See also “controlled pore glass” as an example of silica in the outline

A well described matrix for the purificaiton of proteins is silica. It is commercially available in various particle sizes and different pore radii to enable the user to adapt the adsorbent to the product needs. The glass surface consists of silanol groups that are easily derivatized by silanization reagents, offering a wide variety of possible ligands. Thommes et al. Biotechnology and Bioengineering, 45, pp. 205-211 (1995). 

SiO2 (silica gel or silica) is the most abundant material in the earth’s crust and has been widely used by manufacturers fo chromatographic resins due to its high mechanical strenght, high column efficiency, easily controlled particle size and porosity and low cost.

Types of Silica

Controlled pore glass (CPG), like silica, is another media used as a backbone matrix for chromatographic resins. It is generally produced form a borosilicate base material that is heated to separate the borates and the silicates. The borates are leached out from the material, leaving the silica glass with uniform controlled pores. Thus, CPG is very similar to silica with respect to its surface chemistry. (Ghose, “Preparative Protein Purificaiton on underivatized silica” Biotechnology and Bioengineering, 87(3), 2004.) Interactions with underivatized silica are largely dominated by a combination of hydrophobic and some ionic interactions. The integration of these two modes of interaction confer silica with a unique selectivity that can be effectively exploited for downstream bioprocessing. This combination of interactions makes it possible to employ silica to bind proteins in both high and low ionic strenght environments, something which is not possible in conventional hydrophobic interaciton chromatography. Ghose, “Silica and its application in downstream processing – a blast from the past. Immunex Corporation. 

CPG, which is a silicate containing support material chemically similar to silica for use in liquid chromatography, is commercially available with average particle diameter of 37-177 microns and average pore size of 40-1000 Angstroms. (Crane, US 4,606,825).

Typical silica materials include silicate containing clay minerals including talc, kaolinite, pyrophyllite, serpentine, smectite, montmorillonit, mica. Synthetic silicates include silica gels, powders, porous glass and those prepared by hydrolysis of calcium silicide or sodium silicate. (Hou, US 4,639,513). Packing materials comprising silica as a base material, which are typical chromatography packing materials, are excellent in high flow rate cahracteristics. However, they materia is unstable under alkali conditions (US 13/201,647). 

Bare silica, which is underivatized silica particles is primarily sold as a desiccation agent. Silica is a popular backbone for the production of chromatography media, since it is inexpensive and is sufficiently chemically active to accept the addition of a wide range of substituent groups. Underivatized silica is not generally used as a chromatography medium, although it is used as a filter to remove cellular debris and highly hydrophobic impurities. Silica particles are available in a variety of forms, with different sizes of particle and pore size within the particle. (Builder US 5,451,660). 

Typical carrier materials include polysaccharides, polypeptides and silica (Hou, US 4,639,513). 

Carbonaceous materials

Modified carbonaceous materials:  Kyrildis (US2002/0056686) teaches that carbonaceous materials such as  carbon black, graphite and activated carbon, represent an important class of adsorbents that are used in many field such as purification and waste treatment but have not been used as a standard stationary phase in certain separation systems because carbon is a strong on-specific adsorbent despite carbon’s advantages over commercially available adsorbents (no corrosion problems, stable at a wide pH range unlike silica particles which are stable only in the pH range of 1-8, or are there any swelling problems with carbon products, which are stable in all organic solvents, unlike polysaccharide and/or polymer based chromatographic particles. Kyrildis teaches a chromatography column containing a stationary pahse that is at least a carbonaceous materials having attached at least one organic group such that the carbonaceous material having at least one organic group is capable of adsorbing at least one chemical species present in a mixture. For example, when cationic exchange processes are needed, a sulfonic acid, for instance, can be attached on the carbonaceous material and when anionic exchanges are needed, a quternary amine can be attached onto the carbonaceous material. A preferred process for attaching an organic group to the carbonaceous materials involves the reaction of at least one diazonium salt with a carbonaceous material in the absence of an externally applied current sufficint to reduce the diazonium salt. 

Polyscharides: 

The term “polysaccharide” includes compounds made up of many hundreds or even thousands monosaccharide units per molecule held together by glycoside linkages. Their MW are normally higher than about 5k and up into the millions of daltons. They are normally naturally occurring polymers such as starch, glycogen, cellulose, gum arabic, agar and chitin. (Hou, US 4,639,513).  

Agarose is a typical polysaccharides, but cellulose has a robuster hydrogen bonding netword and is thus advantageous for providng a higher flow rate (US 13/201,647). Cellulose particles can be crosslinked as by adding a crosslinking agent such as epichlorohydrin (US 13/201,647).

Cellulose: Cellulose is a preferred polysaccharide for use as a substrate of an ion exchange matrix. Cellulose is a naturally occuring polysaccharide consisting of (1-4) linked glucose units. Sources include wood, pup, cotton, hemp, ramie or regennerated forms such as raon. Packing materials using polysaccharides have high alkali resistance Hou (US 4,639,513). 

Ookuma (US5,196,527) discloses ion exchange fine cellulose particles which can be used as an affinity carrier. 

Umeda (US2014/0128253) discloses a porous cellulose gel containing crosslinked cellulose particles having a particle diameter of from 1-2,000 um, a swelling degree of from 5-20 mL/g. A method producing the gel by adding to a suspension liquid of cellulose particles a crosslinking agent in an amount of from 4-12 times the amount of the cellulose monomer in terms of moles and an alkali in an amount of from 0.1 to 1.5 times the amount of the corsslinking agent in terms of moles is also disclosed. 

Polypeptides

Polypeptides include compounds made up of many (tens, hundreds or even thousands) of amino acids linked through amide linkages (CONH) with elimination of water. The sequence of amino acids in the chain is of critical importance in the biological functioning of the polypetide and the chains may be relatively straight or coid or helical. In the case of certain types of polypeptides, such as keratins, they are cross linked by disulphide bonds of cysteine. Proteins which are also polypeptides and can be coiled and folded into very complex special patterns can be roughly classified into two groups on the basis of the extent of their coiling and folding. Those arranged as long linear molecules are called “fibrous proteins” and are relatively insoluble in water. Fibrous proteins include collage (the principal fibrous protein of skin, tendons, ligaments, cartilage, bone the cornea of the eye, etc), mysoin (one of the chief proteins in muscle), keratin (the major protein in hair) and fibrin (a protein iportant in blood clotting). Fibrous polypeptide are preferred polypeptides for carrier supports with keratin one of the most preferred. Of the keratinous polypeptides, animal fiber such as wool is preferred (Hou, US 4,639,513). 

Synthetic polymers:

Polyacrylamide (Bio-Gel P)

Polystyrene: 

In the case of polystyrene, a 3 D network is formed first and the functional groups are then introduced into benzene rings through chlormethylation.y

The most typical ion-exchange resins are based on cross-linked polystyrene The required active groups can be introduced after polymerization or substituted monomers can be used. For example, the cross linking is often acheived by adding 0.5-25% of divinylbenzene to sytrene at the polymerization process. Non-cross linked polymers are used only rarely because they are less stable. Cross linking decreases ion exchange capacity of the resin and prolongs the time needed to accomplish the ion exhcange processes. Particle size also influences the resin parameters; smaller particles have larger outer surface, but cause larger head loss in the column processes. Bill (US Patent Applicaiton No: 14/365,449, published as 10/364268).

–Microreticular forms of polystyrene (Styragel)

Poly(vinyl acetate) (Merck-l-Gel OR)

Poly(2-hydroxy ethylmethacrylate) (Spheron)

Polyacryloylmorpholine (Enzacryl)

Silica: See outline

Composite Resins

Polysacharides + other materials:

–-Agarose-Dextran:

The surface of core particles made of agarose has been modified using dextran (US 13/201,647). An commercial example is “SP Sepahrose SL™” from GE Healthcare Sciences.

Dextran-agarose composites are popular because of the widespread acceptance of agarose as a support for biochromatography. These materials are obtained by surface grafting dextran polymers into a macroporous, crosslinked agarose structure, which is then funcitonalized by introducing charged ligands.

Stone (J. Chromatography A, 1146 (2007) 202-215) compared the properties of agarose and dextran-grafted agarose cation exchangers with respect to protein adsorption equilibrium and rates and found that in spite of the reduction pore accessiblity, protein uptake rates were grealy increased with the dextrane grafted sulfopropyl matrices compared to the SP matrixes.

–Cellulose-Dextran: Matsumoto (US 13/201,647) discloses introduction of a cation exchange group such as a sulfone group into a base gel, which is made by adding a predetermined amount of polysaccharides having a limiting viscosity to porous cellulose particles (the free hydroxyl groups of the cellulose particles can be crosslinked with an agent such as epichlorohydrin). Examples of polysaccharides include agarose, dextran, pullulan and starch. According to the procedure, crosslkinked cellulose particles are reacted with epichlorohydrin to introduce an epoxy group, and then reacted with dextran sulfate having a predetermined limiting viscosity thereby adding dextran to the crosslinked cellulose particles. The amount of the polysaccharides to be added to the porous cellulose particles is represented by a change amount of the dry weight per unit volume before and after addition of the polysaccharides. According to the invention, the drug weight per unit volume of the porous cellulose gel is preferably 1.06-1.40 times the drug weight per unti volume of the porous cellulose particles. 

–Pullulan–methacrylate: A strong cation exchange materix was obtained by introducing a ligand into a gel in which pullulan was immobilized to porous methacrylate particles through 2-bromoethanesulfone (US 13/201,647).

–Polysaccharide + synthetic polymer: (WO/1084/003053) discloses a modified polysaccharide material which coprises a 1. polysaccharide such as cellulose covalently bonded to 2. a synthetic polymer made from a polymericable compound which has a chemical group capable of being covalently coupled to said polysaccharide and a chemical group capable of transformation to an ionizable chemical group.

Polyacrylamide within porous silica:

DEAE-dextrane-methacrylate:

Polystyrne-silica-Dextran: It is known that adsorption performance for proteins is improved by using an ion exchagne adsorbent made of dextran derived hydrogel in which an ion exchange group is added to core particles made of polystyrene-silica (Journal of Chromatography A, 679 (1994) 11-22).

–Polystyrne-slica-hydrogel: In the so called HyperD particles, the gel is surrounded by polystyrene-silica composite material giving it the necessary physical hardness for use in HPLC. The soft hydrogel can possess chemical functions or ligands for protein separations. (Harvath, J. Chromatogr. A 679 (1994) 11-22). 

–Polysacharide and synthetic polymer: (WO/1984/003053) discloses an orgnaic synethic polymer which carries chemical groups which are capable of coupling to polysaccharide and also which contains chemical groups which can proviede ion exchange capacity.

Polyethylemimine:  It is known that adsorption characteristics for proteins are improved by using a chromatography packing material made by adding polyethyleniine to methacrylate polymer particles (US 13/201,647).

–Silica – polyethyleneimine (PEI): Chromatography media material, particularly anion exchangers containing primary and secondary amine functionality, have been prepared by coating the internal surface of porous silica materials with polyethyleneimine (PEI) followed by imobilization through crosslinking (Alpert, J. Chromatogr. 185, 275-392 (1979). Deorkar (US20080203029) discloses polymerica media preaapred using polymeric partciles derivatized (not cross-linked) with polyetheyleneimine. 

High Density/Capacity/large scale:

Improvements in the structure of chromatography resin supports have made large scale chromatography a practical alternative to more conventional purificaiton methods. Rigid resins allow alrge volumes to be processed rapidly, and high ligand density gives the increased capacity necessary for large volumen processing (US 2011/0213126).

Type of supports Used

There are many different ion exchanger separation supports for biopolymers on teh market. The most common are of beaded type. The particles can be packed in a column with one inlet and one outlet. By using particles with a large portes, it is possible to achieve very fast separations without diffusion limitations. Pourous bodies are another fast separation system. A porous continous rod or a sponge is packed into the column and is a suitable tool to make separations without diffusion problems. A third gorup is membranes such as stacked flat sheet membranes. Muller (J. Molecules Recognition, 11, 273-278 (1998)

Specific Types of Materials Used

Ion exchange resins were originally limited to use of natural product such as cellulose, clay and other minerals containing mobile ions that would exchange with ionic materials in the surrounding solute phase. Because of the low exchange capacity of these natural products, however, synthetic organic polymers capable of exchanging ions were developed. Among the first generation of synthetic ion exchange materials were the ion exchange resins which are an elastic 3 D hydrocarbon network comprising ionizable groups, either cationic or anionic, chemically bonded to the backbone of a hydrocarbon framework. Typical examples of commercially available ion exchange resins are the polystyrenes cross-linked with DVB (divinylbenzene), and the methacrylatescopolymerized with DVB. (Hou, US 4,663163)

The resistance to flow exhibited by synthetic ion exchange resins is controlled by the degree of crosslinking. With a low degree of crosslinking, the hydrocarbon network is more easily stretched, the swelling is large and the resin exchanges small ions rapidly and even permits relatively large ions to undergo reaction. Conversely, as the crosslinking is increased, the hydrocarbon matrix is less resilient, the pores are the resin network are narrowed, the exchange process is slower, and the exhanger increases its tendency to exclude large ions from entering the structure. The ion exchange resins made from polymeric resins have been successfully applied to the removal of both organic and inorganic ions from aqueous media but are normally suitable for the separation of bioppolymers such as proteins.

The third generation of ion exchange materials were the ion exchange gels. These gels comprise large pore gel structures and include the commerically known material Sephadex, which is a modified dextran. The dextran chains are crosslinked to give a 3D polymeric network.

Ion exchange gels made from synthetic polymers have also been used and include crosslinked polyacrylamdie (Bio-Gel P), microreticular forms of polystyrne (Styragel), poly(vinyl acetate) (Merk-o-Gel OR), crosslinked poly (2-hydroxy ethylmethorrlate) (Spheron) and polyacryloylmorpholine (Enzacryl).

The failure of single components to have both capacity and dimenstional stability led to yet another generation of ion exchange materials comprising composite structures, e.g., hydrid gels. Hybrid gels are made by combining a semi rigid component, for the purpose of convering mechanical stability, with a second component, a softer network, which is responsible for carrying functional groups. Agarose gel, which would otherwise be very soft and compressible, can be made stronger through hydridizing with cross linked polyacrylamide. The crosslinked polyacrylamide component is mechanically stronger than the agarose, improves the gel flow properties and reduces the gel swelling, but it sacrifices molecular fractionation range. Examples of hybrid gels other than polyacrylamide/agarose (Ultrogels) are polyacryloylmorpholine and agarose (Enzacryl) and composite polystyrenes with large pore polystyrenes as a framework filled with a second type of lightly crosslinked polymer.

Yet another composite gel structure is achieved by combining inorgnanic materials coated with organics and are the types known as Spherosil. Porous silica beads are impregnated with DEAE dextran so that the product will have the mechanical properties of silica, with the ion exchange properties of DEAE dextrans. These composites, however, have severe channeling defects arising out of particle packing, and they have capacity limitations on the coated surfaces.

Totally rigid inorganic supports such as porous silica or porous glass which are not susceptible to degradation have also been sued to provide high porosity and high flow rate systems. The major problem, however, is nonspecific adsorption of proteins due to the silanol groups on the silica surface. Since the hydroysis of silica is directly related to the pH conditions, the nonspecific adsorption by silica is minimal at neutral pH, but increases as the pH changes.

Commercially available 

Several commonly employed ion exchange resins are commercially available. The resin matrix backbone includes agarose and dextran (GE Healthcare), glycidyl methacrylate (Macroprep, Bio-Rad), polysyrenedivinylbenzene (Poros, Applied Biosystems) and polymethacrylate (Fractogel, EMD Chemical and Totopearl, Tosoh). (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010). O’Donnel “A high capacity strong cation exchange resin for the chromatographic purificaiton of monoclonal antibodies and other proteins” PREP 2007) teaches “Toyopearl GigaCap S-650M” which is syntehsized on a 1000 Angstrom polymethacryate base with nominal particle size of 75 um. The resin has high capacities for both large (igG) and small (lysozyme) proteins.

Strong CEX:  Mustang S and Sartobind S are strong CEX membranes. The are modified with a form of sulfonic acid. The Mustang Q is made of polyethersulfone (PES) with u pores. The Sartobind S is made of regenerated cellulose with 3-5 um pores. Brown (WO2010/019148)

Fratogel SE Hicap and SP Sepharose Fast Flow resins are strong CEX. Fractogel SE Hicap resin is made of cross linked polymethacrylate particles of 40-90 um diameter with pore size of about 800 A. The functional ligand is covalently attached to the particle with a long, linear polymer chain. The SP Sepharose Fast Flow resin is made of highly cross linked agarose partciles of 45-165 um diameter. Sepharose Fast Flow is a cross linked derivative of Sepharose with a sulfopropyl ligand as the functional group. (Bill, 14,365449). 

Strong AEX: Mustang Q is a strong AEX membrane. It is modified with with a form of quaternary amine. It is made of polyethersulfone (PES) with 0.8 um pores. Brown (WO2010/019148)

Pore Size

In general, IEX membranes have pore sizes of 0.1-100 um. As a reference, Sartobind Q (Sartorius AG) is a strong AEX haing a nominal pore size of 3-5 um and is commercially available in a single or multiple layer format. Mustang Q is a strong AEX having a nominal pore size of 0.8 um. Sartobind S is a strong CEX haivng a nominal pore size of 3-5 um and Mustang S is a strong CEX having a nominal pore size of 0.8 um. Brown (WO2010/019148)

How the Polymer is attached to the support

Polymer coatings can be produced by several routes: (a) by reaction of the surface with tailor-made polymers and crossllinking; (b) by physical adsorption and crosslinking: (c) by graft or block polymerization. Muller (J. Molecules Recognition, 11, 273-278 (1998) discloses that polyamide hollow fibre membranes can be modifed with ion exchanger groups by a block polymerization procedure.

Introduction:

Cation exchange chromatography refers to a solid phase which is negatively charged, and which thus has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Positively charged molecules to be purified are bound to the support matrix which contains negatively charged groups (e.g., carboxy-methyl or sulphonic acid groups). Counterions used are normally sodium or potassium ions which are replaced with the positively charged sample molecules.

Media/Resins: 

Strong Cation exchange matrix: Cation exchanges can be classified as either weak or strong. A strong cation exchanger contains a strong acid (such as a sulfopropyl group, sulfonic acid) that remains charged over a wide pH range (pH 1-14). Suitable strong cation exchangers include charged functional groups such as sulfopropyl (SP), methyl sulfonate (S), or sulfoethyl (SE).

Weak Cation exchange matrix: A weak cation exchanger contains a weak acid (such as a carboxymethyl group) which gradually loses its charge as the pH decreases below 4 or 5.

–Commercial examples: 

Commercially available cation exchange resins include carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW® OR SP-SEPHAROSE HIGH PERFORMANCE®  from Pharmacia) and sulphonyl immobilized on agarose (e.g., S-SEPHAROSE FAST FLOW® FROM pHARMACIA). One exemplary cation exchange material is cross-linked methacrylate modified with SO3- groups available under the name Fractogel EMD SO3- (from Merk).

Cation Exchange Membranes (Anionic Membrane absorbers): See also antibody purification using ion exchange and also filtration, generally.

The majority of established applications are still performed on conventional ion exchangers such as Sepharose Fast Flow Q (GE Healthcare) but charged membrane filtration (Pall Corporation, Sartorius) is becoming increasingly popular (Gagnon, p. 492). See Anion Exchange Chromatography. 

Membrane absorbers with anion exchange ligands have recently seen increased applications in protein and mAb purificaiton processes. Membrane absorbers have large pore sizes and the mass transfer during membrane chromatography is believed to be convective (as monoliths) and not limited by diffusion. AEX membrane absorbers such as Q membrane filters have been used as a polishing step in the flow through mode and removed host cell impurities such as DNA and host cell proteins and provided significant viral reductions. In comparison to resin based conventional AEX chromatography, Q membrane filter was shown to have significantly higher linear flow rate, shorter process time and higher producitvity, and significantly reduced buffer volumes. While mbrane chromatography units are typically used as disposables, eliminating cost for cleaning and reuse validation, multiple reuse of AEX membrane filters has recently been reported, leading to futher reduction of raw material cost. (Lu Current Pharmaceutical Biotechnology, 2009, 10(4)). 

–Commercial Examples of Cation Exchange Membranes:

Mustang S (Pall Corporation) is a strong cation exchange membrane having a nominal pore size of 0.8 um and is commercially available in a single or multiple layer format. (Brown, 14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346).

Natrix S (Natrix Separations, Inc.) is a strong cation exchange membrane commprised of a non-woven highly fibrous durble polymeric substrate encased within a high surface area macro-porous hydrogel. (Brown, 14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346).

Sartobind S (Sartorius AG) is a strong cation exchange membrane having a nominal pore size of 3-5 um and is commercially available in a single or multiple layer format. (Brown, 14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346). 

Conditions/parameters: 

(DePhillips, J, Chromatography A, 933(2001) 57-72) discloses two adsorbent factors to be the dominant determinants of overall protein retenion on cation excahnge adsorbents; the anion type and the adsorbent pore size distribution.

There are 2 critical variables to investigate when developing the wash and elution conditions for antibodies: the buffer pH and the amount of salt in each buffer.  (Fahrner, Biotech. Genetic Eng. Rev. 18, 2001, p. 315 ¶s3-4). (see antibody purification).

–Binding:

Bill US 14/365,449 discloses a method for purification of proteins like antibodies by passing a sample thorugh a CEX under operating conditions of a buffer haivng a pH of about 1-5 pH units below the pI of the polypeptide and a conductivity of less than or equal to about 40 mS/cm, which causes the membrane to bind the polypeptide and at least one contaminant and then collecitng a fraction comprising the polypeptide of interest. 

Prior (US5,118,796) teaches that conditions where a protien such as an immunoglobulin is absorbed or not adsorbed refers to the pH and or salt concentrations. These conditions for a particular protein dpend on its primary structure and most specifically on the number, type and distribution of acidic and basic amino acids residues. In general, the protein will be positively charged at a pH below and hegatively charged at a pH above its isoelectric point (i.e., the pH at which the protein is neutral). Under reasonable conditions of inonic strenght, proteins with a net positive charge will be adsorbed to CEX and hegatively charged proteins to AEX. The major serum proteins components such as albumin have isoelectric points below that of most antibodies and are not sufficiently positivley charged at pH 5-8 to be adsorbed by the resin; however, immunoglobulins are generally adsorbed under these conditions if salt concentration is sufficiently low. In addition, most endotoxins and all nucleic acids are predominatly anions at neutral pH and at pH 5-8. They will therefore not be adsorbed to a CEX resin at that pH range due to electrostatic repulsion.

–Washing: 

Basey (WO99/57134). discloses purifying a polypeptide fro an impurity such as an acidic variant by binding the polypeptide to a CEX using a loading buffer at a first conductivity and pH, washing the CEX with an intermediate buffer at a second condcitvity and/or pH which is greater than that of the loading buffer so as to elute the contaminant, washington the CEX with a wash buffer which is at a third conductivity and/or pH which is less than that of the intermedaite buffer  and washing the CEX with an CEX with an elution buffer at a fourth conductivity and/or pH which is greater than that of the intermediate buffer so as to elute the polypeptide from the CEX. Accordingly, the process invovles changing the conductivity and/or pH of buffers in order to purify the polyeptide. 

–Elution (pH vs salt):

(1) Salt Gradients: 

—Sodium chloride: Traditional CEX is operated at acidic condition with sodium chloride as the elution component. The resulting cation column pool contains a high conductivity. A dilution step frequently is needed to lower the conductivity prior to the next polishing, anion exchange chromatography step. Beside the inconvenience with salt gradient elution, the use of salts in chlorine base at acidic condition has been reported to be problematic to manufacturing facilities. The salts under such condition are the root for metal corrosion for large tanks and column. Zhou describes the development of a pH conductivity hydrbid gradient elution with cation exchange for scale process mAb production(Zho, J. Chromatography A, 1175, 69-80, 2007).

Falkenstein (US13/994673) teaches applying a solution comprising different isoforms of an antibody to a CEX, applying a first solution with a first conductivity to the CEX and then eluting the antibody with a second solution with a higher conductivity. 

(2) Ph Gradients:

The use of a pH gradient as opposed to conventional salt gradient for elution in cation exchagne chromatography was explored by Mhatre “Purification of antibody Fab fragments by cation-exchange chromatography and pH gradient elution” J. Chromatography A, 707 (1995) 225-231. The advantage of using a pH gradient vs. a conventional salt gradient is that the collected fractions contain very low levels of salt thereby eliminating the necesstiy of desalting.

Zwitterions at pHs between 4 and 9 such as glycine have also been used in Protein A elution buffers (Brown, WO 2006/099308).

See also antibody purification using Anion exchange chromatography under antibody purification and then ion chromatography. 

Anion exchange is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal (Strauss, Biotechno. and Bioengineering 102(1): 168-175 2009.)

General Principles: 

In an example of anion exchange, an anion exchange matrix is initially positively charged and in equilibrium wieth a negatively charged counterion (e.g., Cl-). When the negatively charged protein or peptide of interest is applied to the column, the macromolecule displaces the chloride counterion and remains bound to the matrix. To elute the macromolecule, a higher concentration of counterion (e.g., 1MCl-) is added to the column so that the protein is displaced by the strong competition of the concentration counterion and is eluted from the column.

Thus a typical anion exchanger will bind proteins which have a net negative charge (i.e., when the pH of the solution is above the isoelectric point of the protein). In reality, the surface of a protein does not prevent a singular charge; rather it is a mosaic of positive, negative and neutral charges. Surface structure is specific to a given protein and will be affected by solution conditions such as ionic strengh and pH. This uniqueness can be exploited to establish specific donitions where individual proteins will bind or release form the anion exchange resin (US 2011/0213126).

Types of Ligands/Media/Resins

Anion exchange chromatography uses a positively charged group (weakly basic such as diethylamino ethyl, DEAE or dimethylamino ethyl, DMAE; or strongly basic such as quaternary amino ethyl Q or trimethylammonium ethyl, TMAE or quaternary aminoethyl, QAE) immobilized to the resin. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010)

Weak anion exchange Groups: The charge group on a weak anion exchanger is a weak base, which becomes deproteonated and, therefore, loses its charge at high pH. DEAE-sepharose is an example of a weak anion exchanger, where the amino group can be positively charged below pH around 9 and gradually loses its charge at higher pH values. Weak anion exchange groups include N,N diethylamino or DEAE.

Strong anion exchange Groups; A strong anion exchanger, on the other hand, contains a strong base which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). Strong anion exchange groups include trimethylammonium chloride.

–Eshmuno® Q resin: is a strong AEX coupling tentacle structure with a hydrophilic polyvinly etehr base matrix which is avaialbe from Millipore Sigma (now owned by Merck). The functioanl group is TMAE. 

–Fractogel® strong anion exchangers: include Fractogel EMD TMAE, Fractogel® EMD TMAE Hicap which are available from Merck EMD. The structure of the Fractogel® partciles is different form that of other hydrophilic chromatographic resins like dextran, agarose or cellulose. Fractogel® is a synthetic methacrylate based polymeric resin providing excellent pressure stability resulting in high flow rates. In constrast to carbohydrate supports Fractogel® media are also resistant to microbial degradation. (see Fractogel EMD, Process media, D9a form Merck). 

–Q-Sepharose (Q stands for quaternary ammonium) is an example for a strong anion exchanger. 

–Sepharose Fast Flow is a strong anion exchanger sold by GE Healthcare Life Sciences which is composed of crosslinked 6% agarose beads, with quaternary ammonium (Q) strong anion exchange groups. Sepharose Fast Flow Q is a homogeneous agarose based strong anion-exchanger and has been used in the final flow through application for established mAb productions (Wang, J. Chromatography A 1155: 74-84 (2007).

Commercial Suppliers

–Anion Exchange Membranes:

Membrane adsorberts include ChromaSorb, Sartobind.RTM. Q (available from Sartorium BBI Systems GmbH), Sartobind.RTM, Sartobind.RTM, Phenyl, Pall Mustang.RTM available from Pall Corporation). (Wang, US20120264920)

Commercially available anion exchange media are membrane adsorbers such as ChromaSorb™ (imillipore Corporation,), Mustang Q (Pall Corporation), Sartobind Q (Sartorius Stedim, Germany) as well as bead media such as Q Sepharose FF (GE Healthcare), DEAE cellulose, QAE SEPHADEX and FAST Q SEPHAROSE™ (Pharmacia). 

Mustang Q (Pall Corproation) is a strong anion exchange membrane having a nominal pore size of 0.8 um and is afailable in a single or mltiple alyer format. (Brown, 14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346).

Sartobind Q (Sartorius AG) is a strong anion exchange membrane having a nominal pore size of 3-5 um and is commercially available in a single or multiple layer format. (Brown, 14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346).

Sepharose CL, Sepharose Fast Flow, and Sepharose High Performance ion exchange media consist of macroporous, beaded cross-linked agarose to which charged groups are attached. The type of charged group determines the type and strenght of the exchanger, while the total number and availability of charged groups determine the capacity. Sulfonic and quaternary amines form strong ion exchanges, which are completely ionized over a broad pH range. All others form weak ion exchanges, where the degree of dissociation, and thus the exchange capacity, varies markedly with pH. “Strong” and “wek” refer to the extend of ionization with pH, and not to the strengh of binding. (see signma-aldrich.com, product information, 2012).

—-ChromaSorb membrane based anion exchanger: is designed for the removal of trace impurities including HCP, DNA, endotoxins and viruses for MAb and protein purification. (Wang, US20120264920)

Operating Modes

Anion exchange steps operated at very low Kp values (<0.1) are considered to be operating in the “flow through” mode. Conditions with an intermediate Kp, of between 0.1 and 20 are considered to be in the “weak partitioning” mode, and conditions with a Kp values >100 are considered “bind and elute” steps (Iskra, Biotechnology and Bioengineering, 110(4), 2013).

Bind and elute mode: 

Bill (US 14/365,449, published as US 2014/0348845)  disclose a method for purification of a protein such as an antibody by passing a smple thorugh an AEX at operating conditions comprised of a buffer having a pH of about 1-5 units above the pI of the polypeptide and a conductivity of less than or equal to about 40 mS/cm, which causes the membrane to bind the polypeptide and at least one contaminant and then collecting a fraction from the AEX comprising the polypeptide of interest. 

Urthaler (US2004/002081) discloses that in AEX, the material binds to the matrix via electrostatic binding to charge moieties on the surface such as DEAE (diethylaminoethyl) or QA (quaternary ammonium) groups. Normally, binding is achieved at low salt concentrations and elution at increasing salt (usually sodium chloride) concentrations.

Flow-through mode: 

In most cases, AEX chromatography is carried out using flow-through (FT) fashion, in which impurities bind to the resin and the product of interest flows through. However, the use of conventional packed-bed chromatography with FT-AEX requires columns with a very large diameter to permit high volumeric flow rates which are required to avoid a process bottleneck. This disadvantage with AEX columns has led to the development of membrane chromatography or membrane absorbers.

Weak partitioning mode: 

WPC is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding ipurities includes high olecular mass species, which are typically not removed in flow through mode. This increased impurity removal is obtained at the cost of a reduction in step yeild. The step yield can be resotred by extending the columnn load and incorporating a short wash at the end of the load stage. (Iskra, Biotechnology and Bioengineering, 110(4), 2013). 

WPC is defined under conditions where the product Kp (distribution or partition coefficient defined as the ratio of the concentration of the solute bound to the resin divided by the concentration in solution at equilibrium) is between typical B/E and FT modes. The Kp values in WPC mode range from 0.1 to 20. WPC uses more stringest binding conditions than FT during the load and wash stages, often with only modest changes in counterion concentration or pH. This causes an increase in the product Kp, which results in stronger binding of product and impurities. The column loading is increased, and a short wash is included to maximize the product yield. Operationally, a WPC step uses the same stages of operation as an isocratic FT step, but enables higher loading and results in better product purity. In contast to AEX flowthrough, the superior performance of AEX in the WPC mode enables a two column platform. The upper limit of proudct Kp is dictated by a minimum target for the product recovery in the load eluate and wash stages. The lower limit is determined by the minimum impurity LRV required of the separation. Once experience is gained in defining the kp range for a specific resin, one can quickly establish an oeprating window of product Kp that meets the step objectives. (Kelley, Biotech. & Bioeng. 101(3), 2008). 

Combinations of AEX with other Chromatography

AEX-CEX-HIC:

     IL-12:  Deetz (US 5,853,714) teaches a method for purification of IL-12 by loading a mixture onto an AEX in flow through mode, then CEX in bind in elute mode and then HIC in bind elute mode followed by concentrating by tangential flow ultrafiltration. In another embodiment, the mixture is loaded onto an AEX at a pH of 8.0, washed with a solution of a pH of 5.5 and eluted from the AEX, loaded onto the CEX at a pH of 6.0, washed with a solution of pH of 7.2 and eluted followed by HIC. 

AEX-HIC-Protein A-AEX

     CTLA4: Leister (WO/2007/076032) teaches a method of purifying cytotoxic T lymphocyte antigen 4 (CTLA4) – IgG molecules by subsecting a cell culure supernatant to AEX to obtain an eluted protein product and subjecting this eluted protein product to hydrophobic interaction chromatography which is then subjected to affinity chromatography such as Protein A and AEX.

AEX-HIC-Isoelectric focusing/polyacrylamide electrophoresis:

Guild (WO/2006/020622) discloses a method of protein fractionation by applying a protein mixture to AEX. eluting with high salt buffer, HIC and eltuing with a low salt buffer and then iselectric focusing and polyacrylamide electrophoresis.

Agilent Buffering Advisor Software  

General Principles: 

Ion exchange uses an insoluble matrix that carries ionic charges that retard the movement of molecules of opposite charge. There are 2 types, one where the solid phase is negatively charged (cation exchange resin) or positively charged (an anion exchange resin). 

IEX has been a platform for antibody purificaiton for many years. Because antibodies have a more basic isoelectric point that the majority of other serum or contaiminating proteins, IEX is useful in purifying anitobdies regardless of isotype. The general strategy is to keep the pH below the isoelectric point for antibodies so that they will not bind to the AEX such as DEAE modified resin or, alternaitvely to raise the pH above the pI where the antibodies will bind to the DEAE groups. The opposit strategy works for cation exchanges. The bound antibodies are commonly eluted with a salt or pH gradient. (Josic “Analytical and Preparative methods for purification of antibodies” in “methods foor purificaiton of antibodies, Food technol. biotechnol. 39 (3), 215-226 (2001). 

IEX is based on differential adsorption of charged substances at oppositely charged surfaces of porous chromatogrpahic media. The strenght of such interactions and the adsorption capacity of the IEX media are assumed to vary inversely with conductivity.  Protein dynamic binding capacities on IEX are typically expected to decrease with increasing conductivity and decreasing prtoein charge. However, several models describe the complex adsorption mechanisms in IEX and departures from expected IEX behavior have been reported Harinaryayan (Biotechnol Bioeng. 2006, 95(5) 775-87).

Binding conditions: 

a. Generally/optimization: 

In ion exchange chromatography, proteins are separated based on their charge. Proteins consist of various amino acids whose side chains may usually carry, in addition to uncharged radicals, also acidic and basic radicals and which thus contribute to the total charge of the protein. At low pH, below the isoelectric point of the protein, the total charge is positive, due to protonation of the charged side chains. At higher pH it is negative due to deprotonation. Since proteins carry a multiplicity of charged groups whose actual charge depends on the pH and also the environment of the individual amino acid, separation according to charge is a powerful method of separating proteins. In a ion exchange chromatography, the pH is chosen so as to enable the protein of choice to bind to the matirx. In the case of anion exchangers, this pH is usually at least one pH unit above the isoelectric point of the protein (pH at which the protein has a net charge of 0). In the case of cation exchangers it is below the isoelectric point. Binding to the matrix takes place via electrostatic interavctions. Proteins which do not bind to the matrix are washed out with buffer (WO2005/100394). Protein purification using ion-exchange chromatography usually employs positively charged anion exchangers because the majority of proteins are negative charged at neutral pH (i.e., have a low isoelectric point).

IEX chromatography steps are widely applied in protein purificaiton processes because of their high capacity, selectivity, robust operations, and well-understood principles. Optimziation of IEX steps typically involves resin screening and selection of the pH and coutnerion concentraitons of the load, wash, and elution (Kelley, Biotechnology and Bioengineering, 100(5), 2008). 

In summary, the key determinant for adsorption to an ion-exchange matrix is the charge of a peptide or protein. Thus, a protein has an affinity for an anion-exchange matrix if the protein has an overall negative charge, and conversely, a cation exchange matrix binds a positively charge protein. Because of the ionization state of surface amino acids, the net charge of a protein or peptide varies with the pH of the buffer. The pH is referred to as the protein’s isoelectirc point (pI) when the total number of + charges on a protein equals the number of – charges, in other words, when the protein’s net charge is zero. A protein is negatively charged at a pH above its pI and positively charged at a pH below its pI. For most separations, a pH that is 1 U from the pI of the protein is best for achieving the reversible binding required in ion exchange chromatography (see, Bollage, “Ion-Exchange Chromatography” ch. 2 in “Methods in Molecular Biology, Vol 36, Peptide Analysis Protocols”.)

However, the behavior of proteins on ionic exchanges can be unpredictable as shown by Etzel (US 5,986,063). The isoelectric point is 4.2-4.5 for alpha-lactalbumin and 5.1 for beta-lactalbumin. As pH increases, one would the alpha-lactalbumin to elute first from a cation exchange because its net charge switches from positive to hegative at a lower pH than does beta-lactoglobulin which would elute second at a higher pH. However, this does not occur. Alpha-lactalbumin does not fully elute until about pH 6.5 which is 2-2.3 pH units above its PI, whereas the majority of the beta-lactoglobulin elutes at about pH 4.6 as expected, because this pH is close to its PI.

b. pH: 

Ansaldi (WO 99/62936) discloses a method for separating a polypeptide monomer form a mixture comprising dimers and/or multimers, where the method comprises applying the mixture to either a cation exchange or an anion exchange chromatography resin in a buffer, wherein if the resin is cation-exchange, the pH of the buffer is about 4-7, and wherein if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture at a gradient of about 0-1 M of an elution salt.

Hickman (WO 2010/048192) diclsoes the purification of antibodies using anion exchange in flow through mode where suitable equilibration conditions are 25 mM Tris, 50 mM sodium chloride at pH 8.0.

c. Conductivity:

In ion exchange chromatography, adsorbability is well known to decrease by an increase in electrical condutivity of a treatment liquid. Accordingly, in a sample haivng high electrical conductivity, the electrical conductivity needs to be decreased to 5 mS/cm by dilution or deminerlaization before applying adsorption treatment. In order to compesnate for such a disadvantage, achromatography media simultaneotusly haivng hydrophobic itneraction, hydrophilic interaction, a chelate interaction and so forth in addition to the electrostatic interaction has been developed (see mixed mode chromatography). (Matsumoto, US 2015/0344520; see also Matsumoto, US Patent Applicaiton No: 16/349,6127, published as US 2019/0345194) 

d. Addition of chemical compounds:

–EDTA: Charlton (WO2011/073235) disclsoes a method for purifying polypeptides by increasing the mount of polypeptide of interest that binds to an ion exchange matrix relative to the amount of the impurity. This is achieved by adding a chemical compound in the process which by also binding to the IEX due to a charge that is opposit to the charge of the IEX, reduces binding of the impurity more than the binding of the polypeptide of interest. 

Washing:

a. salt concentration/conductivity and/or pH: 

Emery (WO/2004/024866) discloses a method for purifying a polypeptide using an ion exchange resin with an equilibrium buffer having a first salt concentration/conductivity. The exchange is washed with a wash buffer until a predetermiend protein concentration is measured in the flowthrough and during the washing the salt concentration of the wash buffer increases from an initial, second salt concentration/conductivity that is greater than the salt concentration/conductivity of the equilibration buffer to a final third salt concentration.

Basey (WO99/57134) disclsoes a method for purifying a polypeptide by IEX which involves chaing the conductivity and/or pH of buffers during intermediate washings. Thus a polypeptide of interest is bound to the IEX at an initial conductivity or pH and then washed with an intermediate buffer at a different conductivity or pH or both. Then and contrary to standard practice, the IEX is washed with a wash buffer where the change in conductivity or pH or both from the itnermediate buffer is in an opposite direction. 

Elution: 

Elution is generally acheived by increasing the ionic strenght (i.e., conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to acheive elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (ste elution) (Rossi, WO/2005/049649). In IEC, NaCl has been the choice of the salt for elecution (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555).

–Ph and salt gradients: IEX with salt gradient has been used to separate N-terminus variants of recombinant Il-1beta, C-terminus variants of monoclonal antiboides, deamidated variants of mAbs and ribonuclease A. In generaly, a disadvantage of salt gradietn elution is the need for gradient mixing, which can be cumbersome to implement on a process sale. Moreover the salt may have to be removed before the next processing or formulation step. An alternative to salt gradient elution is elution with pH gradients. In this approach, ideally the charge variants are eluted in order of their pI, with more acidic proteins eluting earlier in cation excahngers and later in anion exchangers.  (Pabst, Biotechnol. Prog 2008, 24, 1096-1106).

Types: 

There are two main types of ion exchange chromatography:  a) cation exchange: and b) anion exchange resin: See more information on left panel.A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1-14 whereas a weak cation exchanger contains a weak acid (such as a carboxymethl grou) which gradually loses its charge as the pH decreases below 4 or 5.

General Principles: RPLC separates on the basis of hydrophobicity. As with other HPLC techniques there is a polymeric stationary phase, of for example polystyrene/divinylbenzene. The mobile phase is usually a combination of a weak aqueous buffer or a dilute acid and a water miscible organic solvent. For effective separation of proteins the mobile phase is generally a gradient system required to achieve separation and is preferably linear for convenience. RP-HPLC can efficiently separate molecular species that are exceptionally similar to one another in terms of structure or weight. 

RP-HPLC procedures currently represent the majority of applications for peptide analysis and purification adn over 80% of all analytical sutides with proteins. The dominant effectin refersed-phase chromatography is a hydrophobic interaction between the nonpolar amino acid residues of peptides or proteins and the nonpolar ligands, typically immobilized onto the surface of a spherical porous silica partcile, although nonpolar polymeric sorbents derived, elg., from cross-liniked polystyrene-divinylbenzene can also be employed. (Analysis of Proteins, unit 10:13:1, Current Protocls in Molecular Biology “”HPLC of peptides and proteins: standard operating conditions”). 

A sample is dissolved in mobile phage (1) column: the sample is pumped under high pressure onto a column. Need to consider the mode of separation here (ie., adsorption, reversed phase, ion exchange). (2)elution: the sample is eluted through the column (3) detection: the eluant is monitored with a detector. Detector possibilities include UV, radiochemical, fluorescent, Mass Spec. A new method is evaporative light scattering where there is 1) nebulization (the column effluent passes through a needle, mixes with nitrogen gas and forms a dispersion of droplets, 2) mobile phage evaporation (the droplets pass through a heated drift tube where the mobile phase evaporates, leaving a fine mist of dried sample particles and 3) detection (these sample particles pass through a flow cell where they intercept a laser light beam which scatters the light generating an electrical signal). (4)analysis: Data from the detectors is fed into a computer.

Comparison with HIC: RPLC is closely related to HIC as both are based upon interactions between solvent accessible non-polar groups on the surface of biomlecules and hydrophobic ligands of the matrix. However, ligands used in RPLC are more highly substituted with hdryophobic ligands than HIC ligands. While the degree of substitution of HIC absorbents may be in the range of 10-50 umoles/mL of matrix of C2-C8 aryl ligands, several hudred umoles/mL of matrix of C4-C8 alkyl ligands are used for RPLC. RPLC is also distinct in that HIC is performed in aqueous solvent conditions and changes in ionic strenght are used to elute the column. The protein typically binds in the native state via hydrophobic groups located on the surface of the protein, and the native state is retained during the elution conditions. In contrast, RPLC utilizes a hydrophobic solvent (typically acetonitrile) and the binding of a ligand is a function of the phase partition between the hydrophobic nature of the solvent and column functional group. Proteins are typically denatured to some extent in such solvents and bind due to the hydrophobic nature of the entire polypeptide sequence. Since the majority of hydrophobic groups are located in the core of globular proteins, the binding is related to the extent of denaturation of the protein and accessbility of these groups to the column functional groups.

The Source 30RPC column is a polymeric reverse phase matrix. It is based on rigid, monosized 30 micron diameter polystyrene/divinyl benzemne beads. 

Parameters/Conditions

Mobile Phase:

The organic solvents acetonitrile and methanol are often used in the mobile phase in reversed phase chromatography (“Tips for practical HPLC analysis –Separation Know-how – Shimadzu LC World Talk Special Issue Volume 2.)

In RP_HPLC iscratic eltuion, step elution or gradient elution modes can be utilized to purify peptides and prtoeins. Besides the requirement for an organic solvent to be used as a surface tension modified, ion-pair reagents are utilized at low pH (e.g., pH 2.1) to suppress silanophilic interactions between free silano groups on the silica surface and basic amino acid reisudes.  (Analysis of Proteins, unit 10:13:1, Current Protocls in Molecular Biology “”HPLC of peptides and proteins: standard operating conditions”). 

The price of Protein A affinity chromatography resins is many times the cost of non-affinity supports. Accordingly Protein A resins are typically recyled by treatment with strong chaotropic solutions (urea and guanidinde hydrochloride) or strongly acidic solutions (such as acetic acid) or a combination thereof. Johansson (US2008/0230478)

Particular Agents used for Protein A Regneration

Equilibration buffer: The most commonly used cleaning is a simple wash with buffer, such as the equilibration buffer. Johansson (US2008/0230478)

Equilibration buffers include phosphate and carbonate buffers. Ladiwala (US 13/543650)

Neutralization agents: include phosphate buffers, weak acids and weak bases.

Acid treatment: Washing with an equilibrium buffer can only be used to restore the Protein A matrix a limited number of times. For a more efficient cleaning, treatments with acid and/or base are frequently used, each removing acid and base-sensitive contaminants respectively. Johansson (US2008/0230478)

Acidic solutions include phosphoric and acidic acid. Ladiwala (US 13/543650)

Alkaline protocol (NaOH) (Cleaning In Place (CIP)): In order to even more efficiently restor an Affinity A matrix, an alkaline protocol known as CIP is commonly used with many matrices. The standard CIP involves treatment with 1 M NaOH, pH 14. This harsh treatment will efficiently remove undesired fouling asuch as by protein aggregates and the like, but may on the other hand impair some chromatography matrices. For example, many affintiy matrices, wehre the ligands are proteins cannot withstand standard CIP. Protein A ligands need to be cleaned under milder conditions than conventional CIP in order to maintain selectivity and binding capacity. Thus such a sensitive matrix may be cleaned with standard CIP, if a reduced performance is acceptable. Johansson (US2008/0230478) However, the most extensively used cleaning and sanitising agent is NaOH in a concentration ranging from 0.1 up to 1 M, depending on the degree and nature of contamination. (Ander, 14/385336). 

Chaotropic agents: include guanidine HCl, urea and guanididne acetate. Ladiwala (US 13/543650)

Protein A media can be re-used after cleaning with 6M urea or 6 M guanidine hydrocholoride, which are known as milder cleaning buffers than sodium hydroxide. However, urea is relatively costly and due to its fertilising effect, it cannot be readily disposed of without taking certain precautions. Johansson (US2008/0230478)

Reducing agents: include thioglycerol, 1-4, dithiothreitol and 2-mercaptoethanol. Ladiwala (US 13/543650)

As the skilled person in the field will recognize, adding a reducing agent in a chromatographic process may entail the risk of retaining reducing agent in the matrix, which could potentially harm or contaminate the target molecule. By performing at least one of acidic and alkaline regeneration subsequent to the addition of reducing agent, such risk is minimized. Johansson (US2008/0230478)

Organic soolvents:

Braunger (US 2005/0056592) discloses reeenration of absorbent matrices such as Protein A where the regeneration solution includes an organix solvent such as n- or isoproanol. water-miscile alchols such as methanol, ethanol, t-butanol and secondary butanol, isobutanol.  In one ebmodiment, both an acid solution and an alkaline solution are used consecutively in one, more or all the cycles, preferably with the acid solution preceding the alkaline solution. The organic solvent is prefereable always included in the first of them. 

Chloridne dioxide:

Hamilton (US 2007/0093399) disclsoes that chlorine dioxide is an effective method of cleaning and regenerating chromatography media. 

Regeneration Schemes

Acid solution – Aklaine solution:

Bian (US2016/0193633) discloses methods for cleaning a Protein A chromatography column using both acidic (e.g., H3PO4) and alkaline (eg.g, NaOH) solutions in an alternating manner. 

Braunger (US2005/0056592) discloses a regeneration scheme for adsorbent matrices using an organic sovlent having a pH less than 4 (the acid components as part of the buffer scheme include carboxylic acids such as acetic acid, citric acid, malic acid) and then an alkaline solution with a pH greater than 10 but less than 13. The scheme that is the combination of an acidic solution which contains an organic solvent, preferably isoprobanol or n-propanol, and a second regeneration step with an alkaline solution solution, preferably 5-40 mM sodium hydroxide results in a surprisnly effective regeneration of the matrix with no blockage of the column after more than 100 cycles. 

Mahajan (US 14/479092, published as 2015/0093800 and US 15/920237, published as US 2019/0054396) discloses a method to clean a chromatogrpahy material for reuse by passing two or more material volues of elution buffer comprising about 0.15 M acetic acid at about pH 2.9, statically holding the material in elution buffer for about 10-30 minutes, passing two or more material volumes of elution buffer and then passing about two or more material volumes of regeenration buffer comrpsing about 0.1 N NaOH at about pH 13. 

Chaotropic agent + reducing agent: 

Madani (US6,972,327) teaches a method for regenerating a Protein A chromatography resin by washing the resin with a chaotropic solution such as guanidium HCL containing a reducing agent which is a source of free thils such as glutathione, dithiothreitol (DTT), 2-mercaptoethanol, dithionitrobenzoate (DTNB) and cysteine.

Ladiwaia (US13/543650) also teaches a method for regenerating an antibody binding resin by conducting two separate steps; a first wtep of washing the resin with a reducing solution selected from the group consisting of thioglycerol, 1-4,-dithiothreitol and 2-mercaptoethanol and a second step of washing with a chaotropic solution selected from the group consisting of guanidine HCL, urea and guanidine acetate.

-(acid solution) -(neutralization) -Reducing agent -(equilibration buffer) – chaotropic agent :

Ladiwala (US 13/543650) discloses a method for regenerating chromatography resins which includes a first step of washing the resin with a reducing solution having a reducing agent such as thioglycerol, 1-4-dithiothreitol or 20mercaptoethanol or thioglycerol followed by a second step of washing with a chaotropic solution which includes a chaotropic agent such as guanidine-HCL, urea or guanidine acetate.

Reducing agent -(acid solution) (–alkaline solution) – alkaline agent  -equilibrium: 

Johansson (US 2008/0230478) discloses regeernation of a seapration matrix by reducing regenration by contacting the matrix with a reducing agent, alkaline regeneration and equilibration of the matrix, wherein the steps can be performed in any order. In one embodiment, the process includes acidic regeenration by contacting the matrix with an acidic solution at any time after elution but before quilibration. In a specific embodiment, the acidic regeneration is carried out after the reducing regeneration. In one embodiment, acidic and alkaline regeneration are carried out subsequent to the reducing regeneration.

See also multimers of SpA

In General and Definitions

Cross-Link: is a bond that links one polymer chain to another. They can be covalent or ionic bonds.

Many different types of coupling groups can be introduced into a support. Examples include carboxyl grou pactivated by N-hydroxy succinedimide (NHS), a carboxyl group, a cyanogen bromide activating group, a hydroxyl group, an epoxy group, an aldehyde group and a thiol group. Then the ligand which might for example have a primary amino group can be coupled to the support. For example, a carxoyl group activated by NHS, a carboxyl group, a cyanogen bromide active group, an eopxy group and a formyl group are all capable of coupling with a primary amino group. A sapcer may also be inserted between the support and the coupling group. Koguma (US13/996385)

Method of attaching Ligands to the Support

The ligand may be attached to the support via conventional coupling techniques utilising, e.g., amino and/or carboxy groups present in the ligands. Bisepoxides, epichlorohydrin, CNBR, N-hydroxysuccinimide (NHS) etc are well known coupling reagents. Between the support and the ligand, a molecule known as a spacer can be introduced, which imporves the availability of the ligand and facilitates the cheical coupling of the ligand to the support. Alternatively, the ligand may be attached to the support by non-covalent bonding, such as physical adsorption (Ander, US14/385336)

Immobilising ligands to anyone of the above supports is easily performed by the skilled person in the field followng well known methods (Hermanson, “Immobilized affinity ligand techniques”, Academic Press, INC, 1992).

Protein A can be immobilized via a covalent bond to the supporting medium surface by contacting the protein A with a coupling group immoibilized to the surface of the support. Any method may be employed for introducing the coupling group into the support, however, a spacer is generally introduced between the support and the coupling groups. For example, a graft polymer chain having a coupling group at a temminal and or a side chain may be introduced into the supporting medium. The polymer chain may be previously prepared and then coupled wiht the support or the graft chain can be directly polyemrized onto the support via methods such as a “living radical polymerication method” and a “radiation graft polymerization method”. The “radiation graft polymerization method” is preferably since a reaciton initiator does not need to be introduced in advance into the support and the method is applicable to various types of supports. The graft chain is introduced by the “radiation graft polymerization method” by any means for generating radicals from the support such as ionizing radiation like gamma or beta ray or an electron or neutron beam. Examples of monomers having the coupling group to be used in graft polymerization include monomers such as acrylic and methacrylic acid when a carboxyl group is used as the coupling group. When a primary amino group is used as the coupling group, an allyl amine can be used. When an epoxy group is used as the coupling group, glycidyl methacrylate can be used. The monomer having the precursor functional group can be converted into the coupling group is grafted onto the support and then the precurosor funcitonal group grafter may be coverted into the coupling group. The glycidyl methacrylate (GMA) having an eopoxy group can for example be converted into various functional groups by use of various ring-opening reacitons of the eopoxy group. When a carobxyl group is used as the coupling group, ring opening half esterification reaction can be employed where first, GMA is graft polymerized and then the eopoxy group of the GMA is hydrolyzed into a diol, a cyclic acid anhydride is reactive with the hydroxide group derived form diol through a ring opening half esterification reaction to form a carboxyl group derived from the cyclic acid anhydride. The cyclic acid anydride is desirably succinic anhydride or gltaric anhydride. The catalysist used in the ring opening half esterification includes triethylamine, isobutyl ethylamine, pyridine and 4-dimethylaminopyridine. The ring opening half esterificaiton reaction is preferably performed in an inert organic solvent such as toluene. An NHS activation reactions refers to a step of converting the carboxyl group form by the ring opening half esterificaiton reaction into an active ester. The active ester has a high reactivity compared to the carboxyl group and the protien A can be quickly immobilized onto the carrier.  The Protein A which has an amino group reactive with the active ester to form an amide bond and is thereby immobilized ot the support via a covalent bond. (Koguma, US13/996385). 

Methods of attaching protein ligands such as protein A nd G to a solid support have been described. Typically the media is activated with a reactive functional group such as an epoxide (epichlorohydrin), cyanogens (cyanogens bromide (CNBR)), N,N-disuccinimidylcarbonate (DSC), aldehyde or an activated carboxylic acid (e.g., N-hydroxysuccinimide (NHS) esters, carbonyldiimidazole (CDI) activated esters). These activated groups can be attached directly to the base matrix, as in the case of CNBr, or they can be part of a linker or spacer molecule which is typically a linear chain of carbon, oxygen and nitrogen atoms, such as the ten membered chain of carbon and oxygen found in the linker butaenidiol digycidyl ether (a common epoxide coupling agent). (Bian, US 7,833,723, 7,846,682 and US2011/0105730).

Majima (US 14/916,316, published as US 2016/0215027) teaches a multimeric immunoglobulin binding protein having 6-10 total domains from SpA such as the C domain having the general structure (R1)n-(R2m) or (R2m-(R1)n where the R1 domain is an amino acid sequence in which a non-lysine amino acid has been replaced such as the lysine being substituted with a non-lysine amino acid at 1-3 of positions 4, 7, and 35 R2 is an immunoglbulin binding domain coccuring at the N-temrinus or the C-terminus of the protein and includes an amino acid residue that covalently bonds to an insoluble support and the R2 domain is based on an amino acid sequence in which the lysine residue(s) are substituted with a non-lysine amino acid only at position 35, or at posiiton 35 and one or more positions 4, 7 and 35. In one embbodiment the R2 domain includes substitutions of 1-6 amino acid reisdues with lysine at positions 40, 43, 46, 53, 54, and 56. Because only the (R2) domains are selectively immobilized on the support via a covalent bond, it is possible to acheive a highly selective immobilization reaction trhough for example a lysine or a cysteine residue. The R1 in trun has an amino acid sequence that does not contain an amino acid that is active to the chemical reaction used for immobilization. When the immobilization reaction used to immobilzie the protoien on the support takes place via an amino group, an R2 domain can be produce by substituting the lysine residues contained in the amino acid sequence with non-lysine aino acids only in lysine residues occurring at positiion(s) that interfere with binding to an immunoglobulin upon immobilizing the protein on the support and by substituting some of the non-lysine amino acids not involved in bidning to an immunoglobulin with lysine. In the case of immobilization via a thiol group, a new cysteine residue can be added to the R2 domain. A support immobilization reaction using a disulfide bond or a maleimide group that is highly selective to the thiol group may be used for said immobilization.

Using a disulfide or a thioester bond:

A cystein can be introduced into the carboxy end of a protein A moelcule and the protein A immobilized to a support via a covalent bond at a single site by specifically using a sultheydryl (SH) group which is a side chain of the cystein residue. In a first method of using a dislufide bond, a surface having a sulfhydryl group exposed to the suface such as for example activated Thiol SEPHAROSE 4 B is used as the support and a disulfide bond is formed by the condensation reaction between sulfhydryl grups in both the recombinant protein A and the support.  In a second method of using a thioether bond, a glycopolymer support such as agarose is preliminarily activated with an active epoxy group introducing reagent such as epichlorohydrin and a thioether bond is subsequently formed together with the sulfhydryl group of the protein A. In both methods, protein A can be immobilized at a single site at the carboxy end, which brings about advantages such that the binding stability due to the covalent bond can be ensured and the moelecualr orientation can be uniformed while sustaining the binding sites of the protein A. Iwakura (US 2008/0051555) 

Methods of coupling protein A to a solid matrix such as crosslinked, uncharged agarose (Sepharose, freed from charged fraction of natural agarose) are commonly known in the art and include coupling via primary amino functions of the protein to a CNBr-activated matrix. Recombinant protein A can also be attached by a thiether sulfur bond to the support as is described in US 6399750 from Pharmacia and commercially available as Streamline™ or MabSelect™ from Amersham-biosciences. The thiether can be a thieoether bridge generated by reacting the sulfhydryl group of a cysteine residue of the protein A with an epoxide group on the activated support (WO 2004/076485).  With MabSelect, the ligands comprise recombinant Protein A coupled to a cross-linked agarose support via a C-terminal cysteine. The median particle diameter is 85 um (Berg, US2006/0134805).

Attachment of recombinent Protein A to the column matrix via a single thioester bond allows for higher capacity protein A column. Such single-point attachment by means of suitable reactive residues which further are ideally placed at an exposed amino acid position, namely in a loop, close to the N- or C-temrinus or elsewhere on the outer cirucumference on the protein fold. Suitable reactive groups are sulfihydryl or amino functions. More preferabley, such recombinant protein A comprises a cysteine in its amino acid sequence. Most preferably, the cysteine is comprised in a segment that consists of at least 30 amino acids of the C terminus of the amino acid sequence of the recombinant proteiin A, preferably the protein A is attached by at least 50% via thioether sulfphur bond to the matrix. An example of such an embodiment is described in US 6,399,750 from Pharmacia and is commercially availabe under the tradenames MabSelect™ from Amersham Biosciences. However, the leakae rate of such Protein A matrices if often drastically increased in contrast to tradition, multi-point attached natural Protein A matrices obtained by CNBr coupling (US2008/0312425).

–Thiol groups of arginine or cysteine (e.g., C-terminal cysteine -Thioether bridge coupling):

The protein can be coupled via a C-terminal cysteine provided on the protein. This allows for efficient coupling of the cysteine thiol to electrophilic groups e.g., epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling. (Ander, US14/385336)

Hall (WO2008/039141) discloses that mehtods are readily available for coupling of protein ligands via certain amino acids preferably amino acids that contain nitogen and/or sulphur atoms such as arginine or cysteine. In one embodiment, coupling group is in the C terminal region.

Johansson (US 6,399,750) disclsoes a Protein A based matrix which is attached to the column matrix via a single thioester bond and/or a secondary amin (–NH–)

Ljungquist (Eur. J. Biochem. 186, 557-561 (1989) discloses that a single cysteine residue introduced into the C terminal part of different recombinant protein A molecules can be used for immobilization on a thiol containing solid matrix.

Amino groups of an N-terminal amino acid and lysine: One can use a carrier having an amino group or a carboxyl group and subjecting the carrier to react with a carboxyl group or an amino group of a protein ligand in the presence of a dehydration condesning agent such as a water soluble carbodiimide, to thereby form an amide bond. Tamori (US13/636410

carboxyl grups of a C-terminal amino acid, glutamic acid, and aspartic acid: Protein A can also be immobilized in a C-terminal carboxyl group selective manner via an amide bond on a carrier where a polymer compound having a primary amino group has been introduced (JP 2005-112827A).Using a carrier having a carboxyl group and activating this carboxyl group with N-hydroxysuccinic acid imide to react with an amino group of a protein ligand Tamori (US13/636410).

Epoxy Group – Amino group:

One can also introduce an epoxy group to a carreir by means of bisepoxide, epichlorohydrin or the like and subject the carrier to react with an amino group, a hydroxyl group or a thiol group of a protein ligand. Tamori (US13/636410).

Tamori (US2007/0224424A1) teaches magnetic particles which may contain an epoxy group and reacting substances having a biotin bonding site with the epoxy group.

–ring-opening epoxy group:

The affinity particles can include a ring-opening epoxy group produced by ring-opening of the epoxy group.  The ring-opening epoxy group is produced by ring-opening of an epoxy group as a result of reacting the epoxy group with a nucleophile compound that includes a hydroxide ion, a chloride ion, a mercapto group, an amino group, or the like.  The ring-opening epoxy group may be a substituted or unsubstituted 2,3-dihydroxypropyl group.  An unsubstituted 2,3-dihydroxypropyl group may be produced by ring-opening of a glycidyl group via hydrolysis, as an example. A substituted 2,3-dihydroxypropyl group may be produced by ring-opening of a glycidyl group using a mercapto group-containing blocking agent (e.g., mercaptoethanol) or an amino group–containing blocking agent (e.g., monoethanolamine. (Tamori, US2011/0262748A1) (see also Tamori, US13/636410).

Hydroxyl group on carrier – Amino group:

One can use a carrier having a hydroxyl group and activating the carrier with a cyan halide such as cyan bromide to react with an amino group of a protein ligand. One can also tosylate or tresylate a hydroxyl group of a carrier and subject the carrier to react with an aino group of a protein ligand. Tamori (US13/636410)

Multi-point Attachments of Multi-Domains

Bian (US 12/653888 and EP2202310) discloses chromatography ligands comprising 2 or more B or Z domains of SpA attached to a chromatography resin at more than one site on the resin. In some embodiments the ligands comprising 2 or more domains of SpA are attached to a solid support at more than one site via non-discrimbinate, multipoint attachment. SpA contains abundant free amino groups from numerous lysines in each domain. Thus the attachemt of an SpA domain to more than one site on a solid support can be achieved by reacting the amino group of lysine on SpA, via epoxide ring-opening or reductive amination, respectively. In certain embodiments, multipoint attchment can be acheived by the reaction of one or more naturally occurring amino acids on Sp having free hdyroxyl groups, such as, for example, serine and typosine, with a support containing an epoxide group via a ring opening reaction. Alternatively, multipoint attachment can be acheived, for example, by the reaction of naturally occurring amino acids SpA having free carboxylic acid groups, such as aspartic acid and glutamic acid, with a support containing amino groups via, for example, N, N’-carbonyldiimidazole.

See Linkers for Affinity Chromatography

Types of Linkers:

Flexible Linkers:

Flexible linkers are usually applied when the joined domains require a certain deree of movement of interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (eg., Ser or Thr) amino aicds. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains.. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous wolutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaciton between the linker and the protein moieties. (Chen, “Fusion Protein Linkers: Property, Design and Funcitonality” Adv Drug Deliv Rev: 2013, October 15, 65(1); 1357-1369. )

Polypeptides that form suitable flexible linkers are well known int he art. Flexible linkers typically include glycicne, because this amino acid, which lacks a side chain, is unique in its rotational freedom. Serine or threonine can be interspersed in the linker to increase hydrophilicity. (Kim, US 2007/0042378). 

Unstructured random coil peptide linkers may, but do not necessarily, comprise Gly-rich regions, ntoably unforlded character of any lenght or a combination thereof. (Lin, US 2016/0185826)

Rigid linkers:

While felxible linkers have the advantage to connect the functional domains passively and permitting certain degree of movements, the lack of regidity of these linkers can be a limitation. There are several examples wehre the use of flexible linkers resulted in poor expression yields or loss of biological activity. For instance, a Tf-granulocyte colony stimulating factor (G-CSF) fusion protein failed to be expressed with a felixble (GGGGS)c linker. In anotehr report, the immunoglobulin binding ability of protein G domain in a protein G-Vargula luciferase fusion protein was not recovered after interserting a flexible GGGGS linker. (Chen, “Fusion Protein Linkers: Property, Design and Funcitonality” Adv Drug Deliv Rev: 2013, October 15, 65(1); 1357-1369.

–Alpha helix-forming linkers: with the sequence of (EAAAK)n have been applied to the construction of many recombinant fusion proteins. Many natural linkers exhibit alpha helical structures. The structure is rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone. Therefore, the stiff alpha helical linkers may act as rigid spacers between protein domains. (Chen, “Fusion Protein Linkers: Property, Design and Funcitonality” Adv Drug Deliv Rev: 2013, October 15, 65(1); 1357-1369.)

Rigid linkers may, but do not necessarily comprise at least one alpha-helical structure, Pro-rich sequence or a combination of both. (Lin, US 2016/0185826)

Clevage linkers: 

Clevable linkers may, but do not necessarily, comprise at least one disulfide bond, thrombin-senstive sequence, protease-sensitive sequence or a combination thereof. (Lin, US 2016/0185826)

Non-Covalent Protein Adsorption

Non-covalent methods of protein immobilization are widely employed and involve either passive adsorption onto hydrophobic surfaces or electrostatic interactions with charged surfaces. Here, the use of nitrocellulose membranes or polystyrene microtiter plates for hydrophobic adsorption and polysine coated slides for electrostatic binding are known. (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)

Covalent Immobilziation Methods

For more stable attachment, the formation fo covalent bonds is required which are generally formed through reaction with functional groups present on the protein surface. (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)

Amide bonds: The exposed amine groups of Lys residues readily react with supports bearing active esters, with the most common being N-hydroxysuccinimide (NHS) esters to form stable amide bonds. A disadvantage of using NHS esters is that they are unstable in aqueous conditions and thus attachment of protoein in aqueous buffers will compete with ester hydrolysis, resulting in only modest immobilizaiton yeilds.  (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)

Thioether bonds: The Cys residue bearing the thiol group is often employed for protein immobilziation and readily ungergoes conjugage addtion tihe alpha2beta-unsaturated carbonyls (e.g., maleimides) to form stable thioether bonds.  (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)

Biotinylate proteins & immobilziation onto avidin-functioned substrate:

Streptavidin-biotin binding is a rapid, specific and can occur under conditions where most other ptoeins have denatured, wuch as high temperatures. A breakthrough for the sue of bitoin for protein modifciation was harnessing the cell’s natural machinery for bitoin conjugation, using the E. coli enzyme BirA to acheive precise biotin modificaiton. The natural substarate of BiA is the biotin carboxyl carrier protein (BCCP) requring fusion of at least 75 residues to the target prtoein. However, phage display selection enalbed the development of the AviTag which is superior to BCCp as a BirA substrate but only 15 mino acids in lenght, so estending the range of prtoein sites amenable to site-specific enzymatic biotinylation. (Methods Mol. Bio. 2015, 1266, 171-184). 

Yao (Nature protocols, 1(5), 2006) discloses site-specific biotinylation of proteins using in vitor, in vivo and cellfree systems for teh purpose of fabricating functional prtoein arrays. Biotinylation of recombinant proteins relies on the chemoselective reaction between cystein-biotin and a reactive thioester group at the C temrinus of a protien generated via intein-mediated cleavage. 

Yao “Intein-mediated biotinylation of proteins and its application in a protein microarray” J. Am Chem. Soc. 2002, 124, (2002) discloses  an intein-mediated expression system to express, purify and site-specifically biotinylate prtoeins, followed by immobilization onto avidin-functionalized glass slides. Three model proteins with an intein tag (intein fused to chitin binding domain) at their C-termini) were purified and biotinylated in a single step, by first loading the crude cell lystate onto a column packed with chitin beads adn then flushing the column with biotinylated cysteine. Further improvement may be made by using streptavidin as the immobilization agent on the slide, in place of avidin, which is a glycoprotein and known to have higher nonspecific binding characteristics. 

Commercially availabe Resins

Thermo Scientific SulfoLink Cupoling Resin: is a porous, crosslinked, 6% beaded agarose that has been activated with iodoacetyle groups for covalent immobilization of cystein-peptides and other sulfhydryl molecules. Iodoacetyl groups react specfically with sulhydryls to form irreversible thioether bonds. The resin has been used for example to immoblilize antibody binding prtoeins such as Protein G onto a resin. Kossiakoff (US 2018/0044385