Effect of additives on liquid droplet of protein–polyelectrolyte complex for high-concentration formulations

Abstract

Liquid droplets of protein–polyelectrolyte complexes (PPCs) have been developed as a new candidate for stabilization and concentration of protein drugs. However, it remains unclear whether additives affect the precipitation and redissolution yields of PPCs. In the present study, we investigated the PPC formation of human immunoglobulin G (IgG) and poly-L-glutamic acid (polyE) in the presence of various additives that have diverse effects, such as protein stabilization. Alcohols, including ethanol, successfully increased the PPC precipitation yield to over 90%, and the PPCs formed were completely redissolved at physiological ionic strength. However, poly(ethylene glycol), sugars, and amino acids did not improve the precipitation and redissolution yields of PPCs over those observed when no additives were included. Circular dichroism spectrometry showed that the secondary structure of polyE as well as electrostatic interactions play important roles in increasing the PPC precipitation yield when ethanol is used as an additive. The maximum concentration of IgG reached 100 mg/ml with the use of ethanol, which was 15% higher efficiency of the protein yield after precipitation and redissolution than that in the absence of additives. Thus, the addition of a small amount of ethanol is effective for the concentration and stabilization of precipitated PPCs containing IgG formulations.

Effect of additives on liquid droplets and aggregates of proteins

“…Sugars stabilize the tertiary structure of proteins to increase the interfacial energy and promote the formation of amorphous aggregates. For example, glucose and trehalose dissolve the liquid droplets of immunoglobulin G and polyglutamic acid (Mimura et al 2019). This may be because the addition of the sugars differs the hydration of poly (amino acids), rather than their tertiary structure, resulting in the dissolution of the liquid droplet.…”

“…Solutions with low dielectric constants enhance the electrostatic interactions and hydrogen bonds between the protein molecules; hence, alcohols tend to form α-helix-rich protein structures (Shiraki et al 1995). For example, liquid droplets of immunoglobulin G and polyglutamic acid are stabilized in an ethanol solution (Mimura et al 2019). In contrast, low concentration of 1,6-hexanediol dissolves liquid droplets because alcohol weakens the weak hydrophobic interactions between protein molecules (Lin et al 2016).…”

“…Neutral macromolecules, typically polyethylene glycol and ficoll, stabilize the tertiary structure of proteins via the crowding effect (Kuznetsova et al 2015). The crowding effect prevents the formation of liquid droplets to make the protein structure more compact; however, it stabilizes the droplets once formed (Mimura et al 2019). Crowders accelerate the formation of amyloids (Munishkina et al 2004).…”

“…Proteins comprise 20 naturally occurring amino acids, which have the property to form assemblies, typically liquid droplets and amorphous aggregates in vitro (Iwashita et al 2018b). For example, when an antibody is mixed with a poly (amino acid) at a neutral pH, the protein solution becomes cloudy (Mimura et al 2019). This cloudiness disappears with the addition of hundreds of mM of salt.…”

Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

“…Figure 1E), similarly to our recent studies of cationic protein/anionic polymer pairs. [25,26] Time-lapse images showed rapid, sub-millisecond fusion of the assemblies ( Figure 1F). This behavior indicates that these assemblies are not gel-like aggregates, but instead liquid-like droplets with highly fluid properties, as have been observed for other phase-separating proteins.…”