Proteins polishing

For the purpose of high-resolution fractionation, the gel medium must be tailor made to cope with different separation ranges. The Superdex family is designed for the high resolution of peptides and proteins having a molecular mass of 500 to 100,000. Also, Sephacryl media have found wide applicability as a final polishing step in process scale SEC (see Section III,C). 

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

It is useful to subdivide the downstream processing of recombinant proteins into a few key stages, often referred to as initial processing of the source material and extraction (if necessary), capture, intermediate purification and polishing. These can then be further split into unit operations. In each of these stages, predefined goals have to be achieved, so a well-defined purification protocol will sequentially utilize as... 

Liquid chromatography is the core preparative technique in protein purification, and all supplementary procedures like extraction, centrifugation and filtration, ultimately serve to condition the protein solution for chromatography. A series of chromatographic steps, usually termed capture, intermediate purification and polishing, mak-... 

The pigment, P(s), can, in principle, be any seawater-soluble particulate solid that is being considered for a potential use in a self-polishing antifouling paint. Salts, sugars, and proteins (enzymes, peptides, or hormones) are obvious examples. It should be noted that the pigment can also represent a seawater-... 

Reduction potentials of the S. obliqms His59 Ru(NH3)5-modified protein have been determined by cyclic voltammetry using as electrode the oxidized surface obtained by polishing the edge plane of pyrolytic graphite [137]. The modified protein responds well at the electrode, whereas the native protein requires multi-eharged cations, e.g. Mg or [Cr(NH3)g] as mediators to give satisfactory reversibility. Separate reduction potentials at 1=0.10 M(NaCl) for native S. obliquus plastocyanin (389 mV) and [Ru(NH3)5 (imidazole)]... 

Polishing. This last process step prepares the product for final formulation or for actual sale. It is designed to remove any aggregated protein, remove residual chromatographic eluent(s), and place the product into a specific solvent. These requirements are admirably served by gel filtration. At this point, the sample volume is small and the product fraction to be applied is fairly clean. The gel and column equipment requirements are now within reason and, the clean samples result in much longer gel life. 

Polishing final purification step (invariably using chromatography) to remove dose product-related impurities, residual of host cell proteins (HCPs) and endotoxins. 

A purification strategy for any protein at any scale of operation can be broadly divided into three sequential stages—capture, intermediate purification, and polishing. Each stage represents a set of specific problems that may be encountered during a purification process. The nature of the sample and the scale of operation will dictate what equipment and methodology are appropriate to solve the problem. Many of the procedures discussed here can be found in Coligan et al. (2001). 

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