Chromatographic purification

Once the protein is recovered from its producer source and concentrated it must be purified to homogeneity. In other words, all contaminant proteins and other potential contaminants of potential medical significance (discussed in Chapter 7) must be removed. Purification is generally achieved by column chromatography.

Column chromatography refers to the separation of different protein types from each other according to their differential partitioning between two phases: a solid stationary phase (the chromatographic beads, usually packed into a cylindrical column) and a mobile phase (usually a buffer). With the exception of gel filtration, all forms of chromatography used in protein purification protocols are adsorptive in nature. The protein mix is applied to the column (usually) under conditions that promote selective retention of the target protein. Ideally, this target protein should be the only one retained on the column, but this is rarely attained in practice. After sample application, the column is washed ('irrigated') with mobile phase in order to flush out all unbound material. The composition of the mobile phase is then altered in order to promote desorption of the bound protein. Fractions of eluate are collected in test tubes, which are then assayed for both total protein and for the protein of interest (Figure 6.7). The fractions containing the target protein are then pooled and subjected to the next step in the purification process.

Individual protein types possess a variety of characteristics that distinguish them from other protein molecules. Such characteristics include size and shape, overall charge, the presence of surface hydrophobic groups and the ability to bind various ligands. Quite a number of protein molecules may be similar to one another if compared on the basis of any one such characteristic. All protein types, however, present their own unique combination of characteristics, a protein chro-matographic 'fingerprint'. Various chromatographic techniques have been developed that separate proteins from each other on the basis of differences in such characteristics (Table 6.2). Utilization of any one of these methods to exploit the molecular distinctiveness usually results in a dramatic increase in the purity of the protein of interest. A combination of methods may be employed to yield highly purified protein preparations.

In general, a combination of two to four different chromatographic techniques is employed in a typical downstream processing procedure. Gel-filtration and ion-exchange chromatography are amongst the most common. Affinity chromatography is employed wherever possible, as its high biospecificity facilitates the achievement of a very high degree of purification. Examples include the use of immunoaffinity chromatography to purify blood factor VIII and lysine affinity chroma-tography to purify tPA.

As with most aspects of downstream processing, the operation of chromatographic systems is highly automated and is usually computer controlled. Whereas medium-sized process-scale chro-matographic columns (e.g. 5-15 l capacity) are manufactured from toughened glass or plastic, larger

1. Apply protein-containing sample

2. Irrigate with buffer (wash out unbound material)

3. Apply elution buffer and collect fractions

vy vy vy vy

Collect fractions when elution buffer is applied

Assay for target protein

Assay for target protein

10 15 20 25 30

Fraction number

10 15 20 25 30

Fraction number

Figure 6.7 (a) Typical sequence of events undertaken during an (adsorption-based) protein purification chromatographic step. Note that the chromatographic beads are not drawn to scale, and in reality these display diameters <0.1 mm. Fractions collected during protein desorption are assayed for (i) total protein, usually by measuring absorbance at 280 nm, and (ii) target protein activity. (b) In the case illustrated, two major protein peaks are evident, only one of which contains the protein of interest. Thus, desorption and adsorption steps can result in selective purification

processing columns are available that are manufactured from stainless steel. Process-scale chromatographic separation is generally undertaken under low pressure, but production-scale high-pressure systems (i.e. process-scale HPLC) are sometimes used, as long as the protein product is not adversely affected by the high pressure experienced. An HPLC-based 'polishing step' is sometimes employed, e.g. during the production of highly purified insulin preparations (Chapter 11). Next, we will consider individually the most common forms of chromatography used to purify therapeutic proteins.

Table 6.2 Chromatographic techniques most commonly used in protein purification protocols. The basis of separation is listed in each case

Technique

Ion-exchange chromatography Gel-filtration chromatography Affinity chromatography

Hydrophobic interaction chromatography

Chromatofocusing

Hydroxyapatite chromatography

Basis of separation

Differences in protein surface charge at a given pH

Differences in mass/shape of different proteins

Based upon biospecific interaction between a protein and an appropriate ligand Differences in surface hydrophobicity of proteins Separates proteins on the basis of their isolectric points Complex interactions between proteins and the calcium phosphate-based media; not fully understood

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