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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.

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.

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.

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.

Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Myosin is an important protein resource for food industries and has a bipolar filamentous structure that is composed of subfilaments that occur in vivo. It has been shown that a high ionic strength is required to prevent myosin from forming filamentous structures and to solubilize the protein in aqueous solution. In the presence of 100-200 mM NaCl, 50 mM arginine was more effective than other additives tested, including NaCl, in myosin solubilization. Before reaching equilibrium solubility, the myosin solution was initially supersaturated upon the dilution of a stock myosin solution in 1 M NaCl into the test solvents. Arginine slowed the process of equilibration and stabilized the supersaturated solution more effectively than other additives. No structural changes in myosin caused by arginine were observed, which indicated that arginine enhanced the solubility of myosin in a physiological salt solution without affecting the structure.

More than 50 detergents, including acylated amino acid derivatives, were screened for their ability to solubilize and refold recombinant proteins expressed as inclusion bodies. Two model proteins, human interleukin-6 and microbial transglutaminase, were solubilized by these detergents and the solubilized proteins were rapidly diluted for testing their solubilization and refolding effectiveness. Long chain-acylated amino acid derivatives having dicarboxylic acid moieties were found to be superior to others under the conditions tested. In particular, lauroyl-l-glutamate (C12-l-Glu) showed the highest recovery of the native proteins. The effectiveness of dilution refolding was greatly improved by adding aggregation suppressive arginine into the refolding solvents. To gain understanding how this detergent works, interactions between detergents and proteins were examined using spectroscopic and native gel electrophoretic analyses, showing ideal properties for C12-l-Glu as a solubilzing agent, i.e. highly reversible nature of the detergent binding to the model globular proteins and of the conformational changes. These properties most likely have contributed to the effective protein solubilzation and refolding of inclusion bodies using C12-l-Glu and arginine.

Binding, washing and elution conditions for MEP HyperCel chromatography were examined using two conditioned media (CM) containing monoclonal antibodies (humanized IgG1) and bovine serum albumin (BSA). Monoclonal antibodies derived from mammalian expression system bound to the column without pretreatment, although a majority of contaminating proteins present in the CM also showed binding. Inorganic salts, ethanol and glycerol were ineffective in eluting proteins under the conditions examined, suggesting that electrostatic or hydrophobic interactions are not a major factor for antibody binding to the MEP resin. Ethylene glycol, 2-propanol, urea and arginine were effective, to varying degrees, in elution of the bound proteins. The bound contaminating proteins and BSA were effectively eluted with ethylene glycol and the bound antibodies were finally eluted with aqueous arginine solutions at neutral pH. MEP showed selectivity toward BSA and hence utility for removing BSA from the samples. Interestingly, Fc-fusion proteins derived from silkworm larvae showed no detectable binding. Serum proteins present in silkworm larvae strongly competed with the Fc-fusion proteins and monoclonal antibody for binding to MEP resin, while the same Fc-fusion proteins can be readily purified in one-step by Protein-A resin, again confirming weak selectivity of the MEP resin.

Mildly acidic arginine solution is highly effective in elution of bound proteins from Protein-A columns. Although Protein-A is specific in antibody capture, it does bind other proteins, which must then be removed before elution by aqueous arginine solution. If they are not removed, a strong elution property of aqueous arginine solutions will elute the contaminating proteins along with antibodies. Here we have examined various salt solutions as a column rinse solvent. We screened various solvents for their effects on binding of purified antibodies to Protein-A, instead of their effectiveness to elute the bound contaminants. Those solvents that result in a slight flow-through of the antibodies during loading should be effective in eluting non-specifically bound proteins that have weaker affinity for Protein-A than antibodies: namely, if a particular solvent reduces antibody binding to Protein-A, it is expected to be effective in reducing binding of contaminants and hence eluting them. Such screening showed a few compounds, including arginine and sodium acetate, as potential column rinse agents. A combination of arginine and sodium acetate was tested for a few crude materials containing antibodies.

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein-protein or protein-ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5-1M arginine at pH 3.5-4.4, as reported in this paper.

Virus infection is one of the major threats to human health and can be avoided by minimizing exposure to infectious viruses. Viral clearance of pharmaceutical products and sanitization of skin and mucosal surfaces would reduce such exposures. Even with such care, virus infection does occur, requiring effective treatments by antiviral or virucidal agents. Natural products, in particular ingredients of foods and drinks we normally consume or metabolites present in human body at low concentrations, would have advantage over synthetic drugs as antiviral agents for safety concerns. For this reason, we have been studying natural products for their effects on virus inactivation and growth. Such natural products, which we have been focusing, include gallate derivatives, caffeine present in coffee, caffeic acid present in coffee and various fruits, ascorbic and dehydroascorbic acids and a cell metabolite, arginine. Here we will review our work on antiviral and virucidal activities of these compounds and the mechanism of their antiviral and virucidal effects.

MEP HyperCel resin, one of the Protein-A mimetic columns, is designed to bind antibodies at physiological pH and elutes the bound antibodies at mildly acidic pH. We have tested aqueous arginine solution for washing and elution of the resin. To our surprise, bound antibody and Fc-fusion protein eluted at pH 7.0 using 1M arginine solution. Various solvent additives were then examined at pH 7.0. Among the tested additives, urea and arginine were the only additives that were effective in elution. Thus, urea and arginine at low concentrations were effectively used for washing the resin. NaCl and MgCl(2) at 0.1-1M and ethanol at 5-20% were not effective. Based on these observations, it appears that protein binds to MEP resin through both polar and hydrophobic interactions with some contribution of electrostatic interaction, which can be simultaneously reduced by arginine or urea. On the other hand, Mabsorbent, another Protein-A mimetic column, appears to be more non-specific and non-selective.

Virus inactivation is a critical step in the manufacturing of recombinant therapeutic proteins, in particular antibodies, using mammalian expression systems. We have shown in the previous paper that arginine is effective in inactivation of herpes simplex virus type 1 (HSV-1) and influenza virus at low temperature under mildly acidic pH, i.e., above pH 4.0; above this pH, conformational changes of most antibodies are negligible. We have here extended virus inactivation study of arginine to other enveloped viruses, such as Sendai virus and Newcastle Disease Virus (NDV), and observed that arginine was ineffective against both viruses under the similar conditions, i.e., on ice and above pH 4.0. However, an arginine derivative, butyroyl-arginine, showed a strong virucidal potency against Sendai virus, leading to a 4 log reduction in virus yield at pH 4.0, but not against NDV. In addition, although arginine and butyroyl-arginine were equally effective against influenza virus having a cleaved form of hemagglutinin spike proteins, only butyroyl-arginine was significantly effective against the same virus, but having an uncleaved hemagglutinin spike proteins. Furthermore, butyroyl-arginine was more effective than arginine against HSV-1 at pH 4.5; i.e., it has a broader pH spectrum than does arginine.

A majority of column chromatographies use only selected salts, e.g., ammonium sulfate, NaCl, Citrate and phosphate in hydrophobic interaction chromatography (HIC) and NaCl in ion exchange and dye affinity chromatographies. Alternatively, a pH range below or above the neutral value is often used to reduce affinity interactions, e.g., in Protein-A or dye affinity column chromatography. Although these parameters are easily manipulated, they are not necessarily the optimal conditions for high recovery and resolution of the proteins. So-called co-solvents have been used, although to a limited extent, to manipulate performance of column chromatography. Here the term co-solvent is used to indicate its relatively high concentrations required for these applications, meaning that it also serves as solvent along with water. Ethylene glycol and MgCl(2) have been used to elute specific antibodies from antigen-affinity column. Arginine has also been used for the same purpose. Arginine has much wider applications for various column chromatographies, including size exclusion chromatography (SEC), HIC and affinity chromatography. Polyethylene glycol and glycine have also been used to improve the performance of HIC and hydroxyapatite chromatography. This review summarizes these applications of co-solvents for column chromatographies.

We have successfully developed a novel Package-on-Package (PoP) technology for achieving small package thickness and low package warpage. The novel package structure is based on an ultra-thin coreless substrate, called a Multi-Layer Thin Substrate (MLTS) . The MLTS is fabricated by completely removing the Cu base plate after forming a high-density buildup structure on the base plate. In this paper, however, the Cu posts are formed by selective wet etching of the Cu base plate. Since the novel package has built-in Cu posts as external terminals, the overall substrate can be molded for the peripheral area as well as the chip mounting area. This paper also describes how two types of novel wet etching processes have been developed for obtaining narrower pitch and higher aspect ratio. Through the novel wet etching process, we achieve Cu posts of 0.5 mm pitch and high aspect ratio of 6.0, which cannot be realized by wet etching. A prototype package has 361 pads arranged in 5 rows with 0.5 mm pitch, and the thickness is only 0.23 mm.

The effects of arginine on protein binding and elution in hydrophobic interaction chromatography (HIC) were examined using recombinant human interleukin-6 (IL-6) and activin-A. Binding of IL-6 in the presence of ammonium sulfate (AS) was tested using low- and high-substituted phenyl-sepharose. While inclusion of arginine during loading of IL-6 resulted in incomplete binding to the low-substituted phenyl-sepharose, binding was complete to the high-substituted phenyl-sepharose. Arginine facilitated elution of IL-6 from both columns. These results demonstrate that arginine weakens hydrophobic interactions between IL-6 and the phenyl-sepharose. More drastic results were obtained using activin-A, which showed undetectable recovery from phenyl-sepharose. Although no apparent elution of activin-A was observed from butyl-sepharose in aqueous buffer alone, the addition of arginine to the buffer resulted in partial elution recovery and, together with ethanol, resulted in greatly improved recovery of the protein. Two arginine derivatives, acetylarginine and agmatine, were also effective. These results show that arginine improves protein elution in HIC.

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called "compatible solutes", since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein-protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.

A great deal of attention has been paid to so-called amyloid diseases, in which the proteins responsible for the cell death and resultant diseases undergo conformational changes and aggregate in vivo, although whether aggregate formation is the cause or the result of the cell death is controversial. Recently, an increasing attention is given to protein folding diseases tightly associated with mutations. These mutations result in temperature-dependent misfolding and hence inactivation of the proteins, leading to loss of function, at physiological temperature; at low so-called permissive temperatures, the mutant proteins correctly fold and acquire functional structure. Alternatively, activation can be induced by use of osmolytes, which restores the folding of the mutant proteins and hence are called chemical chaperones. The osmolytes are compatible with macromolecular function and do stabilize the native protein structure. However, chemical chaperones require high concentrations for effective folding of mutant proteins and hence are too toxic in in-vivo applications. This limitation can be overcome by pharmacological chaperones, whose functions are similar to the chemical chaperones, but occur at much lower concentrations, i.e., physiologically acceptable concentrations. Although the research and clinical importance of pharmacological chaperones has been emphasized, the initial and central concept of osmolytes is largely ignored. Here we attempt to bridge the concept of osmolytes to applications of pharmacological chaperones.

A general approach for refolding recombinant proteins from inclusion bodies (IBs) is to screen conditions, that facilitate a conversion of unfolded to folded structure and minimize a conversion of unfolded to misfolded and aggregated structures. In this simplified model, such conditions may be those that stabilize the native protein and/or reduce aggregation. In this paper, a novel screening approach, termed reverse screening, was developed using a native activin. Activin-A, a member of transforming growth factor beta superfamily, is a homodimeric protein with nine disulfide bonds. We examined partial unfolding process of native activin-A dissolved in a buffer containing moderate concentrations of denaturant and reducing reagent (i.e., 1.5 M urea and 0.2 mM dithiothreitol). The recovery of the protein was followed by reverse-phase high performance chromatography analysis. Without additives, activin-A showed about 60% loss of the protein due to aggregation after 12-h incubation in the above condition. We then tested various additives for their effects on the recovery after partial unfolding. One of these additives, sodium taurodeoxycholate (TDCA), greatly increased recovery and suppressed aggregation of the protein. These additives were then tested for refolding activin-A from IBs. TDCA among others is proved to be a highly effective refolding additive. These results strongly suggest that reverse screening using native proteins, if available, may be another approach to discovering effective refolding additives.

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.

Arginine is finding a wide range of applications in production of proteins. Arginine has been used for many years to assist protein refolding. This effect was ascribed to aggregation suppression by arginine of folding intermediates during protein refolding. Recently, we have observed that arginine facilitates elution of antibodies during Protein-A chromatography and solubilizes insoluble proteins from inclusion bodies, which both can be ascribed to weakening of protein-protein interactions. In order to gain understanding on why arginine is effective in reducing protein-protein interactions and suppressing aggregation, the effects of arginine on stability and solubility of pure proteins have been examined, which showed that arginine is not a protein-stabilizer, but is an aggregation suppressor. However, there is no explanation proposed so far on why arginine suppresses aggregation of proteins. This review addresses such question and then attempts to show differences between arginine and strong denaturants, which are also known as an aggregation suppressor.

Arginine suppresses the aggregation of proteins. However, little is known about its mechanism. Here we have used HsNDK (Halobacterium salinarum nucleoside diphosphate kinase) to examine the solvent property of arginine. After exposure to 2 M arginine, HsNDK was diluted to a low salt buffer, resulting in fully active protein. Since unfolded HsNDK cannot refold in such low salt buffer, the observed activity indicates that HsNDK was in the native state in 2 M arginine. Enzyme activity was also examined directly in the presence of arginine, showing that it was active in the presence of 1 M arginine and, to less extent, 2 M arginine. Arginine, however, could not support refolding of heat-denatured HsNDK. HsNDK was stable at 40 degrees C for 19 h incubation in the presence of 1M arginine.

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.

Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inclusion bodies, (2) change in conformation and flexibility or solubility of proteins during refolding upon reduction of denaturant concentration, and (3) the effect of small molecule additives on refolding and aggregation of the proteins.

The recombinant microbial transglutaminase from Streptoverticillium mobaraense var. (rMTGase) was expressed in Escherichia coli. Specific enzyme activity of rMTGase was comparable to native MTGase. However, the gelation of a sodium caseinate solution induced by rMTGase was slower than that induced by native MTGase. In addition, the mechanical property of kamaboko prepared with rMTGase was weaker than that with native MTGase. In SDS-PAGE analysis, α-casein monomers decreased more slowly during the incubation with rMTGase than MTGase. These results confirmed the difference of cross-linking activity between the 2 enzymes. Furthermore, thermal stability of rMTGase was lower compared to native MTGase. These results suggest that the difference of cross-linking activity and thermal stability between the 2 enzymes cause differences in gelation activity of protein.

Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75% of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.

cDNAs encoding human interleukin 6 (hIL-6) and its variants lacking the N-terminal Pro and Pro-Val-Pro-Pro, respectively, were expressed in Bacillus brevis by using the signal peptide fusion approach. The presence of Pro at the N-terminus of the mature protein hindered the action of the Bacillus brevis signal peptidase. hIL-6 lacking the N-terminal Pro-Val-Pro-Pro was most efficiently secreted in a biologically active form and accumulated in the culture medium to a level of 200 mg per liter, which is the highest level reported for the bacterial secretion of hIL-6.

Recombinant human interleukin-6 (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coli as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use. Quantitative reconstitution of the native disulfide bonds of hIL-6 from the fully denatured E. coli extracts could be performed by glutathione-assisted oxidation in a completely denaturating condition (6M guanidinium chloride) at protein concentrations higher than 1 mg/mL, preventing aggregation of reduced hIL-6. Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22°C for 16 h. After completion of refolding by rapid transfer of oxidized hIL-6 into acetate buffer by gel filtration chromatography, residual contaminants including endotoxin and E. coli proteins were efficiently removed by successive steps of chromatography. The amount of dimeric hIL-6s, thought to be purification artifacts, was decreased by optimizing the salt concentrations of the loading materials in the ion-exchange chromatography, and gradually removing organic solvents from the collected fractions of the preparative reverse-phase HPLC. These refolding and purification processes, which give an overall yield as high as 17%, seem to be appropriate for the commercial scale production of hIL-6 for therapeutic use. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 301–310, 1999.

Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Therapeutic potential of immunoconjugates has opened a new window for antibody-based biopharmaceuticals. Greater tissue penetration and hence enhanced cell toxicity are obtained with a smaller version of antibodies. While the whole antibody can be readily produced via mammalian expression system, antibody fragments often require refolding of insoluble proteins. Here we report a new refolding method for antibody fragments using a novel amino acid-based detergent as a solubilizing agent and arginine-assisted refolding. Inclusion bodies of antibody fragments were solubilized by 2.5% lauroyl-L-Glu (C12-L-Glu) and successfully refolded by multi-step dilution into a buffer solution containing arginine hydrochloride and thiol/disulfide-exchange reagents. Adjustment of temperature was found to be critical for increase in the refolding yield. Although each protein requires appropriate optimization, solubilization by C12-L-Glu and dilution refolding assisted by arginine can generate the native, functional antibody fragments. The procedure should enable us to utilize bacterial expression systems for the large-scale manufacturing.

Size exclusion chromatography (SEC) is the most widely used method for aggregation analysis of pharmaceutical proteins. However SEC analysis has a number of limitations, and one of the most important ones is protein adsorption to the resin. This problem is particularly severe when using new columns, and often column preconditioning protocols are required. This review focuses on the role that addition of various cosolvents to the mobile phase plays in suppressing that protein adsorption. Cosolvents such as salt, amino acids, and organic solvents are often used for this purpose. Because the protein interaction with the resin surface is highly heterogeneous, different cosolvents affect the protein adsorption differently. We will summarize the various effects of cosolvents on protein adsorption and retention and describe the mechanism of the cosolvent effects.

Loss of protein aggregates due to matrix protein interaction during SEC often causes underestimation of aggregate content in the quality control of pharmaceutical proteins. Since new columns especially show stronger tendency to bind proteins, an effort should be made to suppress protein adsorption. We examined the effects of arginine on protein binding to SEC columns and found that loss of aggregates was reduced in the presence of arginine, leading to a reliable estimate of aggregate content even with newly used column and small protein load. This is not due to increased ionic strength, as NaCl resulted in increased protein loss.

A number of approaches are available in minimizing aggregation of the final protein products. This chapter describes one such approach, i.e., an attempt to avoid stressful conditions that may eventually lead to protein aggregation. Affinity chromatography uses specific interaction between protein to be purified and ligand attached to the column. Due to high affinity, dissociation of such interaction and hence elution often require harsh solvent conditions. Ion exchange and hydrophobic interaction chromatography also pose certain stressful conditions on proteins. Here we describe development of mild elution buffer using arginine. This chapter covers Protein-A, dye, Protein-A mimetic and antigen affinity chromatography.

It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1alpha, interferon-gamma, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturating solubilization of some of these proteins we have tested.

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-β protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng.ml), notochord- and endoderm-like tissues are induced in a high percentage.

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.

Elevated temperatures have been used to inactivate viruses for plasma-derived biopharmaceuticals. This paper describes the effects of arginine and NaCl in conjunction with elevated temperature for inactivation of two enveloped viruses, i.e., herpes simplex virus type 1 (HSV-1) and influenza virus type A at neutral pH. In phosphate-buffered saline, a significant inactivation of HSV-1 occurred above 40 degrees C, resulting in less than 10% surviving virus (over 90% virus inactivation) at 50 degrees C. Arginine concentration dependently decreased the temperature required for virus inactivation, leading to temperature shift by almost 17 degrees C at 1.2M. NaCl also decreased the inactivation temperature, but to a considerably lesser extent, indicating that virus inactivation effect of arginine is not simply due to ionic strength. Influenza virus was also inactivated by high temperature, but its responses to arginine and NaCl were different from those on HSV-1, suggesting that virus inactivation mechanism is different between these two viruses, i.e., the effects of these reagents are virus specific.

Virus inactivation is a key step for the purification of pharmaceutical proteins derived from recombinant mammalian expression systems and conventionally done using low pH-treatment, which is often harmful to the proteins to be purified. This is particularly true for antibodies, because immunoglobulin proteins undergo conformational changes at acidic pH. We have been developing mild elution solvents using arginine for Protein-A chromatography to minimize the low pH-induced damages on the antibodies. Here we have tested the aqueous solutions containing arginine or butyroyl-arginine at or above pH 4.0 for their effects on virus inactivation, since these solvents are effective above pH 4.0 in elution of bound antibodies from Protein-A columns. When the virus was incubated on ice, 0.1 M sodium citrate was totally ineffective above pH 4.0, but aqueous solutions containing arginine above 0.35 M or butyroyl-arginine above 0.28 M showed extensive virus killing at or even above pH 4.0.

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers.

Arginine is effective in suppressing aggregation of proteins and may be beneficial to be included during purification processes. We have shown that arginine reduces non-specific protein binding in gel permeation chromatography and facilitates elution of antibodies from Protein-A columns. Here we have examined the effects of arginine on binding and elution of the proteins during hydrophobic interaction (HIC) and ion- exchange chromatographies (IEC) using recombinant monoclonal antibodies (mAbs) and human interleukin-6. In the case of HIC, the proteins were bound to a phenyl-Sepharose column in the presence of ammonium sulfate (AS) with or without arginine and eluted with a descending concentration of AS. While use of 1 M AS in the loading buffer resulted in complete binding of the mAb, inclusion of 1 M arginine in loading and equilibration buffer, only when using low-substituted phenyl-Sepharose, resulted in weaker binding of the proteins. While decreasing AS concentration to 0.75 M resulted in partial elution of the mAB, elution was facilitated with inclusion of 0.5-1 M arginine. In the case of IEC, arginine was included in the loading samples. Inclusion of arginine during binding to the IEC columns resulted in a greater recovery and less aggregation even when elution was done in the absence of arginine. These results indicate that arginine enhances elution of proteins bound to the resin, suggesting its effectiveness as a solvent for elution in HIC and IEC.