Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Abstract

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 m urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3–9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of N-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion–ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides. Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 m urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3–9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of N-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion–ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides. Monoclonal antibodies (mAbs) 1The abbreviations used are:mAbmonoclonal antibodyMSmass spectrometryMS/MStandem mass spectrometryHPLChigh-performance liquid chromatographym/zmass-to-charge ratioETDelectron-transfer dissociationCADcollisionally activated dissociationIIPTion-ion proton transferTICtotal ion currentS/Nsignal-to-noise ratioCDRcomplementarity determining regionHCheavy chainLClight chainPTMpost-translational modificationG0Ffucosylated biantennary (-2 galactose) oligosaccharideG1Ffucosylated biantennary (-1 galactose) oligosaccharideG2Ffucosylated biantennary oligosaccharideNAEMN-(2-aminoethyl)maleimideIAMiodoacetamideTCEPtris(2-carboxyethyl) phosphineOMSSAOpen Mass Spectrometry Search Algorithm. and related biological molecules constitute one of the most rapidly growing classes of human therapeutics. These large proteins (Fig. 1) have molecular weights near 150 kDa and are composed of two identical ∼50 kDa heavy chains (HC) and two identical ∼25 kDa light chains (LC) (1.Beck A. Sanglier-Cianferani S. Van Dorsselaer A. Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry.Anal. Chem. 2012; 84: 4637-4646Crossref PubMed Scopus (212) Google Scholar). They also contain at least 16 disulfide bonds that maintain three-dimensional structure and biological activity (2.Liu H. May K. Disulfide bond structures of IgG molecules structural variations, chemical modifications and possible impacts to stability and biological function.Mabs. 2012; 4: 17-23Crossref PubMed Scopus (315) Google Scholar). Although sharing similar secondary protein structures, different mAbs differ greatly in the sequence of variable regions, especially in the complementarity determining regions (CDRs) which are responsible for the diversity and specificity of antibody-antigen binding. Changes to the mAb structure introduced during the manufacturing process or storage may influence the therapeutic efficacy, bio-availability and -clearance, and immunogenic properties and thus alter drug safety (3.Fernandez L. Kalume D. Calvo L. Mallo M. Vallin A. Roepstorff P. Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography.J. Chromatogr. B. 2001; 752: 247-261Crossref PubMed Scopus (32) Google Scholar, 4.Wright A. Morrison S.L. Effect of glycosylation on antibody function: implications for genetic engineering.Trends Biotechnol. 1997; 15: 26-32Abstract Full Text PDF PubMed Scopus (269) Google Scholar, 5.Roberts G.D. Johnson W.P. Burman S. Anumula K.R. Carr S.A. An integrated strategy for structural characterization of the protein and carbohydrate components of monoclonal-antibodies: application to anti-respiratory syncytial virus mab.Anal. Chem. 1995; 67: 3613-3625Crossref PubMed Scopus (88) Google Scholar). Comprehensive characterization of mAbs primary structure, post-translational modifications (PTMs), and disulfide linkages is critical to the evaluation of drug efficacy and safety, as well as understanding the structure/function relationships (4.Wright A. Morrison S.L. Effect of glycosylation on antibody function: implications for genetic engineering.Trends Biotechnol. 1997; 15: 26-32Abstract Full Text PDF PubMed Scopus (269) Google Scholar, 6.Zhang Z. Pan H. Chen X. Mass spectrometry for structural characterization of therapeutic antibodies.Mass Spectrom. Rev. 2009; 28: 147-176Crossref PubMed Scopus (263) Google Scholar). Presented in this work is a novel protein analytical platform that consists of innovative methods for mass spectrometry (MS) characterization of mAbs. The methodology reported here will have a dramatic impact on the whole field of antibody characterization. monoclonal antibody mass spectrometry tandem mass spectrometry high-performance liquid chromatography mass-to-charge ratio electron-transfer dissociation collisionally activated dissociation ion-ion proton transfer total ion current signal-to-noise ratio complementarity determining region heavy chain light chain post-translational modification fucosylated biantennary (-2 galactose) oligosaccharide fucosylated biantennary (-1 galactose) oligosaccharide fucosylated biantennary oligosaccharide N-(2-aminoethyl)maleimide iodoacetamide tris(2-carboxyethyl) phosphine Open Mass Spectrometry Search Algorithm. Typical MS characterization of proteins uses a “Bottom-Up” approach. This method involves tryptic digestion of the protein(s) into small peptides (mostly below 2500 Da) followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses of the resulting peptides (7.Bensimon A. Heck A.J.R. Aebersold R. Mass spectrometry-based proteomics and network biology.Annu. Rev. Biochem. 2012; 81: 379-405Crossref PubMed Scopus (317) Google Scholar). Although sensitive for MS analysis, small tryptic peptides often have issues such as weak retention in liquid chromatography, difficulties in assigning peptides to specific gene products, and loss of combinatorial PTM information (8.Lanucara F. Eyers C.E. Top-down mass spectrometry for the analysis of combinatorial post-translational modifications.Mass Spectrom. Rev. 2013; 32: 27-42Crossref PubMed Scopus (47) Google Scholar). Recent years have seen developments in direct MS analysis of intact proteins (often called “Top-Down” MS). Despite increasing success in characterization of small to medium-sized proteins, MS analysis of intact proteins larger than 50 kDa, including mAbs, is still unsatisfactory because of inefficient gas-phase protein fragmentation and complex fragment ions that restrict efficient data interpretation (9.Fornelli L. Damoc E. Thomas P.M. Kelleher N.L. Aizikov K. Denisov E. Makarov A. Tsybin Y.O. Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation orbitrap FTMS.Mol. Cell. Proteomics. 2012; 11: 1758-1767Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 10.Mao Y. Valeja S.G. Rouse J.C. Hendrickson C.L. Marshall A.G. Top-down structural analysis of an intact monoclonal antibody by electron capture dissociation-fourier transform ion cyclotron resonance-mass spectrometry.Anal. Chem. 2013; 85: 4239-4246Crossref PubMed Scopus (94) Google Scholar). A “compromise” between Bottom-Up and Top-Down approaches is the “Middle-Down” (or “Middle-Up”) method. Middle-Down analysis typically involves proteolysis using proteases (e.g. Lys-C) or chemicals that hydrolyze proteins at a single type of amino acid residue. This approach aims to generate 3–15 kDa peptides which are compatible with high resolution analysis on a The Middle-Down approach of the of Top-Down analysis, with intact protein MS in signal-to-noise ratio of fragment ions for sequence the for mass Mass Spectrom. PubMed Scopus Google Scholar, A. S. capture mass an application for the analysis of Chem. 2013; 85: PubMed Scopus Google Scholar, S. B. analysis a and sensitive platform for high sequence of complex proteins with post-translational modifications analysis of and 4: PubMed Scopus Google Scholar, J.C. L. K.R. M. Thomas P.M. Kelleher N.L. A for 2012; PubMed Scopus (94) Google Scholar, K. Y. analysis using an PubMed Scopus Google Scholar). of for Middle-Down protein analysis are also of the amino is a digestion at amino acid residues still small Da) or large which from the of the Middle-Down approach K. Tsybin Y.O. digestion specificity analysis for of and proteomics 2013; PubMed Scopus Google Scholar). the digestion is often for proteins with folded structure or Although high of such as 8 m urea are often used for protein this used or dissociation analyses a single for gas-phase dissociation and several ions of methods for tryptic ions that typically or are with the analysis of peptides that to high sequence and PTM A. S. capture mass an application for the analysis of Chem. 2013; 85: PubMed Scopus Google Scholar). we a proteolysis method for Middle-Down MS analysis of mAb. hydrolyze the 150 kDa mAb into large peptides for analysis, we a capillary reactor column that a of immobilized and of the sample flow rate to digestion of the protein in the reactor. A short digestion in a small of the protein chain and the of large peptides The a similar using a with or a protein in acid through the reactor in than the protein into large peptides that sequence of and by Y. Y. F. Limited proteolysis in Chem. 2012; 84: PubMed Scopus Google Scholar). The of as well as the in the that for sample flow we a reactor that or native mAb into kDa peptides in a containing 8 m urea with most used and amino acid as well as the disulfide linkages using a used aspergillopepsin for the reactor. also as acid the of proteins at and of but also at of Scopus Google Scholar). are several innovative of this is in 8 m urea at for at least This may the structure of proteins to a and for of the to most regions of the protein the disulfide bonds are with proteases with or amino acid aspergillopepsin an for the protein to with immobilized aspergillopepsin generate large peptides with a because of similar of on peptides. The reactor proteolysis as the sample of the This is in to digestion using proteases that are in the digestion is to or because of the activity in an with or for of the the of by The at for at least a is the of of from one of we introduced a N-(2-aminoethyl)maleimide for protein MS This and protein sequence analysis by electron transfer dissociation mass PubMed Scopus Google of peptides containing residues by a chain to The of strategy to the generation of peptides that cover the murine mAb. and collisionally activated dissociation from the most large peptides by ProSightPC near complete sequence of the mAb and we the native mAb into large of peptides using The proton transfer B. identification using and tandem mass PubMed Scopus Google identification of the and C-terminal sequences of two peptides and of the disulfide different mAb in this work from from was immobilized on by the information for immobilized with in and into a 150 capillary to a reactor. specific column for different information the for protein from was in the digestion containing 8 m at and through the reactor at different flow to different digestion times. the murine IgG1 mAb with the reactor for the disulfide bonds of mAb with and with at in 8 m The mAb was and to in a containing 8 m urea followed by digestion with different digestion times. The same mAb was also and with iodoacetamide as a sample for digestion with aspergillopepsin and digestion with the native mAb for disulfide the intact mAb was in the digestion for digestion with aspergillopepsin I. An of native mAb was with to the peptides. An was with a Mass for of protein of the protein was a capillary with the was connected to an analytical column 50 capillary with 16 of the same that was integrated with a for peptides and proteins on a by electron-transfer dissociation mass PubMed Scopus Google Scholar). at a flow rate of using the for for for acid in and Mass analyses of and mAb sequences of an with MS for sample evaluation followed by a with on the most large peptides from I. For large the ion with the with was as the ion for was on the 50 mAb sequence an was as in a similar as in to generate sequence the location of mAb disulfide bonds, and ion and to sequence by electron transfer dissociation mass Mass Spectrom. 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PubMed Scopus Google and the ions with mass a MS data from the mAb from the murine mAb sequence using Open Mass Spectrometry Search L. M. Open mass spectrometry PubMed Scopus Google Scholar). information for generate kDa from protein we a reactor using with the aspergillopepsin to This is and specificity with for of bonds at the of residues such as and and at the of of Scopus Google Scholar). also the of in 8 m urea for at least most protein disulfide bonds are will denatured and of the reactor involves flowing a of the protein sample in containing 8 m urea through the column This a flow rate and the digestion in the reactor to as a of the flow rate and the of the reactor information for in are total ion current from on peptides from the kDa with different digestion times and in the micro column reactor. peptides the at the intact at the and peptides in the kDa mass are both and well the for the digestion in is the for the peptides with large peptides are with amino acid sequence from on ion the on the ion at that to the residues in the protein ions have to ions by fragment ions of type and are below the sequence for with two and was and with and was For digestion of and mAb in 8 m several times (Fig. as for this a digestion of was because of the resulting product mixture that than peptides with in the kDa the from protein was sequence information for peptides in the kDa we the into 8 and ions the most large peptides at specific in for analysis by during a of a of the ions to of the mass in a of of the of ions into single to the molecular weights of and of a that contain ions from one of the peptides to An of of is in the large peptides on from I. 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