Proteins, desalting

Chlor-alkali production Electrochemical synthesis Water-organic liquid separation Organic liquid mixture separaion Fermentation products recovery and purification Cell harvesting, virus and antibody concentration Protein desalting, concentration and fractionation Blood processing, including artificial kidney Isolation, concentration, and identification of solutes and particulates... 

The simulated moving bed (SMB) technology was developed in the early 1960s by UOP for large-scale hydrocarbon separations in plants [1, 2], Recently, SMB is extensively used for the separation of enantiomers [3, 4], amino acids [5, 6], and protein desalting [7]. The SMB system simulated counter-current movement this is done with a series of packed bed by periodically advancing the input (feed, desorbent) and output port (raffinate, extract) along the solvent flow direction. 

There are two main application areas for size exclusion chromatography of proteins desalting or group separation and fractionation. Both areas are industrially of equal importance but the prerequisites for their ability of being sealed-up are quite different... 

Sephadex. Other carbohydrate matrices such as Sephadex (based on dextran) have more uniform particle sizes. Their advantages over the celluloses include faster and more reproducible flow rates and they can be used directly without removal of fines . Sephadex, which can also be obtained in a variety of ion-exchange forms (see Table 15) consists of beads of a cross-linked dextran gel which swells in water and aqueous salt solutions. The smaller the bead size, the higher the resolution that is possible but the slower the flow rate. Typical applications of Sephadex gels are the fractionation of mixtures of polypeptides, proteins, nucleic acids, polysaccharides and for desalting solutions. 

The selectivity of a gel, defined by the incremental increase in distribution coefficient for an incremental decrease in solute size, is related to the width of the pore size distribution of the gel. A narrow pore size distribution will typically have a separation range of one decade in solute size, which corresponds to roughly three decades in protein molecular mass (Hagel, 1988). However, the largest selectivity obtainable is the one where the solute of interest is either totally excluded (which is achieved when the solute size is of the same order as the pore size) or totally included (as for a very small solute) and the impurities differ more than a decade in size from the target solute. In this case, a gel of suitable pore size may be found and the separation carried out as a desalting step. This is very favorable from an operational point of view (see later). 

Heilman, EJ., Wiksell, E., and Karlsson, B.-M. (1990). A new approach to micropreparative desalting exemplified by desalting a reduction/alkylation mixture, presenred ar Eighr International Conference on Methods in Protein Sequence Analysis, Kiruna, Sweden, July 1-6. 

Steven Carr (SmithKline Beecham) has used microbore columns to desalt proteins prior to ES-MS (32). The pore diameter of PolyHEA used (usually 200 A) was selected so that all proteins of interest would elute at Vo with 50 mM formic acid. Only the Vo peak was allowed to flow into the ES-MS nebularizer the rest of the SEC effluent (including the salts) was diverted to waste by opening a microdumper valve between the column and the nebularizer. The properties of the mobile phase were quite compatible with ES-MS analysis. 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

During cheese making, the coagulated milk or curd is used to make cheese while the supernatant whey is a waste product rich in salts, proteins, and lactose. Whey concentration and desalting by UF produce a retentate product that can be used as an animal feed supplement or food additive. The MMV process (Maubois et al., French Patent 2,052,121) involves concentrating the milk by UF after centrifugation to remove the cream and before coagulation to improve yields and reduce disposal costs. 

The effectiveness of the elution step can be tailored by using a single eluent, pulses of different eluents, or eluent gradients. These systems are generally characterized by mild desorption conditions. If the eluting agent is bound to the protein, it can be dissociated by desalting on a gel filtration column or by diafiltration. 

Remove excess reagent and reaction by-products by dialysis or gel filtration using 0.1M sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.5. For chromatographic separation, use a desalting gel filtration support such as the Zeba desalting spin columns (Thermo Fisher) or the equivalent. The SAMSA-modified protein may be stored at -20°C until needed. 

Separate excess cystamine and EDC (and reaction by-products) from the modified protein by dialysis or gel filtration using 10 mM sodium phosphate, 0.15M NaCl, pH 7.2. A desalting column may be used for the gel filtration procedure (i.e., Zeba spin columns from Thermo Fisher). 

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