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

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

General Principles: 

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

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

Types of Ligands/Media/Resins

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

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

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

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

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

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

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

Commercial Suppliers

–Anion Exchange Membranes:

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

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

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

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

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

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

Operating Modes

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

Bind and elute mode: 

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

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

Flow-through mode: 

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

Weak partitioning mode: 

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

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

Combinations of AEX with other Chromatography

AEX-CEX-HIC:

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

AEX-HIC-Protein A-AEX

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

AEX-HIC-Isoelectric focusing/polyacrylamide electrophoresis:

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