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Characterization Quality Control

Glycan Analysis MALDI-8020

Glycosylation Profile of IgGs Using a Linear Benchtop benefits

MALDI-TOF MS and Affinity Purification of Fc Cell Line Optimization
click here • Enables rapid benchtop glycan profiling with minimal pretreatment.
• Easy maintenance and low running costs
Operating Principle and Features Results
• Ideal for analyzing nucleic acids, proteins, and even polymers.
The MALDI-8020 is a linear-mode MALDI-TOF mass spectrometer with In the mass spectrum of IgG full-length, the IgG molecular weight was
a small installation footprint. It is typically used for quality control or observed near 150 kDa (Fig. 1). Due to inadequate mass resolution
profiling applications for peptides, proteins, polymer or oligonucleic in the m/z range for large molecular weights, the mass spectrum for Culture
acids, and other substances. Despite the benchtop design, the ion about 25 kDa of the Fc region modified by a glycan was analyzed (Fig.
optical system features a large diameter inlet to ensure proper system 2). The mass gap between peaks in each spectrum indicates one sugar
performance levels are maintained for long periods and to reduce the unit. Furthermore, MS measurements of each Fc region were repeated
risk of ion source contamination. The UV laser-based rapid automatic three times and statistically analyzed using eMSTAT Solution. A score
ion source cleaning function (TrueClean) can clean the ion extractor plot was obtained easily by simply making some minimal adjustments
electrode without releasing the vacuum pressure. The system can to a few parameters (Fig. 3).
manage all data and other information in one location and includes a
156695.2
tool for assisting with strict compliance with FDA 21 CFR Part 11.
Mouse myeloma
Measurement Method 147269.0 Purification

Especially for biopharmaceutical development applications, high- Mouse serum
end mass spectrometers are commonly used due to the extreme 100 147795.6
importance of evaluating the properties of N-/O-linkages. However, for % Intensity 80 60
batch analysis intended for screening or QA/QC applications, relatively 40 NIST mAb Specifications
20
inexpensive and user-friendly commercial systems are preferred. 0 100000 150000 m/z 200000 250000
The MALDI-8020 model fits this need, offering more than adequate Instrument MALDI-8020
specifications for general profiling, high throughput, and an excellent Fig. 1 Mass Spectrum of IgG Full-length Mass range m/z 1 to 500,000
value for the price. Sugar unit
The following describes an example of profiling IgG glycan 25363.9 Mass resolution >5,000 FWHM
modifications without releasing any glycans. Human IgG1K monoclonal Sensitivity >250 amol Characterization
25197.0
antibodies (NISTmAb), IgG from mouse serum, and myeloma IgG were 25548.2 Mouse myeloma
dissolved in a Tris-NaCl buffer solution and incubated together with 25552.8 Mass accuracy <20 ppm with internal calibration, <150 ppm with external calibration
IdeZ (IgG-degrading enzyme) for two hours at 37 °C. Then Protein A Acceleration voltage 15 kV
25384.8
magnetic beads were used to recover Fc sections. The Fc regions were Mouse serum
25907.1 Laser Solid-state laser wavelength: 355 nm Repetition frequency: 50, 100, or 200 Hz (variable)
eluted from the beads with an acidic solution and desalted with ZipTip 100 25340.1
C18 tips. Sinapic and ferulic acids were respectively dissolved in a 50 % % Intensity 80 60 25169.6 25512.0 Flight distance 850 mm
acetonitrile solution containing 0.1 % TFA to a final concentration of 40 25689.4 NIST mAb
20 25862.9 Detector Electron Multiplier
20 mg/mL. Then the resulting solutions were used as matrices for MS 0 23000 24000 25000 m/z 26000 27000 28000
analysis. Ion source cleaning Includes automatic cleaning functionality (depending on built-in solid-state laser)
Fig. 2 Mass Spectrum of Fc Region Quality Control
Sample plate Disposable FlexiMass-DS and stainless steel FlexiMass-SR
Mouse myeloma NIST mAb Mouse serum Operating noise <55 dB
A data set of NIST mAb
various Fc Main unit power supply Single-phase 100 to 240 V AC, 50/60 Hz, 1 kVA
Dimensions W 600 mm × D 745 mm × H 1,055 mm (excluding protrusions)
Mouse serum
Save peak lists Weight 86 kg
in each folder Mouse myeloma
Open “IgG” folder Operating environment Temperature: 18 to 28 °C Humidity: Max. 70 % (with no condensation)
in eMSTAT Solution
Data analysis software eMSTAT Solution
Data analysis Univariate analysis t-Test, Mann-Whitney U-Test, ANOVA (analysis of variance) Pharmacokinetics
Generation of Score Plot in a minute functionality
Multivariate analysis PCA (principal component analysis), PLS-DA
Fig. 3 Classification of Fc Region Using eMSTAT Solution
Discriminant analysis Support Vector Machine (SVM), Random Forest
Conclusion Application Examples Other Dynamic grouping
Display functionality Multivariate analysis Peak Matrix, Box Plot, ROC, AUC, Score/Loading Plot, Dendrogram
The MALDI-8020 provides ample MS resolution for recognizing three • Quality control of antibody drugs
Fc regions with different varieties of glycan modifications. In addition, • Synthesis confirmation of nucleic acid drugs Discriminant analysis Discriminant analysis results (Group, Score) superimpose points for unknown samples on a score plot Others
statistical analysis using eMSTAT Solution enabled quick classification of Input/output data Input Peak list (ASCII, JCAMP, or mzML format)
the three Fc types, which could be used for batch analysis, QA/QC, or
other applications. Output Peak list (txt format), data analysis results (xlsm format), graph screenshot

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