Protein purification and chromatography

The need for reagents of high purity in biotechnical applications

Within the broad biotechnical field there is a plethora of applications and systems that in one way or another exploit biomolecular interactions. Medicine and diagnostics are two prominent branches where such applications are pivotal. Developing and manufacturing products and assays, being for example a new drug or a new diagnostic test, without exception requires both chemical and biochemical components supplied in a very pure form with high and even quality. Reagent quality is a central aspect in ensuring function and performance of biotechnical applications.

Getica is active in the field of in vitro diagnostics (IVD), where both antibodies and their corresponding antigens play central roles. As both antibodies and antigens are produced either within host animals or in cultured cells an ever-present task is to successfully extract those from a biologically complex raw material (e.g. animal serum or cell extract). The following section describes the general strategies for protein purification and puts emphasis on the methods of most relevance in Getica’s purification work with antibodies and their corresponding antigens.

Chromatographic techniques

Protein purification is the collective name of the processes used to isolate one or several proteins from a complex mixture. The isolation relies on separation which often in scientific contexts is referred to as chromatography. Generally, chromatographic methods exploit one or several properties of the protein to be isolated. These could for example be solubility, size, charge, or binding specificity. The chromatographic methods Getica mainly utilize are size-exclusion chromatography (SEC), ion-exchange chromatography (IEC) and affinity chromatography. The following sections describe these three chromatographic methods more closely.

Purifying a protein from a complex mixture often requires several chromatographic steps as well as the use of different chromatographic techniques. The number of steps and which techniques to use depends on for example the properties of the protein and the desired purity of the final product. The entire purification procedure also often includes auxiliary steps such as filtration to remove aggregates and particles, protein concentration and/or buffer exchange to create favourable conditions for the different steps in the purification procedure. These auxiliary steps are also often used to adapt the final product to the requirements of the end user.

Size exclusion chromatography (SEC)

Size exclusion chromatography (SEC) separates proteins in a solution based on their respective molecular size. A SEC column consists of a packed resin with non-reactive porous microscopic beads. Larger proteins travel faster through the packed resin as their size do not allow them to travel through the pores of the beads and will therefore elute first. Smaller proteins are able to travel through the porous beads and take a longer route through the column. SEC is the gentlest of the chromatography techniques as it does not require any change in e.g., pH or ionic strength to separate and elute the proteins.

SEC may be used for both purification and analytical purposes, e.g., to determine the ratio of antibody monomers to multimers and the separation of antibody monomers from multimers and degradation products. Analytical SEC requires considerably less sample compared to SEC used for purification.

Figure 1:

Schematic illustration of size exclusion chromatography (SEC); a technique for separating proteins based on their size. The column material, consisting of non-reactive porous beads, causes this separation. Smaller proteins have a longer path length through the column as they can access the pores and cavities of the beads. Larger proteins, that can not access pores and cavities to the same extent has a shorter path length and are therefore eluted earlier.

Schematic illustration of size exclusion chromatography (SEC)

Figure 1: Size exclusion chromatography (SEC)

Ion exchange chromatography (IEC)

Ion exchange chromatography (IEC) is a method that separates proteins based on their charge. The method utilizes the electrostatic interactions between the proteins and the charged column material to achieve this separation. Proteins with a net-charge opposite to that of the column material will adhere to it due to the electrostatic attraction, while proteins with a net-charge of the same polarity as the column material elute due to the electrostatic repulsion.

An effect central to IEC is that the charge of a protein is affected by the pH of its surroundings. In this context the concept of the isoelectric point (pI) is often considered. The pI of a protein is defined as the pH where it has no net-charge. At a pH > pI the protein surface is net negatively charged, and at a pH < pI the protein surface is net positively charged. Therefore, by adjusting the pH of the surrounding medium, in relation to the pI of the protein of relevance, it is possible to dictate its interaction with the column material.

Figure 2:

Schematic illustration of ion exchange chromatography (IEC); a technique for separating proteins based on their charge. In this particular example negatively and positively charged proteins are separated in a column containing a negatively charged resin (so-called cation-exchange chromatography). Negatively charged proteins interacts with the resin in a repulsive way leading to direct elution, while positively charged proteins interacts with the resin in an attractive way making them adhere to it. By increasing the ionic strength (e.g. via addition of salt) this attractive interaction is effectively screened and the positively charged proteins are eluted.

Ion exchange chromatography (IEC)

Figure 2: Ion exchange chromatography (IEC)

Affinity chromatography

Affinity chromatography is a very selective chromatography technique as it utilizes a specific interaction between two biomolecules to achieve the separation. Thus, the biological structure and function are the biomolecular features at play in this separation technique. The interaction could for example be that between an antibody/antigen, an enzyme/substrate, or a receptor/ligand.

The basis for the technique is that the biomolecular entity to be separated out, often from a complex mixture, has its corresponding interaction partner already immobilized to the column material. Upon addition of the complex mixture to the column the interaction partner then effectively captures the entity to be separated out. The other molecules of the mixture, lacking an interaction partner within the column, passes through. Once the separation is complete, and the entities of interest have been captured in the column, the properties of the running buffer can be adjusted (e.g. pH alteration or change of ionic strength) in order to weaken the specific interaction resulting in elution of the entity of interest.

Figure 3:

Schematic illustration of affinity chromatography; a technique for separating biomolecules based on their affinity to an immobilized interaction partner. In this particular illustration a protein is separated from a complex mixture by having its corresponding antibody immobilized to the column material. The antibodies effectively capture the protein of interest while the other passes straight through the column. Once separation is complete the properties of the running buffer are altered (e.g. through an adjustment in pH) to weaken the interaction between protein and antibody, which results in elution of the protein.

Affinity chromatography

Figure 3: Affinity chromatography

If you wish to learn more about how Getica works with purification of antigens and antibodies, please visit our services page or contact us directly.