The thermal aggregation of the biopharmaceutical protein recombinant protective antigen (rPA) has been explored, and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies (ESEMs) and ultraviolet light scattering spectroscopy (UV-LSS). Visual observations and turbidity measurements provided an overall picture of the aggregation process, suggesting a two-step mechanism. Microscopy was used to examine the structure of aggregates, revealing an open morphology formed by the clustering of the microscopic aggregate particles. UV-LSS was used and developed to elucidate the growth rate of these particles, which formed in the first stage of the aggregation process. Their growth rate is observed to be high initially, before falling to converge on a final size that correlates with the ESEM data. The results suggest that the particle growth rate is limited by rPA monomer concentration, and by obtaining data over a range of incubation temperatures, an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation. In doing so, we quantified the susceptibility of rPA aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of UV spectrometry to monitor the particle aggregation quantitatively,
The study of protein aggregation is a burgeoning field of research driven by the urgent need to elucidate the mechanism of neurodegenerative diseases, the desire to understand and mimic natures’ ability to create hierarchical complex nanostructures, and the necessity to understand and minimise product loss during the processing and formulation of biopharmaceuticals. Aggregation is of particular importance for therapeutic proteins as it can lead to a loss of product, reduce efficacy, alter biological activity and pharmacokinetics, and even raise safety concerns such as increased immunogenicity [
The influence of solution pH on aggregation is one of the more studied and important parameters that controls the onset of aggregation and final aggregate morphology [
The literature is awash with many studies that postulate different models for the self-assembly of proteins, and these can generally be divided into two main categories: empirical or mechanistic [
Here, the thermal aggregation of an industrially relevant biopharmaceutical recombinant protective antigen (rPA) (active component in a second-generation anthrax vaccine [
rPA was supplied by Avecia Biologics (UK) at 2 mg mL−1 in a phosphate-buffered saline solution adjusted to pH 7.4. Doubly distilled water was obtained from an Elga PureLab Ultra (18.2 Ω). All other chemicals were purchased from either Sigma-Aldrich (UK) or Acros Organics (UK), where the reagent grade is at least 97% pure and used as recieved.
The rPA samples supplied were treated using the following procedure to provide a consistent starting material for analysis. Initially, rPA was precipitated from solution by heating (50°C for circa 5 min). The resulting gel was centrifuged at 6000 rpm for 1 minute, and the supernatant was discarded. The gel was resuspended by adding doubly distilled water and vortexing for 2 min. The sample was centrifuged as before, and the supernatant was discarded. The gel was resolubilized by adding urea and doubly distilled water to form an 8 M urea solution. rPA was refolded by dilution, using 1 part rPA in 8 M urea to 31 parts refold buffer. This was done in two stages, with a 1 : 7 dilution followed by 1 : 4 dilution 2 minutes later. The refold buffer contained 25 mM TRIS, 25 mM NaCl, 2 mM CaCl2 and was adjusted to pH 7.4 with hydrochloric acid. The final buffered samples contained between circa 0.15 and 0.3 mg mL−1 rPA with 0.25 M urea and were analysed immediately after refolding. This concentration was chosen as it mimics the conditions of storage of some of the formulations of protective antigen vaccines. Samples without urea were prepared using the refolding method followed by dialysis using 3500 Dalton molecular weight cutoff Visking dialysis membrane (Medicell International Ltd) against 10 times excess of chilled refold buffer for 18 hours in the refrigerator (~4°C). After this, the external solution was replaced twice with fresh refold buffer and allowed to dialyse for a further 24 hours. The rPA sample was then recovered from the sealed membrane and stored in the refrigerator (circa 4°C) prior to use. This was done to reduce any degradation of the rPA during storage. Samples, however, were not frozen to avoid potential freeze-thaw damage.
The concentration of the refolded rPA solution was determined from its UV absorbance at 280 nm (Shimadzu UV 2501-PC spectrophotometer), using a molar absorption coefficient of 72769 M−1 cm−1.
The isothermal aggregation of rPA was monitored visually over time by incubating samples over a range of temperatures (43–49°C) close to the denaturation temperature of rPA (50.0°C) by using a recirculating water bath connected to a heating stage that contained the sample cell. The temperature was measured using a calibrated K-type thermocouple placed in the sample solution (accurate to ± 0.3°C).
Images of rPA aggregates were obtained using a Zeiss Axioplan 2 in transmission mode with a 10x magnification objective and a digital camera. The aggregated samples were pipetted onto a microscope slide and a cover slip placed on top.
Aggregates were mounted for ESEM analysis simply by lifting them out of solution on a mica disc and placing them directly onto the sample stage. The samples were examined using a Philips FEI Quanta 200 ESEM with the electron gun accelerating voltage set to 30 kV, the sample stage at 5°C, and the chamber pressure at 6 torr.
Light scattering spectra were recorded using a Shimadzu UV 2501-PC spectrophotometer set to record between 250 and 390 nm every 2 nm at a medium scan rate. The sample was heated
The wavelength dependence of absorbance arising from light scattering by particles in solution follows the relationship [
Refractive index of rPA particles at different wavelengths calculated using the Lorentz-Lorenz molar refraction.
Wavelength/nm | Refractive index, |
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434.0 | 1.684 |
486.1 | 1.677 |
589.3 | 1.665 |
656.3 | 1.661 |
Solutions of rPA (0.31 mg mL−1) were incubated over a range of temperatures (25–50°C), and their visual appearance was monitored over time. This temperature range was selected as the denaturation temperature of rPA is known to be ~50°C [
Photographs taken at different time points of rPA aggregates forming in solution at 47.6°C. Note that each of (a) to (c) and (h) has a black background placed behind the cuvette and (d) to (g) have no background allowing light to enter the sample from behind.
These visual results were complemented by turbidity measurements recorded under identical conditions (see Figure
Absorbance at 320 nm versus time for rPA held at 47.7°C (×). Labels “a” to “h” relate to the photographs shown in Figure
Similar visual and turbidity observations were noted for other samples incubated between 43° and 49°C, as all appeared to have a similar two-step mechanism. The time scales for each step varied widely; however, for the different temperatures, the time to reach peak turbidity ranged from circa 10 minutes at 49°C to 10 hours at 43°C. This dramatic increase in aggregation rate versus temperature could be driven by either increasing the translational kinetic energy of protein monomers, causing more frequent collisions between the particles, or increasing the internal kinetic energy of the protein monomers. The latter will drive monomers toward an unfolded state, with the associated exposure of hydrophobic patches increasing the likelihood that a collision results in self-association.
The morphology of the macroscopic particles formed after incubation was examined using optical and electron microscopy. Figure
rPA aggregates formed after incubation at 47°C for 16 hours: (a) optical micrograph, 10x magnification (170
The rate of aggregation at different isothermal temperatures was explored using UV light scattering spectroscopy (UV-LSS), where spectra were recorded every 20 seconds initially and then every 20 seconds to 40 minutes depending on the aggregation rate. Such fast acquisition times provide a speed advantage over other light scattering techniques for analysing such aggregation kinetics. Typical results for incubation at 45°C are shown in Figure
(a) Change in UV light scattering arising from the growth of rPA aggregates at 45°C, spectra collected in 10-minute intervals for 0–200 minutes, and (b) aggregate diameters calculated from light scattering spectra as a function of time.
As discussed previously, rPA solutions became turbid during incubation at temperatures ≥43°C; therefore, it can be assumed that multiple light scattering was occurring. It can be assumed, however, that the wavelength dependence of light scattering obtained by solving the Mie equations would hold for the scattering occurring here. As such, the UV spectra in Figure
To explore the effect of isothermal temperature on particle growth rate and consequently gain an insight into the aggregation kinetics, particle size was recorded as a function of time for a range of temperatures (43°–49°C) and the results are given in Figure
Comparison of aggregate diameter calculated from light scattering spectra versus time for rPA samples with 0.25 M urea held isothermally at various temperatures (the inset highlights higher temperature runs).
All profiles show an increase in diameter over time; however, the time taken to reach the maximum diameter is markedly different for each temperature: the higher the temperature, the shorter the time scale to reach the maximum diameter. This is reflected in the results by a notable increase in the profile gradient when the temperature is increased, where the maximum gradient increases by 2.7 times on average for every 1°C increase in temperature. Similar results were obtained for a range of samples containing an additive: 0.25 M urea. In this case, similar trends were observed over time for a range of incubation temperatures, and the only difference arising was in the rate of particle growth being slightly faster for each sample in the presence of 0.25 M urea. All samples contained a homogeneous distribution of particle size (confirmed by ESEM) suggesting that the aggregate concentration remained constant over time. This means that the quantity of rPA in the aggregates can be determined by estimating the aggregate concentration and using the ratio of aggregate to monomer volume. From this, the difference between the quantity of rPA incorporated within aggregates and the quantity of monomer present initially gave monomer concentration over time, providing a possible means of assessing the aggregation kinetics. To this end, data over initial incubation times (where the scattering was increasing) were analysed using a generalised scheme of the protein aggregation pathway. This scheme is outlined in Figure
Schematic representing the 4 possible steps in the proposed kinetic model of rPA aggregation: (a) structural perturbation, (b) initial aggregate formation, (c) aggregate growth, and (d) aggregate clustering.
In the case of association-limited aggregation,
(a) Monomer concentration as a function of time for rPA samples with 0.25 M urea held isothermally at various temperatures (the inset highlights higher temperature runs). (b) Second-order association-limited aggregation rate constant versus temperature. (c) Arrhenius plot.
An alternative and more appropriate approach to modelling the temperature dependence of the rate constant is to factor in the behaviour of the equilibrium constant,
Changes in enthalpy (
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rPA without additive | 975.8 ± 40.5 | 3.02 ± 0.13 | 76.1 | 50.2 |
rPA with 0.25 M urea | 949.7 ± 94.6 | 2.92 ± 0.30 | 77.7 | 51.6 |
Natural log of equilibrium constant versus the reciprocal of absolute temperature for the association-limited aggregation model.
To explore the effect of temperature on the extent of disruption of individual proteins, the fraction of protein in the perturbed intermediate state,
Fraction of monomer in the perturbed state as a function of temperature for the association-limited aggregation model. Curves of the best fit follow (
The susceptibility of a biopharmaceutical protein rPA to aggregate as a function of temperature and formulation conditions has been determined, and the kinetics and thermodynamics of the aggregation process have been modelled and quantified. Visual and turbidity experiments showed that the thermal aggregation of rPA occurs at incubation temperatures ≥43°C, which is close to its denaturation temperature. Under these conditions, the protein is likely to have increased translational kinetic energy, hence more collisions will take place, and also be at least partially unfolded, hence, it has some exposed hydrophobic regions which are known to induce rapid and nonspecific aggregation. Such aggregation was found to proceed in a stepwise manner, by first forming spherical microscopic particles followed by clustering to form fractal aggregates. Increasing the temperature more than 43°C increased the rate of aggregation dramatically and also the size of the diameter of the spherical microscopic particles formed from ~360 to 500 nm when increasing the temperature from 43° to 49°C. We went on to show that the growth of the microscopic particles can be monitored using UV-LSS. In particular, we used the increase in scattered light from the sample over time to elucidate aggregate size versus time, giving a quantitative measure of the aggregation. Moreover, the experiments were conducted over a range of temperatures, with and without 0.25 M urea, and as such, the results were analysed to determine the rate constant and the temperature dependence of the thermal aggregation process. Based on this analysis, we proposed that the aggregation process is association limited and that the temperature dependence relates to the equilibrium behaviour between native and perturbed states. We were also able to extract the thermodynamic parameters for aggregation in samples with and without urea, and these indicated that the presence of urea reduces the stability of rPA, hence increases its susceptibility to aggregation. The modelling tools developed here for analysis of data from the easily accessible UV-LSS technique provides a fast between
The authors wish to thank EPSRC and Intertek for financial support and Avecia Biologics for supplying rPA samples. They would also like to thank Dr. Patrick Hill for his help with the ESEM.