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)