Performing native mass spectrometry analysis on therapeutic antibodies.

Since the introduction of "soft" ionization techniques, the role of mass spectrometry (MS) in the field of structural biology has increasingly expanded. With the incorporation of volatile buffers as electrospray ionization (ESI) solvents, non-covalent protein complexes could be efficiently transferred to the gas phase for mass analysis. While native MS has not become a technique used for standard characterization of therapeutic proteins in an industrial setting, it is increasingly used to probe the structural heterogeneity of these complex biomolecules. Here, we describe a detailed sample protocol for the analysis of monoclonal antibodies (mAbs) by native MS and highlight some recent applications of native MS in the analysis of intact mAbs and mAb-based therapeutics.

[1]  A. Pavlou,et al.  Recombinant protein therapeutics—success rates, market trends and values to 2010 , 2004, Nature Biotechnology.

[2]  N. Packer,et al.  Structural analysis of N- and O-glycans released from glycoproteins , 2012, Nature Protocols.

[3]  P. Parren,et al.  Quantitative analysis of the interaction strength and dynamics of human IgG4 half molecules by native mass spectrometry. , 2011, Structure.

[4]  S. Sanglier-Cianférani,et al.  Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS. , 2009, Analytical chemistry.

[5]  A. Heck,et al.  Optimizing macromolecular tandem mass spectrometry of large non-covalent complexes using heavy collision gases , 2007 .

[6]  J. Mora,et al.  Electrospray ionization of large multiply charged species proceeds via Dole’s charged residue mechanism , 2000 .

[7]  Alain Van Dorsselaer,et al.  Characterization of therapeutic antibodies and related products. , 2013, Analytical chemistry.

[8]  Joseph A. Loo,et al.  Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes , 2000 .

[9]  C. Robinson,et al.  The role of mass spectrometry in structure elucidation of dynamic protein complexes. , 2007, Annual review of biochemistry.

[10]  L. Ilag,et al.  Discrimination among IgG1-κ monoclonal antibodies produced by two cell lines using charge state distributions in nanoESI-TOF mass spectra , 2009, Journal of the American Society for Mass Spectrometry.

[11]  P. Schnier,et al.  Resolving disulfide structural isoforms of IgG2 monoclonal antibodies by ion mobility mass spectrometry. , 2010, Analytical chemistry.

[12]  Albert J R Heck,et al.  Ion mobility mass spectrometry of proteins and protein assemblies. , 2010, Chemical Society reviews.

[13]  C. Robinson,et al.  A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. , 2002, Analytical chemistry.

[14]  A. Heck,et al.  The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. , 2001, Rapid communications in mass spectrometry : RCM.

[15]  P. Parren,et al.  Species-Specific Determinants in the IgG CH3 Domain Enable Fab-Arm Exchange by Affecting the Noncovalent CH3–CH3 Interaction Strength , 2011, The Journal of Immunology.

[16]  M. Karas,et al.  Matrix-assisted ultraviolet laser desorption of non-volatile compounds , 1987 .

[17]  Pauline M Rudd,et al.  A systematic approach to protein glycosylation analysis: a path through the maze. , 2010, Nature chemical biology.

[18]  S. Berkowitz,et al.  Conformation and dynamics of biopharmaceuticals: Transition of mass spectrometry-based tools from academe to industry , 2010, Journal of the American Society for Mass Spectrometry.

[19]  John P. Gabrielson,et al.  In vitro stoichiometry of complexes between the soluble RANK ligand and the monoclonal antibody denosumab. , 2012, Biochemistry.

[20]  C. Robinson,et al.  Regional and segmental flexibility of antibodies in interaction with antigens of different size , 2006, The FEBS journal.

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

[22]  N. Leymarie,et al.  Effective use of mass spectrometry for glycan and glycopeptide structural analysis. , 2012, Analytical chemistry.

[23]  I. Chernushevich,et al.  Collisional cooling of large ions in electrospray mass spectrometry. , 2004, Analytical chemistry.

[24]  Natalie J. Thompson,et al.  Exploring an orbitrap analyzer for the characterization of intact antibodies by native mass spectrometry. , 2012, Angewandte Chemie.

[25]  Igor A Kaltashov,et al.  Advances and challenges in analytical characterization of biotechnology products: mass spectrometry-based approaches to study properties and behavior of protein therapeutics. , 2012, Biotechnology advances.

[26]  J. Tibbitts,et al.  Pharmacokinetic Considerations for Antibody Drug Conjugates , 2012, Pharmaceutical Research.

[27]  C. Robinson,et al.  Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. , 2001, Biophysical journal.

[28]  A. Ittner,et al.  Tau‐targeted treatment strategies in Alzheimer's disease , 2012, British journal of pharmacology.

[29]  A. Heck.  Native mass spectrometry: a bridge between interactomics and structural biology , 2008, Nature Methods.

[30]  A. Makarov,et al.  Mass spectrometry of stanozolol and its analogues using electrospray ionization and collision-induced dissociation with quadrupole-linear ion trap and linear ion trap-orbitrap hybrid mass analyzers. , 2005, Rapid communications in mass spectrometry : RCM.

[31]  Damian Houde,et al.  Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding* , 2010, Molecular & Cellular Proteomics.

[32]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[33]  Zhongqi Zhang,et al.  Mass spectrometry for structural characterization of therapeutic antibodies. , 2009, Mass spectrometry reviews.

[34]  Albert J R Heck,et al.  Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. , 2006, Analytical chemistry.

[35]  C. Robinson,et al.  Effects of antibody affinity and antigen valence on molecular forms of immune complexes. , 2009, Molecular immunology.

[36]  A. Heck,et al.  Native protein mass spectrometry: from intact oligomers to functional machineries. , 2004, Current opinion in chemical biology.

[37]  W. Hancock,et al.  Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation. , 2011, Analytical chemistry.

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

[39]  Natalie J. Thompson,et al.  The impact of mass spectrometry on the study of intact antibodies: from post-translational modifications to structural analysis. , 2013, Chemical communications.

[40]  J. Wolchok,et al.  Antibody therapy of cancer , 2012, Nature Reviews Cancer.

[41]  P. Parren,et al.  Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG , 2013, mAbs.

[42]  C. Robinson,et al.  Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. , 2010, Analytical chemistry.

[43]  M. Karas,et al.  Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS. , 2001, Analytical chemistry.

[44]  Christian Bailly,et al.  Strategies and challenges for the next generation of therapeutic antibodies , 2010, Nature Reviews Immunology.

[45]  Aditya A Wakankar,et al.  Analytical methods for physicochemical characterization of antibody drug conjugates , 2011, mAbs.

[46]  Albert J R Heck,et al.  High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies , 2012, Nature Methods.

[47]  P. Kebarle,et al.  Electrospray: from ions in solution to ions in the gas phase, what we know now. , 2009, Mass spectrometry reviews.

[48]  M. Wilm,et al.  Analytical properties of the nanoelectrospray ion source. , 1996, Analytical chemistry.

[49]  R. Aalberse,et al.  IgG4 breaking the rules , 2002, Immunology.

[50]  Natalie J. Thompson,et al.  Qualitative and semiquantitative analysis of composite mixtures of antibodies by native mass spectrometry. , 2012, Analytical chemistry.

[51]  Ton Logtenberg,et al.  Antibody cocktails: next-generation biopharmaceuticals with improved potency. , 2007, Trends in biotechnology.

[52]  Etienne Weiss,et al.  Therapeutic antibodies: successes, limitations and hopes for the future , 2009, British journal of pharmacology.

[53]  P. Parren,et al.  Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo , 2009, Nature Biotechnology.

[54]  A. Heck,et al.  Modern Biomolecular Mass Spectrometry and its Role in Studying Virus Structure, Dynamics, and Assembly , 2011, Angewandte Chemie.

[55]  John F. Valliere-Douglass,et al.  Native intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues. , 2012, Analytical chemistry.

[56]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.