Antibody Characterization

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).

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

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.

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)

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.

""
1
Name
Second name
Company /Affiliation
Message
0 /
Previous
Next
Email this to someoneTweet about this on TwitterShare on LinkedIn