Highperformance liquid chromatography of proteins

Most of the chromatographic techniques described thus far are usually performed under relatively low pressures, where flow rates through the column are generated by low-pressure pumps (i.e. low-pressure liquid chromatography). Fractionation of a single sample on such chromatographic columns typically requires a minimum of several hours to complete. Low flow rates are required because, as the protein sample flows through the column, the proteins are brought into contact with the surface of the chromatographic beads by direct (convective) flow. The protein molecules then rely entirely upon molecular diffusion to enter the porous gel beads. This is a slow process, especially when compared with the direct transfer of proteins past the outside surface of the gel beads by liquid flow. If a flow rate significantly higher than the diffusional rate is used, then 'protein band spreading' (and hence loss of resolution) will result. This occurs because any protein molecules that have not entered the bead will flow through the column at a faster rate than the (identical) molecules that have entered into the bead particles. Such high flow rates will also result in a lowering of adsorption capacity, as many molecules will not have the opportunity to diffuse into the beads as they pass through the column.

One approach that allows increased chromatographic flow rates without loss of resolution entails the use of microparticulate stationary-phase media of very narrow diameter. This effectively reduces the time required for molecules to diffuse in and out of the porous particles. Any reduction in particle diameter dramatically increases the pressure required to maintain a given flow rate. Such high flow rates may be achieved by utilizing high-pressure liquid chromatographic systems. By employing such methods, sample fractionation times may be reduced from hours to minutes.

The successful application of HPLC was made possible largely by (a) the development of pump systems that can provide constant flow rates at high pressure and (b) the identification of suitable pressure-resistant chromatographic media. Traditional soft gel media utilized in low-pressure applications are totally unsuited to high-pressure systems due to their compressibility.

In the context of protein purification/characterization, HPLC may be used for analytical or preparative purposes. Most analytical HPLC columns available have diameters ranging from 4 to 4.6 mm and lengths ranging from 10 to 30 cm. Preparative HPLC columns currently available have much wider diameters, typically up to 80 cm, and can be longer than 1 m (Figure 6.18). Various chemical groups may be incorporated into the matrix beads; thus, techniques such as ion-exchange, gel-filtration, affinity, hydrophobic interaction and reverse-phase chromatography are all applicable to HPLC.

Many small proteins, in particular those that function extracellularly (e.g. insulin, GH and various cytokines) are quite stable and may be fractionated on a variety of HPLC columns without significant denaturation or decrease in bioactivity. Preparative HPLC is used in industrial-scale purification of insulin and of IL2. In contrast, many larger proteins (e.g. blood factor VIII) are relatively labile, and loss of activity due to protein denaturation may be observed upon high-pressure fractionation.

At both preparative and analytical levels, HPLC exhibits several important advantages compared with low-pressure chromatographic techniques:

• HPLC offers superior resolution due to the reduction in bead particle size. The diffusional distance inside the matrix particles is minimized, resulting in sharper peaks than those obtained when low-pressure systems are employed.

• Owing to increased flow rates, HPLC systems also offer much improved fractionation speeds, typically in the order of minutes rather than hours.

• HPLC is amenable to a high degree of automation.

The major disadvantages associated with HPLC include cost and, to a lesser extent, capacity. Thus, for both technical and economic reasons, preparative HPLC is employed almost exclusively

Figure 6.18 Preparative HPLC column (15 cm in diameter) used in processing of proteins required for therapeutic or diagnostic purposes. Column manufactured by Prochrom, Nancy, France (photograph courtesy of Affinity Chromatography Ltd)

in downstream processing of low-volume, extremely high-value proteins, mostly intended for therapeutic use, as opposed to proteins used for industrial preparations.

An alternative chromatographic system to HPLC is also available. Termed fast protein liquid chromatography (FPLC), this technique employs operating pressures significantly lower than those used in conventional HPLC systems. Lower pressures allow the use of matrix beads based on polymers such as agarose. FPLC columns are constructed of glass or inert plastic materials. Conventional HPLC columns are manufactured from high-grade stainless steel. In many cases, FPLC systems are economically more attractive than HPLC. Despite their operation at lower pressures, they still combine high resolution with enhanced speed of operation when compared with traditional low-pressure systems.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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