Antibody Purification Techniques

A single, rapid capture step using affinity chromatography is often sufficient to achieve the level of purity and quantity of product required for research purposes. Antibodies or their fragments can be adequately purified for further use, and a polishing step is sufficient to remove unwanted impurity. If affinity chromatography cannot be used, or if a higher degree of purity is required, alternative techniques need to be combined effectively into a polish strategy. A significant advantage when working with native or recombinant antibodies or fragments is that there is often considerable information available about the product and contaminants, as shown in Table 2.

Table 3. Characteristics of IgG

Antibodies are purified using purification techniques that separate according to differences in specific properties, as shown in Table 3.

Ion-exchange (IEX) chromatography refers to the separation of proteins based on charge. Columns can either be prepared for anion exchange or cation exchange. Anion exchange columns contain a stationary phase with a positive charge that attracts negatively charged proteins. Cation exchange columns are the reverse, negatively charged beads which attract positively charged proteins. Elution of the target antibody is done by changing the pH in the column, which results in a change or neutralization of the charged functional groups of each protein.

Gel filtration (GF) chromatography, also known as size-exclusion chromatography, separates larger proteins from small ones since the larger molecules travel faster through the cross-linked polymer in the chromatography column. The large antibodies do not fit into the pores of the polymer whereas smaller antibodies do, and take longer to travel through the chromatography column, via their less direct route. Eluate is collected in a series of tubes separating antibodies based on elution time. Gel filtration is a useful tool for concentrating an antibody sample since the target antibody is collected in a smaller elution volume than was initially added to the column. Similar filtration techniques might be used during large-scale antibody production because of their cost-effectiveness.

Hydrophobic interaction (HIC) chromatography is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC is based on interactions between hydrophobic (aliphatic or aromatic) ligands on the stationary phase with hydrophobic patches on the surface of proteins. This interaction is enhanced by high-ionic-strength buffer, which makes HIC an excellent purification step after precipitation with ammonium sulfate or elution in high salt during IEX.

Table 4. Protein properties used during purification

Every chromatographic technique offers a balance between resolution, capacity, speed and recovery. as shown in Figure 3.

Fig 3. Considerations in different chromatographic techniques

Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein.

Speed is most important at the beginning of purification where contaminants, such as proteases, must be removed as quickly as possible.

Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavorable conditions on the column.

Resolution is achieved by the selectivity of the technique and the efficiency of the chromatography matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties.

Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step.

A guide to the suitability of each purification technique for polish is shown in Table 5. Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of the next column (see Table 5).

Table 5. Suitability of purification techniques

IEX: Ion-exchange chromatography;HIC: Hydrophobic interaction chromatography;AC:Affinity chromatography;GF: Gel filtration chromatography;RPC:Reverse Phased Chromatography.