Contributions of a disulfide bond to the structure ... - Wiley Online Library

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain ARNOLD MCAULEY, JABY JACOB, CARL G. KOLVENBACH, KIMBERLY WESTLAND, HYO JIN LEE, STEPHEN R. BRYCH, DOUGLAS REHDER, GERD R. KLEEMANN, DAVID N. BREMS, AND MASAZUMI MATSUMURA Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, California 91320, USA (R ECEIVED July 20, 2007; F INAL R EVISION October 4, 2007; ACCEPTED October 5, 2007)

Abstract Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of b-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the CH3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human CH3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized CH3 dimer were similar, but differences were observed. The reduced CH3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer–dimer equilibrium. Interestingly, the dimer–monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human CH3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer–dimer equilibrium of the human CH3 domain. Hence, these results may have implications for the stability of the intact antibody. Keywords: human antibody; CH3 domain; disulfide bond; stability; dimerization

With the recent advances in heterologous expression of antibodies, more than a dozen monoclonal antibodybased drugs have reached the market. Hundreds more Reprint requests to: Masazumi Matsumura, Department of Pharmaceutics, Amgen, Inc., One Amgen Center Drive, Mailstop 2-1-A, Thousand Oaks, CA 91320, USA; e-mail: [email protected]; fax: (805) 375-5794. Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; Cm, midpoints of the transition; DLS, dynamic light scattering; DTT, dithiothreitol; GdmCl, guanidinium chloride; RP-HPLC, reversed-phase high performance liquid chromatography; SE-HPLC, size-exclusion high performance liquid chromatography; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride. Article and publication are at http://www.proteinscience.org/cgi/ doi/10.1110/ps.073134408.

are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. A number of key scientific and technological advancements, such as the generation of fully human antibody by transgenic mice (Green 1999), have contributed to the successful development of antibody-based therapeutics. Stability and homogeneity of therapeutic antibodies are of prime importance for safety and efficacy. Undesired biochemical, structural, and conformational forms can lead to loss of efficacy and risk of adverse side effects. IgG1 (hereafter referred to as antibody) is a complex molecule, composed of two identical light (L) and heavy

Protein Science (2008), 17:95–106. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2008 The Protein Society

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(H) chains. The light chain consists of a variable domain (VL) and a constant domain (CL), whereas the heavy chain is composed of a variable domain (VH) and three constant domains (CH1, CH2, and CH3). Each domain encodes ;100 amino acid residues. One light chain (VLCL) associates with the amino-terminal region (VH CH1) of one heavy chain to form an antigen-binding site (VLVH) through noncovalent interactions and the interchain disulfide bond. The carboxyl-terminal regions (CH2 CH3) of two heavy chains associate with each other through two disulfide bridges in the hinge region between the CH1 and CH2 domains and by strong noncovalent interactions between the two CH3 domains (Huber et al. 1976; Harris et al. 1992). Furthermore, two complex biantennary N-linked oligosaccharides are attached to the antibody through an asparagine residue in the CH2 domain. The recombinant production of therapeutic antibodies requires each chain within the multidomain complex to be accurately synthesized, correctly folded, and self-assembled. As shown in Figure 1, the human CH3 domain has a twofold axis of symmetry that runs through the dimer interface. The intermolecular interface between the two CH3 domains involves 16 amino acid residues located on

Figure 1. Model of the human IgG1 CH3 domain in a dimeric form. The domain structure was truncated from the human antibody IgG1 b12 (Saphire et al. 2001) with a PDB ID of 1HZH. The figures were generated using DS ViewerPro (Accelrys). The viewpoint is parallel (A) and perpendicular (B) to the axis of symmetry with each monomer colored separately, and the Cys and Trp residues are in stick representation. The disulfide linkage of Cys31 and Cys89 in the recombinant CH3 protein corresponds to that of Cys250 and Cys308 in the human immunoglobulin g-1 chain C region (Swiss-Prot entry name, IGHG1_HUMAN; primary accession number, P01857). Similarly, Trp45 and Trp81 correspond to the residues at 264 and 300 of the human IgG1 heavy-chain constant region.

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four antiparallel b-strands (Dall’Acqua et al. 1998). Five of these residues play a key role in the interfacial stability. In contrast, the CH2 domains do not interact with each other, as is evident from the antibody crystal structure (Huber et al. 1976; Deisenhofer 1981), and in fact these isolated domains exist as monomers in solution (Feige et al. 2004). The CH3 domain exhibits a typical immunoglobulin-like fold, which contains seven b-strands folded into an antiparallel b-sandwich. Connecting two b-sheets is a buried, highly conserved disulfide bond between cysteine residues 31 and 89. There is a tryptophan residue at position 45 (Trp45) in close proximity to this disulfide bond. The ˚. distance between Trp45 and the disulfide bond is ;3.5 A Therefore, the fluorescence signal arising from Trp45 is significantly quenched in the folded state by the disulfide bond (Thies et al. 2002). In contrast, another tryptophan (Trp81) is far from the disulfide bond, and hence it is unlikely that its fluorescence is quenched by the disulfide in the folded state. In addition, neither of the tryptophan residues is located near the dimer interface, suggesting that the tryptophan fluorescence signal would not be affected by dissociation of the dimer (Isenman et al. 1979). Disulfide bonds can make a substantial contribution to protein stability. The effect of disulfide bonds on stability has been studied in many small globular proteins, such as ribonuclease (Pace et al. 1988) and lysozyme (Matsumura et al. 1989; Denton and Scheraga 1991; Taniyama et al. 1992). For antibodies, the effect of a disulfide bond on stability has been investigated using various isolated domains (Goto and Hamaguchi 1979; Glockshuber et al. 1992; Proba et al. 1997; Thies et al. 2002). For example, Goto and Hamaguchi (1979, 1986) prepared proteolytically cleaved human IgG CL domain, and Thies et al. (2002) studied the stability of recombinant murine IgG CH3 domain. These studies have shown that the intrachain disulfide bond has little effect on the tertiary structure of the respective domain, but its stability is significantly increased. The origin of the major stabilizing effect of a disulfide bond is thought to be the decrease in conformational entropy in the unfolded state of a protein (Lin et al. 1984; Pace et al. 1988). Furthermore, disulfide bonds have been shown to influence the folding pathways and kinetics in murine antibody CL and CH3 domains (Thies et al. 2002; Feige et al. 2007) as well as in human antibody CL domain (Goto and Hamaguchi 1982). Recombinant human monoclonal antibodies or Fc fragments have been observed to contain some amounts of reduced disulfide bond in the buried regions, especially in the CH3 domain (Zhang and Czupryn 2002; Pipes et al. 2005). To understand the contribution of the CH3 disulfide bond on conformation and stability, we have prepared and conducted experiments on fully reduced and oxidized forms of recombinant human CH3 domain. The reduced CH3 domain spontaneously formed a stable

Stability of human antibody CH3 domain

dimer in solution, and the conformations of reduced and oxidized CH3 were similar as judged by tryptophan fluorescence and circular dichroism (CD). However, the reduced CH3 domain was significantly less stable than the oxidized form against denaturation by GdmCl, acidic pH, and heat. Furthermore, the equilibrium unfolding and dissociation studies using analytical ultracentrifugation (AUC) and size-exclusion HPLC (SE-HPLC) coupled with dynamic light scattering (DLS) revealed that the oxidized CH3 dimer unfolded and dissociated to monomer simultaneously. However, these events were not coupled in the reduced CH3 dimer, as dissociation occurred at a different concentration of denaturant prior to unfolding. Results Escherichia coli-expressed CH3 domain exists in the reduced state The human IgG1 antibody CH3 domain was expressed in the cytosol of E. coli and purified to homogeneity by Niaffinity chromatography. The redox status of the disulfide bond of CH3 domain was investigated by RP-HPLC. As controls, fully reduced (SH) and oxidized (SS) forms of CH3 were prepared by incubating the protein in 3 M GdmCl in the presence of 10 mM DTT or 0.5 mM CuSO4, respectively, where DTT reduces the disulfide bond and copper catalyzes autoxidation to form disulfides (Kachur et al. 1999). RP-HPLC chromatograms of reduced and oxidized CH3 showed that the oxidized and reduced forms eluted at 24.0 and 25.2 min, respectively (data not shown). The oxidized form eluted earlier than the reduced form, presumably due to less exposure of hydrophobic surfaces of the unfolded oxidized form that was induced by the acidic mobile phase pH of 2.0. When the purified CH3 domain from E. coli lysate was analyzed by the same method, the protein eluted at the same time as the fully reduced form, indicating that purified CH3 domain was in the reduced state. The absence of the disulfide bond of purified CH3 was also confirmed by SDS SE-HPLC, tryptic mapping, and ESI-TOF mass spectroscopy. The latter showed a mass difference of 2 Da between oxidized and reduced forms (data not shown). Spectroscopic profiles of reduced and oxidized CH3 To investigate the effect of the disulfide bond on the conformation of human CH3 domain, near- and far-UV CD spectra as well as the fluorescence emission spectra were obtained for both the reduced and oxidized CH3 domains (Fig. 2). The results showed that reduced CH3 domain has intact secondary and tertiary structures and that both the reduced and oxidized forms exhibited similarities, suggesting that their secondary and tertiary

Figure 2. Spectroscopic characterization. (A) Far-UV CD spectra and (B) near-UV CD spectra of reduced (gray) and oxidized (black) forms of human CH3. The measurements were carried out at 20°C at a protein concentration of 230 mg/mL (8.6 mM dimer). The reduced sample was measured in 2 mM sodium acetate, pH 5.0, and the oxidized sample was in 5 mM sodium acetate, pH 5.0. (C) Fluorescence spectra of reduced (gray) and oxidized (black) forms of human CH3. The reduced form is shown under native conditions (0.0 M GdmCl; gray solid line) and denaturing conditions (4.8 M GdmCl; gray dotted line). The oxidized form is shown under native conditions (0.1 M GdmCl; black solid line) and denaturing conditions (5.7 M GdmCl; black dotted line). The measurements were performed at 20°C at a protein concentration of 30 mg/mL (1.1 mM dimer). The excitation wavelength was set at 280 nm. The emission maximum occurs in the region of 357 nm in the unfolded state and is shifted to a lower wavelength (;330 nm) in the folded state. The emission is quenched in the folded state for the oxidized form because of the proximity of the disulfide bond to Trp45 (see Fig. 1).

structures may not be significantly different. The farUV spectrum (Fig. 2A) showed a weak and broad minimum at ;225 nm for the oxidized CH3 domain. At the same wavelength, the magnitude at the minimum was www.proteinscience.org

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lower for the reduced CH3 domain, showing an additional minimum at 218 nm. In the near-UV spectra (Fig. 2B) the main minimum was observed at ;273 nm in both reduced and oxidized forms, and a second minimum was observed at 293 nm. The near-UV spectra were similar for both forms, though the magnitude of the signal was larger for the reduced form. The absolute intensities of both far- and nearUV CD spectra were relatively smaller for human IgG1 CH3 domain compared to murine CH3 (Thies et al. 2002). The fluorescence emission spectra (Fig. 2C) had a lmax at ;330 nm for both reduced and oxidized forms, implying that the tryptophan residues are in similar environments. Upon unfolding, the emission maxima shifted significantly to higher wavelengths (in the region of 357 nm) and the fluorescence intensities increased in both reduced and oxidized forms. The enhanced intensity was more profound for the oxidized form when compared to the reduced form. This dramatic quenching of tryptophan fluorescence signal in the folded, oxidized CH3 is likely due to the proximity of Trp45 to the disulfide bond (Fig. 1) (Hennecke et al. 1997; Thies et al. 2002). Quaternary structure of the reduced CH3 domain To investigate whether the reduced form of human CH3 domain existed as a dimer in solution, the reduced CH3 domain was analyzed by SE-HPLC equipped with a multiangle laser light scattering detector (Fig. 3A). The light scattering result showed that both reduced and oxidized human CH3 formed a dimer with an approximate molar mass of 24 kDa. The elution time of the reduced CH3 dimer was identical to that of the oxidized form. The result was confirmed by size-exclusion chromatography using various molecular weight standards (Fig. 3B). The observation implies that the removal of a disulfide bond in human CH3 domain does not disrupt the dimer interface. Our finding is in good agreement with previous studies conducted on the CH3 domain of murine monoclonal antibody MAK33 (Thies et al. 2002) and the native (oxidized) CH3 domain of human IgG1 (pFc9) (Isenman et al. 1979). Acid- and heat-induced denaturation The effect of acid-induced unfolding of the reduced and oxidized CH3 domain was monitored by fluorescence spectroscopy (Fig. 4A). Both the reduced and oxidized CH3 domains unfolded cooperatively as the pH decreased. The pH values at the midpoint of the acid-induced unfolding transition of reduced and oxidized CH3 were 3.29 and 2.97, respectively. The result showed that the oxidized form was more resistant to acid-induced denaturation than the reduced form by ;0.3 pH unit. Enhanced stability of the oxidized form against pH denaturation was also reported for the constant fragment 98


Figure 3. (A) Molecular mass determination of reduced (gray) and oxidized (black) human CH3 homodimer by dynamic light scattering. X-, left Y-, and right Y-axes represent time, molar mass, and UV absorbance at 280 nm (A280), respectively. The inset SE-HPLC chromatograms show the entire chromatograms monitored by A280. (B) SE-HPLC elution times of reduced and oxidized CH3 (d) were plotted against their molecular masses with the following molecular mass standards: human antibody (IgG, 150 kDa, open squares), bovine serum albumin (BSA, 68 kDa, open triangles), and granulocyte-colony stimulating factor (G-CSF, 14 kDa, open inverted triangles). Both reduced and oxidized CH3 were eluted at identical elution times.

of the immunoglobulin light chain (Ashikari et al. 1985) and endostatin (Zhou et al. 2005). The thermal unfolding of the reduced and oxidized CH3 domain was monitored by CD spectroscopy for changes in secondary structure. The result show that the melting temperatures (Tm) of heat-induced unfolding of reduced and oxidized CH3 domain were 68.5 and 75.5°C, respectively (Fig. 4B), indicating that oxidized CH3 is more thermally stable than the reduced form. It should be noted, however, that the thermal unfolding of reduced CH3 was not reversible. In contrast, the oxidized form showed excellent reversibility during thermal unfolding and refolding cycles. Equilibrium denaturation stability of the reduced and oxidized CH3 domain The stability of both reduced and oxidized CH3 domain was analyzed by GdmCl-induced unfolding transitions at pH 5.0 via tryptophan fluorescence. As seen in Figure 2C, unfolding causes the fluorescence emission wavelength to


both the dissociation of the dimer and unfolding of the monomers. To investigate whether unfolding, as shown in Figure 5A, is coupled with dissociation, and whether the disulfide bond played a role in either of the two events, the molecular weights of both the reduced and oxidized forms at various GdmCl concentrations were determined using sedimentation equilibrium AUC. To make such a comparison feasible, all AUC analyses were conducted under identical experimental conditions as the equilibrium unfolding experiments described previously (1.9 mM dimer in 10 mM sodium acetate buffer at pH 5.0). The AUC results are shown in Figure 5B. For the reduced CH3 domain, at 0.0 M GdmCl the sedimentation data were fit with a single species model with a molar mass of ;26 kDa, which is consistent with a dimer. The dimer was maintained until the GdmCl concentration reached 0.5 M. As the GdmCl concentration increased beyond 0.5 M, the reduced CH3 dimer dissociated to monomer and completed the dissociation at

Figure 4. (A) Acid-induced denaturation of reduced (n) and oxidized (s) forms of human CH3 at pH 2.0–8.0. The fluorescence measurements were performed at 20°C at a protein concentration of 70 mg/mL (2.6 mM dimer). The fraction of folded CH3 was calculated from the red shift of the maximum wavelength of emission spectra at each pH (see details in Materials and Methods). The solid lines were fit to a three-parameter sigmoidal using SigmaPlot software. (B) Thermal unfolding of reduced (n) and oxidized (s) CH3 domain monitored by CD at 218 nm. The solid lines were fit to a three-parameter sigmoidal plot using SigmaPlot software.

red-shift from 330 to 357 nm, which is accompanied by an increase in the fluorescence intensity. Since the equilibrium denaturation curves are expected to be dependent on protein concentration because of the dimeric nature of the native state, the fluorescence measurements were performed at the same protein concentration (1.9 mM dimer) at pH 5.0 for both the reduced and the oxidized forms. This allowed direct comparison of the stability of the two forms. Equilibrium denaturation data are shown in Figure 5A. The results show that the unfolding midpoint was shifted to a higher GdmCl concentration for the oxidized form. The midpoints of the transition (Cm) were 1.3 and 2.2 M GdmCl for the reduced and oxidized CH3 domain, respectively. The loss of secondary structure during unfolding was also monitored by CD, and the results agreed with the loss of tertiary structure monitored by fluorescence (data not shown). Guanidinium chloride-induced dissociation of CH3 homodimer As stated previously, the CH3 domain is dimeric in the native state. Therefore, the addition of GdmCl induces

Figure 5. (A) Guanidinium chloride-induced denaturation of reduced (n) and oxidized (s) forms of human CH3. Measurements were obtained at 20°C at a protein concentration of 50 mg/mL (1.9 mM dimer) in 10 mM sodium acetate, pH 5.0. Data correspond to emission intensities measured at 357 nm at an excitation wavelength of 280 nm. Signals were normalized relative to unfolded and folded values. The solid lines were fit to the data using Equation 3. (B) Sedimentation equilibrium of reduced (n) and oxidized (s) forms of human CH3 at varied concentrations of GdmCl. Ultracentrifugation was performed at 20°C at a protein concentration of 50 mg/mL (1.9 mM dimer) in 10 mM sodium acetate, pH 5.0. Data correspond to the molecular mass of CH3 at equilibrium. Error bars are generated from three data sets.

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1.0 M GdmCl, yielding a molar mass of ;13 kDa. On the other hand, the oxidized CH3 dimer was maintained until dissociation at ;1.5 M GdmCl. Complete dissociation was achieved at 2.5 M GdmCl. At concentrations >2.5 M, the oxidized CH3 was identified as a single species with a molar mass of ;13 kDa. Approximate Cm values for the reduced and oxidized forms were 0.7 and 1.9 M, respectively. Thus, the result showed that the disulfide bond increased the CH3 dimer stability against GdmCl-induced dissociation and implied that the noncovalent interactions at the dimer interface are enhanced by the disulfide bond. Interestingly, for the reduced CH3 domain, the dimer– monomer dissociation transition occurred between 0.5 and 1.0 M GdmCl and did not coincide with the unfolding transition monitored by fluorescence that occurred between 1.0 and 1.8 M GdmCl (Fig. 5, cf. A and B). This is most notable at ;1.0 M GdmCl, where the reduced CH3 domain is monomeric yet the majority of the protein is folded. This implies that the reduced CH3 dimer dissociated before the individual CH3 domains unfold, and therefore the two events may not be coupled. In contrast, dissociation and unfolding of oxidized CH3 domain occurred simultaneously within the same GdmCl concentration window between 1.5 and 2.5 M and therefore looks coupled. Dimer dissociation and unfolding of reduced and oxidized CH3 domain To further characterize the nature of dissociation and unfolding of the CH3 domain, denatured SE-HPLC was conducted at various GdmCl concentrations. The protein was detected by UV absorbance at 214 and 280 nm, and monomer–dimer content was simultaneously monitored by a multi-angle laser light scattering detector. Both reduced and oxidized CH3 were equilibrated at various GdmCl concentrations before being injected onto the column, which had been pre-equilibrated with the respective GdmCl concentration. For the reduced CH3 domain (Fig. 6A), the chromatogram for 1.0 M GdmCl showed a single, homogeneous native dimer peak with a molar mass of 26.5 kDa. At 1.5 M GdmCl, the reduced CH3 domain eluted as two partially resolved species separated by 1 M GdmCl and an oxidative agent, such as Cu2+. Furthermore, we found that the disulfide bond could not be formed efficiently by unfolding and refolding the protein at pH 8.0 or 9.0 without copper or other redox reagents, such as a reduced/oxidized glutathione mixture. Also, it was difficult to fully reduce the disulfide bond using DTT without partially unfolding the protein first with GdmCl. These observations suggest that the structure of folded CH3 is sufficiently stable to prevent action of the oxidative/reductive agents on the hydrophobic core of CH3 where the disulfide bond is located. Several studies with antibodies showed that the formation of interchain disulfide bonds are governed by

proximity of the two thiol groups and by the differences in the ionization constant and reactivity between the two thiol groups (Kishida et al. 1976; Kato et al. 1978; Tanaka et al. 1978). Further studies with the antibody CL domain (Goto and Hamaguchi 1981) and bovine pancreatic trypsin inhibitor (Weissman and Kim 1991) suggested that the lack of disulfide bond formation is due to the burial of two thiol groups in the hydrophobic interior of protein. Although it is clearly demonstrated by these studies that the buried cysteines are not easily oxidized, our observations with a recombinant Fc fragment suggest that the burial of thiol groups alone may not be the single cause. Recently, Pipes et al. (2005) reported that an oxidatively refolded Fc fragment was partially composed of reduced CH3 domain, but CH2 domain is fully oxidized. In spite of the sequence and structural similarities between the CH2 and CH3 domains and the fact that the thiol groups in the both domains were buried in the interior of molecules, the CH3 domain is more likely to exist in a partially oxidized state. This led us to hypothesize that the lack of spontaneous disulfide bond formation in the human CH3 domain may be due to an unfavorable disulfide geometry or distance between the two sulfurs of Cys31 and Cys89. To address this issue, we reduced the disulfide bond of human CH3 by molecular modeling using the Insight II software package (Accelrys) and optimized the simulated reduced structure with 300 cycles of energy minimization. The result revealed that the rearrangement of the overall structure was slight, but noticeable movements were observed around the two cysteine residues. The side chains of Cys31 and Cys89 were rotated ;4° and 22°, respectively, away from their original locations within the oxidized form. In combination, the total movement of the two cysteine side chains (;26° away from each other) put these residues out of proper disulfide bond geometry. The theoretical disulfide bond that could form with the simulated cysteine residues would have a bond x2 of 160°, which is 20° from the low-energy disulfide bond angle of 180° (Thomson et al. 1989). In addition, the oxidized state of the CH3 domain has Sg–Sg atoms that are within a conventional covalent disulfide bond distance ˚ (Saphire et al. 2001). This distance increased to of 2.0 A ˚ in the modeled reduced state. These structural 3.2 A changes could potentially impede the formation of the disulfide bond within a reduced, folded CH3 domain. Comparison with murine CH3 domain The three-dimensional structures of the IgG1 CH3 domain between human and murine species are very similar (Huber et al. 1976; Deisenhofer 1981; Saphire et al. 2001). Both human and murine CH3 domains encode the same numbers of amino acid residues (107 in total), and www.proteinscience.org

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the amino acid sequences are 59% identical and 94% homologous. Nonetheless, the human CH3 domain showed slightly different secondary and tertiary structural characteristics compared to the murine CH3 domain (Thies et al. 2002), as judged by their respective CD spectra. First, the absolute intensities of both far- and near-UV CD spectra were relatively low for the human CH3 domain compared to the murine counterpart. Second, in human CH3 the intensity of far-UV spectra at 220 nm changed very little upon the disulfide bond formation (approximately 1500 deg2 cm dmol1 for both forms). However, the far-UV CD spectra of reduced murine CH3 showed a significant increase in the intensity of the signal at 220 nm (from 2500 to 7000 deg2 cm dmol1) compared to the oxidized domain (Thies et al. 2002). Thies et al. (2002) suggested that the increased far-UV signal may indicate a more compact secondary structure due to the removal of the disulfide spacer between the two b-sheets. In contrast, the far-UV spectra of reduced CL domains of both human and murine are comparable to those of oxidized forms (Goto and Hamaguchi 1986; Feige et al. 2007), as in the case for human CH3 in this study. These observations may suggest that the disulfide formation in the hydrophobic core of CH3 domain may perturb its secondary structure for murine but not human molecule. Third, in human CH3, the intensity of the nearUV CD spectrum of the reduced form was greater than that of the oxidized form. The opposite was observed in the murine CH3. The human CH3 domain contains two tryptophans, five tyrosines, and four phenylalanines, while the murine has three tryptophans, four tyrosines, and four phenylalanines. In addition, some of these aromatic residues are located at different positions. For example, tyrosine at position 37 and phenylalanine at position 68 in human are replaced by phenylalanine and tyrosine in murine, respectively. Different environments around these aromatic residues may contribute to the observed dissimilarity of near-CD spectra between the two species. Disulfide bonds are known to increase the conformational stability of many proteins. For the murine CH3 domain, the reduced protein started to unfold at 0.05 M GdmCl, while the oxidized form was stable up to 0.4 M GdmCl. The midpoints of the transition (Cm) for the murine protein were 0.3 M GdmCl for the reduced form and 1.0 M GdmCl for the oxidized form (Thies et al. 2002). Similarly, the Cm values for the human protein were determined to be 1.3 M GdmCl for the reduced form and 2.2 M GdmCl for the oxidized form. Thus, the disulfide bond stabilizes both murine and human CH3 domains. However, human CH3 is considerably more resistant to GdmCl-induced denaturation than the murine counterpart. For example, at 0.6 M GdmCl, the reduced form of murine CH3 was completely unfolded, whereas 102


the reduced form of human CH3 was predominantly folded. Furthermore, while it has been observed that the unfolding of reduced murine CH3 by GdmCl was not reversible (Thies et al. 2002), unfolding of both forms of human CH3 was reversible under our denaturing conditions (data not shown). Contribution of the disulfide bond to domain dissociation and unfolding Since the CH3 domain exists as a homodimer in the native state, denaturation using GdmCl should lead to both dissociation of the dimer and unfolding of the monomers. For the human CH3 domain, the dissociation and unfolding at equilibrium appears to be uncoupled as shown in Figure 7. The figure depicts both dimer dissociation and monomer unfolding at various concentrations of GdmCl for reduced (Fig. 7A) and oxidized (Fig. 7B) forms. The fractions of unfolded protein and dimer in the figure were calculated using the original data shown in Figure 5, A and B, respectively. For both forms of CH3, the dimer dissociation transition occurred at lower GdmCl concentrations than the monomer unfolding transition, although the two events were distributed over a narrower range of GdmCl concentrations for the oxidized form. A threestate model for dissociation and unfolding of the dimeric

Figure 7. Comparison between the CH3 unfolding monitored by fluorescence (closed symbols; left Y-axis) and the dimer to monomer dissociation monitored by AUC (open symbols; right Y-axis). (A) Reduced form; (B) oxidized form. The fractions of unfolded and dimer were calculated from the data shown in Figure 5, A and B, respectively. The solid lines were fit to a three-parameter sigmoidal plot, (f ¼ a/(1 + exp[(x x0/b)])), using SigmaPlot software.


protein can be expressed by the following equation (Neet and Timm 1994): K1

K2

K1

K2

N2 5 2N 5 2U ðaÞ or N2 5 2I 5 2U ðbÞ K 1 = ½N2 =½N2 ; K 2 = ½U=½N

(1)

entropy in the unfolded state (Schellman 1955), assuming the change in enthalpy (DH) is negligible. According to Pace et al. (1988), the entropic loss by a disulfide bond of an unfolded protein can be estimated using the following equation:

(2) DS ðconformational entropyÞ = 2:1 ð3=2ÞR ln n (4)

The three-state model involves native dimer (N2), native monomer (N) or a monomeric intermediate (I), and unfolded monomer (U). Unfolded dimers are not likely to exist except when they are covalently linked. For convenience, only N is used to display the two equilibrium constants in Equation 2. K1 and K2 are the equilibrium constants for the dissociation and unfolding, respectively, as defined by Equation 2. When K2 is significantly larger than K1, the dissociation dominates the overall transition. In such a case, a native monomer (N) or monomeric intermediate (I) is unstable and does not accumulate significantly during the transition. This scenario (K2 >> K1) may be applied to human CH3 because the accumulation of native monomer was not detected by denatured SE-HPLC (Fig. 6). Hence, Equation 1 resembles a simple two-state model as shown in Equation 3. N2 5 2U

(3)

For the murine CH3 domain, it has been shown that the equilibrium denaturation is described as the two-state model between a folded dimer (N2) and an unfolded monomer (U) expressed by the equation above (Thies et al. 1999). By applying the two-state model (Equation 3) for the dissociation event, the DG values of dissociation were estimated to be 12.4 kcal/mol for reduced and 15.8 kcal/mol for oxidized human CH3. The estimated DG value for the oxidized form is consistent with human CH3 dimers having a dissociation constant of 13 kcal/mol. When the same two-state model was applied for the unfolding event, the DG values of unfolding were calculated to be 12.5 kcal/mol for reduced and 15.2 kcal/mol for oxidized form. The latter compares well to that measured for the oxidized murine CH3 (15.9 kcal/mol) (Thies et al. 1999) (note that the value for reduced murine CH3 is not available, as refolding of the protein was not entirely reversible) and other dimeric proteins (Neet and Timm 1994). Interestingly, the DG values of unfolding coincide well with those DG values of dissociation. Our result also suggests that the net contribution of the disulfide bond to the dimerization and folding is ;3 kcal/mol. The contribution of disulfide bonds to protein stability is presumed to be mainly due to loss of conformational

where n is the number of residues in the loop forming the disulfide bond. For human CH3, n is 57 (residues), and hence DS at 20°C (TDS) is estimated to be 4.1 kcal/mol. This value is close to the observed DG difference (stabilized by ;3 kcal/mol) between reduced and oxidized forms, and thus the majority of stabilization energy provided by the disulfide stems solely from its contribution to the unfolded state, assuming that the difference in free energy change between the two forms in the folded state is small (Matsumura et al. 1989). Our results demonstrated that the disulfide bond not only stabilizes a conformation of monomeric CH3 but also enhances the dimer interactions. This data is well supported by other reports (Azuma et al. 1993; Furukawa et al. 2004) where the shift of the monomer–dimer equilibrium is affected by the disulfide bond. Thus, correct disulfide bond formation in human CH3 domain is critical for both stability and dimerization. This may have possible stability implications for the drug products containing intact antibody.

Materials and Methods Expression and purification of the human IgG1 CH3 domain The CH3 domain of human IgG1 was cloned, expressed, and purified by Abgent, Inc. Briefly, the human IgG1 CH3 domain was cloned into pET21a plasmid (Invitrogen) and expressed in E. coli strain BL21. The amino acid sequence of the human IgG1 CH3 domain used in this study is as follows: MGSSGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH. The theoretical molecular mass is 13,261 Da for the monomeric oxidized form. The cloned sequence contains four (MGSS) and six (HHHHHH) extra amino acid residues at the N- and C-termini, respectively, for expression, cloning, and purification. The glycine (G) at position 5 corresponds to the amino acid position 224 of the human immunoglobulin gamma-1 chain C region (SwissProt entry name: IGHG1_HUMAN, primary accession number, P01857). Likewise, Cys31, Trp45, Trp81, and Cys89 of the recombinant CH3 protein correspond to the residues at 250, 264, 300, and 308, respectively, of IGHG1_HUMAN. The recombinant protein was expressed in E. coli BL21 in the presence of 1 mM isopropyl-b-D-thiogalactopyranoside at room temperature for 4 h. Cells were lysed with 50 mM NaH2PO4,


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300 mM NaCl, 10 mM imidazole, 1% Triton-X100, pH 8.0 buffer with protease inhibitors (Roche Applied Science). Only the soluble fraction of the lysate was purified using Ni-NTA resin (Qiagen GmbH) according to the manufacturer’s protocol. The purified protein was dialyzed against 10 mM sodium acetate, pH 5.0 at 4°C. The protein concentration was determined by UV spectroscopy using an absorption coefficient at 280 nm of 1.417 (mg mL1 cm1) or 37,582 (M1 cm1) for dimer (Pace et al. 1995). As described below, the purified CH3 protein was mostly in the reduced form (SH) and existed as homodimer in solution.

in measuring GdmCl-induced unfolding were prepared in 10 mM sodium acetate buffer at pH 5.0 in 0–6 M GdmCl at a protein concentration of 50 mg/mL (1.9 mM dimer). Both reduced and oxidized samples were equilibrated for a period of ;24 h at room temperature and for 4 d at 4°C prior to measurement. To eliminate the possibility of re-oxidation, 0.5 mM of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to all the reduced samples. The emission spectra were measured for all samples, and the intensity at 357 nm was plotted. TCEP was purchased from Sigma Aldrich.

pH titration by fluorescence spectroscopy Preparation of reduced and oxidized CH3 domain In most cases, the purified CH3 dimer was in the reduced form. However, occasionally the protein was partially oxidized. To prepare fully reduced CH3 domain, the protein was incubated in 3 M GdmCl, 0.1 M Tris-HCl, pH 7.0 with 10 mM dithiothreitol (DTT) for 1 h at room temperature. The protein was refolded by dialyzing against 0.1 M Tris-HCl, pH 7.0 plus 0.45 M L-arginine for 24 h at 4°C (Arakawa and Tsumoto 2003; Umetsu et al. 2003). Subsequently, the protein was extensively dialyzed against 10 mM sodium acetate, pH 5.0 and stored at 4°C until use. High purity grade GdmCl was purchased from MP Biomedicals, Inc. The purified, reduced CH3 protein was denatured and oxidized in 3 M GdmCl, 0.1 M Tris-HCl, pH 7.0, and 0.5 mM CuSO4 for 1 h at room temperature (Winterbourn and Carrell 1977). The final protein concentration during oxidation was adjusted to be #0.6 mg/mL to avoid aggregation. Subsequently the protein was refolded in 0.45 M L-arginine buffer described above, dialyzed against 10 mM sodium acetate, pH 5.0, and stored at 4°C.

Reduced and oxidized CH3 were diluted to a concentration of 70 mg/mL (2.6 mM dimer) in a buffer solution containing 50 mM Tris-HCl, 20 mM sodium acetate, 20 mM MES at pH 2.0–10.0. The samples were left at room temperature for 3 d to reach equilibrium. Aliquots of 1.0 mL sample were placed in a 1-cm cuvette with a stir bar. Spectra were recorded on a PTI fluorometer at a controlled cell temperature of 20°C with gentle mixing. An excitation wavelength of 280 nm was used, and emission spectra were recorded between 290 and 400 nm. The excitation and emission slit widths were 2 nm and 5 nm, respectively. The pH titration data were analyzed by plotting wavelength against fluorescence emission (in counts per second) for each sample. The portion of the curve 620 nm from the wavelength maximum was fitted to a second-order polynomial equation. The first derivative of this equation was then used to solve for the maximum wavelength for each sample at each pH value. The fluorescence maximum was plotted against pH. Pre- and posttransitional baselines were fit from the resulting sigmoidal curve. The baseline equations were used to determine the fraction of folded protein using the equation below:

Reversed-phase HPLC The reversed-phase chromatography was performed with an Agilent 1100 high-performance liquid chromatography (RPHPLC) system using Agilent ChemStation software. An Agilent ZORBAX 300SB-C18 (2.1 3 100 mm, 3.5 mm particle size, 300 ˚ pore size) column was used. Typically 5–10 mg of sample was A loaded on the column, and the chromatography was conducted at 55°C at 0.5 mL/min using a 16.2%–43.2% acetonitrile gradient in the presence of 0.1% trifluoroacetic acid (TFA).

Fraction folded = ðY Y u Þ=ðY f Y u Þ; where Y is the data at each pH value, and Yu and Yf correspond to the denatured and native state linear functions, respectively. The fraction of folded CH3 was then plotted versus pH, and the resulting curve was fit to a three-parameter sigmoid, f ¼ a/{1 + exp[(x x0/b)]}, using SigmaPlot software. In the case of the equilibrium GdmCl denaturation curve measured by fluorescence, the data were fitted to an equilibrium two-state model corresponding to Equation 1 in the Discussion section.

Spectroscopic techniques The circular dichroism (CD) spectra measurements were obtained on a JASCO Model J-810 spectropolarimeter (JASCO, Inc.). A 1 cm path-length quartz cuvette was used for near-UV spectra measurements, whereas a 1 mm path-length quartz cuvette was used for far-UV spectra measurements. All measurements were performed at 20°C. The protein concentration was ;230 mg/mL (8.6 mM dimer) in all cases, and the buffer used was 2 mM sodium acetate (for reduced CH3) and 5 mM sodium acetate (for oxidized CH3) at pH 5.0. The CD spectra of the buffer solutions in the appropriate cuvettes were subtracted from the sample spectra before conversion to absolute CD values. The fluorescence spectra were measured using an AVIV Model ATF 105 spectrofluorometer. The excitation wavelength was set to 280 nm. All measurements were performed at 20°C with the sample in a 1.0 3 0.2 cm quartz cuvette. Samples used

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Thermal unfolding by CD Reduced and oxidized CH3 were prepared to a concentration of 150 mg/mL (5.7 mM dimer) in 10 mM sodium acetate buffer at pH 5.0. The CD spectra measurements were carried out on an AVIV Model 202-01 spectropolarimeter (AVIV Biomedical). A 2-mm path-length quartz cuvette was used for far-UV spectra measurements at 218 nm. All measurements were performed from 25 to 91°C with a 2°C temperature increment and 1 min equilibration time. Two scans were run for each sample, and the data were averaged and plotted as CD signal intensity versus temperature. The fraction of folded CH3 was then calculated and plotted versus temperature, and the resulting curve was fit to a three-parameter sigmoid, (f ¼ a/(1 + exp[(x x0/b)])), using SigmaPlot software.


Analytical ultracentrifugation Equilibrium analytical ultracentrifugation was performed on a Beckman XL-A instrument (Beckman) at 20°C, monitoring UV absorbance at 230 and 280 nm. Reduced and oxidized CH3 proteins at 50 mg/mL (1.9 mM dimer) were incubated in 10 mM sodium acetate, pH 5.0, with various GdmCl concentrations for 5 d at 4°C prior to centrifugation. Optimal sedimentation equilibrium speeds of 34,000, 44,000, and 54,000 rpm were determined using the equilibrium speed calculation module in the Ultrascan Software version 7.3 developed by Demeler (2005). Sigma values for the speeds used were determined to be in the range of 1.5–5.0. The partial specific volume of the CH3 domain was calculated to be 0.7218 mL/g at 25°C. Both partial specific volume and densities of various GdmCl solutions were determined using SEDNTERP software (Laue et al. 1992). Data analysis was performed using both local and global data sets using the Kdalton proprietary software developed by John Philo and Amgen, Inc.

Size-exclusion HPLC with multi-angle laser light scattering An Agilent 1100 HPLC system equipped with a diode array detector, a multi-angle laser light-scattering detector, and a refractive index detector (Wyatt Technology) was used to conduct SE-HPLC with a TSK G2000SWXL (7.8 3 60 mm) column. The mobile-phase buffer used was 100 mM NaH2PO4, 150 mM NaCl at pH 5.0 in the presence of 0–6 M GdmCl. Chromatography was conducted at a constant flow rate of 0.5 mL/min, and the protein was detected at 215 and 280 nm. Prior to chromatography, ;200 mg/mL (7.5 mM dimer) of either reduced or oxidized CH3 protein was incubated in 10 mM sodium acetate buffer at pH 5.0 containing 0–6 M GdmCl for 3 d at 25°C to reach equilibrium. The higher protein concentration (7.5 mM) was necessary to obtain a good signal-to-noise ratio for the light-scattering detector than that used for equilibrium unfolding monitored by fluorescence (1.9 mM). For reduced CH3 samples, 1 mM TCEP was added to ensure the reduced state in the presence of GdmCl during the 3-d incubation. The light-scattering detector was calibrated using toluene and bovine serum albumin. Light-scattering data was collected using the Astra V software (Wyatt Technology), and the corresponding molar mass for each peak was calculated.

Analysis of the equilibrium unfolding and dimer dissociation A two-state model (N2 5 2U, where KU ¼ [U]2/[N2]) was used to fit the data of unfolding (Fig. 5A) experiments to estimate the unfolding free energy. The measured fluorescence intensity (Imeas) at each denaturant concentration is related to fU through fU ¼ (IN Imeas)/(IN IU), where IN and IU are the fluorescence intensities of the native and unfolded molecules, respectively. The equilibrium constant is given by KU ¼ 2cfU2/fN, where c is the total protein concentration (in monomer units) and fU and fN are the fractions of unfolded monomer and native dimer populations, respectively. The unfolding free energy DGu was calculated using DGu ¼ RT ln KU, where R is the gas constant and T is the absolute temperature. The unfolding free energy is assumed to have a linear dependence on the denaturant concentration given by DGu ¼ DGu0 + m[Gdm], where DGu0 denotes

the unfolding free energy in the absence of denaturant and m is a measure of the cooperativity of the transition. The free energy of dissociation (dimer to monomer change monitored by AUC; Fig. 5B) was estimated using the two-state model in the same manner with unfolding as described above.

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