Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

[1]  Wei Zhang,et al.  Free Sulfhydryl in Recombinant Monoclonal Antibodies , 2002, Biotechnology progress.

[2]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[3]  Hongcheng Liu,et al.  Localization and quantitation of free sulfhydryl in recombinant monoclonal antibodies by differential labeling with 12C and 13C iodoacetic acid and LC-MS analysis. , 2009, Analytical chemistry.

[4]  V. Katta,et al.  Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry. , 1991, Rapid communications in mass spectrometry : RCM.

[5]  T. Rejtar,et al.  Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry. , 2010, Rapid communications in mass spectrometry : RCM.

[6]  G. Nakamura,et al.  Isolation and characterization of the B-cell marker CD20. , 2005, Biochemistry.

[7]  T. Dillon,et al.  Interchain disulfide bonding in human IgG2 antibodies probed by site-directed mutagenesis. , 2009, Biochemistry.

[8]  Ulf Göransson,et al.  Disulfide Mapping of the Cyclotide Kalata B1 , 2003, Journal of Biological Chemistry.

[9]  N. M. Gevondyan,et al.  Four free cysteine residues found in human IgG1 of healthy donors , 2006, Biochemistry (Moscow).

[10]  Paul Polakis,et al.  Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index , 2008, Nature Biotechnology.

[11]  C. F. Brewer,et al.  Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins☆ , 1967 .

[12]  Reed J. Harris,et al.  Effect of Copper Sulfate on Performance of a Serum‐Free CHO Cell Culture Process and the Level of Free Thiol in the Recombinant Antibody Expressed , 2008, Biotechnology progress.

[13]  M A Smith,et al.  Specific cleavage of immunoglobulin G by copper ions. , 2009, International journal of peptide and protein research.

[14]  G. Sheldrick,et al.  X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain. , 1997, Folding & design.

[15]  Gerd R Kleemann,et al.  Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. , 2006, Journal of chromatography. A.

[16]  Gang Xiao,et al.  Reversed-phase liquid chromatography-mass spectrometry of site-specific chemical modifications in intact immunoglobulin molecules and their fragments. , 2008, Journal of chromatography. A.

[17]  Hongcheng Liu,et al.  Heterogeneity of monoclonal antibodies. , 2008, Journal of pharmaceutical sciences.

[18]  Wei Wang,et al.  Antibody structure, instability, and formulation. , 2007, Journal of pharmaceutical sciences.

[19]  Randal R Ketchem,et al.  Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype , 2008, Electrophoresis.

[20]  Gerd R Kleemann,et al.  Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis. , 2006, Analytical biochemistry.

[21]  Steven L. Cohen,et al.  Evidence for trisulfide bonds in a recombinant variant of a human IgG2 monoclonal antibody. , 2009, Analytical chemistry.

[22]  D. Ejima,et al.  Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies , 2006, Proteins.

[23]  Y. Hagihara,et al.  Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region* , 2007, Journal of Biological Chemistry.

[24]  D. Brems,et al.  Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column. , 2007, Journal of chromatography. A.

[25]  E. Lacy,et al.  Free sulfhydryl measurement as an indicator of antibody stability. , 2008, Analytical biochemistry.

[26]  Identification and localization of unpaired cysteine residues in monoclonal antibodies by fluorescence labeling and mass spectrometry. , 2009, Analytical chemistry.

[27]  M. Mulkerrin,et al.  Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion. , 1997, Journal of pharmaceutical and biomedical analysis.

[28]  Thomas M. Dillon,et al.  Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass* , 2008, Journal of Biological Chemistry.

[29]  Yanmei Lu,et al.  A high throughput electrochemiluminescent cell-binding assay for therapeutic anti-CD20 antibody selection. , 2006, Journal of immunological methods.

[30]  P. Schnier,et al.  Conformations and folding of lysozyme ions in vacuo. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Burgess.  Refolding solubilized inclusion body proteins. , 2009, Methods in enzymology.

[32]  A. Klibanov,et al.  Thermal destruction processes in proteins involving cystine residues. , 1987, The Journal of biological chemistry.

[33]  P. Parren,et al.  Anti-Inflammatory Activity of Human IgG4 Antibodies by Dynamic Fab Arm Exchange , 2007, Science.

[34]  Alain Balland,et al.  Disulfide connectivity of human immunoglobulin G2 structural isoforms. , 2008, Biochemistry.

[35]  Ziping Wei,et al.  Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains. , 2005, Analytical chemistry.

[36]  O. Blumenfeld,et al.  Reactions of N-ethylmaleimide with peptides and amino acids. , 1964, The Biochemical journal.

[37]  Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth. , 2001, Journal of chromatography. A.

[38]  D. Ouellette,et al.  Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule. , 2010, Analytical biochemistry.