Introduction:

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

Media/Resins: 

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

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

–Commercial examples: 

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

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

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

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

–Commercial Examples of Cation Exchange Membranes:

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

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

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

Conditions/parameters: 

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

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

–Binding:

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

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

–Washing: 

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

–Elution (pH vs salt):

(1) Salt Gradients: 

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

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

(2) Ph Gradients:

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

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