LC/MS of Intact TherapeuticMonoclonal Antibodies UsingAgilent AdvanceBio RP-mAb

Author

Suresh Babu C.V.

Agilent Technologies, Inc.

Application Note

Biologics and Biosimilars

Introduction

Therapeutic proteins such as monoclonal antibodies (mAbs) are gaining muchattention in the biopharmaceutical industry. Post translational modifications duringthe mAb manufacturing process commonly result in microheterogeneity. Hence, it isimportant to monitor and analyze these changes to ensure drug efficacy.Reversed-phase LC/MS is the routine method for the analysis of mAbs at intact orfragment level, which provides accurate molecular mass. The correct choice ofLC column and method is critical to achieve fast analysis times and reproduciblehigh-resolution separations.

We used Agilent AdvanceBio RP-mAb columns for intact mAb analysis todemonstrate the fast analysis time and highly reproducible chromatographicseparation provided by these columns. AdvanceBio RP-mAb columns with C4,SB-C8, and Diphenyl chemistries are based on Agilent Poroshell technology withsuperficially porous particles (3.5 µm) with wide pores (450Å) [1]. These columnsdeliver higher resolution and faster run times compared to columns packed withfully porous particles of the same size. In this work, multiple therapeutic mAbs,including an antibody drug conjugate (ADC), were analyzed using AdvanceBioRP-mAb columns in an LC/MS method. This approach delivered fast analysis timesand accurate mass determination of intact mAbs.

2

Experimental

SamplesTherapeutic monoclonal antibodies mAb1, mAb2, andantibody drug conjugate mAb3 were purchased from a localpharmacy and stored according to the manufacturers’instructions. mAb1, mAb2, and mAb3 (lys-conjugated ADC)were diluted to 1 µg/µL using 0.1% formic acid in 3% ACN,and 1 µL was injected.

InstrumentationLC: Agilent 1290 Infinity LC

MS: Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight(Q-TOF) with Agilent Jet Stream ion source

Results and DiscussionDetermination of therapeutic protein molecular weights andglycosylation profiles is best established by LC/MS. Achievinggood chromatographic peak and high signal-to-noise ratio m/zmass spectra is a tradeoff when working with high molecularweight proteins such as mAbs [2]. In addition, for high-throughput mAb screening in a batch-to-batch variation study,fast and reproducible LC/MS methods are required.AdvanceBio RP-mAb columns are packed with superficiallyporous particles. This particle design reduces diffusiondistances, which allows high linear velocities to be usedwhile still maintaining high chromatographic efficiency. Thisapproach delivers high-speed and high-resolution separations.To improve resolution, different C4, SB-C8, and Diphenylbonded phases are available for flexible method development.

Figures 1 and 2 show the LC/MS data for the three intactmAbs with AdvanceBio RP-mAb C4 and Diphenyl columns,respectively. Both columns provided excellent total ionchromatogram peak shapes with narrow peak width (0.5 minat base) using an isopropanol:acetonitrile:water mobile phase(Figures 1A and 2A). The charge-state envelope ranged from2,000 to 4,000 m/z with a Gaussian distribution (Figure 1Band 2B). The raw mass spectra were converted to zero-chargemass spectra using the maximum entropy and peak modeling(pMod) deconvolution algorithms in Agilent MassHunterBioConfirm software. The deconvoluted spectra are shown inFigures 1C and 2C. Five major glycoforms are seen in themAb1 deconvoluted spectrum, while four major glycoformsare evident in the mAb2 spectrum. Due to the strongheterogeneity of mAb3, split peaks were observed with bothC4 and Diphenyl columns (Figure 2A). These split peakscorresponded to the mAb with different payloads.Deconvoluted mass spectra for mAb3 (Figure 2C) showedincreasing payload trend in steps of one drug load with eightmajor drug conjugations (D0, D1, D2...D8).

ConditionsParameter Agilent 1290 Infinity LC

Column AdvanceBio RP-mAb C4, 2.1 × 50 mm, 3.5 µm(p/n 799775-904 )AdvanceBio RP-mAb Diphenyl, 2.1 × 75 mm, 3.5 µm(p/n 799775-944)

Inj vol 1 µL

Sample thermostat 5 °C

Mobile phase A 0.1% Formic acid in water

Mobile phase B 80% IPA:10% ACN: 9.9% water with 0.1% formic acid

Gradient At 0 min & 20% BAt 4 min & 20% BAt 5 min & 40% BAt 10 min & 70% BAt 11 min & 90% BAt 11.1 min & 20% B

Stop time 11.1 min

Post time 4 min

Column temp 80 °C

Flow rate 0.6 mL/min

Parameter Agilent 6530 Accurate-Mass Q-TOF LC/MS

Ion mode Positive ion mode, dual AJS ESI (profile)

Drying gas temp 350 °C

Drying gas flow 8 L/min

Sheath gas temp 400 °C

Sheath gas flow 11 L/min

Nebulizer 35 psi

Capillary voltage 5,500 V

Fragmentor voltage 380 V

Skimmer voltage 65 V

Oct RF Vpp 750 V

Acquisition parameters MS mode Data acquired at 1 GHz, MS only mode, mass range

2,000 to 6,000 m/z

Data analysis Data from LC/MS were analyzed usingAgilent MassHunter Qualitative Analysis software andAgilent MassHunter BioConfirm software. Max entropyand pMod deconvolution algorithms were used toobtain zero-charge spectra of the drug conjugatedantibody (mAb3) and mAb1/mAb2, respectively.

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Figure 1. Intact mAb mass analysis on an Agilent AdvanceBio RP-mAb C4, 2.1 × 50 mm, 3.5 µm column. A) Total ionchromatogram, (B) mass spectrum, (C) deconvoluted spectrum.

Figure 2. Intact mAb mass analysis on an Agilent AdvanceBio RP-mAb Diphenyl, 2.1 × 50 mm, 3.5 µm column. A) Total ionchromatogram, (B) mass spectrum, (C) deconvoluted spectrum.

×107

×107

×107

×104

×104

×103

×105

×105

×103

0123456

1 2 3 4 5 6 7 8 9 10

0123456

1 2 3 4 5 6 7 8 9 10

012345

Acquisition time (min)1 2 3 4 5 6 7 8 9 10

00.40.81.21.62.02.4

2,200 2,600 3,000 3,400 3,800

00.20.40.60.81.01.21.41.6

2,200 2,600 3,000 3,400 3,800

00.40.81.21.6

22.42.8

Mass-to-charge (m/z)2,200 2,600 3,000 3,400 3,800

00.40.81.21.6

22.4 148,225.23

148,063.92 148,389.73

148,545.04147,917.97

147,800 148,000 148,200 148,400 148,600

00.20.40.60.81.01.21.4 147,244.07 147,405.32

147,079.63 147,564.57

Deconvoluted mass (amu)

147,000 147,200 147,400 147,600

00.40.81.21.62.02.42.8 153,018.05151,101.04 152,058.74

149,183.80150,143.59 153,970.66

154,928.31

156,046.34148,225.52

148,000 152,000 156,000

G0/G0F

G0F/G0F

G0F/G1F

G1F/G1F

G1F/G2F

D0

D1D2

D3 D4D5

D6D7

D8

G0F/G0F

G0F/G1F G0F/G2F

G1F/G2F

A B C

mAb1

mAb3 (antibody drug conjugated)

mAb 2

Coun

ts

Coun

ts

Coun

ts

Coun

ts

Coun

ts

Coun

ts

Coun

ts %

Co

unts

%

Coun

ts %

0

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10

0

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10

01

234

5

1 2 3 4 5 6 7 8 9 10

00.40.81.21.62.02.4

2,200 2,600 3,000 3,400 3,800

0

0.4

0.8

1.2

1.6

2.0

2,400 2,800 3,200 3,600 4,000

00.40.81.21.62.02.42.83.2

2,200 2,600 3,000 3,400 3,800 4,200

00.20.40.60.81.01.21.41.6 148,222.07

148,385.74148,062.43

148,549.88147,916.85

147,800 148,000 148,200 148,400 148,600

00.20.40.60.81.01.21.41.61.82.0 147,241.47

147,402.69

147,081.28 147,565.13

147,000 147,200 147,400 147,600 147,800

00.81.62.43.24.0

152,058.30153,014.75

151,106.88150,142.95 153,974.62

149,187.21154,928.52

156,044.28148,227.09

148,000 150,000 152,000 154,000 156,000 158,000

D0

D1D2

D3D4 D5

D6

D7

D8

G0F/G0F

G0F/G1FG0F/G2F

G1F/G2F

G0/G0F

G0F/G0F

G0F/G1F

G1F/G1F

G1F/G2F

×107

×107

×107

×104

×104

×103

×105

×105

×103

Acquisition time (min) Mass-to-charge (m/z) Deconvoluted mass (amu)

A B C

Counts

Counts

Counts

Counts

Counts

Counts

Counts %

Counts %

Counts %

mAb1

mAb3 (antibody drug conjugated)

mAb 2

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For Research Use Only. Not for use in diagnostic procedures.

Agilent shall not be liable for errors contained herein or for incidental or consequentialdamages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to changewithout notice.

© Agilent Technologies, Inc., 2015, 2017Printed in the USANovember 2, 20175991-6296EN

Conclusions

We successfully analyzed therapeutic mAbs usingAgilent AdvanceBio RP-mAb columns coupled to anAgilent 6530 Accurate-Mass Q-TOF LC/MS. The C4 andDiphenyl AdvanceBio RP-mAb columns, with anMS-compatible method, delivered fast and high-resolutionanalysis for intact and ADC mAbs.

References

1. Gritti, F. Chromatog. Today June 2012, 4-11.

2. Gudihal, R.; Suresh Babu C. V.; Tang, N. Analysis ofMonoclonal Antibody (mAb) Using Agilent 1290 InfinityLC System Coupled to Agilent 6530 Accurate-MassQuadrupole Time-of-Flight (Q-TOF); Application note,Agilent Technologies, Inc. Publication number 5991-4266EN, 2014.

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