Protein A resin is a high-affinity chromatography medium. It’s used for the one-step purification of monoclonal antibody products (mAb) and immunoglobulins from a cell culture media.
Protein A is a 49 kDa protein encoded by the spa gene. Originally, it was found as a type 1 membrane protein in the cell wall of the bacteria Staphylococcus aureus, where it’s regulated by cellular osmolarity, cellular topology, and a two-component system called ArlS-ArlR.
It performs multiple roles in the bacteria, such as:
Other than Protein A, the bacteria also have other immunoglobulin-binding bacterial proteins, such as Protein A/G, Protein L, and Protein G.
Structurally, Protein A contains five Ig-binding domains, each of which can bind a wide variety of mammalian proteins—especially human IgG. Its high selectivity towards antibodies facilitates the effective clearance of host cell proteins, DNA, and viruses.
In the past, wild type proteins were being used as a ligand for lab application processes. However, nowadays, the recombinant protein A, produced in E.coli, is the most prevalent one.
Modification in the structure of the native Protein A, such as single domain multimer and multiple binding sites, results in its high specificity towards the Fc region of antibodies, high binding capacity, and good physicochemical stability.
The modified features have also resulted in the formation of a highly stable and robust Protein A affinity resin, which is extensively used in biotechnology and immunology research studies for downstream processing and purification processes.
By the end of this article, you will learn the types of Protein A resin, its application areas, and the assays that involve the use of the resin.
Protein A resin is attached to a variety of base metrics, such as coated polystyrene, hydrogel filled into a ceramic shell, cross-linked agarose, surface modified porous glass, and other materials based on organic polymers.
Protein A chromatography resin is crucial for the purification process. It allows stringent purity levels during the purification of therapeutic antibodies. For example, it enables the removal of impurities and reduces the host cell proteins to <100 ppm and DNA to <10 pg/dose.
The commercially available protein A, such as MabSelect, MabSelect SuRe, and ProSep Ultra Plus, differ from each other based on the source of Protein A ligand, bead size, base matrix composition, and pore size. Thus, they also vary in their binding capacity, chemical resistance, mass transfer properties, and resin compressibility, affecting the Protein A column performance.
Protein A agarose bead is the most common matrix used in the labs or industries among other matrices. The agarose beads are cross-linked to Protein A resin through amide bond formation. These beads have a high capacity and affinity for antibodies.
Additionally, Protein A agar beads are also resistant to chaotropic agents (such as guanidine hydrochloride and potassium thiocyanate), urea, and a broad pH range (from 2-11). Therefore, it doesn’t break or get damaged even during harsh elution conditions, such as while removing bound antibodies.
Protein A resin has a myriad of applications, ranging from its use in affinity chromatographic assays, and immunoprecipitation, to biomanufacturing and bioprocessing assays. Due to its specificity, the resin is preferred as a standard technique to capture antibodies in purification processes.
Among all the available commercial proteins, Recombinant Streptococcal Protein A expressed in E. coli is the widely used resin. However, it is sensitive to alkaline conditions, and thus, cannot be applied with rigorous and cost-effective CIP and NaOH involved sanitization protocols.
Chromatography is a lab technique for separating components from a mixture. The Protein A affinity chromatography technique is extensively used in labs to capture and purify immunoglobulins, such as IgG and monoclonal antibody products. The process works on the principle of the binding of specific antibodies to immobilized Protein A ligands.
During the assay, Protein A resin is linked to 4% agarose, after which the bonding is optimized to achieve a high binding capacity to immunoglobulins. Several factors determine the dynamic binding capacity of the resin to antibodies, such as flow rate and target antibody.
Immunoprecipitation is an affinity purification technique for antigens. Here, antibodies are immobilized on the agarose resin surface, which allows their efficient binding with specific antigens present in a solution.
Protein A Resin uses purified antibodies in a sandwich format to purify complexes, proteins, and antigenic molecules. Moreover, it also aids in precipitating antibodies from a variety of complex mixtures.
Bioprocessing involves the use of living components, such as cell components and viruses, to build and obtain desired products. Protein A Resin is frequently used by industries as a cost-effective method in downstream processing of normal antibodies and monoclonal antibody products.
Protein A Resins are of high capacity and high affinity. Thus, they are used in labs and industries to obtain antibody products.
Protein A Resin is used in a range of life sciences labs, including biochemistry, biophysics, and immunology labs in assays requiring the purification of a variety of antibodies and antibody products.
Protein A is used in drug discovery and R&D for the healthcare industry, primarily for its high specificity and selectivity for human IgG, which is used to treat diseases like cancer and autoimmune disorders.
Additionally, it’s used in the biopharma industry in the process of manufacturing GMP-compliant biologics that are approved for clinical use and commercialization.
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Protein A resin is a chromatography medium used for the purification of a range of antibodies and monoclonal antibody products. Protein A is isolated from the bacteria Staphylococcus aureus, where it’s involved in many processes including impairing host immune response and the formation of biofilm.
The commercially available resin is a recombinant Protein A expressed in E.coli. In most cases, Protein A resin is employed with agarose beads because they have a high binding capacity, high affinity, and specificity to bind antibodies and facilitate their purification.
The chromatography and immunoprecipitation assays are robust techniques that require high-quality reagents and high-throughput equipment to achieve desired results. However, the instruments involved are pricey, making them difficult to acquire for small and budding laboratories.
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