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Discovery and Characterization of Histidine Oxidation Initiated Cross-links in an IgG1 Monoclonal Antibody Chong-Feng Xu, Rachel Chen, Linda Yi, Tim Brantley, Brad Stanley, Zoran Sosic, and Li Zang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00860 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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Analytical Chemistry

1 Discovery

and Characterization of Histidine Oxidation Initiated 2 Cross-links in an IgG1 Monoclonal Antibody 3 Chong-Feng Xu‡, a, Yunqiu Chen‡, a, Linda Yia, Tim Brantleyb, Brad Stanleyc, Zoran Sosica and Li 4 Zang*, a 5 aAnalytical Development, b Cell Culture Development, c Process Biochemistry, Biogen, Cambridge MA, 02142 6 *Corresponding Author: [email protected] 7 ABSTRACT: Novel cross-links between an oxidized histidine and intact histidine, lysine or cysteine residues were discovered and 8 characterized from high-molecular weight (HMW) fractions of an IgG1 monoclonal antibody (mAb). The mAb HMW fractions 9 were collected using preparative size-exclusion chromatography (SEC) and extensively characterized to understand the mechanism 10 of formation of the non-reducible and covalently linked portion of the HMWs. The HMW fractions were IdeS digested, reduced 11 and analyzed by size-exclusion chromatography coupled with mass spectrometry (SEC–MS). The non-reducible cross-links were 12 found to be enriched in the Fragment crystallizable (Fc) region of the heavy chain, with a net mass increase of 14 Da. Detailed pep13 tide mapping revealed as many as seven covalent cross-links in the HMW fractions, where oxidized histidines react with intact his14 tidine, lysine and free cysteine to form cross-links. It is the first time that histidine-cysteine (His–Cys) and histidine-lysine (His– 15 Lys) in addition to histidine-histidine (His–His) cross-links were discovered in monoclonal antibody HMW species. The histidine 16 oxidation hot spots were identified, which include conserved histidine residues His292 and His440 in the Fc region and His231 in 17 the hinge region of the IgG1 mAb heavy chain. Their cross-linking partners include His231, His292, His440, and Cys233 in the 18 hinge region and Lys297 in the Fc region. A cross-linking mechanism has been proposed which involves nucleophilic addition by 19 histidine, cysteine or lysine residues to the carbonyl-containing histidine oxidation intermediates to form the cross-links.

20 INTRODUCTION 21 The formation of aggregated high-molecular weight (HMW) 22 species in therapeutic proteins is a highly undesirable phe23 nomenon which can occur during bioprocessing, storage and 24 shipping.1 Protein aggregation can promote formation of par25 ticulates and may expose normally unexposed epitopes and 26 lead to increased immunogenicity of the protein. The poten27 tial elicitation of anti-drug antibodies against therapeutic pro28 teins due to presence of protein aggregates can have detri29 mental effects on drug safety, efficacy, and pharmacokinet30 ics.2 Modern cell culture and purification processes are usual31 ly capable of controlling aggregation at a low level, i.e. 0.532 2.0%, in monoclonal antibody (mAb) drugs. However, addi33 tional aggregates can form during storage, especially in case 34 of high concentration formulations (i.e. 50-200 mg/mL), and 35 cause significant challenges during the formulation develop36 ment.3 Due to the highly negative impacts on drug safety and 37 shelf life, aggregation is commonly considered as an obligato38 ry critical quality attribute (CQA) for monoclonal antibodies, 39 and has been studied extensively for the understanding of 40 their chemical structures, physiochemical properties, mecha41 nisms of formation, and changes during storage.1,4-6 42 Aggregation can form for a variety of reasons and may in43 volve both non-covalent molecular interactions and covalent 44 chemical cross-linking between two or more antibodies.5,7,8 45 The covalently linked aggregates can be further divided into 46 reducible aggregates, such as disulfide-linked aggregates 47 which can be dissociated by reducing reagents such as dithio48 threitol (DTT), and non-reducible aggregates, such as the

49 imine cross-link which is formed by the reaction of carbonyls 50 derived from protein oxidation with primary amines of lysine 51 residues.9 Due to the large diversity of potential chemical 52 reactions that may lead to formation of non-reducible cova53 lent aggregates, there is still lack of a comprehensive under54 standing of the mechanism of their formation. So far, only a 55 few non-reducible cross-linked structures have been charac56 terized in detail and reported for mAbs, including a thioether 57 cross-link between light and heavy chains of an IgG1 mAb,10 58 thioether bonded cross-links from an IgG2 mAb,11 and a his59 tidine-histidine (His–His) cross-link from photo-oxidation of 60 an IgG1 mAb.12 Other covalent cross-links discovered from 61 non-antibody proteins include dityrosine cross-links by oxida62 tive cross-linking between two tyrosine residues,13-15 as well 63 as cross-links between primary amines and a carbonyl64 containing oxidation product of tyrosine and phenylalanine.16 65 Besides, dicarbonyls from advanced glycation end products 66 (AGEs), such as methylglyoxal and glyoxal, can react with 67 lysine residues to produce lysine–lysine imidazolium cross68 linked AGEs, called methylglyoxal-lysine dimer (MOLD) 69 and glyoxal-lysine dimer (GOLD), respectively.17,18 70 The challenge in thoroughly characterizing the protein aggre71 gates is mostly attributed to their extremely low abundance, 72 highly heterogeneous nature, and a lack of efficient tools to 73 identify cross-links with unknown structure. In this study, we 74 developed an online size-exclusion chromatography coupled 75 with mass spectrometry (SEC–MS) method for fast identifica76 tion of non-reducible covalent aggregates from HMW species 77 isolated from an IgG1 mAb. The enriched HMW fractions of 78 the IgG1 mAb were first digested by IdeS endoprotease, then

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1 deglycosylated and reduced, and the resulted subu2 nits/fragments were analyzed directly by SEC–MS. The 3 cross-linked species involved in the HMW formation were 4 readily separated and identified by their intact masses, and 5 found to be predominantly cross-linked Fc region of the 6 heavy chain with a net mass increase of 14 Da. Detailed pep7 tide mapping further identified the cross-linked peptides and 8 pinpointed the cross-linked residues of three histidine resi9 dues in the heavy chain, His440, His292 and His231, with the 10 cross-linking partners including histidine, cysteine or lysine 11 residues. A mechanism for cross-linking is proposed based on 12 previous studies on His–His cross-links,12 with new cross13 linking partners, cysteine and lysine residues included. 14 MATERIALS AND METHODS 15 Chemicals. 8M Guanidine hydrochloride, LC/MS grade for16 mic acid (FA), trifluoroacetic acid (TFA), acetonitrile (ACN) 17 and water were from Fisher Scientific (Hampton, NH). DTT, 18 2-mercaptoethanol and iodoacetamide were purchased from 19 Sigma Aldrich (St. Louis, MO). Endoprotease Lys-C was 20 from Wako Chemicals (Japan), and Glu-C was from Promega 21 (Madision, WI). N-glycanase PNGase F was purchased from 22 New England Biolabs (Ipswich, MA). IdeS protease was pur23 chased from Genovis Inc (Cambridge, MA). 18O-water (97%) 24 was obtained from Cambridge Isotope Laboratories (Ando25 ver, MA). Recombinant monoclonal IgG1 antibody was pro26 duced in Biogen (Cambridge, MA). 27 Preparative SEC. The starting material used for SEC frac28 tionation was a drug substance (DS) sample held at 5°C for 29 one year, containing 8.0% HMW species, 91.0% monomer 30 and 1.0% low molecular weight species. This “aged” DS 31 sample was applied to a Superdex 200 Prep Grade column 32 (GE Healthcare Life Sciences, Marlborough, MA) for SEC 33 separation and the HMW (oligomer and dimer, respectively) 34 and monomer fractions were collected. 35 Analytical SEC–UV analysis. Analytical SEC–UV analyses 36 were conducted with a Waters H-class UPLC system. Seven37 ty-five microgram of sample was injected onto an Acquity 38 UPLC BEH SEC column (4.6 mm × 300 mm, 1.7 µm, 200Å, 39 Waters, Milford, MA) heated to 30°C. A mobile phase con40 taining 100 mM sodium phosphate, 200 mM sodium chloride, 41 pH 6.8 was delivered at a flow rate of 0.35 mL/min. UV ab42 sorption at 280 nm was acquired and processed by Empower 43 3 software (Waters, Milford, MA). 44 Non-reducing and reducing capillary electrophoresis– 45 sodium dodecyl sulfate (CE–SDS). Non-reducing and re46 ducing CE–SDS analyses were conducted using a high per47 formance capillary electrophoresis system (PA 800 plus 48 Pharmaceutical Analysis System; AB SCIEX, Framingham, 49 MA). Sample preparation for the reducing CE–SDS involves 50 heating 200 µg of sample mixed with a 10 kDa internal stand51 ard as well as SDS-MW sample buffer and 2-mercaptoethanol 52 at 70 °C for 5 minutes. For non-reducing CE–SDS, heating of 53 the same mixture was performed with iodoacetamide instead 54 of 2-mercaptoethanol. In both methods, the separation was 55 conducted at a constant voltage of –15 kV for 35 minutes 56 with UV detection (λ = 220 nm). 57 Reversed phase LC–MS (RPLC–MS) of the deglycosylat58 ed and reduced mAb. One hundred micrograms of the mAb

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59 SEC fractions were deglycosylated with PNGase F and then 60 reduced at room temperature for 30 minutes using DTT. Two 61 micrograms of the resulted samples were separated on a 62 TSKgel® Phenyl-5PW HPLC column (2.0 × 75 mm, 10 µm) 63 at 55 °C with a flow rate of 0.4 mL/min using an Agilent 64 1290 HPLC connected with an Exactive Plus Orbitrap mass 65 spectrometer (ThermoFisher Scientific, Waltham, MA). Mo66 bile phase A was 0.03% TFA in water and B was 0.024% 67 TFA in ACN. Samples were separated using a gradient of 68 mobile phase B from 28% to 33.5% within 3.5 minutes. The 69 mass spec settings were as following: In source CID: 50 eV; 70 mass range: m/z of 800-3500; Resolution: 17500. Thermo 71 BiopharmaFinder software (v1.0) was used to deconvolute 72 the acquired mass spectra, with the use of Manual RespectTM 73 (isotope unresolved) algorithm. 74 SEC–MS of IdeS digested, deglycosylated and reduced 75 mAb. Fifty micrograms of mAb fractions was mixed with 15 76 µL of 50 mM sodium phosphate buffer, pH 7.2 and 1 µL of 77 IdeS endoprotease (66 IU/µL) and incubated at 37°C for 30 78 minutes, followed by deglycosylation using 1 µL of PNGase 79 F (500 IU/µL) at 37 °C for 3 hours. Reduction was performed 80 at room temperature for 30 minutes with addition of 7.5 µL of 81 100 mM DTT. Six micrograms of the reduced samples were 82 injected for SEC–MS analysis using an Agilent 1290 HPLC 83 connected with an Exactive Plus Orbitrap mass spectrometer. 84 The SEC separation was performed on a Waters Acquity 85 UPLC BEH SEC column (4.6 × 150 mm, 1.7 µm, 200Ǻ), by 86 running an isocratic flow of a mobile phase containing 0.05% 87 TFA, 0.1% FA and 20% ACN at a flow rate of 0.25 mL/min 88 with column temperature at 26°C. Similar mass spectrometry 89 parameters were used as those in the RPLC–MS method, with 90 the following exceptions: in source CID: 60 eV; mass range: 91 m/z of 1000–5000. 92 Lys-C digestion for peptide mapping. SEC fractions (100 93 µg) of IgG1 was first denatured and reduced in the presence 94 of 8 M guanidine and 4 mM DTT for 30 min. Lys-C digestion 95 was performed at 25 ºC for 18-20 hours after 4-fold dilution 96 with digestion buffer (50 mM phosphate buffer, 10 mM 97 EDTA, pH 7.2) and with the addition of 10 µg Lys-C. 98 Combined Lys-C and Glu-C digestion in normal water or 99 18O-water. The early-eluting peak (retention time window of 100 3.6-4.2 min, Figure 5B) from the above SEC separation of 101 IdeS generated mAb fragments was collected and subject to 102 combined Lys-C and Glu-C digestion. After denaturation and 103 reduction, the sample was exchanged to digestion buffer and 104 divided into two aliquots, each completely dried down. The 105 two aliquots were reconstituted in either normal water or 106 heavy isotope 18O-labeled water (H218O). Lys-C and Glu-C (5 107 µg each) were added to both aliquots for overnight digestion 108 at 25 ºC. 109 RPLC–MS of proteolytic digest. The proteolytic digests 110 were analyzed using an Agilent 1290 HPLC system coupled 111 with an Orbitrap Fusion Tribrid mass spectrometer. The sam112 ples were separated on an Acquity HSS T3 UPLC column 113 (2.1 × 100 mm, 1.8 µm, 100 Å, Waters, MA) at 55 °C with a 114 flow rate of 200 µL/min using a gradient of 3.0–35.0% of 115 mobile phase B (0.024% TFA in ACN) vs. mobile phase A 116 (0.03% TFA in water). For the mass spectrometry analysis, an 117 interplay was conducted by full MS scan from m/z 400 to


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Figure 1. Analytical workflow for characterization of HMW factions of an IgG1 mAb. (1) Reducing and non-reducing CE-SDS analyses detected non-reducible covalent cross-links in HMW fractions. (2) Subunit analysis led to the identification of +14 Da in the cross-linked species. (3) Lys-C peptide mapping and database search against theoretical cross-linked peptide library. Cross-linked peptides were tentatively assigned based on the mass matching. (4) The early-eluting SEC peak from the Ides and PNGaseF digested mAb was collected and subject to combined Lys-C and Glu-C digestion. MS/MS was performed to confirm the peptide assignments as well as identify additional cross-linked peptides. The Lys-C and Glu-C digestion was also performed in 18O-water to confirm identification of the cross-linked peptides by a mass shift of 8 Da.

1 1600 with 120,000 resolution power (at m/z 400) followed by 2 data-dependent MS/MS scans. A few selected peptides were 3 fragmented in a targeted way, using collision induced dissoci4 ation (CID) or electron transferred dissociation (ETD) meth5 ods. MS/MS spectra were acquired either in the Orbitrap with 6 15,000 resolution and AGC value of 1E6 or in the ion trap 7 with an AGC target of 5E4. LC–MS data for the proteolytic 8 digest was analyzed using Thermo PepFinder 2.0 followed by 9 manual confirmation or interpretation. The MS/MS spectra of 10 cross-linked peptides were processed by StavroX (v 3.6.0)19 11 to facilitate the identification. 12 RESULTS AND DISCUSSION 13 The HMW fractions of the IgG1 mAb were subject to detailed 14 characterization following the workflow shown in Figure 1. 15 CE-SDS analyses were performed, which led to the detection 16 of covalently cross-linked and non-reducible species from 17 HMW fractions. Subunit analyses led to the identification of 18 +14 Da involved in the cross-links. Detailed peptide mapping 19 analysis resulted in the identification of cross-linked peptides 20 and their identifications were further confirmed by MS/MS 21 analysis. 22 Detection of cross-linked mAb from HMW fractions. In 23 order to fully understand the structural characteristics of 24 HMW species, three fractions of the IgG1 mAb were collect25 ed using a preparative SEC and their purity was assessed us26 ing analytical SEC (Figure S-1). The first fraction contained 27 28.3% oligomer and 60.3% dimer (referred as Oligomer); the 28 second fraction contained 4.7% oligomer and 83.5% dimer 29 (referred as Dimer), and the third fraction contained 99.3% 30 monomer (referred as Monomer) (Table S-1). 31 The SEC fractions were subject to CE–SDS analyses under 32 both non-reducing (Figure 2) and reducing conditions (Fig33 ure 3). From the non-reducing CE–SDS electropherograms, 34 two peaks (peak 3 and peak 4) migrating later than the mon35 omer peaks (peak 1 and peak 2) were observed at significant36 ly higher levels in the oligomer and dimer fractions compared 37 to the monomer fraction (Figure 2). These two peaks ap38 peared to be HMW species which remained stable under the

39 denaturing conditions of the CE–SDS analysis, therefore were 40 most likely formed by either disulfide linkages or other non41 reducible covalent linkages. Relative peak areas revealed that 42 these covalently cross-linked HMW species accounted for

43 44 Figure 2. Non-reducing CE–SDS analysis of (A) monomer, (B) 45 dimer and (C) oligomer fractions. Based on the migration time, 46 peak 1 to 4 was assigned as aglycosylated monomer, monomer, 47 dimer and oligomer, respectively. 48 approximately 74.5% and 81.5% of the dimer and oligomer 49 fractions, respectively. In contrast, these two peaks were un50 detectable in the monomer fraction (Figure 2A). 51 From the reducing CE–SDS electropherograms (Figure 3), in 52 addition to the light chain (LC, migrating at ~15 min), heavy 53 chain (HC, ~19 min) and the aglycosylated heavy chain 54 (~18.5 min), two groups of late-migrating peaks at approxi55 mately 22 min and 24 min appeared at significantly higher 56 levels in the oligomer and dimer fractions compared to the 57 monomer fraction. Based on the migration time, these two 58 groups of peaks were assigned to be heavy chain-light chain 59 dimer (HC–LC) and heavy chain-heavy chain dimer (HC– 60 HC), respectively. In the meantime, these peaks remained



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1 stable under denaturing and reducing conditions, indicating 2 they were formed by covalent linkages other than disulfide 3 bridges. The multiple peaks observed within the two groups 4 of cross-linked species (HC–LC and HC–HC) likely resulted 5 from structural isomers due to the heterogeneity of the cross6 links. Based on the peak area, the non-reducible covalently 7 cross-linked species accounted for 19.7% and 22.2% of the 8 dimer and oligomer fractions, and 2.7% in the monomer frac9 tion. The low level cross-linked species found in the mono10 mer fraction could result from intramolecular cross-linking, 11 which was not detectable by SEC, or could have been intro12 duced during sample preparation.

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43 HC), light chain (LC) and single-chain Fc (scFc) (Figure 44 5A). In the SEC–MS chromatograms of the dimer and oligo45 mer fractions, an early eluting peak in the retention time win46 dow of 3.6–4.2 minute was observed (Figure 5B) and its 47 deconvoluted mass spectrum is shown in Figure 5C. The 48 most abundant species has a molecular weight of 47589 Da, 49 which is 14 Da higher than the mass of two scFc (calculated 50 mass 47575 Da). In addition, masses that are 14 Da higher 51 than the dimer of scFc–Fd, scFc–LC and Fd–Fd were also 52 observed. ScFc appeared to be most enriched in cross-links as 53 it reacted with all other subunits and resulted in 3 out of the 4 54 observed cross-linked species in this early-eluting peak. In the 55 meantime, the scFc–scFc dimer species was present at the 56 highest abundance based on the peak intensity.

57

13 14 Figure 3. Reducing CE–SDS analysis of (A) monomer, (B) di15 mer and (C) oligomer fractions. Peaks corresponding to light 16 chain (LC), heavy chain (HC), aglycosylated heavy chain (HC17 agly) and cross-linked species are labeled. 18 Detection of an unknown +14 Da modification in cross19 linked mAb. As shown in Figure 1, in order to probe the 20 chemical nature of these non-reducible covalent HMWs, the 21 SEC fractions were subject to subunit analyses using RPLC– 22 MS of deglycosylated and reduced antibody and SEC–MS of 23 IdeS digested and reduced antibody. 24 In the RPLC–MS chromatogram (Figure 4), a peak at around 25 4.4 min was observed in the oligomer and dimer fractions but 26 not in the monomer fraction. The dominant mass for each 27 peak was summarized in Figure 4. The late-eluting peak con28 tained a HMW species with molecular weight of 99527 Da, 29 which is 13 Da higher than twice the heavy chain molecular 30 weight (99514 Da). The mass difference is much larger than 31 the mass error typically observed by the high-resolution Or32 bitrap mass spectrometer (within 2 Da for a 100 kDa protein, 33 assuming the mass accuracy is within 20 ppm). Therefore, the 34 13 Da mass difference may reflect the mass addition related 35 to a cross-link formed between two heavy chains in the HMW 36 species in this late-eluting peak. 37 SEC–MS of the IdeS digest of the SEC fractions was next 38 performed. The oligomer, dimer and monomer fractions were 39 first digested using IdeS, which cleaved between two glycine 40 residues slightly below the hinge region, followed by N41 glycan removal and protein reduction. Three fragments were 42 formed from an IgG1 mAb including Fd (Fab region of the

Obs. mass (Da)

Theor. mass (Da)

∆ mass (Da)

LC

23203

23204

-1

HC

49756

49757

-1

cross-linked species

99527

99514*

+13

58 Figure 4. Total ion chromatograms from RPLC–MS analysis of 59 the monomer (green), dimer (red) and oligomer (black) fractions 60 after deglycosylation and reduction. The deconvoluted masses 61 and their differences from the theoretical values for light chain 62 (LC), heavy chain (HC) and cross-linked species are shown in 63 the table. (* Theoretical mass for the cross-linked species was 64 calculated as twice the heavy chain mass). 65 Identification of the cross-linked peptides. In order to 66 achieve a clear understanding of the chemical structure asso67 ciated with the cross-linked species, two separate peptide 68 mapping analyses were performed. Lys-C peptide mapping 69 of the SEC fractions was first conducted and mass matching 70 of the observed peptides with the theoretical cross-linked 71 peptide library resulted in tentative assignments of cross72 linked peptides. Next, combined Lys-C and Glu-C peptide 73 map followed by MS/MS analysis confirmed the assignments 74 from the Lys-C peptide map and identified additional cross75 linked peptides. The workflow is as illustrated in Figure 1. 76 The LC–MS data of Lys-C digest of three SEC fractions were 77 processed through software and manual analysis. First, LC– 78 MS data was analyzed by PepFinder and ions showing over 79 2-fold increase in their intensities in the oligomer and dimer 80 fractions compared to the monomer fraction were extracted. 81 The ion list was then searched against a peptide library con82 taining all possible combinations of two completely digested 83 Lys-C peptides of the mAb with a 14 Da mass increase. A 84 few matches were identified as potential cross-linked peptides 85 out of this effort (Table 1), which showed masses of 13.98 Da 86 higher than combined mass of two original Lys-C peptides. 87 This is in agreement with the results from the RPLC–MS and 88 SEC–MS subunit analyses, described in the previous sections.


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Figure 5. SEC–MS subunit analysis of mAbs after IdeS digestion. (A) IdeS, PNGaseF and DTT treatment produces three subunits from an mAb: light chain (LC), Fd and scFc (deglycosylated). (B) SEC–MS total ion chromatograms showing the three subunits and the crosslinked species from the monomer (green), dimer (red) and oligomer (black) fractions. (C) Deconvoluted mass spectrum of the cross-linked species is shown to be dominated by the scFc–scFc cross-link.

1 The cross-links were found to involve two peptides, S422– 2 K446 and F282–K295, both located in the Fc region of the 3 heavy chain. It was found that these two peptides cross-linked 4 with themselves and with each other. 5 The early-eluting peak (3.6-4.2 min, Figure 5B) from the SEC 6 separation of IdeS generated mAb fragments was collected, 7 which contains enriched cross-linked species. In order to con8 firm the assignments of cross-linked peptides, a combined 9 digestion using both Lys-C and Glu-C was performed for the 10 early-eluting SEC fraction to shorten the peptides for a more 11 effective MS/MS fragmentation. From the analysis, three 12 cross-linked peptides formed between A438–K446 (part of 13 Lys-C peptide S422–K446) and V291–K295 (part of Lys-C 14 peptide F282–K295) were identified (Table 2), which con15 firmed the cross-linked Lys-C peptides in Table 1. A similar 16 data analysis using PepFinder and mass matching with theo17 retical cross-linked peptide library was performed for the 18 combined Lys-C and Glu-C peptide map, and identified addi19 tional cross-linked peptides involving T230–E240 and T296– 20 E301 as listed in Table 2. 21 Confirmation of cross-linked peptides by Lys-C and Glu22 C digestion in 18O-labeled water. To further confirm the 23 identification of the cross-linked peptides, the combined Lys24 C and Glu-C digestion was also performed in 18O-water using 25 the protocol published by Liu et al.11 Protein digested in 18O26 water results in the incorporation of two 18O atoms in the C27 terminus of each peptide, with a total of 4 Da mass shift. 28 Cross-linked peptides have two separate C-termini and theo29 retically should incorporate two 18O atoms at each C-termini, 30 resulting in 8 Da mass shift. All the peptides shown in Table 31 2 showed 8 Da mass shifts when digested in 18O-water, which 32 confirmed that these listed peptides had two C-termini and 33 were cross-linked peptides (Figure S-2). 34 Identification of cross-linked residues by MS/MS. MS/MS 35 spectra of the cross-linked Lys-C and Glu-C digested peptides 36 were analyzed by StavroX software as well as manual exami37 nation, which successfully confirmed the cross-linked peptide 38 identification in Table 2 and pinpointed the amino acid resi39 dues involved in cross-linking. It was found that the cross-

40 links were always associated with a histidine residue present 41 in one of the peptides, and three “hot spots” were identified 42 including His292 and His440 present in the Fc region and 43 His231 in the hinge region of the mAb heavy chain. The CID 44 MS/MS spectra of three cross-linked peptides related to 45 His292 are shown in Figure 6, which led to the identification 46 of His–His, His–Cys and His–Lys cross-links. Figure 6A 47 shows the CID MS/MS spectrum of the cross-linked V291– 48 K295/V291–K295 peptide (m/z = 575.309, z = 2). From the 49 N-terminal b ion series, the observed b2, b3, b4 ions all con50 tained the mass of its cross-linked partner, indicating the 51 cross-linking site is either V291 or H292. From the C52 terminal y ion series, y4 ion contained the cross-linked partner 53 whereas y2 and y3 did not, which confirmed the cross-link 54 occurred between two His292 residues. The CID MS/MS 55 spectrum of the cross-linked peptide V291–K295/T230–E240 56 is shown in Figure 6B. The fragmentation ions generated 57 from T230–E240 (α peptide), including y8α, y9α, y10α and 58 b4α, b7α, b9α, b10α ions all contained the mass of its cross59 linked partner V291–K295, while y2α, y4α and y7α ions did 60 not, indicating Cys233 is one of the cross-linked residues. 61 Since there was no fragmentation ions observed for V291– 62 K295 (β peptide) for this cross-link, we tentatively assigned 63 His292 as the residue cross-linked with Cys233, based on the 64 analysis of other cross-linked peptides. Similarly in the CID 65 MS/MS spectrum of the cross-linked peptide V291– 66 K295/T296–E301 (Figure 6C), fragmentation ions b2β 67 through b4β from the peptide V291–K295 (β peptide) con68 tained the mass of its cross-linked partner T296–E301, indi69 cating His292 is likely the cross-linked residue. Fragmenta70 tion ions b2α and y5α on T296–E301 both contained the 71 cross-linking site, indicating Lys297 is the cross-linked resi72 due that reacted with His292. 73 It was interesting to notice that in most of the CID MS/MS 74 spectra of cross-linked peptides, the masses representing the 75 two peptides participating in the cross-link were found, one of 76 which was intact and the other with 14 Da mass addition (la77 beled in green in Figure 6). These fragmentation ions were 78 generated by cleavage along the cross-linked bond. It sug79 gests the cross-linked bond may be one of the most labile



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1 Table 1. Cross-linked peptides identified from Lys-C peptide mapping. m/z Obs. mass Theor. (Da) mass (Da)a (charge)

∆ Mass (Da)

H440–H440

998.794 (6+)

5986.720 5972.743

13.977

(K)FNWYVDGVEVHNAK(T) (K)FNWYVDGVEVHNAK(T)

H292–H292

842.900 (4+)

3367.571 3353.589

13.981

(K)SRWQQGNVFSCSVMHEALHNHYTQK(S) (K)FNWYVDGVEVHNAK(T)

H440–H292

1170.294 4677.149 4663.166 (4+)

13.981

Peptide Name

Cross-linked peptide sequence

Cross-linked residues

S422–K446/ S422–K446

(K)SRWQQGNVFSCSVMHEALHNHYTQK(S) (K)SRWQQGNVFSCSVMHEALHNHYTQK(S)

F282–K295/ F282–K295 S422–K446/ F282–K295

2 3 Table 2. Cross-linked peptides identified from combined Lys-C and Glu-C peptide mapping. Peptide Name

Cross-linked peptide sequence

Cross-linked residues

m/z (charge)

V291–K295/ V291–K295

(E)VHNAK(T) (E)VHNAK(T)

H292–H292

575.309 (2+)

1148.603

1134.626

13.977

V291–K295/ A438–K446

(E)VHNAK(T) (E)ALHNHYTQK(S)

H292–H440

564.956 (3+)

1691.846

1677.869

13.977

A438–K446/ A438–K446

(E)ALHNHYTQK(S) (E)ALHNHYTQK(S)

H440–H440

746.037 (3+)

2235.089

2221.114

13.975

V291–K295/ T230–E240

(E)VHNAK(T) (K)THTCPPCPAPE(L)

H292–C233

867.389 (2+)

1732.763

1718.787

13.976

V291–K295/ T296–E301

(E)VHNAK(T) (K)TKPREE(Q)

H292–K297

670.848 (2+)

1339.681

1325.705

13.976

A438–K446/ T230–E240

(E)ALHNHYTQK(S) (K)THTCPPCPAPE(L)

H440–C233

759.676 (3+)

2276.006

2262.031

13.975

T230–E240/ T230–E240

(K)THTCPPCPAPE(L) (K)THTCPPCPAPE(L)

773.315 (3+)

2316.923

2302.948

13.975

230

–

230

THTC233 THTC233 b

Obs. mass Theor. mass (Da)a (Da)

∆ Mass (Da)

4 a Theoretical masses are calculated by summing the masses of the two original peptides. b The residues involved in cross-linking is not 5 clear based on currently available data and the cross-linking sites were narrowed down to the region between T230 and C233 in this pep6 tide. All masses shown are monoisotopic masses. Cross-linked residues are shown in red. 7 bonds to cleave during CID fragmentation. This unique 29 His440 not only reacted with His440 or His292 from another 8 fragmentation pattern is especially helpful to identify which 30 Fc molecule, but also reacted with Cys233 in heavy chain 9 one of the peptides contains the 14 Da addition and likely 31 hinge region to form cross-linked peptides. The exact link10 initiates the cross-linking reaction according to the mecha32 age between T230–E240/T230–E240 cross-link is unclear 11 nism described later in the paper. As an example, two chro33 based on the current MS/MS data, but could be narrowed 12 matographically separated peaks both representing the cross34 down to the sequence of 230THTC233. 13 linked V291–K295/A438–K446 peptide formed between 35 Mechanism of cross-linking reaction. The cross-linking 14 His292 and His440 showed different MS/MS fragmentation 36 between two histidine residues in a light-stressed mAb has 15 patterns, as demonstrated in Figure S-3. In one MS/MS 37 been previously reported by Liu et al.12 The proposed 2-step 16 spectrum, the fragment ion representing peptide V291–K295 38 reaction mechanism includes the first step, histidine side 17 +14 Da was found, while in another spectrum fragments for 39 chain undergoing photo-oxidation with a singlet oxygen and 18 A438–K446 +14 Da was detected instead. It indicated that 40 forming an unstable and reactive endoperoxide intermediate, 19 both His292 on peptide V291–K295 and His440 on peptide 41 which then converts to two intermediates, 2-oxo-histidine (220 A438–K446 were capable of carrying the 14 Da addition and 42 oxo-His, +14 Da) and His+32 species (+32 Da) (Figure 7). 21 initiating the cross-linking reaction. Although the final cross43 The second step involves nucleophilic attack at the His+32 22 linked peptides caused by either the oxidized His292 or the 44 intermediate by the imidazole side chain from another histi23 oxidized His440 had the same molecular weight, their chem45 dine, followed by elimination of a water molecule to form 24 ical structures were different and resulted in the chromato46 the final cross-linked product. This reaction involves the 25 graphic separation of the two isomeric cross-linked peptides. 47 incorporation of two oxygen atoms and elimination of a wa26 ETD and CID MS/MS spectra of other cross-linked peptides 48 ter molecule, and results in a net mass increase of 13.979 Da, 27 associated with His292, His440 or His231 are shown in Fig49 which agrees with the mass difference observed in our study. 28 ures S-4 and S-5. These MS/MS spectra confirmed that 50 A similar mechanism that involves histidine oxidation and ACS Paragon Plus Environment

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1 nucleophilic addition is proposed as shown in Figure 7 for 2 the cross-links found in this study. 3 Previously reported histidine related cross-links were mostly 4 found under light-stressed conditions, as photo irradiation is 5 a well-established pathway to generate singlet oxygen, which 6 in turn causes histidine oxidation through peroxide interme7 diates.12,20-22 However, in our study, the multiple histidine 8 initiated cross-links were discovered from an IgG1 mAb 9 manufactured by fed-batch process and stored at 5°C without 10 light stress. The histidine oxidation which has initiated the 11 cross-linking in this study therefore is not a result of light 12 stress. 13 In addition to light stress, histidine could be oxidized by 14 endogenously generated singlet oxygen or other reactive 15 oxygen species (ROS) to form the peroxide intermediates. 16 Formation of singlet oxygen has been reported by UV or 17 visible light radiation through endogenous photosensitizers 18 including vitamins and enzyme cofactors.23,24 Singlet oxygen 19 may also be produced in biological systems through non20 photosensitized mechanisms, such as decomposition of lipid 21 hydroperoxides.25,26 Additionally, the histidine endoperoxide

22 intermediates could also originate from endogenous process23 es other than singlet oxygen. It has been reported that protein 24 peroxides may be produced through enzymatic processes 25 involving peroxidase, lipoxygenase and cyclooxygenase, 26 heme mediated reactions, Fe2+/H2O2 reaction, auto-oxidation 27 of sugars, etc.27 Histidine could also be oxidized by hydroxyl 28 radicals or superoxide anion radicals, through metal cata29 lyzed oxidation mechanisms.28 Histidine is a natural metal 30 binding amino acid due to the presence of an imidazole side 31 chain. His231 and His440 have been reported as Cu2+ bind32 ing sites for an IgG129 and an IgG2 antibody,30 respectively. 33 Histidine residues from various proteins have been reported 34 to be converted to 2-oxo-His through metal catalyzed oxida35 tion.31,32 36 Although it is not clear through which mechanism the histi37 dine residues were oxidized in our samples, a few histidine 38 oxidation products were detected from these HMW fractions, 39 including the 2-oxo-His (+14 Da), His+32 (+32 Da) and 40 His+16 (+16 Da). The Lys-C peptides with masses 14 and 32 41 Da higher than the unmodified peptides S422–K446 and 42 F282-K295 were observed (Table S-2). The MS/MS spectra

43 44 Figure 6. CID MS/MS spectra for cross-linked peptides related to V291–K295 from combined Lys-C and Glu-C digestion. (A) V291– 45 K295/V291–K295 cross-link, precursor ion m/z 575.309, z =2. (B) V291–K295/T230–E240 cross-link, precursor ion m/z 867.389, z = 2. 46 (C) V291–K295/T296–E301 cross-link, precursor m/z = 670.848, z = 2. b ions are shown in red, y ions shown in blue and ions generated 47 by fragmentation along the cross-linked bonds are shown in green. In (B) and (C), the two peptides involved in cross-links were labeled as 48 α and β. The spectra (A) and (B) were acquired at high resolution in the Orbitrap and (C) was acquired in the ion trap. The cross-linked 49 structures were drawn based on the reaction mechanism shown in Figure 7 and m/z of the fragments formed upon cleavage of the cross50 linked structure are marked in green in the structure.



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1 confirmed that the +14 Da and +32 Da modifications oc- 61 In this study, we discovered a series of novel histidine oxida2 curred to His440 and His292 (Figure S-6). 62 tion initiated cross-linked species from the HMW fractions of 3 The His+32Da intermediate contains a conjugated carbonyl 63 an IgG1 mAb through detailed mass spectrometric analysis. 4 that can undergo 1,4-addition reaction with nucleophiles.33 In 64 These cross-links were found involving one of the histidine 5 addition to the side chain imidazole of histidine, both the thiol 65 residues in antibody heavy chain, among His292, His440 in 6 group of free cysteine and the side chain amino group of ly- 66 the Fc region and His231 in the hinge region of the studied 7 sine are capable of nucleophilic addition reaction with the 67 mAb. These histidine residues formed cross-links with anoth8 His+32 intermediate. In this study His–Lys cross-link was 68 er histidine, lysine or cysteine residue, through a 9 identified between His292 and Lys297, and His-Cys cross10 links were identified between His292 and Cys233 as well as 11 His440 and Cys233. To our knowledge, this is the first time 12 that His–Lys and His–Cys cross-links were identified from an 13 mAb, although previous reports suggested histidine and ly14 sine might participate in the photo-induced cross-linking in 15 other protein and peptides.20,34 The reactivity of Cys, Lys and 16 His as nucleophilic attackers for conjugated carbonyls has 17 been confirmed by previous studies on lipid peroxidation, 18 where Cys, Lys and His were reported as the most reactive 19 amino acids for 4-hydroxynoneal (4-HNE), a conjugated car20 bonyl-containing compound from lipid oxidation.35,36 It is 21 well-known that free thiols in cysteine residues are strong 69 22 nucleophiles, while oxidized thiols (in the form of disulfide 23 bonds) are not nucleophilic. The presence of low-level free 70 Figure 7. Proposed mechanism for the formation of histidine 24 cysteine is a common feature of mAbs, usually at 1-5%.37 The 71 initiated cross-links. The mechanism for histidine oxidation is 25 flexible structure and good solvent accessibility of hinge 72 unclear. 26 could have made Cys233 a better partner of cross-linking than 73 mechanism involving oxidation of histidine and subsequent 27 free cysteines at other locations. 74 nucleophilic addition by intact histidine, lysine or cysteine. 28 In this study, we discovered three histidine residues as “hot 75 The discovery of novel cross-links between His–Lys and His– 29 spots” for cross-linking reaction in an IgG1 mAb, which are 76 Cys has advanced our knowledge about the mAb aggregation 30 His292, His440 and His231 of the heavy chain. In addition to 77 formation. As discussed above, the histidine oxidation initiat31 the previously reported His231–His231 cross-link,12 it is the 78 ed cross-links were found in the HMW fractions of two in32 first time that His292 and His440 were found involved in 79 house mAb molecules, indicating this pathway may apply 33 cross-link through the histidine oxidation pathway. The iden- 80 generally to IgG1 mAbs to form non-reducible covalent ag34 tification of His292 and His440 as cross-linking hot spots is 81 gregation. 35 corroborated by a previous report by Cockrell et al., in which 82 In contrast to previous studies, the cross-links were found 36 they suggested two peptides spanning the same regions from 83 from HMWs isolated from an aged mAb drug substance, 37 trastuzumab were involved in the photo-induced cross-link 84 which suggested that the histidine oxidation likely occurred 38 based on their low recoveries in the peptide mapping.20 Fukui 85 either during manufacturing or storage which induced cross39 et al. also reported that histidine residues in equivalent posi- 86 linking of two or more mAb molecules to form the HMW 40 tions of His 292 and His440 were among the most susceptible 87 species. With more sensitive detection tools and more sophis41 ones to oxidation by light stress and singlet oxygen (induced 88 ticated software for data analysis, we expect more cross42 by H2O2 with molybdate).38 89 linked structures involved in protein covalent aggregation to 43 Since the three identified histidine hot spots are located in the 90 be reported in the future. This should lead to a better under44 conserved regions of mAbs, it is expected that this type of 91 standing and control of HMW formation in mAbs, which in 45 cross-links could also occur to other mAbs in addition to the 92 turn will allow for improved bioprocess and formulation de46 particular molecule studied. A different in-house IgG1 mAb 93 velopment of mAb biopharmaceuticals. 47 was found with the same cross-links, indicating that this 48 cross-linking mechanism could apply to other human IgG1 94 ASSOCIATED CONTENT 49 mAbs. In the X-ray structure of the Fc region of human IgG1 50 mAb (Figure S-7), both His440 and His292 are located at the 95 SUPPORTING INFORMATION 51 loop regions and His231 is located in the hinge region. All 96 The Supporting Information is available free of charge on the 52 these three histidine residues have relatively higher flexibility 97 ACS Publications website. 53 and better solvent accessibility than other histidine residues,38 54 which may have led to their higher susceptibility to oxidation 98 AUTHOR INFORMATION 55 and cross-linking than many other histidine residues in an 99 Author Contributions 56 IgG1 mAb. Their cross-linking partners, Lys297 in a loop The manuscript was written through contributions of all authors. 57 region of the Fc and Cys233 in the hinge region, also have 100 101 ‡These authors contributed equally. 58 good solvent accessibility and flexibility for the cross-linking 59 reaction. 102 ACKNOWLEDGMENT 103 The authors thank Yi Pu for his technical support of the studies. 60 CONCLUSIONS 104 REFERENCES ACS Paragon Plus Environment

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