Viscosity of protein solution is one of the most troublesome issues for the high-concentration formulation of protein drugs. In this review, we summarize the practical methods that suppress the ...viscosity of protein solution using small molecular additives. The small amount of salts decreases the viscosity that results from electrostatic repulsion and attraction. The chaotrope suppresses the hydrophobic attraction and cluster formation, which can lower the solution viscosity. Arginine hydrochloride (ArgHCl) also suppresses the solution viscosity due to the hydrophobic and aromatic interactions between protein molecules. The small molecular additives are the simplest resolution of the high viscosity of protein solution as well as understanding of the primary cause in complex phenomena of protein interactions.
Ovotransferrin (OVT) is the main protein component of egg white responsible for initial gelation due to its high thermal susceptibility. Co-aggregation of lysozyme (LYZ) is involved in OVT aggregate ...formation at low temperatures during pasteurization. Undesirable formation of aggregates limits the degree of thermal processing that can be applied to egg white products. However, the characteristics of co-aggregates of OVT and LYZ have not been elucidated. Here, we determined the thermal co-aggregation process of OVT and LYZ in terms of protein composition, structure, intermolecular forces, and morphology. The amount of LYZ involved in co-aggregates was dependent on the amount of aggregated OVT regardless of the mixing ratio. Native LYZ had the capability of precipitating soluble OVT aggregates by non-covalent association. The co-aggregates of OVT and LYZ formed colloidal particles with a large network, which was not observed in systems consisting of either protein alone. The hierarchical co-aggregation of OVT and LYZ started from the aggregation of OVT involving LYZ non-covalently, which suppresses electrostatic repulsion between soluble OVT aggregates, followed by the growth of insoluble aggregates by disulfide bond crosslinkage between the soluble aggregates. These results provide information regarding efficient pasteurization and thermal processing of hen egg white.
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•Co-aggregation of OVT and LYZ was derived from the aggregation of OVT itself.•The aggregation rate of OVT increased in the presence of LYZ.•The amount of LYZ in co-aggregates was proportional to that of aggregated OVT.•Native LYZ precipitated soluble aggregates of OVT by non-covalent association.•The network structure of aggregates depended on the ratio of aggregated proteins.
Unacceptably high viscosity is observed in high protein concentration formulations due to extremely large therapeutic dose of antibodies and volume restriction of subcutaneous route of ...administration. Here, we show that a protein aggregation suppressor, arginine hydrochloride (ArgHCl), specifically decreases viscosity of antibody formulations. The viscosities of bovine gamma globulin (BGG) solution at 250 mg/mL and human gamma globulin (HGG) solution at 292 mg/mL at a physiological pH were too high for subcutaneous injections, but decreased to an acceptable level (below 50 cP) in the presence of 1,000 mM ArgHCl. ArgHCl also decreased the viscosity of BGG solution at acidic and alkaline pHs. Interestingly, ArgHCl decreased the viscosity of antibody solutions (BGG, HGG, and human immunoglobulin G) but not globular protein solutions (α-amylase and α-chymotrypsin). These results indicate not only high potency of ArgHCl as an excipient to decrease the solution viscosity of high concentration antibodies formulations but also specific interactions between ArgHCl and antibodies.
High-concentration and low-viscosity antibody formulations are necessary when administering these solutions subcutaneously (SC) due to limitations on injection volume. Here we show a method to ...decrease the viscosity of monoclonal antibody solution by protein–polyelectrolyte complex (PPC) with poly-l-glutamic acid (polyE). The viscosity of omalizumab solutions was 90 cP at the concentration of 150 mg/mL. In the presence of 20–50 mM polyE, the viscosity of PPC solution of 150 mg/mL omalizumab dramatically decreased below 10 cP due to the formation of crowded solution. The crowded state of PPC, named aggregated PPC (A-PPC), contained water droplets with a diameter of 10 μm or larger with low antibody concentrations. In the presence of 60 mM or more polyE, the omalizumab solution was transparent with the viscosity of 40 cP or less, named soluble PPC (S-PPC). More importantly, the solutions of both A-PPC and S-PPC were fully redissolved by the addition of phosphate saline buffer confirmed by secondary structure, the amount of aggregates, and binding activity to antigen.
Liquid–liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex ...folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that, in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π–π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with a high charge density. Given that DNA quadruplex structures are well-documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.
Plasma medicine is an attractive new research area, but fundamental information related to plasma modification of biomacromolecules in aqueous solution remains elusive. As described herein, we ...investigated the chemical effects of low‐temperature atmospheric pressure plasma on protein in aqueous solution using lysozyme as a model. Plasma treatment decreased enzymatic activity and changed the secondary structure that results from the increased molecular weight of lysozyme with chemical modification. These effects arise neither from UV light nor from plasma heat, suggesting that the reactive species generated by the plasma affect lysozyme. The information presented in this paper represents a crucial first step for elucidating chemical reactions induced by plasma on proteins for biomedical applications.
Low‐temperature atmospheric pressure plasma inactivates lysozyme in aqueous solutions. Decreased enzymatic activity is related to the change of structure and increased molecular weight of lysozyme.
Aromatic interaction plays a crucial role in controlling protein interaction by additives. Here we investigated the interaction of protein salting-in (solubilizing) additives with tryptophan (Trp), ...tyrosine (Tyr), indole, and proteins based on their fluorescence spectra. Five salting-in additives, i.e., arginine (Arg), urea, guanidine (Gdn), ethylene glycol (EG), and magnesium chloride (MgCl2), showed different effects on the fluorescence properties of Trp and Tyr. Arg significantly reduced fluorescence intensity of Trp and Tyr, as was the case for glycine to a lesser extent. MgCl2 and calcium chloride (CaCl2) showed little effect on the aromatic fluorescence spectra. Gdn also showed little effect on the aromatic fluorescence spectra of Trp and Tyr even at high concentrations. EG increased the aromatic fluorescence intensity of Trp and Tyr with blue-shifted emission wavelength. Urea enhanced fluorescence of Trp and Tyr without altering emission wavelength. These results indicate that the protein solubilizing additives interact with aromatic groups differently.
•Arg and Gly quenched the fluorescence of Trp, Tyr, and indole.•MgCl2, CaCl2, and Gdn showed little effect on the aromatic fluorescence.•Urea enhanced fluorescence without altering emission wavelength.•Ethylene glycol increased the aromatic fluorescence intensities with blue-shifted emission wavelength.•Protein solubilizing additives interact differently with the aromatic groups.
Therapeutic antibodies for subcutaneous (SC) injection must be formulated at high concentrations because of the large therapeutic dose and volume restriction. However, concentrated antibody solutions ...have undesirably high viscosity, which hampers SC injection. In this study, we demonstrated that hydantoin and its derivatives suppressed the viscosity of concentrated antibody and bovine serum albumin solutions. Hydantoin derivatives, in particular 1-methylhydantoin, appeared more effective. Both hydantoin and 1-methylhydantoin suppressed the viscosity of proteins more effectively when combined with a physiological concentration of NaCl. Moreover, hydantoin rings exhibited thermodynamically favorable interactions with hydrophobic amino acids, as demonstrated using solubility measurements. Molecular dynamics simulations indicated planar stacking interaction or T-shaped interaction between the hydantoin ring structure and the aromatic rings of tryptophan. Thus, the effects of hydantoin compounds in the presence of NaCl on the high viscosity of concentrated protein solutions result from the combined effects between hydantoin and NaCl in suppressing multiple interactions (electrostatic, hydrophobic, π–π, and cation−π interactions) between protein molecules. The obtained data here should be useful for developing therapeutic antibody formulations.
Effect of additives on protein aggregation Hamada, Hiroyuki; Arakawa, Tsutomu; Shiraki, Kentaro
Current pharmaceutical biotechnology,
06/2009, Letnik:
10, Številka:
4
Journal Article
Recenzirano
This paper overviews solution additives that affect protein stability and aggregation during refolding, heating, and freezing processes. Solution additives are mainly grouped into two classes, i.e., ...protein denaturants and stabilizers. The former includes guanidine, urea, strong ionic detergents, and certain chaotropic salts; the latter includes certain amino acids, sugars, polyhydric alcohols, osmolytes, and kosmotropic salts. However, there are solution additives that are not unambiguously placed into these two classes, including arginine, certain divalent cation salts (e.g., MgCl(2)) and certain polyhydric alcohols (e.g., ethylene glycol). Certain non-ionic or non-detergent surfactants, ionic liquids, amino acid derivatives, polyamines, and certain amphiphilic polymers may belong to this class. They have marginal effects on protein structure and stability, but are able to disrupt protein interactions. Information on additives that do not catalyze chemical reactions nor affect protein functions helps us to design protein solutions for increased stability or reduced aggregation.
Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that ...insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.
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