5 Stage Vacuum System

Agilent Orthogonal

Spray Source(s)

Optically Coupled

Ion Detector

Beam cooling

and guidance

Effective Flight Path

Length of 2.0m Low-expansion

Flight Tube

Design Optimizes Ion Transmission and

Mass Resolving Power

BioPharma – Description and Workflow Description

• Characterization of Proteins at the intact or digest level, or synthetic peptides.

• Provide target sequence(s), and analyze intact protein mixture, or after digestion.

• Find all peptides (MFE) or proteins (LMFE) and match to target sequence while applying

rules for modifications (truncations, PTMs).

Workflow

• Acquire sample with intact protein, protein digest, synthetic peptide or oligo in MS

• Enter sequence of peptide, protein or oligo in MassHunter BioConfirm and set up rules.

• Analyze sample, which matches the found masses to the supplied sequence.

• Optionally acquire MS/MS spectra of compounds in question and use Spectrum Mill

• Print report

Easy Access

Smpl + Seq Setup rules

+sequences

Acq MSMS for

Spectrum Mill Worklist

Sample + Seq

Print

Report Match

sequence

Turnkey Automation

Intact mAb Analysis

Structure and Modifications of Antibody

• Analyze intact antibody

•Analyze deglycosylated

antibody (enzymatic)

•Analyze reduced antibody

(light and heavy chains)

•Analyze Fab and Fc

regions (papain cleavage)

Light Chain

Fc

Fab

Antigen

binding

Hinge

Glycosylation

site Truncation

(lysine)

Disulfide

shuffling

Pyroglutamate

Deamidation/oxidation

Heavy Chain

Fucose – 146Da

Mannose – 162da

N-Acetylglucosamine – 203Da

Galactose – 162Da

1. Mass Spectrum of a mAb – 10 ng on Zorbax SB300-C18

HPLC-Chip

Deconvoluted Mass Spectrum of Intact mAb – 10ng on

Zorbax SB300-C18 HPLC Chip

Mcalc: 148811.95

Mexp: 148812.81

Error: 5.8ppm

Δ1444.87

Δ2890.81

G0F/G0F x104

0

1

2

3

4

5

6

6.5

148812.81

145922.00

147367.94

146329.69

146816.21

147719.65

Counts vs. Deconvoluted Mass (amu)

145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500

5.5

4.5

3.5

2.5

1.5

0.5

Δ 162.2 hexose unit

x104

0

1

2

3

4

5

6

7

8

148812.81

148974.97 148840.65 148916.37

148765.43


148750 148800 148850 148900 148950

G0F/G0F

G0F/G1F

Deconvoluted Mass Spectrum of Deglycosylated mAb –

PNGaseF treated

No glycan-attached species

4 x10

0

1

2

3

4

5

6

7

8

145924.41

146272.08


144500 145000 145500 146000 146500 147000 147500 148000 148500 149000 149500

Mcalc: 145923.24

Mexp: 145924.41

Error: 8ppm

DTT Reduced mAb: Deconvoluted Spectrum of Light Chain

5 x10

0

1

2

3

4

5

23746.50

23727.82 23762.96


23540 23560 23580 23600 23620 23640 23660 23680 23700 23720 23740 23760 23780 23800 23820 23840 23860 23880 23900 23920 23940 23960 23980

Mcalc: 23746.63

Mexp: 23746.50

Error: 5.5ppm

DTT Reduced mAb: Deconvoluted Spectrum of Heavy

Chain

Δ1446.6

Δ162.7

4 x10

0

1

2

3

4

5 50675.58

49228.96

50838.33

49435.14 51023.43 50121.16 49920.85 49009.52


48600 48800 49000 49200 49400 49600 49800 50000 50200 50400 50600 50800 51000 51200

Mcalc: 50675.47

Mexp: 50675.58

Error: 2.2ppm

Deconvoluted Mass Spectrum of Fc Fragment

Δ1445.8

Δ2890.93

Δ162.47

5 x10

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 52755.64

49864.71

51309.87

47485.72

50272.22


44000 44500 45000 45500 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 51000 51500 52000 52500 53000 53500 54000 54500 55000

5 x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2 52755.64

52918.11

52553.59 53081.26


52200 52400 52600 52800 53000 53200 53400 53600

Mcalc: 52755.62

Mexp: 52755.64

Error: 0.4ppm

Deconvoluted Mass Spectrum of FAB fragment

Fab fragment

Mcalc: 48046.18

Mexp: 48046.09

Error: 1.8ppm

Summary – Intact mAb Characterization Results

from HPLC-Chip Accurate Mass QTOF Analyses

Mcalc Mexp Error (ppm)

Intact

major glycoform 148,811.95 148,812.81 5.8

deglycosylated 145,923.24 145,924.41 8

Light chain 23,746.63 23,746.50 5.5

Heavy chain

major glycoform 50,675.47 50,675.58 2.2

Fc

major glycoform 52,755.62 52,755.64 0.4

FAB 48,046.18 48,046.09 1.8

To the Instrument . . .

Let’s inject an intact protein and

perform manual and automated

data analysis

Peptide Mapping of mAb: 1290 Infinity HPLC with

Accurate Mass QTOF

HPLC Parameters for Poroshell-120 SB-C18 2.7µm 3x150 mm

Time %B

0 2

3 2

13 45

15 65

15.1 90

17 90

18 2

Flow – 0.3 mL/min

Stop time – 20min

Initial Solvent – 2% B

Column Temp – 40C

A: Water, 0.1% Formic Acid

B: ACN, 0.1% Formic Acid

5 Replicate Runs of mAb Tryptic Peptide Map on

Poroshell 120 Column

BioConfirm Molecular Feature Extractor of

Stratagene mAb Trypsin Peptide Map

BioConfirm Sequence Coverage of Stratagene

mAb

Glycan Characterization: Agilent 7100 Capillary Electrophoresis-LIF with

Accurate Mass TOF

• Widest choice of detctors

DAD, LIF, CCD, Single-Q, IT, TOF, QQQ, QTOF, ICP-MS

• Only vendor for plug & spray ESI-MS

complete CE-MS portfolio with a single interface solution

• Easy change from LC-MS to CE-MS and back

quick and robust ESI by MS-adaptor & spray needle kit

using Agilent Chemstation software

• Combined detection option

UV-DAD plus MS detection, LIF plus MS detection

• Grounded CE capillary end No compromises between separation- or ESI-spray voltage

Agilent 7100 Capillary Electrophoresis The CE-MS advantage

CE-Electrospray is a powerful method for high resolution separation and identification for a

wide variety of molecules. Additionally it is an orthogonal separation technique to HPLC

The Agilent interface combines the advantage of a grounded CE capillary end with a robust

and easy to use spray needle that fits into the whole portfolio of Agilent MS instruments

Sheath Liquid Addition and Sprayer

Physical Setup

CE

MS

Pump

•Sheath liquid can be added by:

•infusion pump, or

•LC pump + splitter

sprayer tip

CE capillary

sheath liquid line

nebulizing gas line

screw for precise adjustment of the CE capillary

• Important post-translational modification of proteins

• Pharmaceutical relevance – New Chemical Entities New Biological Entities

• Glycosylation pattern strongly influences therapeutic efficacy of antibodies

• Analysis of N-Glycan Important for Research and QA of therapeutic antibodies

N-Glycan Analysis of Monoclonal Antibodies

Negatively Charged Sialylated Glycans

A1F

MW 2077.7455

A2

MW

2222.783

A2F

MW 2367.8336

Extracted Ion Electropherogram of A1F, A2, and A2F

Glycans

A1F

A2F

A2

Mass Spectra of A1F, A2, and A2F Glycans

A2F

A2

A1F

Glycan Characterization: mAb-Glyco HPLC-Chip with

Accurate Mass TOF

Glycoprotein

PNGase F

Glycosylamine

N-Glycan

-NH3

• MALDI-TOF/MS

• LC/MS

• HPLC w/ pulsed amperiometric detection

Anomeric equilibrium

2-AB

labelling agent

Reductant

labelled N-Glycan

• HPLC w/ fluorescence detection

• CE w/ fluorescence detection

Enzymatic N-Glycan cleavage using PNGase F

PNGase F, Enzymatic

N-glycan Release

N-Glycan Concentration

HPLC Analysis

4 minutes

6 seconds

12 minutes

TOF MS Detector

mAb sample

Experim

enta

l Tim

e

LC- or CE- Fluorescence

10-30min

3-8 hours

2-4 days

mAb-Glyco-

Chip-MS

MALDI MS

//

//

On-Chip Glycan Characterization

Time:<30 minutes

Chip Features Inner-outer Rotor Design allows for the reactor isolation from the cap-pump flow path

(a) enzyme reactor

(b) enrichment

(c) separation column

Cap-pump flow rate: 3 ul/min

Empty ER volume: 310 nl

On-chip deglycosylation time: Variable – e.g. 4 min

The experimental workflow of the mAb-Glyco

Chip comprised 5 fully automated steps:

Inner and Outer Rotor Design

Glycan Profile in 12 minutes

Total analysis time of 12 min with

reasonable chromatographic

resolution

A ... Sample loading

B ... ER fill

C ... Deglycolsyation

D ... Glycan Transfer

E ... Glycan Analysis

„Sample Prep“ = 6min

„Sample Analysis“ = 6min

Glycan Quantitation and Report Generation

Glycan database is an accurate mass and structure database with 144 entires of glycans

that are frequently found on mAb‘s.

Reporting templates automatically processes the glycan hits by clustering and merging

isomermic structures and thus significantly helps to deliver fast answers

Glycan

Database

Reporting

Templates

Comparison of mAb-Glyco Chip with CE and MALDI

MS

0

10

20

30

40

50

60

70

Man-5 G0-GlcNAc G0-F G0 G1 G2

Re

lati

ve

mo

lar

dis

trib

uti

on

[m

ol%

]

MALDI-MS (N=34)

CE-LIF (N=43)

Agilent Glycan Workflow Chip (N=50)



mAb-Glyco Chip Reproducibility

Chip Enables Fast Separation of Isomers:

Detection of Immunogenic glycan isomers from mAb made in Mouse NSO Cell

G2 structure

Potential

immunogenic

G2 structure

1786.650 amu

Figure

EIC

EIC

EIC

% G0 = 46 %

% G1 = 41 %

% G2 = 13 %

EIC

EIC

EIC

% G0 = 74 %

% G1 = 24 %

% G2 = 2 %

EIC

EIC

EIC

% G0 = 49 %

% G1 = 36 %

% G2 = 5 %

Biosimilar m-2 Biosimilar m-3 Biosimilar m-1

• Dissimilar glycan distribution within biosimilars m1 – m3

• Biosimilar-m1: Similar glycan distribution as compared to innovator

N-glycan Analysis of Three Biosimilars Using mAb-

Glyco-Chip and Q-TOF

Similarities and Differences – Analyzing the data by principle component analysis (Agilent Mass Profiler Pro)

Biosimilar m-1

Biosimilar m-3

1

2

3

Biosimilar m-2

• Samples from the same

manufacturer clearly cluster together.

• Three clusters obtained for three

biosimilars.

Poster: ASMS 2010: Shiaw-Lin Wu1, Yi Wang1, Ning Tang2, Dayin Lin2, William S. Hancock1, and Barry Karger1 1 Barnett Institute and Department of Chemistry and Chemical Biology, Boston, MA 02115. 2 Agilent Technologies, Waldbronn, Germany 76337

Thank You

References

PUB # Title

5990-

3445EN

Primary Characterization of a Monoclonal Antibody

Using Agilent HPLC-Chip Accurate-Mass LC/MS

Technology

5990-

4587EN

Peptide Mapping of a Monoclonal Antibody using a

Microfluidic-based HPLC-Chip coupled to an

Agilent Accurate-Mass Q-TOF LC/MS

5990-

5190EN

Glycopeptide and Glycan Analysis of Monoclonal

Antibodies using a Microfluidic-based HPLC-Chip

coupled to an Agilent Accurate-Mass Q-TOF

LC/MS

5989-

5893EN

Antibody Analysis by ESI-TOF LC/MS

5989-

7406EN

Accurate Mass LC/TOF MS for Molecular Weight

Confirmation of Intact Proteins

characterization of biologics using agilent tof for the reactor isolation from the cap-pump flow...

Documents