Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains. 2005

Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
Department of Analytical Biochemistry, MedImmune, Inc., One MedImmune Way, Gaithersburg, Maryland 20878, USA. tousg@medimmune.com

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.

UI MeSH Term Description Entries
D007074 Immunoglobulin G The major immunoglobulin isotype class in normal human serum. There are several isotype subclasses of IgG, for example, IgG1, IgG2A, and IgG2B. Gamma Globulin, 7S,IgG,IgG Antibody,Allerglobuline,IgG(T),IgG1,IgG2,IgG2A,IgG2B,IgG3,IgG4,Immunoglobulin GT,Polyglobin,7S Gamma Globulin,Antibody, IgG,GT, Immunoglobulin
D007140 Immunoglobulin Fab Fragments Univalent antigen-binding fragments composed of one entire IMMUNOGLOBULIN LIGHT CHAIN and the amino terminal end of one of the IMMUNOGLOBULIN HEAVY CHAINS from the hinge region, linked to each other by disulfide bonds. Fab contains the IMMUNOGLOBULIN VARIABLE REGIONS, which are part of the antigen-binding site, and the first IMMUNOGLOBULIN CONSTANT REGIONS. This fragment can be obtained by digestion of immunoglobulins with the proteolytic enzyme PAPAIN. Fab Fragment,Fab Fragments,Ig Fab Fragments,Immunoglobulins, Fab Fragment,Fab Immunoglobulin Fragments,Immunoglobulin Fab Fragment,Immunoglobulins, Fab,Fab Fragment Immunoglobulins,Fab Fragment, Immunoglobulin,Fab Fragments, Immunoglobulin,Fragment Immunoglobulins, Fab,Fragment, Fab,Immunoglobulin Fragments, Fab
D007143 Immunoglobulin Heavy Chains The largest of polypeptide chains comprising immunoglobulins. They contain 450 to 600 amino acid residues per chain, and have molecular weights of 51-72 kDa. Immunoglobulins, Heavy-Chain,Heavy-Chain Immunoglobulins,Ig Heavy Chains,Immunoglobulin Heavy Chain,Immunoglobulin Heavy Chain Subgroup VH-I,Immunoglobulin Heavy Chain Subgroup VH-III,Heavy Chain Immunoglobulins,Heavy Chain, Immunoglobulin,Heavy Chains, Ig,Heavy Chains, Immunoglobulin,Immunoglobulin Heavy Chain Subgroup VH I,Immunoglobulin Heavy Chain Subgroup VH III,Immunoglobulins, Heavy Chain
D007147 Immunoglobulin Light Chains Polypeptide chains, consisting of 211 to 217 amino acid residues and having a molecular weight of approximately 22 kDa. There are two major types of light chains, kappa and lambda. Two Ig light chains and two Ig heavy chains (IMMUNOGLOBULIN HEAVY CHAINS) make one immunoglobulin molecule. Ig Light Chains,Immunoglobulins, Light-Chain,Immunoglobulin Light Chain,Immunoglobulin Light-Chain,Light-Chain Immunoglobulins,Chains, Ig Light,Chains, Immunoglobulin Light,Immunoglobulins, Light Chain,Light Chain Immunoglobulins,Light Chain, Immunoglobulin,Light Chains, Ig,Light Chains, Immunoglobulin,Light-Chain, Immunoglobulin
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D010449 Peptide Mapping Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases. Fingerprints, Peptide,Peptide Fingerprinting,Protein Fingerprinting,Fingerprints, Protein,Fingerprint, Peptide,Fingerprint, Protein,Fingerprinting, Peptide,Fingerprinting, Protein,Mapping, Peptide,Peptide Fingerprint,Peptide Fingerprints,Protein Fingerprint,Protein Fingerprints
D003432 Cross-Linking Reagents Reagents with two reactive groups, usually at opposite ends of the molecule, that are capable of reacting with and thereby forming bridges between side chains of amino acids in proteins; the locations of naturally reactive areas within proteins can thereby be identified; may also be used for other macromolecules, like glycoproteins, nucleic acids, or other. Bifunctional Reagent,Bifunctional Reagents,Cross Linking Reagent,Crosslinking Reagent,Cross Linking Reagents,Crosslinking Reagents,Linking Reagent, Cross,Linking Reagents, Cross,Reagent, Bifunctional,Reagent, Cross Linking,Reagent, Crosslinking,Reagents, Bifunctional,Reagents, Cross Linking,Reagents, Cross-Linking,Reagents, Crosslinking
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein

Related Publications

Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
December 1987, Hybridoma,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
May 2004, Journal of biochemistry and molecular biology,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
October 1992, Hybridoma,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
April 2011, Journal of chromatography. A,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
September 1981, Proceedings of the National Academy of Sciences of the United States of America,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
April 1989, American journal of veterinary research,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
January 1977, Methods in enzymology,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
April 1982, Molecular immunology,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
January 1979, Journal of supramolecular structure,
Guillermo I Tous, and Ziping Wei, and Jinhua Feng, and Susanna Bilbulian, and Stephen Bowen, and Jaime Smith, and Robert Strouse, and Patrick McGeehan, and Jose Casas-Finet, and Mark A Schenerman
December 2006, Hybridoma (2005),
Copied contents to your clipboard!