For the Purification of Antibodies using Immunoaffinity of antibodies

Particular types of Antibodies Used

VHHs: 

CaptureSelect technology is proving to be an essential purification platform for many bioterapeutic molecules including viral vectors because it enables a high purity and porductivity process with fewer purificiotn steps than traditional processes while offering process consistency and scalability. Thsi technology is based on the variable domain of Camelid heavy chain only antibodies (VHH) which as single antibody domains provide full functionality in antigen specific recognition and high affinity binding. Becasue of their compact structure, these domains are very robust and can withstand the various conditions as typically run in chromatography. CaptureSelect ahs been validated in many commerical biotherpaeutic downstream purificaiotn processes, including blood coagualtion factors hormones and antibody derived therapeutics. It has also been used to purify andenovirus based vectors (POROS CaptureSelect AAV87 and AAV9 afffinity resins contain an immobilized ligand that specifically binds either AAV8 or AAV9 subtypes). (Terova in “Innovative Downstream Purificaiton Solutions for Viral Vectors” in BioProcess International, October 2016, 14(6), pp. 20-23). 

VHHs against bulk proteins such as HSA and IgG can be used for purification and depletion of these proteins. Klooster (J Immunoglogical Methods 324 (2007) 1-12. 

BAC (the Bio Affinity Company) has developed a proprietary technology (CaptureSelect) based on camelid VHH domains. In the case of the CaputreSelect LC-kappa, the matrix is based on a VHH that recognizes huamn kappa light chains through high affinity binding at the light chain constant domain. Unlike protein L which binds the variable domain and ignores a significant fraction of the kappa light chain population, this product binds 100% of human kappa light chains. In the case of Capture Select LC lambda, the matrix is based on a proprietary ligand that reognizes human lambda light cahins through high affinity binding at the light chain constant domain. BAC also has a CaptureSelect IgG Fc that is designed to purify all human IgGs (including IgG3) and fusion proteins. The amtrix is based on a VHH that recognizes all four subclasses of human IgG through high affinity binding at the Fc region of the heavy chain. (“Novel Affinity Ligands for Bioprocessing” Innovations in Pharmaceutical Technology).

Particular types of Proteins Purified

Glycan-targeting antibodies: 

Glycan targetting antibodies recognizing a specific carbohydrate structure have been used, such as antibodies specific for the Lewis x antigen (Cho, “use of glycan targeting antibodies to identify cancer-associated glycoproteins in plasma of breast cancer patients, Anal Chem. 80:5286-5292 (2008)

Immobilization of Antibodies and their Fragments

The immobilization of antibody and their fragments such as VHH, scFv and Fabs, is one of the key technologies that are used to enhance the sensitivity and efficiency of detection of target molecules in immunodiagnosis and immunospeparation. Polyclonal and monoclonal antiboides (whole antibodies) have been traditionally immobilized by passive adsorption to plastics, covalent coupling via activated functional groups and specific interactions between biomolecules. Passive adsorption is the simplest method used to immobilized proteins, including whole Abs, onto the surfaces of plastic supports such as PS micotiter plates, latex beads and porous hydrophobic membranes such as nitrocellulose. Covlanet coupling of whole Abs to solid supports offers advantgages that can prevent dissociation form the solid phase and enhance the density of antibodies. Several compnies supply a variety of solid phases containing reactive groups such as NH2 and COOH, but these are often not utilized because the density of an immobilized antibody is strongly dependent on the fuctional group introduced on the surface of PS.An amine coupling method is often adopted whereby the primary amine groups of whole Abs are covalently coupled with caroxyl groups introduced on the surface of solid suport. Specific bio-molecular interations such as streptavidin-biotin, IgG protein A/G, and IgG-anti-IgG antibody are soemtimes sueful for indirect immobilizaiton of whole Abs. (Kumada, “Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immuoassay” Biochimica et Biophysica Acta, 1844 (11), 2014).