Takaaki Kurinomaru

Improved Complementary Polymer Pair System: Switching for Enzyme Activity by PEGylated Polymers

The development of technology for on/off switching of enzyme activity is expected to expand the applications of enzyme in a wide range of research fields. We have previously developed a complementary polymer pair system (CPPS) that enables the activity of several enzymes to be controlled by a pair of oppositely charged polymers. However, it failed to control the activity of large and unstable α-amylase because the aggregation of the complex between anionic α-amylase and cationic poly(allylamine) (PAA) induced irreversible denaturation of the enzyme. To address this issue, we herein designed and synthesized a cationic copolymer with a poly(ethylene glycol) backbone, poly(N,N-diethylaminoethyl methacrylate)-block-poly(ethylene glycol) (PEAMA-b-PEG). In contrast to PAA, α-amylase and β-galactosidase were inactivated by PEAMA-b-PEG with the formation of soluble complexes. The enzyme/PEAMA-b-PEG complexes were then successfully recovered from the complex by the addition of anionic poly(acrylic acid) (PAAc). Thus, dispersion of the complex by PEG segment in PEAMA-b-PEG clearly plays a crucial role for regulating the activities of these enzymes, suggesting that PEGylated charged polymer is a new candidate for CPPS for large and unstable enzymes.

Enzyme Hyperactivation System Based on a Complementary Charged Pair of Polyelectrolytes and Substrates

Artificial enzyme activators are of great interest for enzyme applications in a wide range of research fields. Here, we report an enzyme hyperactivation system using polyelectrolytes that are complementary to charged substrates. The enzyme activity of α-chymotrypsin (ChT) for a cationic substrate increased 7-fold at pH 7.0 in the presence of anionic poly(acrylic acid) (PAAc) and for an anionic substrate increased 18-fold at pH 7.0 in the presence of cationic poly(allylamine) (PAA). Analysis of salt and pH effects, enzyme kinetics, dynamic light scattering (DLS), and circular dichroism (CD) indicated that the enzyme activation results from favorable electrostatic interactions between oppositely charged substrates and polyelectrolytes surrounding the enzymes. This hyperactivation system does not require laborious mutagenesis or chemical modification of enzymes and thus is relevant to a number of applications.

Noncovalent PEGylation of l-Asparaginase Using PEGylated Polyelectrolyte

Noncovalent PEGylation has great potential for stabilization of therapeutic proteins. Here, we demonstrated that the noncovalent PEGylation with a PEGylated polyelectrolyte stabilized a therapeutic protein, l-asparaginase (ASNase). Anionic ASNase and cationic poly(ethylene glycol)-block-poly(N,N-dimethylaminoethyl methacrylate) (PEG-b-PAMA) formed a water-soluble protein-polyelectrolyte complex (PPC) without loss of secondary structure and enzyme activity. PPC with PEG-b-PAMA successfully inhibited the shaking-induced inactivation and aggregation of ASNase as well as protease digestion, corresponding to the behaviors of covalently PEGylated ASNase. Thus, noncovalent PEGylation by PEGylated polyelectrolytes is a new candidate for handling of therapeutic proteins.

Protein–Poly(amino acid) Complex Precipitation for High-Concentration Protein Formulation

A method for concentration of protein solutions is required for high-dosage protein formulation. Here, we present a precipitation-redissolution method by poly(amino acid) for proteins, including therapeutic enzymes, antibodies, and hormones. The proteins were fully precipitated by the addition of poly-L-lysine or poly-L-glutamic acid at low ionic strength, after which precipitate was dissolved at physiological ionic strength. The activities and secondary structures of redissolved proteins, especially antibodies, were almost identical to the native state. The precipitation-redissolution method is a simple and rapid technique for concentration of protein formulations.

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.

Bisulfite-free approaches for DNA methylation profiling

The determination of epigenetic modification, especially that of 5-methylcytosine in the CpG sequence in mammals, has attracted attention because it should prove valuable in a wide range of research fields including diagnosis, drug discovery and therapy. Various methods have been developed for recognizing 5-methylcytosine. Most of these methods employ bisulfite conversion, which mediates the deamination of unmethylated cytosine bases to uracil and leaves 5-methylcytosine. However, bisulfite conversion is time consuming with complex multiple operations and it leads to DNA degradation via its acid/base treatment. Therefore, bisulfite-free methods are required for DNA methylation analysis to allow epigenetic research to progress towards clinical applications. In this review, we introduce the recent development of bisulfite-free DNA methylation analysis, which we classify into two categories, namely labelling-based and labelling-free assays.

Stress Tolerance of Antibody-Poly(Amino Acid) Complexes for Improving the Stability of High Concentration Antibody Formulations.

The stabilization of antibodies in aqueous solution against physical stress remains a problematic issue for pharmaceutical applications. Recently, protein-polyelectrolyte complex (PPC) formation using poly(amino acids) was proposed to prepare antibody formulation in a salt-dissociable precipitated state without protein denaturation. Here, we investigated the stabilization effect of PPC of therapeutic antibodies with poly-l-glutamic acid on agitation and thermal stress as forms of mechanical and non-mechanical stress, respectively. The precipitated state of PPC prevented the inactivation and aggregation induced by agitation. Similar results were obtained using the suspension state of PPC, but the stabilizing effects were slightly inferior to those of the PPC precipitate. PPC precipitate and PPC suspension prevented heat-induced inactivation of the antibodies, but showed little effect on heat-induced aggregation. Thus, PPC is a new candidate as a simple storage method for antibodies in aqueous solution, as an alternative state for freeze-drying.

Feasibility of Antibody–Poly(Glutamic Acid) Complexes: Preparation of High-Concentration Antibody Formulations and Their Pharmaceutical Properties

Development of high-concentration antibody formulations for subcutaneous administration remains challenging. Recently, a precipitation-redissolution method was proposed to prepare suspensions or precipitates of salt-dissociable protein-poly(amino acid) complexes. To elucidate the utility of this method for protein therapy, we investigated the feasibility of a precipitation-redissolution method using poly(amino acid) for high-concentration antibody formulation. Omalizumab and adalimumab formulations of 150 mg/mL could be prepared using poly-l-glutamic acid (polyE) from low-concentration stock solutions. Enzyme-linked immunosorbent assay, circular dichroism, and size-exclusion chromatography revealed that the formation of antibody-polyE complex and precipitation-redissolution process did not significantly affect the immunoreactivity or secondary structure of the antibodies. The precipitation-redissolution method was less time-consuming and more effective than lyophilization-redissolution, evaporation-redissolution, and ultrafiltration from the viewpoint of final yield. Scalability was confirmed from 400 μL to 1.0 L. The general toxicity and pharmacokinetic profiles of the antibody-polyE complex formulations were similar to those of conventional antibody formulations. These results suggested that the precipitation-redissolution method using poly(amino acid) has great potential as a concentration method for antibody formulation and medicinal use.

Protein-poly(amino acid) precipitation stabilizes a therapeutic protein l-asparaginase against physicochemical stress.

Long-term storage in aqueous solution has been demanded for the practical application of therapeutic proteins. Recently, a precipitation-redissolution method was proposed to prepare salt-dissociable protein-polyelectrolyte complex (PPC). To elucidate the utility of the complex for storage of proteins, we investigated the stress tolerance of PPC precipitates containing l-asparaginase (ASNase) and poly-l-lysine (polyK). PPC precipitate containing ASNase and polyK was prepared by precipitation-redissolution method. The sample was treated to three types of stress, i.e., heat, shaking, and oxidation. The protein concentration, enzyme activity, and CD spectrum of the supernatants of samples were measured after stressed. PPC precipitate consisting of ASNase and polyK showed tolerance against thermal and shaking stress compared to the native solution. In addition, PPC precipitate protected ASNase from inactivation by oxidation. PPC precipitate of ASNase/polyK complex successfully stabilized ASNase against physicochemical stresses. These results suggest that the PPC precipitate has great potential as a storage method in aqueous solution for unstable proteins.

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-block-poly(N,N-dimethylaminoethyl) (PEG-b-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-b-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-b-PAMA molecule was initially located at six different placements of the x-, y-, and z-axis ±20 Å from the center of α-amylase, which showed that the PEG-b-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-b-PAMA was not inhibited by increasing the ionic strength, consistent with the MD simulation; i.e., PEG-b-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.