Analysis of PEG and a PEGylated Therapeutic Protein Using an Agilent HPLC-Chip Coupled to a Q-TOF Mass Spectrometer

Application Note

Abstract

PEGylated peptides and proteins generate signifi cant challenges for the detailed structural characterization of biotherapeutics mainly due to polyethylene glycol (PEG) heterogeneity. This application note shows the development of a method to analyze PEG and PEGylated protein using an Agilent 1260 Infi nity HPLC-Chip coupled to an Agilent 6520 Accurate-Mass Q-TOF LC/MS System. A charge stripping agent (triethylamine) was used as a post-electrospray addition to aid in obtaining a simpler mass spectrum and hence allow for a better interpretation of the results. The results showed improvement in the mass spectrum quality for PEG and the PEGylated protein.

AuthorsRavindra Gudihal and Suresh Babu CVAgilent Technologies India Pvt. Ltd.Bangalore, IndiaNing Tang Agilent Technologies, Inc.Santa Clara, CA USASundaram Palaniswamy, Umamaheshwari S and Suneel BasingiGangagen Biotechnologies Pvt. Ltd. Bangalore, India

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IntroductionPEGylation is one of the important covalent modifi cations used in biopharmaceutical industry for improving the therapeutic value of proteins.3 PEGylation involves the chemical attachment of PEG to the therapeutic protein. This modifi cation is known to increase the half life of the protein drug. However, the PEGylation reaction leads to product heterogeneity because PEG molecules themselves are heterogeneous. Therefore, it is very important to analyze PEG reagents and PEGylated protein drugs before product release. Liquid chromatography/mass

spectrometry (LC/MS) technology is a powerful and sensitive technique for the characterization of such complex and heterogeneous products. In this study, the intact PEGylated therapeutic protein samples and PEG reagents were analyzed using a C8 packed microfl uidic HPLC-Chip coupled to Q-TOF/MS system. The PEG and PEGylated proteins were separated and eluted from the HPLC-chip, and sprayed through the HPLC-chip nano electrospray tip. A modifi ed version of the ESI-MS based charge stripping approach1,2 was employed in this study. The charge stripping agent, triethylamine (TEA), was delivered

chip

Chip cube

Agilent 6520 Accurate-Mass Q-TOF LC/MS System Triethylamine in acetonitrile/water

was used to spray from referencecalibrant delivery system

Figure 1. Microfl uidic based HPLC-Chip system coupled to an Agilent 6520 Accurate-Mass Q-TOF LC/MS System. The expanded view shows the calibrant delivery system which was used to spray triethylamine solution.

through a calibrant delivery system (CDS) present in the instrument which is generally used to add reference masses (Figure 1). The addition of amine improved the mass spectrum by reducing the charges on the PEGylated protein to yield an interpretable spectrum. The results showed that the PEGylated therapeutic protein under study, as well as PEG reagents, are heterogeneous. After PEGylation, the intact protein showed multiple peaks with each peak differing by approximately 44 Da mass units, which is the mass of the ethylene glycol.

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Experimental

Materials and methods A therapeutic protein sample was obtained from GangaGen Biotechnologies Pvt. Ltd. Para-nitrophenylcarbonate (mPEG-PNP, 10 kDa) was obtained from Creative PEG works. Triethyl amine and formic acid were purchased from Sigma-Aldrich. LC/MS grade water and acetonile were used. The therapeutic protein was PEGylated at a molar ratio of 1:5 using 10 kDa para-nitrophenylcarbonate (mPEG-PNP). Unreacted PEG was removed by applying the reaction mixture onto a cation exchange column. The bound mono-PEGylated protein was eluted using a linear NaCl gradient.

For this study, the Agilent HPLC-Chip/MS Interface (p/n G4240A) was coupled to the 6520 Accurate-Mass Q-TOF LC/MS System. The charge reducing reagent was delivered using the calibrant deliver system (CDS) on the instrument as shown in Figure 1. The LC and MS parameters used for the experiment are shown in Table 1.

Table 1. LC/MS conditions.

HPLC-Chip conditionsHPLC-Chip 5 µm, Agilent ZORBAX 300SB-C8 (300 Å), 40 nL enrichment column and a

75 mm × 43 mm analytical columnInjection volume 1 µL (Needle with wash, fl ush port active for 5 seconds)

Sample thermostat 5 °CMobile phase A 0.1 % formic acid in water Mobile phase B 90 % acetonitrile in water with 0.1 % formic acidSample Loading With Agilent 1260 Infi nity Capillary Pump at 3 % B.Gradient At 0 minutes → 3 % B

At 30 minutes → 50 % BAt 32 minutes → 95 % BAt 34 minutes → 95 % BAt 34.1 minutes → 3 % B

Stop time 36 minutes

Flush volume 4 µLFlow rate 3 µL/min from an Agilent 1260 Infi nity Capillary Pump (p/n G1382A) to the

enrichment column and 600 nL/min from the Agilent 1260 Infi nity Nanofl ow Pump (p/n G2225) to the analytical column.

Q-TOF MS conditionsIon mode Positive ion mode, HPLC-ChipDrying gas temperature 350 °CDrying gas fl ow 5 L/min (nitrogen)Capillary voltage 1,900 VFragmentor voltage 300 V for the PEGylated protein and 250 V for the PEG alone.Skimmer voltage 65 VOct RF Vpp 750 VAcquisition parameters MS mode

Data were acquired on high resolution (3,200 m/z), 4 GHz, MS only mode, mass range 300–2,200 m/z

Data analysis The data obtained from LC/MS were analyzed using Agilent MassHunter Qualitative Analysis software and Agilent MassHunter BioConfi rm software

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different concentrations. Without the TEA post-electrospray addition, PEG was highly charged and all charge state ions were narrowly distributed. It is very diffi cult to obtain the PEG molecular weight distribution from these data by deconvolution (Figure 3A). Inspection of the spectrum shows a poorly resolved

deconvoluted spectrum without well-defi ned peaks. Increasing the concentration of the post-electrospray TEA, lead to a well-distributed charge state of mPEG-PNP. The decreased charge states of PEG resulted in distinct charge state groups in a wide m/z range.

Results and Discussion

LC/MS analysis of PEG with a post-electrospray addition of TEAFigure 2 shows the mass spectra of the main peak of 10 kDa mPEG-PNP obtained using LC/MS with post-electrospray addition of TEA at

×104

×105

×104

00.250.500.751.001.251.501.752.00 705.4490

837.5110

987.50971075.5618

1209.7862 1358.80241537.7975 1798.4864 2154.6377

Mass-to-charge (m/z)

Mass-to-charge (m/z)

Mass-to-charge (m/z)

Coun

tsCo

unts

Coun

ts

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200

No TEA

0.1 % TEA

0.3 % TEA

00.20.40.60.81.01.21.4 1013.6368

924.7440

1126.25421270.7028 1449.8912

1680.82302013.8600

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200

012345678 793.5056

924.8085

1013.61631281.7239

1141.16451474.9009

1695.3382 2040.2013

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200

Figure 2. Mass spectrum of 10 kDa mPEG-PNP at different concentration of TEA.

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PEG masses with different ethylene glycol (44 Da) could also be easily seen under these conditions. Figure 3B shows the deconvoluted mass spectra for 10 kDa mPEG-PNP at 0.3 % TEA concentration. An expanded view (Figure 3C) of the spectra clearly shows well resolved 44 Da oxyethylene unit of PEG.

1.21.11.00.90.80.70.60.50.40.30.20.1

010,300

A

B

C

10,302.30

10,434.82

10,522.79

10,566.76

10,655.11

10,698.5010,787.63

11,040.15

11,179.22

11,256.96

11,337.94

10,400 10,500 10,600 10,700 10,800Deconvoluted mass (amu)

Coun

ts

10,900 11,000 11,100 11,200 11,300

789

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6543210

9700 9800 9900 10,000 10,100 10,200Deconvoluted mass (amu)

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ts

10,300 11,400 11,500 11,600 11,700

789

×105

6543210

10,080 10,120 10,160 10,200 10,240Deconvoluted mass (amu)

Coun

ts

10,280 11,320 11,360 11,400

Figure 3. Deconvoluted mass spectrum of 10 kDa mPEG-PNP without TEA (A), deconvoluted mass spectrum of 10 kDa mPEG-PNP with 0.3 % TEA (B), and expanded view of deconvoluted mass spectrum of 10 kDa mPEG-PNP with 0.3 % TEA (C).

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LC/MS analysis of a mono-PEGylated protein with a post-electrospray addition of TEAFigure 4 shows mass spectra of a PEGylated therapeutic protein obtained by LC/MS with a post-electrospray addition of 0.3 % TEA. The intact molecular weight of the native protein after deconvolution is approximately 26,489 as shown in the inset of Figure 4A. Also seen is a small oxidized native protein at around 26,505. After this protein was modifi ed by 10 kDa PEG, a shift in mass from 26 kDa to 36 KDa was observed as depicted in Figure 4B. Heterogeneity in PEG masses with different ethylene glycol units (44 Da) could be easily seen in the expanded view of the deconvoluted spectrum. The small peak is the oxidated protein as noted earlier with native protein. The LC/MS analysis also confi rmed the monopegylated status of the protein as no other species was observed.

3.0 A

×104

×104

×105

5

4

3

2

1

026.36 26.40

26,460.94 26,505.89

26,489.24

26.44 26.48 26.52 26.56 26.60 26.64 26.68

2.62.8

2.42.22.01.81.61.41.21.00.80.60.40.2

035.8 36.0 36.2 36.4 36.6 36.8 37.0

Deconvoluted mass (amu) ×103

Deconvoluted mass (amu) ×103Coun

ts

Coun

ts

37.2 37.4 37.60 37.80 38.0 38.2 38.4

3

44.03

36,679.5736,723.60

36,767.67

36,811.90

36,855.7936,899.78

36,943.7936,987.76 37,032.07

44.0744.23

43.8943.99

44.0143.97

44.31

2

1

B

036,680 36,720 36,760

Deconvoluted mass (amu)

Coun

ts

36,800 36,840 36,880 36,920 36,960 37,000 37,040

Figure 4. Deconvoluted mass spectrum of 10 kDa PEG modifi ed therapeutic protein with 0.3 % TEA (A); the inset shows the deconvoluted mass spectrum of the unmodifi ed protein. Expanded view of a deconvoluted mass spectrum of a 10 kDa PEG modifi ed therapeutic protein, with 0.3 % TEA, showing a 44 Da mass difference for each ethylene glycol unit (B).

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References 1. Huang, L., Gough, P.C., and DeFelippis, M.R. Characterization of Poly(ethylene glycol) and PEGylated Products by LC/MS with Postcolumn Addition of Amines. Analytical Chemistry, 2009, 81(2):567-577.

2. Bagal, D., Zhang, H., and Schnier, P.D. Gas-Phase Proton-Transfer Chemistry Coupled with TOF Mass Spectrometry and Ion Mobility-MS for the Facile Analysis of Poly(ethylene glycols) and PEGylated Polypeptide Conjugates., Analytical Chemistry, 2008, 80 (7):2408-2418.

3. Veronese, F.M., and Pasut, G. PEGylation, successful approach to drug delivery Drug Discovery Today, 2005,10(21):1451-8.

ConclusionsAn LC/MS method was developed to analyze PEG and PEGylated proteins. Post-electrospray addition of amine caused an improvement in mass spectrum quality and was useful in deconvolution of the complex charge states of the PEGylated proteins. The results showed that the PEGylated therapeutic protein under study, as well as the PEG reagents, are heterogeneous. The average mass and the mass distribution of the PEGylated protein can easily be determined.

HPLC-Chip offers excellent sensitivity for intact protein analysis. An HPLC-Chip coupled to a high resolution high mass accuracy Q-TOF mass spectrometer with post-electrospray addition of amine is a unique confi guration for PEG and PEGylated proteins. This method allows optimization of PEGylation reaction conditions using limited amounts of valuable therapeutic proteins in the early research and development stage of the development process.

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© Agilent Technologies, Inc., 2012Published in the USA, December 12, 20125991-1492EN