Conformational and Colloidal Stabilities of Human Immunoglobulin

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Conformational and Colloidal Stabilities of Human Immunoglobulin G Fc and Its Cyclized Variant: Independent and Compensatory Participation of Domains in Aggregation of Multi-domain Proteins Seiki Yageta, Risa Shibuya, Hiroshi Imamura, and Shinya Honda Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00983 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017

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Molecular Pharmaceutics

Conformational and Colloidal Stabilities of Human Immunoglobulin G Fc and Its Cyclized Variant: Independent and Compensatory Participation of Domains in Aggregation of Multi-domain Proteins Seiki Yageta,†,‡ Risa Shibuya,† Hiroshi Imamura,‡ and Shinya Honda*,†,‡

†

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences,

The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan ‡

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology,

AIST Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ABSTRACT Monoclonal immunoglobulin G (IgG) is a multi-domain protein. It has been reported that the conformational and colloidal stabilities of each domain are different and it is predicted that limited domains participate in IgG aggregation. In contrast, the influence of inter-domain interactions on IgG aggregation remains unclear. The fragment crystallizable (Fc) region is also a multi-domain protein consisting of two sets of CH2 and CH3 domains. Here, we have analyzed the conformational change and aggregate size of an aglycosylated Fc region induced by both acid- and salt-stresses, and have elucidated the influence of inter-domain interactions between CH2 and CH3 domains on the conformational and colloidal stabilities of the aglycosylated Fc region. Singular value decomposition analyses demonstrated that the CH2 and CH3 domains unfolded almost independently from each other in the aglycosylated Fc region. Meanwhile, the colloidal stabilities of the CH2 and CH3 domains affect the aggregation process of the unfolded aglycosylated Fc region in a compensatory way. Moreover, the influence of an additional inter-domain disulfide bond, introduced at the C-terminal end of the CH3 domains to produce the Fc variant, cyclized Fc, was evaluated. This inter-domain disulfide bond increased the conformational stability of the CH3 domain. The stabilization of the CH3 domain in the cyclized Fc successfully improved aggregation tolerance following acid stress, although the size of aggregates produced were comparable to those of the aglycosylated Fc region.

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KEYWORDS Antibody, multidomain, aggregation, unfolding, acid stress


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INTRODUCTION Therapeutic antibodies are superior in their high selectivity and specificity.1 To meet the increasing demand and market for therapeutic antibodies,2 continued improvements in manufacturing and quality control technologies are necessary. A deep understanding of the molecular properties of antibodies is an important foundation for the development of these technologies. Antibody aggregation is one of the important aspects that needs to be considered.1, 3 Contamination of the therapeutic agent with aggregates causes the risk of immunogenic responses and decreases production efficiency.4, 5 The mechanism of aggregation must therefore be understood to maintain the high quality and safety of antibody therapeutics. Immunoglobulin G (IgG), the most common commercially available therapeutic antibody,6 generally forms irreversible aggregates during its unfolding reaction.7-9 Recently, the correlation between IgG unfolding and aggregation has been extensively studied.10-14 For example, a stepwise unfolding reaction of each IgG domain was observed in differential scanning calorimetry (DSC) experiments.10, 15 The CH2 domain exhibits the lowest conformational stability, followed by the Fab region and then the CH3 domain. The heat unfolding of the CH3 domain is mostly irreversible due to the formation of aggregates. The heat unfolding of the CH2 domain is reversible in low ionic solutions, while irreversible in high ionic solutions at pH 4.8.13 These results suggest that the CH2 and CH3 domains clearly possess different conformational and colloidal stabilities, leading to a variety of antibody aggregation behaviors depending on solution conditions. Moreover, since IgG is a multi-domain protein, it is plausible that direct or indirect inter-domain interactions will affect the conformational and colloidal stabilities of each domain and the resulting aggregation of IgG.16, 17 Therefore, the inter-domain interactions as well as the inherent conformational and colloidal properties of domains should be considered in order to fully elucidate the mechanism of IgG aggregation. For isolated IgG constant domains, where inter-domain interactions can be dismissed, the inherent conformational and colloidal stabilities of the CH2 monomer, CL monomer, CH3 homodimer, and CH1-CL heterodimer have been assessed under acid- and salt-stress.18 In this study, the CH2 monomer exhibited the lowest conformational stability, whereas the CL monomer was the most stable. Under acid conditions of pH 2, the colloidal stability of the CH3 homodimer in the fully unfolded state was the lowest among these isolated domains. These data describing the inherent domain properties provide a basis for the elucidation of the inter-domain interactions in the multi-domain structure of IgG.19 The Fc region contains two sets of CH2 and CH3 domains that together form a multi-domain structure. In the native IgG, the CH2 domain is glycosylated at Asn297, which is known to further stabilize the conformational and colloidal stabilities of the Fc region.20, 21 In the present study, we have focused on the stabilization effects which stem solely from the formation of the multi-domain



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structure of the Fc region. To achieve this, an aglycosylated Fc region was synthesized using the Escherichia coli expression system, and used as a simple model to investigate the influence of the inter-domain interaction between the CH2 and CH3 domains on the conformational and colloidal stabilities of the Fc region. The unfolding midpoint pH and the size of aggregates of the aglycosylated Fc region were compared with those of the isolated domains, namely the CH2 monomer and the CH3 homodimer. In addition to these analyses, the influence of an inter-domain disulfide bond artificially introduced at the C-terminal end of the CH3 domains to produce a cyclized Fc variant (cycFc) was examined. Since the Fc region has native disulfide bonds at the N-terminal end of the hinge region, two polypeptide chains of cycFc are connected to each other at both ends. Thus, the variant has a cyclic form. Generally, the introduction of a disulfide bond increases the conformational stability of a protein by decreasing chain entropy.22-24 This decrease is attributed to the restriction of molecular dynamics in the unfolded state, which increases the free energy of the unfolded state. In contrast, the effect of chain entropy on the aggregation process is not well understood. Therefore, in the present study, the effects of restriction of protein dynamics in the unfolded state on the conformational and colloidal stabilities were evaluated by measuring aggregation of the cyclized Fc variant. MATERIALS AND METHODS Gene Cloning, Expression, and Protein Purification Recombinant human IgG1 Fc region (wtFc) and the isolated CH2 and CH3 constant domains were synthesized using the Escherichia coli expression system. The wtFc gene fragment was obtained by polymerase chain reaction (PCR) amplification of the pFUSE-hIgG1-Fc1 plasmid (InvivoGen, San Diego, CA, USA). The N-terminus of the wtFc gene fragment contains a hinge region. The two polypeptide chains translated from the wtFc gene fragment are expected to be connected by two disulfide bonds in the hinge region. The wtFc gene fragment was digested with NdeI/EcoRI and ligated into pET-22b(+) (Merck, Darmstadt, Germany). Escherichia coli strain OrigamiTM B (DE3) (Merck, Darmstadt, Germany) was transformed with the plasmid vector coding wtFc and cultured in Luria-Bertani medium containing 100 µg/mL ampicillin, 20 µg/mL kanamycin, and 20 µg/mL tetracycline. Recombinant gene expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM at 25°C. After overnight culture, cells were pelleted by centrifugation, solubilized in 20 mM phosphate-HCl buffer pH 7.4 containing 150 mM NaCl, and sonicated. The cell lysate was centrifuged at 13,000 g for 30 minutes. The supernatant was applied to a protein G affinity chromatography column (GE Healthcare, Buckinghamshire, England, UK) equilibrated with 20 mM phosphate-HCl buffer pH 7.4 and eluted with 20 mM glycine-HCl buffer pH 2.0. The purified protein was solubilized in 20 mM MES-NaOH buffer pH 6.0 by dialysis and applied to a Resource S cation exchange chromatography column (GE


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Healthcare, Buckinghamshire, England, UK) equilibrated with 20 mM MES-NaOH buffer pH 6.0 and eluted with the same buffer containing 1 M NaCl. The purified protein was concentrated by ultrafiltration and applied to a Superdex 75 (10/300) gel filtration chromatography column (GE Healthcare, Buckinghamshire, England, UK) equilibrated with 20 mM phosphate-HCl buffer pH 7.4 containing 150 mM NaCl. The purities of the samples were confirmed by SDS-PAGE, and mass spectrometry using Synapt G2-Si (Waters, Milford, MA, USA) (Figure S1 and S2). Cloning, expression, and purification of the isolated domains from the CH2 monomer and CH3 homodimer were performed as previously described.18

Introduction of an Inter-Domain Disulfide Bond at the C-Terminus of the Fc Region Based on the report from Wozniak et al.,25 the last three residues (Pro445-Gly446-Lys447) of the C-terminus of the Fc region were replaced with the last three residues of the CL domain (Gly-Glu-Cys) by PCR-based mutagenesis. Cloning, expression, and purification of the cyclized Fc variant (cycFc) was performed as described for wtFc. The formation of the inter-domain disulfide bonds was confirmed using SDS-PAGE and mass spectrometry (Figure S1 and S2).

Preparation of Sample Solutions The two purified Fc regions (wtFc and cycFc) and the two constant domain proteins (CH2 monomer and CH3 homodimer) were solubilized in 20 mM citrate-phosphate buffer pH 7.0 by dialysis and concentrated to 150 µM and 500 µM, respectively, by ultrafiltration. The protein concentrations of these samples were determined by spectrophotometry at 280 nm and a calculation using the molar extinction coefficient. In the case of the CH3 homodimer, the protein concentration was determined using the molar extinction coefficient of the monomer (ε280 = 17,900), regardless of the degree of association in the monomer-dimer equilibrium. Ten solutions of different pH were prepared by mixing 20 mM phosphate solution and 20 mM citrate solution in different ratios. Thirty-one solutions with different NaCl concentrations were also prepared by mixing 20 mM glycine-HCl buffer pH 2.0 and the same buffer containing 400 mM NaCl in different ratios. A Biomek 2000 automated liquid handler (Beckman Coulter, Brea, CA, USA) was used for solution preparations in a 96 well half-area UV plate (Corning, Corning, NY, USA).

Dynamic Light Scattering Measurements Dynamic light scattering (DLS) measurements were carried out using a DynaPro PlateReader II (Wyatt, Santa Barbara, CA, USA). All samples and buffers were filtered through a 0.22 µm centrifugal filter (Merck, Darmstadt, Germany). Before DLS measurements, 10 µL of the concentrated Fc regions or the isolated constant domains were mixed with 90 µL of each prepared buffer solution to give a final volume of the sample solution of 100 µL. The final concentration of



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the Fc proteins was 15 µM, and that of the constant domain proteins was 50 µM. All measurements were performed at 20.5°C. The diffusion coefficient and the particle diameter by the cumulant method were calculated from the autocorrelation function using Dynamics software (Wyatt, Santa Barbara, CA, USA). The diameter after a 10-hour incubation at 20.5°C was used for analysis. After DLS measurements, the pH values and the protein concentrations of the sample solutions were measured and recorded. The pH values of the citrate phosphate buffer solutions ranged from 2.5 to 8.0. The pH value of the glycine-HCl buffer solution with 0–300 mM NaCl was 2.3 ± 0.03.

Circular Dichroism Spectroscopy Circular dichroism (CD) spectroscopy measurements were carried out using a J-1500 spectropolarimeter (Jasco, Tokyo, Japan). Far-UV CD spectra were recorded from 195 nm to 260 nm at 0.1 nm intervals at 20°C in 0.02 cm path length quartz cuvettes. The protein concentrations of the Fc regions and the isolated constant domains were 15 µM and 50 µM, respectively. All spectra were corrected by subtracting the buffer spectrum. The mean residue molar ellipticity was calculated from the mean residue molar concentration and the path length, and used for the following analyses.

Singular Value Decomposition and Two- or Three-State Transition Analyses The measured CD spectra were normalized using the following equation:

[θ] =

[θ] − [θ]

[θ̃]i and [θ]i are the normalized and measured signal intensities, respectively, at wavelength i for each solution condition. [θ̅] is the signal intensity at wavelength i averaged for each set of solution conditions. The symbol σ denotes the standard deviation of the intensities at all wavelengths and all solution conditions in one set. Singular value decomposition (SVD) was conducted by using the following equation:26, 27

= V VT where A is a data matrix containing all normalized spectra in one set of solution conditions. S is a diagonal matrix containing singular values, which quantifies the relative importance of each vector in U and V. U and V are the matrices that contain the left and right singular vectors, respectively. The superscript T denotes transposition of the matrix V. Each U column contains the significant spectrum fractions extracted from data matrix A. Each V column contains a titration profile for the corresponding U column. SVD calculations were carried out using IGOR Pro (Wavemetrics,


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Portland, OR, USA). To evaluate the significance of each component, the contribution ratio of the component and the root mean square deviation (RMSD) between the measured spectrum and the theoretical spectrum reconstructed from only significant components were calculated from the singular value and singular vector of wtFc.28 Based on these values, we assumed that the following reversible sequential three-state transition occurred during the acid unfolding reaction of wtFc: F ⇄ I ⇄ U where F, I, and U are the folded state, an intermediate state, and the unfolded state, respectively. The equilibrium constants (K) for each transition can be derived from the Henderson-Hasselbalch equation with Hill coefficient:29 F-I = 10( ) I-U = 10!( !) where m1 and m2 correspond to Hill coefficients, which reflect the cooperativity of the conformational transition. pKa1 and pKa2 are microscopic acid dissociation constants.30 The equilibrium constants were estimated using nonlinear curve fitting of the following equation to the first and second right singular vectors.

V" =

V"F + V"I F-I + V"U F-I I-U (" = 1, 2) 1 + F-I + F-I I-U

where Vn (n = 1, 2) are the n-th right singular vector calculated from singular value decomposition. VnF, VnI, and VnU are the baseline vectors of the folded state, the intermediate state, and the unfolded state, respectively. The fractions of the conformational states (fF, fI, and fU) can be expressed with equilibrium constants as follows:31

&F =

[F] 1 = [F]+[I]+[U] 1 + F-I + F-I I-U

&I =

[I] F-I = [F]+[I]+[U] 1 + F-I + F-I I-U

&U =

[U] = 1 − &F − &I [F]+[I]+[U]



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The data matrix A containing all normalized spectra is also expressed with the following two matrices.26 A = D DFT where D is a matrix that contains the normalized pure spectra of the folded, intermediate, and unfolded states. F is a matrix that contains fractions of the conformational state. The matrix D was obtained by rearranging the above equation as follows: D = (AF AF)(F AF FT F) Since the pure spectra in D are expressed as normalized forms, the results were restored to a pre-normalized form, i.e., a measured form, using the following equation: [θ],) = *θ+,) + [θ] (, = F, I, U) where [θ]i,j (j = F, I, and U) are pure CD spectra of the folded state (j = F), the intermediate state (j = I), and the unfolded state (j = U) at wavelength i. The pure CD spectra [θ]i,j are expressed as a measured form. The pure CD spectra of three conformational states calculated from wtFc were used for the determination of equilibrium constants and fractions for cycFc. The following equation was fitted to the measured CD spectra of cycFc:

[θ] =

[θ],F + [θ],I F-I + [θ],U F-I I-U 1 + F-I + F-I I-U

Based on the singular value and singular vector of the isolated CH2 monomer, we assumed that the reversible two-state transition occurred during the acid unfolding reaction: F ⇄ U The equilibrium constant KF-U for the transition between the folded and unfolded states of the CH2 monomer is represented by the following equation: F-U = 10-( -)


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The first light singular vector was fitted using the following equation:

V1 =

V1F + V1U F-U 1 + F-U

The fractions of the folded and unfolded state were calculated from the following equations:

&F =

1 1 + F-U

&U = 1 − &F For the CH3 homodimer, we also assumed that the reversible two-state transition occurred during the acid unfolding reaction:32-34 F! ⇄ 2U where F2 and U are the folded state of the homodimer and the unfolded state of the monomer, respectively. The equilibrium constant is: ./!0 = 101( 1) The fractions of the folded and unfolded state were calculated from the following equations:

&0 =

[U] −A + √A! + 4A = 23 2

&. =

2[F! ] = 1 − &0 23

where Pt and A are total protein concentration and a dimensionless parameter, respectively, defined as: 23 = 2[F! ] + [U] A=

./!0 223

The first light singular vector was fitted using the following equation:



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V1 = V1. &. + V10 &0 RESULTS The CH2 and CH3 domains in the Fc region unfold almost independently in accordance with their inherent conformational stabilities To investigate the acid unfolding behaviors of the CH2 and CH3 domains in the Fc region, conformational changes in the aglycosylated Fc region (wtFc), the CH2 monomer, and the CH3 homodimer were analyzed by measuring their far-UV CD spectra under ten different solution conditions at pH 2.5–8.0 containing either 0 or 300 mM NaCl (Figure 1 and S5). Singular value decomposition (SVD) analyses were performed to determine the number of significant conformational transitions. A two- or three-state conformational transition analysis was performed to estimate the fractions in every conformational state. By calculating the contribution ratio and the RMSD from the singular value and the singular vector of wtFc, we considered the first and second components as being significant (Table 1 and Figure 2a–c), furthermore the titration profiles in these right singular vectors corresponded to the conformational transition. The third left and third right singular vectors had large variations reflective of noise (Figure 2b, c). While the titration profile of the first right singular vector showed a continuing decrease from pH 3.8 to pH 2.6, that of the second right singular vector showed a maximum at pH 3.1, indicating that an intermediate conformation accumulated around pH 3.1 (Figure 2c). Therefore, the acid unfolding reaction at pH 2.5–8.0 contained two conformational transitions reflective of three conformational states (i.e., the folded state, the intermediate state, and the unfolded state). SVD was also performed against the CH2 monomer and CH3 homodimer. The results suggested that only the first component was significant (Table 1 and Figure S6a–f). Therefore, we conclude that both the isolated CH2 monomer and the CH3 homodimer unfolded through a two-state transition between the folded and unfolded states. Although the unfolding reaction of the CH3 homodimer apparently involve two processes, namely dissociation and unfolding, previous studies suggest that the two-state conformational transition model can be applied to the unfolding reaction of the CH3 homodimer.33, 35 Thies et al. showed that the equilibrium unfolding of the murine CH3 homodimer is highly cooperative and that an intermediate state cannot be detected.33 Also, McAuley et al. demonstrated that the accumulation of the native monomer could not be observed by SEC-HPLC during unfolding of the human CH3 homodimer.35 The conformational transition of the CH3 homodimer in the present study was also highly cooperative, and the SVD analysis revealed only one significant component. Therefore, we conclude that the conformational transition of the CH3 homodimer can be described using the two-state transition model in the present condition. The three-state conformational transition model was applied to the first and second right singular vectors of wtFc, and the equilibrium constant of each transition and the fractions of the folded, intermediate,


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and unfolded states were calculated (Table 2 and Figure 2c, d). In contrast, the CH2 monomer and CH3 homodimer were analyzed using the two-state transition model against the first right singular vector, and the equilibrium constant and the fractions of the folded and unfolded states were calculated (Table 2 and Figure S6e–h). As a result, the transition curve and the midpoint pH of the folded state of wtFc overlapped with those of the folded state of the CH2 monomer, indicating that the first transition curve of wtFc corresponds to the unfolding reaction of the CH2 domain, and the remaining second transition curve of wtFc corresponds to the unfolding reaction of the CH3 domain (Table 2 and Figure 2d). However, the second transition curve and the midpoint pH of wtFc did not exactly overlap with those of the CH3 homodimer (Table 2 and Figure 2d). This can be reasonably explained as follows; The conformational transition of the CH3 homodimer is a second-order reaction, involving two reaction processes namely dimerization and folding. In principle, the dimerization process should depend on protein concentration so that the midpoint pH should shift to a higher pH range as protein concentration decreases. Therefore, the disagreement between the two transition curves is not merely derived from difference in changes in the free energy (∆∆G) of the folding reaction between the CH3 domain in wtFc and the CH3 homodimer. Taken together, we speculate that the CH2 and CH3 domains in wtFc unfold almost independently, suggesting that there are no significant inter-domain interaction between the CH2 and CH3 domains during the unfolding process of wtFc. In principle then, the unfolding pathway of wtFc may contain at most four conformational species namely CH2(F)-CH3(F), CH2(U)-CH3(F), CH2(F)-CH3(U), and CH2(U)-CH3(U) as shown in Figure 3. However, only three conformational states were observed experimentally. The reason for this disagreement could be due to the negligible amount of CH2(F)-CH3(U) under the measured conditions. Since the CH2 domain unfolds at a higher pH than the CH3 domain, the reaction pathway surrounded by a dotted line in Figure 3 would become dominant. In solutions containing 300 mM NaCl, two-step unfolding was also observed for wtFc (Figure 1b, c). These steps correspond to the unfolding reactions of the CH2 and CH3 domains, as described above. However, the unfolding reactions of the CH2 and CH3 domains in wtFc at 300 mM NaCl occurred at pH 4.2 and pH 3.2, respectively, which were higher than those observed in solutions without NaCl. The CH2 monomer and CH3 homodimer also unfolded at a higher pH (Figure S5c–f). These apparent increases in pH may arise as a result of irreversible aggregation (see next section). Since the unfolding reaction in 300 mM NaCl involved an irreversible aggregation process, we did not perform the three-state conformational transition analysis, which should only be applied to reversible equilibrium phenomena. In the present study, wtFc was synthesized using an E. coli expression system, so the CH2 domain was not glycosylated at Asn297. Glycosylation is generally known to enhance the conformational stability of the CH2 domain.20, 21, 36 We also analyzed the glycosylated Fc region



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prepared by papain-digestion and compared the results among the glycosylated and aglycosylated Fc regions (glycoFc and wtFc, respectively), and the isolated domains (Figure S5–7, 1a,c, 2d). Then, we reconsidered the stabilization effects by the glycosylation in terms of the multi-domain architecture. The data suggested that unfolding of the CH2 domain in glycoFc did not occur to any significant extent at pH 3–4, whereas, in contrast, independent unfolding of the CH2 monomer and the CH2 domain in wtFc did occur. The glycan attachment may simply enhance the stability of the CH2 domain through the same mechanism generally observed in glycoproteins.37-40 Interestingly, the CD spectrum of glycoFc at pH 2.6 did not show the complete random coil spectrum, suggesting that the CH3 domain in glycoFc exhibited a higher conformational stability than in wtFc. The glycan chain attached to Asn297 in the CH2 domain does not contact with the CH3 domain,41-43 implying that an indirect interaction between the glycan chain and the CH3 domain through the stabilized CH2 domain may affect the conformational stability of the CH3 domain, although further studies will be necessary to understand clearly this complex effect.

Higher colloidal stability of the CH2 domain counterbalances an insufficient stability of the CH3 domain in the Fc region To elucidate the influence of the inter-domain interaction between the CH2 and CH3 domains on the colloidal stability of the Fc region, the wtFc aggregation process triggered by the acid unfolding of the CH2 and/or CH3 domains was analyzed. The particle sizes of wtFc aggregates after 10 hours of incubation were compared with those of the isolated CH2 monomer and CH3 homodimer under ten different solution conditions at pH 2.5–8.0 containing 300 mM NaCl and thirty-one different conditions at pH 2.3 containing 0–300 mM NaCl. In solutions without NaCl, the particle sizes of the wtFc, the CH2 monomer, and the CH3 homodimer remain almost the same during a 10-hour incubation, indicating that obvious aggregation did not occur (data not shown). In solutions containing 300 mM NaCl, a two-step change in aggregate size was observed for wtFc. wtFc formed aggregates of moderate size at pH 4.2 and pH 3.6 (Figure 4a). At pH 3.2, the largest aggregate size was obtained. Below this pH, the aggregate size decreased according to the decrease in pH. In the isolated domains, the unfolded CH2 monomer and CH3 homodimer aggregated below pH 3.6 (Figure 4c, e). The size of the unfolded CH3 homodimer aggregates was far larger than those of the unfolded CH2 monomer. Considering the unfolding pH of the CH2 and CH3 domains in wtFc described above and the aggregate sizes of the CH2 monomer and CH3 homodimer presented here, the formation of moderate-sized wtFc aggregates at pH 3.6–4.2 was mainly led by the unfolded CH2 domain, while the unfolded CH3 domain participated in the formation of large aggregates at pH 2.5–3.2. To evaluate the effect of NaCl concentration on the aggregation of fully unfolded proteins, aggregate particle sizes were also measured under conditions of 0–300 mM NaCl at pH 2.3 (Figure


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4b, d, and f). Aggregation of the wtFc, the CH2 monomer, and the CH3 homodimer was observed at concentrations higher than 50, 200, and 50 mM NaCl, respectively. Because aggregation of the wtFc and the CH3 homodimer occurred at similar NaCl concentrations, this suggests that aggregation of the Fc region at around 50 mM NaCl was mainly caused by the inter-molecular interaction between unfolded CH3 domains. In addition to this mechanism, we speculate that the unfolded CH2 domain also has a significant influence on wtFc aggregation. The aggregate sizes of the wtFc, the CH2 monomer, and the CH3 homodimer in 300 mM NaCl at pH 2.3 are shown in Table 3. The size of the wtFc aggregates was clearly smaller than that of the CH3 homodimer over a range of protein concentrations from 7.5–50 µM, indicating that wtFc aggregation was not solely led by the most aggregation prone CH3 domain. The poor colloidal stability of the unfolded CH3 domain would be counterbalanced by the higher stability of the unfolded CH2 domain in the aggregation of the fully unfolded Fc region. This compensatory mechanism is supported by the evidence that aggregate size of the isolated CH2 monomer was the smallest. The introduction of a C-terminal inter-domain disulfide bond increases the conformational stability of the cyclized Fc variant and restricts aggregation To evaluate the effects of unfolded protein chain entropy on conformational and colloidal stabilities, the conformational change and aggregate size of the cyclized Fc variant (cycFc), in which an inter-domain disulfide bond was artificially introduced at the C-terminal end of the CH3 domain, were measured using far-UV CD spectroscopy and DLS in solutions containing 0 or 300 mM NaCl at pH 2.5–8.0, and the results compared with those obtained with wtFc measured under the same conditions (Figure 5). In solutions without NaCl, the CD spectra of cycFc at neutral pH were identical to those of wtFc, indicating that the secondary structure is not affected by the introduction of the inter-domain disulfide bond (Figure 5a, b). To analyze the conformational stability, an SVD analysis was attempted using the CD spectra of cycFc (Figure 6a–c). However, as the second right singular vector showed a noisy titration profile (Table 1 and Figure 6c), we could not fully complete the three-state conformational transition calculation (data not shown). Because cycFc did not completely unfold at pH 2.6, spectral information on the fully unfolded state could not be extracted from the measured data. As an alternative approach, the pure spectra of the folded, intermediate, and unfolded states obtained from wtFc CD spectral data were used for further analyses. The equilibrium constants and fractions for cycFc were calculated by nonlinear curve fitting of the pure spectra of wtFc to the measured CD spectral data of cycFc (Table 2 and Figure 6d–f). The transition midpoint pHs of the CH2 and CH3 domains in cycFc were lower than those in wtFc (Table 2), indicating that the inter-domain disulfide bond increased the conformational stability of both the CH2 and CH3 domains. Like wtFc, in solutions containing 300 mM NaCl, cycFc also unfolded at a higher pH than in solutions without



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NaCl (Figure 5b, d). Acid unfolding of the CH2 and CH3 domains in cycFc was observed around pH 4.2 and pH 2.9, respectively. The particle size of cycFc aggregates was measured using DLS (Figure 7a). While aggregation was not identified in solutions without NaCl, aggregation that accompanied the unfolding reactions of the CH2 and CH3 domains was observed in solutions containing 300 mM NaCl at pH 4.2 and pH 2.9, respectively. Aggregation caused by the CH3 domain in cycFc was observed at a lower pH (pH 2.9) than that in wtFc (pH 3.2), indicating that the increase in conformational stability, caused by the introduced disulfide bond, restricted the acid unfolding reaction and the resulting aggregation. The increase in conformational stability caused by glycosylation also restricted the acid unfolding and aggregation of glycoFc (Figure S7). In contrast, the cycFc aggregate sizes at pH 4.2 and pH 2.9 were 12.1 nm and 19.7 nm, respectively, which were similar to the aggregate sizes of wtFc (12.1 nm and 19.5 nm at pH 4.2 and 2.9, respectively), suggesting that the colloidal stability was very similar between cycFc and wtFc. In DLS measurements of cycFc at pH 2.3 and 0–300 mM NaCl, aggregation occurred at 50 mM NaCl, and the aggregate size was similar to that of wtFc under the same conditions (Figure 7b). Taken together, the introduction of the inter-domain disulfide bond successfully increased the conformational stability of the CH3 domain and restricted aggregation, while the colloidal stability of the unfolded state of cycFc was comparable to that of wtFc. DISCUSSION In the present study, we demonstrated that the CH2 and CH3 domains in wtFc unfolded almost independently, and there was no significant cooperativity between the CH2 and CH3 domains in wtFc during their conformational transitions. Latypov et al. have reported changes in the chemical shifts of the Fc region between pH 2.5 to pH 4.7 using NMR spectroscopy.12 They suggested that the global unfolding reactions of the CH2 and CH3 domains are largely uncoupled, which agrees with the observation in the present study. Their NMR study also captured several minor changes in the chemical shifts of the CH3 domain around the CH2-CH3 interface at pH 3.5 accompanied by independent unfolding of the CH2 domain. The independency of conformational stability could be attributable to a weak inter-domain enthalpic interaction between the CH2 and CH3 domains. The number of inter-domain contacts at the CH2-CH3 interface calculated from the crystal structure (PDBID: 3D6G41) is smaller than that at the CH3-CH3 interface (Table 4). Therefore, a small number of inter-domain contacts leads to a weak inter-domain enthalpic interaction. This weak interaction may enhance the folding efficacy of each domain and thereby avoid aggregation by promoting the folding reaction.44-47 Budyak et al. proposed that the early barrel closure of intracellular lipid-binding proteins is a structural feature that results in successful folding and minimization of aggregation.48 This independency attributed to a weak inter-domain enthalpic interaction could also provide domain-based flexibility to the Fc region.49, 50 A recent study showed that this type of


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flexibility plays a critical role in the formation of a strong interaction between the Fc region and FcγRI 51. Such a domain-based flexibility arising from a weak inter-domain interaction has been also reported in other multi-domain proteins.52 The midpoint pH for the unfolding reaction of the CH2 domain in wtFc was almost identical to that of the CH2 monomer. This indicates that the formation of the multi-domain structure of wtFc has little influence on the conformational stability of the CH2 domain. On the other hand, the midpoint pH for the unfolding reaction of the CH3 domain in wtFc was shifted to a more acidic pH compared to that of the CH3 homodimer. As described in the RESULTS section, this can be explained by the concentration dependency of the dimerization reaction of the CH3 homodimer. Since the CH3 domains in wtFc are covalently connected to each other through the original disulfide bonds at the N-terminal end of the hinge region, an effective concentration effect must promote the dimerization reaction between the domains as compared with the isolated homodimer. The introduction of an inter-domain disulfide bond at the C-terminal end of the Fc region enhanced the conformational stability of the CH3 domain in cycFc. Wozniak et al. previously demonstrated that the introduction of an inter-domain disulfide bond in the C-terminus of the Fc region increased the thermal stability against heat stress using differential scanning calorimetry,25 indicating that the inter-domain disulfide bond can enhance the stability against acid stress as well as heat stress. It is generally accepted that the introduction of disulfide bonds at appropriate positions increases the conformational stability of a protein mainly due to restricting molecular dynamics and decreasing chain entropy in the unfolded state, which ultimately increases the free energy of the unfolded state.22-24, 35, 53, 54 The additional inter-domain disulfide bond at the C-terminal end of the CH3 domain in cycFc should be exposed at the surface, and therefore not change either the intra-domain interaction or the dimer interaction interface of the CH3 domain. Therefore, CH3 domain stabilization would be principally caused by restriction of its dynamics in the unfolded state and in addition by facilitation of dimerization allowing the two CH3 domains to come into close proximity. Interestingly, the introduction of the inter-domain disulfide bond also shifted the transition midpoint pH of the CH2 domain in cycFc. The CH2 domain is physically distant from the C-terminal region of the CH3 domain which contained the inter-domain disulfide bond. To try and understand this surprising result, we calculated the free energy changes (∆G) of each conformational transition using the measured equilibrium constants (Figure 8). The difference in ∆GF-I between wtFc and cycFc (∆∆GF-I) was smaller than that of ∆∆GI-U, indicating that the effect of the inter-domain disulfide bond on the CH2 domain is relatively small. Nevertheless, the slope of ∆GF-I against the pH was very shallow meaning that the midpoint pH (i.e., ∆GF-I = 0) will change dramatically even with a slight fluctuation in free energy. Based on these considerations we conclude that the introduced disulfide bond mainly stabilized the CH3 domain. A second reason that could explain the shift of the



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transition midpoint pH of the CH2 domain in cycFc is that there is a rearrangement of chemical equilibrium according to Le Châtelier's principle. According to this idea, stabilization of the CH3 domain decreases the molecular fraction of the unfolded state fU, which causes an increase in the molecular fractions of not only the intermediate state fI, but also of the folded state fF. In wtFc aggregation, the poor colloidal stability of the unfolded CH3 domain was counterbalanced by the higher stability of the unfolded CH2 domain. It is known that tag proteins such as glutathione-S-transferase (GST) and maltose-binding protein (MBP) can increase the solubility of a fused protein.55, 56 Therefore, it is not unreasonable to hypothesize that the existence of colloidally stable CH2 domain could improve the whole colloidal stability of wtFc. Wright et al. proposed that since sequence homology among structurally analogous domains in a multi-domain protein is relatively low, a domain adjacent to an unstable domain has evolutionarily acquired the ability to enhance the stability of the whole multi-domain protein.57 In this regard, sequence homology among the constant domains of IgG is less than 30%.18 Although the CH3 domain of IgG forms a homodimer and plays a critical role in maintaining the whole antibody structure, the large hydrophobic moiety at the dimer interface would be expected to reduce colloidal stability. Thus, the higher colloidal stability of the CH2 domain may have been evolutionarily enhanced to compensate for the colloidal stability of the whole Fc region. It has been reported that an isolated murine CH1 monomer is an intrinsically unfolded protein58 and is also highly aggregation prone.59 However, the formation of a CH1-CL heterodimer provided superior colloidal stability in the unfolded state.18 Therefore, we consider it likely that the colloidal stability of whole IgG could be regulated by the compensatory participation of domains, where domains having higher colloidal stability (i.e., CH2 and CL) counterbalance domains with poor colloidal stability (i.e., CH3 and CH1). In the present study, the unfolding reactions and aggregation processes of the multi-domain Fc region were uncovered using a domain-based approach. This approach opens up the possibility of developing optimized therapeutic antibodies based on domain properties. In particular, aglycosylated antibodies and their engineered variants have been recently developed as next-generation therapeutics,60-62 and it is likely that the results of the present study, which describe the behavior of the aglycosylated antibody in acidic conditions, will contribute to improvements in manufacturing and quality control of these aglycosylated antibodies. During the manufacturing of therapeutic antibodies, the protein A affinity chromatography step and the virus inactivation step require that antibodies are exposed to acidic conditions between pH 2.5–4.0 for several hours to overnight. Under these conditions, the CH2 and CH3 domains in the aglycosylated Fc region would unfold and form aggregates based on their conformational and colloidal stabilities. The data presented here show that the rapid formation of large aggregates mediated by the unfolded CH3 domain could be avoided by maintaining the solution pH above 3.6. This would also restrict aggregation during the neutralization step following acid exposure. A lower NaCl concentration could maintain the Fc


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region in a mono-disperse form even in the fully unfolded state at pHs lower than pH 3.6. CONCLUSIONS The conformational and colloidal stabilities of IgG could be optimized by adapting its multi-domain architecture through independent and compensatory participation of domains. Domain-based analyses revealed that the conformational stabilities of the CH2 and CH3 domains in the aglycosylated Fc region were almost independent of acid stress, and that the unfolded domains drive the formation of aggregates. The aggregate size was found to be mainly dependent on the colloidal stability of the unfolded domains. However, the whole colloidal stability of the fully unfolded Fc region was compensated for by combination of domains exhibiting high colloidal stability (CH2) with those exhibiting poor colloidal stability (CH3). Restriction of protein dynamics to decrease the chain entropy in the unfolded state successfully increased the conformational stability in the folded state and confined any aggregation. Knowledge of the conformational and colloidal stabilities that can be deduced from domain-based investigations will contribute to optimizing and rationalizing the manufacturing process and development of therapeutic antibodies. ASSOCIATED CONTENT Supporting Information Additional Materials and Methods, Results and Discussion, Table S1, and Figures S1–S7. This material is available free of charge via Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel: +81 29 862 6737. Fax: +81 29 861 6194. E-mail: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We thank Dr. Hisashi Takahashi, Chieko Igarashi, Reiko Kojima, Dr. Yuichi Takeda, Tomoko Oshikata, and Dr. Yan Wen Feng for technical support and valuable advice. This research is partially supported by “The Developing Key Technologies for Discovering and Manufacturing Pharmaceuticals Used for Next-Generation Treatments and Diagnoses” both from the Ministry of Economy, Trade and Industry, Japan (METI) and from Japan Agency for Medical Research and Development (AMED).



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ABBREVIATIONS USED CD, circular dichroism; cycFc, cyclized Fc variant; DLS, dynamic light scattering; DSC, differential scanning calorimetry; Fab, fragment antigen-binding; Fc, fragment crystallizable; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; MBP, maltose binding protein; MES, 2-Morpholinoethanesulfonic acid; IgG, immunoglobulin G; PCR, polymerase chain reaction; RMSD, root mean square deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; SVD, singular value decomposition; wtFc, recombinant protein of human IgG1 Fc region

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COCOMAPS: a web

application to analyze and visualize contacts at the interface of biomolecular complexes.

Bioinformatics 2011, 27, (20), 2915-6.


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Table 1. Contribution ratios from singular value decomposition Contribution ratio (%)

wtFc

cycFc

CH2 monomer

CH3 homodimer

First component

98.7

98.7

99.7

99.8

Second component

1.27

0.84

0.12

0.08

Third component

0.02

0.15

0.08

0.03


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Table 2. Parameters for the acid unfolding reaction of proteins Parameters

wtFc

cycFc

CH2 monomer

CH3 homodimer

m1 or m3

1.4 ± 0.2

1.4a

2.4 ± 0.1

-

pKa1 or pKa3

3.3 ± 0.1

3.1 ± 0.0

3.3 ± 0.0

-

a

m2 or m4

7.3 ± 3.8

7.3

-

6.5 ± 0.2

pKa2 or pKa4

2.9 ± 0.0

2.5 ± 0.0

-

2.3 ± 0.0

midpoint pH1

3.3

3.1

3.3

-

midpoint pH2

2.8

2.5

-

3.0

a

The parameter was fixed in the non-linear curve fitting.



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Table 3. Particle diameters at pH 2.3 in the presence of 300 mM NaCl Proteins

Diameter (nm) Protein concentration (µM) 7.5

wtFc

N. D.a

CH2 monomer

N. D.

CH3 homodimer

24.0

a

15 a

50

14.0 N. D.

20.5 a

27.8

Diameters were not determined because of insufficient scattering intensities.


6.9 35.9

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Table 4. The number of inter-domain contacts in wtFc Number of contacts CH2-CH2 inter-domain contacts

1

CH2-CH3 inter-domain contacts

99

CH3-CH3 inter-domain contacts

249

CH2 intra-molecular contacts

2,295

CH3 intra-molecular contacts

2,235

The inter-domain contacts in the crystal structure of the Fc region (PDBID: 3D6G41) were calculated using COCOMAPS.63 The cut-off distance was set to 8 Å. The CH2-CH2 inter-domain contacts does not include the contact of sugar chains.



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Figure Legends

Figure 1 Circular dichroism (CD) spectra of wtFc as a function of pH and salt concentration. (a) CD spectra in a 0 mM NaCl solution. (b) CD spectra in a 300 mM NaCl solution. (c) CD intensities at 200 nm in a 0 mM NaCl solution (red filled circle) and at 210 nm in a 300 mM NaCl solution (blue filled square).

Figure 2 Output of the singular value decomposition and the three-state conformational transition analysis of wtFc in a 0 mM NaCl solution. (a) Singular values and root mean square deviations (RMSD). (b) The first to third left singular vector (U1–U3). (c) The first to third right singular vector (V1–V3). The dashed lines represent the calculated fitting results for V1 and V2. (d) pH-dependence of the conformational state fractions for wtFc (solid lines), CH2 monomer, and CH3 homodimer (broken line).

Figure 3 Possible conformational transition paths for the Fc region. Four conformational states (CH2(F)-CH3(F), CH2(U)-CH3(F), CH2(F)-CH3(U), and CH2(U)-CH3(U)) are depicted. The folded and unfolded states of the domains are represented as an ellipse and a diamond, respectively. Each conformational transition occurs depending on the corresponding equilibrium constant (K). The area surrounded by the dotted line represents the major conformation paths suggested by the three conformational transition analyses in this study.

Figure 4 Aggregation propensity of proteins as a function of pH and salt concentration. (a, c, e) pH-dependence of the particle size is represented by the blue bar. CD intensities at the same pH are represented in red. Salt concentration was maintained at 300 mM NaCl. (b, d, f) Salt concentration-dependence of particle size. (a, b) wtFc, (c, d) CH2 monomer, and (e, f) CH3 homodimer.

Figure 5 CD spectra of cycFc as a function of pH and salt concentration. (a) CD spectra in a 0 mM NaCl solution. (b) CD spectra in a 300 mM NaCl solution. For comparison the CD spectra of wtFc are also shown as broken lines. (c) CD intensities at 200 nm in a 0 mM NaCl solution (red circle). (d) CD


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intensities at 208 nm in a 300 mM NaCl solution (red circle). The CD intensities of wtFc are also represented as blue squares for comparison.

Figure 6 Output of the singular value decomposition and the three-state conformational transition analysis of cycFc in a 0 mM NaCl solution. (a) Singular values and root mean square deviations (RMSD). (b) The first to third left singular vector (U1–U3). (c) The first to third right singular vector (V1–V3). (d) The pure spectrum of folded (F, red), intermediate (I, green) and unfolded (U, blue) states of wtFc. (e) The calculated fitting results for the CD spectra of cycFc. The symbols and the solid lines represent the measured CD spectra and the calculated fitting results, respectively. (f) pH-dependence of the conformational state fractions for cycFc (solid line) and wtFc (broken line).

Figure 7 Aggregation propensity of cycFc as a function of pH and salt concentration. (a) pH-dependence of aggregate size for cycFc (red bar) and wtFc (blue bar). CD intensities at the same pH for cycFc (green circle) and wtFc (orange circle) are shown. Salt concentration was maintained at 300 mM NaCl. (b) Salt concentration-dependence of the particle size for cycFc (red filled circle) and wtFc (blue open square). The pH was maintained at 2.3.

Figure 8 Free energy changes through the conformational transitions for cycFc and wtFc. ∆GF-I and ∆GI-U indicate the free energy changes between the folded and intermediate states, and the intermediate and unfolded states, respectively.



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Figure 1. Circular dichroism (CD) spectra of wtFc as a function of pH and salt concentration. Figure 1 102x227mm (300 x 300 DPI)


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Figure 2. Output of the singular value decomposition and the three-state conformational transition analysis of wtFc in a 0 mM NaCl solution. Figure 2 238x151mm (300 x 300 DPI)



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Figure 3. Possible conformational transition paths for the Fc region. Figure 3 223x187mm (150 x 150 DPI)


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Figure 4. Aggregation propensity of proteins as a function of pH and salt concentration. Figure 4 231x230mm (300 x 300 DPI)



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Figure 5. CD spectra of cycFc as a function of pH and salt concentration. Figure 5 220x152mm (300 x 300 DPI)


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Figure 6. Output of the singular value decomposition and the three-state conformational transition analysis of cycFc in a 0 mM NaCl solution. Figure 6 233x228mm (300 x 300 DPI)



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Figure 7. Aggregation propensity of cycFc as a function of pH and salt concentration. Figure 7 127x151mm (300 x 300 DPI)


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Figure 8. Free energy changes through the conformational transitions for cycFc and wtFc. Figure 8 101x68mm (300 x 300 DPI)



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Conformational and Colloidal Stabilities of Human Immunoglobulin

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