Aggregative protein–polyelectrolyte complex for high-concentration formulation of protein drugs

Aggregative protein–polyelectrolyte complex (PPC) has been proposed as a concentrated state of protein with a great potential for biopharmaceutical application. In this review article, we introduce a unique concentration method of protein formulation using PPC for a dozen types of pharmaceutical antibodies, hormones, and enzymes. Aggregative PPC can be obtained only by mixing poly(amino acid)s with proteins under low salt concentration conditions at an ambient temperature. The aggregative PPC is in a stabilized state against shaking, heating, and oxidation. More importantly, the aggregative PPC can be fully redissolved by the addition of physiological saline without denaturation and activity loss for many proteins. In addition, the general toxicity and pharmacokinetic profiles of the aggregative PPC are identical to those of the control antibody formulation. Thus, the protein formulation produced by aggregative PPC would be applicable for biomedical use as a kind of concentrated-state protein

Noncovalent PEGylation through Protein–Polyelectrolyte Interaction: Kinetic Experiment and Molecular Dynamics Simulation

Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein handling technique to control protein function and stability. A diblock copolymer containing PEG and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent attachment of PEG to a protein surface because of the binding through multiple electrostatic interactions without protein denaturation. To obtain a deeper understanding of protein–polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic PEGylated polyelectrolyte binds to anionic α-amylase in enzyme kinetic experiments and molecular dynamics (MD) simulations. Cationic PEG-<i>block</i>-poly­(<i>N</i>,<i>N</i>-dimethylaminoethyl) (PEG-<i>b</i>-PAMA) inhibited the enzyme activity of anionic α-amylase due to binding of PAMA chains. Enzyme kinetics revealed that the inhibition of α-amylase activity by PEG-<i>b</i>-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-<i>b</i>-PAMA molecule was initially located at six different placements of the <i>x</i>-, <i>y</i>-, and <i>z</i>-axis ±20 Å from the center of α-amylase, which showed that the PEG-<i>b</i>-PAMA nonspecifically bound to the α-amylase surface, corresponding to the noncompetitive inhibition manner that stems from the polymer binding to an enzyme surface other than the active site. In addition, the enzyme activity of α-amylase in the presence of PEG-<i>b</i>-PAMA was not inhibited by increasing the ionic strength, consistent with the MD simulation; i.e., PEG-<i>b</i>-PAMA did not interact with α-amylase in high ionic strength conditions. The results reported in this paper suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random electrostatic interaction between protein and polyelectrolyte