About mAbsolute Kits

The mAbsolute kits, are low- and high-throughput glycan analysis kits, based on highly pure synthetic isotope-labeled internal standards for the absolute quantification of mAb glycosylation by MALDI-TOF MS. Minimal handling and time are required for sample preparation and within seconds, all relevant glycans of a deglycosylated mAb sample are quantified by comparison of MALDI profiles with the corresponding internal reference standards.

mAbsolute kits have been optimized for the MALDI-TOF based absolute glycan quantification using Bruker Autoflex and UltrafleXtreme III equipments. Validation is on-going for the application of the kits to different MALDI-TOF instruments and to other mass spectrometry techniques.

Video shows how to proceed to analyze glycan content of mAb using mAbsolute Kit.

Each Kit Contains:

Quantified mixture of 8-11 13C-standards

Covering all major glycans present in therapeutic monoclonal  antibodies expressed in CHO cell lines. Complete kit also includes 2,3-sialylated standards. Also, a humanized mixture of standards has been developed covering also bisected-biantennary standards as well as 2,6-sialylated structures, for applications in human IgG glysosylation analysis, both Fc and Fab regions.

CarboClip PNGase F enzyme

Proprietary peptide N-glycosidase F (PNGase F). The glycan release from the proteic backbone is performed by CarboClip, an enzyme that cleaves the amide bond between the N-glycan and the asparagine residue in the protein. The enzyme included in the kit has been developed in-house and is provided as lyophilized powder to maintain a high deglycosylation activity.

Sialic acid stabilization procedure

Despite the mild ionization conditions of MALDI-TOF techniques, the sialylated N-glycans can be partially hydrolized during the laser induced-desorption step concerning the quantification of the sample. A simple derivatization step included in the sample preparation protocol avoids the desialylation and also helps distinguising the different 2,3- and 2,6-sialyl regioisomers, that can be present in a sample.

Proprietary Quantification Software

The MALDI-TOF based quantification method is compatible with a high-troughput performance, yielding a complex data set which can be treated easily with the quantification software provided with the kit. Despite it was developed for Bruker data formats, it is very simple to import data in general and common formats as .csv, analyzing tenths or hundreds of experiments automatically. The software generates also a customized report regarding absolute and relative quantification data.

Applications

in Glycobiology

  • Rapid Absolute glycan quantification
  • Measure of glycan recovery for in sample preparation
  • Internal standards for the quantitative glycan profiling by MALDI-Tof MS employing mAbsolute kits

Biopharmaceutical development

  • Rapid high-throughput glycan analysis in clone selection and process development
  • Quality control
  • Biosimilar optimisation

Clinical Chemistry Biomarkers

  • Measure glycan levels in serum samples with picomole precision
  • Establish cut-off values for glycan biomarkers
  • Quantify aberrant glycosylation in cancer samples
  • Internal performance calibrants for method transfer and multiple laboratory studies

References

1. Synthesis and use of isotopically-labelled glycans. EP 2981541 A1

2. Anal Chem. 2015 Nov 17;87(22):11460-7

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