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). 

Hydrophobic interaction chromatography (HIC) is frequently used for the purificaiton and analytical characterization of proteins. This chromatographic mode relies on interactions between hydrophobic patches on the protein’s surface with immobilized hydrophobic ligands. The specific interaction is influenced by the addition of highly concentrated electrolytes and additives. In contrast to other chromatographic modes, the salt type and potential additves can be varied in a wide range. This offers additional modulation opportunities for the separation of proteins. (Muller, J. Sep Sci, 36: 1327-1334 (2013).

Factors which affect Dynamic Binding Capacity:

The term “dynamic capacity” of a HIC column refers to the maximum amount of protein in solution which can be loaded without significant breaktrhough or leakage of the protein into the solution phase of the column before elution. 

The dynamic binding capacity (DBC) of HIC resins is affected by many factors such as mobile phase composition and resin characteristics. Both binding affinity and DBC are higher with stronger salting-out salts and increase with salt concentration (Senezuk, Biotechnology and Bioengineering, 103(5), 2009)

For antibodies: Traditional HIC media typically exhibit dynamic IgG binding capacities ranging from about 15 to 40 g/L at 10% breakthrough, with most monoclonals exhibiting capacities of 20-30 g/L typically at 5% break-through. Improved HIC resins such as Toyopearl® has been shown to have DBC of about 40 g/L at 10% break-through for mAbs and 58 g/L for lysocyme. Monoliths are denoted as the fourth generation chromatography material. (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)). 

Effect of pore size:  HIC resins with optimized pore size have significantly imporved binding capacity which can increase HIC purificaiton unit operation efficiency (Chn, J Chromatogr A 2008, 1177(2), 272-81, 2007).

Effect of temperature: Temperature is known to affect HIC binding capacity and increased temperature generally leads to higher binding capacity due to the entropy driven characteristics of the hydrophobic interactions. However, the response (ie., change in retentin time, of any given protein to temperature is highly individual (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)).

Use of Dual Salts: during HIC have been reported to increase dynamic binding capacity to up to 3 times over that of single salt. (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)). For example, Senzuk (Biotechnology and Bioengineering, 103(5), 2009; see also US 2010/01311953) report that combining two salting out salts had a remarkable benefit on protein solubility and binindg of a given protein to HIC resin. 

 Operating Conditions:

HIC seperates molecules based on hydrophobicity of molecules. Hydrophobic regions in the molecuel of interest bind to the HIC resin through hydrophobic interaction. Strenght of the itneraction depends on operating conditions such as pH, ionic strengh, and salt. (Welsh, US 20230077205)

Binding Conditions:

Salts:

Some additive like alcohols and polymers greatly impact the protein soluility as well as adsorption to HIC media. They can either increase or decrease the binding apacity and their use may lead to an increase in recvoery. Ammonium sulfate is freequenly employed for protein preciptiation and HIC because it is readily soluble at high concentraitons. Further, it precipitates proteins at high pH and it is availabe at relatively hihg purity and in large quantites. Several other salts like sodium citrate, sodium phosphate, or sodium chloride provide an alternative. the most prominanet example for salt classificaiton is the Hofmeister series. Sales are classified as “kosmotripc” or chaotropic”. Kosmotropic salts are usually emmployed for precipitation of proteins. In constrast, chaotropic salts support protein solubilziation, In HIC, mianly kosmotropic salts are used because they convey the interaction between protein and resin thereby increasing the binding capacity. The Hofmister series may vary from different prtoeins. Further, the relative salting out effectiveness also depends on the isoelectric point of a certain protein and the experimentally applied pH value. (Muller, J. Sep Sci, 36: 1327-1334 (2013).

Compared to the single salt solution, simialr or higher DBCs can be achieved for salt mixtures of chatropic and kosmotropic. For lysozyme and mAb appropriate concetnraitons of the salt with the low surface tension to be added is around 1M whereas the concetnraiton of salt with the high surface tension depends on the solubility of the protein. In addition to sodium sulfate-sodium acetate, the salt combiantions of sodium citrate-sodium chloride, ammonium sulfate-sodium chhloride and ammonium sulfate-glycine elasd to the most promising resutls regarding increase in resin capacity. (Muller, J. Sep Sci, 36: 1327-1334 (2013).

(compare hydrophobic charge induction chromatography under “mixed mode chromatography”); See also antibody purificaiton by HIC

HIC is useful for the purification and separation of molecules such as proteins based on differences in their surface hydrophobicity. Hydrophobic groups of a protein interact non-specifically with hydrophobic groups coupled to the chromatography matrix. Differences in the number and nature of protein surface hydrophobic groups results in differential retardation of proteins on an HIC column and, as a result, separation of proteins in a mixture of proteins. HIC was first developed following the observation that proteins could be retained on affinity gels which comprised hydrocarbon spacer arms but lacked the affinity ligand. 

Applications: Hydrophobic interactions are strongest at high ionic strenght. Thus this form of separation is conveniently performed following salt preciptiations or ion exchange procedures (Shadle, US5429,746). A description of the general principles of HIC can be found in US 3,917,527 and 4,000,098. The application of HIC to the purificaiton of specific proteins include human growth hormone (US4,332,717), toxin conjugates (US4771128), antihemolytic factor (4,743,680), TNF (4,894,439), IL-2 (US4,908,434), human lymphotoxin (US4,920,196). HIC steps are generally performed to remove protein aggregates, such as antibody aggregates and process related impurities. 

Mechanism of Action: Whereas ion exchange chromatography relies on the charges of the antibodies to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of the antibodies. Hydrophobic groups on the antibody interact with hydrophobic groups on the column. The more hydrophobic a protein is the stronger it will interact with the column.

Types of HIC Resins/Ligands

In general, HIC resins contain a base matrix (e.g., cross-linked agarose or synthetic copolymer) to which hydrophobic ligdands (e.g., alkyl or aryl groups) are ocupled. Examples include Phenyl SEPHAROSE 6 FAST FLOW (Pharmacacis LKB Biotechnology, AB, Sweden), Phenyl SEPHAROSET high performance, Octyl SEPHAROSET High performance, Fractogel EMID Propyl or fRACTOGEL EMD Phenyl (Merck), MACRO-PREP Methyl or MACO_Prep t-Butyl (Bio-Rad, CA), WP HI-Propyl (C3) (J.T. Baker, NJ), TOYPEARL either phenyl or butyle (TosoHaas, PA) and Tosoh-Butyl-650M (Tosoh Corp. Tokyo). (Welsh, US 20230077205)

HIC column normally comprises a base matrix (e.g., cross-lined agaros or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. Suitables column include an agarose resin substituted with phenyl groups (e.g., a Phenyl SepharoseTM column). Many HIC column are commercially available (Hickman, US 12/582556). The most extensively used chromatogrpahic supports for HIC are agarose, silica and organic polymer or co-polymer resins. Useful hydrophobic ligands include groups having from about 2-8 carbon atoms such as butyl, propyl, or octyl or aryl groups such as phenyl. Conventional HIC products for gels and columns may be obtained commercially from suppliers such as Pharamcia LKB AB, Upsala, Sweden (butyl-SEPHAROSE), phenyl or butyl-SEPHAROSE CL-4B, butyl-SEPHAROSEFF, octyl-SEPHAROSE FF and phenyl-SEPHAROSE FF or Tosoh Corporation, Tokyo Japon as TOYOPEARL either 650, phenyl 650 or butyl 650 (Factogel); Miles-YEda, Rehovot, Israel as aklyl-agarose, wherein the alkyl group contains from 1-10 C atoms and J.T. Baker, Phillipsburg, N.J. as Bakerbond WP-HI-propyl. (Shadle, WO 95/22389) It is also possible to prepare the desired HIC column using conventional chemistry. (Biochem.Biophys. Res. Comm, 49:383 (1972)) 

A suitable column is one whose stationary phase comprises hydrophobic groups such as a phenyl Sepharose column. US 2010/0075375 A1 lists common HIC resins. 

Membrane HIC: Cellulose membranes bonded with four commonly used hydrophobic lgiands, octyl, butyl, pheny and polyethylene glycol have been evaluated for protein and enzyme purification, reportedly resulting in faster purification. Both polyvinylidene fluoride (PVDF) and paper-PEG membranes have also been examined for purificaiton for mAbs. For mAb purification it is advantageous to carry out the membrane HIC in flow through mode so that the majority of the product of interest will flow through, leaving the limited capacity for absorption of aggregates and other impurities. Since mAb aggregates show higher hydrophobicity and elute later than the monomer species, it is conceivable that membrane HIC can be designed to be carried out in flow through mode for aggregate removal. (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)).

While HIC has been presented as an efficient mode of removing dimers and higher MW aggregates when used as a polishing step in mAb purification, the flow rate and diffusion limitation associated with packed-bed HiC can increase the risk of protein denaturation due to the long contact time on the hydrophobic surface and high concentration of lyotropic salt. The use of a convective chromatography technique such as membrane chromatography reduces these problems by allowing much faster processing time.  In this respect, Phenyl™ membrane adsorber has been developed for large scale purificaiton of biomolcules based on HIC principles. (Kuczewski, Biotechnology & Bioengineering, 105(2), 2010).

Conditions/parameters  see outline

In Combination with Other Purification Techniques

HIC-IEX (CEX or AEX): Daniel (US7,138,262) teaching a method of purifying high mannose glucocerbrosidase (hmGCB) by subjecting the hmGCB to HIC such as MEP Hypercel and further purification by at least one ion exchange chromatography step such as AEX or CEX.

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

The large-scale, economic purification of proteins is an increasinly important problem for the biopharmaceutical industry. Manufactuers of protein based pharmaceutical products must comply with strict regulatory standards, including extremely strinent purity requirements.

Hycrophobic interaction chromatography (HIC) – anion/mixed-mode/cation exchange: Defrees (US 2010/0075375 A1) discloses processes for the isolation of polypeptide conjugates using HIC in conjuction with at least one other chratography step selected from AEX, mixed mode chromatography such as hydroxyapatite or fluorapatite chromatography and CEX.

Introduction:

In this mode of chromatography, the product of interest is loaded beyond the dynamic binding capacity of the chromatography material for the product, thus referred to as overload. 

In displacement mode chromatography which is covered in a different section on the site, the sample mixture in a carrier solvent that has a low affinity for the stationary pahse is loaded and the bound components are displaced by a solution of displacer which has a greater affintiy for the stationary pahse than any of the sample components. In “sample displacement mode” during loading of the sample there is competition among the sample components for the hydrophobic adsorption sites of the stationary phase. The more hydrophobic components compete more successfully for these sites than the more hydrophilic components, which are displaced and eluted from the column. Finally, the adsorbed components are eluted with an aqueous organic eluent (Veeraragavan J. Chromatography, 541 (1991) 207-220.

Particular Modes of Overload Chromatography:

Gradient mode elution in combination with Displacement Separation

Martin (WO2006/116064) discloses a method for separating a target comound by injecting an overloaded amount of the sample into a chromatographic conduit thereby forming a displacement train in which the target compound is substantially separated and then flowing a solvent causing an eluent of the at least one target compound.

Brown (WO 2010/019148) (see also below under IEX) discloses purifying a polypetpide from a contaminant which includes the steps of passing the composition through an ion exchange membrane where the polypeptide and the membrane have opposite charge at operating conditions comprised of a buffer having a pH sufficiently distinct form the pI of the polypetpide to enhance the carge of the polypeptide and a low ionic strengh effective to prevetn the shielding of charges by buffer ions which cause the membrane to bind the polypeptide and the at elast one contaminant and recoveirng the purified polypetpide form the effluent. In one emboidment the compoistion is passed thorugh a CEX membrane at operating conditions that include a buffer having a pH of about 1-5 pH units below the pI of the polypetpide and a conductivity of less than about 40 mS/cm which casues the membrane to bind the polypeptide and the at least one contaminant and recvoeirng the purifed polypeptide form the effluent. In anotehr alterrnative, the composition is passed through an AEX membrane at operating condtiions that include a buffer having a pH of aobut 1-5 pH units above the pI of the polypetpide and a condtuvitiy of less than or equal to about 40 mS/cm, which causes the membrane to bind the polypetpide and the at least one contaminant and recvovering the purifed polypetpide from the effluent. Accordingly, the purificaiton of plypeptide/antibody using the IEX membrane is in indigenous protein displacement mode. This is accomplished by operating at low inonic strengh and at a pH above the mAb pI during anion exchange and below the mAb pI during cation exchange. At these conditions, an attractive force is established between membrane and mAb resulting in product adsorption. Feedstream loading continues beyond the breakthrough capacity and the membrane effluent is collected in a purified form.

Particular Types of Media Run in Overload Mode:

In General:

Ayers (WO02/28194) discloses the separation of CMP (caseinomacropeptide, also known as GMP and CDP) which is rich in sialic acid by containing an anion exchanger with an excess of feedstock under conditions where the sialic acid rich glyco-CMP is selectively adsorbed and eluting the sialic acid rich glyco-CMP. Preferably the anion exchange is overloaded with respect to CMP so that non-sialic acid bearing aglyco-CMP which may initially bind to the exchanger is displaced by the more acidic sialic acid rich glyco-CMP.

Gilar (WO2006/116064) discloses separation of one or more compounds using displacement separation (overloaded sample condition) in combination with gradient mode elution. Additional embodiments also utilize a mass spectrometer or other suitable detector to provide a good resolution of eluting compounds to permit accurate observation of boundaries between the eluting sample compounds. In one embodiment, the method includes injecting an overload amount of the sample into a chromatographic conduit, causing an eluent including the at least one target compound to elute from an exit of the conduit.

Liu (US 2013/0079272) teaches overload chromatography using CEX, MM and IEX with a loading densitiy between about 150 g/L and about 2000 g/L

Nadarajah, US14/355818 (US2014/0301977)) discloses methods for purifying a polypeptide by a) loading the composition onto a chromatography material such a mixed mode material, an anion exchange material, a hydrophobic interaction material and an affintiy material in an amount in excess of the DBC of the material for the polypeptide, b) eluting the polypeptide from the chromatography material under conditions wherein the one or more contaminants remain bound to the material and pooling factions comprising the polypeptide in the chromatography eflluent form steps a and b. In some embodiments the partition coefficient of the chromatography material for the polypeptide is greater than 30 or greater than 100. 

Zarbis-Papastoitsis (WO/2012/030512) discloses a process for isolating a protein of interest from a sample which includes applying the sample to a chromatography material under conditions that highly bind the protein of interest but where the amount of protein of interest exceeds the binding capacity of the chromatography material such that a substantial amount of the protein of interest does not adsorb to the material and collecting at least a porition of the sample that does not adsorb to the material to thereby isolate the protein of interest.

Ion Exchange:

–AEX

Ahn (US2007/0264710) discloses separation of hTPO analogues according to their sialic acid content by loading eluted fractions obtained by reverse phased chromatography onto an AEX, washing the column and then eluting with 10 mM sodium phosphate buffer along with a 0-0.3 M sodium chloride gradient. The hTPO analogues with low sialic acid content were found at the fractions eluted with below 0.15 M sodium chloride whereas the analogues with high sialic acid content were found at the fractions eluted with 0.15 – 0.3 M sodium chloride. 

Ladiwala (US13/885446 and WO2012/068134) discloses an “overload bind and elute” mode of chromatography operation where a product such as an Fc-fusion protein is contacted with a chromatography medium at a concentration which exceeds the static of dynamic binding capacity of the medium. During the overlaod and bind step, the product having a selected characteristic (such as a high overall net-negative charge or a high sialic acid content) binds to the support while other impurities having less of the characteristic is excluded. Under an overloaded bind and elute process, higher sialytated glycoforms having higer net negative chage and binding affinity to TMAE HiCap compelted effectively for binding sites with the lower affinity lesser sialyated and non-sialyated glycoforms, thus displacing these lower affinity species.Subsequent to the overlaod and bind step, the target it eluted and recovered. In one embodiment, a strong anion exchange chromatography (TMAE HiCap) was used for separating Fc-fusion protein glycoforms. The more sialylated glycoforms were preferentially absorbed onto the TMAE HiCap, while the lower sialyated and non-sialyated glycoforms flowed through. Subsequently, the sialyated glycoforms were eluted form the column.

—-AEX-CEX (overloaded):

Pliura (US5,439,591 and 5,545,328) discloses a process for purifying hemoglobuilinwhere in the first stage the sample is applied to an anion exchange such that Hb and contaminants having lower affinity for the column are displaced such that certain contaminants which are more acidic than the Hb species are absorbed onto the solid phase and are thereby separated form the Hb which appears in the eluent along with the more acidic contaminants. Then, in a second stage, the eluent is applied to a CEX until saturation loading is execeeded so as to create an overload condition in the column so that at the overload condition, the Hb species is eluted from the column by displacement by the contaminants of greater affinity under the chosen conditions and the hb is thereby collected in the eluent at a very high state of purity.  A typical procedure starts with adult human red blood cells which are filtrated to remove leukocytes, then lysed to extract the hemoglobin, washed to remove plasma proteins and RBC proteins present, diafiltered and concentrated. The first stage of the process (anionic stage) is run at a high pH under conitions of low ionic concentration so that the desired Hb species will initially bind to the column medium along with all other speces but as the feed of impure solution continues, those species of greater acidity which show greater affinity than Hb gradually displace the Hb and more basic contaminants form the column. Eventually, an overload of the column is achieved so that all the Hb and more basic contaminants are displaced leaving the more acidic contaminant bound to the medium. The second stage is where the eluent is also run in overload mode. The desired Hb and other more basic impurities are bound in overloaded amounts. The contaminants of higher affinity (those more basic than Hb) displace the Hb from the column and Hb is thus eluted and recovered.

CEX-MM/HIC

Allen and Davies (WO2009135656) discloses cation exchange in displacement mode followed by by either a thiophilic interaction step or HIC step or mixed mode chroamtgoraphy step.

Liu (US2013/0079272; see also Liu, “Exploration of overloaded cation exchange chromatography for monoclonal antibody purification, 2011, 39, 1218) teaches methods for purifying a polypeptide from a composition using an overloaded CEX. For example, the methods comprise loading onto a CEX at a loading denstiy of greater than about 150 g/L of cation exchange material. The methods may further comprsies loading onto a mixed mode material.

Myers “out with the old and in with the new: Mixed-mode chromatography as an alternative to anion exchange as a polishing step”  BIOT, March 27, 2011) discloses a continous process of mixed mode chromatography in flow through mode with DEX chromatography in overlaoded mode. High throughput of over 300 g antibody/L of resin can be achieved.

Ion Exchange Membranes:

Brown, (14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346) discloses overloading an ion exchange membrane such that at least one contaminant remains bound to the membrane while the polypeptide of interest is primarily in the effluent and collecting the effluent from the IEX compirsing the popypeptide of interest and subjecting this polypeptide to a purification step of similar charge as the previous step. For membrane Cation exchange chromatography run in overlaod mode, the pH of the load material is adjusted to aobut 1-5 pH units below the pI of the antibody and the conductivity of the load material is adjjusted to less than about 40 mS/cm. Becasue the pH of the load is less than the pI of the antibody, the antibody (which has becopositively charged) will not flow thorugh initially. Rather, the antibody will be electrostatically bound to the negative funcitonal groups of the cation exchanger. Since the pI of many contaminants such as host cell proteins that elute with the antibody during protien A affinity chromtogrpahy is only slightly different from the pI of the antibody, these contaminants liek the basic antibodies will also bind to the membrane. But when run in overlaod mode, the pH and conductivity conditions induce charge with minimal ionic shielding and competitive adsorption occurs with the contaminants preferentially binding and displacing the antibody form the membrane. For membrane anion exchange chromatogrpahy run in overload mode, the pH of the laod material is adjusted to about 1-5 pH units above the pI of the antibody and the conductivity of the laod mateiral is adjsuted to less than about 40 mS/cm. Becasue the pH of the laod is greater than the pI of the antibody, the antibody (which has become negatively charge) will not flow thorugh initially. Rather the antibody will electrostatically bind to the positive functional grups of the anion exchanger. This is becasue the antiobdy (negative) and membrane (positive) have opposite charge. Since the pI of many contaminants such as host cell proteins like CHOP that elute with the antibody druing protien A affinity chromatography is only slightly different from the pI fo the antibody, these contaminants, like the “acidic” antibodies, will also bind to the membrane. When the membrane AEX is run in overlaod mode at pH and conductivity conditions that induce charge with minimal ionic sielding, competitive adsorption occurs and the contaminants preferentially bind to the membrane and displace the antibody form the membrane. Brown teaches that unlike applications that use IEX membrane primarily as a sole purificaiton step ofr final polishing step, the membranes can be used to protect a similarly charged ion exchange column and thus reduce or eliminate the impurities going onto the column. The impurities can also displace the polypeptide/antibody so taht it eventually makes it way onto the column. The membranes can be used either continusouly or non-continueously with the column. 

Brown (Biotechnol. Appl. Biochem (2010) 56, 59-70) disclsoes overloading cation and anion exchange membranes in a normal flow mode resulting in retention of impurities and breakthrough of purified antibody. Althouh some amount of the product also binds to the membrane, yields of greater than 99% were acheived by loading more than 3 kg mAb/l membrane. 

Brown (WO 2010/019148) discloses purifying a polypetpide from a contaminant which includes the steps of passing the composition through an ion exchange membrane where the polypeptide and the membrane have opposite charge at operating conditions comprised of a buffer having a pH sufficiently distinct form the pI of the polypetpide to enhance the carge of the polypeptide and a low ionic strengh effective to prevetn the shielding of charges by buffer ions which cause the membrane to bind the polypeptide and the at elast one contaminant and recoveirng the purified polypetpide form the effluent. In one emboidment the compoistion is passed thorugh a CEX membrane at operating conditions that include a buffer having a pH of about 1-5 pH units below the pI of the polypetpide and a conductivity of less than about 40 mS/cm which casues the membrane to bind the polypeptide and the at least one contaminant and recvoeirng the purifed polypeptide form the effluent. In anotehr alterrnative, the composition is passed through an AEX membrane at operating condtiions that include a buffer having a pH of aobut 1-5 pH units above the pI of the polypetpide and a condtuvitiy of less than or equal to about 40 mS/cm, which causes the membrane to bind the polypetpide and the at least one contaminant and recvovering the purifed polypetpide from the effluent. Accordingly, the purificaiton of plypeptide/antibody using the IEX membrane is in indigenous protein displacement mode. This is accomplished by operating at low inonic strengh and at a pH above the mAb pI during anion exchange and below the mAb pI during cation exchange. At these conditions, an attractive force is established between membrane and mAb resulting in product adsorption. Feedstream loading continues beyond the breakthrough capacity and the membrane effluent is collected in a purified form. 

–AEX membrane:

Brown (US 10,927,144) discloses a method for purifying an antibody from CHO cell culture composition be subjecting the composition to AEX perforemd in idigenous protein displacement mode by laoding the composition onto an AEX membrane, wehrein the atnibody and the membrane have opposite charge as a result of the laoded composition haivng a pH of 1-5 pH units above the pI of the antibody and a conducitvity of less than 40 mS/cm which prevents the shelding of the charges by buffer ions and casue the membrane to bind both the antibody and CHOP and continuing to load the membrane beyond the breakthrough capacity to a load denstiy of at leats 1000 g/L wherein the boudn antibody is displaced form the membrane by the binding of CHOP. 

—-AEX Membrane-AEX:

Brown (14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346) discloses that for membrane anion exchange chromatogrpahy run in overload mode, the pH of the load material is adusted to about 1-5 pH units above the pI of the antibody and the conductivity of the load material is adjusted to less than or equal to about 40 mS/cm. Becasue the pH of the load is greater than the pI of the antibody, the antibody (which has become negatively charged) will not flow through initially. Rather, the antibody will be electrostatically bound to the positive functional gruops of the anion exchanger. This is becaseu the antibody (negative) and membrane (positive) have opposite charge. Since the pI of many contamininants, eg., host cell prtoeins, aminoglycoside antibiotics sucha s gentamicine and ionic polymer additives such as polyethyleneimine (PEI) that elute with the antibody during protien A is only slightly different form the pI of the antibody, these contaminants like the acidic antibodies will also bind to the membrne. Accordingly, when the membrane AEX is run in overlaod mode, at pH and conductivity conditions that induce charge with minimal ionic shielding, competitive adsorption occurs and the contaminants preferentailly bind to the membrane or otehrwise effectively displace the antibody from the membrane, allowing the antibody to elute form the matrix or flwo through after binding and be recovered in the effluent.

–CEX Membrane:

Brown (US 10,927,144) discloses a method for purifying an antibody from CHO cell culture composition be subjecting the composition to CEX perforemd in indigenous protein displacement mode by loading the composition onto an CEX membrane, wehrein the antibody and the membrane have opposite charge as a result of the laoded composition haivng a pH of 1-5 pH units below the pI of the antibody and a conducitvity of less than 40 mS/cm which prevents the shelding of the charges by buffer ions and casue the membrane to bind both the antibody and CHOP and continuing to load the membrane beyond the breakthrough capacity to a load denstiy of at leats 2000 g/L wherein the boudn antibody is displaced form the membrane by the binding of CHOP.

–—CEX Membrane-CEX:

Brown (14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346) discloses that for membrane cation exchange chromatogrpahy run in overlaod mode, the pH of the laod material is adjsuted to about 1-5 pH units below the pI of the antibody and the conductivity of the load material is adjusted to less than or equal to about 40 mS/cm. Becasue the pH of the load is less than the pI of the antibdoy, the antibody (which has become postivively charged) will not flow through initially. Rather, the antibody will be electrostatically bound to the negative functional gruops of the cation exchanger. This is becaseu the antibody (postive) and membrane (negative) have opposite charge. Since the pI of many contamininants, eg., host cell prtoeins, aminoglycoside antibiotics sucha s gentamicine and ionic polymer additives such as polyethyleneimine (PEI) that elute with the antibody during protien A is only slightly different form the pI of the antibody, these contaminants like the basic antibodies will also bind to the membrne. Accordingly, when the membrane CEX is run in overlaod mode, at pH and conductivity conditions that induce charge with minimal ionic shielding, competitive adsorption occurs and the contaminants preferentailly bind to the membrane or otehrwise effectively displace the antibody form the membrane, allowing the antibody to elute form the matrix or flwo through after binding and be recovered in the effluent. 

Weak partitioning mode of operation enhances the F/T mode by identifying solution conditions wehre there is weak binding of MAb to the resin (2 to 20 g/L). Under these conditions the impurities bind stronger than in the F/T mode and thus enhanced purificaiton is obtained. However, load conditions are targeted to have a low product partition coefficient (Kp) in the range of 0.1-20. (Nadarajah, US 14/355,818)

Kelley ( US8,067,182, US2007/0060741A1, equivalent foreign filing is Brown (WO 2006/099308); see also “Weak Partitioning Chromatography for Anion Exchange purification of Monclonal Antibodies” Biotechnology and Bioengineering 101(3): 553-566 (2008) describes a third mode for the operation of chromatography columns referred to as a “weak partitioning mode” where at least one product and at least one contaminant both bind to the chromatographic resin. However, the impurities bind more tightly to the medium than the product and loading continues, unbound product passes through the medium and is recovered from the column effluent. The medium is optionally subsequently washed under isocratic conditions to recover additional weakly bound product from the medium and the purified product is pooled with the product from the column effluent. The more stringent load conditions under a weak partitioning mode are disclosed to result in much stronger impurity binding, which improves the product pool purity. Weak partioning mode may be used in conjuction with any chromatographic resin or medium for separation of a product form impurities. Emobdiments include ion exchange resins, HIC resins, hydroxyapatite chromatography, immobilized metal affinity chromatography. Before loading, it may be necessary to identify the region of weak partitioning by finding the operating conditions which cause the meidum to bind at least 1 mg of product per mL of medium. 

As applied to specific Resins

AEX:

Weak paritioning AEX chromatography requires a precisely formulated mobile phase to accomplish retention of stronger binding impurities, such as HMW species and HCP at the expense of a weaker binding product. The binding strenght is determined by a thermodynamic partition coefficient, Kp, which is calcualted form batch binding experiments as the ratio of absorbed protein concentration to free protein cocnetrations at equilbirum. Kp is a function of pH and concentration of anions (typically, Cl- in the AEX equilibration loading and wash buffers. The Kp=f(pH, [Cl-]) function is empirically dervied from batch binding experiments. Coffman (US13/811178)

ProA–AEX: In traditional batch chromatography mode, a pro A peak is eluted with a low-pH buffer, containign glycine as a buffering agent and salt (NaCL) which is a major source of chloride ions that control the binding strenght to AEX downstream The pooled peak is titrated with a concentrated neutralizing agent (high pH Tris buffer) until the desired pH is reached. Thus, Kp of the AEX load is controleld by accurate formulation and titration of the proA elution buffer. Coffman (US13/811178) teaches a tandem arrangement of ProA-AEX where pumps are synchronized to deliver a pre-programmed variable ratio of buffers. The first eluate produced from the ProA is continuously titrated with a pH buffer and at least 150 mM of a salt and the titrant is added at a target volumetric ratio such that there is a change in partition coefficient of less than 205 when the actual volumetric ratio of the first eluate to the titrant varies up to about 40% from the target volumetric ratio. 

Distinguish Overload chromatography (see outline). 

See also purification of antibodies using CEX displacement mode chromatography and overload chromatography 

See also purification of antibodies using AEX displacement mode chromatography and overload chromatography

Displacement chromatogrphy employs a displacer compound to remove components of a mixture from a column. The displacer compound generally has a much higher affinity for the stationary pahse than do any of the components in the mixture to be seaprated. This is in contrast to elution chromatography, where the eluent has a lower affinity for the stationary phase than do the components to be separated. A key operational feature that distinguishes displacement chromatography from elution or desorption chromatography is the use of a displacer compound. (Little, US2007/0102363). 

Displacement mode (displacement chromatography): means operating a known chromatography column/assembly in a displacement mode (using a displacer molecule) instead of operating it in the elution mode (using an elution buffer/buffer solution). It is a method of eluting species bound to a chromatography column on the basis of competition between bound species and a molecule (“the displacer”) that strongly binds to the column (Davies, WO 2009/135656).

Displacement chromatography is fundamentally different from other modes of chromatography in that the solutes are not desorbed in the mobile phase modifier and separated by differences in migration rates. In displacement mode, molecules are forced to migrate down the chromatogrpahic column by an advancing shock wave of a displacer molecule that has a higher affinity for the stationary phase than any component from the feed stream. This forced migration results in higher product concnetrations and purifites compared to other modes of operation (Spitali, WO2012/013682) . 

In dsplacement mode, the displacer binds at the top of the column and displaces any high affinity porduct/impurities bound in this region which then re-bind further down the column in turn displacing other species with a slightly lower affinity for the resin. This cycle of binding/displacement/re-binding continues down the chromatography column as more displacer is loaded and results in different species being resolved in to distinct bands which are then eluted from the column in reverse order of affinity (weakest binding first). The technique allows high loading concentrations of product (about 100g/L resin). Davies (WO2009/135656).

An example of a commercially avaialbe displacer molecule is Expell (SP1 (Sachem Inc., Texas, WO 2007/055896) and other potential displacer molecules are described in WO99/47574, WO 03074148 and WO963420).

Types of Chromatography used with Displacement Mode

Cation-exchange:

Torres (US5,028,696) discusses using displacement chromatography in a CEX column by the use of anionic displacers. 

Monoliths:

One area that should profit tremendously from the use of monolithic rather than particle based columns is displacemente chromatography. Other than in gradient elution, in displacement chromatography a certin distance is needed for the devleopment of the displacement train. (UNO Q and UNO S respectively). (Ruth, “Novel approaches to the chomatography of proteins” Biotechnoly and Bioprocessing/Biotechnol. Bioprocess. 27, 2003 455-502).

Paticular Types of Displacer Compounds/molecules

A displacer molecule should dissolve well in the mobile phase as high displacer oncentrations are required for fast and efficient separations. The molecule should be homogeneous, nontoxic, biocompatible, cheap and detectable. A polyion may be a displacer candidate for IEX. Small molecules have certain advantages over true polymers as protein displacers because they are more homogeneous and thus less likely to contain low affinity fracitons that may pollute the displacement train. Concomitantly, their separation from the protein zones is easier since the difference in size can be exploited (e.g., in gel filtraiton or dialysis). A problem is their inherently lower affinity, which can be ameliorated by either increasing the charge desnity or by introducing secondary nonspecific forms of interaction. (Ruth, “Novel approaches to the chomatography of proteins” Biotechnoly and Bioprocessing/Biotechnol. Bioprocess. 27, 2003 455-502). 

Examples of displacer molecules are disclosed in WO99/47574, WO03074148. 

Polyelectrolytes

The design and applicaiton of polyelectrolytic dispalcers requires good knowledge of the interaction between the charged molecules and the oppositely charged stationary phase surface. In addition to the predominant interaction mode, electrostatic in the case of ion exchange phases, a variety of secondary interactions may influence the displacer binding. (Schmidt J Chromatography A 1018 (2003) 155-167). 

Quaternary ammonium salts:

(Little, US2007/0102363) discloses a class of compounds known as organic quaternary ammonium salts (quat salts) which have advantages as displacer compounds. . Quat salts comprise positively-charged nitrogen atoms. 

Displacement of Particular Types of Proteins

Antibodies:

Wan (US13/803,808 and US9,067,990; US14/635,505 and US2015/0166653);  US14/079,076 and US9,017,687) teaches a method for producing a low acidic species comprising an antibody by contacting a first sample comprising the antibody with a chromatography media wehrein the antibody binds to the media, displacing the antibody with a displacing buffer comprising at least one displacer molecule and collecting the chromatography sample which comprising a composition of the antibody.

Gradient mode elution in combination with Displacement Separation

Martin (WO2006/116064) discloses a method for separating a target comound by injecting an overloaded amount of the sample into a chromatographic conduit thereby forming a displacement train in which the target compound is substantially separated and then flowing a solvent causing an eluent of the at least one target compound.

Batch Chromatography

Traditionally, capture chromatography is run in single column, discontinous mode which is characterized in that the column is consecutively (i) equilibrated, (ii) loaded with feed containing the target product, (iii) washed, (iv) eluted for recoveirng the desired target product, (vi) cleaned and (i) re-equilibrated for the next run. The possible maximum load of the column with product is strongly dependent on the stationary phase capacity (a distinction needs to be made between static capcity which corresponds to the occupancy of all ligands with target molecules obtained under equilibrium conditions and dynamic binding capacity under flow conditions). Depending on factors such as linear flow rate and mass transfer properties, the DBC is typically significantly lower than static binding capacity. This entails that under flow conditions in single column chroamtography, the stationary pahse capacity is not fully and efficiently utlized since the loading has to be stopped far before the static capacity is reached in order to avoid product loss. In order to increase the stationary phase capacity utilization and increase the process producitvity, continous countercurrent chromatgoraphy has found application (Muller-Spatch (US2014/0299547).

See also antibody purification

Definitions

Ion-exchange chromatography: retention is based on the attraction between solute ions and charged sites bound to the stationary phase. 

High-perfromance liquid chromatography (HPLC): is a technique in analytical chemistry used to separate, identify and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causig different flow rates and leading to the separation of the components as they flow out of the column. HPLC is distingusihed from traditional low pressure liquid chromatography becasue operational pressures are significantly higher whereas ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. 

–Normal phase HPLC (NP-HPLC): separates analytes based on their affintiy for a polar stationary phase such as silica. It is based on analyte ability to egnage in polar interations (such as hydrogen-bonding or dipole-dipole types of itneractions) with the sorbent surface. 

–RP-HPLC: oeprations on the principale of hydrophobic interactions. 

Reversed-phase chromatography (RPC): has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary pahse is a slica. With such a sationary phase, retention time is longer for molecuels whihch are less polar, while polar molecuels elute earlier.

Size-exclusion chromatography: also known as gel permeation chromatography separates particles based on their molecular size. It is generally a low resolution chromatogrpahy and thus often reservef for the final “polishing” step of the purificaiton. 

Modes of Operation

In complete flow through chromatography, the partition coefficient (Kp) is less than 0.1 and there is no protein binding to the resin. In weak partitioning chromatography, Kp is 0.1-20 and there is weak partitioning between the product and the chromatogrpahy media. In a bind and elute mode, product is tightly bound to the resin, and the Kp is greater than 100 but the load desnity is limited to the product binding capacity. However, in an overload and elute mode of chromatogpahy, load conditions are such that the product and the impurities Kp are greater than 100 and although the product flows through after reaching its binding capacity, the impurities keep binding to the resin and does not break through until they reach their binidng capacity, which could be higher than the product binding capacity. (Nadarajah, US 14/355,818).

Bind-elute Mode: Under B/E chromatography the product is usually laoded to maximiz DBC to the chromatography material and then wash and elution conditions are identified such that maximum product purity is attahed in the eluate. A limitation of B/E chromatography is the restriction of the load density to the actual resin DBC. (Nadarajah, US2014/0301977)

Flow through mode:  Using F/T chromatography, load conditions are identified where impurities strongly bind to the chromatography material while the product flows through. F/T chromatography allows high load density for for standard MAbs but may not be implementable for non-platform MAbs or the solution conditions that enable F/T operation for these non-platform MAbs may be such that they are not implementable in existing manufacturing plants (Nadarajah, US2014/0301977). 

–Recyling of flow-through: 

Rose (WO 2017/140081) disloses a method for the purificaiton of a protein of interest which includes loading a first mixture containg the protein onto a chromatography materix such that the protein binds until 40-100% of the maximum static binding capacity of the matrix is reached (the extend of overloading material need not be to the point of saturation) , collecting flow-through containig unboudn protein of interst and recyling the collected flow-through by re-loading it and a second volume of the protein of interest mixture. 

Weak partitioning mode: See outline