As to Antibody purification by affinity chromatography see antibody purification .   As to affinity purication of antibodies by FcyRs see antibody purification by affinity chromatography

As to antibody purification by immunaffinity see immuaffinity under antibody purification

For protocols of antibody purification by Protein A/G see (Darcy , chapt 20 from Protein Chromatography: Methods and Protocols, Methods in Molecular Biology, vol. 681 (2011).

Affinity chromatography refers to any purification or assaying technique which involves the addition of a sample containing a target analyte or molecule (e.g., an immunoglobulin or Ig) to a solid support which carries on it a protein which binds the analyte (e.g., PrA, PrG or PrL). Subsequent to the binding of the target analyte to the protein and the flowthrough of the undersired impurities, the target analyte may be eluted. The method takes advantage of a protein’s binding site and its ligand. The ligand, when immobilized, attracts the protein from a mixture, while other molecules are washed away. The protein of interest is reversibly and specifically bound to a biospecific ligand. Most proteins have specific ligands; For example, haveand cofactors. The interactions are not due to the general properties of the molecule such as isoelectric point, hydrophobicity or size but are a result of specific interactions from the molecule of interest and the ligand such as the hydrophobic and precise protein domain fit for protein A. This is to be contrasted with “non-specific binding” interactions as with ionic exchange chromatography and hydrophobic charge induction chromatography where interactions between a protein of interest and a ligand or other compound rely on non-specific interactions such as electrostatic forces, hydrogen bonding, hydrophobic forces and/or van der Waals forces. 

In conventional affinity chromatography the dissociation constant, KD, between the ligand and the target protein is in the range of about 10 -5 – 10 -7 M. Interactions with dissociation constants exceeding 10-10 – 10-11 M are often impossible to use, as the conditions required to dissociate the complex are then the same as those that will result in denaturation of the target proteins (US 12/740940). 

Type of Supports Used and Attachment of Ligands (See also Protein A supports)

An ideal insoluble support should possess the following properties (1) it must interact very wealy with proteins in general, to imnimize the nonspecific adsorption of proteins, (2) it should exhibit good flow properties, (3) possess chemical groups which can be activated or modified to allow the chemical linkage of a variety of ligands, must be mechanically and chemically stable to the conditions of coupling and the varying conditions of pH, ionic strenght, termpature and presence of denaturants (e.g., urea) which may be needed for adsorption or elution and it should form a very loose, porous work which permits uniform and unimpaired entry and exist of large macromolecules throughout the entire matrix; the gel particles should preferably be uniform, spherical and rigid. For successful purification by affinity chromatography, the inhibitor groups critical in the interaction with the macromolecule to be purified must be sufficiently distant from the solid matrix to minimiz steric interference with the binding process. Of the beaded agarose derivative commercially available, Sepharose 4B (Pharmacia) is the msot useful for affinity chromatography. Chemical compounds containing primary aliphatic or aromatic amines can be coupled directly to agarose beads after activation of the latter with cyanogen bromide at alkaline pH. The quantity of ligand coupled to agarose can be controlled by varying several parameters such as the amount of ligand added to the activated agarose. When highly substituted derivatives are desired, the amount of inhibitor added should, if possible be 20-30 times greater than that which is desired in the final product. For ordinary procedures, 100-150 mg of cyanogen bromide is used per milliliter of packed agarose. An important consideration in the covalent attachment of a biologically active protein to an insoluble support is that the protein should be attached to the matrix by the fewest possible bonds. This will enhance the probability that the attached macromolecule will retain its native teriary structure. Proteins react with yanogen bromide activated agarose through the unprotonated form of their free amino groups. Since most proteins are richly endowed with lysyl residues, which are exposed to the solvent, it is likely that such molecules will have multiple points of attachment to the resin when the coupling reaciton is performed at pH 9 or higher. The number of linkage points and the resultant biologic activity of the insolubilized protein an be controlled by manipulating the pH of the coupling reaction. Since selective adsorbents must be tailored to the special characteristics of the individual protein and to the equally stringent ligand requirements of that protein, it is useful to have alternative methods of attaching ligands to agarose. In many cases it is much easier to modify the matrix support than the ligand. These derivatizations provide procedures especially applicable to cases in which (1) an amino group on the lgiand is not avaialbe or its synthesis is difficult, (2) hydrocarbon hains of varyin glenght need to be interposed between the matrix and the ligand and (3) only the mildest eluting conditions (eg., neutral pH, absence of protein denaturants) are tolerated by the adsrobed enzyme. (Durkee, Protein Expression & Purificaiton, 4, 405-411 (1993). 

Types of Ligands Used

High affinity ligands such as those between antigens and antibodies are exploited in immunoaffinity chromatography (IAC). Receptors and receptor binding substances are also highly specific affinity systems. For example, recombinant IL-2 is purified to homogeneity on its immobilised receptor. Useful high affinity systems are also those based on the avidin/streptavidin biotin complexes. General or group specific affinity ligands such as Triazine dyes, amino acid based ligands (antisense peptides, oligopeptides, lysine, histidine), nucleotide based ligands (e.g., nucleotides, coenzymes, polyU and poly A, DNA), Heparain, chelated metal cations, carbohydrate binding lgiands (lectins) and protein ligands (SpA, SpG) exhibit wide spectrum of interaction with protein molecules and thus their selectivity is reduced. Affinity chromatography remains the most widely used affinity technology. However, several other chromatography based approaches have been developed such as affinity tails which is a genetic engineering approach to protein purificaiton via affinity chromatography. An affinity tails is attached to the target protein by covalent bond. For example, binding domains from SpA is attached to the target protein and the fusion is then purified by affinity chromatography on an IgG affinity column which binds specifically the affinity tail of the fused produce. Afterwards, the affinity tail is disconnected. Support materials suitable for affinity chromatography applications usually consist of macroporous hydrophilic beaded partciles, usually bearing free hydroxyl groups for ligand attachment. When the support is mode of non-compressible speherical particles of small diameter and anrrow size distribution, the ternique is termed high-performance affinity chromatography (HPAC). Silica based supports are used widely in HPAC. Centrifugal affinity chromatography (CAC) combines the high flow rate, created by centrifugation foce, which the specificity of affinity chromatography. Several non-chromatographic approaches are also increasing attention. In continous affinity recycle extraction (CARE), iestead of packing conventional absorbent particles in a fixed bed column, soli/liquid contact is acheived in well mixed reactors. The absorbent is continously recirculated between two contactors (tanks). Affinity partitioning is also regarded as a non expensive approach to affinity based purification. When aqueous solutions fo two high MW polymers such as dextran and PEG are mixed thwo pahses may be formed (aqueous two phase affinity partitioning). Covalent attahcment of an affinity ligand to a filtration membrane has also been employed in membrane affinity filtration purificaitons. In reversible soluble affinity polymer purification the target protein is bound to a suitable ligand precoupled to a polymer. Following a change in a property such as termpature, pecipitation/separation of the ligand-polyer conjugate occurs. Affinity precipitation is another technique wehre an insoluble protein netword is formed by cross linking a multifunctional affinity ligand with the target rpotein, resulting in selective precipitation of the target protein from solution. Such a ligand, in principle, consists of two recognition moieties which are able to bind siultaneously. (Labrou “Minireview: The affinity technology in downstream prcoessing” J. Biotechology 36, 1994, 95-119). 

Antibodies:  (for the purification of antibodies using antibodies, see Antibody purification and “immunoaffinity of antibodies”

–VHH: 

Herman (“Implementation of novel affinity ligands for biotherapeutic purification”) dislcose a ligand technology based on a special class of Camelid antibodies that are devoid of light chains. The variable domains of these so called heavy chain antibodies have evolved to compensate for the lack of light chains. For rexample, in order to maintain antibody diversity, the CDR regions of these singel domain antibody fragments show a higher variability to amino acid composition compared to classical antibodies. In addition, a nubmer of conserved hydrophobic residues, normally interacting with light chains, are replaced by charged amino acids, which prevents aggregation and increases solubility. The single domain antibody fragments are ideal molecules to serve as affinity ligands in chromatography. The ligand technology is applicable to a wide variety of target molcules. Target specific ligands are isoalted form expression libraries that represent the single domain antibody repertoire of a llama perviously immunized with a target molcule of interest. These immune libraries are screened at the monoclonal level for ligands that specifically bind to the target molecule. Subsequently, ligands are slected and tested for affinity, selectivity and stability in chromatography experiments. Finally, the ligands are recombinantly produced on any scale in Saccharomyces cerevisiae (Baker’s yeast).  For how VHH fragments are produced (immunization, screening, etc) see Haaft “Separations in Proteomics, Use of Camelid Antibody Fragments in the Depletion and Enrichment of human plama proteins for proteomic applications”) Separations in Proteomics, Chapter 3

VHH fragments have been used successfully as affinity ligands in chromatography processes for purifying many types of protein, including antibodies, blood factors, and adenoassociated viruses. For example, VHH ligands has been used to purify alpha-1 antitrypsin (AAt) from human plasma.  (Detmers, “Novel affinity ligands provide for highly selective primary capture” BioProcessInternational, 2010). 

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)

Aptamers

Classical antibody based affinity reagents have been challenged by novel types of binding proteins developed by combinatorial protein engineering principles. One of these classes of binding proteins of non-Ig origin are the so-called affibody binding proteins, functionally selected from libraries of a small (6 kDa), non-cysteine three-helix bundle domain used as a scaffold. The small size offers the possibility to produce functional affidobdy binding prtoeins also be chemical synthesis production routes, which has been found to be advantageous for the site-specific introduction of various labels and radionuclide chelators. (Nygren “Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold” FEBS Journal 275 2668-2676, 2008). 

An “aptamer” refers to a singel stranded nucleic acid, DNA or RNA that binds to a protien of interest. The protein of interest can be for example an immunoglobulin by binding to the conserved Fc fragment of the IgG. Aptamers generally includes between 5-120 nucleotides and may be selected in vitro by the SELEX (systematic evolution of ligands by exponential enrichmetn) method. (Bataille, US 15/125,483, published as US 2017/0073396)

Avidin: 

One often used method of affinity chromatography is to use a column containing avidin-coated beads. A sample is incubated with an antibody that is conjugated to biotin. When the sample is passed through the column the biotin binds to the avidin. This has been used to isolate by conjugating CD34 to biotin and passing them through the column.

De novo designed three-helix bundle proteins:

The three-helix bundle occurs buiquitously in nature as a robust scaffold for molecular recognition. First observed in the helical IgG-binding domains of Staphylococcal aureus, this family has grown to include DNA -binding protiens, enzymes and structural proteins. A de novo threee-helix bundle protein was designed using as a starting point he crystal strucutre of a de novo designed antiiparallel three stdnded coiled coil “Coil-Ser”.  (Wash, Proc. Natl. Acad. Sci. USA, 92, pp. 5486-5491). Lafleur (US 15/564,319, published as ) also discloses surface exposed residue modifications of this reference scaffold so as to create novel polypeptides that bind to targets of interest. 

PrA, PrG and PrL based affinity Chromatography resins: 

PrA based affinity chromatography, for example, refers to the separation or purification of molecules such as immunoglobulins, using protein A (PrA). PrA may be recovered from a native source such as from the bacterium Staphylococcus aureus or be synthetically produced. PrA has the ability to bind proteins which have a CH2/CH3 regions such as immunoglobulins. The PrA is generally immobilized on a solid support such as an affinity resin. Several types of chromatography media are available including Sepharose Fast Flow (crosslinked agarose), Poros 50 (polystyrene-divinylbenzene), and Prosep (controlled-pore glass). The ligand (PrA) is capable of binding to a molecule of interest (e.g., an immunoglobulin) which is to be purified from a mixture. The eluted IgG may be subjected to additional purification steps prior to, or after the protein A affinity chromatography step such as filtration, HPLC, etc. The purification using protein A or protein G affinity chromatography vary according to the IgG isotype. Human Ig1, IgG2 and IgG4 bind strongly to Protein A and all human IgGs includeng IgG3 bind strongly to Protein G. 

–Protein A: See Protein A 

–Protein C: Hall (US8,772,447) discloses chromatography ligands which comprises 1 or more Domain C units from SpA which is capable of binding to the Fab part of antibodies. 

–Protein G: See Protein G

–Protein L: is a cell surface protein expressed by Peptostreptoccocus magnus, which binds to the variable light chains of immunoglobulins without interfering with antigen binding. It can be used for purification of mammalian antibodies of all classes in contrast to the Ig-binding proteins protein A and protein G.

Commercial supplies:  Capto L (Data file 29-0100-08 AC GE, 2012-2014) discloses a recombinant Protein L with 4 binding domains that binds to the variable region of the kappa light chain of immunoglobulins and their fragments. 

–—Where Protein L binds:

Protein L binds to antibodies from a wide range of species including about 50% of human and 75% of mouse antibodies through the Vk region. The location of this unique binding site in the framework region of the light chain of antibodies allows protein L to bind an alternative subset of immunoglobuilins compared to protein A and G and also to bind the range of antibody fragment such as scFv, Fab and single domains, disulphide bonded Fv, a Fab fragment and a F9ab)2 fragment used in antibody engineering, if they have the correct K framework. Protein L has been found to bind to Vk of subgroups I, III and IV. (Enever 9WO/2005/033130).

Protein L binds human Fab via VL kappa 1, 3, and 4 but does not bind to VL kappa 2 and none of the VL domains of the lambda isotype. (Hermans US13/982970, published as US 2013/0337478)

—–Structure of Protein L:

Protein L (PpL) contains a plurality of VL-k-binding domains consisting of 70-80 residues. Yoshida Hyogo (US 15/660,365, published as US 2018/0016306). The number of FL-k-binding domains and the amino acid sequence of each domain are different depending on the kind of Protein L strain. For example, the number of Vl-k-binding domains in PpL of Peptostrepococcus magnus 312 strain is five, and the number of VL-k-binding domains in PpL of Peptostreptoccus mangus 3316 strain is four. Yoshida, Hyogo, (US 15/660, 373, published as US 2017/327535). Each of the VL-k-binding domains of PpL312 are referred to as B1, B2, B3, B4 and B5 domain in the order from the N-terminal and each of the VL-kbinding domains of PpL3316 are referred to as a C1, C2, C3, C4 domain in teh order from the N-terminal Yoshida Hyogo (US 15/660,365, published as US 2018/0016306),

Bjorck (US 5,965,390) discloses sequences of protein L including multiples of the domains B1-B5 which bind to light chains in Ig. 

Yoshida, Hyogo, (US 15/660, 373, published as US 2017/327535) discloses an affinity matrix based on the beta5 domain of Protein L dervied from Peptostreptococcus magnus 312 strain. Yoshida (US 16/176040, published as US 2019/0119562) also teaches using the B5 domain for affinity chromatography purification of an antibody with a kappa chain variable region. In one preferred embodiment, the beta 5 domain of Protein L has an N terminal deletion. 

Yoshida Hyogo (US 15/660,365, published as US 2018/0016306) also discloses a substitution of one of the amino acid residues at positions 15-18 in one of the Vl-k-binding domains B1-B5 and C1-C4 of PpL which results shifting the pH to a neutral side for dissociation on an affinity resin of the VL-k. 

Hydrophilic interaction chromatography (HILIC) (also known as hydrophilic-iteraction chromatography) : See also characterization of antibodies: HILICis a variatn of normal phase liquid chromatogrpahy that partly overlaps with other chomatographic applications such as EX and reversed phase liquid chromatography. The stationary phase of HILIC is a polar and hydrophilic phase which results in enhanced rention for polar anlyses. The mobile phase of HILIC is a reversed-phase type high organic eluent, for example, a mixture of water and acetonitrile. A methanism of separating anlytes in HILIC can be a comination of partitioning, ion exchagne and reverse-phase chromatography. HILIC has been used for the separation and analysis of large proteinaceous biomolecules, such as glycoproteins. (Lauber US 2015/0316515)

HILIC using a hydrophilic stationary phase and a hydrophobic eluent was first described in 1990, although it had been applied to the separation of sugars 15 years earlier. Since then, it has been successfully applied in all types of liquid chromatogrpahic separations, include small molecules, pharmaceutical compounds, metabolites, drugs of abuse, peptides and prtoeins. A variety of stationary phases have been described. The most commonly used are Atlatnis HILIC Xilica, ZIC-HILIC (sulfobeetaine-functionalized silica) and Luna HILIC (aminopropyl phase) colums which are classified as neutral, zwitterionic (weak electrostatic interactions) and charge (strong electrostatis interactions) columns. (Area “Hydrophiblic interaction chromatography in drug analysis” Centr. Eur. J. Chem, 10(3) 2012, 534-553). 

Affinity/Epitope tagging: is a technique that uses genetic engineering to attach a short peptide sequence, called an affintiy tag, either to the C or N terminus of a recombinant protein to facilitate affinity purificaiton. An ideal affinity tag should be short and as inert as possible so taht it does not alter protein folding and functionality and to limit potenntial interaction with the protein or proteins that might be present in the culture medium. (Sikka “Advancing Vaccine development with novel chromatogrpahy solutions” BioProcess international, 21(9), 2023) 

–E-P-E-A tag:

the CaptureSelect C-tagXL affintiy tag systems offers selectivity for a four amino acid peptide tag (glutamic acid-proline-glutamic acid-alanine: E–E-A) simplifying purificaiton of proteins tagged at their C-termini. This sequence is the smallest affinity tag that can be fused at the C-temrinus of any recombinant protein. The CaptureSelect C-tagOX affinity resin binds the E-P-E-A sequence . (Sikka “Advancing Vaccine development with novel chromatogrpahy solutions” BioProcess international, 21(9), 2023)

Hydrophobic Interactive Chromatography (HIC): see outline

Histidine Ligand Affinity chromatography (His-tags) (IMAC):  See IMAC under “Affinity Chromatography”

Small Molecule Affinity Chromatography: (As to the use of for purifying antibodies see affinity chromatography under “antibody purificaiton”

Synthetic affinity ligands (As to the use of for the purificaiton of antibodies see affinity chromatography under purificaiton of antibodies

Immunoaffinity (Antibody (immunosobent) Affinity Chromatography): See outline

Lectin Affinity Chromatography See outline

Receptor Affinity Chromatography

–Fc Receptors: As to Rc receptors for purification of antibodies see Antibody purificaiton and affinity chromatography