Perturbation of Thermal Unfolding and Aggregation of Human IgG1 Fc

Dec 20, 2012 - A single cell cuvette holder (Quantum Northwest Inc., Liberty Lake, WA, United States) thermostated at 72 °C was placed between the li...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Perturbation of Thermal Unfolding and Aggregation of Human IgG1 Fc Fragment by Hofmeister Anions Jian Zhang-van Enk,† Bruce D. Mason,‡ Lei Yu,‡ Le Zhang,‡ Wael Hamouda,‡ Gang Huang,‡ Dingjiang Liu,‡,§ Richard L. Remmele, Jr.,‡,∥ and Jifeng Zhang*,‡,⊥ ‡

Department of Analytical and Formulation Sciences and †Systems Informatics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States S Supporting Information *

ABSTRACT: The thermal unfolding and subsequent aggregation of the unglycosylated Fc fragment of a human IgG1 antibody (Fc) were studied in the salt solutions of Na2SO4, KF, KCl and KSCN at pH 4.8 and 7.2 below and at its pI of 7.2, respectively, using differential scanning calorimetry (DSC), far ultraviolet circular dichroism (far-UV CD), size exclusion chromatography (SEHPLC) and light scattering. First, our experimental results demonstrated that the thermal unfolding of the CH2 domain of the Fc was sufficient to induce aggregation. Second, at both pH conditions, the anions (except F−) destabilized the CH2 domain where the effectiveness of SO42− > SCN− > Cl− > F− was more apparent at pH 4.8. In addition, the thermal stability of the CH2 domain was less sensitive to the change in salt concentration at pH 7.2 than at pH 4.8. Third, at pH 4.8 when the Fc had a net positive charge, the anions accelerated the aggregation reaction with SO42− > SCN− > Cl− > F− in effectiveness. But these anions slowed down the aggregation kinetics at pH 7.2 with similar effectiveness when the Fc was net charge neutral. We hypothesize that the effectiveness of the anion on destabilizing the CH2 domain could be attributed to its ability to perturb the free energy for both of the native and unfolded states. The effect of the anions on the kinetics of the aggregation reaction could be interpreted based on the modulation of the electrostatic protein−protein interactions by the anions. KEYWORDS: thermal unfolding, DSC, CD, aggregation, protein−protein interactions, Hofmeister salt series, Fc

■

INTRODUCTION Protein thermal unfolding and the subsequent aggregation are important research topics for understanding protein stability. In the arena of the development of recombinant therapeutic proteins, control and prevention of thermal unfolding and aggregation are essential for ensuring efficacy and safety.1 The well-known Lumry−Eyring model could be applied to describe the kinetics of the aggregation pathways, in which a reversible conformational change occurs and then irreversible assembly of the structure-perturbed species leads to aggregation.2 Experimentally, it has been shown that a partially unfolded state is a prerequisite for irreversible protein aggregation in some cases.3 In addition, it is well recognized that, for a natively folded globular protein, thermal unfolding could expose its hydrophobic core and result in irreversible aggregation.4 Salts could significantly affect the thermal unfolding and aggregation behavior of natively folded proteins in complex ways. Salt ions could either increase or decrease protein thermal stability. The Hofmeister series is often used to define the effectiveness of salt ions in stabilizing proteins at high salt concentrations (more than 0.5 M).5 However, at low salt © 2012 American Chemical Society

concentrations ( |ΔG1+| (decrease in free energy) would result in ΔG12* < ΔG12 and a decrease in Tm. (B) Increase in the free energy state for both N and U due to preferential hydration of proteins when the kosmotropic anions are added. This scenario of |ΔG2+| > |ΔG1+| (rise in free energy) would result in ΔG12* > ΔG12 and an increase in Tm. On the other hand, disruption of ion-pairing in N* leads to ΔG12* < ΔG12 and a decrease in Tm.

°C proceeded in a two-state manner. Then the second step is the process to form a dimeric aggregate (D), which can lead to further aggregation. We considered the dimeric aggregation primarily a kinetic process driven by the aggregation rates (rate constant of k′) of the unfolded protein molecules.16 Furthermore, the extent of the irreversibility is correlated with the magnitude of this aggregation rate. In this work we aimed at modeling the predominant underlying physical processes on a macroscopic level in order to extract the relevant thermodynamic and kinetic parameters that help to provide insight into the influence of the anions on the thermal unfolding of the CH2 domain and unfolding-mediated aggregation of the Fc. The model system used is a simplified version of the theoretical model set up in an earlier work.17 Briefly, the kinetics for the unfolding and aggregation reaction can be described as follows:

Figure 1. General schemes for the thermal unfolding in a two-state transition and the following aggregation step. N, U and D represent the native state, the unfolded state and the aggregate state of a dimer. k1, k2 and k′ are the rate constants for the unfolding, refolding and aggregation reactions.

unfolded state throughout this article refers to the state in which the CH2 domain is unfolded to some extent while the CH3 domain remains intact. In our studies, the thermal unfolding temperatures of 65 and 72 °C at pH 4.8 and 7.2, respectively, were well below Tapp (the apparent unfolding temperature) for the CH3 domain (an example of thermogram up to 90 °C at pH 4.8 is shown in Figure I in the Supporting Information). In the first step, a two-state unfolding process can be approximately described by a reversible process characterized by ΔG, the difference between the free energies of the native (N) and unfolded states (U), as described in Figure 2, independent of the subsequent aggregation process. In the first DSC scan for the Fc in 12.5 mM KF at pH 4.8, our calculation of ΔHVan’tHoff = ΔHcal supports the idea that the transition to 65

dN = −k1N + k 2U dt

(1)

dU = k1N − k 2U − k′U 2 dt

(2)

dD = k′U 2 dt

(3)

where N, U and D are the concentrations of the native, the unfolded and the aggregate proteins normalized by the initial concentration of the Fc, and k1, k2 k′ are the unfolding, 621

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

used as an accurate measurement. But the assumption that the drift is negligible within each group of repeated scanned data seems to be valid based on the results. Combining the parameters, the thermodynamic stability of the native protein can be described by the unfolding Gibbs free energy:

refolding of the CH2 domain and the aggregation kinetic coefficients, respectively. Since N, U and D are normalized by the initial concentration, the choice of the form of the aggregation kinetic in eq 3 implies that the rate constant k′ is a linear function of the initial concentration of the Fc. Since the initial aggregation rate is proportional to this rate constant, it is therefore also proportional to the initial concentration of the Fc. The validity of this assumption can be substantiated by the measured initial aggregation rate as a function of the initial Fc concentration, as shown in Figure II in the Supporting Information. The kinetic coefficients have the following temperature dependence:

⎛ ⎛ T ⎞ T ⎞ ΔG(T ) = ΔHm⎜1 − ⎟ ⎟ + ΔCP ⎜T − Tm − T ln Tm ⎠ Tm ⎠ ⎝ ⎝ (5)

At a given temperature, the higher the Gibbs free energy, the more stable the protein.

■

RESULTS AND DISCUSSION Shown in Figure 3A are the DSC thermograms from the repetitive scans for the Fc in 12.5 mM KF at pH 4.8 up to 65

k1 = k1m exp[ − E1/R(1/T − 1/Tm)] k 2 = k 2m exp{ − E2 /R(1/T − 1/Tm) − ΔCP /R[Tm/T − 1 + ln(T /Tm)]}

k′ = k′m exp{ − E′/R(1/T − 1/Tm) + CP /R[Tm/T − 1 + ln(T /Tm)]}

where R is the gas constant, ΔCP is the change in heat capacity between the unfolded and the folded state, T is the temperature in kelvins, Tm is the “melting” temperature at which the kinetic coefficients are k1m, k2m and k′m, respectively, and E1, E2 and E′ are the activation energies of the unfolding, refolding and aggregation, respectively. With this approach we ignore the difference between heat capacities in the folded and the aggregated states. The reason for this simplification is that the rescanning baselines which include the aggregates did not show a significant difference from the first scan. Moreover, the sensitivity analysis of the model also shows that fitting to the DSC data we collected cannot determine the extra parameter within the error of the data. Given Tm, ΔCP and the unfolding enthalpy ΔHm, the folding kinetic coefficient k2 can be completely determined via the following relations: k2m = k1m and ΔHm = E2 − E1. Therefore the system is described by seven parameters: Tm, ΔHm, ΔCP, k1m, E1, k′m and E′. And the corresponding heat capacity change measured by the DSC experiment can then be modeled by CP(v , T ) = [ΔHm + ΔCP(T − Tm)](k1N − k 2U )/v + E′ k′U 2/v

(4)

where v is the DSC temperature scan rate, N and U can be calculated from eqs 1−3 with the initial condition N(t=0) = 1, U(t=0) = D(t=0) = 0, and T = T0 + vt for the first scan. For the repetitive scans N(t = 0) is the remaining portion of the native state after the cumulative aggregate amount from previous scans is subtracted. Between the scans, a cooling period of 13 min is also modeled where the exponential cooling constant is approximated to be 2/13 min−1 and the target temperature is set to be 10 °C. Since the DSC data, after the reference scan is subtracted, includes a baseline representing the heat capacity of the native protein, we include a linear baseline as well to eq 4 and then fit it to the DSC data with a nonlinear least-squares routine in Matlab for the seven model parameters and the two baseline parameters. Note that although the baseline in theory represents the native protein heat capacity, it does contain an arbitrary error resulting from the drifting of the baseline from different experimental runs. So the absolute value cannot be

Figure 3. Representative DSC thermograms for the Fc at pH 4.8 in (A) 12.5 mM KF and (B) 10 mM Na2SO4.

°C, with Tapp of ∼58.1 °C for the CH2 domain. This temperature is significantly lower than that for the glycosylated CH2 domain, i.e., 71.4 °C, which suggests the additional contribution to the thermal stability afforded by the glycosylation to this domain.18 In other experiments, we noticed that the thermal scan was irreversible after the CH3 domain was unfolded at higher temperature (data not shown). Visually as shown in Figure 3A, the thermal scans are almost 100% reversible up to 8 repetitive scans. The thermal unfolding of the CH2 domain at 65 °C was in agreement with the disappearance of the negative minimum in the far-UV CD spectrum at approximately 217 nm (Figure 4A), the typical signature for the presence of β-sheet structure. The analysis of the spectral similarity shows that ∼60% of the structure features 622

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

Na2SO4 at pH 4.8 was irreversible with the concomitant formation of aggregates. Anion-Dependent Perturbation of the Thermal Stability of Fc. Shown in Figure 5 is the perturbation of the model-fitted theoretical Tm for the CH2 domain in the different salt solutions at pH 4.8 and 7.2 under which the protein should have a net positive charge and be charge neutral, respectively, because of its pI of 7.2. Tm is often used as an indicator for the thermal stability of proteins in solutions.17 At pH 7.2 close to the pI of the Fc, the Tm was higher than at pH 4.8. This is in agreement with the general observations that proteins are more stable at pH conditions close to their respective pIs.19 In addition, the increased thermal stability near the pI is consistent with the model proposed by Linderstrom-Lang,20 which predicts the maximal physical stability at the pI. As shown in Figure 5, the addition of the salts, except KF, monotonically decreased the Tm, suggesting that the CH2 domain became less stable. This is consistent with the trends of the free energy required for unfolding at 25 °C under the influence of different salts at different concentrations as depicted in Figure III in the Supporting Information. Furthermore, the anions demonstrated specific perturbations on the Tm under both pH conditions. At pH 4.8, the anions followed the order of SO42− > SCN− > Cl− > F− in their effectiveness at reducing the Tm at the salt concentrations below ∼50 mM (inset in Figure 5A). KF had the weakest effect, as suggested by a change of ∼2 °C as the Tm decreased to ∼57 °C at 25 mM. The effectiveness of the monovalent anion for reducing the Tm is consistent with the Hofmeister series.5 It is interesting to note that Na2SO4, a strong kosmotrope, behaved like a denaturant. It decreased the Tm more effectively on a per molar basis than KSCN. At pH 4.8 in Figure 5A, as the concentration of the different salts increased, their effects on the Tm began to diverge. The further addition of KF gradually increased the Tm to ∼62 °C at 500 mM while the addition of KSCN monotonically decreased the Tm by almost ∼10 °C to ∼48 °C at 50 mM. KCl had the intermediate effect among the three monovalent salts as the Tm gradually decreased to ∼48 °C at 200 mM and then leveled off. The destabilizing effect by Na2SO4 diminished at as low as ∼85 mM, even though it destabilized the protein more effectively than KSCN at lower salt concentrations. At pH 7.2, the destabilization of the Fc by the anions was less dramatic than at pH 4.8, as shown by their subtle effect on Tm and the free energy of the unfolding in Figure 5B and Figure IIIB in the Supporting Information, respectively. It seems that, below 100 mM, the anions follow SCN− ≅ SO42− > Cl− > F− in effectiveness at destabilizing the Fc. It is interesting to note that the Tm decreased in the KF solutions, in comparison to the gradual increase at pH 4.8. Anion-Dependent Effects on the Thermodynamics of Thermal Unfolding of Fc. To explain our experimental observations, we are proposing the following thermodynamic picture for the thermal unfolding of the CH2 domain. First, during the DSC thermal scan the CH2 domain is unfolded through a two-state transition (from N to U) as presented by the equilibrium in Figure 1, supported by the observation of ΔHVan’tHoff = ΔHcal ≅ 103 kcal/mol for the Fc in 12.5 mM KF at pH 4.8. Second, modulation of the Tm for the CH2 domain may be explained by the Wyman linkage function,21 in which anions could perturb the free energy of both native and unfolded states to a different degree as shown in Figure 2. * < ΔG12 Specifically, Tm is expected to decrease when ΔG12

Figure 4. Far-UV CD spectra for the Fc acquired at 20 °C, 65 °C and then cooled to 20 °C at pH 4.8 in (A) 12.5 mM KF and (B) 10 mM Na2SO4.

in the unheated condition were lost when the sample was heated to 65 °C. Then, the same analysis indicated that after the sample was cooled to room temperature, the spectrum was ∼100% identical to that of the unheated sample. This result suggests that the native secondary structure of the protein was regained upon cooling to room temperature as shown in Figure 4A, supporting the notion that the thermal unfolding was reversible. The thermograms in the solution condition containing 10 mM Na2SO4 at pH 4.8 are shown in Figure 3B where the Tapp was ∼53.5 °C, suggesting that SO42− decreased the thermal stability of the CH2 domain. In addition, the results from the repetitive DSC scans in Figure 3B show that the thermal unfolding was not fully reversible. The SE-HPLC analysis of the post-DSC samples revealed significant amounts of aggregation, i.e., >50%. All these observations indicate the loss of the reversibility was caused by the aggregation formation as presented in Figure 1. This notion was in agreement with the appearance of the minimum at approximately 217 nm in Figure 4B, in comparison to that in Figure 4A. This suggests that there was formation of some new β-sheet structure after thermal unfolding. This observation is consistent with the idea that the aggregation process is accompanied by the formation of intermolecular β-sheet structure.3 The analysis of the spectral similarity shows that ∼60% of the structure features in the original condition were lost when the heated sample was cooled to room temperature. The observation further supports the hypothesis that the thermal unfolding of the Fc in 10 mM 623

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

Figure 5. The data points are the fitted Tm parameter values for the CH2 domain versus salt concentration: (A) pH 4.8. The Tm for the CH2 domain in 100 and 200 mM KSCN is not shown since they were below 44 °C and the reversibility was close to zero. (B) pH 7.2. The lines connecting points are simple straight lines drawn to facilitate visual inspection. Each model fitting procedure was performed with the group of repeat scans under one salt condition. The number of repeat scans ranged from 2 to 8. In the case where the aggregation was almost 100% after the first scan, the second scan showed no structure, thus no further scan was performed.

(Figure 2A) and increase when ΔG*12 > ΔG12 (Figure 2B), where the subscript 1 and 2 refer to the native and unfolded state, respectively. Mechanistically as described in Table 1, the observed effect on the thermal stability by the anions could be attributed to the net effects on the free energy for both the native and unfolded states in the salt-perturbed system (denoted by the * in Figure 2) with respect to the original system from preferential hydration of the protein (mechanism I) and the specific interactions (or binding) of the anions on the protein surface through mechanisms II and III.22 The interactions between the cations, i.e., Na+ and K+, and the Fc are not considered at this point because it has been shown that these monovalent cations interact with proteins rather weakly and typically the anions dominate the interactions between proteins and these salts, especially when the pH of a solution is below or near the pI of a protein.5 The extraordinary destabilizing effect by SO42− below 85 mM at pH 4.8 suggests the dominance of mechanism II in Table 1 by SO42−, i.e., its binding to the Fc, over mechanism I, and the important role of electrostatic interactions in maintaining the thermal stability of the CH2 domain. The divalent SO42− could have stronger electrostatic interactions with the positively charged side chains, i.e., guanidinium ion, on the protein surface than all the monovalent anions. This binding could potentially lead to the destabilization of the CH2 domain.

Table 1. Illustration of the Possible Interaction Mechanisms and Strength between the Anions and Fc at pH 4.8a mechanism I: preferential hydration

mechanism II: through positively charged side chain

mechanism III: through polar and nonpolar side chains, and exposed amide backbone

possible dominant mechanism for the individual anion interacting with the Fc at pH 4.8

F− Cl− SCN−

strong medium weak

weak medium strong

weak weak strong

SO42−

strong

strong

weak

mechanism I mechanisms I and II mechanisms II and III mechanism II

a

For mechanism I, the kosmotropic (strongly hydrated) anions are expelled from the amide backbone and hydrophobic patches on protein surface. This mechanism of preferential hydration for proteins in general helps stabilize protein folding9b,25 and the Tm could increase. In mechanism II, the anions could interact with the protein surface at the positively charged side chains, such as Arg, His and Lys. Based on the law of matching water affinities, the monovalent anions follow the order of SCN− > Cl− > F− for interacting with the counterparts on protein surface.9a,26 It has been shown that SO42− could bind to positively charged protein strongly,27 possibly even more strongly than SCN−. In mechanism III, the weakly hydrated anions, e.g., SCN−, could interact with polar and nonpolar side chains and exposed amide backbone on the protein surface.9b,25a This type of interaction typically destabilizes the protein, and the Tm could decrease. The final effect on the Tm is the overall outcome from all these three mechanisms.

624

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

Figure 6. The reversibility for the thermal unfolding of the CH2 domain in KF, KCl, KSCN and Na2SO4 solutions. (A) pH 4.8. The reversibility for the CH2 domain in 100 and 200 mM KSCN is not shown; it was close to zero. (B) pH 7.2.

This observation is consistent with the notion that SCN− could interact not only with the positively charged side chains through mechanism I but also with nonpolar patches and dipolar amide backbones on protein surface as described by mechanisms II and III, respectively in Table 1. As mentioned earlier, protein unfolding exposes more hydrophobic surface and amide backbones than the native state. As more SCN− continues to bind to the unfolded state and drive down the free energy for the U* state as illustrated in Figure 2A, this would result in ΔG12 * < ΔG12 and a monotonic drop in the Tm. The intermediate destabilizing effect by Cl− versus F− and SCN− on the CH2 domain is consistent with the fact that the strength of Cl−’s interactions with proteins is generally located in the middle of the three monovalent anions for mechanisms I and II. The presence of a plateau for the Tm in KCl and Na2SO4 at pH 4.8 suggests that their electrostatic interactions with the CH2 domain, e.g., disruption of ion-pairing or binding to the exposed positively charged side chains, reached saturation. The fact that the saturation point was reached at 85 mM for Na2SO4 and ∼400 mM KCl is consistent with the hypothesis that SO42− interacts with the protein more strongly than Cl−. The anions can be ranked as SO42− > SCN− > Cl− > F− for their effectiveness in decreasing the Tm at pH 4.8 at low salt concentrations while their effects were much less at pH 7.2. This ranking is in agreement with the proposed order of their interaction strength with the protein surface electrostatically through mechanism II as illustrated in Table 1 and supports the hypothesis that electrostatic interactions contribute to the stability of the CH 2 domain at both pH conditions. Furthermore, the contribution from electrostatic interactions

Currently, there is limited understanding for the role of electrostatic interactions, such as the stabilizing ion-pairing and destabilizing charge−charge repulsion, for maintaining the folding of the native state (N) of the Fc. It is difficult to pinpoint the exact mechanism for how SO42− decreased the free energy cost for the thermal unfolding of the CH2 domain. There are a few possible scenarios where SO42− could destabilize the CH2 domain. First, at pH 4.8 the unfolding process could reveal positive-charge side chains, i.e., histidine, for SO42− to interact with. Now there will be a drop for the free energy of U*. This will result in a drop of ΔG+12 more than ΔG+1 , resulting in ΔG*12 < ΔG12 as shown in Figure 2A and a decrease in Tm. Second, SO42− could decrease the thermal stability through the disruption of ion-pairing in the native state, where the free energy of N* is raised more than that of U* because it is expected that there is no ion-pairing in U* (shown in Figure 2B). Therefore ΔG*12 < ΔG12 and the Tm is expected to decrease. In fact, this mechanism was proposed to explain the destabilization of human prion protein by Na2SO4.7 In contrast to SO42−, F− has been shown to impose weak electrostatic interaction toward the positively charged side chains and other sites on the protein surface as described for mechanisms II and III in Table 1, respectively. This is consistent with the observation that it was the least effective at reducing the Tm, suggesting that the exclusion of the strongly hydrated F− from the backbone amide bond and hydrophobic surface (mechanism I in Table 1) could be the dominant factor, resulting in the raised Tm at pH 4.8 as described in Figure 2(B). SCN−, the most chaotropic anion used in our study, monotonically decreased the Tm without reaching saturation. 625

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

Figure 7. The data points are the fitted values of the rate constant, k′, for the aggregation reaction in KF, KCl, KSCN and Na2SO4 solutions. (A) pH 4.8. At KSCN concentrations more than 50 mM, k′ is out of the scale depicted in the figure. (B) pH 7.2. The straight lines connecting points are drawn to facilitate visual inspection.

might be more important at pH 4.8 than at pH 7.2 as suggested by the observation that the Tm was more sensitive to salt concentration at pH 4.8 than at pH 7.2. DSC Scan Reversibility Correlated with Aggregation Kinetics. The reversibility of the thermal unfolding in our DSC experiment is defined as the ratio of the enthalpy of the second to the first scan. It has been shown that the reversibility of the thermal unfolding for proteins is closely related to the kinetics of the aggregation step,17 as suggested in Figure 1. Specifically, a faster aggregation reaction leads to worse reversibility. We further suggest that the rate constant for the aggregation reaction, k′, is correlated to protein−protein interactions between unfolded proteins. Namely, the more attractive protein−protein interactions are, the faster the aggregation reaction is, and the worse the reversibility becomes. It is anticipated that the anions can affect the reversibility of the thermal unfolding because they can significantly modulate electrostatic protein−protein interactions. Reversibility and Rate Constant for Aggregation at pH 4.8. Shown in Figure 6A is the reversibility of the DSC scans plotted against the salt concentration at pH 4.8. At the low salt concentrations, the thermal unfolding of the CH2 domain of the Fc was fully reversible up to 65 °C. However, as the salt concentrations increased, the reversibility of the DSC scans deteriorated. Because the Fc has a net positive charge at pH 4.8, repulsive electrostatic protein−protein interactions may dominate at the low salt concentrations. It is then expected that the anion could bind to the positively charged Fc, neutralize its net charge and weaken the repulsive intermolecular interactions.

Consequently, the disappearance of the thermally unfolded state due to accelerated aggregation resulted in the worsening of the reversibility of the thermal unfolding process. In addition, there is an anion-specific effect on the reversibility as shown in Figure 6A. Below 50 mM, the drop in the reversibility was steepest for Na2SO4, followed by KSCN, KCl and KF. Then the lowest reversibility, close to 0, was achieved in the KSCN solution at 100 mM. On the other hand, the worst reversibility in the KF solutions was only ∼60%. In the Na2SO4 and KCl solutions, the worst reversibility was ∼10%. Furthermore, the results from the SEC analysis (data not shown) of the post-DSC samples with low reversibility suggest the following: (1) there was significant formation of aggregates after thermal unfolding; and (2) there were more aggregates in the samples with the lowest reversibility. In addition, the far-UV CD spectra for the thermal unfolding of the Fc in 10 mM Na2SO4 at pH 4.8 as shown in Figure 4B suggest that this process was not fully reversible and aggregates formed. Our observations suggest that the reversibility of the thermal unfolding of the CH2 domain was correlated with the kinetics of the aggregation reaction. Specifically, the fast aggregation reaction leads to poor reversibility of the thermal unfolding. By assuming that the aggregation reaction follows a second order reaction, the rate constant as shown in Figure 7A was obtained as described above for each salt condition from the repetitive DSC scans. The validity of the model was evaluated by the kinetic study of the isothermal unfolding-induced aggregation as described. The experiments of the thermal 626

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

Figure 8. The aggregation kinetics measured by the SEC HPLC method for the Fc incubated at 65 °C in KF, KCl, KSCN and Na2SO4 solutions at pH 4.8. Solid lines with stars are the experimental data. The dashed lines with circles are the predictions. Symbols mark the points of measurement or computation. The curves from bottom to top in each frame correspond to increasing salt concentration.

unfolding and aggregation kinetics at pH 4.8 monitored by SEHPLC were performed to measure the total amount of aggregates as a function of time under different solution conditions. The aggregation results can be then directly compared with the theoretical prediction using the parameters obtained from fitting the DSC data. Specifically, to predict the results of aggregation in the SEC experiments, the extracted parameters from the DSC data are substituted back into the expressions for k1, k2 and k′ with the temperature (T) fixed at the experimental temperature of 65 °C. We then solve eqs 1−3 for D for the measured time range. As shown in Figure 8, the determined rate constant in Figure 7A could predict the kinetics of the aggregation in each salt condition reasonably well. In the salt solutions at the salt concentration below 50 mM as shown in Figure 7A, k′ is the fastest in the Na2SO4 solutions, followed by the KSCN, KCl and KF solutions. This trend suggests that the more strongly an anion interacts with the positively charged Fc, the faster the aggregation reaction becomes. This observation indicates that the increased aggregation kinetics could be a direct consequence of the anion binding, which neutralizes the positive charge on the Fc surface23 and weakens the repulsive electrostatic protein− protein interactions. The above events seem to support that the aggregation reaction is diffusion controlled.24 These notions of anion binding and charge neutralization were evaluated by the zeta-potential measurement. As shown in Figure 9A, the positive zeta-potential is consistent with the fact that the Fc is net positively charged at pH 4.8. The gradual decrease in the zeta-potential with the addition of KCl is in

Figure 9. The results from zeta-potential measurement for the Fc at pH 4.8 in (A) KCl solutions and (B) in different salt solutions. The standard deviations were from the measurement of three separate samples.

627

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

Figure 10. Light scattering measurement and model of aggregation at pH 7.2. The dashed lines are the relative size of the particles, δ. The markers (and the lines connecting them) are the measured scattering data in an arbitrary unit. The smooth lines are from eq 6. From top to bottom the curves represent the increasing salt concentration.

disrupting the attractive interactions and thus slowing down the aggregation reaction and improving the reversibility according to their ranking of interaction strength with the protein molecule. We noticed that the aggregation formed at pH 7.2 seemed to continue to grow in size rapidly and precipitated out quickly. In addition we noticed that the aggregates could dissociate in the SE-HPLC analysis (data not shown). This made the SE-HPLC experiments difficult to perform with accuracy for assessing aggregation. Therefore, the in situ light scattering measurement was utilized during the isothermal unfolding experiment. Although it is difficult to extract a detailed aggregation mechanism from the light scattering data because the scattered light intensity depends on both the number and size of the aggregates, we can gain some insight from the following phenomenological model. Since the wavelength of 633 nm used in the experiment may be much larger than the initial size of the aggregates ( SCN− > Cl− > F− at pH 4.8, possibly matching their ranking of binding to proteins through electrostatic interactions. The thermodynamic data implied that electrostatic interactions play an important role in maintaining the thermal stability of the CH2 domain. At pH 4.8, the anions accelerated the aggregation reaction by neutralizing the net charge of the Fc and weakening the electrostatic repulsive protein−protein interactions, following the effectiveness of SO42− > SCN− > Cl− > F−. But at pH 7.2, the thermal unfolding of the CH2 domain was less sensitive to the salt perturbation than at pH 4.8. The anions slowed down the aggregation reaction with the same ranking of effectiveness by possibly disrupting the attractive electrostatic protein− protein interactions.

(7)

where g is the relative size growth constant without salt. Note that although we have observed visible particles in the solution, implying that aggregates with size larger than the wavelength have formed, the exponential precipitation factor renders their contributions to be small. As shown in Figure 10, the increase in the scattered light intensity and the relative size of the aggregate for the Fc samples suggested that the Fc was aggregating after the thermal unfolding at 72 °C. Also, the addition of the salts in general slowed down the aggregation reaction. This is consistent with the trending of the improvement in reversibility from the DSC experiments. In addition, the phenomenological binding constant Ksalt was determined to be approximately 0.28, 0.20, 0.08 and 0.04 mM−1 for Na2SO4, KSCN, KCl and KF, respectively, from eq 7. This is consistent with the notion that the anions bind to the Fc specifically at pH 7.2, following the ranking of SO42− > SCN− > Cl− > F− for binding strength to the protein surface. The above discussion, to a large extent, depends on the information we extracted and interpreted based on the model and the fitted parameters we presented. Given the uncertainties associated with the limited mechanisms represented in the model and the numerical errors in fitting the model to the data, we would like to point out that the results of the modeling were consistent with the evidence we have presented, and the independent check of the model prediction against the SEC and light scattering experiments further support the validity of the model. However, as the experimental conditions go beyond the range in our work, the applicability of the model needs to be re-examined and possibly the model reformulated. Another limitation of the model was that, given the set of data, it cannot accurately fit for all parameters as some parameters are not sensitive enough to the data. The cases in point are the parameters k1m and E1. It is well-known that, as long as the DSC scan is slow enough compared to the unfolding kinetics, the shape of the thermogram peak before unfolding does not depend on the kinetic parameters. In the experimental work, we have performed a check with slower scanning rate and found the peak shape to be unchanged. Thus in our model fitting procedure we chose k1m and E1 to be some arbitrary but physically constrained values. Similarly the data are also not sensitive enough to the activation energy of the aggregation kinetic rate constant E′. So it cannot be obtained from this set of data.

■

ASSOCIATED CONTENT

S Supporting Information *

DSC thermogram, plot of initial aggregation rate as a function of initial Fc concentration, plots of free energy for Fc unfolding and figure depicting phenomenological binding constants. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Amgen, Inc., Department of Analytical and Formulation Sciences, Mailstop: 2-2-A, One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States. Phone: 317-4330120. Fax: 317-276-1271. E-mail: [email protected] or [email protected]. Present Addresses §

Formulation Development, Regeneron Pharmaceuticals, Tarrytown, NY 10591, United States. ∥ Vaccine Formulation Development, MedImmune, 319 Bernardo Ave, Mountain View, CA 94043. ⊥ Bioproduct Research & Development, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN 46285. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank Dr. Linda Narhi for providing valuable comments when reviewing the manuscript and Ms. Cynthia Li for providing the analysis of similarity comparison for the far-UV CD spectra.

■

REFERENCES

(1) Wang, W. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm. 2005, 289 (1−2), 1−30. (2) Lumry, R.; Eyring, H. Conformation Changes of Proteins. J. Phys. Chem. 1954, 58 (2), 110−120. (3) Uversky, V.; Fink, A. L. Protein Misfolding, Aggregation, and Conformational Disease Part A: Protein Aggregation and Conformational Diseases; Springer Science+Business Media, Inc.: Singapore, 2006. (4) Brandts, J. F. Thermobiology; Academic Press: New York, 1967. (5) Curtis, R. A.; Lue, L. A molecular approach to bioseparations: protein-protein and protein-salt intractions. Chem. Eng. Sci. 2006, 61, 907−923. (6) Ramos, C. H. I.; Baldwin, R. L. Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect. Protein Science 2002, 11, 1771−1778.

■

CONCLUSIONS The thermodynamics for the thermal unfolding and the kinetics for the subsequent aggregation reaction of the Fc were studied in the KF, KCl, KSCN and Na2SO4 solutions at pH 4.8 and 7.2 where the protein was net positively charged and charge neutral respectively. The results from our experiments reveal that the unfolding of the CH2 domain could induce aggregation. Furthermore, our results suggest that the anions have specific effects on both the thermodynamics of the thermal unfolding of the CH2 domain and the kinetics of the aggregation reaction. 629

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630

Molecular Pharmaceutics

Article

(26) (a) Collins, K. D. Charge density-dependent strength of hydration and biological structure. Biophys. J. 1997, 72, 65−75. (b) Collins, K. D. Ions from the Hofmeister series and osmolytes: effect on proteins in solution and in the crystallization process. Methods 2004, 34, 300−311. (c) Collins, K. D. Ion hydration: Implications for cellular function, polyelectrolytes, and protein crystallization. Biophys. Chem. 2006, 119 (3), 271−281. (d) Collins, K. D.; Washabaugh, M. W. The Hofmeister effect and the behaviour of water at interfaces. Q. Rev. Biophys. 1985, 18 (4), 323−422. (27) Wernersson, E.; Heyda, J.; Kubíčková, A.; Křížek, T.; Coufal, P.; Jungwirth, P. Effect of Association with Sulfate on the Electrophoretic Mobility of Polyarginine and Polylysine. J. Phys. Chem. B 2010, 114 (36), 11934−11941.

(7) Apetri, A. C.; Surewicz, W. K. Atypical Effect of Salts on the Thermodynamic Stability of Human Prion Protein. J. Biol. Chem. 2003, 278 (25), 22187−22192. (8) Kumar, R.; Mauk, A. G. Atypical Effects of Salts on the Stability and Iron Release Kinetics of Human Transferrin. J. Phys. Chem. B 2009, 113 (36), 12400−12409. (9) (a) Collins, K. D.; Neilson, G. W.; Enderby, J. E. Ions in water: characterizing the forces that control chemical processes and biological structure. Biophys. Chem. 2007, 128, 95−104. (b) Zhang, Y.; Cremer, P. S. Interactions between macromolecules and ions: the Hofmiester series. Curr. Opin. Chem. Biol. 2006, 10, 658−663. (10) Nimmerjahn, F.; Ravetch, J. V. Fc[gamma] receptors as regulators of immune responses. Nat. Rev. Immunol. 2008, 8 (1), 34− 47. (11) Roopenian, D. C.; Akilesh, S. FcRn: the neonatal Fc receptor comes of age. Nat. Rev. Immunol. 2007, 7 (9), 715−725. (12) Tischenko, V. M.; Abramov, V. M.; Zav’yalov, V. P. Investigation of the Cooperative Structure of Fc Fragments from Myeloma Immunoglobulin G. Biochemistry 1998, 37 (16), 5576−5581. (13) Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B. L. Design of therapeutic proteins with enhanced stability. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (29), 11937−11942. (14) Liu, D.; Ren, D.; Huang, H.; Dankberg, J.; Rosenfeld, R.; Cocco, M. J.; Li, L.; Brems, D. N.; Remmele, R. L. Structure and Stability Changes of Human IgG1 Fc as a Consequence of Methionine Oxidation. Biochemistry 2008, 47 (18), 5088−5100. (15) Li, C. H.; Nguyen, X.; Narhi, L.; Chemmalil, L.; Towers, E.; Muzammil, S.; Gabrielson, J.; Jiang, Y. Applications of circular dichroism (CD) for structural analysis of proteins: qualification of near- and far-UV CD for protein higher order structural analysis. J. Pharm. Sci. 2011, 100 (11), 4642−4654. (16) Joly, M. A. Physico-Chemical Approach to the Denaturation of Proteins; Academic Press: London, 1965. (17) Remmele, R. L., Jr.; Zhang-van Enk, J.; Dharmavaram, V.; Balaban, D.; Durst, M.; Shoshitaishvili, A.; Rand, H. Scan-RateDependent Melting Transitions of Interleukin-1 Receptor (Type II): Elucidation of Meaningful Thermodynamic and Kinetic Parameters of Aggregation Acquired from DSC Simulations. J. Am. Chem. Soc. 2005, 120 (23), 8328−8339. (18) Mimura, Y.; Church, S.; Ghirlando, R.; Ashton, P. R.; Dong, S.; Goodall, M.; Lund, J.; Jefferis, R. The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms. Mol. Immunol. 2000, 37 (12−13), 697−706. (19) (a) Tanford, C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv. Protein Chem. 1970, 19, 223− 286. (b) Tanford, C. Physical chemistry of macromolecules; Wiley: New York, 1961; pp 238−253. (20) Linderstrom-Lang, K. The ionization of proteins. C. R. Trav. Lab. Carlsberg, Ser. Chim. 1924, 15, 1−29. (21) Timasheff, S. N. Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components. Proc. Natl. Acad. Sci U.S.A. 2002, 99 (15), 9721− 9726. (22) Pace, C. N.; Alston, R. W.; Shaw, K. L. Charge-charge interactions influence the denatured state ensemble and contribute to protein stability. Protein Sci. 2000, 9, 1395−1398. (23) Zhang, Y.; Cremer, P. S. The inverse and direct Hofmiester series for lysozyme. Proc. Natl. Acad. Sci U.S.A. 2009, 106, 15249− 15253. (24) Solc, K.; Stockmayer, W. H. Kinetics of diffusion-controlled reaction between chemically asymmetric molecules. I. General theory. J. Chem. Phys. 1971, 54, 2981−2988. (25) (a) Zhang, Y.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. Specific Ion Effects on the Water Solubility of Macromolecules: PNIPAM and the Hofmeister Series. J. Am. Chem. Soc. 2005, 127 (41), 14505−14510. (b) Zangi, R.; Hagen, M.; Berne, B. J. Effect of Ions on the Hydrophobic Interaction between Two Plates. J. Am. Chem. Soc. 2007, 129 (15), 4678−4686. 630

dx.doi.org/10.1021/mp300378y | Mol. Pharmaceutics 2013, 10, 619−630