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.