Boxu Yan and Daniel BoydBreaking the light and heavy chain linkage of - PDF document

1 Boxu Yan and Daniel BoydBreaking the lig
Boxu Yan and Daniel BoydBreaking the light and heavy chain linkage of human IgG1 by radical reactions published online May 23, 2011J. Biol. Chem.    10.1074/jbc.M111.255026Access the most updated version of this article at doi:  Alerts:   When a correction for this article is posted•  When this article is cited•  to choose from all of JBC's e-mail alertsClick here by guest on January 11, 2021http://www.jbc.org/Downloaded from 23256.923360.623391.7 2.315e42.335e42.355e4Mass, Da Fig. 7C23360.223256.323391.523407.523440.123423.1 2.315e42.335e42.355e4Mass, Da 100 Fig. 7D by guest on January 11, 2021http://www.jbc.org/Downloaded from 27 8.0 16.0 24.0 32.0 milliAbsorbance Units (mAU)ControlPartialMainFig. 7A Time (min) 2.315e42.335e42.355e42.375e4Mass, Da 23360.623392.0100Fig. 7B by guest on January 11, 2021http://www.jbc.org/Downloaded from b5-HFig. 6EFull ms2 929.54 300 400 500 600 700 800 m/z 100RelativeAbundance 600.33814.42582.34825.41686.31471.23330.29558.27797.49697.39D K K V E P K SCONH y7 y6 y5 y3 b4 b5 b6 b7 by guest

2 on January 11, 2021http://www.jbc.org/Do
on January 11, 2021http://www.jbc.org/Downloaded from [MH-SOy7-SOy6-SOy4-SO Full ms2 557.25[2+] 300 400 500 600 700 800 900m/z 100Relative Abundance 517.33600.22434.23417.28790.29870.34825.35663.69288.92Fig. 6DD K K V E P K S CCONH y7 y3 y2 b5 b7 S F N R G E-CONH2 Fig. 6Cy3 Full ms2 708.34 400 500 600 m/z Relative Abundance 691.31505.20562.16474.29360.35621.29MH-NH b4 b5y5 y4 y3 by guest on January 11, 2021http://www.jbc.org/Downloaded from S F N R G E CFig. 6AMH-NHFull ms2 860.32 400 600 700 800m/z Relative Abundance 691.27843.19626.19505.25562.33512.21457.32 b4 b5 b6y5 y4 SOH500[MH-HO] Fig 6BFull ms2 844.32 300 400 500 600 700m/z 100Relative Abundance 413.82610.19592.16234.98691.19505.24283.10y5-HS F N R G E C b4 b5 b6y5 y4 SB by guest on January 11, 2021http://www.jbc.org/Downloaded from Fig. 5 16.0 20.0 24.0 28.0 ab 16.0 20.0 24.0 28.0 abc milliAbsorbance Unit (mAU)Time (min) by guest on January 11, 2021http://www.jbc.org/Downloaded from 23375.723557.4Fig. 4 C 2.31e42.33e42.35e42.37e4Mass, Da 10047082.147182.947

3 319.6469540.046839.1Fig. 4D 4.67e44.69e4
319.6469540.046839.1Fig. 4D 4.67e44.69e44.71e44.73e44.75e4Mass, Da %0100D by guest on January 11, 2021http://www.jbc.org/Downloaded from Fig. 4 A 2.325e42.345e42.365e4Mass, Da 23408.423392.123360.523439.823256.223424.2100 Fig. 4B 2.325e42.345e42.365e4Mass, Da 23364.023412.723260.023396.010023379.4 B by guest on January 11, 2021http://www.jbc.org/Downloaded from 20 Time (min) 30 34 38 42 milliAbsorbance Units (mAU) Partial FabFig. 3AMainFig. 3B 10 14 18 22 26 30 200 400 Time (min)milliAbsorbance Units (mAU) by guest on January 11, 2021http://www.jbc.org/Downloaded from 19 12345ufFig. 2ARelative Fluorescence Units% CleavagentEDSODSOSODSOD+ PO 123456789Fig. 2B by guest on January 11, 2021http://www.jbc.org/Downloaded from Fig. 1 HC: TKVDKKVEPKS C D K T H T C231LC: SSPVTKSFNRGE C215 by guest on January 11, 2021http://www.jbc.org/Downloaded from Peptide Adduct Domain C-terminus % 215 -SOH LC ND ~50 215 214 LC -CONHDKKVEPKSC -SO Fd -CONHDKKVEPKS Fd -CONH ~75 The est

4 imation of abundance for each peptidperc
imation of abundance for each peptidpercent peak area relative to the total integrated peak area of the C-terminal peptide derivatives from the LC or Fd. Since the amino acid sequence SFNRGEC is the C-terminal peptide of the LC, no--CONH was determined. by guest on January 11, 2021http://www.jbc.org/Downloaded from TABLE 1. The average masses of the Fab fragments analyzed by RP-HPLC-TOF/MS. Residues Theoretical Observed Difference Proposed (Start-end) mass (Da) (Da) modification Non-reducing conditions Asp-Cys215 23359.9 23360.5 0.6 23392.1 32.3 +S 23408.4 48.5 +SOH 23424.2 64.3 +SO 23439.8 79.9 +SOH Asp214 23256.0 23256.2 0.2 224 23376.1 23375.7 -0.4 Glu1-Cys225 23479.4 23557.4 78.0 +SOFab -Cys225 46839.2 46839.1 -0.1 226 46954.3 46954.0 -0.3 -Lys 47082.5 47082.1 -0.4 47183.6 47182.9 -0.7 229 47320.7 47319.6 -1.1 LC (Reducing conditions) Asp-Cys215 23363.9 23364.0 0.1 23379.

5 4 15.5 -OH 23396.0 32.1 -OH
4 15.5 -OH 23396.0 32.1 -OH 23412.7 48.8 -OH214 23260.0 23260.0 0.0 Note: a: masses listed are for fragments with –CONH at C-terminus. by guest on January 11, 2021http://www.jbc.org/Downloaded from the C-terminalthe C-terminalDa) of the LC comprising a -sulfur at the side chain of Cys (SFNRGEC). at its C-terminus (DKKVEPKS the C-terminal peptide (1113.50Da) of Fd at its C-terminus (DKKVEPKSC) of Fd from the Asp-N peptide map. rtial IgG1 and main peak purified by SEC. A. reducing conditions in comparison with the control reference sample (Ref). Partial: the partial IgG1; Main: the main peak purified by SEC. The LC was eluted at ~16 min as spectrum of the LC from the control. C. the deconvoluted spectrum of the LC from the parthe LC from the main peak. by guest on January 11, 2021http://www.jbc.org/Downloaded from FIGURE LEGENDS Schematic illustration of the inter-chain disulfide bond between the heavy and light chain of an IgG1. The hinge cleavage sites that were reported previously (3-9) are indicated by the ain. The inter

6 -chain disulfide H is the primary radica
-chain disulfide H is the primary radical site for hydroxyl radical attack. The amino acid semapping (see the text for details). The correlation between Hproduction in PBS buffer at pH 7.4 and 37production in the presence of EDTA (20mM)base level. The production of H was determined by the AmplexRed method using G1 under the same incubation C for 15 days in the PBS buffer. The cleavage can (10mM) or EDTA (20mM). Results represent the mean of six replicates. Analysis of the hinge cleavage products. at 37 then the Fab and partial molecule were purifiethree separated peaks, labeled by main peafrom analysis of the Fab under non-reducing conditions. The peaks 1 (L), 2(Fd) and 3 and the results were summarized in Table . The deconvoluted mass spectra of peaks 1–3 from Fig. 3A are presented in Panelsdeconvoluted mass spectrum of the reduced LC. Each major peak is labeled with the observed mass, and corresponding fragments are summarized in Table 1. r the cleaved Fab under non-reducing The Fab fragments were denatured proteases as described under “Experimental P

7 rocedures.” The digest was analyzed
rocedures.” The digest was analyzed on a Phenomenex Proteo column (2.0 x150 mm) and monitored at 214 nm. Panel Aview of the Lys-C peptide map with C-termpeptide of SFNRGEC: truncated peptide (SFNRGE). Panel B, expanded view of the Asp-N peptide map with C-terminal peptide of the LC. peak a truncated peptide (DKKVEPKS at its C-terminus; peptide DKKVEPKSC by guest on January 11, 2021http://www.jbc.org/Downloaded from 29. Poole, L. B., and Claiborne, A. (1989) 30. Poole, L. B., Karplus, P. A., and Claiborne, A. (2004) 31. Takamoto, K., and Chance, M. R. (2006) 32. Stadtman, E. R., and Levine, R. L. (2003) Amino Acids33. Stadtman, E. R., and Berlett, B. S. (1997) Chem Res Toxicol 34. Cross, A. R., and Jones, O. T. (1991) 35. Chance, B., Sies, H., and Boveris, A. (1979) Physiol Rev36. Boveris, A., and Chance, B. (1973) 37. MacManus-Spencer, L. A., and McNeill, K. (2005) 38. Zhao, Y., Wang, Z. B., and Xu, J. X. (2003) J Biol Chem39. Fridovich, I. (1997) 40. Reece, S. Y., and Nocera, D. G. (2009) 41. Gray, H. B., and Winkler, J. R. (2003) 42. Gray, H. B., and Wi

8 nkler, J. R. (2005) 43. Jacob, C., Knigh
nkler, J. R. (2005) 43. Jacob, C., Knight, I., and Winyard, P. G. (2006) 44. Winyard, P. G., Moody, C. J., and Jacob, C. (2005) 45. Davies, M. J., Donkor, R., Dunster, C. A., Gee, C. A., Jonas, S., and Willson, R. Biochem J46. Davies, M. J., Forni, L. G., and Shuter, S. L. (1987) 47. Chan, P. C., Bielski, B.H.J. (1973) J Am Chem Soc48. Barton, J. P., Packer, J.E. (1970) Int'l J Rad Phys Chem 49. Shimazu, F., Kumta, U. S., and Tappel, A. L. (1964) Radiat Res50. Shimazu, F., and Tappel, A. L. (1964) 51. Shimazu, F., and Tappel, A. L. (1964) 52. Hiller, K. O., Masloch, B.,Gobl, M., Asmus, K.D. (1981) critical reading and comments on the manuscript. by guest on January 11, 2021http://www.jbc.org/Downloaded from REFERENCES 1. Reichert, J. M., and Va2. Carter, P. J. (2006) 3. Ouellette, D., Alessandri, L., Piparia, 4. Cordoba, A. J., Shyong, B. J., Breen, D., and Harris, R. J. (2005) 5. Gaza-Bulseco, G., and Liu, H. (2008) 6. Cohen, S. L., Price, C., and Vlasak, J. (2007) J Am Chem Soc7. Dillon, T. M., Bondarenko, P. V., Rehder, D. S., Pipes, G. D., Kleemann, G. R.,

9 8. Yan, B., Yates, Z., Balland, A., and
8. Yan, B., Yates, Z., Balland, A., and Kleemann, G. R. (2009) 9. Nathan, C. (2003) J Clin Invest 10. Nathan, C. (2003) Nature11. Jack Schubert, V. S. S., E. R. White, Linda S. Bergelson. (1968) 12. Lin, S., Gurol, M. (1998) 13. Davies, M. J., Dean R.T. (1997), Radical Mediated Protein Oxidation, pp. 50–120, Oxford University Press, Oxford, 14. Garrison, W. M. (1987) 15. Fridovich, I. (1978) 16. Imlay, J. A. (2003) 17. Imlay, J. A. (2008) 18. Yates, Z., Gunasekaran, K., Zhou, H., H19. Wentworth, P., Jr., Jones, L. H., WentWilson, I. A., Xu, X., Goddard, W. A., 3rd, Janda, K. D., Eschenmoser, A., and Science20. Wang, P. X., and Sanders, P. W. (2007) 21. Rosen, G. M., Pou, S., Ramos, C. L., Cohen, M. S., and Britigan, B. E. (1995) 22. Datta, D., Vaidehi, N., Xu, X23. Zhu, X., Wentworth, P., Jr., Wentworth, A. D., Eschenmoser, A., Lerner, R. A., and Wilson, I. A. (2004) Proc Natl Acad Sci U S A24. Wentworth, A. D., Jones, L. H., WentwoProc Natl Acad Sci U S A25. Shimazu, F., and Tappel, A. L. (1964) 26. Buxton, G. V., Greenstock, C.L.,Helman, W.P., Ross, A.B. (19

10 88) 27. Hayward, M. A., Campbell, E. B.,
88) 27. Hayward, M. A., Campbell, E. B., and Griffith, O. W. (1987) 28. Stipanuk, M. H., Hirschberger, L. L., and De la Rosa, J. (1987) by guest on January 11, 2021http://www.jbc.org/Downloaded from 11e NHCHCOAsp Ser COOH Glu CH2 Asp Ser COOH Glu CH2 H ChainL Chain Asp Ser COOH Glu CH2 S NHCHCOAsp Ser CH2 COOH Glu CH2 S (1)(2a)(2b) NHCOAsp Ser COOH Glu CH2 CHS + Asp Ser CH2 COOH Glu CH2 S (2c) (2d) CONHCH P + e C(O ) NHCH P C(OH ) NHCH(R) P CONH + CH(R) P ~CONHC(=CH)CO~ ~CON=C(CH)CO~~CON=C(CH)CO~ + HO ~CONHCOCO (5)(4) by guest on January 11, 2021http://www.jbc.org/Downloaded from 2:1 for the fragments of Glu225which is not in a good agreement with the Garrison (20), an electron radical addition certain residues could occur and also lead for the electron addition, and the formed dehydroalanine amide at the N-1 position. As indicated thermal incubation conditions also alter local conformation or conformational dynamic of an IgG1. between Cys17 Å is greater than a typical electron the new local confor

11 mation or conformational dynamic in the
mation or conformational dynamic in the upper hinge region could bring the H-L bond more proximal to , which would allow interaction and the H-L bond or electron transfer from the transient radical 229Collectively, our results demonstrated that the thermal incubation induced degradation of the IgG1 hinge follows radical reaction mechanisms with a combination of pathways taking place under high and low Implication of H in the development of mAb therapeuticsthermal incubation induced hinge degradation presents great challenges for the stability programs in the development of mAb therapeutics.molecules, radical reaction mechanisms that may compromise the stability, safety species are the inevitable by-products of the reduction chain reaction needed to minimize the impact of these species to the stability of an IgG1. Preventing metal ions from redox cycling is one mechanism to inhibit the production of hydrogen peroxide in the formulation development and during storage; this can least content of redox metal ions or by using metal chelating reagents. Alternatively, su

12 bstitutimeans of resisting radical induc
bstitutimeans of resisting radical induced fragmentation based on our previous substitution results (8). Further hinge residues and evaluation of some promising mutants could enrich our capable of resisting such degradation by guest on January 11, 2021http://www.jbc.org/Downloaded from the L and Fd with an extra sulfur at Cys225 and fragments without these two reaction pathways for the thermal It is reasonable the H-L linkage also initiates by breaking formation in one of the first hinge Cys and electron transfer leads to localization of the range electron transfer has been observed in some proteins, none has shown an fragments must be generated by new Garrison, Davies and Jacob (13,14,43,44), reaction of Cystine (a disulfide bond) with an electron is known to be extremely rapid constant of 1.6 × 10, faster than with -1 (13,26). Similar to other proteins with elevated energy transferred from to the H-L of an IgG1 results in residue leads to the formation of a radical-reactions depending on the conditions and substrate (47,48). Although the chemical attachment

13 to the understood (14), it has been demo
to the understood (14), it has been demonstrated that electron addition to the thiol groups of Cys and Met leads to efficient cleavage of the C-S bond with elimination of –SH a thiol anion (13,26,49). Such reactions are slow with low yields of minor products Based on the results presented in this study, we propose the following pathways IgG1. In the first stethe H-L bond results in the formation of a Decomposition of the anion results in 215LC that is in the H-L bond (reactions 2a- 2d). With the low oxygen tension, some sulfur groups are oxidized, the rest remain in a reduced form. In the counterpart, the Cys that lost the sulfur group forms a carbon-centered radical that is the same species as a result of hydrogen abstraction from the side chain of Ala, forming unstable dehydroalanine. amide at the N-1 position of new C-terminus (reaction 3). Meanwhile, as demonstrated by solutions occurs in competition with competition results in the formation of a new intermediate radical species –(OH)NH- at Cys (reaction 4). Dissociative deamidation of this

14 new at new C-terminus (reaction 5). Such
new at new C-terminus (reaction 5). Such mechanisms have been described by Shimazu and Buxton etc (25,26,49-52). It should be pointed out that reaction 2d only releases the LC without any cleavage of the HC, as no radical remains at the side after decomposition of the radical anion. This reaction could also LC fragment in the partial IgG1 and main by guest on January 11, 2021http://www.jbc.org/Downloaded from Analysis of the partial IgG1 and main also release the LC species by the reduction place under the thermal incubation LC from the partial molecule or the main peak would be expected. To this end, the partial IgG1 and main peak were analyzed under non-disulfide bonded LC eluted at 16 min in the varied in the samples, the control sample (non treated sample) 1.6%, whereas the main peak and the and 2.5% respectively. TOF/MS analysis indicated molecular masses of 23360.5 Da and 23392.0 Da for the control sample, ced form of the LC The main peak revealed three species with masses of 23256.9 Da, 23360.6 Da and a reduced form of the L and the reduced 7C). H

15 owever, the partial IgG1 showed multiple
owever, the partial IgG1 showed multiple species that are similar to the LC of the cleaved Fab, with the main component as the reduced form of the LC partial IgG1 and main peak that are similar the thermal incubation induced radical of the backbone. Radical reaction mechanism for the environmental processes can lead to the formation of Hfound responsible for degradation of many mechanisms (31-33). However, the O is a relatively unreactive species whose major of an electron(15,21). Estimated in vivo -11respectively (34-36), whereas these two OH)(37). In the chain reaction of Thus, low oxygen tension may allow thermal incubation inducproducts of a different nature than those Like all chemical reactions that generate a product, the accumulation of the its further production. Even in the presence of a catalyst (e.g. SOD, or redox metal limited unless there is a continuous supply of oxygen and sequential consumption of of scheme 2, the enables the degradation of an IgG1 hinge, which in turn consumes the Hmay explain the reason why an accumulation of the cleavage pr

16 oducts was a limited capacity to produce
oducts was a limited capacity to produce H by guest on January 11, 2021http://www.jbc.org/Downloaded from If these fragments are derived from radical reactions, the C-terminal residues at some products should be an amide (-NH2), rather than –COOH according to radical reaction mechanisms (8, 17). To this end, Lys-C and Asp-N peptide mapping were conducted under non-reducing conditions to monitor the C-terminal peptides at the except for the C-terminal Cys from the LC or Fd. As shown in Fig. 5A, three forms of the C-terminal peptide of the LC eluting at ~ 21, 22 and 23 min in the Lys-C map, +48 Da adduct at 21 min, the same peptide with a +32 Da adduct at 22 min, and the at 23 min, respectively. the necessary data to assign the location of , as shown in Fig 6A. Similarly, the +32 Da was assigned to the side chain of ions (y2 and y5) (Fig. 6B). Combining the . Fig. 6C shows the MS/MS spectrum of the peptide SFNRGEtruncated form of SFNRGECCOOH, at the C-terminus. Among these three forms of the C-terminal peptide of the LC, it appears to be that the truncated fo

17 rm modified forms account for ~75% of th
rm modified forms account for ~75% of the C-terminal peptide (Table 2). In contrast to the LC, the C-terminal peptide of Fd contains more of the truncated form DKKVEPKS, approximately ~75%, which eluted at 17.8 min in the Asp-N map as shown in Fig. 5B. The minor form of the C-terminal peptide DKKVEPKSC, eluting at 20.7 min, The MS/MS spectrum provides clear ) is . The most dominant ion of m/z 557.33 was observed in the full spectrum, which corresponds to the [MH- form. In addition, the observed b indicates that the -SO at the C-terminus (Fig. 6D). Fig. 6E shows the MS/MS spectrum of the truncated peptide DKKVEPKS, a series of y ions (y3, an amide at the C-terminus. Table 2 summarizes the quantitative information of each form of the C-terminal peptides. The fact that more truncated forms of the L corresponded to more Fd fragments with an extra –S at Cyswere generated through a loss or gain of the Cys residues. In other words, a fragment rminus could be the result of the gain of a sulfur group at the side chain of Cys in the counterpart. The truncate

18 d fragment may be explained by formed by
d fragment may be explained by formed by the loss of the sulfur group at mixture of –SOH and –S adducts at the implied that these products were likely generated by limited oxidation under low oxygen tension. Collectively, these observations indicated cleavages of the C-terminal residues in the non-disulfide bonded LC and Fd fragments by guest on January 11, 2021http://www.jbc.org/Downloaded from other peaks correspond to the masses of the adducts, respectively (Fig. 4A, Table 1). However, under reducing conditions, the L showed four peaks with molecular masses Da, respectively(Fig. 4B), which correspond to the fragment of Gluthe LC and the LC with +16 Da, +32 Da were disulfide bond related, implying that reducing conditions may be the result of at was attached to the side chain of Cys. The peptide mapping confirmed this conclusion (see below). It has been known that atmospheric oxygen is Cys-SH is heat-labile and can be oxidized into Cys-SOH and Cys-SOcan be converted into Cys-SOultimate product (29,30). It is possible that +48 Da derive from oxidation

19 during RP-HPLC-TOF/MS or sample processi
during RP-HPLC-TOF/MS or sample processing, and these adducts were added on to the extra Analyzing Fab portion of the H (Fd) under non-reducing conditions revealed very similar results. The Fd species eluting at 23.3 min was a mixture of two components with masses of 23375.7 Da and 23557.4 Da (Fig. 4C), corresponding to 224 with a +78 Da analysis indicated that the +78 Da extra mass was attributed to an additional SOthe C-terminus of Cys Unlike the peaks 1 and 2, the Fab of the peak 3 eluting at 24 min in the RP-HPLC 227228 at the C-terminal ends (Fig. 4D). The UV signal for the peak 3 (Fab) to the sum of the peaks esting that half of thermal incubation conditions. RP-HPLC-TOF/MS Analysis of the Fab treatment Compared to the Hmediated radical tension, thermal incubation induced and consumption of O Thus it remained to be determined if the H-L bond could be leading to break the inter-chain linkage at s. To explore this purified Fab was examined to determine if any additional breakage of the H-L or cleavage of residues around C-terminal Cys occurred. Th

20 e Fab at ~0.47 mg/ml was treated with 20
e Fab at ~0.47 mg/ml was treated with 20 mM H in a molar ratio : Fab at 2000:1 at 25 C for 6 days, and then analyzed by RP-HPLC-TOF/MS. before and after the Htreatment (not shown), implying that Cysin the Fab may be not the direct target for mediated radical reaction. Characterization of the products of the fragments of Glu-Glu and Asp of the LC with an by guest on January 11, 2021http://www.jbc.org/Downloaded from produced via the Fenton-like chemistry (reaction 5). Scheme 2: Thermal incubation generates hinge Critical evidence hinge cleavage of an IgG1 was obtained from analysis of an IgG1 incubated in C, using the same conditions asof the hinge cleavage were measured by size exclusion chromatography (SEC). As is produced resulted in more in PBS for 15 days induced a 3.7 % level of into the reaction resulted in an increase of cleavage to 9.7 %. A similar increase in cleavage was observed with (not shown). In addition, superoxide dismutase (SOD) was also SOD resulted in a cleavage level of ~17%, further increased the cleavage to ~32%. However, EDTA

21 was �found capable of inhibiting
was �found capable of inhibiting 95% of the metal ion induced cleavage, implying an involvement of the transitional metal ions ce of 5,5-dimethyl-1-e results suggest an involvement of H in the reactions and that the radical reaction mechanism may be An IgG molecule is itself capable of (19,20), which may lead to radical reactions that result in hinge cleavage. However, the self-produced Hhas been determined to generate at the interface of the heavy and light chains with 24). Thus, the fact that redox metal ions and/or SOD mediate the Hand the hinge cleavage excluded the the main force to drive the hinge cleavage. Analysis of the cleaved Fab by RP-While the radical mechanism may be responsible for the hinge cleavage, it remains to be determined if the thermal induced degradation generates the same products as those Shimazu, Buxton and Garrison etc (14,25,26) had demonstrated that the different under different reaction would help to address these questions. To exclusion chromatography (SEC) (Fig. profile with three marevealed that the peak 1 eluting at 16

22 .5 min derived from the LC, which was ch
.5 min derived from the LC, which was characterized by multiple peaks with masses of 23256.2, 23360.5, 23392.1, to the mass of a fragment of 214 by guest on January 11, 2021http://www.jbc.org/Downloaded from detection at 215 nm and then directed in-line to a TOF mass spectrometer. The initial mobile phase was 25% solvent B for 5 min, and then a two-stage gradient was applied: 2 % solvent B per min from 25 to B per min from 30 to 42% solvent B. The separation was performed at 75 °C at a flow rate of 0.5 ml/min. Electrospray ionization TOF/MS was performed on Applied Biosystems QSTAR Elite XL mass spectrometer equipped with an Agilent 1200 HPLC system. The electrospray ionization mass spectra were Biosystems). The IgG1 was denatured in the presence of 4 M GuHCl for 5 min at 75 °C. Prior to digestion, samples were buffer-exchanged into 50 mM Tris-HCl, pH 8.0, using Bio-Spin 6 columns (Bio-Rad) according to the manufacturer’s instrusequencing grade Asp-N or Lys-C (Roche an enzyme to protein ratio of 1:10 (w/w), overnight. Analytical peptide maps onto a Phenome

23 nex Jupiter Proteo C18 column, 2.0 mm x
nex Jupiter Proteo C18 column, 2.0 mm x 250 mm, heated at 55 °C. The separation was performed by gradient system. The column was held at the initial trifluoroacetic acid in water) at a flow rate of 0.3 ml/min for 5 min, and then the digest was eluted with a linear gradient to 60% acetonitrile) over 160 min. The peptides MS fragmentation using a Thermo LTQ Orbitrap mass spectrometer. Detection of HMolecular one on each oxygen atom. With the is a facile process (reaction 1 in scheme 2). In fact, the reduction of Ointo more reactive species OH is has been determined in a biological system (16,17,21). and its derivatives in a an easy and direct assay to measure the O We found that the chain reaction of the this reaction was obtained from the measurement of Hmethod as described previously (19,20). The rate of the H to a level that can be easily detected in PBS buffer is very limited, as no evidence for a continuing accumulation of buffer age. Similar observations were obtained from other buffers such as Tris- shown). We found reactions in scheme 1 can take place

24 via the following reactions in scheme 2:
via the following reactions in scheme 2: the Ounder catalysis of redox metal ions (reactions 2-3) or by SOD (reaction 4). by guest on January 11, 2021http://www.jbc.org/Downloaded from HC and dehydroalanine amide at new C-terminus. And, as Hthe inevitable by-prreactions in buffer systems, related strategies of how to improve the stability of EXPERIMENTMaterialis a recombinant fully human antibody of the IgG1 subclass. The molecule was expressed in Chinese Hamster Ovary (CHO) cells and chromatographically purified in Genentech, Inc. Examination of the primary sequence indicates that the antibody contains glutamic acid (Glu) at the N-terminus of the heavy chain (HC), and an aspartic acid (Asp) at the N-terminus of the light chain (LC). Thermal incubation of the IgG1phosphate buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Phosphate, C, with or or Feunits). Catalase (1000 mU, Sigma) was the reaction. In some cases, EDTA (50 mM) or DMPO (100mM) was added into the sample prior to addition production was determined using Amplex mixed with Amplex Red working solut

25 ion and incubated at room temperature, p
ion and incubated at room temperature, protected from light. The H 2 O 2 was measured by fluorescence signals. Fluorescence emission excited at 545 nm performed in six replicates, and the results were reported as the average.The degraded chromatography (SEC) on TSK G3000SWxl dual-columns, 7.8 mm 300 mm, at a flow rate of 0.5 mL/min. Eluting protein was monitored at 280 nm. The SEC running buffer contained 250 mM potassium phosphate, 200 mM potassium chloride at pH 6.2. The cleavage was measured by the relative percentage of integrated peak area of partial molecules. SEC fractionation was Agilent 1200 HPLC system (Agilent Millipore Centriprep YM-30 filter units with a 10-kDa molecular weight cutoff (Millipore, Billerica, MA). TOF/MS)RP-HPLC-TOF/MS was performed as described previously (7). Briefly, antibody samples were diluted to 1 mg/mL in 50 mM Tris-HCl (Sigma, St. Louis, MO) at pH 8.0 with a final hydrochloride (GdnHCl, Mallinckrodt). RP-HPLC was performed on an Agilent 1200 HPLC system. The mobile phase (Burdick Jackson) with 0.09% TFA as solvent B. A Varian PL

26 RP-S (Varian, Inc., 50 mm, 8 µm particl
RP-S (Varian, Inc., 50 mm, 8 µm particle size, 1000-Å pore size column was used for the RP-HPLC-TOF/MS analysis. The column eluent was analyzed by UV by guest on January 11, 2021http://www.jbc.org/Downloaded from found, interestingly, as the same as those mediated radical induced observation of the same cleavage sites under different conditions, e.g. thermal production, implies a common mechanism may exist to drive this unique degradation. Two types of the inter-chain disulfide mediated radical subsequent formation of radical species at which functions as the primary radical center in the hingefrom the radical center to upper hinge, radical cleavage per molecule basis to release Fab domain. Unlike the cell various cell surface receptors (9,10), there in buffers under thermal incubation conditions. This seems to cast a doubt if radical reaction mechanisms contribute significantly to thermal stress degradation. In addition, by catalytic decomposition of Hredox metal ions in Hmediated radical reactions either in the cell culture or in vitro reaction

27 systems(11,12), whereas it remains uncl
systems(11,12), whereas it remains unclear if such oxygen tensions exist under thermal incubation conditions. These factors are important, as Garrison demonstrated that radical reactions can take these reactions could facilitate the development efforts to improve the stability of an IgG1 under thermal stress Scheme 1: In this paper, we present experimental evidence that demonstrates the thermal radical reaction mechanisms with new that were not determined previously. Our results first show the existence of detectable Hin buffer systems, which is ) under thermal incubation conditions. ·- is the first intermediate product in molecular oxygen to water (Scheme 1)(15-nverted to hydrogen ) by redox active metal ions and / or superoxide dismutase (SOD) introduced into buffer systems. In addition to the previous observed Fab fragments (8,18), new fragments were observed, e.g. 224-Cys and Asp of the light chain (LC). Among them, an additional sulfur group was ain of the C-terminal fragments, respectively.termini of the fragments of the Glu224214determined. These f

28 ragments were not the products of simple
ragments were not the products of simple reduction of the inter-chain disulfide bond, rather, they were formed by radical reaction pathways that were not determined in mAbs previously. The results from analyzing the products by guest on January 11, 2021http://www.jbc.org/Downloaded from BREAKING THE LIGHT AND HEAVY CHAIN LINKAGE OF HUMAN IGG1 BY RADICAL REACTIONS From Department of Pharma Technical Development, Genentech, Oceanside, CA 92056 , Phone: 760 231 3029; Email: We report that the production of water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The be accelerated by superoxide dismutase or redox active metal ions, or inhibited correlates well with the hinge cleavage. mechanisms described previously, new degradation pathways and products were observed. These products were bond between CysDecomposition of the resulting disulfide The hydrolysis of the unsaturated an amide at the C-terminus of the new fragment. Meanwhile, the competition between the carbonyl (-Cthe side chain of Cys allows an electron

29 carbon, forming a new intermediate radi
carbon, forming a new intermediate radical species -. Dissociative deamidation occurs along the N-Cperoxide is a commonly observed role in mediating the unique degradation of an IgG1, strategies for improving Recombinant monoclonal antibodies (mAbs) represent one of the fastest-growing segments of biotechnology. More than 20 mAbs had been approved by the US Food and Drug Administration (FDA) ation is available in the research and development of therapeutic mAbs. Despthere are still unmet demands in improving the stability, efficacy, pharmacokinetic mAbs(1,2). New mechanisms that govern mechanisms underlying the instability of mAbs remain to be elucidated. As one of needs to be monitored in the entire development course because it can common observations in the stability testing program is the so-called “hinge cleavage”, which generates a Fab domain and a partial IgG1 that is missing the Fab. These products contain ladder cleavage nge residues at new termini of the products (3-7), which were by guest on January 11, 2021http://www.jbc.org/Downloaded f