The Profinia TM Protein Purification System Simplifies Antibody Purification With Protein A

Introduction Immobilized Protein A from Staphylococcus aureus has been used for many years to purify antibodies from a variety of species (Hjelm et al. 1972). The high selectivity and stability of protein A has made it a popular choice for the purification of antibodies from a wide range of sample sources, including serum, ascitic fluid, and hybridoma cell culture supernatants. Mammalian antibodies are categorized into five major classes: IgA, IgD, IgE, IgG, and IgM. IgG is the predominant class of antibody in serum and is generated in large amounts during the secondary immune response. The IgG class of antibody is further divided into subclasses that vary depending upon the species and the properties of the heavy chain component. There are four subclasses of IgG in humans (IgG1, IgG2, IgG3, IgG4) and in mice (IgG1, IgG2a, IgG2b, IgG3). The affinity of protein A for IgG varies considerably between species and IgG subtypes and has been extensively characterized (Duhamel et al. 1979, Schwartz 1990). In humans, protein A binds with high affinity to IgG1, IgG2, and IgG4, but poorly to IgG3. Among the four IgG subtypes in mice, protein A has the weakest affinity for IgG1. The binding of antibodies to protein A is mediated, at neutral or alkaline pH values, through hydrophobic interactions involving a highly conserved histidyl residue located in the protein A binding site of IgG. The elution of IgG from immobilized protein A is commonly achieved by lowering the pH using an acidic buffer. Protein A-purified antibodies are then typically neutralized with a base, dialyzed against a neutral buffer, or desalted using a gel-filtration column to avoid acid-mediated hydrolysis and denaturation. The use of protein A affinity chromatography offers a number of advantages to alternative separation techniques based on ion exchange, immobilized metal affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC), due to its simplicity and high recoveries of the purified product. The Profinia protein purification system is an automated chromatography instrument designed for the purification of affinity-tagged proteins and antibodies. An intuitive touch screen interface allows users to access preprogrammed chromatography methods or to customize any method for a particular application. With the Profinia system, antibodies can be purified and immediately desalted, precluding the need for neutralization, dialysis, or additional chromatography steps. The system is specifically designed for ease of use and for routine purification of 1–100 mg of IgG, using either 1 ml or 5 ml prepacked protein A cartridges. In this study, the purification, yield, and purity of IgG recovered from human, rabbit, mouse, rat, and goat sera using protein A-based separations are described. In addition, the effect of varying the elution buffer composition on the elution of IgG from protein A cartridges was investigated. Lastly, the purification of a monoclonal antibody from a hybridoma cell culture supernatant using the Profinia system and its subsequent functional analysis by an ELISA is presented.