Analysis of Polyethylene Glycol (PEG) and a Mono and Di-PEGylated Therapeutic Protein Using HPLC and Q-TOF Mass Spectrometry

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 describes a method for analyzing PEG and PEGylated protein using an Agilent HPLC coupled to an Agilent 6520 Accurate Mass Q-TOF LC/MS System. A charge stripping agent (triethylamine) was used as a post column addition to aid in obtaining a simpler mass spectrum and hence a better interpretation of the results. The results showed signifi cant improvement in the mass spectrum quality for PEG and the PEGylated protein.

AuthorsRavindra Gudihal and Suresh Babu CVAgilent Technologies India Pvt. Ltd.Bangalore, India

Ning TangAgilent Technologies, Inc.Santa Clara, CA, USA

Sundaram Palaniswamy, Umamaheshwari S and Suneel BasingiGanGagen Biotechnologies Pvt. Ltd. Bangalore, India

2

IntroductionLiquid chromatography/mass spectrometry (LC/MS) technology is a powerful and sensitive technique for the characterization of proteins. LC/MS methods are used in both qualitative and quantitative analysis of protein pharmaceuticals for regulatory approval. PEGylation is one of the important covalent modifi cations used in the biopharmaceutical industry for improving the therapeutic value of proteins.1 The PEGylation process involves the chemical attachment of polyethylene glycol (PEG) to the therapeutic protein. This modifi cation

is known to increase the half-life of the protein drug. The PEGylation reaction leads to product heterogeneity because PEG molecules are themselves heterogeneous. Therefore, it is very important to analyze the PEG reagents and PEGylated protein drugs before product release. In this study, the intact PEGylated therapeutic protein samples and the PEG reagents were analyzed using an Agilent 1260 Infi nity LC coupled to an Agilent 6520 Q-TOF Mass Spectrometer. The PEG and PEGylated proteins were separated and eluted from the HPLC, while the charge stripping agent, triethylamine (TEA), was delivered using a syringe

pump through a tee junction, as shown in Figure 1.2,3 TEA was mixed with the PEGylated sample before it went into the mass spectrometer, where it acted as a charge reducing reagent. The addition of amine improved the mass spectrum and was useful for interpreting the results. The results showed that the PEGylated therapeutic protein under study, as well as PEG reagents, is heterogeneous. After PEGylation, the intact protein showed multiple peaks, with each peak differing by approximately 44 Da mass units which is the mass of ethylene glycol.

Tee

MS

Syringe pumpColumn

Figure 1. Instrument setup consisting of an Agilent 1260 Infi nity LC System and an Agilent 6520 Accurate-Mass Q-TOF Mass Spectrometer. The syringe pump was used to deliver triethylamine solution through a T connector.

3

Experimental

Materials and methods The therapeutic protein sample was obtained from GangaGen Biotechnologies Pvt. Ltd. Para-nitrophenylcarbonate (mPEG-PNP, 10 kDa) was obtained from Creative PEG works. Triethyl (TEA) and formic acid was purchased from Sigma-Aldrich. LC/MS grade water and acetonile were used. The therapeutic protein (P128) 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 to a cation exchange column. The bound mono and di-PEGylated protein was eluted using a linear NaCl gradient.

For this study, the 1260 Infi nity LC System was coupled with the 6520 Accurate-Mass Q-TOF Mass Spectrometer. Post column addition of amine solution (1 % TEA in 50:50 acetonitrile/water), using a syringe pump continuously with steady fl ow rate of 0−10 μL/min during the LC/MS runs, through a tee junction as shown in Figure 1. Table 1 shows the LC and MS parameters used for the run.The data obtained from LC/MS were analyzed using Agilent MassHunter Qualitative Analysis software and Agilent MassHunter BioConfi rm software.

Table 1. LC/MS conditions.

LC conditionsColumn mRP Hi-recovery protein column (p/n 5188-5231), 4.6 mm × 50 mm

Injection volume 1 µL for PEG, 2 µL for di-PEGylated protein, 5 µL for mono-PEGylated protein (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 acidGradient At 0 minutes      →  3 % B

At 8 minutes    →  75 % BAt 15 minutes    →  100 % BAt 16 minutes    →  100 % BAt 17 minutes →  3 % B

Stop time 25 minutesColumn temperature 60 °CFlow rate 0.2 mL/min from an Agilent 1260 Infi nity Binary Pump (p/n G1312B)Q-TOF MS conditionsIon mode Positive ion mode, ESI (Profi le)Drying gas temperature 350 °CDrying gas fl ow 6 L/min (nitrogen)Nebulizer 40 psiCapillary voltage 3,800 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 minimum storage (20,000 m/z), 1 GHz, MS only mode, mass range 300–10,000 m/z

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Results and Discussion

LC/MS analysis of PEG with a post column addition of TEA Figure 2 shows the mass spectra of the main peak of 10 kDa mPEG-PNP obtained by LC/MS with a post column addition of TEA at different concentrations. When no TEA was added, the PEG was highly charged and all charge state ions were narrowly distributed. It is diffi cult to obtain the PEG molecular weight distribution from these data by deconvolution (Figure 3A). The deconvoluted spectrum shows no well-defi ned deconvoluted peaks. Increasing concentration of post-column addition of TEA leads to well distributed charge-states of mPEG-PNP (Figure 2). The charge states of PEG had decreased resulting distinct charge state groups in a wide m/z range. Figure 3B shows the deconvoluted mass spectra for 10 kDa mPEG-PNP at 10 µL/min TEA infused. The ethylene glycol unit (44 Da) is clearly detected.

4.03.6

A

B

3.22.82.42.01.61.20.80.4

8,934.379,153.76

9,497.929,896.24

10,068.8210,367.15

10,,596.1410743.71

11,038.77 11,513.3712,046.08 12,488.97

0

×105

2.8

2.4

2.0

1.6

1.2

0.8

0.48,704.53 9,145.06

9,585.87

9,806.21

10,026.3710,158.32

10,335.11

10,642.85

10,951.90 11,083.5211,259.28

11,479.76

11,788.1111,919.56

12,138.2312,448.64

0 8,800 9,200 9,600 10,000 10,400 10,800Deconvoluted mass (amu)

Coun

tsCo

unts

11,200 11,600 12,000 12,400

×104

No TEA

10 µL TEA

Figure 3. Deconvoluted mass spectrum of 10 kDa mPEG-PNP with no TEA added (A). Deconvoluted mass spectrum of 10 kDa mPEG-PNP, with 10 µL TEA added (B).

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

1.2 916.4317 No TEA0.8

1.4

0

×105

2.5 1,249.80662.5 µL TEA

1.01.52.0

0.50

×104

1.2 1,337.5795 5 µL TEA

0.40.60.81.0

0.20

×104

1.01,341.2730

10 µL TEA

0.40.60.8

0.2

500 1,000 1,500 2,000 2,500Mass-to-charge (m/z)

Coun

tsCo

unts

Coun

tsCo

unts

3,000 3,500 4,000 4500 5,000 5,5000

×104

5

LC/MS analysis of a mono-PEGylated protein with a post column addition of TEA Figure 4 shows the deconvoluted mass spectra of a mono-PEGylated and a di-PEGylated therapeutic protein obtained by LC/MS with a post column addition of 10 µL/min TEA. The intact molecular weight of the native protein after deconvolution was approximately 26,489, which is shown in the inset of Figure 4A. Figure 4A shows a shift in mass from 26 kDa to 36 KDa after this protein was modifi ed by the addition of a 10 kDa PEG molecule. Heterogeneity in PEG masses with different ethylene glycol units (44 Da) is easily seen in the deconvoluted spectrum.

The LC/MS analysis also confi rmed the mono-PEGylated status of the protein as no other species was observed except for a small fraction of unmodifi ed native protein (data not shown). Figure 4B shows the deconvoluted mass spectrum of the di-PEGylated form of P128 with a 10 kDa PEG modifi cation. The mass of the di-PEGylated protein is approximately 46 kDa confi rming that two 10 kDa PEG molecules are attached to the 26 kDa protein. Here, heterogeneity in PEG masses with different ethylene glycol units (44 Da) is easily seen in the deconvoluted spectrum.

Figure 4. Deconvoluted mass spectrum of 10 kDa mono-PEGylated therapeutic protein, with 10 µL/min TEA addition (A); inset shows the deconvoluted mass spectrum of unmodifi ed protein. Deconvoluted mass spectrum of 10 kDa di-PEGylated therapeutic protein, with 10 µL/min TEA addition (B).

5

35,971.11

36,324.27

36,587.84

36,765.30

36,897.28

36,983.63

37,072.6237,,248.79

37424.7754321026,360 26,440

26,460.94

26,489.24

26,505.89

26,520 26,600 26,680

37,557.5937,644.09

38,042.8438,174.62

38,303.8938,526.66

2

3

4

1

35,800 36,200 36,600 37,000Deconvoluted mass (amu)

Deconvoluted mass (amu)

Coun

ts37,400 37,800 38,200 38,600

0

×105

45,061.1545,148.39

45,499.41

45,676.02 45,852.8546,028.97

46,293.06

46,556.41

46,645.0346,733.07

46,997.85

47,129.95

47,439.1347,615.02

2

3

4

1

44,800 45,200 45,600 46,000Deconvoluted mass (amu)

Coun

ts

46,400 46,800 47,200 47,6000

×103

×103

A

B

ConclusionsAn LC/MS method with post column addition was developed on an Agilent 6520 Q-TOF LC/MS System to analyze PEG and PEGylated proteins. Post column addition of an amine resulted in an improvement in mass spectrum quality and was useful in deconvolution of the complex charge states of PEG and PEGylated proteins. The average mass and the mass distribution of the PEGylated protein was easily determined. The results showed that the PEGylated therapeutic proteins under study, as well as PEG reagents, are heterogeneous. The developed method is useful for studying the PEGylation process during the formulation stages of therapeutic drug development.

References 1. Veronese, F.M., and Pasut, G. PEGylation, successful approach to drug delivery, Drug Discovery Today, 2005, 10(21):1451-8.

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

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© Agilent Technologies, Inc., 2012Published in the USA, December 3, 20125991-1509EN