See also Immunoaffinity generally under Affinity Chromatography

Particular Types of Antibodies Used

VHH antibodies:

VHH which are naturally devoid of light chains can be used to generate immunosorbent materials for the purification of human IgG antibodies. Advantages of VHH fragments are that they are single domain peptides, which are exceptionally stable even at higher temepratures. They are also small and easily produced in cost-efficient host organisms such as Saccharomyces cerevisiae. In addition, due to the sequence similarity between these VHH fragments and the human VH3 domain family, immunogenecity is expected to be very low compared to bacterial surface proteins like Protein A and G (WO2006/059904; EP-A-656946).(Low, J Chromatography B, 848 (2007) 48-63)

VHH are basically a single variable heavy chain fragment. They are very stable, low molecular weight (12 kDa) sequences. Antibodies are raised in camels or LLamas and the VHH genes are then cloned and expressed in yeast. The VHH fragmetns contain three CDRs which allows for both high selectivity and customization. The resulting molecules show good stability in caustic (0.1N NaOH). Because of their small size they are able to penetrate deeply into molecules and reognize unique conformational epitopes, alhough this capability may be limited when immobilized on a chromatography resin. Unlike Protein A, however, VHH fragments are monomeric and do not have the capability to bind multiple IgG molecules, which may impose a limitation on capacity. (Low, J Chromatography B, 848 (2007) 48-63)

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.

Frenken (US 2016/0207986) disclsoes immobilisation of wngle domain antigen bidning fratments of antibodies naturally devoid of light chains onto a solid surface. Ffater coupling of both tagged an untagged antimouse Fc VHH, complete mouse serum was loaded onto the columns. An elution buffer of Ha2HPO4/NaH2PO4 150 mM NaCl with addition of 12 mM HCL, final pH 2 was used. Immobilised tagged VHH and untaggged VHH has essentially the same results. 

Hermans (US 2015/037627) discloses immobilization of IgG-Fc domain binding VHH fragments onto sepharose, loading human IgG in PBS pH 7.4 on the column with a lienar flow of 150 cm/h, washing with PBS pH 7.4 and eluting with 0.1 M glycine buffer pH 3.0. 

FAB antibodies

—using MgCL elution buffer:

Moorenweis (US 15/166761, published as US 2016/0347826) discloses using the HuCAL Platinum library that includes the CysDisplay selection technology in order to generate Fab antibodies against human IgM, IgA and IgE that bind at neutral pH but can be eluted from the antigen under mild condition (e.g., pH 4-5). Selection of the anitgens using elution under mild conditions resulted in 14 anitobdies against IgM, IgA and 17 antibodies against IgE. Recombinant antiboides were isolated from the HuCAL Platinum library of human antibody genes by three rounds of panning with the antigens. For pannings, some antigens were passively adsorbed to microtiter plates and other antigens were coupled to beads. The pahge antibody library was incubated with the antigens and specific antibody pahge were eluted by elution with 1-2 MgCL. 

Single-chain Antibodies (scFv)

Kuman (US 15/774,398, published as US 2018/0327803) discloses immobilization of a single-chain antibody as a separation ligand for a human serum-derived IgG polyclonal antibody. In one embodiment, the single-chain antibodies are produced by phage display of a produced antibody library to serum-derived IgG or IgA polyclonal antibody coupled to multilamellar liposomes. Several clones were identified which bound to not only a human IgG antibody but also a human IgA antibody coupled to the multilamellar liposomes, suggesting that the single-chain antibodies bind to the L chain, specifically to the kappa chain, of a human antibody which is a region common to both a human IgG antibody and a IgA antibody. The single chain antibody was coupled to a carrier (the carboxyl group of Sepharose/agarose carreir in the form of beads, was esterified with NHS and the purified single-chain antibody was contained with the coolumn so that an amino group of the single-chain antibody forms an amide bond with teh caroxyl group). To the column, a human serum-derived IgG polyclonal antibody was supplied, the column washed and the human serum-derived IgG polyclonal antibody eluted with 0.5 M arginine (pH 1.5). 

pH Dependent antibodies (binding affinity higher at higher verus lower pH):

The purification of an antigen by affinity chromatography using serum antibodies, produced by a host animal’s response to the antigen which are bound to a solid support as an immunoadsorbent has been used for many years. Two serious shortcomings are that if antibodies of high affinity are used to extract the antigen from a sample, harsh conditions are requried to dissociate the antigen form the antibodies after non-adsrobed impurities have been washed away. The required conditions for this include for example a pH of less than 3 or greater than 11 or a concentrated chaotrope such as guanidine or urea solution which can dentautre the antigen and the antibodies. To avoid this, it has become common practice to use immobilized antiobdies of low affinity as an immunoadsorbent which permits elution of the antigen from the body of the immunoadsorbent using mild, non-denaturing conditions. However, the requisite step of washing the column to elute impurities also elutes some of the antigensuch that efficiency of separation is reduced. In addition, low affinity antibodies cannot efficiently bind antigens which are present in the media at relatively low concentrations (e.g., less than 10 ng/ml). Bartholomew WO 83/03678). 

Bartholomew (WO 83/03678) describes a different approach which is using antibodies having a high affinity for the antigen at a first pH, usually at or near pH 7, but with a much lower affinity iat a second lower pH, such as in the range of pH 4.5-4.0. It is believed that the change in binding affinity is likely due to protonation of histidine residues or deprotonation of lysine or possibly tyrosine or aginine residues in either the antibody, the antigen or both which alters the aility of the antigen and antibody to complex with each other. 

Ito (FEBS 11483, August 1992) “The His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values”) discloses that Hisitdine residues can be introduced into various sites in the amino acdi sequences of antibodies by site-directed mutagenesis so as to alter the binding constants of the antibodies with antigen (e.g., lysozime) at various pH values and at two ionic strenghts. Binding constants at low ionic strengh were higher than those at high ionic strenght at all pH values tested. Binding constants at higher pH were higher than those at lower pH. Differences between the binding constants at pH 5.2 nad those at pH 7.8 at low ionic strenght are large than those at higher ionic strenght. 

Domains/Regions on the Antibody bound

Binding to light chains: 

The amino acid sequences as described in WO2006/059904 relate to VHH fragments that bind to the light chain of human antibodies of either the kappa or lambda isotype.

A CaptureSelect biotin anti-labda binds to constant domain of human lambda light chains. 

A Capture Select anti-LC-kappa binds to constant domain of human kappa light chains. 

Binding to Fc part of IgG: 

The amino acid sequences as described in WO2009/011572 relate to VHH fragments that bind to the Fc part of human IgG. Thus they do not allow purification of fragments of human IgG that do not comprise the Fc domain, such as Fab of F(ab’)2 fragments of human IgG.

Several mouse monoclonal antibodies have been described that are capable of binding to the Fc domain of human IgG antibodies (Nelson PN, “Characterisation of anti-IgG monoclonal antibody A57H by epitope mapping. Biochem Soc Trans 1997; 25: 373). A problem associated with the use of mouse Mabs or antibodies from non-human species is the release of Mabs form the matrix which causes contaimination in the purified preparations that is difficult to remove. (Hermans US13/982970)

–Binding to CH3 domain of Human IgG-Fc:

A captureSelect biotin conjugate from TheroScientific binds to CH3 domain of human IgG-Fc. 

Binding to CH1 domain: 

–CaptureSelect™ Biotein Anti-IgG-CH1 Conjugate: (BAC, which was aquired by ThermoFisher Scientific)

Hermans (US13/982970) discloses antigen-binidng proteins which comprise one or more single binding domains which do not comprise a light chain but has full antigen-binding capacity. Examples of such ABP are are VHHs obtained by immunisation of camelids (e.g., llama, camels, dromedaries, Bacterian camles, alpacas, vicunas and guanacos) or sharks). The ABPs bind to an epitope of the CH1 domain that involves one or more of the amino acids: a phenylalamine at position 122, none or a single cysteine at either one of positions 127 and 128, a serine or a lysine at position 156 and/or an asparagine or a serine at position 216. Hermans discloses that the molecules are useful for purifying a target molecule having a human IgG-CH1 domain such as a Fab fragment. The ABPs are advantageous in that they bind the CH1 domain of each of human IgG1, IgG2s, IgG3 and IgG4. The ABPs can be used to purify any human IgG dervied Fab fragment independent of its IgG subclass, light chain isotype and VH subclass. The ABPs do not bind to human non-IgG related antibody isotypes or to the Fc or Fv domain or to free light chainsof a human IgG (accordingly there is no copurification of free light chains).  

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)

Commonly Used Affinity Resins for Antibody Purification  (see also Protein L under Affinity Chromatography)

KappFabSelect: is a highly corss-linked agarose with a recombinant protein/ligand produced in S. crevisiae with affinity for the constant domain of the immunoblogulin kappa light chain (See GE Heealthcare Life Scince “KappaSelect, 25 ml”

LambdaFabSelect: is a highly corss-linked agarose with a recombinant protein/ligand produced in S. crevisiae with affinity for the constant domain of the immunoblogulin lambda light chain (See GE Heealthcare Life Scince “LambdaFabSelect Data file 28-9448-22AA”

Capto L: is an affinity chromatography medium (resin) for the purificaiton of antibodies and antibody fragments. It combines a rigid, high-flow agarose matrix with the immunoglobulin binding recombinant protein L ligand, which has strong affintiy to the variable region of antibody’s kappa light chain. (GE Healtchare Life Sciences “Capto L, HiTrap Protein L Hiscreen Capto L”). Native Protein L is a 76-106k protein containing 4-5 highly homologous consecutive extracellular domains responsible for the protein’s interaction with Ig Kappa light chains. Given that is target is the akppa light chain, Protein L will bind to representaitves of most antibody classes, including IgG, IgM, IgA, IgE, and IgD. However, it does not recognize antibodies for related fragments from certain animal species. As the binding site for Protein L is located in the framework region 1 (a less variable region than the CDRs of the variable domain) of the kappa light chain, fragments derived form antibodies that have the kappa light chain can be purified using Protein L. About 60% of mammalian IgG light chains are kappa chains, with the remaining 40% being lambda chains that lack binding sites for Protein L. (GE Healthcare “a platform approach to purificaiton of antibody fragments” 

Protein L is able to bind to some kappa light chain variable domains without interfering with the antigne binding site. This characteristic has the great advantage of allowing purificiaton not only of IgGs, but also antibody fragments, such as single-chain variable fragments (scFv) which are devoid of constant comains. However, PpL is only effective in binidng certain subtypes of kappa light cahins. For example, it binds human VkI, VkIII and VkIV subtypes, but does not interact with VkKK subtype. (Lakhrift, MABS 2016, 8(2), 379-388).