Molecular Origins of Surfactant Stabilization of a Human Recombinant Factor VIII

Molecular Origins of Surfactant Stabilization of a Human Recombinant Factor VIII

Recombinant Factor VIII (rFVIII) is a genetically engineered protein therapeutic used to treat Hemophilia A, a genetic blood disorder found in 1 out of every 5,000 males in the United States. rFVIII is the largest molecule ever successfully cloned by genetic engineering techniques and is the largest and most complex protein currently manufactured. The rFVIII molecule is sensitive to both chemical and physical degradation. Surface adsorption of rFVIII is rapid, and about 50% of rFVIII product may be lost due to adsorption during sterile filtration alone. Like many other biopharmaceuticals it is stabilized downstream by the surfactant polysorbate 80 (PS 80) to protect against activity loss caused by aggregation and adsorption. In its final formulation buffer, the protein loses activity within a few hours and therefore must be packaged in a lyophilized form. This inherent instability makes rFVIII one of the most expensive drugs in the world.

Surfactants are able to stabilize proteins through two different mechanisms (Fig. 1). One mechanism involves the preferential location of surfactant at nearby interfaces, creating a steric barrier that prevents protein adsorption. The other mechanism involves association of protein and surfactant molecules into complexes that prevent proteins from interacting with surfaces as well as with each other. In general, both mechanisms must be at play for effective protein stabilization against activity loss, but selection of surfactants for protein stabilization is not currently made using any quantitative, predictive information to ensure that this requirement is met.

Figure 1. Surfactants can stabilize proteins by (a) preferentially accumulating at an interface to prevent adsorption or (b) creating a surfactant-protein complex to inhibit aggregation [1].

PS 80 is effective at preventing rFVIII activity loss (denaturation) due to adsorption at hydrophobic surfaces by preferentially adsorbing to the surface and creating of a steric barrier between the protein and the surface. However, PS 80 has been shown to provide little protection against rFVIII aggregation in solution [2] and little protection against rFVIII adsorption at surfaces for which it has low affinity, e.g., hydrophilic surfaces [3]. On the other hand, we have recorded air-water tensiometry results suggesting that the relatively large, triblock polymer surfactant Pluronic^® F68, used primarily upstream for its protective effect on mammalian cells, is less effective at blocking protein adsorption to surfaces than it is at entering into stable protein-F68 associations that strongly resist aggregation and activity loss.

An interesting characteristic observation of surface tension depression by surfactant-protein mixtures is that the kinetics of surface tension depression (by surfactant) in such mixtures tends to be uniformly greater than that recorded for a surfactant alone at the same concentration. We hypothesize that parameters that determine whether surfactant stabilization occurs mainly by association with the interface or by association with protein ought to be quantifiable for different surfactants by analysis of such “accelerated adsorption” data. We are testing this through comparison of surface tension depression by F68 and PS 80, in the presence and absence of rFVIII. We have seen that accelerated surfactant adsorption in the presence of rFVIII will occur with surfactants that stabilize protein mainly by adsorption (e.g., PS 80); we expect the absence of accelerated surfactant adsorption will occur with surfactants that form stable surfactant-protein complexes. In particular, formation of a “structural intermediate” between surfactant aggregates and adsorbed surfactant monomers will either facilitate aggregate destabilization by increasing the concentration of surfactant monomers and accelerating adsorption (Fig. 2), or by producing stable surfactant-protein complexes and having less effect on adsorption rate.

Figure 2: Hypothetical mechanism for transport of surfactant from an aggregated state to the surface in the case of protein-mediated acceleration of surfactant adsorption. In the absence of protein (top), surfactant unimers must dissociate and migrate through the liquid to the interface. In the presence of protein (bottom), formation of a structural intermediate between surfactant aggregates and adsorbed unimers reduces the thermodynamic barriers associated with their location at the interface [1].

Information gained on the rFVIII stabilization mechanism adopted by each surfactant is being validated using optical waveguide lightmode spectroscopy (OWLS). In these experiments surfactant and surfactant-protein solutions are brought into contact with hydrophilic, silica-coated surfaces for which protein adsorption affinity is high while surfactant affinity is low. While we expect OWLS will show high rFVIII adsorption in the presence of PS 80, if rFVIII stabilization by F68 occurs through formation of F68-rFVIII complexes, OWLS will show low rFVIII adsorption in the presence of Pluronic^® F68, even though surface affinity of F68 is low.

This research will provide direction for surfactant selection and application to address issues surrounding aggregation and adsorption loss in practically relevant circumstances. Eventually, for a given surfactant we believe its “favored” mechanism (i.e., surfactant adsorption vs. formation of protein-surfactant associations), if any, will be predictable based on its structure and colloidal properties in solution.

References

1.       Lee, H.J., McAuley, A., Schilke, K.F., McGuire, J. Molecular origins of surfactant-mediated stabilization of protein drugs. Adv. Drug Deliv. Rev. 63:1160-1171. 2011.

2.       Joshi, O., Chu, L., McGuire J., Wang, D.Q. Adsorption and function of recombinant Factor VIII at the air-water interface in the presence of Tween 80. J. Pharm. Sci. 98:3099-3107. 2009.

3.       Joshi, O., McGuire, J., Wang, D.Q. Adsorption and function of recombinant Factor VIII at solid-water interfaces in the presence of Tween 80. J. Pharm. Sci. 97:4741-4755. 2008.