Specific Racemization of Heavy-Chain Cysteine-220 in the Hinge

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Specific Racemization of Heavy-Chain Cysteine-220 in the Hinge Region of Immunoglobulin Gamma 1 as a Possible Cause of Degradation during Storage Masato Amano,† Jun Hasegawa,† Naoki Kobayashi,† Naoyuki Kishi,† Takashi Nakazawa,‡ Susumu Uchiyama,§ and Kiichi Fukui*,§ †

Analytical & Quality Evaluation Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan Department of Chemistry, Nara Women’s University, Nara 630-8506, Japan § Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Osaka, Japan ‡

bS Supporting Information ABSTRACT: Therapeutic antibodies often suffer from degradation due to various modifications during storage. We detected a degradation of immunoglobulin gamma 1 (IgG1) stored for 6 month at 40 °C, and identified the modification as the racemization of Cys220 in the hinge sequence S219CDKTHT225 of the heavy chain by tryptic peptide mapping and tandem mass spectrometry. The rate of racemization at Cys220 was enhanced deliberately by incubating the protein at 50 °C and pH 9.0, while all the other cysteine residues were not affected. The racemization of Cys220 was confirmed by mass spectrometry in conjunction with extracted ion chromatography of the tryptic digest of IgG1 forced to degrade in D2O and a series of synthetic hinge fragments containing D-amino acid, as well as the detection of D-cysteine in the acid hydrolysate. To rationalize the possible relationship between the racemization of Cys220 and isopeptide formation at the neighboring residue of Asp221, we suggest a new reaction mechanism that assumes a base catalyst to initiate these reactions by activating the amide nitrogen of Lys222. Due to the highly flexible nature of the hinge region, Lys222 can participate in either the formation of a cyclic imidazoline intermediate involving the R-carbonyl carbon of Cys220 to facilitate the racemization of Cys220 or that of a succinimide structure leading to the isomerization of Asp221.

onoclonal antibodies including γ-globulins have been the main products of biotherapeutics over the past decade.1,2 Special care should be taken to protect therapeutic antibodies from physical and chemical degradation during the entire process of manufacturing and storage because of their use as medicines.3,4 A variety of degradations that have been identified in monoclonal antibodies,57 include oxidation of methionine8 and tryptophan,9 glycation of lysine,1012 pyroglutamic acid formation of N-terminal glutamine,13 deamidation of asparagine, and isomerization of aspartic acid.1416 In particular, native or artificial post-translational modifications of chimerized/humanized/human immunoglobulin gamma 1 (IgG1) occur frequently in the hinge sequence S219CDKTHT225 (EU numbering) of the heavy chain, referred to as a “hot spot” of degradation and fragmentation1721 (Figure 1). These modifications have been identified in IgG1 stored for several weeks or months, as well as a much shorter period of time under a forced condition to accelerate degradation. For example, the cleavage of the peptide bond between Ser219 and Cys220 was detected in IgG1 stored for 17 days at 45 °C, whereby the involvement of β-elimination at the side chain of Cys220 was suggested by the identification of a

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peptide with a thioether linkage in the reaction products.20 In addition, Asp221 was found to undergo isomerization via a succinimide intermediate, leading to an isoaspartic acid residue.2124 Although the mechanisms of these reactions have been proposed individually, the site-specificity of each reaction that occurs in the hinge sequence still remains to be interpreted. If there is a common feature in these reactions, the degradation of therapeutic antibodies could be avoided or suppressed effectively by establishing a strictly controlled storage condition. We report here the identification of Cys220 being subjected to racemization as a novel modification in the “hot spot” of IgG1 during storage for 6 months at 40 °C. To characterize anomalous peptides detected by peptide mapping,25,26 we employed extracted ion chromatography (XIC) in LC/MS for the analysis of isobaric peptides presumably containing isomeric or racemic amino acid residue(s). Degradation in D2O has proved effective to monitor racemization by using the method based on Received: February 7, 2011 Accepted: April 5, 2011 Published: April 05, 2011 3857

dx.doi.org/10.1021/ac200321v | Anal. Chem. 2011, 83, 3857–3864

Analytical Chemistry

Figure 1. Three-dimensional structure of intact human IgG1 (PDB code 1HZH). The hinge region is highlighted in bold lines and the enlarged view of the “hot spot” (enclosed in dashed rectangle in the overall structure) is illustrated below as a stick model.

CR-hydrogen/deuterium (H/D) exchange,27 because the CRH bond of amino acids is intrinsically nondissociable but undergoes exchange reaction only when the covalent structure is altered. This technique contrasts sharply with the conventional amide-H/D exchange approach dealing with labile amide protons for the studies of protein conformation, dynamics, and interactions.28 The sitespecificity of the racemization in Cys220 was such that the other 30 cysteine residues were kept intact in this storage condition. Conventional racemization schemes include a base-catalyzed abstraction of CR-proton at the initial step, as proposed for the less-sitespecific racemization of serine residues in IgG1, -2, and -4 stored at pH 8 and 40 °C for 4 weeks.27 Apparently, this mechanism is incompatible with racemization occurring exclusively at Cys220. Allowing for the involvement of adjacent residues Cys220 and Asp221 in the formation of thioether-linked peptide and isopeptide, respectively, we suggest a new mechanism that assumes the highly nucleophilic amide nitrogen of Lys222 to be created commonly at the initial step of reactions, leading to racemization, β-elimination, and isopeptide formation. We will also discuss the site-specificity of racemization in connection with the possible formation of highly nucleophilic amide nitrogen in the hinge region, where conformational flexibility is remarkably high in IgG1.

’ EXPERIMENTAL SECTION Materials. Guanidine hydrochloride (Gdn 3 HCl), acetonitrile, trifluoroacetic acid (TFA), formic acid, ammonium hydrogen carbonate, lysylendopeptidase (mass spectrometry grade), sodium hydroxide, dithiothreitol (DTT), iodoacetamide (IAM), and o-phthalaldehyde (OPA) were purchased from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan). Tris(hydroxymethyl)aminomethane (Tris) was purchased from Bio-Rad Laboratories, Inc. (Tokyo, Japan). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Dojindo Laboratories (Kumamoto, Japan). Trypsin (sequencing grade) was purchased from Promega Corp. (Tokyo, Japan). Deuterium oxide (D 99.9%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). 2,3,4,6-Tetra-O-acetyl-1-thioβ-glucopyranoside (TATG) was purchased from Sigma Aldrich K. K. (Tokyo, Japan). Hydrochloric acid (6 M, sequencing grade)

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was purchased from Thermo Fisher Scientific K. K. (Tokyo, Japan). Borate buffer was purchased from Agilent Technologies, Inc. (Tokyo, Japan). Pharmaceutical Stability. The recombinant human IgG1 antibody solution (8.0 mg/mL) was prepared in a 10 mM sodium phosphate buffer (pH 7.0) containing 140 mM sodium chloride and 0.01% polysorbate 80. Under sterile conditions, 3 mL of the antibody solution was dispensed into polyethylene terephthalate glycol (PETG) vials and stored at 40 °C for 6 months. In a forced degradation experiment, the buffer of the antibody was exchanged to 500 mM Tris 3 HCl buffer (pH 9.0) with Amicon Ultracel-30K (Millipore, Bedford, MA), followed by incubation for 7 days at 50 °C. Mass Spectrometry. We employed an LC/ESI-MS system consisting of a Q-Tof Premier (Waters Milford, MA) equipped with an LC1200 instrument (Agilent, Santa Clara, CA). All the measurements were performed in the positive-ion mode. The capillary and cone voltages were set to 3.0 kV and 25 V, respectively. MS/MS analysis was carried out on a Thermo Fisher Scientific (Waltham, MA) LTQ XL operating in the positive-ion mode. The capillary voltage was set to 5.0 kV, and CID fragmentation of the isolated precursor ion in a width of 5.0 Da was carried out using helium at the collision energy of 35.0 eV. The injection time of the ion was set to 100 ms, and the CID reaction time was 30 ms. Peptide Mapping. The samples were diluted to 1.8 mg/mL using 8 M Gdn 3 HCl and 500 mM Tris 3 HCl buffer (pH 8.5), followed by the addition of DTT to a final concentration of 50 mM, and let stand for 30 min at 37 °C. IAM was added to a final concentration of 100 mM and allowed to react for 30 min in a dark place, and then the reaction was quenched with DTT (final concentration of 80 mM). The samples were desalted using a TSKgel BioAssist DS, 4.6 150 mm column (Tosoh, Tokyo, Japan) with an eluent of 20% aqueous acetonitrile containing 0.05% TFA. The desalted sample was dried up, redissolved to 10 mM ammonium bicarbonate buffer, and incubated with trypsin at the protein/enzyme weight ratio of 73:1 for 16 h at 37 °C. The digestion was stopped by the addition of 5% TFA. To analyze the hinge region peptide, the tryptic digest (29 μg) was separated with reversed-phase HPLC in an isocratic mode of 0.1% TFA on an Agilent LC1200 equipped with a Waters Xbridge BEH130 C18 column (2.1 250 mm, 5 μm). The flow rate was 0.2 mL/min at the column temperature of 40 °C. As comprehensive peptide mapping, the tryptic digest was separated using a linear gradient from the initial at 100% mobile phase A (0.2% TFA, 0.8% formic acid) to 120 min at 43% mobile phase B (90% acetonitrile, 0.2% TFA, 0.8% formic acid). The eluted peptides were directly subjected to ESI-MS. Forced Degradation in D2O. The IgG1 was degraded in D2O (500 mM Tris buffer) incubated for 7 days at 50 °C. The antibody degraded in D2O was analyzed with the peptide mapping as described in the preceding section, which used H2O as a solvent to ensure back-exchange of mobile deuterons, and mass spectrometry with the same procedures as those performed in H2O. Reduction and Alkylation of Chemically Synthesized Model Peptides. The chemically synthesized peptides L-Ser-LCys-L-Asp-L-Lys (S-LCDK) and L-Ser-D-Cys-L-Asp-L-Lys (S-DCDK) in their reduced (thiol) forms were purchased from Takara Bio Inc. (Shiga, Japan). Each peptide was dissolved in 50 mM ammonium bicarbonate buffer at the concentrations of 5 mg/mL and incubated with an equal volume of 50 mM DTT for 15 min at 3858

dx.doi.org/10.1021/ac200321v |Anal. Chem. 2011, 83, 3857–3864


Figure 2. Extracted ion chromatograms of the hinge peptide fragments corresponding SC(CAM)DK ([M þ H]þ, m/z 509.2 ( 0.1) of IgG1 and the synthetic hinge peptides. The digested peptide from (a) the native (freshly prepared) IgG1, (b) the sample stressed at 40 °C for 6 months, and (c) the sample forced to degrade in 500 mM Tris, pH 9, at 50 °C for 7 days. The reduced and alkylated synthetic peptide of (d) S-LC(CAM)DK and (e) S-DC(CAM)DK. The corresponding TIC chromatograms are shown in Figure S-1 of the Supporting Information.

37 °C, followed by alkylation with 120 mM IAM for 15 min in a dark place. Amino Acid Analysis with Chiral Separation. The reduced and alkylated model peptide and the fractionated hinge peptides were hydrolyzed with 6 M hydrochloric acid overnight at 110 °C in a Waters Pico-Tag Workstation. The hydrolysate was dried up and dissolved in 28 μL of 400 mM borate buffer (pH 10.2). To the solution was added 2 μL of 8 mg/mL OPA and 44 mg/mL TATG in acetonitrile with vigorous mixing.29 The OPATATG derivatized amino acids were separated by reversed-phase HPLC with an Agilent Eclipse Plus C18 column (2.1 250 mm, 3.5 μm) using a linear gradient from the initial at 20% mobile phase A (20 mM ammonium acetate) to 30 min at 60% mobile phase B (50% acetonitrile, 50% methanol) at a flow rate of 0.2 mL/min. The column temperature was set at 40 °C and the eluent was detected with a UV detector at 340 nm. The observed peaks were identified with MS. Nonreduced Peptide Mapping. The samples were dried up and redissolved into 100 mM HEPES, 6 M GdnHCl, pH 6.8, followed by an immediate dilution with 100 mM HEPES to a final Gdn 3 HCl concentration of 1.5 M. Digestion with lysylendopeptidase was performed at the protein/enzyme ratio of 40:1 at 37 °C for 16 h and stopped with 5% TFA. The digest (145 μg) was separated with reversed-phase HPLC on a LC1100 equipped with a Zorbax 300SB-C18 column (4.6 150 mm, 5 μm; Agilent Technologies, Inc.) using a linear gradient from 10 min at 100% mobile phase A (0.1% TFA) to 130 min at 40% mobile phase B (90% acetonitrile containing 0.1% TFA) at a flow rate of 1.0 mL/ min and a column temperature of 70 °C. The eluent was infused to the Q-Tof Premier with a split at the ratio of 1:5.

’ RESULTS AND DISCUSSION Detection of Modification in the Hinge Region. The tryptic digest of degradation products of IgG1 antibody, in which cystine residues were successively modified with reduction and S-carbamidomethylation after storage at 40 °C for 6 months, was characterized with LC/MS and LC/MS/MS. A few peaks attributable to the hinge region fragment SC(CAM)DK, which contains S-carbamidomethylcysteine (C(CAM)), were detected in

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Figure 3. MS/MS spectra of the three peaks observed in the extracted ion chromatogram (m/z 509.2) of the sample stored at 40 °C for 6 months. The precursor ions were m/z 509.2 for (a) peak I (assigned as aspartyl isopeptide), (b) peak II (unmodified peptide), and (c) peak III (peptide possibly containing D-Cys(CAM)).

XIC (Figure 2ac).30,31 The sole peak II that eluted at 8 min in the chromatogram of nontreated IgG1 preparation (Figure 2a) appeared to degrade to give peaks I and III after storage at 40 °C for 6 months (Figure 2b). Peak I eluted earlier than peak II and was assigned to a peptide containing an isoaspartyl residue, as reported previously.14 Peak III eluted later than peak II by 30 s, as shown in Figure 2b, and was also observed in the chromatogram shown in Figure 2c for the antibody that was degraded under a forced condition of pH 9 (in 500 mM Tris 3 HCl buffer) at 50 °C for 7 days. All of these peptide fragments were indistinguishable with respect to the molecular mass of 509 Da, which was consistent with the sequence of SC(CAM)DK. In MS/MS analysis of peak III, all the y-series ions (y1, m/z 147; y2, m/z 262; y3, m/z 422) and two b-series ions (b2, m/z 248; b3, m/z 363) were observed, in good agreement with those of peak II (Figure 3). The unknown peptide fragment in peak III is isobaric to the original peptide SC(CAM)DK, suggesting the possibility of either racemization in one of the four amino acid residues or isomerization of the peptide linkage such as the N f O acyl shift at Ser219 and the R f β shift at Asp221 to an isoaspartyl peptide. In fact, we have detected the isoaspartyl peptide eluted in peak I in the LC separation (see Figure 2). Thus, this excludes the identity of the peptide in peak III as the product of isomerization at Asp221, unless the isoaspartyl residue has D-configuration32,33 Identification of the Racemization by the Forced Degradation in D2O. To pursue the possibility of racemization as well as to locate the racemized residue (if any), the IgG1 antibody was subjected to forced degradation in D2O, where the R-hydrogen of an amino acid could be replaced with a deuterium atom during racemization. Compared with the XIC of the hinge peptides obtained from the IgG1 with and without the degradation in D2O, the mass of the peptide in peak III increased by 1 Da. In the MS/MS spectrum of the peptide in peak III (Figure 4), the peaks at m/z 147 and 262 for y1 (K) and y2 (DK) ions, respectively, were unaffected by the forced degradation in D2O. On the other hand, the fragment ions of y3 (C(CAM)DK) at m/z 423, b2 (SC(CAM)) at m/z 249, and b3 (SC(CAM)D) at m/z 364 were shifted by þ1 Da compared with those from peak II, clearly indicating that the CR-H/D exchange has occurred at Cys220. 3859



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Figure 4. MS/MS spectra of the hinge peptide SCDK peaks with CR-H/D exchange experiment. The precursor ions were m/z 509.2 ( 5 for (a) peak II from freshly prepared IgG1, (b) peak II from IgG1 forced to degrade in D2O, and (c) peak III from IgG1 forced to degrade in D2O.

Figure 5. UV chromatograms of the OPATATG derivatized amino acids from (a) a fractionation of peak II, (b) a fractionation of peak III, (c) synthetic S-LC(CAM)DK, and (d) synthetic S-DC(CAM)DK.

The racemization of Cys220 was confirmed by identifying in the peptide fragment SCDK, which was isolated from the degraded IgG1 samples in peak III. The chirality of each amino acid resulting from the acid hydrolysis of this peptide fragment was determined by derivatization with OPA and TATG,29 followed by separation with reversed-phase HPLC equipped with an ESI mass spectrometer (Figure 5). Eventually, the derivative S-carboxymethyl-D-cysteine (D-Cys(CM)) was detected in the acid hydrolysate of peptide in peak III (Figure 5).34 To exclude the possibility of racemization during the acid hydrolysis, synthetic peptides (S-DCDK and S-LCDK) were also submitted to chiral amino acid analysis, confirming that the enantiomers of Cys(CM) retained the respective configurations. No D-enantimer of amino acid other than cysteine was identified. The identity of the peptide in peak III as S-DCDK was finally confirmed by the finding that its elution profile in reversed-phase HPLC was identical to that of the authentic S-DCDK (Figure 2d,e). These results unambiguously indicate that the racemization of Cys220 is at least one of the major causes of degradation to occur D-cysteine

Figure 6. Results of the nonreduced peptide mapping. (1) Extracted ion chromatograms of the nonreduced hinge fragment SCDK-{SS}SFNRGRC ([M þ 2H]2þ, m/z 631.3 ( 0.1) from (a) native (freshly prepared) IgG1, (b) the sample stored at 40 °C for 6 months, and (c) the sample forced to degrade in 500 mM Tris with D2O, pH 9, at 50 °C for 7 days. (2) Mass spectra of (a) peak IV from freshly prepared IgG1, (b) peak IV from IgG1 forced to degrade in D2O, and (c) peak V from IgG1 forced to degrade in D2O. The corresponding TIC chromatograms are shown in Figure S-1 of the Supporting Information.

in IgG1 stored at 50 °C, pH 9, for 7 days. In contrast, the formation of isoaspartyl peptide appeared predominantly in the protein stored at 40 °C for 6 months, as judged from the XIC shown in Figure 2c. 3860



Figure 7. Extracted ion chromatograms of the thioether-linked hinge region fragment SCDK-{S}-SFNRGEC by the nonreduced peptide mapping ([M þ 2H]2þ, m/z 615.2 ( 0.1). The digested peptide from (a) the native (freshly prepared) IgG1, (b) the sample stressed at 40 °C for 6 months, and (c) the sample forced to degrade in 500 mM Tris with D2O, pH 9, at 50 °C for 7 days. The corresponding TIC chromatograms are shown in Figure S-1 of the Supporting Information.

This suggests that the conditions for deliberately promoting the degradation of proteins might not exactly reproduce those in natural or nonforcible degradation. Nevertheless, S-DCDK did prove to occur in IgG1 stored in the ordinary condition of 40 °C for 6 months, while the yield was lower than the isoaspartyl peptide. Specificity of Racemization at Cys220. In this section, we focus on the specificity of modification at Cys220 in all the 32 cysteine residues in IgG1 stored at 40 °C for 6 months. For the ease of detecting the incorporation of deuterium in cysteine residue(s), however, we used the forcibly degraded sample, in which racemization was deliberately accelerated, while side reactions including deamidation of asparagine were also enhanced. The number of peptides to be analyzed could be reduced to half by conducting the peptide mapping without cleaving disulfide bonds (nonreduced peptide mapping). We again detected only one clear difference in the elution pattern of digested peptides between freshly prepared and degraded IgG1 preparations on the map (Figure 6-1). The newly appeared peak was identified to be the peptide containing one cystine residue linking SCDK (heavy chain) and SFNRGEC (light chain), according to the mass value of the peak at m/z 631 ([M þ 2H]2þ). The molecular mass of this peptide fragment increased by 1 Da by the degradation in D2O (Figure 6-2). Along with this peptide, we also identified two isobaric peptides with peaks at m/z 615.2 ([M þ 2H]2þ), indicating that one sulfur atom is missing from the cystine-connecting peptide described above (Figure 7). The formation of these thioether-linked peptides containing lanthionine in place of cystine has been anticipated from the literature, assuming the β-elimination of one cysteine residue to give dehydroalanine, followed by the Michael addition of the thiol group of another cysteine residue.7,20 The formation of a pair of isobaric peptides could correspond to the diastereomerization involving the L- and D-enantiomers of residue-220. In the peptide mapping of reduced and alkylated IgG1, we did not detect CR-H/D exchange of cysteine residues except for Cys220. These results taken together, it is concluded that the racemization occurred specifically at Cys220 in the heavy chain. Reaction Pathway Leading to Racemization. There are at least two suggested reaction pathways leading to the racemization of a cysteine (cystine) residue (Scheme 1): (1) base-catalyzed exchange reaction of the CR-proton through a carbanion intermediate

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and (2) β-elimination of the thiol group, followed by the Michael addition of the thiol group to dehydroalanine formed by the elimination. As depicted in Scheme 1C, ketoenol tautomerism concerning the CRHCO moiety in the reaction pathway 1 can proceed by acid catalysis, in which the carbanion intermediate does not appear explicitly. It should be noted that the formation of a carbanion in pathway 1 could also be involved in the mechanism of β-elimination. It is possible to distinguish between these pathways by examining the reaction product(s) other than the racemic cysteine residue. In IgG1 stored for 17 days at 45 °C, a β-elimination mechanism has been proposed for the hydrolysis of the peptide bond between Cys220 and Asp221, on the basis of the detection of a thioether-linked antibody as well as that of reaction products expected to be derived from dehydroalanine.20 Indeed, we could identify the relevant peptide, SCDK-{thioether}-SFNRGEC (cystine residue connecting two peptides is replaced by lanthionine), in the tryptic digest of the degraded IgG1 (Figure 7). The appearance of two isobaric peaks suggests the formation of diastereomeric peptides involving the L- and D-enantiomers of residue-220. However, the finding of a thioether-linked peptide does not exclusively support a mechanism that assumes the initial formation of a carbanion intermediate, because it still insufficiently explains the site-specificity of racemization, which occurs solely at Cys220 linked to Asp221 in the hinge region, a putative hot spot of degradation. Even though a high solvent accessibility of that region is a favorable factor for the suggested mechanism assuming a base-catalyzed formation of a carbanion intermediate, it is necessary to elaborate an alternative mechanism that could solve this problem. We note that Cys220 is situated next to Asp221, which has been reported to undergo isomerization. This implies that these modifications have a common feature in the reaction mechanism. We thus suggest here a new reaction mechanism that assumes the nucleophilic attack of the amide nitrogen of Lys222 to the R-carbonyl carbon of Cys220 to give an imidazoline intermediate (Scheme 1A). In this mechanism, racemization of Cys220 could be facilitated by extending the conjugate system of the imidazoline ring through the spontaneous deprotonation at the CRposition in the manner of ketoenol tautomerism. Under a solution condition near pH 7, where degradation of IgG1 occurs, protonation of the ring nitrogen to initiate the enolization is more favorable than that of the carbonyl oxygen, which is apparently less basic than the ring nitrogen, thus making it unnecessary to assume a strongly acidic group to promote the racemization (Scheme 1B,C). One of the main features of this mechanism is that it also allows the nucleophilic amide nitrogen of Lys222 to attack the β-carboxyl carbon of Asp221 to form a succinimide intermediate, leading to an isoaspartyl residue. Note that both the imidazolone and succinimide derivatives thus formed have five-membered rings and that the conformational flexibility of the hinge region can be associated with the versatility of the reaction to yield different products. As shown in Scheme 1A, the β-elimination of the disulfide can occur through the migration of the CdC double bond to form an R,β-unsaturated dehydroalanine residue, which might be involved in the imidazolone ring. It is reasonable to consider that the racemic thioether- and disulfide-linked peptides are produced by the Michael-type addition of thiol and disulfide, respectively, to the dehydroalanine moiety. According to the present reaction mechanism, racemization does not require the successive β-elimination and the Michael addition but a much 3861



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Scheme 1. (A) Reaction Pathways Assumed to Involve a Mechanism of Racemization, β-Elimination, and Isoaspartyl-Peptide Formation Comprehensively in the Sequence Ser-Cys-Asp-Lys of IgG1, (B) A Possible Reaction Mechanism of Racemization, Which Corresponds to Box B in the Upper Part of A, Through an Imidazolone Intermediate,a and (C) Conventional Nonspecific Racemization Scheme27 Contrasting with B, in Which Neither Strong Acid to Protonate the Carbonyl Oxygen nor Strong Base to Abstract the CR-Proton Is Necessaryb

a

Incorporation of deuterium atom in the CR-position of Cys220 during the H/D-exchange is depicted. b RSH stands for cysteine.

simpler process of imidazolone-ring formation preceding the elimination. Spontaneous cyclization of peptide backbone to form an imidazolone ring has been suggested as one of the possible mechanisms of nonenzymatic biosynthesis of a chromophore from the sequence Ser65-Tyr-Gly67 in green fluorescent protein (GFP).35 In GFP, the side-chain carboxylate anion of Glu222 is considered to initiate the requisite cyclization by indirectly activating the amide nitrogen of Gly67 through one or two water molecules with the assistance of Arg96 to protonate the amide carbonyl oxygen of Tyr66. Provided that the degradation in the hinge region of IgG1 follows the reaction mechanism illustrated in Scheme 1A, there should be a base catalyst to enhance the nucleophilicity of the amide nitrogen of Lys222. There is an imidazole nitrogen atom (ND1) of His224 within the distance of

4 Å from the amide nitrogen of Lys222, according to one possible three-dimensional structure (PDB code 1HZH) of the hinge region of IgG1 (Figure 1).36 Generally, an imidazole group has a characteristic pKa value near 7 in proteins,37 so it can catalyze reactions as both an acid and a base. The short distance between His224 and Lys222 suggests that the imidazole group of His224 could act as a base in the present racemization mechanism (Scheme 1A). Alternatively, the β-carboxyl group of Asp221 could also serve as a base to react with the amide group of Lys222 at the longer distance of 56 Å, because this distance is comparable to that of 5.7 Å between the γ-carboxyl oxygen of Glu222 and the amide nitrogen of Gly67 in GFP.35 In any case, it is essential that racemization and isomerization would occur in the folded state of IgG1, provided the requisite stereochemistry. Note that only the 3862


Analytical Chemistry hinge region in the protein molecule is flexible enough to be able to take various conformations, rendering the characteristic nature as being the “hot spot” of degradation. On this ground, we are undertaking to identify the functional group(s) responsible to catalyze racemization and isomerization in the hinge region of IgG1. The results will be reported elsewhere. Although D-amino acid residues have been detected in longlived proteins and play a critical role in aggregation,3841 the relationship between the racemization of the hinge region and the pharmaceutical potency of the antibody is not clear. Nevertheless, degradation of any drug should be avoided at all cost. Once the mechanisms of racemization and isomerization are established, it is possible to suppress degradation effectively by avoiding the optimal conditions for enhancing these reactions. More importantly, IgG proteins can be genetically engineered to remove undesirable functional groups by taking the reaction mechanism into account.

’ CONCLUSION As a cause of degradation of IgG1 in a pharmaceutical stress testing, the specific racemization of Cys220 in the hinge region was identified through mass spectrometry in conjunction with extracted ion chromatography, chiral amino acid analysis, and chromatographic comparison with synthetic peptides. We confirmed this by detecting the CR-H/D exchange at Cys220 in IgG1, which was subjected to the degradation in D2O. Allowing for the concomitant isopeptide formation at Asp221 next to Cys220, we suggest a reaction mechanism involving a base catalysis, which activates the amide nitrogen atom of Lys222 to attack either the carbonyl carbon of Cys220 to form an imidazolone ring or the β-carboxyl group of Asp221 to form a succinimide. The specificity of these reactions is associated with the high flexibility of the hinge region, known to be the “hot spot” of degradation, as well as the intervention of base catalysis. ’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as discussed in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ81-6-6879-7440. Fax: þ81-6-6879-7441. kfukui@ bio.eng.osaka-u.ac.jp.

’ ACKNOWLEDGMENT Authors highly acknowledge Emi Komori and Kei Kubota for their kind support and helpful discussions. ’ REFERENCES (1) Weiner, L. M.; Surana, R.; Wang, S. Nat. Rev. Immun. 2010, 10, 317–327. (2) Maggon, K. Curr. Med. Chem. 2007, 14, 1978–1987. (3) Daugherty, A. L.; Mrsny, R. J. Adv. Drug Delivery Rev. 2006, 58, 686–706. (4) Kozlowski, S.; Swann, P. Adv. Drug Delivery Rev. 2006, 58, 707–722. (5) Liu, H.; Gaza-Bulseco, G.; Faldu, D.; Chumsae, C.; Sun, J. J. Pharm. Sci. 2008, 97, 2426–2447.

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