Application Note

Biologics

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

Anne Blackwell and Maureen Joseph Agilent Technologies, Inc. 2850 Centreville Rd Wilmington DE 19808 USA

AbstractThis Application Note describes the use of Agilent AdvanceBio Desalting-RP cartridges for the mass spectrometric analysis of salt-containing monoclonal antibody (mAb) and antibody drug conjugate (ADC) samples.

Efficient Monoclonal Antibody and Antibody Drug Conjugate Desalting Prior to Mass Spectrometry Using AdvanceBio Desalting-RP Cartridges

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IntroductionAlmost 30 years ago, the 2002 Nobel laureate, John Fenn, described for the first time an elegant way to present proteins to the mass spectrometer (MS)1. The principle known as electrospray ionization (ESI) revolutionized the analysis of proteins. In combination with high-resolution MS systems, ESI is now widely applied for the characterization of protein biopharmaceuticals such as monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). The ESI process generates multiply charged ions from proteins, resulting in a characteristically charged envelope. This can readily be converted to the molecular mass by applying, for example, a maximum entropy algorithm. Such a deconvoluted spectrum provides information on molecular weight, structural integrity, and modifications. The success of ESI measurements largely depends on the sample composition. Samples encountered in biopharmaceutical analysis contain a high amount of nonvolatile salts, detergents, and so forth.This comes as a result of formulation or preceding chromatographic separations (for example, Protein A chromatography, cation-exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography). Since ESI can only tolerate a certain amount of these nonvolatile constituents, desalting is required. This Application Note describes the use of the Agilent AdvanceBio Desalting-RP cartridges for the online reversed-phase (RP) desalting of mAbs and ADCs before time-of-flight (TOF) MS measurement.

Experimental

MaterialsAcetonitrile, formic acid, and water were acquired from Biosolve (Valkenswaard, the Netherlands). Dithiothreitol, cysteine, EDTA, sodium phosphate, and papain were purchased from Sigma-Aldrich (St. Louis, MO, USA). Immunoglobulin-degrading enzyme from Streptococcus equi ssp. zooepidemicus (IdeZ) came from Promega (Madison, WI, USA). Tris-HCl was from Thermo Fisher Scientific (Waltham, MA, USA). Monoclonal antibody trastuzumab, marketed as Herceptin, and ado-trastuzumab emtansine, marketed as Kadcyla, were obtained from Roche (Basel, Switzerland).

Sample preparation• Reduction: 100 mM DTT was

added to the sample to a final concentration of 10 mM DTT following sample dilution to 0.21 mg/mL using 100 mM Tris pH 8.0. Reduction was performed at 60 °C for one hour.

• Papain digest: Herceptin was diluted to 2 mg/mL using papain digest buffer. Activated papain was added to the diluted sample at an enzyme:protein ratio of 1:60 (µg/µg). Digestion was performed at 37 °C for two hours.

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

• Intact measurement: Proteins were diluted to 2 mg/mL using 100 mM Tris, pH 7.5.

InstrumentationOnline desalting MS measurements were performed with the following configurations:

• Agilent 1290 Infinity LC equipped with:

• Agilent 1290 Infinity binary pump (G4220B)

• Agilent 1290 Infinity autosampler (G4226A)

• Agilent 1290 Infinity thermostat (G1330B), and

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

• Agilent 6550 iFunnel Q-TOF LC/MS (G6550A) with Jet Stream ESI source, or

• Agilent 6560 ion mobility Q-TOF LC/MS (G6560A) 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)

• Agilent MassHunter DAR calculator software

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Online desalting LC/MS method

Parameter Value

Cartridge Agilent AdvanceBio Desalting-RP, 2.1 × 12.5 mm, 10 µm, 1,000 Å (p/n PL1612-1102)

Mobile phase A 0.1 % Formic acid in water (v/v)

Mobile phase B 0.1 % Formic acid in acetonitrile (v/v)

Flow rate 400 µL/min

Injection volume Volume corresponding to 1 µg of material, unless otherwise mentioned

Needlewash solvent 60 % Acetonitrile, 35 % water, 5 % isopropanol

Autosampler temperature 7 °C

Gradient program

0 minutes, 5 %B 0.5 minutes, 5 %B 3 minutes, 80 %B 4 minutes, 80 %B 4.1 minutes, 5 %B 6 minutes, 5 %B

Q-TOF source

Positive ionization mode Drying gas temperature: 300 °C Drying gas flow: 8 L/min Nebulizer pressure: 35 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 (light chain (Lc), heavy chain (Hc), Fab, Fc, F(ab’)2, Fc/2)

Q-TOF detection

Mass range: 3,200 amu Data acquisition range: m/z 500 to 3,200 High resolution mode: 4 GHz Data acquisition rate: One spectrum per second Profile acquisition

Diverter valve0 minutes Waste 1 minute MS 4.5 minutes Waste

Results and discussionThe AdvanceBio Desalting-RP cartridge, packed with wide-pore (1,000 Å) polymeric RP particles, is installed between the LC injector valve and the MS diverter valve. The latter directs the flow either to waste or the ESI needle. Following sample loading at low acetonitrile concentration, salts are diverted to waste for one minute. The diverter valve is subsequently switched to the ESI needle. The mAb or ADC is eluted at increased acetonitrile concentration and presented as gas-phase ions to the TOF mass analyzer.

Figures 1, 2, and 3 show the online desalting TIC chromatograms, with raw and deconvoluted spectra obtained from various Herceptin middle-down measurements. The data correspond to the MS analysis of DTT-reduced, papain-digested, and IdeZ-treated Herceptin. Reduction of the mAb with DTT generates the Lc and Hc, digestion with papain results in a Fab and Fc fragments, and IdeZ treatment gives rise to an F(ab’)2 and Fc/2 fragments. Although the aim of the cartridge is mainly to desalt proteins, partial separation is obtained, as shown by the different peak shapes (Figure 1) and the different spectra captured as a function of time (Figure 2). As shown in Table 1, samples contain a wide range of non-MS compatible chemicals at relatively high concentrations. Nevertheless, their presence does not prevent highly informative spectra that enable the determination of molecular weight, confirmation of the gene-derived sequence, and protein modifications such as glycosylation. Tables 2 and 3 also show that measurements using the AdvanceBio Desalting-RP cartridge are highly precise in terms of peak area and glycoform assessment.

Figure 1. Online desalting TIC chromatograms obtained from the analysis of reduced, papain-digested, and IdeZ-treated Herceptin. Data were acquired on an Agilent 6540 Q-TOF LC/MS.

0

0.5

1.0

1.5

2.0 1 1 2 2 3 3

1 1 2 2 3 3

1 1 2 2 3 3

0

0.5

1.0

1.5

2.0

0

0.5

1.0

1.5

2.0

1 2 3 4 5

Reduced Herceptin

Papain digested

Herceptin

IdeZ-treated

Herceptin

WASTE WASTEMS×108

×108

×108

Acquisition time (min)

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600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000 2,100 2,200 2,300 2,400 2,500 2,600 2,700 2,800

Lc + Hc

Fc

Fab

F(ab’)2

Fc/2Retention time window: 1.9 to 2.0 minutes

Retention time window: 2.2 to 2.4 minutes

Papain-digested Herceptin

Papain-digested Herceptin

IdeZ-treated Herceptin

IdeZ-treated Herceptin

Retention time window: 1.9 to 2.0 minutes

Retention time window: 2.1 to 2.4 minutes

DTT-reduced Herceptin

Retention time window: 2.0 to 2.4 minutes

×104

×104

×104

×104

×103

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Mass-to-charge (m/z)

Figure 2. Raw spectra retrieved from the online desalting MS experiments of reduced, papain-digested, and IdeZ-treated Herceptin (Figure 1). Lc and Hc are not separated by the cartridge, Fc, Fab, Fc/2, and F(ab’)2 are partially separated, providing discrete spectra. Data were acquired on an Agilent 6540 Q-TOF LC/MS.

Table 1. Composition of injected samples (Figures 1–3).

ReducedHerceptin

Papain‑digested Herceptin

IdeZ‑treated Herceptin

Formulation‑derived

mAb (mg/mL) 0.189 2 0.5

L-Histidine HCl (mg/mL) 0.004 0.045 0.011

L-Histidine (mg/mL) 0.003 0.029 0.007

Trehalose (mg/mL) 0.172 1.818 0.455

Polysorbate (mg/mL) 0.001 0.008 0.002

Sample preparation‑derived

Tris-HCl pH 8 (mM) 99 – –

DTT (mM) 10 – –

Papain (µg/µg) – 0.0167 –

Na2HPO4.2H2O (mM) – 45.2 48.3

EDTA (mM) – 0.9 –

Cysteine (mM) – 9.0 –

IdeZ (unit/µg) – – 1

NaCl (mM) – – 145

5

0

0.5

1.0

23,440.1

23,602.1

23,200 23,300 23,400 23,500 23,600 23,700 23,8000

1

2

3

4

5

6

7

8

50,597.4

50,759.6

50,922.250,451.6

50,200 50,400 50,600 50,800 51,000 51,200

0

1

2

3

4

47,638.2

47,200 47,400 47,600 47,800 48,000 48,2000

1

2

3

4 52,982.1

52,820.2

53,144.7

53,306.752,675.3

52,200 52,400 52,600 52,800 53,000 53,200 53,400 53,600 53,800

G0F

G1F

G2FG0

G0F/G0F

G0F/G1F

G1F/G1F

G1F/G2FG0/G0F

Lc

Fc

Fab

F(ab’)2

Hc

×106 ×105

×105×106

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Deconvoluted mass (amu)

Deconvoluted mass (amu)

Deconvoluted mass (amu)

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Deconvoluted mass (amu)

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Deconvoluted mass (amu)

Deconvoluted mass (amu)

Fc/2

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1

2

3

4

5

6

7

8 97,630.6

96,600 97,000 97,400 97,800 98,200 98,600 99,0000

0.5

1

25,232.8

25,394.9

25,556.525,086.6

24,800 25,000 25,200 25,400 25,600 25,800

G0F

G1F

G2FG0

Figure 3. Deconvoluted spectra of the Herceptin Lc, Hc, Fab, Fc, F(ab’)2, and Fc/2 fragments desalted on the AdvanceBio Desalting-RP cartridge (Figure 1). Data were acquired on an Agilent 6540 Q-TOF LC/MS.

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As demonstrated in Figure 4, the cartridge also successfully allows the measurement of mAbs at the intact protein level even in the presence of high salt concentrations. When combined with the 6550 iFunnel Q-TOF LC/MS, a 50-ng load on the cartridge still enables decent spectra (Figure 5).

Table 2. Peak area precision data obtained from the five-fold analysis of reduced, papain, and IdeZ-treated Herceptin. Extracted ion chromatograms were integrated to retrieve the peak area.

DTT reduced HerceptinPapain digested

Herceptin IdeZ treated Herceptin

Lc Hc Fab Fc F(ab’)2 Fc/2

Extracted ion 1,303.2 1,177.7 1,362.1 1,325.6 2,219.9 1,329.0

Peak area repl. 1 52,353,329 22,318,745 72,745,026 21,710,454 44,178,915 44,884,582

Peak area repl. 2 51,276,011 21,017,616 72,282,429 22,262,514 40,960,959 40,937,521

Peak area repl. 3 48,808,842 20,974,828 72,108,434 21,943,232 39,402,122 41,508,316

Peak area repl. 4 54,928,024 21,317,775 71,773,149 21,782,185 45,129,358 45,387,243

Peak area repl. 5 50,095,342 20,588,954 72,334,985 21,841,694 43,973,079 46,013,420

% RSD 4.5 3.1 0.5 1.0 5.7 5.4

Table 3. Glycoform precision data obtained from the five-fold analysis of reduced, papain, and IdeZ-treated Herceptin.

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 %RSD

Hc (DTT‑reduced Herceptin)

% G0 6.3 6.2 5.9 6.1 6.4 3.5

% G0F 46.7 47.1 47.4 47.5 46.5 0.9

% G1F 39.2 38.6 38.8 38.6 39.1 0.7

% G2F 7.8 8.1 8.0 7.8 8.0 1.4

Fc (Papain‑digested Herceptin)

% G0/G0F 5.1 5.2 4.9 4.9 5.3 3.4

% G0F/G0F 29.8 27.7 28.7 29.1 28.4 2.7

% G1F/G0F 34.0 35.5 36.3 34.4 35.4 2.6

% G1F/G1F or G2F/G0F 24.9 24.6 23.1 24.9 25.2 3.3

% G2F/G1F 6.2 7.0 7.0 6.6 5.7 8.4

Fc/2 (IdeZ‑digested Herceptin)

% G0 6.3 6.5 5.9 6.8 6.5 5.1

% G0F 47.6 47.8 51.0 48.0 47.5 3.0

% G1F 38.8 38.2 36.1 38.1 38.7 2.9

% G2F 7.2 7.5 7.0 7.2 7.3 2.5

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Figure 4. Deconvoluted spectra of intact Herceptin in the presence of 0, 150, and 300 mM NaCl, demonstrating the effectiveness of the AdvanceBio Desalting-RP cartridge (4 µg load). Data were acquired on an Agilent 6540 Q-TOF LC/MS.

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1

2

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4

5 148,223

148,385148,061

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148,223

148,385148,062

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148,383148,062

146,750 147,000 147,250 147,500 147,750 148,000 148,250 148,500 148,750 149,000 149,250 149,500 149,750 150,000

Herceptin

0 mM NaCl

150 mM NaCl

300 mM NaCl

Herceptin

Herceptin

G0F/G0F

G0F/G1F

G1F/G1F

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Figure 5. Deconvoluted spectra of intact Herceptin desalted on the AdvanceBio Desalting-RP cartridge at 5 µg (black) and 50 ng (blue) load. The red trace corresponds to a blank injection. Data were acquired on an Agilent 6550 iFunnel Q-TOF LC/MS.

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147,000 147,500 148,000 148,500 149,000 149,500 150,000

Herceptin 5 µgG0F/G0F

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G1F/G1F

G1F/G2F

G0/G0F

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Deconvoluted mass (amu)

Herceptin 50 ng

Blank

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The AdvanceBio Desalting-RP cartridge has also been used to measure the lysine-conjugated ADC Kadcyla at the intact level and following IdeZ treatment (Figures 6 and 7). Kadcyla combines the anti-HER2 antibody

trastuzumab (Herceptin) with the cytotoxic microtubule-inhibiting maytansine derivative, DM1, by a nonreducible thioether linker (Figure 6). This measurement allows an accurate determination of the drug-to-antibody

ratio (DAR), a critical quality attribute of ADCs, obtained using the MassHunter DAR calculator. A DAR of 3.6 is retrieved, which is in accordance with the DAR value reported in literature for this particular ADC2.

Figure 6. Deconvoluted spectrum of intact Kadcyla desalted on the AdvanceBio Desalting-RP cartridge. When combined with the MassHunter DAR calculator, a DAR of 3.6 is retrieved. Red stars correspond to the small molecule cytotoxic drug (4 µg load). Data were acquired on an Agilent 6540 Q-TOF LC/MS.

Trastuzumab

G0FG0F

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1

2

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4

151,099

152,054

150,141

153,013

149,179

153,984

148,217154,976

148,000 149,000 150,000 151,000 152,000 153,000 154,000 155,000 156,000

DAR 0

DAR 1

DAR 2

DAR 3

DAR 4

DAR 5

DAR 6

DAR 7

DAR 8

G0F/G0F

G0F/G1F

G1F/G1F

DM1 Thioether linker

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Figure 7. Deconvoluted spectra of IdeZ-treated Kadcyla desalted on the AdvanceBio Desalting-RP cartridge. Linker without drug is observed in the data as well, indicating that the reaction between the linker and the drug is not driven to completion (8 µg load). Data were acquired on an Agilent 6540 Q-TOF LC/MS.

0

1

2

3 25,233.0

25,395.1

26,190.8

26,352.5

25,086.725,557.3

27,147.726,044.0 26,514.4

24,600 25,000 25,400 25,800 26,200 26,600 27,000 27,400

0

1

2

99,545.6

98,588.4100,503.7

101,460.8

97,631.4

102,418.3

97,000 98,000 99,000 100,000 101,000 102,000 103,000

G0F

G1F

G2FG0

G0F

G1F

G2FG0G0F

G1F

DAR 0

DAR 1

DAR 2

DAR 1

DAR 2

DAR 3DAR 4

DAR 5

Fc/2

F(ab’)2

+ linker+ linker

+ linker

+ linker

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Deconvoluted mass (amu)

DAR 0

B

A

www.agilent.com/chem

This information is subject to change without notice.

© Agilent Technologies, Inc. 2018 Printed in the USA, October 31, 2018 5994-0302EN

The aim of the desalting cartridge is not to perform protein separations, for example, Lc and Hc. Nevertheless, when combining the online AdvanceBio Desalting-RP cartridge with a 6560 ion mobility Q-TOF LC/MS, which incorporates an ion mobility (IM) drift cell into the TOF analyzer, separation of the Lc and Hc can be governed at a millisecond timescale (Figure 8). This puts fewer constraints on the maximum entropy algorithm, where there are overlapping Lc and Hc m/z values.

ConclusionThe AdvanceBio Desalting-RP cartridge has successfully been implemented in our daily routine to allow the MS measurement of challenging molecules such as intact and fragmented mAbs and ADCs.

AcknowledgementsThe authors acknowledge Maryann Shen, Ning Tang, and David Wong (Agilent Technologies, Inc., Santa Clara, CA, USA).

References1. Fenn, J. B., et al. Science 1989, 246,

64-71.

2. Kim, M. T.; et al. Bioconjug. Chem. 2014, 25, 1223-1232.

1,000 1,500 2,000 2,50025

30

35

40

10+11+12+

13+ 9+

15+14+

12+

13+

16+17+

18+

40+38+

36+

42+44+

Heavy chain Light chain 1

Light chain 2

1,950 1,952 1,954 1,956 1,958 1,96025

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35

40

1,290 1,295 1,300 1,305 1,310 1,315

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Light chain 1 (18+)

G0G0F

G1FG2F

Light chain 2 (12+)

Light chain 1 (12+)

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2.0

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3.5

4.01,954.3517

2,131.92991,466.0237

1,303.1962 1,563.69031,804.0944

2,345.0023

2,605.4370

1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000 2,100 2,200 2,300 2,400 2,500 2,600 2,700

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Mass-to-charge (m/z)

Mass-to-charge (m/z) Mass-to-charge (m/z)

Mass-to-charge (m/z)

Dri

ft t

ime

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s)

Dri

ft t

ime

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Dri

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C

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Figure 8. Raw MS spectrum (A) and IM-Q-TOF MS profile (B) of Herceptin Lc and Hc simultaneously desalted on the AdvanceBio Desalting-RP cartridge. Zoomed views are shown on the right. Two Lc conformations with identical m/z and molecular weight are also highlighted. Data were acquired on an Agilent 6560 ion mobility Q-TOF LC/MS.