Biopharma

MALDI-Tof MS employing internal stable isotope labeled glycan standards is genuinely suited for clone selection and process development where highthroughput glycoprofiling is required. Our fast sample preparation and data acquisition routines permit the analysis of hundreds of samples with unparalleled speed.

Comparability studies of biosimilars reveal that products differ widely in composition and not always meet self-declared specifications. Glycosylation profile can exhibit batch-to-batch variation affecting the activity of a recombinant protein directly, and products that are similar from a qualitative perspective often differ quantitatively in the glycosylation profile, i.e. the clones are undergalactosylated.
Customized mAbsolute kits allow the absolute quantification of  the glycans attached to a given specific biosimilar in a few hours.

Most eukaryotic proteins, both membrane bound and soluble, and the large majority of commercial recombinant therapeutic proteins are modified with N-glycans that can have a major impact on protein solubility, structure, immunogenicity, circulatory half-life, and consequently drug efficacy.

Isotopic dilution mass spectrometry has been the golden standard for absolute metabolite quantification in newborn screening, quantification of immunosuppressor levels, in screening for illicit drugs, and in various approaches for quantitative proteomics. An application of isotopic dilution to glycan analysis, however, has been hampered largely by a lack of heavy isotope labeled glycan standards.

Changes in protein glycosylation are also a hallmark of many cancers, infectious and autoimmune diseases, and the growing number of congenital disorders of glycosylation (CDG)4 suggesting an increasingly important role of glycans as biomarkers.

Consequently, robust and quantitative methods for the analysis of glycans are not only required for mapping glycan structure to function but also highly relevant in biopharmaceutical quality control and in the development of glycans as selective and complementary disease markers.

Relative Quantification methods

Many profiling methods require the enzymatic or chemical release of the glycans from the peptide backbone. The resulting mixture of glycans can then be chemo-selectively derivatized with a fluorescent label8 like 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), or 9-aminopyrene-1,4,6-trisulfonic acid (APTS), separated by HPLC or capillary electrophoresis and analyzed by fluorescence and/or mass spectrometry detection. Alternatively, glycans can be profiled, often after permethylation, directly by mass spectrometry although isobaric structures remain unresolved, unless diagnostic fragment ions can be produced by tandem MS. While the chromatographic methods are sensitive and provide relative quantification of glycans via uniform labeling, they are more time-consuming, expensive, and in general more prone to error due to additional sample preparation steps. More importantly, current methods only provide relative but no absolute quantification of individual glycans, e.g., for diagnostic applications or quantification of immunogenic glycan levels. While the relative quantification of glycans is thought to be sufficient to track changes in glycosylation between samples in many biopharmaceutical applications, a clinical use of glycans as disease markers that goes beyond glycan ratios would require methods that measure absolute concentrations of individual glycans.

A new Absolute Quantification method

Isotopic dilution mass spectrometry has been the golden standard for absolute metabolite quantification in newborn screening, quantification of immunosuppressor levels, in screening for illicit drugs, and in various approaches for quantitative proteomics. An application of isotopic dilution to glycan analysis, however, has been hampered largely by a lack of heavy isotope labeled glycan standards.

Monoclonal Antibody N-glycan Characterization Kit

Human IgG N-glycan Absolute Quantification

Glycans are detached from the glycosylated peptides using PNGase F (CarboClip) and occupancy is determined by comparing the abundance of deamidated asparagine against the intact asparagine (non glycosylated)

Qualitative and quantitative determination of the amino acid composition of antibodies, proteins, peptides and others for research and pharmaceutical applications

  • Clinical research in Universities and veterinary labs.
  • Quality Control and compositional analysis in biosciences and pharmaceutical industries.

Measurement of absolute heat capacities of biopolymers in dilute solution

  • Biopolymer Solution Conformation & Solvation (Absolute heat capacities)
  • Biopolymer Stability (Protein denaturation)
  • Biopolymer Structure (Domain organization)
  • Bioengineering (Mutant proteins)
  • Ligand Interactions (Drug binding to proteins or nucleic acids)
  • Membrane Structure (Lipid Bilayers, membrane proteins)
  • Polynucleotides (Helix to coil transitions).

MALDI-TOF MS based glycan structure identification, including α-2,3/ α-2,6 differential sialylation following sialic acid stabilization procedure and using CarboQuant glycan standards

N-acetyl and N-glycolyl neuraminic acid (NANA and NGNA) quantification by RPLC, using DMB labeling.

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Glycomics

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.

CarboQuant Glycan Standards are patented synthetic 13C-labeled N-glycans, produced entirely in our laboratory for use as internal standards in absolute glycan quantification by mass spectrometry. They are available as single standards or in kit form including a quantification software and all reagents and consumables required for glycan analysis.

Other applications in the field of glycobiology research and proteomics:

  • Increase reproducibility in lab-to-lab method transfer (internal calibration standard)
  • De-convolute and quantify co-eluting peaks in LC-MS
  • Quantify glycan recovery after sample preparation

CarboClip is a recombinant peptide-N-Glycosidase F (PNGase F), from Elizabethkingia sp, overexpressed in E. coli that contains a hexahistidine tag in its C-term and cleaves between the GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. CarboClip is provided glycerol-free as a lyophilized powder containing Tris-HCl, NaCl2, and Na2EDTA.

Activity: After reconstitution in 150 μL of UltraPure H2O, 1 μl of CarboClip can deglycosylate > 95% of ribonuclease B (1 mg) in less than 20 minutes. Activity is calculated on a 12 % SDS-PAGE and percent of deglycosylation using densitometry.

Storage: Lyophilized powder: -80 °C and -20 °C >1 year. At room temperature 10 days. Reconstituted protein should be stored at 4 °C and is stable for 2-3 months

Characterization of glycosylation in proteins, antibodies, hormones, and other glycoconjugates using lectin microarray

Glycans are detached from the glycosylated protein using PNGase F (CarboClip) and occupancy is determined by comparing the abundance of deamidated asparagine against the one containing aspartic acid (non glycosylated)

Quick determination of the glycosylation profile of a expressed recombinant protein depending on the heterologous host. Correlation of the differences in glycosylation and protein function

MALDI-TOF MS based glycan structure identification, including α-2,3/ α-2,6 differential sialylation following a sialic acid stabilization procedure and using CarboQuant glycan standards

N-acetyl and N-glycolyl neuraminic acid (NANA and NGNA) quantification by RPLC, using DMB labeling.

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Proteomics

Qualitative and quantitative determination of the amino acid composition of antibodies, proteins, peptides and others for research and pharmaceutical applications

  • Clinical research in Universities and veterinary labs.
  • Quality Control and compositional analysis in biosciences and pharmaceutical industries.

Measurement of absolute heat capacities of biopolymers in dilute solution

  • Biopolymer Solution Conformation & Solvation (Absolute heat capacities)
  • Biopolymer Stability (Protein denaturation)
  • Biopolymer Structure (Domain organization)
  • Bioengineering (Mutant proteins)
  • Ligand Interactions (Drug binding to proteins or nucleic acids)
  • Membrane Structure (Lipid Bilayers, membrane proteins)
  • Polynucleotides (Helix to coil transitions).

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics

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