See also “smart polymers” and “partitioning” right hang panel.

Polyelectrolytes are water-soluble polymers comprised of charged monomer units. The electrolyte (charged functionality) group undergoes protein dissociation (ionization) in aqueous solutions according to acidity (pH). Negatively charged, anionic polyelectrolytes and positively charged, cationic polyelectrolytes may be used in a precipitation reaction to purfiy antibodies.

When the solution pH is less that the pI of a particular antibody, the antibody is positively charged. Under these conditions, a cationic polyelectrolyte may precipitate impurities and leave the antibody of interest in solution. Conversely, an anionic polyelectrolyte may precipitate the antibody forming a protein-polyelectrolyte precipitate, leaving impurities in solution (WO2008/091740A2). 

Polyelectrolyte Precipitation Methods In General

Jaber, Moya, etc. (US 8,691,918; see also WO 2011/146394) disclose separating a target molecule by providing a sample containing the target molecule, contacting the sample with a soluble stimulate responisve polymer comprising a polyelectrolyte backbone comprising one or mroe hydrophobic groups attached to the backbone to form a complex of the polymer and one or more impurities, adding a stimulus to the sample to precipitate the complex out of solution to thereby separate the target molecule from one or more impurities. The resulting clarified cell culture fluid may be subsequently passed through a capture step using a chromatography media to bind the desired target molecule. The selection of the particular stimulus responsive polymer to sue is based on what the polymer is intended to bind. For example, in case of a biomolecule that has a net hegative charge at a pH above its pI (e.e.g, whole cells, cellular debris, DNA, endotoxins and proteins), a stimulus responsive polymer having a polyelectrolyte backbone that is cationic (i.e., positively charge) is desirable to use. On the other hand, in case of a biomolcule which has a net positive charge at a pH below its pI (.e.g. certain proteins), a stimulus responsive polymer compirsing a polyeletrolyte backbone which is anionic (i.e., negatively charged) is desirable to use.

Moya (US 8,362,217 and and WO2008/079280A1) (See also Moya, US2008/0255027 and US 13/747495) discloses a Ph dependent polymer such as poly(4-vinylpyridine-co-styrene) which has an affinity for a desired biomolecule such as an antibody in the insoluble state. In a first step, a mixture is harvested from cell broth, then the mixture is conditioned to the correct pH (e.g., below 5) to maintain the polymer in solution, then the mixture conditions are changed to cause the polymer to precipitate out of solution by altering the pH (e.g., 7.0) and the antibody recovered such as by elution. Moya discloses that preferred pH sensitive soluble polymers include cationic polyelectrolytes such as chitosan, polyvinylpyridines, primary amino containing polymers, secondary amine containing polymers and teriary amino containing polymers and anionic polyelectrolytes such as acrylic acid, methacrylic acid and methyl methacrylate. The polymers have the capability even when precipitated to selectively and reversible bind to one of more biomolecules of interest. Preferred polymers have electrostatic and hydrophobic ability. As some of these polymers may not have the ability to selectively bind or elute the desired molecules, they need to be modified with ligands of chemical groups that will complex with the desired molecule and hold it in complex and then release it under appropriate elution conditions. Such chemical groups can include carboxylated and pyridine groups formed as part of or attached to the polymer.

Anionic polyelectrolytes (polyanion polyelectrolytes): See outline

Cationic polyelectrolytes (polycation polyelectrolytes): See outline

For stimuli responsive polymers (smart polymers) see outline.

Affinity Precipitation In General

In comparison to nonspecific precipitation using as for example ammonium sulphate precipitation, affinity precipitation makes use of the selective binding an an affinity molecule to a target molcule. 

A distinction is sometimes drawn between first and second order affinity precipitation.

(I)  In first order affinity precipitation, both the affinity molecule and the target molecule have two binding sites, so that it is possible for a network to form between the two molecules and an affinity complex is formed that sediments at a specific size.

Ambrosius (US 14/151949, published as US 2014/0128578) disclose affinity precipitation using a binding protein with two binding sites such as a dimer of protein A or protein G. Preferably, the monomers of the dimer are linked to one another via a disulphide bond.

(II) In second order affinity affinity precipitation, the affinity molecule is bound to a stimulable substance, usually a polymer, which can change in response to various conditions like pH or temeprature, usually a polymer. The stimulatable substance changes its solubility characteristics as a result of a change in the ambient conditions, such as e.g. a shift in the pH or temperature, and precipitation occurs. Unlike first order affinity precipitation, there is no need for a bifunctional affinity molecule or for a bifunctional target molecule. 

For example, Stayton (US 7,625,764) discloses precipitation of IgG (Fc-protein) using Protein A-conjugates. The conjugation partner for the Protein A is stimulus responsive polymer which includes pH and temperature sensitive polymers.

Specific Affinity Precipitation Molecules

Protein A/G:

SpA forms large networks with IgG to form a precipitate and both the dimeric and tretrameric domain B proteins form precipitates with IgG as well. (Saito (Protein Engineering 2(6), 481-487, 1989)

Ambrosius (US 14/151949, published as US 2014/0128578) discloses a process for the selective concentration of immunoglobuilins or other proteins that contain an Fc domain by using an Fc-binding protein such as Protein A or G with precisely two binding sites under conditions that allow binding to occur, separating the precipitate fromt he liquid phase and undoing/eluting the binding of the target protein from the Fc binding protein. In one embodiment the Fc binding protein is a dimer of an Fc binidng domain of Protein A or G. 

Ligand (e.g., Protein A) attached to coodinating moiety (e.g., M+):

Patchornik (US 2006/0121519) discloses ligands such as protein A, which are attached to at least one coordinating moiety such as a bis-chelator capable of directing the composition of matter to form a non-coavlent complex when co-incubated with a coordinator ion or molecule such as M+, M2+, M3+. The target protein of interest can then be recoved form the precipitate formed. In one embodiment preicpitation of a target molecule is obtained using biotin as the coordinating moeity. A ligand with a covelaently bound bi-biotin or biotin derviative sucha sDSB-X Biotein is incubated in the presence of the target molecule. Introducstion of avidin (or its derivatives) creates a netword comprising ligand-coorindating moiety (biotin) target molecuel avidin. In anotehr embodiment purification of an antibody is demonstrated using a modified Protein A (ProA-CAT) adn Fe3+ions. Binding of the ProA-CAT to teh target IgG leads to the formation of the ProA-CAT target IgG) soluble complex. Additon of Fe3+ ions to the complex geenrates insoluble macro-complexes containing the target IgG. Impurities eleft in the supernatant are discarded via centrifugation. Target IgG is eluted under acidic conditions without dissociating the ProA-CatFe3″ macro-complex of the insoluble pellet. 

See also ethanol fractionation under plasma proteins.  See also precipitation of plasma proteins such as antibodies under “IVIG Preparation” see also Affinity precipitation  See also Polyelectrolyte Precipitation 

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subject of proteins from a protein solution, or as a very specific method to recover a single protein of interest form a purification step ((Current Protocls in Protein Science, “Extraction, Stabilization, and Concentration” Unit 4.5) Typical precipitation agents are ethanol, polyetheylene glycol, lyotropic (i.e., anti-chatropic) salts such as ammonium sulphate and potassium phosphate and carpylic acid (WO 2007/073311).

Precipitation methods can be classified in 2 grouping; those that selectively precipitate antibodies, mainly by altering the properties of the solvent and those that selectively precipitate contaminants by complexation with agents that reduce their solubility. The first group that selectively precipitates antibodies includes precipitation with salts such as ammonium sulfate, with organic polymers such as polyethylene glycol (PEG) and by electrolyte depletion. The second group includes precipitation by organic complexants such as short chain fatty acids and organic bases (Gagnon, “Purificaiton Tools for Monocloanl Antibodies” Validated Biosystems 1996, pp. 1-269). 

Salt Precipitation (“salting out”) See also isolation of IgY by ammonium sulfate precipitation

High salt concentraiton promotes protein aggregation and precipitation. Alhtough the mechanisms is not well understood, the salt is thought to remove the water of solution from the protein, thereby reducing protein solubility. The Hofmeister series represents decreasing anion effectiveness: citrate>phosphate>sulfate>acetate>chloride>nitrate>thiocynate. The salts at the low end of theis series casue structural damage to proteins. The high solubility of ammonium sulfate in water and the position of sulfate in the Hofmeister series make it the most popular choice for slating out proteins. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Precipitation is widely used for product recovery of biomolecules such as immunoglobulins. The most common type of precipitation of proteins is salt induced precipitation. (WO 2007/073311)

Temponi (Hybridoma, (8)(1), 1989, 85-95) discloses that the sequence (NH4)2SO4 – caprylic acid precipitation and caprylic acid precipiation of IgG mAbs from acitic fluid was comparable. 

Caprylic Acid (CH3(CH2)6COOH) See also Precipitation of Plasma proteins with fatty acids

Cell Culture-Caprylic Acid

Arunakumari (WO2010/151632) disclose methods for purifying a protein of interest such as an antibody from a cell culture by precipitation of the contaminants such as host cell DNA with 1% caprylic aicd. Mixtures that were thus depleted of contaminants can then be sued directly in downstream chromatography applications such as IEX. 

Jiao (US 2005/0272917) discloses a method of purifying IgG from a feedstock which can be supernatant from a cell culture that includes adjusting the pH to 4-5.5 then contacting the pH-adjusted feedstock with a mono or polyakanoic acid haivng between 4-12 carbon atoms, preferably caprylic acid. After the addition of alkanoic acid, the supernatant containing the IgG is separated form the precipitate as by centrifugation or filtration. The supernatant is then dialzyed against a buffer haivng a pH of about 4.5-6.0. 

—-Cell Culutre -Caprylic Acid – Allantoin

Gagnon (US 15/121,676, published as US 20160362446) discloses a method of purifying an antibody by contacting a propertin preparation such as a cell culture harvest having an antibody with at least one fatty acid such as enanthic (heptanoic) acid, caprylic acid, pelargonic (nonanoic) acid or capric (decanoic) acid having 7 to 10 carbon atoms to form a mixture, contacting this mixsture with allantoin and then removing solid materials form the IgG containing liquid. 

—-Cell Culture -Caprylic acid – PEG and/or CaCl2:

Jungbauer (US 15/029,682 published as US 2016/0272675; see also WO2015/056237) discloses precpitating a clarified cell culture supernatant using 100 mM sodium citrat, pH 4.5, 1% caparylic acid and removing the precipitate to provide a secondary supernatant to which 21% PEG at pH 7 was added so as to precipitate the antibody. This precipitate can be resuspended and treating using CaCl2 to precipitate further impurities such as DNA which can be removed from the solution coprising antibodies. 

Wien, “Continous precipitation of therapeutic proteins, with an emphasis on monoclonal antibodies” Thesis, December 2013) discloses various sequences for mAb purificaiton from CHO cell culture sueprants using caprylic acid (CA), calcium chloride (CaCL2), polyethyle glycol (PEG) and ethanol (CEP). In one embodiment, a two step precipiation using CA/PEG was performedfwhere CA was used for precipitating HCP and dsDNA and PEG was used for mAb capturing from the supernatant. 

 –Cell Culture – Electropositive multivalent ions  (e.g., Tren) + Fatty Acids (electronegative)

Gagnon (US 14/769,098, published as US 2016/0009762 and US 9,994611) also discloses a method of purifying a protein such as an antibody by conditioning an impure protein preparation by contacting it with (i) at least one electropositive organic additive such as methylene blue, ethacridine, chlorhesidine, benzalkonium chloride, cetyl trimethyl ammonium bromide, (Tris(2-aminoethyl) amine (TREN) AND (ii) at least one electronegative organic additive such as  octanoic acid and subsequently removing solides thereby removing at least 95% of chromatin and then applying the conditioned preparation to an adsorptive chromatography medium.   In one embodiment the method includes adding allantoin to carify chromatin from a cell culture harvest, then methylene blue or ethacridine or cetyl trimethyl ammonium bromide or chlorhexidine is added. Then particles bearing an electropositive metal affinity ligand tris (2-aminoethyl)amine (TREN) is added. In a separate embodiment allantoin is added to a cell culture and then heptanoic acid  or octanoic acid or pelargonic (nonanoic) or capric acid is added. The mixture is then stirred for 2 hours and the electropositive metal affinity ligand tris(2-aminoethyl)amine (TREN) are added. In some embodiments the method reduces the contaminants that interfere with a subsequet purificaiton Protein A purificaiton step. 

–Cell Culture — Electropositive multivalent ion (e.g., ethacridine) OR Fatty (electronegative such as caprylic acid) — PEG

Gagnon (US 14/766,131, published as US 2015/0376231 and US 9,988419) discloses a method for purifying a desired protein such as an antibody by conditioning the preparation with ethacridine or an electronegative organic additive such as octanoic acid, thereby removing at elast 90% of chromatin and then precpitating the antibody with PEG in the presence of non-protien-recpitating salts to provide a precipitate with the antibody.  

–Cell Culutre -Fatty acid (e.g., caprylica cid) –substrate with metal binding functional group + allantoin

Gagnon (14/894,993 published as US 2016/0115194 and US 10,174,076) also discloses pufication of a protein such as an antibody with caprylic acid followed by porous particle treatment and further purificaiton by precipitation with polyethylene glycol. In one embodiment a cell culture harvest is contacted with caprylic acid and with a functionalized substrate such as a metal binding funcitonal group such as tris(2-aminoehyl)amine). “TREN’ refers to Tris(2-aminoethyl) amino and is an electropostive compound that is known for strong affinity for metal ions. It may be chemically affixed to various soluble and insoluble materials. In one embodiment allantoin is also present. 

Protein A – Caprylic Acid

Brodsky (Biotechnolgy and Bioengineering, 109(1), 2012) discloses subjecting samples of protein A purified mAbs having been eluted at pH 3.6 which were then dialyzed into 50 mM acetate or 5-0 mM citrate at different pHs between 4.5-5.5 and then precipitation of contaminants by adding 10 mL of caprylic acid to 10 mL of dialyzed mAb solution at the same pH, and removing the precipitate.

–Protein A — Caprylic acid -IEX:

Zheng (US 15/523632), published as US2107/0313742) disccloses a process of purifying a prtoien of interest such as an antibody by subjecting a mixture containing the antibody to Protein A affinity chromatography, recvocering the protein in an eluate, adding caprylic acid to precipitate contaiminants, removing the contaminant precipitate and subjecting the solution to a second chromatography step such as IEX. 

Di and Trivalent Metal Cation Precipitants (see also removing aggregates)

Metal ions such as Mn2+, Fe2+, Ca2+, Mg2+ and Ag+ bind to different protein funcitonal groups and can cause them to preciptiate. They act at much lower concentrations than the ions of Hofmeister series (see aobve) and are easily removed by ion-exchange adsorption or chelating agents. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Di and trivalent metal cations (e.g., Zn2+, Mn2+, Ca2+ and Al3+) provide two means for precipitating proteins; they may act directly or often are used as auxiliary agents for other precipitation methods such as the crowding method using neutral polymers ((Lovrien, Unit 4.5 from Current Protocols in Protein Science). Flocculation reagents such as calcium chloride and potassium phosphate have been studies to vaoid potential issues related to polymer or residual monomer toxicity and the need for their removal through subsequent purification steps. The amount required can be very low (e.g., 20-60 mM calcium chloride with an equimolar amount of phosphate added to form clacium phosphate. The mechanism is thought to be related to the co-precipitation of clacium phosphate with cells, cell debris and impurities. (Liu, mAbs, 2:5, 480-499, 2010)

Thio-heterocyclic cations: refers to an organic cation having 3 rings in a coplanar arrangement bearing at least one thiol group and at least one amino goup, bearing at least one positive charge where the positive charge may reside with the thiol or with an amino group. 

–Methylene blue (IUPAC name: [7-(dimethylamino)phenothiaziin-3-ylidene]-diemthylazanium] (AKA “basic blue 9): 

Gagnon (US 14/766, 123, published as US 2015/0376230) and WO 2014/123486) discloses a method of reducing aggregate content in a protein preparartion haing a target protein such as an antibody by contacting the preparation with a thio-heterocyclic cation such as methylene blue to form a mixture and then contacting the mixture with at least one functionalized solid such a negatively charged solid used for CEX to remove excess thio-heterocyclic cations. In certain embodiments, the sample is additionally contacted with an antiviral agent such as benzalkonium chloride, chlorhexidine or ethacridine and/or a soluble organic modulator such as a noionic organic polymer along with an electropositive organic additie. 

Multivalent metal ions with monovalent/multivalent anions

Calcium/Potassium Phosphate

Coffman (US7,855280; See also WO 2007/035283) discloses using calcium and phosphate as precipitants of mAbs produced in CHO cells. 2M potassium phosphate is added to the culture medium and 5M calcium chloride was added to a concentration of 30 mM. The pH of the mixture was 7.3. The flocculated culture was incubated while mixing and then treated through centrifuge  and the antibody recovered in the supernatant to separate the solid from the antibody target. Coffman discloses that examples of first cations inclue calium (Ca2+), magnesium (Mg2+), aluminum (AL3+), copper (Cu(I) or Cu(II), etc) and examples of first anion include those preferred ligands to the metal ion used such as fluoride, phosphate, carbonate, sulfite, etc. 

Divalent Cation/Alcohol – Alchohol/Divalent Cation

Jungbauer (US14/423802, published as US 2015/0225473) discloses a method for isolating a protein such as an antibody by combining a cell culture supernatant with a divalent cation salt to precipitate impurities and then combining the supernatant with an aliphatic alcohol such as ethanol to precipitate the antibody, resuspending the antibody containing precipitate in a buffer comprising a divalent cation salt suitable for the precipitation of impurities therefrom to produce a protein contianing solution and combining the protein containing solution with an aliphatic alcohol such as ethanol to precipitate the antibody/protein and isolating the protein containing precipitate. In another embodiment the aliphatic alcohol is added first to form the protein containing precipitate  which is resuspended in a buffer comprising the divalent cation salt to precipitate impurities and the antibody containing solution is combined with the aliphatic alcohol to form an antibody precipitate.

Other Precipitating Agents

Allantoin (2,5-dioxo 4-imidazolidinyl urea):

Allantoin cyrstalling pwoder can be added to a protein solution at suerpstarated conentraitons, endotoins bind and undissolved cyrsals with bound endotoxins are removed by filtration or centrifugation. The method has been shown to remove an average of 99.98% endotoxin for 20 test proteins (Vagenende, J Chromatography A, 2013, 15-20). 

DNA intercalating Agents

–Ethacridine (7-ethaoxyacridine-3,9-diamine): 

Gan (J of Chromatography A, 1291 (2013) 33-40) discloses that IgM mAbs and aggregates in mammalian cell culture supernatants are associated with contaminants such as nucleosomes, DNA and histone proteins drived form nucleic of host cells and that for this purpose adding the DNA intercalating agent ethacrine and allantoin and then flowing the supernatant through a column of mixedmode particles bearing metall affinity, AEX and CEX functionalities was effective for removing the contaminants. 

Ethacridine (2-ethoxy-6.9-diaminoacridine lactate)

Ethacridine is one of several organic bses used for commecial fractionation of plasma proteins and the only one that has been adapted systematically for purificaiton of mAbs. It is strongly hydrophobic, positively charged and used at alkaline pH. This might lead one to believe that the precipitaiton mechaism is similar to octanoic acid but in fact the complexant is applied at much lower ionic strenght than octanoic acid and it precipitates contaminants by forming stable insoluble ionic complexes. Functionally ethacridine operates like a liqui phage anion exchanger, with selectivity to match. (Gagnon, “Purificaiton Tools for Monoclonal antibodies” Vaoidated Biosystems, 1996, pp. 1-269). 

Organic Solvents

Organic solvents are often used for fractional precipitation of proteins. One of the largest scheme for sepration of therpaeutic proteins from human plasma is based on a series of precipitation steps based on changes in temperature and addition of ethanol. In general, however, protein separation by precipitation lacks specificity which has led in the late 1970s to the development of affinity precipitation (see below).(Hibrig, J. Chromatography B, 790 (2003) 79-90).

Addition of a mild organix solvent to an aqueous protein solution reduces the solvent dielectric constant, thereby indcing protein precipitation. The solvent must be completely miscibe with water (e.g., ethanol and acetone). Solvent precipitation is typically performed at low temperature (<10C) because conformational regidity then prevents irreversible denaturation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Ethanol: Fractionation of antibodies by precipitation using ethanol has been in practice for many years. In plasma fradtionation, precipitaiton is still the method choice. In the case of the Cohn fractionation, immunoglobulins are precipitated in the so-called precipitate II+III with 25% ethanol, at pH=6.8, and the termpature of -5C (Josic, Food Technol. Biotechnol. 39(3) 215-226 (2001).

20% ETOH — 25% ETOH

(Methods in Immunogly and Immunochemistry, Curtis Williams and Merrill W. Chase, Volume 1) discloses a rapid approach where serum is diluted with distilled water, pH is adjustd to 7.7 and sufficient ethanol slowly added with vigrous stirring to give a final concentraiton of 20%. The insoluble proteins designated as precipitate A are removed by centrifugation. This precipitate A is suspended in cold NaCl, the pH lowered so that yG immunoglobulins remain in the supernatant while precipitating yA and yM immunoglobulins are in the precipitate (precipitate B). The supernatant is raised to about pH 7.4 and thanol added to concentraiton of 25% to precipitate yG immunoglobulins. 

Phillips (J Immunol Methods, 1984, 30; 74(2): 385-93) discloses using the method of Coh (1946) as modified by Deutsch (1967) for purifcation of IgG by ethanol precipitation. Antserum is deluted in deionised water and pH adjusted to 7.5, cooled to 0C and sufficient ethanol added to final concentraiton of 20% with vigorous stirring adn same time reducing temperature to about 0%c. The resulting recipitate is removed by centrifugation at 0% and resuspended in 15 mM NaCL, pH lowered to 5.2. The precipitated material contains IgA and IgM which can be removed by centifiguation. The pH of the suerpnatant is brought to 7.4 and 95% ethanol added to overall concentraito of 25% to precipitate IgG which can be removed by centrifugation. 

Polymer precipitation: (See outline)

Salting-Out Procedures

Salting out procedures have certain advantages over ethanol fractionation for preliminary concentration and partial purification of immunoglobulins. Among these are simplicity and relatively slight danger of denaturation. (Methods in Immunogly and Immunochemistry, Curtis Williams and Merrill W. Chase, Volume 1). 

Salting-out invoves the precipitation of a protein by the addition of high concentrations of neutral salts or amino acids under appropriate conditions of pH, total prtoein concentration and temperature. (Rothstein, “Differential Precipitation fo proteins” in Dekker, Bioprocess technology, 1994, 115-160).

Ammonium sulfate (NH4)2SO4 precipitation 

Ammonium sulfate fractionation, is generally employed as the initial step in the isolation of crude antibodies from serum or ascitic fluid. Lorette C. Javoid “Immunocytochemical Methdos and Protocols” Second Edition, volumen 115 in “methods in Molecular Biology”, 1999.

Ammonium sulfate precipitation also known as “salting out” is still widely used for protein separation based on the fact that the solubility of most proteins decreases at high electroyte concentration. Sulfate is used because multivalent ions are more effective than monovalent ions. This procedure is usually carried out in the cold with control of pH close to neutrality. In sequential combination with caprylic acid, precipitation can even achieve a crude antibody purification by precipitation alone.eted polypeptide.

Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins form targeted polypeptid(s). The preferred salt is ammonium sulfate. It precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discared (unless the targeted catalytic polyptide is hydrophobic) and ammonium sulfate is added to the supernantant to a concentration known to precipitate the target protection. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other method that rely on solubility of proteins, such as cold ethanol precipitation, are well known in the art and can also be used to fractionate complex protein mixtures (US 20060088883).

 By Lowering the pH: 

Proteins are soluble in water due to the interaction of their charged groups with ionized water molecuels. Adjustment of the pH to the isoelectric point (pI) yields minium solubility, since the net charge of the protein is eliminated. Most proteins have a pI<7, and the relatively low cost of acids make pH adjustment with acid a popular method of protein preciptiation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Binder (WO2011/110598) teaches a method for purifying cell cultivation supernatants by adjusting the pH value in the acid range so that nucleic acid and host cell protein is precipitated by the targeted polypeptide remains in solution.

Liddel (US 13/122676) disclsoes a process of purifying a fragment antibody from a culture by separating the fragment antibody from the medium by lowering the pH so that the fragment antibody is souble, but one or mroe of the impurities are insoluble. 

Lydersen (“Acid precipitation of mammalian cell fermentation broth” Annals New York Academy of Sciences, 745, 222-231, 1994) discloses a process for the purificaito of an antibody from impurities in a cell culture medium by reducing pH. Accordingly to the procedure, the antibody remains soluble and the impurities are precipitated. The precipitate is then removed during centriguation or microfiltration. 

Takeda (EP1561756A1) also discloses a method for removing impurities in a sample by converting the sample into an acidic aqueous solution of low conductivity at a pH below the isoelectric point of the active protein which can be an antibody and then removing the resulting particles. 

Lower pH + Divalent Cations:

Romero (WO 2008/127305) discloses a method of isolation a macromolecule such as an antibody by lowering the pH of the composition, adding a divalent cation such as Ca2+ and then separating the antibody from the impurity. 

See also partitioning in general under “partitioning”

Both polyer/polymer and polymer/salt systems have been used for the purificaiton of antibodies. Using polymer/salt systems, IgG can be partially purified without using any type of ligands. 

Aqueous Two-Phase Systems 

Co-precipitation of non-IgG contaminants with positvely charged polymers parallels the selectivity of anion exchange chromatography. These reagents selectively coprecipitate acidic host cell proteins, DNA and various cell culture additives. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70)

Aqueous two-phase system (ATPS) are formed spontaneously upon mixing two aqueous solutios of structurally different components, such as two polymers or a polymer and a salt, above a certain critical concentration. Extraction of antibodies in an ATPS constitues an interesting alternative to the traditional downstream processing of antibodies, with most of the research utilizing either a polymer-salt system or a functionalized polymer-polymer system. Most of the strategies have relied on enhancing the partitioning of antibodies towards the upper phase (PEG-rich) because most impurities partition preferentally to the bottom phase. (Azevedo, “Chromatography-free recovery of biopharmaceutical through aqueous two-phase processing” Trends in Biotechnology, (2009), 27(4):240-247

PEG/Salt Systems:

PEG/Nacitrate: Azevedo (J Chromatography A, 1213 2008, 154-161) discloses aqueous two phase extraction of antibodies using PEG and sodium citrate, allowing the concentration of the antibodies in the citrate rich phase and removal of most hydrophobic compounds in the PEG rich phase. 

PEG/NaCl/phosphate system: The addition of NaCl to polyethylene glycocl (PEG)/phosphate systems was shown to increase the partition coefficient of IgG from a CHO cell supernatant, from 0.06 to 36.4% (Azevedo, J. Biotechnol. 132, 2007 209). 

Tran (WO/2010/062244) discloses an aqueous two phase extraction precipitation process for antibodies using a forward extration PEG-Phosphate ATPE system in which the antibody is partitioned to the polymer rich phase and then a second ATPE back extraction system by introducing the polymer rich phase form the forward extraction to a new phosphate salt rich phase, cuasing the product to precipitate at the interface between the two phases. 

PEG/Dextran: PEG/dextran systems have been reported fro the purificaiton of IgG in the presence of affinity ligands. The addition of ligands containing carboxylic functionalities has been shown to direct the partition of IgG to the PEG rich phase. 

EOPO Systems:

EOPO-dextran: Aires-Barros (J. Chromatography A, 1995 (2008) 94-100 disclose using aqueous two-phase sytem composed of ethylene oxide/propylene oxide (UCON) and dextran for the purificaiton of IgG from CHO cells supernatant. In UCO/dextran systems IgG partitions preferentailly to the less hydrophobic dextran rich phase. The addition of triethylene glycol-diglutaric acid (TEG-COOh) shifted the IgG partition to the upper phase. Using an ATPS coposed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified in two steps with a global yield of 85% and 88% purity. 

By Hydrophobin – fusion molecules

Joensuu (US 14/902851, published as US 2016-0159854) discloses a method of purifying an antibody by means of a hydrophobin-Protein A fusion and a continous phase separation. In the method a surfactant or detergent is added to the protein liquid whih contains at least one hydropbin to form an extraction solution. Non-ionic surfactants are preferred. Hydrophobins can include HFBI, HFBII, Sc3p, certo-ulmin and cryparin. The mixture is then transferred to a separation vessel and phases are allowed to separate. After intial phase separation, mixed extraction solution is continously added to the separation vessel. Simultaneously a separated strem of the surfactant pahse and a separated stream of the aqueous phase are continsouly removed from the extraction solution. Pferably, the thoroughly mixed extraction solution is pumped into a separation vessel with a first flow speed, in which vessel a surfactn phase and an aqueous phase is allowed to separate. A part of the separated surfactant phase and a part of the aquous phase are continously removed from the separated vessel with a second flow speed, which keeps the volumes of the phase states in the separation vessel unchangeable. 

Introduction

Hydroxyapatite is commonly used for purification of antibodies Gagnon (US2008/0177048). 

Hydroxyapatite is chemically similar to the mineral component of bones and hard tissues in mammals. it is seldom pure in nature but rather often occurs mixed with fluorapatite. 

Along withe crystalline form of hydroxylapatite it is also possible to sue a creamic form which can be obtained by sintering. HA can be bought for example from Bio-Rad. Its ceramic HA is provided in two forms (type 1 and type 2). On account of larger surface areas, the type 1 material has a greater binding capacity for relatively small molecuels whereas the type 2 has larger pores which enalbe the penetration and thus better bidning of large molecules such as DNA or large proteins. (Kretschmar, US 7.939,643)

Other names: hydroxyapatite, hydroxylapatite, calcium hydroxide phosphate

Complexity: Due to the complexity of the mechanisms involved, antibody separations on HA remain difficult to predict and are not very widespread. However, HA can be efficiently used when applied after Protein A chromatography for the recovery of therapetuic antibodies form cell culture supernatants. (Roque, J. Chromatography A, 1160 (2007) 44-55. 

Modes of Operation

Bind and Elute mode:

Gagnon (US2009/0270596) disclsoes methods of pruifying antibodies by containing a sample containing the antibodies to a mixed mode chromatography matrix such as ceramic hydroxyapatite (CHT) or ceramic fluoroxyapatite (CFT), spearating the matrix with the bound antibodies from the solution and eluting the antibodies by chaing the pH or salt concentration.

–single-hydroxyapatite chromatographic step:

Ahn (1989, 17(3), 269-72) discloses development of purification of monoclonal antibody from ascites fluid using a single hydroxyapatite step.

Engelhorn (US 4745183) discloses purification of monoclonal antibodies of the same isotype but different light chain composition by hydroxylapatite and concentration gardient elution phosphate buffer, pH 6.0-8.0.

–Ceramic hydroxyapatite: IgG1 can be resolved form an IgG1-Protein A complex in unfractionated media on CHT ceramic hydroxyapatite (Chromatography, tech Bio-Rad note 2849; S.G. Franklin, Bio-Rad laboratories, Inc., 2000 Alfred Nobel Drive, hercules CA 94547 USA).

Flow through mode:

Vedantham (US 7,476,722) disclsoes a method of separating a protein such as an antibody from a second protein such as Prtoein A that is affixed to an affinity chromatography support which has been used to previously purify the antibody where the second protein/Protein A binds to the hydroxyapatite but the antibody/protein does not. The protein of interest which comprises an Fc region may also be TNFR:Fc. 

Weak Partitioning Mode:

Kelly (US 8,067,182) teachings using weak partitioning mode with hydroxyapatite chromatography for product purification.

Euilibration:

The column is usually equilibrated and the sample applied in a buffer that contains a low concentrate of phosphate. Adsorbed antibodies are often eluted in an increasing gradient of phosphate salts. Gradients of phosphate combined with non-phosphate salts such as sodium choloride have also been used for protein purification, including antibody purification. One such approach involves the application of a gradient of sodium chloride or potassium chloride while a low level of phosphate is held constant. Both hydroxyapatite and fluroapatite have been shown to be effective for removal of aggregates from many antibody preparations. Antibody aggregates usually elute after antibodies. (Gagnon (US2008/0177048)

Native hydroxyapatite and fluorapatite can be converted to calcium-derivatized forms by exposure to soluble calcium in the absence of phosphate. This converts P-sites into secondary C sites, agolishing phosphoryl cation exchange interactions, increasing the number of C sites and fundamentally altering the selectivity of the apatite support. Calcium derivatized apatites are restored to their native forms by exposure to phosphate buffer, at which poiht they may be eluted by methods commonly applied for elution of antive apatite supports. (Gagnon, US 8,093,364). 

Binding

Organic polymers: 

Gagnon discloses that the use of an aqueous-soluble (i.e., hydrophilic) nonionic organic polymer such as polyethylene glycol (PEG), polypropylene glycol, polyvinylpyrrolidone, dextran, cellulose and starch) enhances retention of antibody on mixed mode chromatography supports in comparison to most contaminating proteins. In one embodiment, a protein preparation may be applied to the mixed mode chromatography support under conditions that prevent the binding of non-aggregated protein while permitting the binding of aggregated protein and other large molecules (i.e., “flow-through” mode). In other embodiment, the preparation is applied under conditions that permit the binding of virus or non-aggregated protein and contaminants with fractionation of the virus or non-aggregated protein being achieved by changing the conditions such that the non-aggregated protein (.e.g, antibody) is eluted while contaminant remain bound to the support (e.g., “bind-elute” mode). (US13/178970). 

Gagnon (US2008/0177048) also discloses methods of purifyig intact non-aggregated antibody from an antibody preparation by contacting the preparation with a mixed mode chromatography support in the presence of an aqueous-soluble (i.e., hydrophilic) nonionic organic polymer such as PEG. The presence of a nonionic organic polymer enhances binding capacity of antibody on the support, thereby enabling higher levels of productivity to be acheived. The presence of nonionic organic polymer also preferentially enhances the retention of antibody on the support.

–Salt (NaCL):

Sun (WO/2005/044856 and US2005/0107594) discloses method for removal of HWMA from antibody preparations by contacting a mixture with a hydroxyapatite resin and selectively eluting the antibody. Alternatively, the antibody preparation may be buffer-exchanged into an equilibration buffer and then allowed to flow through a hydroxyapatite resin. A combination of these binding/flow through hydroxyapatite chromatography methods may also be used. In one procedure an elution buffer or load buffer that contains from 1-20 mM sodium phosphate and from 0.2-2.5M NaCl, wherein the elution bufer or load buffer has a pH from 6.4-7.6 was used.

Washing:

Morii (EP0333474) discloses removal of endotoxin contaminants from an aqueous solution of a valuable protein such as an antibody using hydroxyapatite which includes a wash with pH in the range of 6-8 and an amino compound such as arginine at a concentration 1-100 mM.

Elution:

Most non-antibody protein contaminants elute before antibodies on HA but different clones elute in different areas of pI of the elution profile and may therefore overlop to varying degrees with containating proteins. Antiibody aggregates usually elute after antibodies but may coelute to varying degrees Gagnon (US2008/0177048).

The effects of different salts on the selectively of a given apatite are unpredictable. For example, in the absence of phosphate, sodium chloride is unable to elute most IgG monoclonal antibodeis from native hydroxyapatite, even at concentrations in excess of 4 moles per liter. This iplies extremely strong binding. In exclusively phsophate gradients, IgG is typcially one of the latest eluting proteins, usually requiring 100-150 mM phosphate. When eluted with a combination of lower concentrations of both salts, such as 0.25 M sodium chloride and 50 mM phospahte however, IgG is one of the earliest eluting proteins. Other paradoxxes reinforce the point: increasing the sodium chloride concentration in the presence of phosphate, which casues IgG to bind less strongly, has the opposite effect on DNA. Additionaly, lysozyme elutes at a higher phosphate concentration than BSA in the absence of sodium chloride but fails to bind in the presence of 1 M sodium chloride. (Gagnon, US 14/819,334)

Sulfate, borate, and certain monocarboxylic acids or zwitterions are able to elute antibodies form apatite supports in the absence of phsophate and such elution produces unique selectivities that permit effective fractionation of, cinluding removal of aggregates, that may not be adequately served by elution with phosphate or by combinations of phsophate and chloride. (Gagnon, US 14/819,334)

Isocratic Elution:

Mazzola (WO 2009/017491) discloes a emthod of purifying antibody using ceramic HA wehre the ab is eluted by isocratic elution that simultaneously removes at least one impurity. 

Phosphate:

—-Phosphate gradients:

Hydroxyapatite is most commonly eluted with phosphate gradients. The strong calcium affinity of phosphate suspends calcium chelation and coordination interactions, while its ionic character suspends phosphoryl cation exchange interactions  (Gagnon, US 14/819,334, now US 9,938,317). Hydroxyapatite has been used in the chromatographic separation of proteins, nucleic acids as well as antibodies. The column is normally equilibrated adn the same applied in a low concentraiton of phosphate buffer and the absorbed proteins are then eluted in a concentraiton gradient of phosphate buffer ((Sun, US 2013/0107198) 

(Gagnon, US 14/819,334, now US 9,938,317) discloses mAb antibody cpature on calcium derivatized HA, conversion to native HA, and elution in a phosphate gradient. Specifically, HA was eluilibrated with CaCl2, cell culture supernatant continaing a mAb was equilibrated to 5 mM calcium by addition of 1 M calcium chloride and the sample applied to the column. The column was washed with 5 mM sodum phosphate and eluted with a linear gradient to 300 mM phsophate, pH 6.7. The antibody eluted in a single peak at about q165 mM dosium phosphate.  

—-Sodium Phosphate – Salt (NaCL): 

Some applications elute hydroxyapatite with combinations of phosphate and chloride salts. Chlorides preferentially elute the phosphoryl cation exchange interaction while having relatively little effect on calcium affinity interactions (Gagnon, US 14/819,334, now US 9938317). Elution of hydroxyapatite and fluorapatite with chloride gradients at low fixed concentraiotns of phosphate has been shown to be more effective than simple phosphate gradients for antibody preparations. (Gagnon US 7,691,980). 

Mazzola (US2010/0234577) discloses that a limitation of the repeated re-use of hydroxyapatite is the instability of hydroxyapatite in calcium chelating buffers, such as citrate. The elimiation of citric acid or alternatively filtration though an anion exchange filter so that a Protein A eluate can be directly loaded onto a cHA column can be ahevied by eluation from Protein A using sodium phosphate at pH 2.1-3.5. 

Nadkarni (US 15/778,290, published as US2019/0367556) disclsoes methods for purifying antibody drug conjugate monters form aggregates using HA using a low concentration of sodium phosphate buffer for the wash and eluting the ADC with a dosium phosphate buffer increasing concentration. 

Sun (WO/2005/044856 and US2005/0107594) discloses method for removal of HWMA from antibody preparations by contacting a mixture with a hydroxyapatite resin and selectively eluting the antibody. Alternatively, the antibody preparation may be buffer-exchanged into an equilibration buffer and then allowed to flow through a hydroxyapatite resin. A combination of these binding/flow through hydroxyapatite chromatography methods may also be used. In one procedure an elution buffer or load buffer that contains from 1-20 mM sodium phosphate and from 0.2-2.5M NaCl, wherein the elution bufer or load buffer has a pH from 6.4-7.6 was used.

NaCl gradients:

(Gagnon, US 14/819,334, now US 9,938,317) discloses intermediate purification of a mAb by binding in the presence of calcium, converstion to native apatite and elution in a sodium chloride gradient. Specifically, HA was equilibrated with CaCl2, about 50 mg of protein A purified mAb was equilibrated with CaCl2 and the sample was applied to the column. The column was washed with buffer having sodium phosphate and then eluted with a linear gradeint to 20 mM Hepes, `0 MM phosphate, 2 M soidum chloride, pH 6.7. The antibody eluted in a single peak at 0.6 M soidum chloride, follwed by a well separate aggregate peak. 

Schubert (J. Chromatography A, 1142 (2007) 106-113) discloses binding antibody at pH 6, 7, 7,8 and 8.2 and to HA and elution in a NaCL gradient.

Ionic Species

–Borate

(Gagnon, US 14/819,334, now US 9938317) discloses purificaitona of an IgG mAb from cell culture supernatant on native HA, eluted with a broate gradeint. The column was eluted with a linear gradient fo 1M sodium borate, 5 mM sodium phosphate, pH 7.0. The majority of contaminating proteins eluted before the antibody. Non-aggregated antibody eluted at an average conductivity of about 5 mS/cm. Aggregates eluted later. 

–Monocarboxylic acid (lactate)

(Gagnon, US 14/819,334, now US 9938317) discloses purificaiton of an IgG mAb from cell culture supernatant which includes elution onf HA using a linear gradient of sodium lactate. The majority of contaminating proteins eluted before the antibody. 

–Monocarboxylate (e.g., acetate) or Monocarboxylate zwitterion (e.g., glycine) 

Gagnon (US 14/819,334, now US 9938317; US 15/910,702, published as US 2018/0186831) discloses elution from apatite using a monocarboxylic which refers to organic acid salts having a single carboxylic acid moeity such as sodium or potassium salts of formic, acetic, propionic, lactic, pyruvic, gluconic or glucuronic acid OR with a monocarboxylic zwitterion which refers to a molecule containing a site carboxyl moiety and at least one moiety with a positive charge such as glycine, proline, lysine and histidine. In one embodiment, the elution is conducted in the presence of a nonionic organix polymer such as dextrans, starches, celluloses, polyvinylpyrrolidones, polypropylene glycols and polyetheylene glycol (PEG). 

–Sulfate

Gagnon, (US 14/819,334, now US 9938317) discloses purification of an IgG mAb form a cell culture sueprnatant on calcium derivatized HA, eluted with a borate gradient. The majority of contaminating proteins eluted before the antibody. Antibody aggregate eluted after non-aggregated antibody. The column was eluted with a linear gradient to 1M sodium borate, 2.5 mM calcium chloride, 10% PEG 600 pH 7.0. 

Gagnon (US 8,093,364) discloses methods of fractionating a desired antibody or fragemtn using a native or calciu-derivatized apatite chromatography support which incuddes eluting the support in the presence of an ionic species which is a sulfate, borate, monocarboxylic organic acid salt or monocarboxylic zwitterion. In certian embodiments the ionic species is the primary eluting ion in the eluent. In certain embodiments the eluent is substantially free of phosphate as an eluting ion. 

Non-ionic Polymers:

–Polyethylene glycol (PEG):

PEG modulates elution of antibodies in hydroxyapatite purificaiton processes (Protein & Peptide Letters, 2008, 15, 544-555). PEG imposes a size discriminating selectivity on HA which dramatically enhances aggregate removal efficiency. (Gagon “Nonionic polymer enhancement of aggregate removal in ion exchange and hydroxyapatite chromatography” 12th Annual Waterside condference, San Juan, Puerto Rico, April 23-25, 2007). 

Gagnon (US 7,691,980) disclsoes a bind-elute mode enhanced revmoal of non-antibody protein contaimants and aggregates from a preparation of unpurified mAb which includes HA, CHT Type II that includes elution with a gradient to 5 mM sodium phosphate, 2.0 M sodium chloride, pH 6.7. The run is repeated but eluted with linear gradient to about 5 mM sodium phosphate, 2.0 M sodium chloride and about 5% PEG-6000. 

Hydroxyapatite in Combination with other Chromatography

Affinity chromatography – hydroyapatite: 

Vedantham (US 2003/0166869 and WO/2003/059935) discloses a method for separating a protein such as an antibody by affinity chromatography and then subjecting the protein to HA in flow through mode.

–Affinity (Protein A) – Ion Exchange – Hydroxyapatite: Mazzolo teaches a method of capturing IgG using protein A followed by at least one ion exchange prior to adsorbing the IgG to HA and selectively eluting.

–HCPLC – hdroxyapatite: Yamakawa (J. Liquid Chromatography, 1988, 11(3) 665-81) teach HPLC on spherical beads of hydroxyapatite to purify moncolconal antiobdies secreted into mouse ascitic fluid.

Resins with multimodal ligands such as Capto MMC and Capto Adhere (GE Healthcare) can be used as polishing steps for antibody purificaiton. The multimodal functionality of the resin provides selectivity that is different from standard ion exchange ligands, which makes them suitable for solving purification problems at both high and low conductivity or pH. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Lali (US2010/0113746) discloses a process for purification of IgG using an affinityligand  which is ionic, hydrophobic and/ or mixed mode (combinations of groups). In one embodiment the IgG is eluted with a buffer having a pH of 2.5 to 9, containing an organic or inorganic salt and containing an additive such as ethanol, ethylene glycol, glycerol, polyethylene glycol, surgars so as to ehance recover of the IgG in elution.

Types of Mixed Mode Chromatography

Affinity Mixed Mode

–Affinity – Cation Exchange (CEX)

Kihara (US Patent application No: 15/952,627) disclsoes an affinity chromatography carrier which includes a substrate that is composed of a polysaccharide (e.g., crosslinke agarose-based substrate such as Sepharose 6 Fast Flow (GE Healthcare GMbH, (“SEPHAROSE”), an acrylate-based polymer, a methacrylate-based polymer or a styrene-based polymer, an affinity ligand that includes an antibody-binding polypeptide such as Protein A and a carboxy group that is introduced at 15-60 mmol/L-gel. (1 meq/L-gel- 1 mmol/L-gel). Examples of introducing a carboxy group into the carrier include reacting the carrier with chloroacetic cid under alklaine onditions, a carboxymethyl group is introduced into a part of hydroxy groups on the surface of the carrier, an aldehyde group is introduced into a carrier, a carboxy roup is introduced into the aldehyde group by reductive animation through an amino group by reductive amination through an amino group of a compound having a carboxy groupand an amino group such as amino acid. An example of introducing an affinity ligand into the carboxylated carrier is not particularly limited and includes a method in which a part of carboxy groups is converted into N-(3-dimethylaminopropyl)_-N’-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) and reacted with an affintiy ligand such as protein A and the urneacted EDC/NHS converted carboxy group is regenerated after the reaction and a method in which in the case of an affinity lgiand having an amino group such as protein A, a carboxy group is protected, an aldehyde group is introduced, adn then the affinity ligand is introduced into the aldehyde group by reductive animation through an amino group. The substrate also includes a hydrophilic polymer such as 13 g of desxtran 270 (intrinsic viscosity: 0.23 dL/g, weight-average molecular weight: about 70,000). 

Anion Exchange Mixed Mode Chromatography

Engstrand (US2007/0259453 and WO 2006/043895 A1) discloses a method of separating antibodies with a multi-modal separation matrix to adsorb undersired compounds while the antibodies remain free in the liquid (flow-through mode). The matrix comprises first groups which are capable of interacting with negatively charged sites of the target compounds (e.g., strong anion exchangeres such as quaternary amines (“Q groups”)) and second groups which are capable of at least one interaction other than charge-charge interaction with said target compounds (e.g., aromatic groups and/or hydrogen bonding groups). Ligands useful in the method include N-benzyl-N-methy ethanolamine. The method is run under conditions conventional for anion exchange chromatography which commonly involves adsorption at a relatively low salt concentration.

Gagnon teaches removing a virucidal agent such as methylene blue from a target protein such as an antibody using hydrophobic, negatively charged mixed mode support that has high affinity for both the target protein and the virucidal agent. The antibdoy is then eluted from the support. 

Suitable annion exchange columns include Capto adhere. Althouse (WO 2015/070068)

–Capto Adhere.RTM. is a strong anion exchange with multimodal functionality which confers different selectivity to the resin compared to traditional anion exchanges. Its ligand is N-Benzyl-N-methyl ethanolamine. (Nti-Gyabaah (US14355014). useful multi-modal anion-exchange ligands include N-benzyl-N-methl etahnolamine, N,N-demethylbenzylamine, 2- amino benzimidazole and thiomicamine Engstrand (US 2007/0259453).  The Capto Adhere ligand, N-benzyl-N-methyl ethanol amine, has a high ion-exchange capacity mixed with an aromatic moeity, which also promotes its stability. The Capto Q ImpRes ligand has a high ion-exchange capacity due to a quaternary amine. (see Chromatographia). 

Cation Mixed Mode Chromatography

Johansson (US2007/0112178) teaches separating antibodies from contaminants in a solution by contacting the solution with a chromatography resin having a support to when multi-modal ligands have been immobilised. The multi-modal ligand comprises at least one cation-exchanging group and at least one aromatic or heteroaromatic ring system. In one emobdiment, the ring forming atoms of the aromatic or hteroaromatic entity are C, S or O and the cation exchanging group is a weak cation exchanger. The method may be used as a single step or as a polishing step following Protein A chromatography.

Suitable cation exchange column include Capto MMC and nuvia cPrime (Biorad). Althouse (WO 2015/070068). Capto MMC is a multimodal weak cation exchange that is salt tolerant (see Cytiva). 

Conditions/Parameters:

Binding:

(i) Bind and elute mode:

(a) binding

—-Amino acids. Suenaga (US2011/0251374) discloses a method of purifying an antibody by treating a solution containing the antibody in the presence of an amino acid by mixed mode chromatography. The amino acid can be a basic amino acid such as arginine, a hydrophobic amino acid, an acidic amino acid or citrulline. In one embdodiment the antibody was applied to a Capto adhere column quilibrated with a citrate buffer containing proline.

—-Salts & Denaturants:

Falkenstein (US13883243, now US 9422329 and WO/2012/059495; see also 15/216,099 published as 2017/0015702) discloses applying a buffered solution comprising an inorganic salt to a multimodal weak cation exchange chromatography material such as Streamline CST and/or Capto MMC and then applying a cell culture containing an antibody to a multimodal weeak cation exchange chromatography material. In one embodiment, the buffer containing the inorganic salt also contains a denaturant such as guanidinium hydrochloride and urea.

—-Non-ionic organic polymer:  Gagnon (US2009/0247735; US20080177048WO2008/086335) teaches a method of purifying proteins such as antibodies from a mixture by contacting the mied with a mixed mode chromatography support in the presence of an aqueous soluble (i.e., hydrohpilic) non-inoic organic polymer. The presence of the polymer enhances binding capacity for virus or protein on mixed mode chromatography supports and enhances the retention of antibody in comparison to most contaminating proteins, thereby enabling novel selectivity for improved removal of non-antibody proteins. 

(i) flow through mode

Engstrand (US 7,714,112) discloses a method of separating antibodies from other compounds in a sample using a multi modal seapration matrix to absorbed undesired ocmpounds while the antibodies remain free in the liquid.

(Nti-Gyabaah, (US 14/355014) discloses using MM  for the purificaiton of monoclonal antibodies from heterogeneous aggregates. In certain embodiments, the MM is conducted in flow through mode using Capto adhere at operating conditions of pH 6.8-7.7, conductivity of 4-25 mS/cm, protein loading of 100-205 gm/protein liter of resin and 20-200 mM salt concentration. In one embodiment, the column is equilibrate with 3 CV of 100 mM sodium citrate pH 3 followed by 5 CVs of 20 mM Tris-HCL, 110 mM NaCl pH 7.2. The pH and conductivity of the feed are adjusted to the desired values then loaded to the column at 300 cm/hr. Upon completion of the loading, the column is washed with 2-10 CV of the equilibration buffer at hte same flow rate. Becasue the product is in the flowthrough, the product collection begins at 100 mAU into the loading and ends at 350 mAU during hte wash step. Product pool pH is 7.2 upon elution and is quenced with 1M acetic acid to a final pH of 5.5 prior to analysis. 

(ii) Overload mode

(Nadarajah, US2014/0301977) discloses loading a composition onto a chromatography material such as HIC such that the product like a polypeptide is loaded onto the material at an amount exeeding the DBC of the material for the product. In some embodiments the chromatography conditions are choses such that even if product breaks through aftering binding most of the impurities do not. The product found in the eluate can be pooled. Upon completion of laoding, the product (e.g., polypeptide/antibody) is eluted. In some embodiments, the overload and elute chromatography (OEC) is performed where the partition coefficient (Kp) of the product is greater than about 30, 50, 75 or 100. Nadarajah exemplifes using Capto Adhere resins and loading Protein A pools on such mixed mode resinsfollowed by an elution buffer to elute off bound protein. The flowthrough or eluate during the load, overload and eltuion phases were collected as fractions.

(b) Elution:

Althouse (WO 2015/070068) disclsoses a mixed mode step that follows Protein A affinity chromatography. The mixed mode can be either cation or anion or a combination. This step can also be based on a single type of ion exchange mixed mode or can include multiple ion exchanger mixed mode steps such as a cation excahnge mixed mode step followed by an anion exchange mixed mode step or vice versa. Both the AEX and CEX mixed mode chromatographys can be run in bind and elute mode. 

Arakawa (US 2014/0072560) discloses mixed mode chromatography for seaprating correctly folded proteins such as etanercept. In one embodiment, the bound prtoein is eluted by a simultaneous gradient of both pH and NaCL concentration. 

Gronberg (WO2005082483) discloses process for the purification of antibodies using multi modal ligands having at least one CEX group and at least one aromatic or heteroaromatic ring system immobilised to absorb the antibodies to the resin, adding an eluent to release the antiboides and contacting the eluate with a second chromatography resin. The CEX group is preferably a weak cation exchanger.

Johansson (US2007/0167613 and WO2005/082926) discloses purification of antibodies using multi-modal ligands immobilised to adsorbe the antibodies to the resin whicherein each multi-modal ligand has at least one cation exchanging group and at least one aromatic or heteroaromatic ring system. The eluate is commonly a buffer such as a phosphate buffer and in one embodiment a step elution by increase of pH and/or salt concentration. The CEX group is preferably a weak cation exchanger (group which can be protonated at certain pH values). The multi modal chromatgoraphy can be followed by a second chromatography step such as IEX, HIC and affinity chromatography.

–Ethylene glycol + Inorganic salt

Falkenstein (US13883243, now US 9422329; see also US 15/216,099, published as 2017/0015702; see also WO/2012/059495) discloses applying a cell culture containing an antibody to a multimodal weeak cation exchange chromatography material such as Streamline CST and/or Capto MMC and recovering the antibody by applying a buffered solution comprising ethylene glycol and an inorganic salt such as ammonium, potassium or soldium chloride. In one embodiment, the buffer further comprises a denaturant such as guanidinium hydrochloride or urea. In one embodiment, the buffer includes sodium chloride, aobut 20% (w/v) ethylene glycol at pH 7.1-7.3.

In Combination with Other Types of Chromatography

Affinity (e.g., Protein A/G) – Mixed Mode

Althouse (WO 2015/070068) disclsoses a mixed mode step that follows Protein A affinity chromatography. The mixed mode can be either cation or anion or a combination. This step can also be based on a single type of ion exchange mixed mode or can include multiple ion exchanger mixed mode steps such as a cation excahnge mixed mode step followed by an anion exchange mixed mode step or vice versa. 

Anion-MM

Perlasca Islas (US 2014/0187749) disclsoes a method for the purificaiton of anitobides which includes intermedaite and polishing step that includes in-line AEX and mixed mode chromatography. 

MMC-HIC:

Dong (US2012/0238730) teaches purification of antibodies using Protein A followed a fine purification step which included mixed mode based separation using CaptoAdhere followed by either CEX or HIC. 

Ma (US14208043) discloses a method for reducing high molecular weight (HMW) species and/or high mannose species by starting a starting solution having a desired protein such as an antibody to MMC to form a first eluate and subjecting the firust eluate to HIC . In one embodiment the MX is conducted in flow through mode and the HIC is conducted in bind/elute mode. 

Mccue (US2011/0129468) discloses a method for purifying Ig (constant domain of Ig) fusion proteins by MM in bind and elute mode followed by HIC in bind and elute mode.

Gagnon (US2009/0247735) teaches the use of mixed mode chromatography for purificaiton of an antibody from other materials such as aggregated antibodies which can also be integrated with other fractionation methods such as HIC. 

Hunter (US 14/890,791, published as US 2016/0083453) discloses a emthod of separating antibody multimers with minimal separation of monomers by subjecting a mixture cmprising a plurality of mAbs to mult-modal chromatography, apatite chromatography and HIC. 

Herigstad (US9,249,182) disclsoes subjecting a sample media comprising an antibody such as adlimumab to a mixed mode chromatography and then hydrophobic interaction chromatography. The HIC can remove a variety of process related impurities (e.g., DNA) as well as product related species (e.g., high and low MW proudct related species such as aggregates and fragments).

Johansson (US7,750,129) dicloses a process for the purification of antibodies using multi-modal chromatography which includes at least one cation exchaing group and at least one aromatic or heteroaromatic ring system to adsorb the antibodies and adding an eluent to release the antibodies and contacting the eluate with a second chromatography which can be HIC. 

Ramasubramanyan (14/077,871; (US2015/0110799); 14/575,691 (US2015/0132801) and US 14/614,311 (2015/0140006) and US14/584,619 (US2015/0166650) teaches a method for producing a low acidic species composition comprising an antibody by contacting a sample comprising an antibody to an AEX, CEX or mixed mode mediaand eluting the antibody and further contacting the leuted sample to an HIC media.

See also particular antibodies purified

Introduction and What it is used for

CEX has become a preferred method for IgG purificaiton. The relatively mild buffer solution conditions of CEX help preserve the IgG native structure, while purifying it from host cell proteins and IgG variants. (Dillon, US14/363735)

CEX step removed charged antibody variants, host cell proteins, and low molecular weight antibody variants and antibody aggregates. Antibody aggregates are more tightly bound than the antibody monomer and elute after the amin peak. Blank (Bioseparation 10: 65-71, 2001) As to types of impurities purified see sources and types. 

Cation exchange is generally more effective than anion exchange for reduction of leached protein A (Gagnon, p. 493). Cation exchange is used as an intermediate purification step for recombinant antibodies. Although the protein A affinity step greatly reduces the amount of host cell proteins, DNA and endotoxin, these impurities must be further removed. In addition, the protein A affinity step does not reduce the level of aggregate and it introduces protein A molecules into the purified antibody. Cation exchange chromatography reduces the level of host cell proteins, DNA, endotoxin, aggregate, and leached protein A. It will also reduce the level of any misformed antibody (for example, antibody with 2 heavy chains and one light chain) (Fahrner, Biotec. Genetic Eng. Reviews, 18, 2001, p. 315, last ¶).

Types of CEX resinds used

Weak Cation exchange:

Falkenstein (WO2006/125599) discloses a method for purifying an immunoglobulin from aggregated immunoglobulin molecules using a weak ion exchange (e.g., CM-Toyopearl) where the immunoglobulin is recorvered from the weak IEX in a single step or linearly  by using a buffer such as sodium citrate  and a salt such as sodium chloride. In certain embodiments the immunoglobulin is an IL-1R antibody or an anti-HER-2 antibody.

Modes of Operation

Bind and elute mode:  See below under operating condition

Flow through mode:

Kozlov (US2013/0245139) disclsoes CEX operated in flow through mode wehre a monomeric protein of interest is loaded from a mixture having aggregates onto a CEX at a density of about 1-30mM and CEX binds the aggregates.

Displacement and Overload Modes: 

–Indigenous protein displacement mode: 

Bill (US 14/365449, published as US 2014/0348845; see also Bill (US Patent Applicaiton No: 14/365,449, published as 10/364268) disclsoes a method for purifying antibodies from various contaminants such as ionic polymers like polyethyleneimine (PEI), polyvinylamine, polyarginine, polyvinylsulfonic acid or polyacrylic acid, which includes loading the sample through a CEX under buffer conditions having pH about 1-5 pH units below the pI of the antibody and a conductivity of about 40 mS/cm which causes the membrane to bind the antibody and at least one contaminant and collecting the fraction that includes the antibody. In one embodiment, the CEX is run in overload mode followed by final polishing steps. The impurities displace the antibdoy so it is contained the eluent. 

Brown (WO2010/019148) is closes CEX run in indigenous protein displacement mode where the pH of the load material is adjusted to about 1-5 pH units below the pI of the antibody, the conductivity of the load is adjusted to less than about 40 mS/cm, depending on the pH. Because the pH of the load is less than the pI of the antibody, the antibody (which has beomce positively charged) will not flow through initially. Rather, it will be electrostatically bound to the negative funcitonal groups of the CEX. Since the pH of many contaminants (e.g.,, host cell proteins, such as CHOP) that elute with the antibody druing protein A affinity chromatography is only slightly different from the pI of the antibody, these contaminants like the basic antibodies will also bind to the membrane. However, the contaminants preferentially bind to the membrane or otherwise effectively “displace” the antibody from the membrane allowing the antibody to elute from the matrix or flow through after binding and be recovered in the effluent. 

Liu (J. Chromatography A, 1218 (2011) 6943-6952) discloses using CEX in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L (dynamic binding capacity of CEX is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAB break through). All of the matrices effectively retained host cell protein and DNA during the loading and wash steps while antibody flowed through each matrix after its dynamic binding capacity was reached. 

McCooey (“exploration of overloaded cation exchange chromatography” BIOT-59, 2011) discloses that CEX is typically operated in bind and elute mode with loading capacity of up to 100 g/L chromatography resin. But McCooey was able to load 1,000 g monoclonal antibody per L matrix in flow through mode under isocratic conditions in overload mode to remove the majority fo host cell prtoein, DNA, Protein A leachate and reducing aggregate, while maintaining high yields. The removal of impurities during the overloaded step may be the result of impurities displacing monomer.

–Displacement Mode

—-Particular displacing Compounds/molecules:

Expell SP1:

Davies (WO2009/135656) discloses using a packed bed chromatography in displacement mode uisng a CEX chromatography that has high DBC at operating conditions of pH less than the pI of the protein/antibody, preferably 2 pH units below the pI such that the DBC of the resin is high enough to allow formation of a displacement train. The compound Expell SP was used as the displacer. 

Zhang “Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography” J. Chromatography A, 1218 (available online May 27, 2011) 5079-5086) discloses isolation and enrichment of charge variants of a mAb IgG1 using CEX displacement chromatography. Zhang discloses separation of acidic, main and basic charge variants with high recovery and purity by using a Expell SP1 displacer stock buffer. 

Gentamicin: (Bill, US 14/365449, published as US 2014/0348845) discloses using Mustang S membrane (CEX) where the membrane was washed with equilibration buffer and gentamicin was used to elute the membrane. 

Conditions:

Generally: here are 2 critical variables to investigate when developing the wash and elution conditions: the buffer pH and the amount of salt in each buffer. The pH of the separation will be determined in part by the stability of the antibody, and before finalizing the separation pH the stability of the antibody at that pH should be evaluated. A pH of 5.5 is often optimal for bind and elute cation exchange of antibodies. The typical column capacity at pH 5.5 and a load conductivity <8 mS/cm is about 40 g/l. The elution conditions are optimized in series of experiments where the column is loaded to capacity and eluted using varying concentrations of sodium chloride or another salt. Generally, at lower salt concentration the antibody may not completely elute and yield will be low, and at higher salt concentrations the yield will be high but aggregate and host cell proteins will being eluting with the antibody. This balance between purity and yield will then be the focus (Fahrner, Biotech. Genetic Eng. Rev. 18, 2001, p. 301-27).

Falkenstein (US 2010/0311952; see also WO2008/145351A1) teaches a method for purifying an immunoglobulin where the method comprises applying a buffered solution comprising the immunoglobulin in monomeric and in aggregated form to a cation exchange under conditions where the immunoglobulin in monomeric form does not bind to the cation exchange material).

Pre-treatment/equilibration of sample

–filtration/lowering pH

Yoon (US 14/365,027) teaches the expression of trastuzumab antibody in CHO cells and in method 1, the culture supernatant was recovered by removal of the cells through primary filtration using a depth filter and then the pH value of the culture supernatant was reducedto 5 followed by re-filtration wehreas in method 2, the pH of the culture broth was reduced to 5 and then culture broth was subjected filtration using depth filter so as to prepare the sample for loading on a CEX. Yoon reports that method 1 showed a yield of 84% and final HCP content of 47.3 ppm after CEX whereas method 2 of direclty reducing the pH of the culture broth showed the final yeild of 82% and HCP content of 110.6 ppm.

–conductivity and pH

Basey (EP1308455) disloses using CEX for purifying a polypeptide of interest which is bound at an iniitial conductivity or pH, then the CEX is washed with an intermedaite buffer at a different conductivity or pH or both and then washed with a wash buffer where the change in conductivity or pH or both is different and in the opposit direction then the previous steps.

Eon-Duval teaches that before loading a fluid containing an Fc containing protein on a CEX, the fluid is preferably either adjusted to a pH of less than 5, preferably about 4 or as an alternative diluted with water to a conductivity of less than about 4 mS/cm at bout pH 7. This is essential to allow binding of the Fc containing prtoein to the CEX.

Shanghai (US 15/034,821, published as US 2016/0289264) disclsoes a method for purifying an anitbody using multiple euilibration/washing/elution buffers having distinct conductivity and pH. For example, in one embodiment the wash buffer is at a third conductivity and a gradually increased pH and the subsequent elution buffer is at a fifth conductivity and pH. The change in pH of the wash buffer and first elution buffer is achieved by adjusting the mixed ratio of two salt containing buffers that have different pH as for example by changing 25%Na2HPO4 pH7.5 + 75%Na2HPO4 pH9.3-94 to 15%Na2HPO4pH7.5 + 85%Na2HPO4 pH9.3-9.4. 

Absorption/binding: 

The dynamic binding capacity of mAbs on cation exchange resins depends on pH and conducitvity. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010)

Graf (Ion exchange resins for the purification of monoclona antiobides from animal cell culture” Bioseparation 4, 7-20, 1994) discloses a one purification step based on cation exchange chromatography with well defined conditions of adsorption (20 mM MES buffer, pH 6.50).

Ansaldi (WO99/62936) teaches separating a polypeptide monoer from a mixture of dimers and/or multimers by applying the mixture to a CEX in a buffer with pH of about 4-7 and eluting at a gradient of about 0-1 M of the elution salt. 

Stein, J. Chromatography B, 848 (2007) 151-158) investigated the influence of pH on the static binding capacity of two strong CEX exchangers (Fractogel EMD SO3- and Fractogel EMG SE Hicap) for a mAb. both resins examined displaed broad binding optimum around pH 6.3

–Cationic buffers:  Gagnon (US 2013/0210164) discloses methods for purifying proteins/antibodies using CEX where a displacement counter ion which is an cationic buffer such as Tris, imidazole, histidine or histamine is used to compete with the hydrogen ions associated with the neatively charged functionality of the CEX. Other possible displacement counter ions inclue lysine or ariginine. Following the displacement of the hydrogen ion, the target molecule can be eluted such as by increasing the conducitvity (ionic strenght) in the solution. In one example, an antibody sample was loaded on a CEX at pH about 6 with conductivity about 12 mS/cm. The column is washed with equilibration buffer. hydrogen ions were replaced by re-equilibrating with Tris, adjusted to pH 5.5 with acetic acid, thereby replacing hydrogen ions on the surface of the column. Elution was with a linear gradient of 2 M NaCL.

Washing: 

–PH

Chumsae (US 2014/0275486) disclsoes a method for purifying a composition containing an antiboy by loading the composition onto a CEX using a loading buffer with pH of the loading buffer lower than the pI of the target protein, washing with a buffer wherein the pH of the washing buffer is lower than the pI of the target protein and leuting with buffer. In one embodiment, the conductivity of the elution buffer is here than the conductivity of the washing buffer. 

–Ph and Salt Concentration

Basey (US 6,339,142) discloses a method for purifying an antiboy by CEX which involves chaning the conducttivity and/or pH of the buffers in order to resolve the antibody from contaminants. 

Eon-Duval (US 2012/0202974) discloses a method for separating an antiboy by binding the antiboy a a CEX, washing with a buffer at a pH about 1 unit below the isoelectric point of the antiboy, the buffer having a conductivity of about 2-6 mS/cm and eluting the antiboy with a buffer at a pH about 1 unit below the pI of the antiboy with an increasing salt gradient. 

Incheon (US 15/033,335, published as US 2016/0264618) discloses purification of an antibody using CEX with a washing buffer haivng .a pH which is at least 1.0 lower than the pI of the antibody such as a pH 5-8 and a salt concentration of 10-300 mM. 

–By change in conductivity:

Emery (WO2004/024866) discloses a method of purifying an antibody using CEX with wash buffers such that the salt concentration of the wash buffers increases and then eluting the antibody with a salt concentration that is greater than the final salt concentration of teh wash buffer.

Falkenstein (US2014/0162317; see also US 14/934866, published as US 2017/0066814) discloses a method for proudcing an antibody preparation by applying a solution that includes different isofrom of the antiboy to a CEX, applying a first solution with a first conducitvity whereby the isofroms remoin bound and apply a second solution with a second conductivity that exceeds the conductivity of the first solution by not mroe than 10% so as to obtain the antibody using a citrate buffered solution.. 

–By change in pH

Coan (US 5,110,913) discoses a method of purifying an anti-TNF antibody using a set of conditions were the column was equilibrated at a 1st pH value so that the antibody binds and then increasing the pH to an intermedaite pH where the protein (such as anti-TNF ab) will not bind to the CEX but does not elute it if already bound, and then raising the pH to a third value which elutes the antibody. In a particular embodiment S-Sepharose.TTM. colum was equilibrated with sodium acetate at pH 4.6, the anti-TNF solution was applied to the column, the column was washed with a buffer of sodium acetate, pH 5.5 and the antibodies eluted with pH of 6.5

–Multiple Washes with different pH

Arunakumari (US 2012/0178910) discloses purificaiton of antibody using CEX which includes contacting an antibody with the resin at a first pH that is less than the pI of the most acidic isofrom of the antibody and washing at a second pH that is greater than the first pH but less than the pI of the most acidic isoform and eluting at a third pH that is about euqal to or less than the first pH. 

Lebreton (US2009/0148435 and WO2009/058812) discloses antibody purification by CEX using a high pH wash step followed by a second wash at a pH which is less than that of the first wash buffer and then eluting the antibody with a buffer at a conductivity which is substantially greater than that of the second wash buffer.

Ram (US 2015/0152179) discloses using CEX for removal of HCPs and other contaimaintans where the conductivity and pH can be reduced, or maintained or increased in wash buffers used in subsequent wash steps. 

Yoon (US14/365027, published as US 2014/0316115 and US 9,683,012)) discloses multiple CEX wash. In one embodiment, a carboxymethyl sepharose having a COO- group may be used to remove acidic antibody isoforms. In this case, the column is washed with a buffer 20-30mM sodium acetate pH 4.5-5.5 and 35-45 mM sodium chloride, washing the column with 20-30 mM Tris HCL, pH7-7.5, washing the column with 20-30 mM Tris hydrogen chloride, pH 7-7.4 and 20-30 NaCL and washing the column with 20-30 Tris-HCL, pH 7-7.5. In another embodiment, acidic antibody isoforms and basic antibody isoform may be done using a fractogel COO- composed of a synthetic methacrylate polymer resin as a support unlike the sepharose based support for CM. The multiple wash is with a buffer having 20-30 mM sodium acetate, pH 4.5-5.5 and 35-49 mM NaCL, then washing with 25-35mM sodium acetate, pH 5.5-6.5, then washing with 25-35 sodium acette, pH 5.5-6.5 and 45-55 mM NaCL and then washing with 25-35 mM sodium acetate, pH 5.5-6.5. 

–Gradients

Baek (WO 2013/089477) disclosess loading an antibody sample onto a Fractogel COO- resin and using various washes to separate acidic antibdoy isoforms. In particular, washing 1 (20 mM sodium acetate pH 5, 40 mM NaCL) after loading is a step of attaching the antibody and removing the supernatant form the culture supernatant, washing 2 (30 mM sodium acetate pH 6) is a re-equilibration step of increasing the pH from 5 to 6 to separate antibdoy isoforms, washing 3 (30 mM sodium acetate pH 6, 50 mM NCL (10 CV) is a step of removing acidic antibody isoform and washing 4 (30 mM sodium acetate pH 6) is a step of re-adsorbing the acidic antibody isorm after removing a part of it. 

Incheon (US 15/033335, published as US 2016/0264618) discloses purifying an antibody which includes a single step gradient washing at variying ratios (e.g., 70:30, 65:35 adn 60:40) of buffer A (20 mM Na phosphate, pH 6) to buffer B (20 mM Na phosphate, pH 8.0).  The method is advantagous in that various isoforms weakly attached to the CEX were detached in the washing step. 

Elution: 

–pH Gradients:

Compared with salt gradient, pH gradient elution provides equivalent product purity with a higher yield, a smaller pool volume (up to 50% of volume by salt gradient) and low pool conductivity. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

Kaltenbrunner (J of Chromatography 639 (1993) 41-49) discloses separation of isoproteins of monoclonal antibodys using a strong CEX and ascending pH gradient combined with a linear descending salt gradient suing a buffer consisting of borate, mannitol and different concentrations  of NaCL.

–Salt /conductivity Gradients:

The separation of proteins by IEX is usually made with a salt gradient, going from a pure buffer solution to a solution of salt and buffer. The elution strengh of an eluent is the ability to elute solutes form the column. For a given stationary phase, the elution strenght is determined by the concentration and type of ionic species in the eluent. The elution strenght is hus lower in the starting buffer than in the eluting buffer. This leads to an increase in elution strenght during the gradient. (Malmquist, J. Chromatography, 627 (1992) 107-124).

Graf (Biosecparation, 4, 7-20, 1994)iscloses that on cation exchangers, MAbs were eluted between 0.10 and 0.25M NaCl. The eltuion of MAb from Sp Spherodex cation exchanger required higher salt concentration than from S Sepharose FF.

Burg (US2012/0177640) teaches using CEX for purifying antibodies such as HER2 antibodies using either a gradual increased of conductivity and/or pH or a step wise increase of conductivity and/or pH based on a threshold ratio. of the amount of antibody molecule or variant thereof to the sume of the amounts of the antibody molecule and the variant thereof. Below a definted threshold ratio the ralative amount of unwanted variant is relatively high and the amount of the antiboedy is relaitvely low. In this case, a gradient elution mode followed by step elution is preferred . The step elution mode is less suitalbe in this case due to the lower degree of purification of the antibody moleucle from the variant. On the other hand, if the ratio of the amount of the antibody to the sum of the amounts of antibody and variant is higher than the threshold ratio, then a step elution mode is preferred sinice it results in higher yields and still a drug product can be obtained which is suitable for the market.

Emery (WO2004/024866) teaches a method for purifying a polypeptide such as an antibody using CEX by eluting the polypeptide with an elution buffer that has a salt concentration that is greater than the final salt concentration of the wash buffe.

Eon-Duval (WO2008/087184) teaches a method for the purificaiton of an Fc containing protein such as an antbiody on CEX by washing the CEX with a buffer at a pH about 1 unit below the pI of the antibody and a conductivity of about 2-6 mS/cm and eluting the protein with an increasing salt gradient.

Falkenstein (US13/994673) teaches applying a solution to a CEX with a conductivity that exceeds the conductivity of the binding buffer to elute different isoforms of an antibody. 

Falkenstein (WO2006/125599) also discloses a method of purifying an immunoglobulin using a weak ion exchange resin and a single step elution where the immunoglobulin is eluted by increasing the conductivity of the buffer/solution.

–pH-conductivity Hybrid:

Traditional CEX is operated at acidic condition with sodium chloride as the elution component. The resulting cation column pool contains a high conductivity and a dilution step is often needed to lower the conductivity prior to the next polishing AEX step. Beside the inconvenience, the use of salts in chlorine base at acidic condition has been reported to be problematic to manufacturing facilities. Zhou (J. Chromatography A, 1175 (2007) 69-80) disclose a pH-conductivity hybrid gradient elution with CEX for scale process mAb production. The resulting CEX pool contains minimal conductivity going into the next step, making the process more manufacture friendly. In one embodiment, the an equilibration buffer of sodium acetate pH 5.0 followed by a wash step using equivalent pH and conductivity and then an elution step using a linear gradient of 200 mM NaCl in the equilibration buffer at pH 5.0 or various concentrations of sodium acetate at pH 6.0 was used.

–Addition of Additives/Stabilizers during Elution:  (see also multi-muldal CEX under Mixed Mode)

—–Polymers (e.g., PEG)/sugars (e.g., sorbitol)/amino acids (e.g., glycine):

Non-ionic Polyether + Additives:   (See also multi-modal weak CEX)

Gagnon (US 2009/0318674) discloses an antibody purificaiton process that includes aggregate removal which inludes the use of a nonionic polymer such as polyethylene glycol (PEG) in at elast one step to enhance removal of aggregates. In certain emobdimetns, the fraction which contains the protein product collected from the first chromatography step is collected into a composition that includes a solubility enhancing additve such as a zwitterion like glycine. 

Neumann (US14/025622, published as US 2014/008100, now US 9,394,337); see also US 16/450,827, published as US 20200148719) discloses recovering an antibody in monomeric form from an ion exchange material such as CEX by applying a solution comprising a non-ionic polymer which a non-ionic polyethe such as poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), PEG-PPG copolymers, and triblock copolymers composed of poly (oxypropylene) (poly (propylene oxide) flanked by poly (oxyethylene) (poly (ethylene oxide). an additive such as a zwitterion, or an amino acid (such as glycine, bicine, tricine, alanine, proline, Betaine), or urea or a sugar (e.g., glucose, sucrose, raffinose), alkylene glycol, an ampholyte (e.g., MES or MOPS or HEPES or PIPES or CAPS or a polyol (such as glycerol, xylitol, or sorbitol) and an elution compound such as sodium chloride.  In one embodiment, the method of purifying the antibody with CEX includes applying a solution comprising a non-ionic polymer and an additive to a resin to which an antibody has been adsorbed wehreby the antibody in monomeric form remains adsorbed to the resin and recoveirng the antibody in monomeric form by applying a non-ionic polymer, an additive and an elution compound. 

Laursen (US7138120 and US2001/0051708) discloses adding a stabilizing agent such as sorbitol, mannose, glucose, trehalose (maltose), proteins (such as albumin), amino acids (such as lysine, glycine) and organic agents (such as PEG). 

–PEG + Glycine:

Neumann (US 15/184883, published as US 20170107249) disclsoes a method for producing an antibody of the IgG class by equilibating a CEX, optionally with poly (ethylene glycol) and glycine and then eluting the IgG in monomeric form by applying a solution to the CEX comprising poly (ethylene glycol) and glycine. 

–PEG + Sorbitol:

Laursen (US7138120 and US2001/0051708) discloses adding a stabilizing agent such as sorbitol, mannose, glucose, trehalose (maltose), proteins (such as albumin), amino acids (such as lysine, glycine) and organic agents (such as PEG). In one preferred emobdiment, the stabilizing agent is sorbitol, preferably at a final concentration within the range of 2-15 (w/v) sorbitol, eg. abou 2.5 %, to an IgG fraction immediately after or during the elution. 

Nuemann US 14/025,622, now US 9,394,337) disclsoes a method for producing an antibody of the IgG class by equilibrating a CEX, optionally with poly (ethylene glycol) and sorbitol, applying the IgG solution and then eluting the antimbody in monomeric form by applying a solution comprising poly (ethylene glycol) at abtout 0% by weigh and sorbitol at 5-20 by weight. 

Changing Conductivity and/or pH buffers:

Basey (US6,339,142 & WO99/57134) discloses a method of purifying an antibody by CEX by loading the antibody onto a CEX using a loading buffer that is at a pH and/or conductivity (e.g., low conductivity such as 5.2-6.6 mmhos and pH about 5), washing with an intermediate buffer at a second conductivity and/or pH (increasing conductivity or pH or both such as conductivity 7.3-8.4 mmhos and pH about 5.4) so as to essentially elute the contaiminant but not a substantial amount of the antibody such that the antibody, washing with a buffer that has a conductivity or pH or both which is less than the intermediate buffer (i.e., changed in opposed direction such as a wash buffer that has a conductivity about the same as the loading buffer 5.2-6.5 mmhos and pH about 5.0) and then eluting the antibody using an elution buffer that has a pH and/or conductivity that generally exeeds the pH and/or conducitvity of the laiding, intermediate and wash buffer used in the previous steps (e.g., conducitvity 10-11 mmhos or pH about 6.0).

Cation followed by secondary steps:

Follman (J Chromatogr A, 2004 1024(1-2):79-85 teaches that for combinations of CEX, AEX, HA, HIC, HICH, the order of the process steps affected purification performance significantly.

Cation-Anion:

Arunakumari (WO/2007/108955) discloses purifying antibodies that does not include an affinity chromatography step by subjecting the mixture to CEX followed by AEX where there is no in process TFF step. 

Davies (WO2009/135656) discloses Capto S resin (CEX) to capture antibody from conditions cell culture sueprnatant. The column was operated in displacemenodeDisplacement was made using the molecule Expell SP1. The displacement chromatogrpahy was followed by AEX. 

Kim (US 14/896,380 published as US2016/0122384) discloses a method for purifying an antibody from HCP using CEX in bind and leute mode, passing the eluate througha multilayer filter, and passing the filtrate through an AEX in flow through mode. 

Prior (WO 89/05157) discloses purification of immunoglobulins from cell culture using cation exchange so as to adsorb the product but not the contaminants. The eluted product is then recycled or is applied to anion exchange for further purification.

Humphreys (WO2004/035792) discloses purifying Fab’ fragments expressed in the perioplasmic extracts of E coli by CEX at pH 4.5 during which the Fab’ fragment binds, eluting the Fab’ fragment and then run on AEX at pH in flow through mode. A final Hydrophobic interaction chromatography (HIC) step was removed by altering the physical properites of contaminating E coli proteins such that they would no longer co-purify with the Fab’ on IEX. For example the phosphate binding protein PhoS is a very abudnant protein in some fermentation process due to the phosphate depletion that can occur in defined media. PhoS has a functional pI very close to that of Fab’. Humphreys (see also Protein Expression and Purificaiton 37, 2004, 109-118) produced a version of PhoS that would not purify at all with any Fab’ during IEX.

Spitali (US13/812654, published as US2013/0184439, now US 9,309,280; WO2012/013682; see also 14/977688, published as US 2016-01018119; see also 16/248845, published as US 2019/0135909) discloses purification of an antibody fragment from a periplasmic cell extract using CEX in bind and elute mode followed by AEX in flow through mode. In one embodiment, the CEX is performed in elution mode. In one embodiment the antibody was expressed in E coli host cells at a concentration of 2.5 g/L, released form the periplasmic space with Tris-EDTA and heat treatment, cell material removed through centrifugation and the extract ajdusted to pH 4.5, calrified by centrifugation and DF, eluted with water and fed to a Capto S cation exchange column from GE Healthcare, washing and eluting, then UF/DF and Capto Q AEX.

Cation-Anion-HIC:

–Viral Inactivation/CEX/AEX/HIC: 

Eon-Duval (US2012/0202974; see also WO 2008/087184) discloses s a method for purifying an Fc containing protection using CEX in bind and elute mode followed by AEX or an HIC. In one embodiments, the protein is eluted from the AEX and applied to an HIC column in bind and elute mode.

Hickman (US 12/582556; see also US 12/582,434) discloses a method for antibody purificaiton by subjecting the mixture to a reduction in pH of 3.8 using citric or phosphoric acid, adjusting the primary recovery sample to a pH of about 5.0 and applying the primary recovery sample to a CEX, followed by AEX and then HIC. 

Wan (WO2007/117490) discloses a method for producing HCP reduced antibody preparation from a mixture comprising an antibody comprising an IEX step such as CEX and then eluting the antibody from the CEX and subjecting the first eluate to a viral inactivation step followed by application the preparation to an AEX.

–CEX-UF/DF/AEX-HIC/Filtration-UF/DF: Hickman (WO/2010/048183) discloses purifying anti-Il-18 antibodies by CEX-UF/DF-AEX-HIC-Filtration-UF/DF. 

–Cation/Viral Inactivation/Anion/Hydrophobic interaction: Wan (US2007/0292442; WO2007/117490; see also US9,102,723) teaches antibody purification using cation exchange, viral inactivation by pH viral inactivation, anion exchange and then hydrophobic interaction column (HIC). Preferably, the antibody mixture has not been subjected to protein A capture prior to applying to the cation exchange resin.

CEX-HIC-AEX Combination:

–CEX/HIC/HPTFF or –CEX-AEX/HPTFF: 

Fahrner (US 2003/0229212 and WO03/012132) discloses antibody purification using two non-affinity purification steps followed by high performance tangential flow filtration (HPTFF) in the absence of an affinity chromatography step. In a particular embodiment, the first and second non-affinity chromatography purification steps consist of ion exchange chromatography and hydrophobic interaction chromatography.

Gagnon (“use of hydrophobic interaction chromatography with a non-salt buffer system for improving process economics in purificaiton of monoclonal antibodies” presented at waterside conference on monoclonal and recombinant antibodies, Miami April 30-May 3, 2000) discloses a three step purification begining with filtered ascites, first step CEX, then non-salt buffer (glycine) HIC and final step AEX, without requirement for itnermediate sample preparation steps suf as buffer exchange chromatogrpahy or diafiltration.

–Pretreatment of culture broth(debth filtration)/CEX/HIC/AEX:

Eon-Duval (WO2008/087184) discloses purification of Fc containing proteins such as an antibody comprising the steps of binding the protein to CEX and then binding the elute to HIC followed by AEX.

Yoon (US14/365027) discloses discloses a method of antibody purification which includes the steps of pretreatment of culture broth by removal of cells from the culture broth through a primary filtration using a depth filter, reducing the pH to 5 followed by refiltration, loading a sample containing a mixture of antibodies onto a CEX. optionally washing the column, eluting the antibodies from the CEX thereby removing antibody isoforms, loading the sample prepared by mixing the sluate with salt onto an HIC, eluting the antibodies to remove HCPs and loading the eluate onto an AEX such as a Fractogel COO- column and collected the flow through. 

CEX/HCIC combinations: 

Arunakumari (WO 2006/110277 A1) (see also US 14/193731) teaches the steps of cation exchange chromatography and hydrophobic charge induction chromatography, without regard to their order and without the use of affinity chromatography in order to yield high purify protein/antibody compositions.

Boschetti (TRENDS in Biotechnology, 20(8), 2002) teaches cation exchange capture step followed by HCIC for purification of antibodies.

Follman (J. Chromatography A, 1024, pp. 79-85 (2004). teaches a three spec process of CEX-AEX-HCIC without protein A chromatography for antibody purification. Follman teaches that the order of the process steps affected purificaiton performance significantly.

 Particular Antibodies purified by Cation Exchange

–Single Domain antibodies (VHH)  See outline: domain antibodies 

Brown (US12/608964) also teaches purification of VHHs using action exchange under conditions which allow the VHH to bind to the suport and then selecting eluting the VHH.

Jonniaux (US2011/0183861) discloses purification of nanobides via CEX with a wash buffer of 10 mM citric acid, pH 4.0 and elution buffer of 10 mM citric acid/1M NaCL, pH4 followed by size exclusion chromatography. The nanobdy was produced intra cellularly or from an inclusion body in a bacterial cell or produced extracellularly and isolated form the medi7um in which the host cell is cultivated.

Silence (US2006/0115470) discloses purification of VHH on a cation exchange column which had been equilibrated in 25 mM citric acid pH 4.0 and then eluted with 1M NaCL.

Particular Variants elimintated

Charge variants: Zhng (J. Chromatography A, 1218 (2011) 5079-5086) teaches isolation of charge variants from a monoclonal antibody IgG1 using cation exchange displacement chromatography. 

See also cation exchange for purification of antibodies

Anion exchange is nearly as universal as protein A in the purificaiton of monoclonal IgG. It is s employed frequently as the last chromatography step because of its ability to scavenge endotoxins that may have entered the process via contaminated manufacturing materials or inappropriate sample handling.

Different Modes of Operation:

Flow through mode: 

–Operating Conditions for AEX in Flow through Mode:

Anion exchange is frequently applied in flow-through mode. Buffer conditions are set so that the antibody passes through the column while strongly electronegative contaminants are captured. The operating pH is normally 8 to 8.2 with a conducitvity of up to 10 mS/cm (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010)

Kulkarni (13/518532) also disclose purification of antibody using anion exchange chromatography performed in flow-through moade where the eluate obtain from a protein A chormotography step without substantial adjustment of pH. In some embodiments the pH values are about 3.3 to about 6. 

Pompiati (US 13/141306, published as US 2011/0257370 and US Patent No. 9631008; see also 15/461,198, published as US 2018/0162930; see also 16/538,604, published as US 2020/0207839) discloses an anion exchange chromatography step, in which the immunoglobulin in monomeric form can be obtained from an anion exchange material in a flow through mode which is performed in a narrow pH range of from, for example, pH 7.8-8.8.

Wan (US6177548) teaches purification of an antibody from aggregates and other impurities using anion exchange chromatography. The pH is adjusted to near the isoelectric point of the products/antibody which is loaded onto the ion exchange column so that the more charged aggregates bind to the column whereas the antibody is collected in the flow through.

Bind-and-elute mode: The isoelectric point of many antibodies is high (often >8 and sometimes >9), so anion exchange chromatography run in flow through mode offers a high yield method for final purification of antibodies with a high pI. For antibodies with a pI lower than about 8, the anion exchange chromatography step may be run in bind and elute mode, which may offer advantages over the flow through mode, such as additional clearvance of leached protein A. (Fahrner, Biotec. Genetic Eng. Reviews, 18, 2001, p. 322, ¶1).

Hana (“removing specific cell culture contaminants in a MAb purificaiton process” Biopharm, 4(9), pp. 33-37, 1991) disclsoes purifying a MAb where AEX is part of a schedule with Protein A affinity.

Kaisha (US 15/108017, published as 2016/0326253) discloses purifying an antibody with a low pI such as 5-7.5 which includes treating a composition containing the antibdoy with an acidic condition such as virus inactivaiton, neutralization of the composition and removing aggregates with AEX used in bind and elute mode. 

Takao (JP 2010241761) discloses purifying an antibody monomer using AEX in bind and elute mode. In one embodiment, the isolectric point of the antibody monomer is 6-9, the salt concentraiton 0-0.1M, the antibody absorbs to the membrane and elution is carried out with lower pH. 

–-With Tentacle AEX: (see also purification of antibody fusion proteins).

Perry (US 16/042,965, published as US 2019/0085021) disclsoes a method of purifying a recombinant protein having a CH2/CH3 region of an antibody such as a TNF rectpor F fusion protein using as tentacle AEX mdium under condtions swhere the recombinant protein binds the AEX and eluting the protein. The tentacle AEX was particularly effective in reducing leached protein A and also in yeild of the recombinant protein. 

Weak partitioning mode: 

As with flow-through, the process is run isocratically, but in contrast to flow through mode, the conductivity and pH are chosen such that the binding of both the product and impurities are enhanced, attaining an antibody partition coefficient (Kp) between 0.1-20 and preferable between 1 and 3. This takes advantage of the fact that the impurities to be removed are more acidic than the product. Both antibody and impurities bind to the anion exchange resin, but the impurities are much more tighly bound than in flow through mode, which can lead to an increase in impurity removal. Thus, eaker binding impurities that are not removed efficiently in flow through mode can be removed to a greater degree under conditions wehre their partition coeefficient (Kp) has been increased. Pfouct yield in weak partitioning mode is maximized by including a short wash at the end of the load. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs, 2:5, 480-499 (2010).

One aspect of weak partitioning chromatography is that the pH and counterion conditions need to be optimzied for each product. This is in contrast to some platform chromatography processes that are able to use standardized conditions on an anion exchange matrix for most products. In order to rapidly define the opimal operating conditions for WPC, high through put screening methods have been developed. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Coffman (US2013/0317198 and WO2012/014183) discloses an in tandem Protein-A AEX scheme where the AEX is operated in a “weak partinioning mode”. 

–With Tentacle AEX:

Corbett (J of Chromatography A 1278 (2013) 116-125) disclsoes that tentacle type anion exchanges, comprising charged polymers grafted toa macroporous matrix ahve been found to be particularly effective in 2 stage Protein-AEX, purification processes, especially when used in the weak-partitioning mode. 

Displacement/Overload mode:

–Indigenous Displacement Mode: 

Brown (WO2010/019148) discloses a method for purifying an antibody using AEX under conditions of a buffer having a pH of about 1-5 pH units above the pI of the antibody and a conducitvity of les than about 40 mS/cm which case the membrane to bind the antibody and recovering the purified antibody from the elluent. For embrane AEX run in indigenous protein displacement mode, the pH of the laod material is adjusted to aobut 1-5 pH units above the pI of the antibody, the conductivity of the load is adjusted to less than about 40 mS/cm, depending on the pH. Because the pH of the load is greater than the pI of the antibody, the antibody (which has become hegatively charged) will not flow through initially. Rather, the antibody will be electrostatically bound to the positive funcitonal groups of the AEX. This is becasue the antibody (negative) and membrane (positive) have opposite charge. Since the pI of many contaiminatns (e.g., host cell proteins such as CHOP) is only slightly different form the pI of the antibody, these contaminants like the “acidic” antibodyes, will also bind to the membrane. However, the contaminants preferentially bind to the membrane or otherwise effectively displace the antibody from the membrane. 

(Nadarajah, US2014/0301977) discloses loading a composition onto a chromatography material such as HIC such that the product like a polypeptide is loaded onto the material at an amount exeeding the DBC of the material for the product. In some embodiments the chromatography conditions are choses such that even if product breaks through aftering binding most of the impurities do not. The product found in the eluate can be pooled. Upon completion of loading, the product (e.g., polypeptide/antibody) is eluted. In some embodiments, the overload and elute chromatography (OEC) is performed where the partition coefficient (Kp) of the product is greater than about 30, 50, 75 or 100. Ndarajah exemplifes using AEX chromatgoraphy in an overload and elute mode of chromatography. Load conditions were found such that the product and the impurities Kp were >100 and although the product flows through after reacing its binding capacity, the impurities keep binding to the resin and do not break until they reach their binding capacity, which could be higher than the product binding capacity.

Void Volume-partitioning:

(Gagnon, US patent applicaiton 14/555060, published as  US Patent No: 9890205) discloses providing a packed chromatographic column having positively charged porous particles (i.e., AEX), equilibrating the packed column to conditions to which the desired antibody/protein is to elute, contacting the sample with the column such that the sample volume applied to the column is less than or equal to the interparticle space of the positively charged porous particles within the column and eluting the desired protein from the column. As a result of the electropositive porous particles, the antibody are restricted to the inter-particle or void space (the space between particles) by the force of electrostatic republision which may cause the antibody to transit the column substantially if not exclusively through the void space. The unique oeprating features are acheived by limiting sample application to a volume not exceeding the inter particle volume of the positively charged particles within the column. 

Operating Conditions

Equilibration:

Gagnon (US 14/555060) discloses using an organic cation such as ethacridine or arginine as an aggregate dissociating agent prior to the step of contacting an antibody sample with an AEX.

Binding:

Ansaldi (WO99/62936) discloses a method of separation a monomer from a mixture of dimers/multimers by applying the mixture to a AEX wehre the pH of the buffer is about 6-9 and eluting at a gradient of about 0-1 M of an elution salt.

–Anionic buffers: Gagnon (US 2013/0210164) discloses methods for purifying proteins/antibodies using AEX where a displacement counter ion which is an anionic buffer such as acetate, phosphate, citrate, formate, succinate, malate or lactate is used to compete with the hyroxide ions associated with the positively charged functionality of the AEX. In some embodiments the solid phase is euqilibrated to a pH of 6.5-8.5.

Wash:

–Isocratic wash:

Yao (US2012/0282654) discloses purifying an antibody which incorporates AEX in the scheme where the antibody is applied to a strong quater-nary ammonium (Q) AEX equilibrated with 20 mM tris(hydroxymethyl) aminomethan hydrochloride pH 80 and washed with the same buffer wherein the soltuon washes the unbound antibody through the column and collecting the antibody in the washed through eluate. 

Elution: 

—-Elution with salt gradient:

The separation of proteins by IEX is usually made with a salt gradient, going from a pure buffer solution to a solution of salt and buffer. The elution strengh of an eluent is the ability to elute solutes form the column. For a given stationary phase, the elution strenght is determined by the concentration and type of ionic species in the eluent. The elution strenght is hus lower in the starting buffer than in the eluting buffer. This leads to an increase in elution strenght during the gradient. (Malmquist, J. Chromatography, 627 (1992) 107-124).

In bind and elute mode, the antibody product pool is loaded onto an anion exchange column and the product of interest is then eluted with a higher salt concentration in a step or linear gradient, leaving the majority of impurities bound to the column (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs, 2:5, 480-499 (2010).

Ansaldi (WO99/62936) discloses a method for separating a polypeptide monomer from a mixture of dimers or multimers by applying the mixture to an anion-exchange resin in a buffer of about 6 and eluting the mixture at a greadient of about 0-1 M of an elution salt.

Graf (Bioseparation, 4, 7-20, 1994) discloses elution of antibodies from anion exchangers performed at around 0.13 NaCl.

Liu (WO2005077130) disclsoes a “bind-washout” process for the purificaiton of anitobdy monomers from a sample containing aggregates by determing the pH value and salt concentration such that the antibody monomers and the aggregates bind to a chosen resin such as anion exchange and then loading the sample onto the chosen resin  and eluting the antibody monomers from the resin using a step gradient.

  –For removal of Protein A contaminant: Protein A has also been selectively isolated from a liquid contianing antibody and Protein A by exposure thereof to an anion exchange material. Both components are absorbed to the anion exchange and the antibodies and Protein A are then sequentially eluted under conditions of increasing ionic strenght (US 4,983,722).

—-Elution with amino acids:

gillespie (US2012/0149878) discloses methods of reducgin HMW in a sample containing a mAb by loading the sample onto an AEX and eluting with amino acids such as arginine and glycine.

Changing Conductivity and/or pH buffers:

Basey (US6,339,142 & WO99/57134) discloses a method of purifying an antibody by AEX by loading the antibody onto a AEX using a loading buffer that is at a pH and/or conductivity , washing with an intermediate buffer at a second conductivity and/or pH that at a pH which is less than the equilibration buffer so as to essentially elute the contaiminant but not a substantial amount of the antibody such that the antibody, washing with a buffer that has a conductivity or pH or both which is increased compared to the intermediate buffer  and then eluting the antibody using an elution buffer that has a pH and/or conductivity that is less the pH and/or conducitvity of the loading, intermediate and wash buffer used in the previous steps.

In Conjunction with Other Purification Steps (see also Protein A followed by secondary steps like AEX under affinity chromatography)

Anion Exchange as a First Capture Step

Since most foulants are negatively charged and most antibodies are strongly electropositive, foulant removal can be achieved by anion exchange at physiological pH and ionic strenght. The simplest removal can be obtained by adding microgranular cellulosic anion exchange media directly to the raw materail. (Josic “analytical and preparative methods for purificaiton of antibodies” Methods for purificaiton of antibodies, Food technol. biotechnol. 39(3), 215-226 (2001). 

Luhrs (J. Chromatography B, 877 )2009) 1543-1552) discloses a prcoess of separating anti-histone antibodies from histone-DNA complexes by adjusting the salt concentraiton to mM to dislodge any H1 proteins and their attachedd antibodies from the chromatin matrixand then to separate the soluble antibody and histone H1 from the remaining DNA bound chromatin debris using a quarternary amin column (HiTrap q) attached in tandem in front of Protein A. The strong AEX captures the negatively charged NA and its accompanying binding proteins. 

Zarbis-Papastoitsis (WO2011/110598) discloses a method to remove host ccell protein from a cell broth from a secreted desired biological substance having an overall positive cahrge by tcontacting the broth with an anion exchanger, allowing incubation to result in th fomation of a cell pellet and supernatant layer and separating the resulting cell pellet from the supernatant layerand detereming the reduction of the HCP content in the supernatant layer.

AEX-Protein A affinity chromatography or Mixed Mode – CEX

Felfoldi (US 15/120359, published as US 2017/0058019) disclsoes a method of purifying an immunoglulin by exposing a sample with the antibody to AEX and then contacting the flow through from the AEX to Protein a affinity chromatography or to mixed mode chromatography and then exposing the eluate to CEX. 

Anion Exchange followed by Secondary steps:

AEX-CEX: Ishihara (US 2006/0257972) teaches purification of antibodies via AEX and then CEX. 

AEX-HIC: 

Guse (J chromatography A, 661 (1994) 13-23) discloses purification of m anti-CD4 using (NH4)2SO4 precipitation, AEX on MonoQ or Q Sepharose, hydrophobic interaciton chromatography on phenyl-Sepharose and gel filtration.

Damasceno (Protein Expression and Purification 37 (2004) 18-26) discloses a two step chromatography procedure for purificaiton of a single chain variable domain fragment antibody (scFv) using anion exchange in bind in elute mode followed by hydrophobic interaciton chromatography.

Kremer (US2013/0131318; see also WO2011/098526) discloses purification and polishing steps for antibodies which includes serial in line AEX in flow through followed by HIC. The separation mixture prior to HIC is supplemnted with an adequate amount of lyotropic salt. 

Ntiglyabaah (US2014/0288278) disclsoes methods of purifying an antibody such as adalimumab by binding the antibody to Protein A then to AEX in flow through mode and a polishing step which can be mixed mode resin or HIC. 

Wan (WO 2007/117490) disclosea a emthod for proudcing a HCP reduced antibody preparation using AEX in flow through mode followed by HIC in bind and elute mode.

AEX-MM

Islas (US/14/126677, published as US2014/0187749) discloses a method for the purification of antibodies using serial in-line (single unit operation) AEX followed by MM both in flow through mode. 

Nti-glyabaah (US2014/0288278) discloses methods of purifying an antibody such as adalimumab by binding the antibody to Protein A then to AEX in flow through mode and a polishing step which can be mixed mode resin or HIC.

Anion Exchange which itself follows others steps  See also other types of chromatography such as affinity chromatography where AEX is a subsequent step 

Crystallization –AEX:  Wilkins (WO 2009/085765) discloses a method of purifying a CD20 anitbody from harvested cell culture fluid of ammalian cells by concentrating the HCCF, dafiltering the HCCF with a high salt concentraiton at a pH that inhibits crystallization, crystallizing the CD20 antibody by raising the pH dissolving the antibody crystals to obtain a CD20 antibody solution, AEX. 

Types of Resins

Membrane Absorbers (Membrane chromatography): The use of conventional packed-bed chromatography with flow-through anion exchange (FT-AEX) requires columns of very large diameter to permit high volumetric flow rates to prevent a prcoess bottleneck at the polishing step. Also, proper flow distribution in production columns requires a significantly large bed volume. These disadvantages seen with AEX columns have led to the devleopment and utilization of membrane chromatography or membrane adsorbers such as the Sartobind Q membrane adsorber. (Zou, J. Chromatography A, 1134 (2006) 66-73)

Ion exchange has several characteristics that make it particularly suitable for large scale purificaiton regimes, especially when compared to affinity based methods. IEX can be significantly cheapter than affinity matrices, are also more physically robust and so can be cleaned in place with harsh reagents such as 1M NaOH without loss of performance. Humphreys (see also Protein Expression and Purificaiton 37, 2004, 109-118)

Conditions/Parameters Generally

Binding:

Brown (WO2010/019148) discloses purifying an antibody using CEX where the polypeptide and the membrane have opposite charge, at operating conditions comprised of a buffer having a pH of about 1-5 pH below the pI of the polypeptide and a conductivity of less than about 40 mS/cm, which cause the membrane to bind the polypeptide and at least one contaminant and then recovering the purified antibody/polypeptide from the effluent. 

Elution: (see also CEX and AEX chromatography)

–pH Gradients

By generating a lienar pH gradient antibody isoforms can easily be eluted near their isoelectric points. Kaltenbrunner “Isoprotein analysis by ion-exchagne chromatogrpahy using a linear pH gradient combined with a salt gradient) J. Chromatography, 639 (1993) 41-49)

–Salt Gradients:

The separation of proteins by IEX is usually made with a salt gradient, going from a pure buffer solution to a solution of salt and buffer. The elution strengh of an eluent is the ability to elute solutes form the column. For a given stationary phase, the elution strenght is determined by the concentration and type of ionic species in the eluent. The elution strenght is hus lower in the starting buffer than in the eluting buffer. This leads to an increase in elution strenght during the gradient. The choice of displacing salt has a significant impact on the separation.  (Malmquist, J. Chromatography, 627 (1992) 107-124).

Ansaldi (WO99/6936) discloses separating a polypeptide monomer from a mixutre of dimers/multimers by applying the mixture to eitehr a CEX or AEX and eltuing at a gradient of about 0-1 M of elution salt, wherein the monomer is separated from the dimers and/or multimers.

Combined pH – Salt Gradient

Wang (WO 2014/078729) discloses methods for analyzing/purifying polypeptides such as antibodies by ion exchange such as a CEX or AEX chromatography where a combination of pH gradients and ionic strengh gradients are used to separate the polypeptide from charge variants of the polypeptide. In some embodimetns, the pH is either a linear or step gradient, and the gradient includes a decrease from about pH8-5. In other embodiments, the ionic strenght gradient is a step gradient. 

–-Inverse pH -Salt Gradients

Joehnck (SU Patent Application No: 15/776,941, published as US 2018/0327447) discloses a method for purifying a protein such as an antibody using IEX and running an opposite pH-salt gradient by an ascending pH and descending salt concentration to separate the protein(s) or vice versa running a descending pH and an ascenidng salt concentration. The opposite pH-salt hybrid gradient is generated by externally mixing two buffers (i.e., A and B) with different pH values and sodium chloride concentrations (i.e., A with low pH and high salt concetnration; B with high pH and low salt concentration) at the column inlet, which then travels through the column. 

Kaltenbrunner “Isoprotein analysis by ion-exchagne chromatogrpahy using a linear pH gradient combined with a salt gradient) J. Chromatography, 639 (1993) 41-49) discloses separation of isoproteins of human monoclonal antibodies by ion-exchange chromatography with a linear ascending pH gradient combined with a linear decending salt gradient using borax mannitol and salt. 

Changing Conducitivy and/or pH buffers:

Basey (US6,339,142 & WO99/57134) discloses a method of purifying an antibody by IEX which includes chaing the conductivity and/or pH of the buffers in order to eliminate contaminants. The protein is bound to the IEX using a loading buffer at a first conductivity and pH, washed using an intermediate buffer at a second conductivity and/or pH so as to elute the contaminant, washed at a third conductivity and/or pH at a conductivity and/or pH in an opposite direction to the conducitvity and/or pH from the loading buffer to the intermediate buffer and then eluted at a fourth conductivity and/or pH.

Particular Impurities Separated

Antibody monomers form dimers and/or multimers

Ansaldi (US 2002/0010319) disclose separating an antibody monomer from a mixture comprising dimers and/or multimers by applying the mixture to either a cation-exchange or an anion-exchange chromatography resin in a buffer, wherein if the resin is CEX, the pH of the buffer is about 4-7 and if the resin is AEX the pH of the buffer is about 6-9 and eluting the mixture at a gradient of about 01 M of an elution salt, wherien the monomer is separated from the dimers and/or multimers presentin in the mixture. 

Antibody Variants

Melter (J of Chromatography A, 1200 (2008) 156-165) discloses that small changes in the isoelectric point )oI) of proteins can induce largely different retention behaviors in ion exchange chromatography which is particularly true for MAb variants having different numbers of C terminal lysine groups. C terminal lysine groups contain an amino group and thus an increase in the number of lysine group increases the net positive charge of the MAb and its pI.

HCP:

Wan (WO2007/117490) discloses a method for producing a host cell protein (HCP) reduced antibody preparatio from an antibody mixture using an ion exchange separation step such that a first eluate having a reduced level of HCP is obtained. 

Ion Exchange in combination with other Techniques Generally

IEX-HIC: 

Basey (WO99/57134) disclsoes a method of purifying a polypeptide such as an antibody by ion exchange chromatography which involves changing the conductivity and/or pH of buffers. In one emboidment the ion exchange is CEX and the antibody is binding using a loading buffer at a first conductivity and pH, using a washing buffer at a second conductivity and/or pH which is greater than that of the laoding buffer so as to elute the contaminant, washing the CEX with a wash buffer at third conducitvity and/or Ph which is less than that of the intermediate buffer and finally washing with an elution buffer at fourth conductivity and/or pH which isgreater than that of the itnermediate buffer so as to elute the polypeptide. In an AEX embodiment, the conductivity are generally as with CEX but the direction of change in pH is different. For example, the loading buffer has a first pH and the pH is decreased in the interemdiate buffer so as to elute the contaminant, then the column is washing with a buffer in opposite direction to the previous step(pH may be increased to that of the intermediate buffer), then the antibody is eluted using an elution buffer at a fourth conductivity and/or pH. The method is usefuly for resolving a polypeptide which differs only slightly in ionic charge from acontaminant. Additional purification steps include HIC as well as further AEX or CEX steps.

Hickman (WO2010/048183) discloses a methods of purifying antibodies by adjusitng a primary recovery sample to pH 4.5-5.5 followed by applying it to an ion exchange resin which can be AEX or CEX followed by applyting the flow through fraction to hydrophobic interactive chromatography (HIC) and then eluting the antibodies from the HIC. 

Wan (WO2007/117490) discloses an ion exchange separation step such that a first eluate having a reduced level of HCP is obtained, a second ion exchange step such that a first flow through is obtained and a hydrophobic interaction  (HIC) separation step comprising loeading the first flowthrough onto a column comprising a first HIC material such that a second eluate is obtained.

Activated Carbon Filters

Activated (active) carbon (“activated charcoal) is a form of carbon processed to have small, low-volume pores that inc4rease the surface area available for adsoption. it is produced from carbonaceous source materials such as nutshells, coconut husk, peat, wood, coal and petroleum pitch.

Activated carbon is an inexpensive natural material having extensive non-specific adsorption properties and is used as an adsorbent or as a decolorant in the industrial fields such as production of chemicals and foods, sewage or waster water treatment, and water filtration.

How Activated Carbon is Produced

Activated carbons are characterized by a large specific surface area typically in the range of 500-2500 m2/g, Commercial grades are designated as either gas phase or liquid phase adsorbents. Liquid phase carbons generally may be powdered, granular or shaped; gas phase are hard granules. Commercial activated has been made from material of plant origin such as hardwood and softwood, corncobs, kelp, coffee beans, rice hulls, fuit pits, nutshells and wastes such as bagasse and lignin. AC also has been made from peat, lignite, soft and hard coals, tars and pitches, aslphalt, petroleum residues and carbon black. Activation of the raw material is accomplished by one of two distinct processes: 1) chemical activation or (2) thermal activaiton. (Tolles &S 5,204,310)

Use in Antibody Purification

Activated carbon (AC) is compatible with all common solution conditions found throughout the downstream process. It is capable of operating in acidic and basic pH ranges as well as high and low olution condcitivties. Since AC can be operated in flow through mode under typical process pH and conductivities, the possibility to connect it with adjacent IEX chromatogrpahy steps becomes attractice. Millistak+ CR40 media is formulated with AC retained in a rigid structure by a cellulose matrix and can be used to reduce HCP present in Protein A elution pools. (“The use of Millistak+ Activated Carbon (AC) for downstream purification of monoclonal antibodies” 2015). 

Skudas (US 15/524824, published as US 2017/033741) discloses a method for puriying an antibody away from exctables/leachables using activated carbon in a filtration device.