N-terminal Glutamate to Pyroglutamate Conversion in Vivo for Human IgG2 Antibodies

Abstract

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute. Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute. IntroductionPyroglutamate (pE), 2The abbreviations used are: pEpyroglutamateHCheavy chainsPGAPpyroglutamate amino peptidaseFAformic acid. or pyrrolidone carboxylate, is a cyclic amino acid found at the N termini of some proteins and biological peptides (1Blomback B. Hirs C. Methods in Enzymology. 11. Academic Press, New York1967: 389-411Google Scholar). Formation occurs through the rearrangement of the originally synthesized glutamate or glutamine residues at this position (Fig. 1). Although this reaction proceeds spontaneously at reasonable rates, glutaminyl cyclase, an enzyme that catalyzes this reaction, is found in many plants and animals, including humans (2Schilling S. Wasternack C. Demuth H.U. Biol. Chem. 2008; 389: 983-991Crossref PubMed Scopus (78) Google Scholar). Reaction rates for both the spontaneous reaction and the enzymatically catalyzed reaction appear to be much faster with glutamine than with glutamate residues (2Schilling S. Wasternack C. Demuth H.U. Biol. Chem. 2008; 389: 983-991Crossref PubMed Scopus (78) Google Scholar).Many antibodies contain pE on the light chain or heavy chain (3Dayhoff, M. O., (ed) (1972) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Silver Spring, MD.Google Scholar). Although pyroglutamate formation is more prevalent at N-terminal glutamine residues (4Ouellette D. Alessandri L. Chin A. Grinnell C. Tarcsa E. Radziejewski C. Correia I. Anal. Biochem. 2010; 397: 37-47Crossref PubMed Scopus (54) Google Scholar, 5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar), it can occur at glutamate residues as well (6Chelius D. Jing K. Lueras A. Rehder D.S. Dillon T.M. Vizel A. Rajan R.S. Li T. Treuheit M.J. Bondarenko P.V. Anal. Chem. 2006; 78: 2370-2376Crossref PubMed Scopus (159) Google Scholar, 7Yu L. Vizel A. Huff M.B. Young M. Remmele Jr., R.L. He B. J. Pharm. Biomed. Anal. 2006; 42: 455-463Crossref PubMed Scopus (53) Google Scholar, 8Liu H. Gaza-Bulseco G. Sun J. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2006; 837: 35-43Crossref PubMed Scopus (99) Google Scholar). Antibody pE forms spontaneously in vitro (5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar), although it is not known whether glutaminyl cyclase accelerates this rate in blood.For therapeutic monoclonal antibodies, pE can be one of many post-translational modifications observed during production and storage. Because of the loss of a primary amine in the glutamine to pE conversion, antibodies become more acidic. Incomplete conversion produces heterogeneity in the antibody that can be observed as multiple peaks using charge-based analytical methods, such as ion exchange chromatography or isoelectric focusing (4Ouellette D. Alessandri L. Chin A. Grinnell C. Tarcsa E. Radziejewski C. Correia I. Anal. Biochem. 2010; 397: 37-47Crossref PubMed Scopus (54) Google Scholar). Differences in pE formation have been considered a concern in drug production, because heterogeneity differences may indicate a lack of process control (5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar). Interestingly, heterogeneity as the result of glutamate to pE conversion would not be observable by these analytical methods, because antibodies with these N termini share the same charge state.Recently, more effort has been placed on understanding how heterogeneity impacts product quality (9Chirino A.J. Mire-Sluis A. Nat. Biotechnol. 2004; 22: 1383-1391Crossref PubMed Scopus (312) Google Scholar). Those attributes of particular concern are ones that affect the safety or efficacy of the drug. In contrast, attributes not impacting drug safety or efficacy would appear to be less of a concern. Focusing process control on critical quality attributes is the cornerstone of Quality by Design, an emerging paradigm in biotechnology development (10Rathore A.S. Trends Biotechnol. 2009; 27: 546-553Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 11Rathore A.S. Winkle H. Nat. Biotechnol. 2009; 27: 26-34Crossref PubMed Scopus (607) Google Scholar). As part of an effort to ascertain the impact of N-terminal heterogeneity on the safety and efficacy of therapeutic antibodies, studies were conducted to monitor pE formation in vivo. Here we describe N-terminal glutamate to pE conversion, as opposed to the more prevalent glutamine to pE conversion, in vivo and how these studies are used, in part, to assess criticality of this attribute.DISCUSSIONIn previous studies, glutamate on the N termini of antibodies was shown to spontaneously convert to pyroglutamate under fairly mild conditions in vitro. Here we observed that similar conversion occurs on antibodies in vivo. For two antibodies containing N-terminal Glu on both heavy and light chains, conversion could be observed in circulation, although the rates differed between antibodies and between the polypeptide chains on the same antibody. A simple PBS model under physiological pH and temperature replicated the in vivo conversion rates reasonably well. These results strongly suggest that Glu to pE formation in vivo is nonenzymatic. Differences in the conversion rates in vitro could be attributed to structural differences between the proteins. The addition of denaturant increased the conversion rates and eliminated differences between polypeptides.Pyroglutamate formation has been proposed as a stabilization mechanism for proteins and peptides in vivo. Because this cyclized residue is resistant to amino peptidases (18Rink R. Arkema-Meter A. Baudoin I. Post E. Kuipers A. Nelemans S.A. Akanbi M.H. Moll G.N. J. Pharmacol Toxicol Methods. 2010; 61: 210-218Crossref PubMed Scopus (103) Google Scholar), it is reasonable to propose that its presence could slow physiological turnover. In this study, the turnover of antibodies appeared unaffected by the presence of pE. This conclusion is based on the observation that pE formation rates in vivo matched those measured in vitro. If pE-containing antibodies were cleared at a different rate than the original form, differences in the apparent conversion rates would have been expected. Thus, pyroglutamate appears to be a spontaneous and naturally occurring post-translational modification of antibodies in vivo that does not impact protein turnover.The analysis and monitoring of pE during the manufacturing and storage of therapeutic antibodies can be achieved with charged based methods, such as cation exchange (4Ouellette D. Alessandri L. Chin A. Grinnell C. Tarcsa E. Radziejewski C. Correia I. Anal. Biochem. 2010; 397: 37-47Crossref PubMed Scopus (54) Google Scholar), but only if it originates from glutamine, not glutamate. Glutamate conversion to pE does not result in a charge state difference, so this change might be missed using simple chromatography-based release assays. To monitor glutamate cyclization, more complex assays, such as peptide mapping, would be required (5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar, 6Chelius D. Jing K. Lueras A. Rehder D.S. Dillon T.M. Vizel A. Rajan R.S. Li T. Treuheit M.J. Bondarenko P.V. Anal. Chem. 2006; 78: 2370-2376Crossref PubMed Scopus (159) Google Scholar). Whether pE is monitored and controlled for a given therapeutic antibody should be based on its impact to safety and efficacy. Because the N-terminal residues of both the light chains and the heavy chains are in the variable region and near the CDR, pE formation potentially could impact target binding, but this may vary for individual antibodies. An impact on efficacy would be expected to increase the criticality of this attribute. Other results, described in this study, could also bear on the criticality assessment. In particular, pE exposure in patients, through its natural occurrence in endogenous antibodies and through pE formation on the therapeutic antibody in vivo, may lessen the safety concerns for this attribute. In cases where pE impacts efficacy, patient exposure profiles can be used to estimate how initial levels of pE could affect activity over time in vivo and help guide rational manufacturing limits on such labile attributes. IntroductionPyroglutamate (pE), 2The abbreviations used are: pEpyroglutamateHCheavy chainsPGAPpyroglutamate amino peptidaseFAformic acid. or pyrrolidone carboxylate, is a cyclic amino acid found at the N termini of some proteins and biological peptides (1Blomback B. Hirs C. Methods in Enzymology. 11. Academic Press, New York1967: 389-411Google Scholar). Formation occurs through the rearrangement of the originally synthesized glutamate or glutamine residues at this position (Fig. 1). Although this reaction proceeds spontaneously at reasonable rates, glutaminyl cyclase, an enzyme that catalyzes this reaction, is found in many plants and animals, including humans (2Schilling S. Wasternack C. Demuth H.U. Biol. Chem. 2008; 389: 983-991Crossref PubMed Scopus (78) Google Scholar). Reaction rates for both the spontaneous reaction and the enzymatically catalyzed reaction appear to be much faster with glutamine than with glutamate residues (2Schilling S. Wasternack C. Demuth H.U. Biol. Chem. 2008; 389: 983-991Crossref PubMed Scopus (78) Google Scholar).Many antibodies contain pE on the light chain or heavy chain (3Dayhoff, M. O., (ed) (1972) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Silver Spring, MD.Google Scholar). Although pyroglutamate formation is more prevalent at N-terminal glutamine residues (4Ouellette D. Alessandri L. Chin A. Grinnell C. Tarcsa E. Radziejewski C. Correia I. Anal. Biochem. 2010; 397: 37-47Crossref PubMed Scopus (54) Google Scholar, 5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar), it can occur at glutamate residues as well (6Chelius D. Jing K. Lueras A. Rehder D.S. Dillon T.M. Vizel A. Rajan R.S. Li T. Treuheit M.J. Bondarenko P.V. Anal. Chem. 2006; 78: 2370-2376Crossref PubMed Scopus (159) Google Scholar, 7Yu L. Vizel A. Huff M.B. Young M. Remmele Jr., R.L. He B. J. Pharm. Biomed. Anal. 2006; 42: 455-463Crossref PubMed Scopus (53) Google Scholar, 8Liu H. Gaza-Bulseco G. Sun J. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2006; 837: 35-43Crossref PubMed Scopus (99) Google Scholar). Antibody pE forms spontaneously in vitro (5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar), although it is not known whether glutaminyl cyclase accelerates this rate in blood.For therapeutic monoclonal antibodies, pE can be one of many post-translational modifications observed during production and storage. Because of the loss of a primary amine in the glutamine to pE conversion, antibodies become more acidic. Incomplete conversion produces heterogeneity in the antibody that can be observed as multiple peaks using charge-based analytical methods, such as ion exchange chromatography or isoelectric focusing (4Ouellette D. Alessandri L. Chin A. Grinnell C. Tarcsa E. Radziejewski C. Correia I. Anal. Biochem. 2010; 397: 37-47Crossref PubMed Scopus (54) Google Scholar). Differences in pE formation have been considered a concern in drug production, because heterogeneity differences may indicate a lack of process control (5Dick Jr., L.W. Kim C. Qiu D. Cheng K.C. Biotechnol. Bioeng. 2007; 97: 544-553Crossref PubMed Scopus (97) Google Scholar). Interestingly, heterogeneity as the result of glutamate to pE conversion would not be observable by these analytical methods, because antibodies with these N termini share the same charge state.Recently, more effort has been placed on understanding how heterogeneity impacts product quality (9Chirino A.J. Mire-Sluis A. Nat. Biotechnol. 2004; 22: 1383-1391Crossref PubMed Scopus (312) Google Scholar). Those attributes of particular concern are ones that affect the safety or efficacy of the drug. In contrast, attributes not impacting drug safety or efficacy would appear to be less of a concern. Focusing process control on critical quality attributes is the cornerstone of Quality by Design, an emerging paradigm in biotechnology development (10Rathore A.S. Trends Biotechnol. 2009; 27: 546-553Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 11Rathore A.S. Winkle H. Nat. Biotechnol. 2009; 27: 26-34Crossref PubMed Scopus (607) Google Scholar). As part of an effort to ascertain the impact of N-terminal heterogeneity on the safety and efficacy of therapeutic antibodies, studies were conducted to monitor pE formation in vivo. Here we describe N-terminal glutamate to pE conversion, as opposed to the more prevalent glutamine to pE conversion, in vivo and how these studies are used, in part, to assess criticality of this attribute.