Gel Filtration and Size Exclusion

It is useful to use the term size exclusion to describe the type of gel filtration used to separate proteins by molecular weight. Size exclusion chromatography can be a useful purification tool in separating by size two proteins that may have similar charges and so co-purify on ion exchange. However, for this technique to be efficient, it is reliant on a concentrated feedstock, and a very large column may be required as the load volume is often only about 1-2 % of the column volume.

This technique is generally difficult to scale-up and the column bed height is an important factor in this. To separate well, a size exclusion matrix may need a bed height of 70 to 90 cm. Columns for production scale may need to be split into sections in order to give the matrix sufficient support. There are rigid size exclusion matrices, such as Toyopearl from Tosoh Bioscience, which may be scaled more easily than agarose-based matrices.

As columns are scaled-up, the bed height is kept constant and their diameter is increased. This can cause the flow distribution to be uneven over the cross section of the column, with flow at the edges lagging behind. The resolution between protein peaks is reduced and the required separation at large scale may not be achieved, see Figure 18.7.

Separation is determined by the size and shape of the proteins, and selecting the matrix by its nominal separation range for model globular proteins may be misleading. A variety of matrices should be screened.

Size exclusion is also normally a lengthy process step due to the low flow rates employed, and in terms of process economics in manufacture, it can be costly.

Gel filtration can also be used to separate protein from buffer solutes to effect a buffer exchange, also called desalting, and is a useful alternative to diafiltration. This method requires columns with relatively short bed-heights of approximately 20 cm. It is generally scaleable and the column load can be around 30 % of the column volume. The relatively high flow rates that can be used contribute to making this a useful method. However, when used in this mode it does not separate different proteins. Buffer exchange can also be achieved during size exclusion chroma-tography, as above. Gel filtration is a more rapid and more convenient method of buffer exchange when size separation is not required.

Absorbance i>

Elution Volume

Absorbance i>

Absorbance

Elution Volume

Figure 18.7 The scale-up of a size exclusion step can cause loss of resolution. The flow distribution on a small column allows the protein to be uniformly applied to the matrix. With a much wider column it is likely that the flow distribution will be imperfect, spreading the peak and reducing resolution. This can be critical in size exclusion separations where resolution is a key factor for purification. Loss of purity can result, or, if less of the product peak is collected where overlap occurs, recovery will be reduced.

Elution Volume

Figure 18.7 The scale-up of a size exclusion step can cause loss of resolution. The flow distribution on a small column allows the protein to be uniformly applied to the matrix. With a much wider column it is likely that the flow distribution will be imperfect, spreading the peak and reducing resolution. This can be critical in size exclusion separations where resolution is a key factor for purification. Loss of purity can result, or, if less of the product peak is collected where overlap occurs, recovery will be reduced.

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