Interleukin-6 (IL-6) is an aggregation-prone cytokine whose recombinant production from Escherichia coli inclusion bodies remains challenging because of inefficient refolding. Here, we present a limited batch-mode process-analytical study comparing two practical buffer-exchange operations, “fast” and “slow”, during batch dilution refolding of IL-6. Using SDS-PAGE, size-exclusion chromatography (SEC), dynamic light scattering (DLS), and far-UV circular dichroism (CD) spectroscopy, we examined dimerization and aggregation under acidic and guanidine denaturation workflows. The data suggest that IL-6 forms predominantly non-covalent dimers and that batch buffer-exchange operation and timescale are associated with aggregation outcome. Because the acidic and guanidine workflows differed in starting solvent composition, residual denaturant, and final protein concentration, the results should be interpreted as comparisons of complete workflow conditions rather than isolated denaturant effects. Overall, the combined use of SEC, DLS, and far-UV CD provides a practical framework for monitoring IL-6 refolding outcomes, while mechanistic assignments remain beyond the scope of this study.

Nonspecific adsorption of proteins onto solid surfaces is a fundamental phenomenon in interface science, influencing the physicochemical properties and functional performance of materials in bioanalytical applications. In this study, we developed a common characterization technique for nonspecific reaction factors to provide a foundation for the rational control of specific protein interactions and the development of novel blocking agents, enabling tailored surface engineering strategies. Proteins adsorbed onto insoluble carriers, using magnetic particles as the model system, were explored from approximately 24,000 human proteins and subsequently characterized in silico. Statistical analysis revealed common features of highly adsorptive proteins, including distinct clustering of specific amino acid residues, such as arginine (Arg) and alanine. Adsorption was governed by structural flexibility and charge distribution, particularly in denatured regions. These findings support the hypothesis that protein charge blockiness, resulting from Arg residues in highly mobile denatured regions, affects adsorption. Comprehensive analyses, combining protein array-based interaction profiling and in silico characterization, provided new insights into the molecular mechanisms underlying protein-surface interactions. This methodology facilitates the rational design of advanced blocking agents and surface modifications to control protein adsorption and is applicable to a wide range of materials beyond magnetic particles. Overall, this work advances the understanding of nonspecific adsorption at biomaterial interfaces and contributes to the development of tailored surface engineering strategies in interface science.

Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.

We review here several niche downstream purification processes that are not covered by other articles in this special issue. The first is the use of activated carbon to capture contaminants and clarify culture medium for purification of proteins, including antibodies. Flow-through operation of the activated carbon filter showed over 80 % recovery of antibodies with 10--fold reduction of host cell proteins and effective lipopolysaccharide and virus removal. The second is salt or arginine-tolerant column chromatography, in which the respective resins, such as hydroxyapatite and mixed-mode resins, can bind proteins in the presence of salt or arginine at high concentrations. Effective use of arginine was also suggested in size exclusion and affinity chromatography. The last is the isolation of proteins using gel electrophoresis and simple extraction procedure. These technologies offer simple and cost-effective methods for purifying proteins and protein complexes in the native state.

Current research investigating chimeric antigen receptor (CAR)-T cell therapeutics indicates that CAR properties other than intracellular signaling systems, such as high association rate constants and enhanced self-interaction of the antigen-binding domain (ABD), elicit enhanced therapeutic efficacy and are key factors for CAR-T candidates. However, rational strategies to achieve these features are to be established. To develop novel CAR-T cells with these characteristics, ABD was engineered by introducing several arginine residues into the light-chain framework region-3 of the single-chain fragment variable. The mutant CAR-T cells exhibited a higher cell-killing efficacy than that of wild-type cells and superior antitumor effects in mice, prolonged persistence in vivo, and decreased interferon-γ secretion. RNA sequencing revealed differential gene expression profiles between the mutant and wild-type CAR-T cells before and after antigen stimulation. In conclusion, we proposed a design strategy to generate CAR-T cells with high therapeutic efficacy by modulating CAR properties using an arginine cluster.

Sodium dodecyl sulfate (SDS) is one of the most widely used detergents. Here, we discuss current knowledge regarding applications of SDS and its modes of interaction with proteins, particularly at low concentrations. SDS at 1-2 %, which is well above the critical micelle concentration, is commonly used to extract fully denatured and dissociated proteins and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in various applications, especially proteomics. In contrast, low concentration SDS may have been relatively underutilized. Here, we demonstrate the use of 0.1 % SDS for decellularization and protein fractionation. Why is 0.1 % SDS unique? The interaction between SDS and proteins is complex and depends on both the conditions and the proteins involved. At 0.1 %, the effects of SDS appear to be intermediate between negligible and extensive binding, highlighting its potential for novel applications. Two milder anionic detergents, Sarkosyl and sodium N-lauroyglutamate, whose effects are similar in certain applications to those of low concentration SDS, were briefly discussed.

Protein refolding is vital for protein production; howev-er, a scalable method has not yet been developed. We introduce an innovative refolding approach using a flow microreactor (FMR) that allows precise control of buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed gram-scale production. This FMR system allows flash changes in reaction conditions, ef-fectively circumventing protein aggregation during re-folding. To the best of our knowledge, this is the first study to use an FMR for protein refolding, which offers a more efficient and scalable method for protein produc-tion . The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The novelty of this approach is derived from the unique ability of the FMR to control both spatial and temporal aspects of protein refolding.

Interleukin-6 (IL-6) is a multifunctional cytokine involved in essential biological processes, including cell proliferation, differentiation, and maturation, making it a key target in therapeutic development, particularly for COVID-19 treatments. Given its complex biological properties, a thorough understanding of IL-6’s aggregation behavior is critical to optimize the production and stability of anti-IL-6 therapeutic candidates. Recombinant human IL-6 (hIL-6) is typically expressed in Escherichia coli, resulting in significant formation of inclusion bodies, which presents challenges in solubility and refolding. Furthermore, IL-6 exhibits a strong propensity for aggregation, reducing the overall yield of biologically active protein after refolding. This study aimed to elucidate the underlying mechanisms of IL-6 aggregation and to identify refolding conditions that mitigate this phenomenon. Using a combination of SDS-PAGE, size-exclusion chromatography (SEC), Dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy, we analyzed the dimerization and aggregation behavior of IL-6 under varying denaturation and refolding conditions. Our results suggest that IL-6 dimerizes primarily through non-covalent interactions, with its secondary structure largely preserved in the dimeric state. Comparative refolding studies under acidic and guanidine-induced denaturation conditions revealed that the rate of solvent exchange is a critical factor influencing aggregation propensity. These findings provide mechanistic insights into IL-6 aggregation during refolding and highlight strategic parameters for reducing aggregation, thereby enhancing the production yield of refolded IL-6. This work establishes a foundation for refining refolding protocols for IL-6 and potentially other aggregation-prone proteins used in therapeutic applications.

Recombinant protein production is an essential aspect of biopharmaceutical manufacturing, with Escherichia coli serving as a primary host organism. Protein refolding is vital for protein production; however, conventional refolding methods face challenges such as scale-up limitations and difficulties in controlling protein conformational changes on a millisecond scale. In this study, we demonstrate the novel application of flow microreactors (FMR) in controlling protein conformational changes on a millisecond scale, enabling efficient refolding processes and opening up new avenues in the science of FMR technology. FMR technology has been primarily employed for small-molecule synthesis, but our novel approach successfully expands its application to protein refolding, offering precise control of the buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed for gram-scale production. This FMR system allows flash changes in the reaction conditions, effectively circumventing protein aggregation during refolding. To the best of our knowledge, this is the first study to use FMR for protein refolding, which offers a more efficient and scalable method for protein production. The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The FMR technique offers a promising approach for enhancing protein refolding efficiency and has demonstrated its potential in streamlining the process from laboratory-scale research to industrial-scale production, making it a game-changing technology in the field.

Gel electrophoresis, a transport technology, is one of the most widely used experimental methods in biochemical and pharmaceutical research and development. Transport technologies are used to determine hydrodynamic or electrophoretic properties of macromolecules. Gel electrophoresis is a zone technology, where a small volume of sample is applied to a large separation gel matrix. In contrast, a seldom-used electrophoresis technology is moving boundary electrophoresis, where the sample is present throughout the separation phase or gel matrix. While the zone method gives peaks of separating macromolecular solutes, the moving boundary method gives a boundary between solute-free and solute-containing phases. We will review electrophoresis as a transport technology of zone and moving boundary methods and describe its principles and applications.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro refolding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. Non-natural L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin-biotin system, even for in vivo use.

The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro folding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin-biotin system, even for in vivo use.

We have here observed that the differential scanning calorimetry profiles and melting temperatures of a humanized antibody were unchanged over a 10-year span when stored at 4°C and at different pH values, even at pH 2.7. This is somewhat surprising, as this particular antibody undergoes conformational changes below pH 4.0. Differential scanning calorimetry analysis showed that melting of the antibody at pH 2.7 was highly reversible, suggesting a possibility that the observed reversibility is at least in part responsible for a 10-year stability at low pH. Conversely, it showed thermal unfolding followed by aggregation at higher pH.

We have tested here whether or not arginine, a well-known aggregation suppressor, is effective in removing bacterial cells, which may present a potential risk of accidental pneumonia infection in aged individuals, from the oral mucosal membranes. This is based on the ability of arginine to suppress protein-protein interaction and surface adsorption and increase the solubility of organic compounds. Twelve student volunteers were subjected to mouthwashes with saline, citrate buffer (pH 3.5), arginine (pH 3.5) and a commercial Listerine. Insignificant effects were observed with saline and citrate buffer, whereas arginine and Listerine mouthwashes led to significant reduction of bacterial cells from the dorsal side of the volunteer's tongue. Arginine also appeared to disrupt biofilms present in the mouth.

Local transient high protein concentration or high density condition can occur during processing of protein solutions. Typical examples are saturated binding of proteins during column chromatography and high protein concentration on the semi-permeable membrane during ultrafiltration. Both column chromatography and ultrafiltration are fundamental technologies, specially for production of pharmaceutical proteins. We summarize here our experiences related to such high concentration conditions. (C) 2016 Elsevier B.V. All rights reserved.

Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0 M NaCl at 25 °C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution.