Chromatography – Purification of Antibodies and Antigens
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.
Methods for protein purification
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.
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.
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.
Ion exchange chromatography (IEC)
Ion exchange chromatography separates proteins based on charge. The charge of the protein to be purified may be manipulated by using knowledge about the specific proteins’ isoelectric point (pI). The pI of a protein is the pH where the protein has no net charge. Above pI, the protein surface is net negatively charged, below pI, the protein surface is net positively charged. Consequently, the pH of the surrounding solution influences the net charge of the protein surface.
The IEC resin is charged and when running the sample onto the column, oppositely charged protein may bind to resin. The attached proteins are eluted by changing pH and/or ionic strength of the running buffer.
Affinity chromatography is the most selective of the chromatography techniques used at Getica and utilizes a specific interaction between two molecules, e.g., the interaction between an antibody and an antigen or a hormone and its’ corresponding receptor. The ligand (e.g., an antigen or a receptor) is attached to a column resin, a sample containing the analyte of interest (e.g., an antibody or hormone) is added to the column, and the analyte attaches reversibly to its’ ligand. By altering the conditions in terms of pH and/or ionic strength, the interaction between the ligand and analyte is reversed and the analyte of interest is eluted.
Protein Purification with several chromatography techniques
One or several of the described chromatography techniques is used for each protein purification at Getica. Intermediate and final steps in the protein purification scheme include filtration to remove aggregates and particles, protein concentration and/or buffer exchange to create favourable conditions in the next purification steps or finalize a product for sale.