Application Note

Biologics

AuthorsIsabel Vandenheede, Lucie Jorge, Pat Sandra, and Koen Sandra Research Institute for Chromatography (RIC) President Kennedypark 26 B-8500 Kortrijk

Linda Lloyd, Anne Blackwell, and Maureen Joseph Agilent Technologies, Inc.

AbstractThis Application Note describes how size exclusion chromatography (SEC) coupled to high-resolution mass spectrometry (HRMS) can be applied to the detailed characterization of monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs).

SEC Coupled to High-Resolution Mass Spectrometry for Detailed Characterization of mAbs and ADCs

2

IntroductionMonoclonal antibodies (mAbs) are being developed at an explosive rate and have attracted great interest from both smaller biotech firms and big pharma companies. Developing mAbs and next-generation antibody drug conjugates (ADCs) is highly demanding in many ways. From an analytical perspective, handling mAbs and ADCs presents new challenges for chromatographers. As a result, the field of biochromatography is advancing rapidly as new and higher-resolution techniques for characterizing these biomolecules are evaluated and better understood1,2.

The current contribution describes how size exclusion chromatography (SEC) with high-resolution mass spectrometry (HRMS) can be applied to the detailed characterization of mAbs and ADCs. These measurements provide insights into mAb identity and structural integrity, post-translational modifications (for example, glycosylation) next to drug distribution, and drug-to-antibody ratios (DARs). MS-compatible SEC conditions allowing separation and ionization in the denatured state are described.

Experimental

MaterialsAcetonitrile and water were obtained from Biosolve (Valkenswaard, the Netherlands). Formic acid (FA), trifluoroacetic acid (TFA), dithiothreitol (DTT), sodium chloride, sodium phosphate, cysteine, EDTA, and papain were from Sigma-Aldrich (St. Louis, MO, USA). Immunoglobulin-degrading enzyme from Streptococcus equi ssp. zooepidemicus (IdeZ) was from Promega (Madison, WI, USA). Tris-HCl was acquired from Thermo Fisher (Waltham, MA, USA). Trastuzumab (Herceptin) and ado-trastuzumab emtansine (Kadcyla) were obtained from Roche (Basel, Switzerland). mAb Chinese hamster ovary (CHO) cell culture supernatant was kindly provided by a local biotechnology company.

Sample preparation• Intact mAb: mAb was diluted to

2 mg/mL using 100 mM Tris pH 7.5.

• IdeZ treatment: The mAb or ADC was diluted to 0.5 mg/mL using 50 mM sodium phosphate, and 150 mM sodium chloride, pH 6.6. Then, IdeZ was added in an enzyme:protein ratio of 1:1 (U/µg), and digestion was performed at 37 °C for 60 minutes.

• Papain treatment: The mAb was diluted to 2 mg/mL using 50 mM sodium phosphate, and 1 mM EDTA, 10 mM cysteine, pH 7. After activation, the papain was added in an enzyme:protein ratio of 1:60 (µg/µg), and digestion was performed at 37 °C for two hours.

• Reduction: After dilution of the sample to 0.2 mg/mL using 100 mM Tris pH 8, DTT was added to a final concentration of 10 mM. Reduction was performed at 60 °C for one hour.

Before injection, all samples were centrifuged at 2,000 g for one minute.

InstrumentationSEC/MS measurements were performed on the following instrument configuration:

• Agilent 1290 Infinity LC equipped with:

• Agilent 1290 Infinity binary pump (G4220B)

• Agilent 1290 Infinity autosampler (G4226A)

• Agilent 1290 Infinity thermostat (G1330B)

• Agilent 1290 Infinity thermostatted column compartment (G1316C)

• Agilent 1290 Infinity diode array detector (G4212A)

• Agilent 6540 Q-TOF LC/MS (G6540A) equipped with Jet Stream ESI source

Software• Agilent MassHunter Acquisition

instrument control (B05.01)

• Agilent MassHunter Qualitative Analysis software (B07.00)

• Agilent MassHunter BioConfirm software (B07.00)

3

Results and discussionSEC chromatography typically uses buffers containing nonvolatile salts such as a phosphate buffer. These conditions are not compatible with MS. Figure 1 compares the separations of the humanized mAb Herceptin on a Bio SEC-3 column using a nonvolatile buffer (150 mM phosphate pH 7.0) and an MS-compatible mobile phase (20 % ACN, 0.1 % FA, and 0.1 % TFA). When using a phosphate buffer, sharp peaks are obtained, and aggregates and fragments can be detected at levels as low as 0.1 %. Spraying a phosphate buffer into the MS, however, results in a dirty source and lack of MS signal. When using a mobile phase containing volatile constituents, proteins can be sprayed effectively into the MS, generating high-quality MS data.

SEC/MS Method

Parameter Value

Column Agilent Bio SEC-3 (7.8 × 300 mm, 3 µm, p/n 5190–2511)

Mobile phase 20 % acetonitrile, 0.1 % FA, 0.1 % TFA in water

Flow rate 1 mL/min

Column temperature 24 °C (uncovered)

Injection volume 20 µL (unless noted)

Needlewash solvent Water

Autosampler temperature 5 °C

DAD detection 214 and 280 nm

Q-TOF source

Jet Stream positive ionization mode

Drying gas temperature 350 °C

Drying gas flow 10 L/min

Nebulizer pressure 50 psig

Sheath gas temperature 350 °C

Sheath gas flow 11 L/min

Nozzle voltage 1,000 V

Capillary voltage 3,500 V

Fragmentor voltage 200 V (papain, reduced, IdeZ), 350 V (intact)

Q-TOF detection

Mass range: 3,200 amu Data acquisition range: m/z 1,000 to 3,200 High resolution mode (4 GHz) Data acquisition rate: One spectrum per second Profile acquisition

Diverter valve Bypassed Column outlet directly connected to ESI needle via PEEK tubing (0.12 mm)

Figure 1. SEC/UV chromatograms (214 nm) of Herceptin obtained using different mobile phase compositions with an Agilent Bio SEC-3 column. To the right are pictures of the MS source. Injection volume: 2.5 µL.

mAU

0

50

100

150

200

250

300

350

min0 2 4 6 8 10 12 14 16

mAU

0

50

100

150

200

250

300

350

150 mM phosphate pH 7.0

20 % ACN, 0.1 % TFA, 0.1 % FA

Sharp peak

Dirty MS source

No MS signal

Broader peak

Clean MS source

Good MS signal

4

SEC using a mobile phase containing 20 % ACN, 0.1 % FA, and 0.1 % TFA has successfully been applied on intact mAbs as well as fragments. These fragments originate from reduction (light and heavy chain (Lc and Hc)), papain digestion (Fab and Fc), or IdeZ digestion (F(ab)’2 and Fc/2) (Figure 2).

The SEC/UV/MS analysis of papain-digested Herceptin is shown in Figure 3. UV and MS chromatograms are similar in terms of peak widths. The path from the UV detector to the MS system does not contribute substantially to peak dispersion where the MS diverter valve is bypassed. Deconvoluted MS data associated with the annotated peaks are shown in Figure 4. Fab, Fc,

and smaller fragments are identified and glycosylation revealed. The measured molecular weight values are well below 0.005 % different from the theoretical molecular weight values. Peaks c, d, and e are resolved, though their molecular weights are similar. Therefore, the MS data of peaks c, d, and e show that the separation is not purely driven by size for these components.

Reduction

Papain

5 6 7 8 9 10 11

Intact

IdeZ

Fc/2

F(ab‘)2

Fc

Fab

5 6 7 8 9 10 11

Hc

2 × Hc2 × Lc

2 × Fc2 × Fab

2 × Fc/21 × F(ab’)2

Lc

Acquisition time (min)

5 6 7 8 9 10 11Acquisition time (min)

5 6 7 8 9 10 11Acquisition time (min)

Re

sp

on

se

un

its

Re

sp

on

se

un

its

Re

sp

on

se

un

its

Acquisition time (min)

Re

sp

on

se

un

its

Figure 2. SEC/UV chromatograms (214 nm) of intact, DTT-reduced, papain-digested, and IdeZ-treated Herceptin. Mobile phase composition: 20 % ACN, 0.1 % FA, 0.1 % TFA. Column: Agilent Bio SEC-3. Injection volume: 20 µL except for intact mAb (5 µL).

5

Figure 4. Deconvoluted Q-TOF MS spectra associated with the annotated peaks in Figure 3.

G0F/G0F

G0F/G1F

G1F/G1F

G1F/G2F

G2F/G2F

G2FG0/G0F

0

1

52,982.2

52,820.1

53,144.3

52,673.8 53,306.3

52,000 53,000 54,000

0

0.5

1.0 47,637.6

46,000 47,000 48,000 49,000

0

2

425,516.0

26,412.5

23,000 25,000 27,000

0

4

823,439.4

22,000 23,000 24,000 25,000

0

2

4

24,200.6

23,819.7

23,000 24,000 25,000 26,000

G0F

G1F

×105×104

×104

×104

×106

AC

D

E

B

Co

un

ts

Co

un

tsC

ou

nts

Co

un

tsCo

un

ts

Deconvoluted mass (amu)

Deconvoluted mass (amu) Deconvoluted mass (amu)

G1F

G0F

G0

N-AcetylglucosamineGalactoseMannoseFucose

Figure 3. SEC/UV/MS chromatograms of papain-digested Herceptin. Mobile phase composition: 20 % ACN, 0.1 % FA, 0.1 % TFA; column: Agilent Bio SEC-3.

a

b

cd e

Fc

FabMS

UV

00.51.01.52.02.53.03.5

0

0.2

0.4

0.6

0.8

1.0

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

×107

×103

A

B

Acquisition time (min)

Re

sp

on

se

Re

sp

on

se

6

Figure 5 shows the SEC/UV/MS data of intact Herceptin. Several peaks are annotated, and the corresponding deconvoluted spectra are shown in Figure 6. The intact mAb with a molecular weight of approximately 150 kDa is successfully measured and the glycosylation pattern is detailed. Fragments of the mAb are also highlighted.

a

b

c d

MS

UV

1.5

2.0

2.5

3.0

3.5

0

0.2

0.4

0.6

0.8

1.0

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

×106

×102

A

B

Acquisition time (min)

Re

sp

on

se

Re

sp

on

se

Figure 5. SEC/UV/MS chromatograms of intact Herceptin. Mobile phase: 20 % ACN, 0.1 % FA, 0.1 % TFA; column: Agilent Bio SEC-3.

×104

×104×103

×103

A

B

Co

un

tsC

ou

nts

Co

un

tsC

ou

nts

Deconvoluted mass (amu)

Deconvoluted mass (amu)

Deconvoluted mass (amu)

Deconvoluted mass (amu)

G0F

N-AcetylglucosamineGalactoseMannoseFucose

G0F/G0F

G0F/G1F

G1F/G1F

G1F/G2FG0/G0F

G0F

G0

G1F

G2F

0

0.5

1.0

1.5

148,226

148,066 148,387

147,918

147,000 148,000 149,000

0

1

2

3

74,031

74,193

74,353

73,879

73,000 74,000 75,000

0

1

2

3

446,877.1

46,000 47,000

0

0.5

1.0

23,558.7

23,744.2

23,000 24,000

C

D

Figure 6. Deconvoluted Q-TOF MS spectra associated with the annotated peaks in Figure 5.

7

Figure 7 compares the SEC/UV/MS measurements of IdeZ-treated Herceptin and the lysine conjugated ADC Kadcyla. Deconvoluted spectra associated with the annotated peaks are shown in Figure 8. Insight into glycosylation, DAR, and drug distribution is provided.

Figure 7. SEC/UV chromatograms (280 nm) of IdeZ-digested Herceptin and Kadcyla; column: Agilent Bio SEC-3.

MS

UV

×101

×101

A

B

Acquisition time (min)

Re

sp

on

se

Re

sp

on

se

a b

c

d

Herceptin

Kadcyla

0

0.5

1.0

1.5

2.0

0

0.2

0.4

0.6

0.8

1.0

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Figure 8. Deconvoluted Q-TOF MS spectra associated with the annotated peaks in Figure 7.

×105

×104

×105

×104

A

B

C

D

Co

un

tsC

ou

nts

Deconvoluted mass (amu) Deconvoluted mass (amu)

Deconvoluted mass (amu) Deconvoluted mass (amu)

G0F

N-AcetylglucosamineGalactoseMannoseFucose

0

0.5

1.097630

96,000 98,000 100,000 102,000

0

0.5

1.0

1.598,588

99,546

97,630

100,503

96,000 98,000 100,000 102,000

DAR 0

DAR 1

DAR 2

DAR 3

DAR 4

+ li

nke

r

+ li

nke

r

0

1

2

325,232.7

25,394.9

25,000 26,000 27,000 28,000

0

1

2

3

4

5

6

7 25,232.6

25,394.9

26,190.2

26,352.5

27,147.6

25,000 26,000 27,000 28,000

DAR 0

DAR 1

DAR 2G0

G0F

G1F

G2F

G0

G0F

G1F

G2F

www.agilent.com/chem

This information is subject to change without notice.

© Agilent Technologies, Inc. 2018 Printed in the USA, October 31, 2018 5994-0303EN

The SEC/UV/MS data of the supernatant of a mAb-producing CHO clone and the corresponding Protein A purified sample (intact and DTT reduced) are shown in Figure 9. Protein A binds the Fc region, and can be used to selectively enrich mAbs from cell culture supernatants. The corresponding MS data allow the annotation of the different peaks. Figure 10 presents the deconvoluted MS spectra associated with the Lc and Hc of the reduced Protein A purified sample. Insight into Hc glycosylation, Hc C-terminal lysine truncation, and Lc glycation is obtained. These data illustrate the level of detail of mAb characteristics that can be obtained with the described SEC/MS method.

ConclusionSEC/UV/MS represents a powerful addition to the analytical toolbox and opens new possibilities for the detailed characterization of mAbs and ADCs. Intact, reduced, papain-digested, and IdeZ-treated mAbs and ADCs can successfully be analyzed. Conditions shown in this Application Note do not maintain mAbs and ADCs in their native state, and are expected to disturb noncovalent interactions.

Important note: All SEC/MS experiments were performed on an SEC column with an internal diameter of 7.8 mm operated at a flow rate of 1 mL/min. Sensitivity enhancement or reduced sample consumption is expected when performing these experiments on an SEC column with an internal diameter of 4.6 mm operated at a flow rate of 0.35 mL/min.

×102

Acquisition time (min)

Re

sp

on

se

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5

Figure 9. SEC/UV chromatograms (214 nm) of the supernatant of a mAb-producing CHO clone (black) and the corresponding Protein A purified sample (red: intact, blue: DTT reduced) produced using an Agilent Bio SEC-3 column.

G0F

G1F

G0F + Lys

Lc + hexose

Lc

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8 50,596.7

50,759.0

50,919.850,393.9

50,100 50,300 50,500 50,700 50,900 51,100 51,300

00.20.40.60.81.01.21.41.61.82.02.22.42.6

23,439.7

23,602.2

23,000 23,100 23,200 23,300 23,400 23,500 23,600 23,700 23,800 23,900 24,000

×105

×105

A

B

Co

un

tsC

ou

nts

Deconvoluted mass (amu)

Deconvoluted mass (amu)

Figure 10. Deconvoluted Q-TOF MS spectra associated with the Hc and Lc peaks observed in the Protein A purified and reduced sample shown in Figure 9 (blue trace).

References1. Sandra, K.; Vandenheede, I.;

Sandra, P. Modern chromatographic and mass spectrometric techniques for protein biopharmaceutical characterization. J. Chromatogr. A 2014, 1335, 81–103.

2. Fekete, S.; et al. Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals. Anal. Chem. 2016, 88, 480–507.