Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody

Molecular Immunology 45 (2008) 701–708

Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody Hongcheng Liu ∗ , Georgeen Gaza-Bulseco, Tao Xiang, Chris Chumsae Process Sciences Department, Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, United States Received 19 June 2007; received in revised form 5 July 2007; accepted 10 July 2007 Available online 24 August 2007

Abstract Methionine (Met) is one of the most susceptible amino acids to oxidation. Met256 (CH2-Met15.1) and Met432 (CH3-Met107) of a recombinant humanized monoclonal IgG1 antibody are located in the CH2 and CH3 domains, respectively. In three-dimensional structure, these two Met residues are close to the CH2-CH3 interface. In close proximity, oligosaccharides on the conserved asparagine (Asn) residues are enclosed in the CH2 domains. The relationship of Met oxidation with oligosaccharides and their effect on the structure of the antibody was investigated. Removal of oligosaccharides did not alter the oxidation rates of Met256 and Met432, however it caused significant structural changes as evidenced by the susceptibility of the deglycosylated antibody to trypsin and chymotrypsin. Oxidation of Met256 and Met432 did not cause significant conformational changes of the antibody with oligosaccharides, however oxidation of these Met residues accelerated degradation of the deglycosylated antibody. Analysis by mass spectrometry indicated that most of the protease cleavage sites were in the CH2 domains, which suggested that conformational changes induced by the removal of oligosaccharides and further by Met oxidation were local to the CH2 domains. © 2007 Elsevier Ltd. All rights reserved. Keywords: Recombinant monoclonal antibody; Glycosylation; Oxidation; Mass spectrometry

1. Introduction Glycosylation of the conserved asparagine (Asn) residue in the CH2 domain is the most common enzymatic modification of antibodies. Removal of oligosaccharides can induce significant structural changes. For example, it has been demonstrated that deglycosylated IgG-Fc cannot be crystallized under the conditions used for glycosylated Fc (Krapp et al., 2003). Structural changes have also been demonstrated by the increased susceptibility of deglycosylated and non-glycosylated antibodies to proteases (Dwek et al., 1995; Tao and Morrison, 1989) and decreased thermal stability of antibodies and their Fc fragments (Ghirlando et al., 1999; Liu et al., 2006; Mimura et al., 2000, 2001). The most dramatic differences between antibodies with and without oligosaccharides are Fc effector functions. Near complete loss of binding affinity of antibodies to the first component of complement (C1q) and Fc receptors have been reported in various antibodies without oligosaccharides (Boyd et al., 1995; Mimura et al., 2000, 2001; Nose and Wigzell, 1983; Tao and ∗

Corresponding author. Tel.: +1 508 849 2591; fax: +1 508 793 4885. E-mail address: [email protected] (H. Liu).

0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.07.012

Morrison, 1989). The loss of Fc functionality is most likely a reflection of conformational changes as there are only minimal direct interaction between Fc oligosaccharides and the effector molecules including C1q and Fc receptors (Jefferis et al., 1998; Kato et al., 2000; Sondermann et al., 2000). Oxidation of Met residues is a common non-enzymatic modification, which has been demonstrated to change protein structure, function and stability (Brot and Weissbach, 1983; Carp et al., 1982; Chugha et al., 2006; Gao et al., 1998; Kim et al., 2001; Kornfelt et al., 1999; Lu et al., 1999; Teh et al., 1987). There are several reports on the oxidation of Met residues in monoclonal antibodies. Although Met residues of different regions of antibodies have been oxidized under various conditions (Chumsae et al., 2007; Kroon et al., 1992; Lam et al., 1997; Matamoros Fernandez et al., 2001; Roberts et al., 1995; Shen et al., 1996), two Met residues in the Fc region of recombinant humanized monoclonal antibodies are most susceptible to oxidation (Chumsae et al., 2007; Lam et al., 1997; Shen et al., 1996). The two susceptible Met residues are located in the CH2 and CH3 domains in the primary structure, respectively, however in the three-dimensional structure, they locate close to the CH2-CH3 interface and the oligosaccharides, which are

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Fig. 1. Crystal structure of human IgG1-Fc drawn from published coordinates. Met256 (IMGT CH2-15.1) and Met432 (IMGT CH3-107) are labeled. The glycosylation site is Asn301 (IMGT CH2-84.4).

enclosed within the two CH2 domains (Fig. 1). Therefore, it is intriguing to study the relationship of Met oxidation and the presence or absence of oligosaccharides and their effect on the structure and stability of the antibody. In this study, a recombinant humanized monoclonal antibody (IGHG1*01 with a kappa light chain) was used. Incomplete processing of the C-terminal lysine (Lys) residues results in the antibody with zero, one or two C-terminal Lys. The five major forms of oligosaccharides include three biantennary complex structure with core fucose with either zero (Gal 0), one (Gal 1) or two (Gal2) terminal galactose and two oligomannose with either five or six mannose residues. Each light chain has one Met (IMGT (Lefranc et al., 2003), FR1-Met4) residue. Each heavy chain has four Met residues with two in the Fab region (IMGT (Lefranc et al., 2003), CDR1-Met35 and FR3-Met91) and two in the Fc region (IMGT (Lefranc et al., 2005), CH2-Met15.1 and CH3-Met107). The antibody with and without oligosaccharides was oxidized with different amounts of tert-butyl hydroperoxide (tBHP). Oxidation sites and percentage were determined by liquid chromatography-mass spectrometry analysis (LC-MS). Conformational changes caused by the removal of oligosaccharides and Met oxidation were probed by limited trypsin and chymotrypsin digestion. 2. Materials and methods

Formic acid (FA) was purchased from EMD (Madison, WI). PNGaseF was purchased from Prozyme (San Leandro, CA). Lys-C, chymotrypsin, phenylmethylsulfonyl fluoride (PMSF) and N-octylglucoside were purchased from Roche (Indianapolis, IN). Acetonitrile, trifluroacetic acid (TFA), guanidine hydrochloride and 1N hydrochloride acid (HCl) were purchased from J.T. Baker (Phillipsburg, NJ). Trypsin was purchased from Worthington (Lakewood, NJ). 2.2. Deglycosylation of the recombinant monoclonal antibody The recombinant monoclonal antibody at approximately 70 mg/mL in formulation buffer (5.57 mM sodium phosphate monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM sodium chloride, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol and 0.1% Tween) at pH 5.2, was diluted to 10 mg/mL using 10 mM sodium phosphate, pH 7.5. PNGaseF (2.5 mU/␮L) was added to the diluted sample based on the ratio of 1 ␮L enzyme:500 ␮g antibody. N-Octylglucoside was included in the sample preparation to a final concentration of 1% to facilitate the removal of N-linked oligosaccharides. Digestion was allowed to proceed at 37 ◦ C for 18 h. The same antibody diluted to 10 mg/mL using 10 mM sodium phosphate, pH 7.5, with 1% N-octylglucoside but without PNGaseF was also incubated at 37 ◦ C for 18 h and used as a control.

2.1. Materials 2.3. Oxidation of methionine residues The recombinant humanized monoclonal antibody was produced by transfected Chinese hamster (CHO) ovary cell lines and purified at Abbott Bioresearch Center (Worcester, MA). tert-Butyl hydroperoxide (tBHP), dithiothreitol (DTT), and iodoacetic acid were purchased from Sigma (St. Louis, MO).

The deglycosylated and control antibody samples were buffer exchanged to 10 mM sodium phosphate, pH 7.5 using Amicon Ultra-4 centrifugal filter device (Millipore, Billerica, MA) with a molecular weight cut-off of 10 kDa and then diluted to 10 mg/mL

H. Liu et al. / Molecular Immunology 45 (2008) 701–708

using 10 mM sodium phosphate, pH 7.5. tBHP was included in the sample preparations with final concentrations of 0, 0.1, 0.5, 1, 2 and 5% (v/v). The samples were incubated at room temperature (25 ◦ C) for 18 h. Unreacted tBHP was removed using NAP-5 columns (GE Healthcare, Piscataway, NJ). 2.4. Lys-C digestion and LC-MS analysis To determine the sites of oxidation, aliquots of each oxidized sample were diluted to 0.5 mg/mL using 10 mM sodium phosphate, pH 7.5, in a final volume of 500 ␮L. Lys-C was added to each sample at an enzyme:antibody ratio of 1:500 (w/w). The samples were incubated at 37 ◦ C for 40 min. The digested samples were then reduced using DTT at a final concentration of 20 mM at 37 ◦ C for 5 min. Molecular weights of the Lys-C digested and reduced samples were determined by LC-MS. An Agilent HPLC (Santa Clara, CA) and a protein C4 column (Vydac, 150 mm × 1 mm ˚ pore size) were used to desalt, i.d., 5 ␮m particle size, 300 A separate and introduce samples into the mass spectrometer. Five micrograms of each sample was loaded at 95% mobile phase A (0.02% TFA, 0.08% FA in Milli-Q water) and 5% mobile phase B (0.02% TFA, 0.08% FA in acetonitrile). After running at 5% mobile phase B for 5 min, proteins were eluted off the column by increasing mobile phase B to 65% within 35 min. The column was washed by increasing mobile phase B to 95% in 5 min and then decreasing to 5% in another 5 min. The column was equilibrated at 5% mobile phase B for 10 min before the next injection. The flow rate was set at 50 ␮L/min. The column oven was set at 60 ◦ C. The mass spectrometer scan range was set at a range of m/z 800–2500. IonSpray voltage was set at 4500 V. Source temperature was set at 350 ◦ C. 2.5. Peptide map with LC-MS detection Samples were denatured with 6 M guanidine hydrochloride in 100 mM Tris, pH 8.0, reduced with 10 mM DTT at 37 ◦ C for 30 min and then alkylated with 25 mM iodoacetic acid at 37 ◦ C for another 30 min. The samples were then buffer exchanged to 10 mM Tris, pH 8.0, using NAP-5 columns. Protein was eluted off the columns using 900 ␮L of 10 mM Tris, pH 8.0, and collected. Samples were digested with trypsin at a 1:20 (w/w) trypsin:antibody ratio at 37 ◦ C for 4 h. Digestions were stopped by adding 1N HCl to lower the pH. The Agilent HPLC and a C18 column (Vydac, ˚ pore size) 250 mm × 1 mm i.d., 5 ␮m particle size, 300 A were used to separate and introduce peptides into the Q Star mass spectrometer. Approximately 20 ␮g of each sample was loaded at 98% mobile phase A and 2% mobile phase B, then eluted by increasing mobile phase B from 2 to 35% in 140 min. IonSpray voltage was set at 4200 V. Source temperature was set at 75 ◦ C. m/z was scanned from 250 to 2000.

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1 mg/mL using 10 mM sodium phosphate, pH 7.5, in a final volume of 200 ␮L. Four micrograms of trypsin or chymotrypsin was added to each sample to achieve an enzyme:antibody ratio of 1:50 (w/w). Digestions were allowed to proceed at 37 ◦ C for 2 h. The digested samples were analyzed by reducing SDSPAGE and reverse-phase high performance chromatography (RP-HPLC). 2.7. SDS-PAGE Twenty microliters of each digested sample was mixed with 20 ␮L of 2× Tris-glycine SDS sample buffer (Invitrogen, Carlsbad, CA) with 100 mM DTT and immediately incubated at 100 ◦ C for 5 min. Ten microliters of each sample was loaded into the wells of 12% Tris-glycine precast gels (Invitrogen). SeeBlue plus 2 was loaded as the molecular weight standard. The gel was run at 75 constant volts until samples were stacked, then run at 125 V until the dye front reached the bottom of the gels. The running buffer was 0.025 M Tris, 0.19 M glycine, 0.1% SDS, pH 8.6. Gel was stained with colloidal blue using a colloidal blue staining kit (Invitrogen) and then destained using Milli-Q water. 2.8. Reverse-phase chromatography and fraction collection A Shimadzu HPLC and a protein C4 column (Vydac, ˚ pore size) were 250 mm × 4.6 mm i.d., 5 ␮m particle size, 300 A used to analyze the digested samples. One hundred micrograms of each sample was reduced with 10 mM DTT in the presence of 2 mM PMSF, and then loaded at 95% mobile phase A and 5% mobile phase B at a flow-rate of 1 mL/min. After running at 5% mobile phase B for 5 min, samples were eluted off the column by increasing mobile phase B from 5% to 65% in 30 min. The column oven temperature was set at 60 ◦ C. Elution of protein peaks was monitored by UV at 214 and 280 nm. Peaks from samples oxidized with either 0% or 5% tBHP and digested with either trypsin or chymotrypsin were collected for mass spectrometry analysis. 2.9. Identification of the protease cleavage sites To identify trypsin and chymotrypsin cleavage sites, the digested samples used in the previous section and the collected fractions after concentration using speed-vacuum were analyzed using the Agilent HPLC and the Q star mass spectrometer. The same column, mobile phases, gradient, column oven temperature and mass spectrometer parameters as described in the “Lys-C digestion and LC-MS analysis” section was used for the analysis of larger fragments. For identification of peptides, mass spectrometer parameters used in the section of “peptide map” were used. 3. Results

2.6. Limited protease digestions

3.1. Lys-C digestion and LC-MS analysis

Aliquots of each sample with and without oligosaccharides oxidized with different amounts of tBHP were diluted to

The recombinant monoclonal antibody after oxidation with various amounts of tBHP was digested with Lys-C, which

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Fig. 2. MS spectra of the recombinant humanized monoclonal antibody after the removal of oligosaccharides oxidized with 0% (A) or 5% (B) tBHP. Peaks corresponding to Fc fragments are enlarged to show Met oxidation. Numbers in parenthesis indicate the number of oxidation sites. Peak with the molecular weight of 24,327 Da was not identified.

cleaves the hinge region between amino acids 226 and 227, to generate heavy chain fragments of amino acids 1–226 and 227–450. After reduction, the digested samples were analyzed using LC-MS. Representative mass spectra are shown in Fig. 2. The molecular weights of approximately 23,409 and 24,038 Da observed in the samples treated with either 0% or 5% tBHP are in good agreement with the calculated molecular weights of the light chain (23,408 Da) and the heavy chain amino acids 1–226 (24,037 Da), respectively, which indicated that Met residues in the light chain and the heavy chain of amino acids 1–226 were not oxidized. The observed molecular weights of 25,175 Da and 25,303 Da of the 0% tBHP treated sample (Fig. 2A) are in good agreement with the calculated molecular weight of the heavy chain amino acids 227–450 without (25,174 Da) or with a C-terminal Lys residue (25,302 Da), respectively, which indicated that no significant levels of oxidation were present in the absence of tBHP treatment. On the other hand, molecular increases of approximately 15 Da (25,190 Da) and 32 Da (25,207 Da) (Fig. 2B) over the molecular weight of the unoxidized heavy chain amino acids 227–450 (25,175 Da) (Fig. 2A) were observed in the sample treated with 5% tBHP. The molecular weight increases, which corresponded to oxidation of one (16 Da) or two (32 Da) Met residues, indicated that Met residues (Met256 (CH2-15.1) and Met432 (CH3-107)) in the Fc region were oxidized. Peaks corresponding to the heavy chain amino acids 227–450 with C-terminal Lys with oxidized Met residues were also detected but the signals were very low. 3.2. Peptide map To quantify the percentage of oxidation, tryptic peptides of the samples were analyzed by LC-MS. The percentage of oxidation of each residue was calculated using the total ion current

(TIC) chromatogram peak area, which is summarized in Table 1. Similar oxidation percentage was observed for antibody with and without oligosaccharides for Met256 (CH2-15.1) as well as Met432 (CH3-107). Therefore, the removal of oligosaccharides did not change the susceptibility of these two Met residues to oxidation. Met256 was more susceptible than Met432. No significant amount of oxidation was detected in the control, which indicated that oxidation of Met did not occur to a significant level during sample preparations. 3.3. Limited trypsin digestion and SDS-PAGE To probe the structural changes caused by deglycosylation and oxidation, the antibody with and without oligosaccharides after oxidation with different concentrations of tBHP were digested with trypsin or chymotrypsin. Oxidation alone did not cause any damage to the molecule as the heavy chain and the light chain were the only two bands observed on SDS-PAGE (data not shown). Hardly any bands were visible between the heavy chain and the light chain after trypsin or chymotrypsin digestion of the antibody with oligosaccharides and with different degrees of Table 1 Oxidation of Met256 and Met432 %tBHP

0 0.1 0.5 1 2 5

Met256

Met432

Native

Deglycosylated

Native

Deglycosylated

0 25.5 47.1 50.9 84.1 95.0

0 27.6 49.8 63.0 79.7 94.9

0 25.9 36.1 41.7 61.7 78.7

0 19.2 37.2 49.2 60.6 81.0

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Fig. 3. SDS-PAGE of the recombinant humanized monoclonal antibody with (A) and without (B) oligosaccharides digested with trypsin at 37 ◦ C for 2 h. SeeBlue plus 2 was used as molecular weight markers (Lane 1). The samples were oxidized with 0% (Lane 2), 0.1% (Lane 3), 0.5% (Lane 4), 1% (Lane 5), 2% (Lane 6) and 5% (Lane 7) tBHP.

Met oxidation (Figs. 3A and 4A), which indicated that antibody with oligosaccharides was resistant to protease cleavage. Oxidation of Met256 and Met432 did not alter the susceptibility. In contrast, deglycosylated antibody was susceptible to trypsin or chymotrypsin digestion as new bands appeared above the light chain (Figs. 3B and 4B, Lane 2). Therefore, removal of the oligosaccharides alone can render the antibody to protease cleavage. The intensity of the new bands increased with the increase of oxidation (Figs. 3B and 4B, Lanes 3–7), which suggested that oxidation enhanced the enzymatic susceptibility of the deglycosylated antibody. Degradation was mainly on the heavy chain because the intensity of the heavy chain band decreased, while the intensity of the light chain remained the same.

3.4. Identifications of cleavage sites Chromatograms of the deglycosylated antibodies oxidized with 5% tBHP digested with either trypsin or chymotrypsin are shown in Fig. 5. Trypsin and chymotrysin cleavage sites were identified using LC-MS and are summarized in Tables 2 and 3. All the potential trypsin cleavage sites in the CH2 domain were cleaved. The major cleavage sites are populated in the CH2 domains except one trypsin cleavage site and three chymotrypsin cleavage sites, which were in the CH3 domain (Plate 1), which indicated that conformational changes caused by deglycosylation and further by Met oxidation was only limited to CH2 domains. Interestingly, oxidation accelerated enzymatic digestion, but it did not change the cleavage sites as the same

Fig. 4. SDS-PAGE of the recombinant monoclonal antibody with (A) and without (B) oligosaccharides digested with chymotrypsin at 37 ◦ C for 2 h. SeeBlue plus 2 was used as molecular weight markers (Lane 1). The samples were oxidized with 0% (Lane 2), 0.1% (Lane 3), 0.5% (Lane 4), 1% (Lane 5), 2% (Lane 6) and 5% (Lane 7) tBHP.

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Table 2 Identification of fragments from trypsin digestion Peaks

a b c d e f g h Other peptidesb

a b

Identitiesa

Molecular weight (Da) Observed

Calculated

1676.8 2227.2 1807.0 12162.3 11963.4 23409.2 49226.1 26751.8

1676.8 2227.2 1807.0 12162.5 11963.2 23408.1 49223.4 26750.2

1670.7 1188.4 850.3 659.3 837.4 653.3

1670.8 1188.5 850.4 659.3 837.5 653.3

HC279-292 (39-79) HC306-324 (85-103) HC306-321 (85-100) HC343-450 (Met432 oxidized) (CH2 123-CH3 127) HC345-450 (Met432 oxidized) (CH2 125-CH3 127) Light chain HC with Met256 and Met432 oxidized HC1-252 (FR 1-CH2 12) HC293-305 (80-85.1) HC297-305 (84-85.1) HC253-259 (Met256 oxidized) (CH2 13-17) HC444-450 (CH3 121-127) HC331-338 (CH2 101-120) HC333-338 (Chymotrypsin like cleavage) (CH2 102-120)

The amino acid numbers in parenthesis are IMGT numbers. Other peptides refer to peptides are not shown in Fig. 3A and B.

Table 3 Identification of fragments from chymotrypsin digestion Peaks

Identitiesa

Molecular weight (Da) Observed

Calculated

1

1216.5 1601.7 1010.5 1285.6

1216.6 1601.8 1010.5 1285.7

2

11291.9 11379.4 11492.6 11593.6 12413.7 12541.8

11291.6 11378.7 11491.8 11592.9 12413.0 12541.1

HC342-440 (Met432 oxidized) (CH2 124-CH3 116) HC341-440 (Met432 oxidized) (CH2 123-CH3 116) HC340-440 (Met432 oxidized) (CH2 122-CH3 116) HC339-440 (Met432 oxidized) (CH2 121-CH3 116) HC331-440 (Met432 oxidized) (CH2 110-CH3 116) HC330-440 (Met432 oxidized) (CH2 109-CH3 116)

3 4 5 6

23408.8 49221.0 25398.7 25942.1

23408.1 49223.4 25397.5 25942.4

Light chain Intact heavy chain with Met256 and Met432 oxidized) HC1-239 (FR1 1-CH2 2) HC1-245 (FR1 -CH2 5)

459.2 722.4 753.4 481.2 1048.5 562.3 1288.6 897.5 1066.6 697.3 671.4 540.3 1154.7 1522.8 2584.4 2731.6

459.2 722.4 753.4 481.2 1048.5 562.3 1288.6 897.4 1066.5 697.3 671.4 540.3 1154.7 1522.8 2584.4 2731.5

Other peptidesb

a b

The amino acid numbers in parenthesis are IMGT numbers. Other peptides refer to peptides are not shown in Fig. 3A and B.

HC280-289 (CH2 40-45.1) HC311-323 (CH2 90-102) HC310-317 (CH2 89-96) HC247-257 (CH2 7-15.2)

HC446-450 (CH3 123-CH3 127) HC318-323 (CH2 97-CH2 102) HC283-289 (CH2 43-CH2 45.4) HC280-282 (CH2 40-CH2 42) HC256-264 (Met256 oxidized) (CH2 15.1-CH2 22) HC240-245 (CH2 1.1-CH2 5) HC314-323 (CH2 93-CH2 102) HC311-317 (CH2 90-CH2 96) HC319-327 (CH2 98-CH2 106) HC314-318 (CH2 93-CH2 97) HC305-310 (CH2 85.1-CH2 89) HC427-431 (CH3 102-CH3 106) HC246-255 (CH2 6-CH2 15) HC307-319 (CH2 86-CH2 98) HC246-268 (Met256 oxidized) (CH2 6-CH2 26) HC245-268 (Met256 oxidized) (CH2 5-CH2 26)

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Fig. 5. UV 280 nm chromatograms of the deglycosylated antibody oxidized with 5% tBHP and digested with either trypsin (A) or chymotrypsin (B) at 37 ◦ C for 2 h. Peaks a–h as shown in (A) were identified and are summarized in Table 2. Peaks 1–6 as shown in (B) were identified and are summarized in Table 3.

Plate 1. Diagram of the heavy chain to show the cleavage sites after either trypsin (A) or chymotrypsin (B) digestion. No cleavage sites were observed in the light chain and thus it is not shown. Each heavy chain contains one variable (VH) and three constant (CH1, CH2 and CH3) domains. CH1 and CH2 are connected by the hinge region. Arrows indicate the cleavage sites.

fragments were identified in the samples with and without Met oxidation. 4. Discussion Oligosaccharides on the conserved Asn residues in the Fc region of antibodies are enclosed between the two CH2 domains. Interactions between the oligosaccharides and the amino acids are critical for the structural integrity of the CH2 domains (Deisenhofer, 1981; Dwek et al., 1995; Krapp et al., 2003; Rosen et al., 1979; Saphire et al., 2002; Sutton and Phillips, 1983).

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Removal of oligosaccharides has been shown to cause a significant conformational change in the CH2 domains (Dwek et al., 1995; Krapp et al., 2003; Liu et al., 2006; Rosen et al., 1979). This observation is further supported by the current study, where increased susceptibility of the CH2 domains to proteases was observed. Conformational changes of the CH2 domain caused by the removal of oligosaccharides have been localized to the hinge proximal region of the CH2 domains (Krapp et al., 2003; Liu et al., 2006). Thus, enzymatic cleavage sites in the hinge remote region of the CH2 domains as observed in this study may be secondary to the cleavage in the hinge proximal region of the CH2 domains. On the other hand, distribution of the cleavage sites in the entire CH2 domain may suggest an overall conformational change of the CH2 domain. In the three dimensional structure, oligosaccharides and Met 256 (CH2-15.1) and Met432 (CH3107) are in close proximity, however conformational changes in the CH2 domains of the deglycosylated antibody did not alter oxidation rates of Met256 (CH2-15.1) and Met432 (CH3-107). This is likely due to the fact that Met256 (CH2-15.1) and Met432 (CH3-107) are exposed even before the removal of oligosaccharides. Based on the crystal structure of human IgG-Fc, Met256 (CH2-15.1) is at the end of a loop (IMGT, AB-LOOP (Lefranc et al., 2005)), while Met432 (CH3-107) is at the end of a ␤sheet structure (Deisenhofer, 1981) (IMGT, FG-LOOP (Lefranc et al., 2005)). Furthermore, Met 432 (CH3-107) is in the CH3 domain, and conformational changes in the CH2 domain may not be sufficient to affect the CH3 domain because of the presence of a relatively flexible CH2-CH3 interface (Connell and Porter, 1971; Deisenhofer, 1981; Ellerson et al., 1976; Krapp et al., 2003; Matthews et al., 1971; Turner and Bennich, 1968). Met oxidation results in the replacement of the smaller and hydrophobic side chain of Met with a larger and hydrophilic side chain of Met sulfoxide, which has been considered as mutagenesis (Kim et al., 2001). However, oxidation of Met256 (CH2-15.1) and Met 32 (CH3-107) did not cause significant conformational changes of the antibody with oligosaccharides. These data further demonstrated the importance of the oligosaccharides and their interactions with the protein moieties for the structure integrity of the CH2 domains. Oxidation of Met256 (CH2-15.1) and Met432 (CH3-107) accelerated the degradation of the deglycosylated antibody, which suggested that loss of the oligosaccharides and their interactions with proteins destabilized the molecules significantly, which allowed oxidation of Met to exert its effect to further destabilize the CH2 domains. The same protease cleavage sites of the deglycosylated antibodies without and with oxidation indicated that oxidation did not cause different conformational changes, but it only exaggerated the structural changes caused by the removal of oligosaccharides. In summary, removal of the oligosaccharides caused significant conformational changes in the CH2 domains of the recombinant monoclonal antibody as probed by limited trypsin and chymotrypsin digestion. The conformational changes had no effect on the oxidation rates of the two susceptible Met residues located in the CH2-CH3 domain interface. However, oxidation of these Met residues caused further conformational changes of the deglycosylated antibody.

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