Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MSE), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MSE peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.

[1]  C. Damen,et al.  Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab , 2009, Journal of the American Society for Mass Spectrometry.

[2]  A. Lim,et al.  Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals. , 2007, Mass spectrometry reviews.

[3]  R. Jefferis,et al.  Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. , 2009, Trends in pharmacological sciences.

[4]  S. Misbah,et al.  Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. , 2008, The New England journal of medicine.

[5]  J. Gebler,et al.  A rapid sample preparation method for mass spectrometric characterization of N-linked glycans. , 2005, Rapid communications in mass spectrometry : RCM.

[6]  J. B. Bogardus,et al.  Chemical and Physical Stability of Chimeric L6, a Mouse−Human Monoclonal Antibody , 1994, Pharmaceutical Research.

[7]  Daniel J. Kroon,et al.  Identification of Sites of Degradation in a Therapeutic Monoclonal Antibody by Peptide Mapping , 1992, Pharmaceutical Research.

[8]  Liang-Yu Shih,et al.  An improved trypsin digestion method minimizes digestion-induced modifications on proteins. , 2009, Analytical biochemistry.

[9]  J. Reichert,et al.  European Medicines Agency workshop on biosimilar monoclonal antibodies , 2009, mAbs.

[10]  H. Jörnvall.  Differences between Alcohol Dehydeogenases , 1977 .

[11]  Douglas S Rehder,et al.  Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. , 2005, Analytical chemistry.

[12]  M. Gorenstein,et al.  Absolute Quantification of Proteins by LCMSE , 2006, Molecular & Cellular Proteomics.

[13]  J. Gebler,et al.  Characterization of protein impurities and site-specific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry. , 2009, Analytical chemistry.

[14]  P. Rudd,et al.  Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent. , 2010, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[15]  J. Bennetzen,et al.  The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase. , 1982, The Journal of biological chemistry.

[16]  Roy Jefferis,et al.  Glycosylation as a strategy to improve antibody-based therapeutics , 2009, Nature Reviews Drug Discovery.

[17]  Alain Van Dorsselaer,et al.  Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells. , 2004, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[18]  Yow-Ming C Wang,et al.  Development of Biosimilars—Pharmacokinetic and Pharmacodynamic Considerations , 2009, Journal of biopharmaceutical statistics.

[19]  C. Wills,et al.  The two major isozymes of yeast alcohol dehydrogenase. , 1979, European journal of biochemistry.

[20]  Janice M Reichert,et al.  Monoclonal antibodies as innovative therapeutics. , 2008, Current pharmaceutical biotechnology.

[21]  Bethan Hughes.  Gearing up for follow-on biologics , 2009, Nature reviews. Drug discovery.

[22]  T. Wurch,et al.  Editorial [Hot Topic: Therapeutic Antibodies and Derivatives: From the Bench to the Clinic(Guest Editor: Alain Beck)] , 2008 .

[23]  A. Chakraborty,et al.  Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies. , 2007, Rapid communications in mass spectrometry : RCM.

[24]  S. Clarke,et al.  Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: Effects of the solvent dielectric , 1993, Protein science : a publication of the Protein Society.

[25]  T. Wurch,et al.  Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. , 2008, Current pharmaceutical biotechnology.

[26]  T. Wurch,et al.  Therapeutic antibodies and derivatives: from the bench to the clinic. , 2008, Current pharmaceutical biotechnology.

[27]  P. Rutgeerts,et al.  IgG1 heavy chain-coding gene polymorphism (G1m allotypes) and development of antibodies-to-infliximab , 2009, Pharmacogenetics and genomics.

[28]  J. Ravetch,et al.  Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation , 2006, Science.

[29]  Hongcheng Liu,et al.  Method to differentiate asn deamidation that occurred prior to and during sample preparation of a monoclonal antibody. , 2008, Analytical chemistry.

[30]  P. Bondarenko,et al.  Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs , 2008, Journal of the American Society for Mass Spectrometry.

[31]  R. Borchardt,et al.  Stability of Protein Pharmaceuticals , 1989, Pharmaceutical Research.

[32]  D. Brems,et al.  The effects of alpha‐helix on the stability of Asn residues: Deamidation rates in peptides of varying helicity , 2008, Protein science : a publication of the Protein Society.

[33]  B. Kabakoff,et al.  Identification of multiple sources of charge heterogeneity in a recombinant antibody. , 2001, Journal of chromatography. B, Biomedical sciences and applications.

[34]  Alain Beck,et al.  Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. , 2009, Analytical biochemistry.

[35]  J. Vliegenthart,et al.  Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas. , 1989, Biochemistry.

[36]  W. Ens,et al.  Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis. , 2006, Analytical chemistry.

[37]  Quynh-Thu Le,et al.  Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. , 2008, The New England journal of medicine.

[38]  M. Gorenstein,et al.  The detection, correlation, and comparison of peptide precursor and product ions from data independent LC‐MS with data dependant LC‐MS/MS , 2009, Proteomics.

[39]  M. Gorenstein,et al.  Quantitative proteomic analysis by accurate mass retention time pairs. , 2005, Analytical chemistry.

[40]  E. Waltz.  Western biotechs ponder follow-on possibilities , 2008, Nature Biotechnology.

[41]  N. Robinson,et al.  Prediction of primary structure deamidation rates of asparaginyl and glutaminyl peptides through steric and catalytic effects. , 2004, The journal of peptide research : official journal of the American Peptide Society.

[42]  T. Griffin,et al.  Trade-off between high sensitivity and increased potential for false positive peptide sequence matches using a two-dimensional linear ion trap for tandem mass spectrometry-based proteomics. , 2006, Journal of proteome research.