ITABLE 412 Some proteinprecipitation agents and their mechanisms of action

Agents

Mechanisms of Actions

Ethanol and acetone

Ammonium sulfate

Charged polymers

(e.g., polyacrylic acid) Non-ionic polymers

(e.g., polyethylene glycol) pH (isoelectric point)

Temperature

At low temperature, reduce dielectric constant and enhance electrostatic interactions between protein molecules Removes protein-bound water molecules and promotes hydrophobic interactions between protein molecules Neutralize charge of protein, which decreases protein solubility in aqueous environment Reduce water molecules available for protein solvation

Lowers solubility of protein at or near isoelectric point or pI, where the protein is in net unionized form; some protein may become denatured under these conditions Heat-sensitive proteins expose hydrophobic domains, which exhibit decreased solubility. This process must be reversible if original conformation is required for protein activity

Figure 4.16. Precipitation and extraction of proteins in stainless steal stir-tanks in pharmaceutical scale preparations. (NIH Fredrick facility, with permission)

of the agent used for protein precipitation, the precipitant and protein solution are mixed in a stir tank (Figure 4.16). Aggregates are allowed to settle to the bottom of the tank, and the aggregate slurry contain ing concentrated protein is collected for further processing.

A less-destructive approach to protein concentration has been developed using membrane separation or ultrafiltration

ITABLE 4.13. Some matrices used for industrial-scale protein purification chromatography

Column Matrix or Media

Trade Name

Agarose

Agarose and dextran composite Agarose and polyacrylamide composite Agarose and porous kieselguhr composite Cellulose

Dextran, cross-linked

Dextran and polyacrylamide composite

Ethyleneglycol-methacrylate copolymer

Hydroxyacrylic polymer

Hyroxymethacrylate polymer

Polyacrylamide

Polyacrylamide, cross-linked

Polystyrene-divinyl benzene

Porous silica

Rigid organic polymer

Sepharose, Sepharose CL, HP, FF, Suprose, Ultrogel,

Superdex

Ultrogel AcA

Macrosorb KA

Whatman TM, Cellufine, Sephacel, Cellex

Sephadex

Sephacryl

Fractogel TSK, Toyopearl

Trisacryl

Spheron

Eupergit C

BioGel P

Poros

Spherosil, Accell Monobeads, TSK-PW

technologies [6,10,11] that are similar to those described for solid-liquid separation steps. The key difference is the pore size of the filter used for this purpose. In most cases, pore size is selected to retain the recombinant macromolecule while allowing the passage of water and other small molecules. In practice, however, the selection of pore size is more an art than a science, especially the choice of design and sizing configurations of the filtration system. The selection of a proper filtration system may lead to additional benefits in terms of an increase in the yield of recombinant protein and its purity.

Purification

Further purification is needed to increase the purity of the recombinant protein found in the concentrated solution. The additional steps are termed intermediate purification. In this stage, increased purity is achieved through removal of most contaminant proteins, nucleic acids, endotox-ins, and viruses. This is accomplished by means of chromatography [12,13].

In the chromatography technique, proteins bind differentially to solid matix supports or media with various functional groups to provide hydrophobic, ionexchange, and affinity interactions. Some of the matrices used for intermediate purification that provide sufficient flow rate for large-scale purification are listed in Table 4.13. Some of the functional groups attached to matrix supports and examples of proteins purified by these matrix supports are listed in Table 4.14. The chro-matography technique should provide high capacity and selectivity. The matrix material must withstand multiple purification cycles with minimum loss of efficiency.

On completion of intermediate purification, most contaminants are removed. For example, endotoxin concentrations are reduced by more than two orders of magnitude, while the specific activity of recombinant protein, a measure of purity, is increased several-fold. The purified material emerging from this stage of downstream processing is composed of recombinant

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