©2007 Waters Corporation v1

Petra Olivova, Weibin Chen, John C. Gebler Biopharmaceutical Sciences, Waters Corp., Milford, MA 01757

RAPID STRUCTURAL ANALYSIS OF MONOCLONAL ANTIBODY AND SYNTHETIC POLYMERS USING

ELECTROSPRAY ION-MOBILITY TIME-OF-FLIGHT MASS SPECTROMETRY

THEORY OVERVIEW

Structural characterizations of intact MAb molecules are

challenging due to their high molecular mass, hydrophobic

nature, and presence of sugar moieties. Conversely, the analysis

of intact protein reduces dramatically the time for sample

preparation and data interpretation, and it also minimizes the

chance of introducing putative modifications, which are often

observed during peptide mapping.

The aim of this study is to perform structural characterization on

a monoclonal antibody (IgG1, κ), and synthetic polymers. The

presented study covers the results in the following areas:

♦ analyzing intact antibodies to map the glycosylation profile

of the antibody

♦ separating the light and heavy chains of the reduced

antibody by ion mobility to yield further characterizations

♦ fragmenting the intact light chain subunit to obtain partial

amino acid sequence of the light chain

♦ characterizing and differentiating PEGs by their sizes and

conformations in a quick infusion experiment

INTRODUCTION

• A new class of mass spectrometer has been developed by Waters (Synapt™ HDMS™) with the unique capability to fractionate biomolecules in the gas phase by size, shape, and charge prior to mass spectrometric detection.

• The Synapt HDMS system enables researchers to

simplify complex samples, simplify or eliminate chromatographic separations, or permit the in-depth characterization of hybrid structures within a single analysis.

• This poster presents the design and theory behind

Synapt HDMS, and shows specific applications.

• The ion-mobility section is comprised by three Traveling Wave-enabled Stacked Ring Ion Guides (SRIG).

• The TRAP ion guide is used to accumulate ions and release them as packets for ion mobility separation.

• The TRANSFER ion guide conveys the mobility separated ions to the oa-TOF for mass analysis.

• Fragmentation can take place either in the TRAP or in the TRANSFER ion guide or both.

RESULTS

Figure 1. (A) Positive ion ESI mass spectrum of an intact monoclonal antibody acquired in V mode with resolution 10,000. (B) Positive ion ESI mass spectrum for 53+ charged ion of the intact monoclonal antibody. Peaks represent various glycoforms. (C) Deconvoluted ESI mass spectra of the intact monoclonal antibody IgG1 containing multiple peaks due to the sugar heterogeneity.

m/z1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800

%

0

10053+

56+

46+

49+

60+

A

m/z2780 2790 2800 2810 2820 2830

%

0

100

G0/G0F

G1F/G2F

G1F/G1FG0F/G2F

G0F/G1F

G0F/G0F

G2F/G2F

B

mass147700 148100 148500 148900 149300

%

0

100

148067

148213

148376

148535

148697

148868

G0/G0F

G1F/G2F

G1F/G1FG0F/G2F

G0F/G1F

G0F/G0F

G2F/G2F

C

Intact IgG1

A B C

CONCLUSIONS ♦ Synapt HDMS (ESI-IMS-TOF MS) has been successfully used for the structural characterization

of a mouse monoclonal antibody.

♦ Synapt offers unique, enabling functionalities for characterization and differentiating PEGs and its derivatives by their sizes and conformations in a quick infusion experiment. In addition, the impurities and low molecular mass species in PEG material can be promptly observed from 3D heat maps by simple eye-view.

♦ The system combines high-efficiency, ion-mobility based measurements and separations with high-performance quadrupole, time-of-flight mass spectrometry to deliver enhanced specificity and sample definition beyond that achievable by conventional mass spectrometers.

♦ All of the results demonstrate that Synapt HDMS mass spectrometer is a superior tool to characterize MAb and other complex protein pharmaceuticals.

Figure 3. (A) Ion mobility DriftScope of the fragment ions from fragmentations of the intact light chain. Singly charged species are clearly separated from the other multiply charged fragments by ion mobility. These fragments contain partial amino acid sequence of the light chain (B) The summed mass spectrum of all the singly charged ions generated from the intact light chain fragmentation process.

REDUCED IgG1

m/z200 400 600 800 1000 1200

%

0

100 187.111

789.373

328.192215.099

459.228688.365560.285

626.309739.383 840.435

886.43 1086.55

927.45

1181.62

999.55

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Drift Time (msec)

m/

z

A

B

Figure 2. (A) Ion mobility DriftScope showing the separation of heavy chains and light chains from the reduced IgG1. (B) The summed ESI mass spectrum of light chain (C) The summed ESI mass spectrum of heavy chain. (D, E) Deconvoluted ESI mass spectra of light chain and heavy chain.

mass24050 24150 24250 24350 24450

%

0

10024195

24249

mass49600 49800 50000 50200 50400

%

0

10050077

49916

50239

49769

m/z800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

%

0

10020+

17+

15+

12+23+

26+

10+

m/z800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

%

0

100 40+

36+

47+

44+

30+

33+

36+

51+

32+

B C

D E

Light Chain Region A

Light Chain Region B

Drift Time

m/z

Heavy Chain

Light Chain

A

B

A

B

C

D

E

+1 +2

Higher Charge State

Fragment Ions

PEG classification by IM-TOF MS

Infusion-IM-TOF MS of PEG-class surfactant

Drift Time

m/

z

A

B

3 1

2 4

Infusion-IM-TOF MS of PEG 4450 Infusion-IM-TOF MS of PEG 1000

IM-TOF MS Differences between different PEG-class surfactant

Summed spectra obtained by combining ions with drift profiles shown above.

The chemical nature of PEG makes characterisation by conventional methods extremely challenging (determining the extent, and site of PEGylation; characterisation of low level components). IM-TOF MS is capable of characterizing and differentiating PEG by their sizes; also quick determining of impurities.

Impurities

AAA

BBB

EXPERIMENTAL METHODS

• The LC/MS system was configured with a Waters nanoACUITY UPLC™

chromatography system and a Waters Synapt™ HDMS™ quadrupole ion-mobility time-of-flight mass spectrometer. Synapt HDMS was operated in mobility-TOF mode for all analyses. MassLynx 4.1 software was used for instrument control and data processing.

• On-line desalting was accomplished on a prototype 2.1 x 5 mm desalting

cartridge. A step gradient was used for bolus elution of the desalted intact and reduced antibody.

Protein introduced into the ion-source. Select charge states for fragmentation using quadrupole. Intact protein subjected to high energy in the TRAP portion of

triwave. Separation of +1, +2, +3, and multi- charged peptide

fragments.

Drift Time

Selected charge states

m/z

TO

F A

naly

zer