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.