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OVERVIEW

CHARACTERIZATION AND PEGYLATION SITE DETERMINATION OF A PEGYLATED BIOTHERAPEUTIC BY CID MS/MS COUPLED WITH ION-MOLECULE REACTIONS

Asish B. Chakraborty1, Weibin Chen1, StJohn Skilton1, Isabelle Gusev2, Jesse Dong2, and Jeff Mazzeo1 1Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757; 2IPSEN/Biomeasure Inc., Milford, MA 01757

CONCLUSIONS

Polyethylene glycols (PEGs) and its derivatives are widely used in the biopharmaceutical industry for the delivery of therapeutic drugs.

Mass spectrometric analysis of high molecular weight

PEGs and PEGylated biotherapeutics is very challenging due to the great complexity and diversity of the materials.

The use of gas phase ion-molecule reactions for the

analysis of PEG, PEGylated proteins and peptides has precedent within the biopharmaceutical industry today1.

This presentation describes: 1) An improved method to

accurately measure the average molecular weight of PEGs, PEGylated peptide and protein products using ion-mobility time-of-flight mass spectrometry coupled with gas-phase ion–molecule reactions; and 2) An improved methodology to elucidate the PEGylation sites in PEGylated-biotherapeutics by sequential in-source fragmentation and CID MS/MS.

The method is developed based on a simple and

flexible modification to a Waters SYNAPT® HDMS™ System with off-the-shelf components, thus enabling a gas-phase ion–molecule reaction to be effectively coupled to the high-performance tandem mass spectrometer without sacrificing any high performance attributes of the original instrumentation2.

We have demonstrated a method to accurately measure the average MWs and MW distributions of large PEG using ion-molecule reaction inside a Waters SYNAPT HDMS Mass Spectrometer.

The combination of in-source fragmentation and traditional CID MS/MS has provided a mechanism for confirming the PEGylation site of the PEGylated growth hormone.

With many unique capabilities of the instrument, such as ion–mobility separation, the ability to perform ion–molecule reaction inside SYNAPT HDMS has truly expanded the applicability of the instrument. It is conceivable that this configuration can be readily applied to many challenging analytical tasks in pharmaceutical and biopharmaceutical industries.

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.

Gas phase ion-molecule reactions can take place either in the TRAP cell or in the TRANSFER cell.

ADDING ION MOBILITY TO A Q-TOF MS

SYSTEM CONFIGURATION FOR ION/MOLECULE REACTIONS

Charge Stripping Analysis of PEG 20kDa

Charge Stripping Analysis of PEG 4450

Figure 3. ESI IMS TOF analysis of PEG 4450 using SYNAPT HDMS. Driftscope™ shows the gas-phase separation power of SYNAPT in the analysis of PEG 4450. Components with different charge states (1+ to 4+) are separated via ion-mobility, thus enabling the examinations of different (minor) components in the PEG materials.

Figure 4. ESI-TOF mass spectrum of PEG 4450 after reaction of all charge states in the trap cell of the instrument with super base, 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU). The inset shows the expanded view of the best fit to a Gaussian distribution.

m/z1000 2000 3000 4000 5000

%

0

100

1600 2000 2400 2800

2+

1+

1+

ESI-IMS-TOF ANALYSIS OF PEG 4450

Figure 1. Instrument (SYNAPT HDMS) schematic illustrating the Triwave™ Technology embedded in a high-performance oa-TOF tandem mass spectrometer (A). Schematic diagram showing the modified gas line configuration for performing ion/molecule reaction inside the TRAP cell of SYNAPT HDMS Mass Spectrometer (B).

MS Conditions MS System: Waters SYNAPT HDMS Ionization Mode: ESI Positive Capillary Voltage: 3.0 kV Cone Voltage: 40V Source Temp: 100 °C Desolvation temp: 250 °C IMS Cell Pressure: 0.5 mbar (N2) Wave Pulse Height: 5-14 V RF Amplitude: 320 V (Peak-to-Peak) IMS Cell Velocity: 240 m/s

Figure 5. ESI-TOF mass spectrum of PEG-aldehyde 20 kDa before (A) and after (B) reaction with super base, DBU, in the TRAP cell of the Triwave. Reaction conditions were optimized to strip all the charge

m/z

1000 2000 3000 4000 5000 6000 7000 8000

%

0

100A

After Charge Stripping

Before Charge Stripping

B

Rel

ativ

e Rel

ativ

e In

tensi

ty

4+

References: 1. Bagal et.al Anal. Chem. 80, 2408–2418 (2008). 2. Chakraborty et.al Pharmaceutical Technology JULY 2008

LC-MS PEPTIDE MAPS FROM NATIVE AND PEGYLATED GROWTH HORMONE

Native T1 Peptide Present

No native T1 peptide present after

pegylation of GH

A

B

C D

PEGylated T1 peptide (multiple forms)

Figure 7. Mass spectra from PEGylated peptides from a regular LC-MS analysis and from a LC-MS analysis coupled with charge stripping (DBU).

Before Charge Stripping difficult to distinguish by eye

Peak B

Peak A

Peak A

Peak B

Figure 6. LC-MS peptide mapping of tryptic digest from native and PEGylated growth hormone.

Differences betn Peaks A & B are more

salient with charge stripping

6+

Automated deconvolution shows different distribution of control (PEG) sample and

Analyte (PEG -Peptide)

30 kD PEG (‘control’)

30 kD PEGylated Tryptic Peptide

(‘analyte’)

Figure 8. BiopharmaLynx™ Software view of TIC (After Charge Stripping, inset). Biopharmalynx processed (MaxEnt 1) deconvo-luted 30kD PEG and PEGylated Tryptic fragment.

Determination of PEGylation Site by MS/MS

PEGylated T1-PEG (m/z 670.387, precursor ion,+2) selected for

traditional CID MS/MS.

PEGylated T1 Peptide Series separated by 22

Figure 9. MS/MS spectrum of PEGylated growth hormone after in-source fragmentation (ISF) confirms PEGylation at the N-terminal. The m/z 670.387 (+2) of the precursor ion was selected for CID.

MS/MS spectrum of PEGylated T1 after ISF

CID Mass spectrum

y7 MH22+

XFPTIPLSR

y6 y5 y4

y3 y2 y1

30 kD PEG (‘control’)

30 kD PEGylated Tryptic Peptide (‘analyte’)

TIC

ESI-TOF ANALYSIS OF PEG 4450

m/z200 600 1000 1400 1800 2200 2600

%

0

100

Intensi

ty

2+

3+4+

1008 1016 1024 1032

Figure 2. ESI-TOF mass spectrum of PEG 4450 without IMS separation. The isotopic resolution for peaks in the spectrum (inset) showing the sufficient resolving power of the instrument at the collected m/z window. The isotopic resolution permits the determination of the number of charges that each oligomer holds and their charge states distribution in the spectrum. Spectrum contains several charge states ranging from 2+ to 4+ that are due to the addition of multiple cations (e.g., Na+, K+, and H+) to each PEG oligomer, generating many different ion series in the spectrum. Consequently, the average molecular weight cannot be readily determined.

K

K

K

K

K

K

K

10

40

50

90

70

80

120

100 110

130

140

150160

170

180

PEG

PEG

PEG

PEG

PEG

PEG

PEG

COOHPEG-N

H2

XFPT

IP

Ls

R

N-term is thetarget

K

K

K

K

K

K

K

10

40

50

90

70

80

120

100 110

130

140

150160

170

180

PEG

PEG

PEG

PEG

PEG

PEG

PEG

COOHPEG-N

H2

XFPT

IP

Ls

R

N-term is thetarget

Possible PEGylation Sites

OverviewConclusionSAdding ION MOBILITY TO A Q-TOF MSSystem configuration for Ion/molecule reactionsCharge Stripping Analysis of PEG 20kDaCharge Stripping Analysis of PEG 4450ESI-IMS-TOF analysis of Peg 4450LC-MS PEPTIDE MAPS FROM NATIVE AND PEGYLATED GROWTH HORMONEDetermination of PEGylation Site by MS/MSESI-TOF analysis of Peg 4450

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