Abstract
Purpose. The potential to estimate protein solubilities, with limited protein, by excluded-volume interactions was evaluated using polyethylene glycols (PEG) and recombinant bovine Somatotropin (rbSt).
Methods. Solutions of rbSt were prepared at concentrations significantly below saturation solubility. Subsequently, varying amounts of PEG were added to force protein precipitation. Following centrifugation, the protein concentration in the supernatant was assayed by reversed-phase HPLC, where a logarithmic relationship between solubility and % PEG was observed.
Results. An apparent protein solubility in the absence of PEG was determined by extrapolation and compared well with values measured by conventional approaches. Slopes of log solubility versus % PEG curves were consistent with excluded-volume principles and depended on the molecular weight of the PEG used. Furthermore, the precipitation process proved to be reversible, allowing for recovery of intact protein. Solubility-pH profiles obtained in the presence of PEG greatly reduced the quantities of protein needed and compared favorably with profiles in the absence of PEG.
Conclusions. Thus, it appears feasible and practical, with certain limitations, to obtain solubility estimates of proteins by volume-exclusion methods with limited supplies of protein.
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REFERENCES
C. R. Middaugh and D. B. Volkin. Protein Solubility. In T. J. Ahern and M. C. Manning (eds.) Stability of Protein Pharmaceuticals. Plenum Press, New York, 1992, pp. 109–134.
A. Polson, G. M. Potgieter, J. F. Largier, G. E. F. Mears and F. J. Joubert. The fractionation of protein mixtures by linear polymers of high molecular weight. Biochim. Biophys. Acta 82:463–475 (1964).
I. R. M. Juckes. Fractionation of proteins and viruses with polyethylene glycol. Biochim. Biophys. Acta 229:535–546 (1971).
E. Edmond and A. G. Ogston. An approach to the study of phase separation in ternary aqueous systems. Biochem. J. 109:569–576 (1968).
K. C. Ingham. Polyethylene glycol in aqueous solution: solvent perturbation and gel filtration studies. Arch. Biochem. Biophys. 184:59–68 (1977).
K. C. Ingham. Precipitation of protein with PEG: characterization of albumin. Arch. Biochem. Biophys. 186:106–113 (1978).
O. Zschornig, H. Machill, D. Wiegel, J. Arnhold and K. Arnold. Aggregation of human plasma high density lipoproteins induced by poly(ethylene glycol). Biomed. Biochim. Acta. 50:959–966 (1991).
K. Arnold and O. Zschornig. Aggregation of human plasma low density lipoproteins by means of poly(ethylene glycol). Biomed. Biochim. Acta. 47:949–954 (1988).
D. H. Atha and K. C. Ingham. Mechanisms of precipitation of proteins by polyethylene glycols. J. Biol. Chem. 256:12108–12117 (1978).
F. Hasko, R. Vaszileva and L. Halasz. Solubility of plasma proteins in the presence of polyethylene glycol. Biotechnology and Bioengineering 24:1931–1939 (1982).
C. L. Stevenson and M. J. Hageman. Estimation of protein solubilities by excluded-volume interactions with polyethylene glycols. Pharm. Res. 5:S-30 (1988).
S. A. Charman, M. Snoswell and W. N. Charman. Solubility assessment of recombinant porcine growth hormone as a function of pH and ionic strength. Pharm. Res. 7:S-47 (1990).
T. Arakawa and S. N. Timasheff. Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry 24:6756–6762 (1985).
C. R. Middaugh, W. A Tisel, R. N. Haire and A. Rosenberg. Determination of the apparent thermodynamic activities of saturated protein solutions. J. Biol. Chem. 254:367–370 (1979).
R. N. Haire, W. A. Tisel, J. G. White and A. Rosenberg. On precipitation of proteins with polymers: the hemoglobin-polyethylene glycol system. Biopolymers 23:2761–2779 (1984).
S. I. Miekka and K. C. Ingham. Influence of self-association of proteins on their precipitation by poly(ethylene glycol). Arch. Biochem. Biophys. 191:525–536 (1978).
S. R. Davio and M. J. Hageman. Characterization and formulation considerations for recombinantly derived bovine somatotropin. In Y. J. Wang and R. Pearlman (eds.) Stability and Characterization of Protein and Peptide Drugs. Plenum Press New York, 1993, pp. 59–90.
G. A. F. Seber. Linear regression analysis. John Wiley and Sons, New York, 1977, pp. 197–203.
J. L. Cleland and T. W. Randolph. Mechanism of polyethylene glycol interaction with molten globule folding intermediates of bovine carbonic anhydrase B. J. Biol. Chem. 267:3147–3153 (1992).
D. N. Brems, S. M. Plaisted, E. W. Kauffman and H. A. Havel. Characterization of an associated equilibrium folding intermediate of bovine growth hormone. Biochemistry 25:6539–6543 (1986).
D. N. Brems. Solubility of different folding conformers of bovine growth hormone. Biochemistry 22:4541–4546 (1988).
A. P. Gast, C. K. Hall and W. B. Russel. Polymer-induced phase separation in nonaqueous colloidal suspensions. J. Coll. Interf. Sci. 96:251–267 (1983).
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Stevenson, C.L., Hageman, M.J. Estimation of Recombinant Bovine Somatotropin Solubility by Excluded-Volume Interaction with Polyethylene Glycols. Pharm Res 12, 1671–1676 (1995). https://doi.org/10.1023/A:1016201319136
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DOI: https://doi.org/10.1023/A:1016201319136