Protein materials solvent process

Thermodynamically stable microemulsions and kinetically stable emulsions may be utilized to bring water and nonvolatile hydrophilic substances, such as proteins, ions, and catalysts, into contact with a SCF-continuous phase (e.g. CO2) for separation, reaction and materials formation processes. Reactions between hydrophilic and hydrophobic substrates may be accomplished in these colloids without requiring toxic organic solvents or phase transfer catalysts. CO2 and aqueous phases may be mixed together over a wide range in composition in w/c and c/w emulsions. The emulsion is easily broken by decreasing the pressure to separate the water and CO2 phases, facilitating product recovery and CO2 recycle. Reaction rates can be enhanced due to the considerably lower microviscosity in a w/c as compared to a water-in-alkane microemulsion or emulsion. 

The low solubility of water in SFCO2 has forced workers to use organic solvents including dimethylformamide (DMF) and dimethylsulfoxide (DMSO) as nonaqueous media for biological materials such as proteins ]. Such solvents have limitations because proteins have low solubility and potential loss of secondary and tertiary protein structure in solution of these agents. Nevertheless, although extensive perturbation was evidenced in DMSO solutions and was partially present in the solid protein particles, micrometer-sized particles of insulin, lysozyme, and trypsin prepared by the SAS process essentially recovered biological activity on reconstitution ]. 

The formation of a 3D network stabilised by new interactions or bonds, after removal of the intermolecular bond scission agent. Two different technological strategies can be used to make protein-based materials the wet process or solvent process involving a protein solution or dispersion, and the dry process or thermoplastic process using the thermoplastic properties of the proteins under low-hydration conditions (Figure 11.4). 

Silk fibroin (SF) polymers consist of repetitive protein sequences and provide structural roles in cocoon formation, nest building, traps, web formation and egg protection. Silks are generally composted of P-sheet structures due to the dominance of hydrophobic domains consisting of short sidechain amino acids in the primary sequence. Silk is biocompatible, degradable and shows superior mechanical properties. Silk materials are amenable to aqueous or organic solvent processing and can be chemically modified to suit a wide range of biomedical applications [249-251]. 

The problems associated with conventional size reduction methods, with respect to biologicals, make particle formation processes using SCF technology an attractive single-step alternative. In particular, the application of the GAS-type processes has demonstrated significant promise for the preparation of micropar-ticulate protein material (10). The low solubility of water in supercritical CO2 has required many workers to use nonaqueous media, such as dimethylsulfoxide (DMSO) and dimethylformamide (DMF), as solvents for protein material. These are not ideal choices due to the relatively low solubility of proteins in such sol-... 

As with organic solvents, proteins are not soluble in most of the ionic liquids when they are used as pure solvent. As a result, the enzyme is either applied in immobilized form, coupled to a support, or as a suspension in its native form. For production processes, the majority of enzymes are used as immobilized catalysts in order to facilitate handling and to improve their operational stability [24—26]. As support, either inorganic materials such as porous glass or different organic polymers are used [27]. These heterogeneous catalyst particles are subject to internal and external... 

It appears that none of these process techniques is dominant, at least with the lactide/glycolide materials. Researchers have considerable choices available in regard to fabrication of microspheres from these polymers. The most commonly used procedures employ relatively mild conditions of pH and temperature and are usually quite compatible with the bioactive agents to be entrapped, including proteins and other macromolecules. Only in the case of live virus and living cell encapsulation have serious deactivation problems been encountered and those problems were due to solvents used in the process. 

Diafiltration is a process whereby an ultrafiltration system is utilized to reduce or eliminate low molecular mass molecules from a solution and is sometimes employed as part of biopharmaceuti-cal downstream processing. In practice, this normally entails the removal of, for example, salts, ethanol and other solvents, buffer components, amino acids, peptides, added protein stabilizers or other molecules from a protein solution. Diafiltration is generally preceded by an ultrafiltration step to reduce process volumes initially. The actual diafiltration process is identical to that of ultrafiltration, except for the fact that the level of reservoir is maintained at a constant volume. This is achieved by the continual addition of solvent lacking the low molecular mass molecules that are to be removed. By recycling the concentrated material and adding sufficient fresh solvent to the system such that five times the original volume has emerged from the system as permeate, over 99... 

Relative molecular mass distributions for components of biochemical and polymer systems can be determined with a 10% accuracy using standards. With biochemical materials, where both simple and macro-molecules may be present in an electrolyte solution, desalting is commonly employed to isolate the macromolecules. Inorganic salts and small molecules are eluted well after such materials as peptides, proteins, enzymes and viruses. Desalting is most efficient if gels with relatively small pores are used, the process being more rapid than dialysis. Dilute solutions of macro-molecules can be concentrated and isolated by adding dry gel beads to absorb the solvent and low RMM solutes. 

Treatment with hot organic solvents was the next step in the tissue fractionation, to remove lipid-phosphorous and breakdown lipid-protein interactions. In the Schneider procedure, nucleic acids were then extracted in hot dilute trichloroacetic or perchloric acid, leaving a protein residue with any phosphoprotein links still intact. This method was to become particularly useful when 3H thymidine became the preferred label for DNA in the early 1960s. For investigations where both RNA and DNA were to be examined the Schmidt-Thannhauser process was often chosen. Here the lipid-extracted material was hydrolyzed with dilute sodium hydroxide releasing RNA nucleotides and any hydroxyamino acid bound phosphorus. DNA could be precipitated from the extract but the presence in the alkaline hydrolysate of the highly labeled phosphate released from phosphoprotein complicated... 

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