Isothermal experiments characterize time-dependent aggregation and unfolding

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1 1 Energy Isothermal experiments characterize time-dependent aggregation and unfolding Technical ote Introduction Kinetic measurements have, for decades, given protein scientists insight into the mechanisms involved in the self-assembly of higher order protein structures. ovel experimental measurements have pushed the boundaries of the temporal resolution meaning that faster and faster events that occur as a protein changes conformation can be observed via changes in an observable metric indicative of protein structure. Some of the techniques best suited to this type of measurement are optical methods. In particular, fluorescence and light scattering measurements allow very rapid measurements of spectroscopic parameters that can indicate tertiary structure changes (such as the fluorescence emission peak maximum wavelength), the degree of fluorescence quenching (via integrated fluorescence intensity) or the change in average molecular weight via the intensity of scattered light. The value of kinetic measurements Methods for determining protein stability are often focused on their equilibrium stability and how the delicate balance between unfolded and folded states changes under a variety of stresses. Protein conformational stability can be challenged by addition of various chemical compounds, change of temperature or application of mechanical forces. For a simple system where a protein exists in equilibrium between only two conformations, a folded state and an ensemble of unfolded states, the energetics of the system can be described using a difference in the Gibbs free energy ( G) of the two states (Figure 1). A negative value indicates that the folded state is more stable than the unfolded state, and it can be seen that conditions promoting stabilization of the native state will result in larger negative values. Application of a stress may affect the balance of the equilibrium such that the difference in the free energies of the two states changes until it is zero. At this point the protein is equally likely to be in either conformation, and this condition is often described as the midpoint of a reversible transition the T m if the stress applied is temperature, or [D] ½ if the stress applied is a chaotropic chemical. Further stress then shifts the balance until the probability of having unfolded protein increases towards near certainty. ΔG A B C Figure 1: A protein with a free energy difference (ΔG) between the unfolded protein () and native protein () that results in A: a significantly populated and stable folded state with having a lower free energy than, B: equal probabilities of the presence of both folded and unfolded states as and having the same free energy and C: predominantly unfolded species as the folded state is unstable with having a lower free energy than. An important consideration is that a bias in the balance of the equilibrium between the two states does not infer that the least probable state is never occupied, just that it is statistically less likely to be. What these equilibrium experiments do not do is provide any information about the rate at which the protein molecules switch between the two possible states as this is not only governed by the free energy difference between them. Instead, transition state theory suggests that between each state there is a transition state barrier which must be overcome to change from one state to the other. The height of this barrier determines the rate at which it is crossed. Figure 2 shows a schematic energy diagram, where a small barrier between the unfolded protein and ΔG

2 Technical ote the folded protein is overcome at a faster rate than a larger barrier going the other way. Energy kf ΔG ku steady-state equilibrium is re-established. This means that incubation of a range of protein samples under constant temperature conditions can provide useful insights into the rank order of their stabilities. Example study A polyclonal antibody (IgG, I5506 Sigma) was formulated in a variety of different guanidine hydrochloride (GuHCl) concentrations (from 0 6 M), in citrate phosphate buffer at ph 8. GuHCl is a chemical denaturant that is expected to reduce the stability of the native state relative to that of the ensemble of Figure 2: An energy barrier containing a transition state,, that divides the unfolded () and folded (native, ) state of a protein leading to a slower rate of unfolding (ku) (as the barrier is larger from ) than folding (kf). Experimental methods measure the ensemble of many protein molecules in solution going both ways, so an average of the conformational change is observed. This observed rate constant, k obs, can be judged to be equal to the sum of the folding and unfolding rate constants in a simple two state model of the system. In this situation the population of the native state can be described by [] = c 1 exp( k obs t) + c 2 where c 1 and c 2 are constants based on the initial native state population, the total number of molecules and the equilibrium constant and t is time. For single proteins which visit more than two states during folding the mathematical solutions are a little more involved and include multi-exponential functions. It is useful to note that there are always one fewer nonzero rate constants than the number of states involved in the process. It has also been demonstrated that for more complex mechanisms alternative examinations of the kinetic data may prove valuable. Such analyses include fitting linear regions of the plots, integration under the curve and measurement of observable lag times. It is therefore possible to gain information about the conformational stability of a protein by observing the kinetics of protein folding or unfolding upon application of a stress condition and measuring the rate at which a Figure 3A: Thermal ramp of IgG samples mixed with a range of GuHCl concentrations (0 6 M at 0.2 M increments). The lowest GuHCl concentration is shown in red, with increasing denaturant levels represented by colours of the rainbow. The data is analysed by taking the differential of the barycentric mean (BCM) of the intrinsic fluorescence spectra at each temperature studied (see the it Technical ote entitled Intrinsic fluorescence monitors conformational changes in proteins ). The maximum value of this differential is taken as Tm. 3B: The value of BCM of each IgG sample at 60 C plotted against the GuHCl concentration of the samples. 2

3 Technical ote unfolded states. The effect of heating upon the conformational state of the protein was probed via changes in the intrinsic fluorescence emission, which is typically dominated by tryptophan (Trp). The barycentric mean (BCM) provides a measure of the shift in emission maximum that is usually observed as Trp residues buried in the hydrophobic core of native globular proteins are exposed to the surrounding polar solvent upon unfolding. An initial thermal ramp experiment was performed (Figure 3) as described in the it Method ote entitled Thermal ramp experiments measure protein conformational stability and propensity to aggregate. This allows determination of the T m values of the samples, which relate to their conformational stabilities. Such data permit selection of an optimal temperature for isothermal experiments on these samples. Practical consideration must be given to the fact that above the T m the protein will unfold very quickly, whereas below Figure 4A: Isothermal unfolding curves at 50 C of IgG in a range of GuHCl concentrations (0-6 M at 0.2 M increments). The lowest GuHCl concentration is shown in red, with increasing denaturant levels represented by colours of the rainbow. 4B: Rate constants extracted from monoexponential fits to the unfolding curves, the data are fit to a linear function. 4C: The area underneath the isothermal curves corrected for the initial BCM wavelength for each sample. The peak around 1 M GuHCl suggests the biggest amplitude change occurs here. Data are fit to a Gaussian function. 3

4 Technical ote the T m it will unfold more slowly. In this case, the range of melting temperatures means that the choice of isothermal temperature is critical to the validity of the data interpretation A wide range of melting temperatures are observed depending upon GuHCl concentration (Figure 3A), and it can be seen, for instance, by looking at the equilibrium BCM wavelengths at 60 C extracted from the thermal ramp experiment (Figure 3B) that, at this temperature, the protein is fully unfolded at ~2 M GuHCl and it is unlikely that any kinetic data will be determinable above this concentration. Therefore, picking an isothermal temperature of 50 C would allow observation of a range of different behaviors as it is above the T m of many of the samples meaning that observed kinetics will be dominated by the unfolding of the protein. The data obtained from the isothermal experiment (Figure 4A) at this temperature indeed show that the samples unfold as indicated by a shift in BCM to longer wavelengths. It is clear that the rate of unfolding increases with GuHCl concentration, and that at higher levels of the denaturant early stages of the unfolding occur during the dead time of the measurement. This correlation between unfolding rates and GuHCl concentration was confirmed when the data were fit to the exponential equation shown earlier and the resulting rate constants plotted against denaturant levels (Figure 4B). This demonstrates that the rate at which proteins unfold provides information about their stability. These experiments can yield further information, such as the degree to which proteins unfold, which can be obtained by calculating the amplitude change between the initial and final BCM value. Alternatively, similar results can be achieved by integrating underneath the curve (Figure 4C), which is non-parametric and accounts for changes that occur in the response of one sample and not another. This analysis demonstrates that, at this temperature, the largest changes in amplitude, and hence the most observable flux through the transition state, occur at around 1 M GuHCl for this sample. Clearly such isothermal experiments afford valuable information which can be used to screen protein formulation stability. However, care should be taken to choose a temperature that optimizes the resulting output. The full story aggregation and unfolding A therapeutic protein may encounter a number of stresses during bioprocessing which pose a challenge to the maintenance of a soluble, native-like state. The protein may have to withstand physical forces such as sheering, stirring or pressure applied during filtrations as well as changes in temperature designed to reduce viscosity and improve filter flux. Furthermore, it may encounter chemical stresses such as addition of chaotropes during refolding from inclusion bodies; changes in ionic strength during elution from ion exchange columns and incubation at low ph to inactivate viruses. ICH guidelines require accelerated stability measurements as a specification for observing biophysical and chemical stability of proteins used in biotechnology. These studies are performed at a range of elevated temperatures under the justification that these studies can correlate with monomer fraction and activity at 4 C, albeit after a reduced period of time. These analyses can be used to demonstrate optimal formulation and minimize aggregate formation, or to improve production and maximize yields. Data were presented earlier to describe thermal unfolding of protein formulations at an elevated temperature, however, this process does not necessarily correlate with aggregation propensity over time for all systems. The kinetics of aggregation will depend on the degradation pathway of the protein. What about the aggregation? There are two primary routes for protein aggregation in a simple two state system (Figure 5): 1. ative state aggregation the folded protein forms intermolecular interactions via surfaceexposed residues (green arrow in Figure 5) 2. on-native state aggregation hydrophobic patches, typically buried in the core of a native state, become exposed and stick to other similar molecules to minimise unfavourable interactions with water (red arrows in Figure 5) 4

5 Technical ote Energy A a2 k u-agg ΔG 2 Figure 5: Energy diagram indicating unfolded state aggregation and native state aggregation. ative state aggregation results in cases where the protein will aggregate at temperatures below the unfolding onset point. Therefore, an isothermal experiment would yield significant changes in the light scattering signal, accompanied by either little observable change in the conformational state or slow unfolding kinetics. nder these conditions the barrier between the native state and the aggregated state is significantly lower than the barrier between the folded and unfolded state. Aggregation is often promoted by only short stretches of primary amino acid sequence, and in this regime these aggregation-prone regions are located in surface-exposed positions. Therefore, formation of favorable protein-protein interactions that result in aggregates are mediated by solution conditions and can often be mitigated by formulation optimization. k u k n-agg a1 A For the second regime the most likely result of an isothermal experiment is that there would be an observed conformational change followed by an aggregation response. Characterizing SLS data in such cases involves taking into consideration the lag phase associated with a requirement to occupy the unfolded state before the onset of the aggregation, the duration of which depends on the ratio of the activation barrier to unfold and the activation barrier to the aggregated state. nder most conditions unfolding of proteins leads to formation of aggregates. This is due to the fact that intramolecular interactions between hydrophobic residues facilitate entropically-driven folding and generate a buried hydrophobic core in the native state. These same interactions can drive aggregation, thus upon unfolding, these moieties become exposed to unfavorable interactions with aqueous solvent and form intermolecular interactions with hydrophobic regions of other denatured protein molecules. Observation of unfolding and aggregation rates under different formulation conditions To provide an example where kinetics of both unfolding and aggregation are used, a monoclonal antibody (MAb1) was incubated at 65 C under different formulation conditions. Data for 1 mg ml -1 MAb1 stored in a phosphate-citrate buffer at ph 3.6 and ph 4.6 is shown in Figure 6. Figure 6A: nfolding (monitored by BCM, shown by filled circles) and aggregation (monitored by SLS of 266 nm laser, shown by line) of a monoclonal antibody incubated at 65 C and stored in formulations at ph 3.6 (red) and ph 4.6 (blue). 6B: Reaction rates extracted from these data 5

6 Technical ote As monoclonal antibodies are multi-domain proteins they have more than one unfolding transition. As judged by thermal ramp experiments (data not shown) MAb1 displays two observable unfolding transitions under these conditions. It is known that MAb1 undergoes aggregation only upon unfolding of the higher stability domain, indicating it undergoes nonnative state aggregation as described above. Presumably unfolding of the lower stability domain results in a state where aggregation-prone regions are still well protected from intermolecular interactions within the complex structure of the antibody. For the sample formulated at ph 3.6, the incubation temperature used in this isothermal experiment (65 C) is above the T m value of the higher stability domain and the aggregation onset (T agg) value. This is reflected in the fast rates observed in both the unfolding and aggregation kinetics. Conclusions Isothermal experiments can provide multiple kinetic parameters relating to protein stability. These measurements are particularly useful in optimizing formulation of biologics and are recommended by regulatory agencies. The it provides the ability to obtain such data over a wide temperature range on multiple samples (up to 48 simultaneously) using only small sample volumes (9 L). The flexibility provided by simultaneously taking different measurements for protein conformational change and aggregation mean that varied degradation pathways can be probed using this system. At ph 4.6 the T m value for the low stability regions of MAb1 is lower than the incubation temperature used in this isothermal experiment. However, the higher stability domain has a T m value of 71.6 C (which correlates well with a T agg value of 71.5 C) that is above the incubation temperature used here. The unfolding rate under these conditions is lower than observed for the protein stored at ph 3.6, and two phases are observed. It is possible that the fast phase corresponds to unfolding of the low stability domain, whereas the slower phase is due to unfolding of the higher stability domain in a fraction of the proteins present. It is clear that aggregation rates under these conditions are reduced from those during incubation at ph 3.6 which confirms that the low stability domain has a low aggregation propensity, even in the unfolded state. The observed increase in SLS at ph 4.6 thus could be due to either slow kinetics of aggregation for the unfolded low stability domain or aggregates formed by the fraction of protein molecules where the higher stability domains gradually unfold. This data illustrates the utility of multi-parameter isothermal experiments in gaining information on the mechanisms of protein degradation. Toll-free: (800) Tel: (925) orders@unchainedlabs.com unchainedlabs.com 6