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Exposure of a Monoclonal Antibody, IgG1, to UV-Light Leads to Protein Dithiohemiacetal and Thioether Cross-Links: A Role for Thiyl Radicals? Olivier Mozziconacci,† Bruce A. Kerwin,‡ and Christian Scho¨neich*,† Department of Pharmaceutical Chemistry, 2095 Constant AVenue, UniVersity of Kansas, Lawrence, Kansas 66047, and Department of Analytical and Formulation Science, Process and Product DeVelopment, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119 ReceiVed June 2, 2010
Recently, we characterized a thiyl radical-dependent mechanism for the photolytic conversion of a disulfide bond in a model peptide into dithiohemiacetal and subsequently into thioether (Mozziconacci et al. (2010) J. Phys. Chem B 114, 3668-3688). This mechanism is of potential relevance for the photodegradation of disulfide-containing proteins, which may be a problem for the production and formulation of diagnostic and therapeutic protein pharmaceuticals. In this Rapid Report, we show that similar products are also formed when an antibody (IgG1) is subjected to photoirradiation at 253.7 nm, suggesting the involvement of thiyl radicals also in these processes. A series of dithiohemiacetal and thioether cross-links were identified in photoirradiated IgG1 through HPLC-MS/MS analysis. Therapeutic and diagnostic antibodies are a rapidly growing class of biotechnology products (1-4). They are sensitive to an array of chemical and physical degradation processes, including deamidation, fragmentation, oxidation, β-elimination, and aggregation (5, 6). These degradation processes may lead to various consequences such as reduced potency (7), aggregation (8), and/or immunogenicity (9), which are of great concern to both the biotechnology industry and regulatory agencies. In order to fully realize the pathways leading to antibody degradation and their potential consequences, a thorough characterization of chemical products is necessary. Here, we report on the photolytic (λ ) 254 nm, Ar-saturated solution) formation of novel dithiohemiacetal products in IgG1, which may serve as precursor for thioether formation. UV irradiation at 254 nm is of concern to IgG1 stability because of its potential use for viral decontamination of biotechnology products (10). Earlier, thioethers have been detected in antibodies as a result of accelerated stability studies (5, 11, 12) but have been mechanistically rationalized to date predominantly through β-elimination followed by the addition of Cys to dehydroalanine. We recently characterized a novel free radical mechanism underlying the formation of dithiohemiacetal and thioether products in a small intrachain disulfide-containing model peptide (Scheme 1, reactions 1-5) (13). Here, the dithiohemiacetal serves as precursor for photolytic thioether formation via the elimination of H2S, the formation of which was also reported earlier (14). Important for dithiohemiacetal formation is the disproportionation reaction 2 leading to thiol and thioaldehyde, followed by reaction 4 (Scheme 1). These mechanisms are probably responsible for photolytic product formation in IgG1, presented in this Rapid Report. The key to our product analysis is a distinction between photochemically generated Cys residues and/or thiols in IgG1, * To whom correspondence should be addressed. E-mail: schoneic@ ku.edu. † University of Kansas. ‡ Amgen Inc.
and Cys residues formed through chemical reduction of the remaining disulfides after photolysis. In the following, photochemically generated thiols are always derivatized with Nethylmaleimide (NEM), while the remaining unreacted disulfides are subsequently reduced with dithiothreitol (DTT) and then derivatized with diethylmaleate (DEM) (see Materials and Methods provided in Supporting Information). At lower doses of 254 nm photoirradiation (20 and 3 min of irradiation with fluxes of 2.96 × 10-6 einstein min-1 and 1.91 × 10-5 einstein min-1, respectively), the interchain disulfide bonds of the IgG1 hinge region, Cys[222]-Cys[222] and Cys[225]-Cys[225], are converted into thiol and aldehyde, respectively, analogous to reactions 1-3 (Scheme 1). However, it appears that both interchain disulfide bonds are not damaged photolytically simultaneously. Either Cys[222]-Cys[222] undergoes photoreaction and subsequently Cys[225]-Cys[225] is chemically reduced and tagged with DEM or vice versa. Hence, Cys[222] and Cys[225] in the proteolytic peptide THTC(222)PPC(225)PAPE are present in different combinations of Cys-DEM and Cysaldehyde/Cys-NEM, respectively (Supporting Information, Figure S1-S3). In addition, the intrachain disulfide bonds Cys[140]-Cys[196] and Cys[32]-Cys[96] convert into aldehyde and thiol, respectively. The aldehyde and thiols, derivatized with NEM, are detected for Cys[140] and Cys[96], respectively (Figure S4-S5, Supporting Information). The lack of product detection for Cys[32] may be due to incomplete deglycosylation prior to mass spectrometry analysis. A higher dose of photoirradiation (20 min of irradiation with a flux of 1.91 × 10-5 einstein min-1) generates a significant yield of cross-links I and II (Scheme 1) in addition to the products observed at lower dose. Cross-link I is formed between two pairs of cysteine residues of the heavy chain Cys[216]-Cys[257], and Cys[257]Cys[317] (Figure 1 and Figure S6 (Supporting Information)). Cross-link II is exclusively observed between a Cys residue of the light chain (LC) and one of the heavy chain (HC), Cys[217,LC]-Cys[222,HC] (Figure S7, Supporting Informa-
10.1021/tx100193b 2010 American Chemical Society Published on Web 07/06/2010
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Scheme 1. Reaction Scheme Demonstrating the Photolytic Formation of Cross-Links I and II in a Simple Intrachain Disulfide Bonda
a In the antibody, the presence of numerous disulfide bonds can lead to the simultaneous homolytic photochemical cleavage of more than one disulfide bond. This may lead to cross-link I and/or II between Cys residues of originally different disulfide bonds. Note that this scheme has been developed for a small synthetic peptide containing an intrachain disulfide bond (13); however, it represents the reactions important for product formation even for interchain disulfides in the antibody, in which individual chains are held together through additional disulfide bonds.
Figure 1. CID mass spectrum of the digested peptide corresponding to the region HC: 215-218 and HC: 255-270 involving Cys[216] and Cys[257] in cross-link I.
tion)1. The cysteine residues involved in these different photoproducts are depicted in Chart 1. A description of radical mechanisms leading to the formation of cross-links I and II has recently been given for intrachain disulfides of model peptides (13). Here, for the first time, we identify such photochemical pathways for a large protein. In the model peptides, photolysis of cross-link I led to the formation of cross-link II (13). For IgG1 photolysis, no cross-link I was detected between Cys[217,LC] and Cys[222,HC], indicating that either cross-link I between these Cys residues is very sensitive to further photolysis or that cross-link I is not formed and that cross-link II between Cys[217,LC] and Cys[222,HC] forms via an alternative route. An important observation is the formation of cross-links I and II between Cys residues which are not part 1 Cross-link II was absent in the nonirradiated control, indicating that it is not formed during sample preparation for analysis. The labeling of the cysteine residues in our IgG1 is shifted by 9 and 1 residues compared to the sequences given in the reports of Yan et al. (6) and Tous et al. (11), respectively.
of an original disulfide bond, i.e., Cys[217]-Cys[222], Cys[216]-Cys[257], and Cys[257]-Cys[317]. This can be rationalized by the fact that in an antibody containing 14 disulfide bonds the simultaneous homolytic photochemical cleavage of more than one disulfide bond can lead to Cys radical disproportionation products between radicals of different original disulfide bonds. This may lead to cross-links I and/or II between Cys residues of originally different disulfide bonds. An alternative pathway for Cys thiyl radical formation is the photoionization of Tyr and/or Trp (15-17), leading to a solvated electron, followed by one-electron reduction of the disulfide to thiyl radical and thiolate. This pathway may in part be responsible for cross-link formation between Cys residues which are not part of original disulfide bonds. Dithiohemiacetal and thioether formation in protein pharmaceuticals have the potential for biochemical and therapeutic consequences. For example, for human growth hormone the conversion of an intrachain disulfide into thioether resulted in significant changes in receptor binding (18). Analogous con-
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Chart 1. Disulfide Bridges Damaged after Lower Doses of Photoirradiation (5.8 × 10-5 Einstein) (Labeled in Red) and Cysteine Residues Involved in Cross-Links of Types I (O, 0) and II (∆), Formed Additionally after a Higher Dose of Irradiation (3.8 × 10-4 Einstein)
sequences may be expected for therapeutic antibodies but must await further pharmacological characterization. Acknowledgment. We are grateful to Amgen Inc. for financial support and thank Dr. Nadya Galeva for operating the FT-MS instrument. Supporting Information Available: Materials and Methods section and MS/MS spectra of the photoproducts. This material is available free of charge via the Internet at http://pubs.acs.org.
References (1) Costa, A. R., Rodrigues, M. E., Henriques, M., Azeredo, J., and Oliveira, R. (2010) Guidelines to cell engineering for monoclonal antibody production. Eur. J. Pharm. Biopharm. 74, 127–138. (2) Rodrigues, M. E., Costa, A. R., Henriques, M., Azeredo, J., and Oliveira, R. (2010) Technological progresses in monoclonal antibody production systems. Biotechnol. Prog. 26, 332–351. (3) Kelley, B. (2009) Industrialization of mAb production technology: the bioprocessing industry at a crossroads. MAbs 1, 443–452. (4) Shukla, A. A., and Thommes, J. (2010) Recent advances in largescale production of monoclonal antibodies and related proteins. Trends Biotechnol. 28, 253–261.
Mozziconacci et al. (5) Qi, P., Volkin, D. B., Zhao, H., Nedved, M. L., Hughes, R., Bass, R., Yi, S. C., Panek, M. E., Wang, D., Dalmonte, P., and Bond, M. D. (2009) Characterization of the photodegradation of a human IgG1 monoclonal antibody formulated as a high-concentration liquid dosage form. J. Pharm. Sci. 98, 3117–3130. (6) Yan, B., Yates, Z., Balland, A., and Kleemann, G. R. (2009) Human IgG1 hinge fragmentation as the result of H2O2 mediated radical cleavage. J. Biol. Chem. 284, 35390–35402. (7) Rehder, D. S., Chelius, D., McAuley, A., Dillon, T. M., Xiao, G., Crouse-Zeineddini, J., Vardanyan, L., Perico, N., Mukku, V., Brems, D. N., Matsumura, M., and Bondarenko, P. V. (2008) Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity. Biochemistry 47, 2518–2530. (8) Singh, S. K., Afonina, N., Awwad, M., Bechtold-Peters, K., Blue, J. T., Chou, D., Cromwell, M., Krause, H. J., Mahler, H. C., Meyer, B. K., Narhi, L., Nesta, D. P., and Spitznagel, T. (2010) An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J. Pharm. Sci. 99, 3302–3321. (9) Rosenberg, A. S. (2006) Effects of protein aggregates: an immunologic perspective. AAPS J. 8, E501-507. (10) Lorenz, C. M., Wolk, B. M., Quan, C. P., Alcala, E. W., Eng, M., McDonald, D. J., and Matthews, T. C. (2009) The effect of low intensity ultraviolet-C light on monoclonal antibodies. Biotechnol. Prog. 25, 476–482. (11) Tous, G. I., Wei, Z., Feng, J., Bilbulian, S., Bowen, S., Smith, J., Strouse, R., McGeehan, P., Casas-Finet, J., and Schenerman, M. A. (2005) Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains. Anal. Chem. 77, 2675–2682. (12) Liu, H., Gaza-Bulseco, G., and Chumsae, C. (2009) Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry. J. Am. Soc. Mass Spectrom. 20, 2258– 2264. (13) Mozziconacci, O., Kerwin, B. A., and Scho¨neich, C. (2010) Photolysis of an intrachain peptide disulfide bond: primary and secondary processes, formation of H2S, and hydrogen transfer reactions. J. Phys. Chem. B 114, 3668–3688. (14) Grossweiner, L. I. (1976) Photochemical inactivation of enzymes. Curr. Top. Radiat. Res. Q. 11, 141–199. (15) Vanhooren, A., De Vriendt, K., Devreese, B., Chedad, A., Sterling, A., Van Dael, H., Van Beeumen, J., and Hanssens, I. (2006) Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin. Biochemistry 45, 2085–2093. (16) Bent, D. V., and Hayon, E. (1975) Excited state chemistry of aromatic amino acids and related peptides. I. Tyrosine. J. Am. Chem. Soc. 97, 2599–2606. (17) Bent, D. V., and Hayon, E. (1975) Excited state chemistry of aromatic amino acids and related peptides. III. Tryptophan. J. Am. Chem. Soc. 97, 2612–2619. (18) Lispi, M., Datola, A., Bierau, H., Ceccarelli, D., Crisci, C., Minari, K., Mendola, D., Regine, A., Ciampolillo, C., Rossi, M., Giartosio, C. E., Pezzotti, A. R., Musto, R., Jone, C., and Chiarelli, F. (2009) Heterogeneity of commercial recombinant human growth hormone (rhGH) preparations containing a thioether variant. J. Pharm. Sci. 98, 4511–4524.
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