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Characterization Quality Control

Characterization of Monoclonal Antibodies LCMS-9030

Molecular Weight Analysis of Monoclonal Antibodies Using benefits

the LCMS-9030 Quadrupole Time-of-Flight Mass Spectrometer Cell Line Optimization
click here • Trace quantities of impurities contained in pharmaceutical ingredients can be
measured with high sensitivity.
Operating Principle and Features Results
• Accurate mass information can be used to identify impurities in products.
The LCMS-9030 is a Q-TOF mass spectrometer that includes both Fig. 1 shows the TIC chromatogram, deconvoluted MS spectrum, and • Deconvoluted spectra can be used to check the molecular weights in antibody drugs.
quadrupole and time-of-flight separation systems, two types of the MS spectrum measured from the intact mAb. A comparison of the
mechanisms for separating ions. The system includes unique Shimadzu mass values measured from the intact mAb to theoretical mass values Culture
technologies in a variety of locations for acquiring data with both high confirmed that measured values were consistent with theoretical values
sensitivity and high resolution, while also ensuring mass accuracy is to a precision level of 25 ppm or less (Table 2). Protein Metrics Intact
always stable. For example, it includes technology for increasing the ion Mass Workflow software was used for the deconvolution of intact
usage rate, machining technologies for manufacturing powerful and mAb. The TIC chromatogram, MS deconvoluted spectrum, and MS
finely detailed grating electrodes, technology for precision temperature spectrum were also similarly analyzed for mAb subunit H and L-chains.
control, and technology for optimizing the distribution of electric Results from checking their molecular weights provided good values.
potential values.
Table 2 Comparison of Measured and Theoretical Mass Values
Analytical Conditions Sample name NIST mAb
Peak#
1 Purification
Human IgG1K monoclonal antibodies (mAb) were dissolved in 50 Name Expected mass Mass
mmol/L aqueous ammonium hydrogen carbonate solution to prepare a G2F/G2F+Hex 148848 148850 1.8
1 mg/mL standard solution (intact mAb). mAb subunits were prepared G2F/G2F 148686 148688 1.9
148652
148653
G1F/G2F+Lys
1.6
by adding 8 mol/L urea and a 50 mmol/L Tris-HCl buffer solution G1F/G2F 148524 148525 1.0
containing 50 mmol/L DL-dithiothreitol to 100 μg of the intact mAb to G1F/G1F+Lys 148490 148489 -0.9
reduce the antibodies to H and L-chains. Measurement conditions for G1F/G1F 148362 148362 0.7
the intact mAb and mAb subunits are indicated in Table 1. G0F/G1F-GluNAc 147996 148000 3.5
G0F/G1F+Lys 148328 148326 -1.4
Table 1 Analytical Conditions G0F/G1F 148199 148200 0.7
[LC] G0F/G0F-GlcNAc 147834 147837 3.2
Column: Restek C4 G0F/G0F-2GlcNAc 147631 147631 0.0 Characterization
(150 mm × 2.1 mm I.D., 5 µm)
G0F/G0F+Lys 148165 148164 -1.5 Specifications
Column oven: 50 °C (Intact), 85 °C (subunits)
G0F/G0F 148037 148039 2.0
Solvent A: 0.1 ％ formic acid/water
Instrument LCMS-9030
Solvent B: 0.1 % formic acid/acetonitrile
Intact Gradient: 0 % (0.5 min) → 5 % (3.0 min) → Mass range Quadrupole mass range: m/z 10 to 2,000
(Conc. B) → 60 % (5.5 – 5.6 min) → 5 % (10 min)
Subunits Gradient: 0 % (0.5 min) → 15 % (1.5 min) → TOF mass range: m/z 10 to 40,000
(Conc. B) → 30 % (2.5 – 3.5 min) →
→ 45 % (7.5 – 8.5 min) → Sensitivity ESI positive 1 pg reserpine
→ 50 % (10.5 – 10.6 min) →
→ 15 % (15 min) S/N > 1,000:1 (RMS) in MS mode
Flowrate: 0.4 mL/min S/N > 10,000:1 (RMS) in MS/MS mode Quality Control
[Q-TOF]
ESI negative 1 pg chloramphenicol
Mode: MS mode
Intact Subunits S/N > 1,000:1 (RMS) in MS mode
TOF Start m/z: 1000.0000 800.0000 TIC Chromatogram Deconvoluted Spectrum
TOF End m/z: 4000.0000 4000.0000 S/N > 10,000:1 (RMS) in MS/MS mode
Event Time (s): 5.0 2.0 Resolution (Quadrupole) R < 0.8 u FWHM
Pulser Inj. Times: 9993 3993
Interface: 300 °C Resolution (TOF) ESI positive 30,000 FWHM at m/z 1,972
Nebulizer gas: 3 L/min
ESI negative 30,000 FWHM at m/z 1,626
Drying gas: 10 L/min
Heating gas: 10 L/min Mass accuracy MS mode < 1 ppm (peak to peak) at m/z 622.5662, Pharmacokinetics
Mass Spectrum NaI cluster (internal calibration)
Fig. 1 NIST mAb Intact Results MS/MS mode < 2 ppm (peak to peak) at m/z 1072.2489 > 472.6719
NaI cluster (external calibration)
Conclusion Application Examples Mass accuracy temperature stability 1 ppm/24 h, 18 to 28 °C at constant temperature
Maximum acquisition rate 100 Hz
Using the LCMS-9030 Q-TOF mass spectrometer with Protein Metrics • Quantitative analysis of pharmaceuticals Polarity switching time 1 sec Others
software enables evaluation of molecular weights in biopharmaceuticals • Identification of impurities
with high sensitivity and high resolution to achieve consistently high • Peptide mapping Interface Standard: ESI
precision. Optional: APCI, DUIS, CDS, Nano-ESI

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