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, in situ, in a nondestructive and time-resolved manner. 1. Introduction 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 [1–3]. Aggregation can be induced by solution conditions such as protein concentration, pH, salinity, temperature, and the presence of additives [4, 5]. These variables are also known to affect the quantity and morphology of aggregate formed [5]. Stresses to the protein such as over-expression, refolding, freeze-thaw cycles, agitation, or exposure to hydrophobic surfaces or air (including foaming) can also lead to the formation of aggregates [2, 4–6]. Each of these environmental factors is typically encountered during
References
[1]
E. D. B. Clark, “Protein refolding for industrial processes,” Current Opinion in Biotechnology, vol. 12, no. 2, pp. 202–207, 2001.
[2]
J. L. Cleland, M. F. Powell, and S. J. Shire, “The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation,” Critical Reviews in Therapeutic Drug Carrier Systems, vol. 10, no. 4, pp. 307–377, 1993.
[3]
A. Braun, L. Kwee, M. A. Labow, and J. Alsenz, “Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-α) in normal and transgenic mice,” Pharmaceutical Research, vol. 14, no. 10, pp. 1472–1478, 1997.
[4]
W. Wang, “Instability, stabilization, and formulation of liquid protein pharmaceuticals,” International Journal of Pharmaceutics, vol. 185, no. 2, pp. 129–188, 1999.
[5]
W. Wang, S. Nema, and D. Teagarden, “Protein aggregation—pathways and influencing factors,” International Journal of Pharmaceutics, vol. 390, no. 2, pp. 89–99, 2010.
[6]
J. R. Clarkson, Z. F. Cui, and R. C. Darton, “Protein denaturation in foam: I. Mechanism study,” Journal of Colloid and Interface Science, vol. 215, no. 2, pp. 323–332, 1999.
[7]
M. R. H. Krebs, G. L. Devlin, and A. M. Donald, “Protein particulates: another generic form of protein aggregation?” Biophysical Journal, vol. 92, no. 4, pp. 1336–1342, 2007.
[8]
C. M. Dobson, “Protein folding and misfolding,” Nature, vol. 426, no. 6968, pp. 884–890, 2003.
[9]
M. R. H. Krebs, K. R. Domike, and A. M. Donald, “Protein aggregation: more than just fibrils,” Biochemical Society Transactions, vol. 37, no. 4, pp. 682–686, 2009.
[10]
W. S. Gosal and S. B. Ross-Murphy, “Globular protein gelation,” Current Opinion in Colloid and Interface Science, vol. 5, no. 3-4, pp. 188–194, 2000.
[11]
A. M. Morris, M. A. Watzky, and R. G. Finke, “Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature,” Biochimica et Biophysica Acta, vol. 1794, no. 3, pp. 375–397, 2009.
[12]
C. J. Roberts, “Nonnative protein aggregation: pathways, kinetics, and stability prediction,” in Misbehaving Proteins, R. M. Murphy and A. M. Tsai, Eds., pp. 17–46, Springer, New York, NY, USA, 2006.
[13]
S. E. Bondos, “Methods for measuring protein aggregation,” Current Analytical Chemistry, vol. 2, no. 2, pp. 157–170, 2006.
[14]
M. E. M. Cromwell, C. Felten, H. Flores, J. Liu, and S. J. Shire, “Self-association of therapeutic proteins: implications for product development,” in Misbehaving Proteins, R. M. Murphy and A. M. Tsai, Eds., pp. 313–330, Springer, New York, NY, USA, 2006.
[15]
R. M. Murphy and C. C. Lee, “Laser light scattering as an indispensable tool for probing protein aggregation,” in Misbehaving Proteins, R. M. Murphy and A. M. Tsai, Eds., pp. 147–165, Springer, New York, NY, USA, 2006.
[16]
S. Jendrek, S. F. Little, S. Hem, G. Mitra, and S. Giardina, “Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants,” Vaccine, vol. 21, no. 21-22, pp. 3011–3018, 2003.
[17]
K. Chen, A. Kromin, M. P. Ulmer, B. W. Wessels, and V. Backman, “Nanoparticle sizing with a resolution beyond the diffraction limit using UV light scattering spectroscopy,” Optics Communications, vol. 228, no. 1–3, pp. 1–7, 2003.
[18]
A. J. Cox, A. J. DeWeerd, and J. Linden, “An experiment to measure Mie and Rayleigh total scattering cross sections,” American Journal of Physics, vol. 70, no. 6, pp. 620–625, 2002.
[19]
H. Fang, M. Ollero, E. Vitkin et al., “Noninvasive sizing of subcellular organelles with light scattering spectroscopy,” IEEE Journal on Selected Topics in Quantum Electronics, vol. 9, no. 2, pp. 267–276, 2003.
[20]
J. R. Mourant, T. Fuselier, J. Boyer, T. M. Johnson, and I. J. Bigio, “Predictions and measurements of scattering and absorption over broad wavelength ranges in tissue phantoms,” Applied Optics, vol. 36, no. 4, pp. 949–957, 1997.
[21]
A. M. K. Nilsson, C. Sturesson, D. L. Liu, and S. Andersson-Engels, “Changes in spectral shape of tissue optical properties in conjunction with laser-induced thermotherapy,” Applied Optics, vol. 37, no. 7, pp. 1256–1267, 1998.
[22]
L. B. Scaffardi, N. Pellegri, O. De Sanctis, and J. O. Tocho, “Sizing gold nanoparticles by optical extinction spectroscopy,” Nanotechnology, vol. 16, no. 1, pp. 158–163, 2005.
[23]
S. M. Scholz, R. Vacassy, J. Dutta, H. Hofmann, and M. Akinc, “Mie scattering effects from monodispersed ZnS nanospheres,” Journal of Applied Physics, vol. 83, no. 12, pp. 7860–7866, 1998.
[24]
C. F. Bohren and D. R. Huffman, Absorption and Scattering of Light by Small Particles, Wiley, New York, NY, USA, 1983.
[25]
P. Schiebener, J. Straub, J. M. H. L. Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” Journal of Physical and Chemical Reference Data, vol. 19, pp. 677–717, 1990.
[26]
A. Weissberger, “Physical methods of organic chemistry—part 2,” in Techniques of Organic Chemistry, Wiley, New York, NY, USA, 1960.
[27]
L. Willard, A. Ranjan, H. Zhang et al., “VADAR: a web server for quantitative evaluation of protein structure quality,” Nucleic Acids Research, vol. 31, no. 13, pp. 3316–3319, 2003.
[28]
C. Petosa, R. J. Collier, K. R. Klimpel, S. H. Leppla, and R. C. Liddington, “Crystal structure of the anthrax toxin protective antigen,” Nature, vol. 385, no. 6619, pp. 833–838, 1997.
[29]
H. M. Berman, J. Westbrook, Z. Feng et al., “The protein data bank,” Nucleic Acids Research, vol. 28, no. 1, pp. 235–242, 2000.
[30]
H. Fischer, I. Polikarpov, and A. F. Craievich, “Average protein density is a molecular-weight-dependent function,” Protein Science, vol. 13, no. 10, pp. 2825–2828, 2004.
[31]
D. A. Chalton, I. F. Kelly, A. McGregor et al., “Unfolding transitions of Bacillus anthracis protective antigen,” Archives of Biochemistry and Biophysics, vol. 465, no. 1, pp. 1–10, 2007.
[32]
P. Meakin, “Fractal aggregates,” Advances in Colloid and Interface Science, vol. 28, pp. 249–331, 1987.
[33]
C. J. Roberts, “Kinetics of irreversible protein aggregation: analysis of extended Lumry-Eyring models and implications for predicting protein shelf life,” Journal of Physical Chemistry B, vol. 107, no. 5, pp. 1194–1207, 2003.
[34]
B. S. Kendrick, J. L. Cleland, X. Lam et al., “Aggregation of recombinant human interferon gamma: kinetics and structural transitions,” Journal of Pharmaceutical Sciences, vol. 87, no. 9, pp. 1069–1076, 1998.
[35]
P. W. Atkins, Physical Chemistry, Oxford Unveristy Press, Oxford, UK, 2nd edition, 1982.
[36]
R. L. Thommarson, “Alkyl radical disproportionation,” Journal of Physical Chemistry, vol. 74, no. 4, pp. 938–941, 1970.
[37]
R. Jaenicke, “Protein stability and protein folding,” in Ciba Foundation Symposium 161—Protein Conformation, D. J. Chadwick and K. Widdows, Eds., pp. 206–221, Wiley, Chichester, UK, 2007.
[38]
G. Jiang, S. B. Joshi, L. J. Peek et al., “Anthrax vaccine powder formulations for nasal mucosal delivery,” Journal of Pharmaceutical Sciences, vol. 95, no. 1, pp. 80–96, 2006.
