Resins with multimodal ligands such as Capto MMC and Capto Adhere (GE Healthcare) can be used as polishing steps for antibody purificaiton. The multimodal functionality of the resin provides selectivity that is different from standard ion exchange ligands, which makes them suitable for solving purification problems at both high and low conductivity or pH. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Lali (US2010/0113746) discloses a process for purification of IgG using an affinityligand  which is ionic, hydrophobic and/ or mixed mode (combinations of groups). In one embodiment the IgG is eluted with a buffer having a pH of 2.5 to 9, containing an organic or inorganic salt and containing an additive such as ethanol, ethylene glycol, glycerol, polyethylene glycol, surgars so as to ehance recover of the IgG in elution.

Types of Mixed Mode Chromatography

Affinity Mixed Mode

–Affinity – Cation Exchange (CEX)

Kihara (US Patent application No: 15/952,627) disclsoes an affinity chromatography carrier which includes a substrate that is composed of a polysaccharide (e.g., crosslinke agarose-based substrate such as Sepharose 6 Fast Flow (GE Healthcare GMbH, (“SEPHAROSE”), an acrylate-based polymer, a methacrylate-based polymer or a styrene-based polymer, an affinity ligand that includes an antibody-binding polypeptide such as Protein A and a carboxy group that is introduced at 15-60 mmol/L-gel. (1 meq/L-gel- 1 mmol/L-gel). Examples of introducing a carboxy group into the carrier include reacting the carrier with chloroacetic cid under alklaine onditions, a carboxymethyl group is introduced into a part of hydroxy groups on the surface of the carrier, an aldehyde group is introduced into a carrier, a carboxy roup is introduced into the aldehyde group by reductive animation through an amino group by reductive amination through an amino group of a compound having a carboxy groupand an amino group such as amino acid. An example of introducing an affinity ligand into the carboxylated carrier is not particularly limited and includes a method in which a part of carboxy groups is converted into N-(3-dimethylaminopropyl)_-N’-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) and reacted with an affintiy ligand such as protein A and the urneacted EDC/NHS converted carboxy group is regenerated after the reaction and a method in which in the case of an affinity lgiand having an amino group such as protein A, a carboxy group is protected, an aldehyde group is introduced, adn then the affinity ligand is introduced into the aldehyde group by reductive animation through an amino group. The substrate also includes a hydrophilic polymer such as 13 g of desxtran 270 (intrinsic viscosity: 0.23 dL/g, weight-average molecular weight: about 70,000). 

Anion Exchange Mixed Mode Chromatography

Engstrand (US2007/0259453 and WO 2006/043895 A1) discloses a method of separating antibodies with a multi-modal separation matrix to adsorb undersired compounds while the antibodies remain free in the liquid (flow-through mode). The matrix comprises first groups which are capable of interacting with negatively charged sites of the target compounds (e.g., strong anion exchangeres such as quaternary amines (“Q groups”)) and second groups which are capable of at least one interaction other than charge-charge interaction with said target compounds (e.g., aromatic groups and/or hydrogen bonding groups). Ligands useful in the method include N-benzyl-N-methy ethanolamine. The method is run under conditions conventional for anion exchange chromatography which commonly involves adsorption at a relatively low salt concentration.

Gagnon teaches removing a virucidal agent such as methylene blue from a target protein such as an antibody using hydrophobic, negatively charged mixed mode support that has high affinity for both the target protein and the virucidal agent. The antibdoy is then eluted from the support. 

Suitable annion exchange columns include Capto adhere. Althouse (WO 2015/070068)

–Capto Adhere.RTM. is a strong anion exchange with multimodal functionality which confers different selectivity to the resin compared to traditional anion exchanges. Its ligand is N-Benzyl-N-methyl ethanolamine. (Nti-Gyabaah (US14355014). useful multi-modal anion-exchange ligands include N-benzyl-N-methl etahnolamine, N,N-demethylbenzylamine, 2- amino benzimidazole and thiomicamine Engstrand (US 2007/0259453).  The Capto Adhere ligand, N-benzyl-N-methyl ethanol amine, has a high ion-exchange capacity mixed with an aromatic moeity, which also promotes its stability. The Capto Q ImpRes ligand has a high ion-exchange capacity due to a quaternary amine. (see Chromatographia). 

Cation Mixed Mode Chromatography

Johansson (US2007/0112178) teaches separating antibodies from contaminants in a solution by contacting the solution with a chromatography resin having a support to when multi-modal ligands have been immobilised. The multi-modal ligand comprises at least one cation-exchanging group and at least one aromatic or heteroaromatic ring system. In one emobdiment, the ring forming atoms of the aromatic or hteroaromatic entity are C, S or O and the cation exchanging group is a weak cation exchanger. The method may be used as a single step or as a polishing step following Protein A chromatography.

Suitable cation exchange column include Capto MMC and nuvia cPrime (Biorad). Althouse (WO 2015/070068). Capto MMC is a multimodal weak cation exchange that is salt tolerant (see Cytiva). 

Conditions/Parameters:

Binding:

(i) Bind and elute mode:

(a) binding

—-Amino acids. Suenaga (US2011/0251374) discloses a method of purifying an antibody by treating a solution containing the antibody in the presence of an amino acid by mixed mode chromatography. The amino acid can be a basic amino acid such as arginine, a hydrophobic amino acid, an acidic amino acid or citrulline. In one embdodiment the antibody was applied to a Capto adhere column quilibrated with a citrate buffer containing proline.

—-Salts & Denaturants:

Falkenstein (US13883243, now US 9422329 and WO/2012/059495; see also 15/216,099 published as 2017/0015702) discloses applying a buffered solution comprising an inorganic salt to a multimodal weak cation exchange chromatography material such as Streamline CST and/or Capto MMC and then applying a cell culture containing an antibody to a multimodal weeak cation exchange chromatography material. In one embodiment, the buffer containing the inorganic salt also contains a denaturant such as guanidinium hydrochloride and urea.

—-Non-ionic organic polymer:  Gagnon (US2009/0247735; US20080177048WO2008/086335) teaches a method of purifying proteins such as antibodies from a mixture by contacting the mied with a mixed mode chromatography support in the presence of an aqueous soluble (i.e., hydrohpilic) non-inoic organic polymer. The presence of the polymer enhances binding capacity for virus or protein on mixed mode chromatography supports and enhances the retention of antibody in comparison to most contaminating proteins, thereby enabling novel selectivity for improved removal of non-antibody proteins. 

(i) flow through mode

Engstrand (US 7,714,112) discloses a method of separating antibodies from other compounds in a sample using a multi modal seapration matrix to absorbed undesired ocmpounds while the antibodies remain free in the liquid.

(Nti-Gyabaah, (US 14/355014) discloses using MM  for the purificaiton of monoclonal antibodies from heterogeneous aggregates. In certain embodiments, the MM is conducted in flow through mode using Capto adhere at operating conditions of pH 6.8-7.7, conductivity of 4-25 mS/cm, protein loading of 100-205 gm/protein liter of resin and 20-200 mM salt concentration. In one embodiment, the column is equilibrate with 3 CV of 100 mM sodium citrate pH 3 followed by 5 CVs of 20 mM Tris-HCL, 110 mM NaCl pH 7.2. The pH and conductivity of the feed are adjusted to the desired values then loaded to the column at 300 cm/hr. Upon completion of the loading, the column is washed with 2-10 CV of the equilibration buffer at hte same flow rate. Becasue the product is in the flowthrough, the product collection begins at 100 mAU into the loading and ends at 350 mAU during hte wash step. Product pool pH is 7.2 upon elution and is quenced with 1M acetic acid to a final pH of 5.5 prior to analysis. 

(ii) Overload mode

(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 laoding, 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. Nadarajah exemplifes using Capto Adhere resins and loading Protein A pools on such mixed mode resinsfollowed by an elution buffer to elute off bound protein. The flowthrough or eluate during the load, overload and eltuion phases were collected as fractions.

(b) Elution:

Althouse (WO 2015/070068) disclsoses a mixed mode step that follows Protein A affinity chromatography. The mixed mode can be either cation or anion or a combination. This step can also be based on a single type of ion exchange mixed mode or can include multiple ion exchanger mixed mode steps such as a cation excahnge mixed mode step followed by an anion exchange mixed mode step or vice versa. Both the AEX and CEX mixed mode chromatographys can be run in bind and elute mode. 

Arakawa (US 2014/0072560) discloses mixed mode chromatography for seaprating correctly folded proteins such as etanercept. In one embodiment, the bound prtoein is eluted by a simultaneous gradient of both pH and NaCL concentration. 

Gronberg (WO2005082483) discloses process for the purification of antibodies using multi modal ligands having at least one CEX group and at least one aromatic or heteroaromatic ring system immobilised to absorb the antibodies to the resin, adding an eluent to release the antiboides and contacting the eluate with a second chromatography resin. The CEX group is preferably a weak cation exchanger.

Johansson (US2007/0167613 and WO2005/082926) discloses purification of antibodies using multi-modal ligands immobilised to adsorbe the antibodies to the resin whicherein each multi-modal ligand has at least one cation exchanging group and at least one aromatic or heteroaromatic ring system. The eluate is commonly a buffer such as a phosphate buffer and in one embodiment a step elution by increase of pH and/or salt concentration. The CEX group is preferably a weak cation exchanger (group which can be protonated at certain pH values). The multi modal chromatgoraphy can be followed by a second chromatography step such as IEX, HIC and affinity chromatography.

–Ethylene glycol + Inorganic salt

Falkenstein (US13883243, now US 9422329; see also US 15/216,099, published as 2017/0015702; see also WO/2012/059495) discloses applying a cell culture containing an antibody to a multimodal weeak cation exchange chromatography material such as Streamline CST and/or Capto MMC and recovering the antibody by applying a buffered solution comprising ethylene glycol and an inorganic salt such as ammonium, potassium or soldium chloride. In one embodiment, the buffer further comprises a denaturant such as guanidinium hydrochloride or urea. In one embodiment, the buffer includes sodium chloride, aobut 20% (w/v) ethylene glycol at pH 7.1-7.3.

In Combination with Other Types of Chromatography

Affinity (e.g., Protein A/G) – Mixed Mode

Althouse (WO 2015/070068) disclsoses a mixed mode step that follows Protein A affinity chromatography. The mixed mode can be either cation or anion or a combination. This step can also be based on a single type of ion exchange mixed mode or can include multiple ion exchanger mixed mode steps such as a cation excahnge mixed mode step followed by an anion exchange mixed mode step or vice versa. 

Anion-MM

Perlasca Islas (US 2014/0187749) disclsoes a method for the purificaiton of anitobides which includes intermedaite and polishing step that includes in-line AEX and mixed mode chromatography. 

MMC-HIC:

Dong (US2012/0238730) teaches purification of antibodies using Protein A followed a fine purification step which included mixed mode based separation using CaptoAdhere followed by either CEX or HIC. 

Ma (US14208043) discloses a method for reducing high molecular weight (HMW) species and/or high mannose species by starting a starting solution having a desired protein such as an antibody to MMC to form a first eluate and subjecting the firust eluate to HIC . In one embodiment the MX is conducted in flow through mode and the HIC is conducted in bind/elute mode. 

Mccue (US2011/0129468) discloses a method for purifying Ig (constant domain of Ig) fusion proteins by MM in bind and elute mode followed by HIC in bind and elute mode.

Gagnon (US2009/0247735) teaches the use of mixed mode chromatography for purificaiton of an antibody from other materials such as aggregated antibodies which can also be integrated with other fractionation methods such as HIC. 

Hunter (US 14/890,791, published as US 2016/0083453) discloses a emthod of separating antibody multimers with minimal separation of monomers by subjecting a mixture cmprising a plurality of mAbs to mult-modal chromatography, apatite chromatography and HIC. 

Herigstad (US9,249,182) disclsoes subjecting a sample media comprising an antibody such as adlimumab to a mixed mode chromatography and then hydrophobic interaction chromatography. The HIC can remove a variety of process related impurities (e.g., DNA) as well as product related species (e.g., high and low MW proudct related species such as aggregates and fragments).

Johansson (US7,750,129) dicloses a process for the purification of antibodies using multi-modal chromatography which includes at least one cation exchaing group and at least one aromatic or heteroaromatic ring system to adsorb the antibodies and adding an eluent to release the antibodies and contacting the eluate with a second chromatography which can be HIC. 

Ramasubramanyan (14/077,871; (US2015/0110799); 14/575,691 (US2015/0132801) and US 14/614,311 (2015/0140006) and US14/584,619 (US2015/0166650) teaches a method for producing a low acidic species composition comprising an antibody by contacting a sample comprising an antibody to an AEX, CEX or mixed mode mediaand eluting the antibody and further contacting the leuted sample to an HIC media.