![]() L –1 osmolytes or >CMC for detergents such as Tween).These additives must be added in amounts largely exceeding the 1:1 w/w protein/additive ratio, (e.g., 1 mol Currently, most proteins are formulated in solutions containing osmolytes, such as polyols (6) or arginine, (7) added in molar amounts and neutral detergents at micellar concentration. Noncovalent binding of proteins to synthetic molecules is another approach explored to enhance the stability of proteins. (3-5) These beneficial factors are mitigated by the commonly observed decrease in biological activity associated with protein PEGylation. (3, 4) Covalent attachment of water-soluble macromolecules, such as poly(ethylene glycol) (PEG), shields the proteins from their environment, which can improve the proteins stability in vivo, decrease their immunogenicity, and prolong their plasma half-life. (1, 2) Several approaches to enhance the stability of therapeutic proteins are actively pursued, either by tailoring the sequence of antibodies (2) or by chemical modifications. Together with the excitement generated by the efficacy of therapeutic proteins come concerns related to their biological half-life, insufficient stability, and aggregation. Their medical use and commercialization are predicted to increase rapidly over the next few years. Therapeutic proteins, including antibodies, enzymes, or growth factors, play an increasingly important role in the treatment of viral infections, autoimmune disorders, cancers, and several other human diseases. They do not modify IgG permanently, which is an asset for applications in therapeutic protein formulations since the in vivo efficacy of the protein should not be affected. This study leads the way toward the design of new formulations of therapeutic proteins using noncovalent 1:1 polymer/protein association that are transient and require a markedly lower additive concentration compared to conventional osmolyte protecting agents. Amphiphilic PAA derivatives (1:1 w/w IgG/polymer) are able to prevent thermal aggregation (preserving IgG monomers) or retard aggregation of IgG (formation of oligomers and slow growth), revealing the importance of both hydrophobic interactions and modulation of the Coulomb interactions with or without NaCl present. These interactions are screened in 0.1 M NaCl and, consequently, PAAs lose their protective effect. The complexes exhibit a remarkable protective effect against IgG aggregation and maintain low aggregation numbers (average degree of oligomerization <12 at a temperature up to 85 ☌). In salt-free solutions, the hydrophilic PAA chains form complexes with IgG upon thermal unfolding of the protein (1:1 w/w IgG/PAA), but they do not interact with native IgG. ![]() ![]() The IgG/polyanion interactions were monitored by static and dynamic light scattering, fluorescence correlation spectroscopy, capillary zone electrophoresis, and high sensitivity differential scanning calorimetry. They were derived from two poly(acrylic acid) parent chains of M w 5000 g The polyanions selected were sodium poly(acrylates), random copolymers of sodium acrylate and N- n-octadecylacrylamide (3 mol %), and a random copolymer of sodium acrylate, N- n-octylacrylamide (25 mol %), and N-isopropylacrylamide (40 mol %). Using a polyclonal immunoglobin G (IgG) as a model system for antibodies, we have studied the mechanisms by which this multidomain protein interacts with polyanions when incubated at physiological pH and at temperatures below and above the protein unfolding/denaturation temperature, in salt-free solutions and in 0.1 M NaCl solutions. Prevention of thermal aggregation of antibodies in aqueous solutions was achieved by noncovalent association with hydrophobically modified poly(acrylate) copolymers.
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