secondary steps – Ypatent

See also ion exchange and Particular types of antibodies

Affinity A followed by AEX Chromatography

A typical platform for purificaiton of mAbs derived from recombinant cell culture employes three chromatographic steps, with a Protein A capture column followed by two chromatogrpahic polishing steps. The polishing steps almost invariable employ anion exchange operated in a flow through mode, and one additional adsorptive step (either cation exchange, eramic hydroxyapatite or hydrophobic interaction). (Kelley, Biotech. & Bioeng. 101(3), 2008).

AEX is almost always used in the purification of mAbs to remove acidic host cell proteins (HCPs), DNA, virus and other negatively charged contaminants. When used in tandem with a selective capture step such as Protein A, AEX can serve as the basis for a two column purificaiton platform. In this case, the bulk of the impurities are cleared trhough the selective Protein A step while remaining traces are removed by adsorption on the AEX resin. (Corbett, J of Chromatography A, 1278 (2013) 116-125).

Affinity-AEX:

Bonnerjea (US2006/0194953) discoses a method for selectively removing leaked protein A from antibody purified by Protein A affinity chromatography by first purifying an antibody by means of protein A , secondly, loading the antibody onto an anion exchange under conditions that allow for binding of the Protein A.

Corbett (J of Chromatography A, 1278 (2013) 116-125) diclsoes that tentacle type anion exchangers, comprising charged polymers grafted to a macroporous matrix, have been found to be particularly effective in using AEX as part of a two-column purification platform following Protein A capture step, especially wehn used in the so-called weak-partitioning chromatoraphy mode.

Wan (US 6,177,548) teaches a method for removing aggregates from partically purified monoclonal antibody using anion affinity A followed by anion excahnge. The pH of the sample is adjusted to be lower than the isolectiric point of the product.

–Affinity-AEX (flow through mode)

Kulkarni (13/518532) also disclose purification of antibody using anion exchange chromatography perforemd in flow-through moade where the eluate obtain from a protein A chormotography step without substantial adjustment of pH. In some embodiments the pH values are about 3.3 to about 6.

–Affinity-AEX (weak partitioning mode)

Coffman (US13/811178) teaches purifying a protein/antibody using Protein A in bind/elute mode to from a first eluate which is then titrated continously as it passes to an AEX. The columns are arranged in tandom (in-line) and the titration is done with a pH buffer and at least 150 mM of a salt and added at a target volumetric ration to the first eluate such that there is a change in partition coefficient of less than 20% when the actual volumetric ration of the first eluate to the titrant varies up to about 405 from the target volumetric ratio. The operating conditions brings the resulting prcoess stream to a specific range of pH levels and salt constration levels that promote weak partitioning chromatography on the AEX.

Corbett, J of Chromatography A, 1278 (2013) 116-125) discloses that AEX is typically used in conjunction with Protein A chromatography to remove remaining traces of impurities such as HCPs, DNA virus and other negatively charged contaminants and that in this respect tentacle type anion exchanges have been found to be particularly effective expecially when used in weak-partitioning chromatography mode.

Iskra, Biotechnology and Bioengineering, 110(4), 2013) discloses that excellent removal of host-cell proteins, leached Protein A, DNA, HMW species and model virus can be achieved using AEX oeprated in WPM following Protein A chromatography.

Kelley ( “Weak Partitioning Chromatography for Anion Exchange purification of Monclonal Antibodies” Biotechnology and Bioengineering 101(3): 553-566 (2008) describes Pro A – AEX sequence but in contrast to the standard flowthrough conditions used for AEX chromatography a significant amount of product binds to the resin. These more stringent load conditions result in much stronger impurity binding, which improves the product pool purity.

Affinity-AEX-CEX:

Most antibody manufacturers currently use a three-column platmform comprising Protein A affinity chromatography for product capture, followed by anion exchange (AEX) chromatography in flow-through mode to extract negatively charged contaminants, and then cation exchange (CEX) chromatography or hydrophobic interaction chromatography (HIC) in retention mode to remove positvely charged contaminant species (Giovannoni, “Antibody Purification using Membrane Adsorbers”, BioPharm International, 21(12), 2008 pp. 48-52).

Bonnerjea (US2008/0312425) (see also US2006/0194953) discloses antibody purification by protein A and ion exchange chromatography and exemplifies protein A followed by AIX (equilibration/loading buffer pH 6.5-9, conductivity 0.5-5 mS/cm) followed by CEX (loading buffer 4-7; 0.1-1.2 M salt).

Goklen (WO 2012/135415) discloses a method for purifying an antibody using Protein A, low pH treatment, AEX, CEX, NF and then UF/DF. The method advantgeously employes a simplified sodium chloride free buffer system that consists of two componants and acid such as acetic acid and an organic bse such as Bis Tris.

Falkenstein (US2010/0311952; see also WO2008/145351A1) disclsoes a method for purifying an immunoglobulin using CEX in flow through mode. In one embodiment, a mixutre comprising the immunoglobulin is applied to an affintiy column under conditions where the antibody binds to the column, applying the eluate to an AEX in flow through mode and then applying this eluate to a CEX under conditions where the antibody in monomeric forms does not bind to the CEX.

Ishihara (US 2006/0257972) teaches purification of antibodies via protein A affinity chromatography, then AEX and then CEX.

Kremer (US13881201) discloses method for purification of antibodies using serially connected AEX followed by CEX, both in flow through.

Lonza (WO 2004/076485A1) discloses combination of a protein A chromatography step followed by a first anion exchanger and a second cation exchanger for antibody purification. Preferably, the loading or equilibration buffer for the frist anion exchange step has a conductivity 0.5-5 mS/cm, more preferably of from 1-3 mS/cm, most preferably of from 1.25-2.5 mS/cm. The antibody sought to be purified is collected in the flow through and then loaded onto the cation exchange.

Tugcu (“Maximizing productivity of chromatography steps for purificaiton of monoclonal antibodies, Biotechnology and Bioengineering, 99(3), 2008) discloses a multi step purification method for antibody purification using a MabSelect column (protein A) (elution at pH 2.3 then neutralized to pH 6.5) followed by an AEX step (Q Sepharose XL and Biorad UNOsphere Q) (flow through mode) and CEX (Fractogel SO3-S and Biorad UNOsphere S) (elution pH 4.5). Tugcu states that generally, running AEX following Protein A brings some operational advantages. The pH titration of the Protein A which is already a part of the pH adjustment required following the low pH hold viral inactivation step can easily be extended to pH 7.0 or higher. Because in most cases Protein A product will have a low conductivity (generally 10-15 mS/cm), it is usually possible to prepare the AEX feed by simple batch dilution. Having CEX follow AEX will diminish the problem of further reducign the conducitvity of the feed stream and a simple pH adjustment to lower the pH will be sufficient to preare the feed for CEX loading.

Yao (WO2010/127069) discloses a method for purifying an antibodyt that includes protein A chromatography, inactivating virus particles, CEX, AEX, filtering, ultrafiltering, diafiltering and fine filtering.

Protein A – AEX – CEX – HIC:

Hickman (WO 2010/048192; US2010/0135987) (see Protein A – CEX – AEX – HIC below).

Protein A -AEX – HIC: See also “antibody fusion proteins” under Purification of particular types of antibodies

Mendiratta (US2016/0115195) discloses a method for the purificaiton of a monoclonal antibody which includes Protein A chromaotgraphy, hydrophobic interaction chromatography (HiC) and AEX. The HIC and AEX may be carried out in eitehr order. In one embodiment, the column matrix for AEX is selected from DEAE sepharose, Mono Q and Q Sepharose XL. Purification of the antibody from the HIC is in bind and elute mode wehre the antibody is eluted with down the gradient salt concentration (i.e., with decreased conductivity compared to that of the equilibration buffer conductivity). Elution takes place in the form of a single broad peak.

Ntigyabaah (US2014/0288278 and WO2013/066707) discloses Protein A followed by viral inactivation, AEX chromatography and a polishing step such as HIC. In one embodiment the HIC such as Phenyl Sepharose HP is doncuted in flow through mode using operating conditions of pH 7, conductivity of 110 mS/cm and protein loading of at least 425 grams of protein per liter of resin. Preferred salts include but are not limited to sodium phosphate, tris-HCL and ammonium sulfate. In another embodiment, the HIC is conducted in bind and elute mode where adalimumab antibody elutes for teh first seven column volumes with a 95% yieldindicating that the aggregates contain a higher hydrophobicity than the anti-TNF mAB monomer and can be easily separted form the monomer peak with minimal yield. While the HIC could be utilized in a bind and elute mode, the low step throughput coupled with ammonium sulfate disposal costs and unkown protein stability at high salt concentrations rendered the HIC polishing step unsuitable for a robust commercial purificaiton strategy. On the other hand, this significant amount of 100% pure monomer in the majority of the elution peak indicated HIC could be oeprated in a flowthrough mode to acheive higher productivities. Product pools in flwothrough mode contained solely 100% pure monomer with 85% yeild. However, limitations of HIC still exist such as ammonium sulfate disposal costs and unkown protein stabiliyt at higher sale concetnration.

Trejo (US2016/0024144) discloses purification of a recombinant protein such as etanercept by affinity chromatography over Protein A, subjecting the eluate to a tentacle AEX where either before or after the AEX an additional chromatography step can be performed such as HIC in either flow through or bind and elute mode.

—-Plant extraction:

Hamorsky (Antimicrobial Agents and Chemotherapy, p. 2076-2086, May 2013) discloses production of an anti-HIV-1 mAb in Nicotiana benthmiana plants which includes homogenization of the leaf in an ice cold extraction buffer that includes phosphate, NaCl, ascorbic acid, filtering and then GE Healtcare High Trap protein A column with elution using a gradient f glycine, L-arginine pH 3.0 followed by HiTrap Phenyl HP column.

Protein A – virus inactivation -AEX – HA:

Mazzola (WO2009/017491) teaches purifying antibody by affinity chromatography, eluting the antibody and inactivating with a pH step, adjusting the eluate to pH 6.0 to 8.5 and filtering through an AEX and applying the filtrate to cermaic hydroxyapatite.

Morton (US 15/110200) discloses a method of purifying mAB from a plant source using Protein A chromatography followed by Capto Q column in flow through mode and then ceramic Hydroxyapatite Type II.

Ntigyabaah (WO2013/066707) discloses Protein A followed by viral inactivation, AEX chromatography and a polishing step such as as HA such as Capto adhere operated in bind and elute mode.

—-Plant extraction:

Oligner (PNAS, October 30, 2012, 100(44)) discloses production of anti-Ebola virus mAbs in whole plant cells (Nicotina benthamiana). Plants were brown 24-26 d, equal volumes of Agrobacterium cultures were mixed in infiltraiton buffer and plants inverted into the solution. At 7 days postinfiltration, left tissue was extracted with Tris, ascorbic acid, EDTA pH 8.5 buffer, extract was clarified with filter press, antibody was pautre using MabSelect SurRe, further purified with Mustang Q membrane and further purified with MEP HyperCell.

Pettitt (Science Translational Medicine, 5(199) August 2013) discloses manufacture of mAbs in plans by growing plants, equal volumes of Agrobacgerium cultures were grown overnight and mixed with infiltration buffer. At 7 days after infiltration .eft tissues were extraced with buffer of tris, ascorbin acid, EDTA, pH 8.5, the extract was adjusted to pH 8.0, clarified with filter press and the antibody caputres on a MabSelect SuRE, washed and eluted with acetic acid and further purified using a Mustang Q membrane, Pall with a final polishing step using MEP Hypcell.

Affinity A followed by Cation Exchange (CEX)

Affinity-CEX:

Falkenstein (US2010/0311952) teaches a method for purifying an immunoglobulin by affinity chromatography and then CEX under conditions where the antibody in monomeric form does not bind to the CEX (flow through mode).

Hua Zhou (US2007/0167612) disclsoses a method for removing impurities from a target antibody solution by collecting the prtoein from an affinity chromatography resin and soading it onto a CEX wehre it is eluted using a buffer in which a time dependent or eluant volume dependent pH gradient is established.

Kulkarni (13/518701) teaches a process for purification of antibodies using protein A hcromatography and then CEX wherein the eluate from the Protein A chromatography is loaded onto the CEX without substantial adjustment of pH.

Mele (WO 95/16037) discloses purification of a bispecific monoclonal antibody from hybrid hybridoma by protein A cation exchange chromatography.

Wang (US2012/0264920) discloses a method for purifying a protein using a scheme of clarification by depth filtration, Protein A capture chromatography, low pH viral inactivaiton cation exchange chromatography, viral filtration, UF/DF.

Affinity-CEX-AEX:

Since no single chromatography step can achieve the necessary anitbody purity for scale purification of antibodies, a 3 step recovery process using 3 chromatography steps: protein A affinity chromatography, followed by cation exchange chromatography, followed by anion exchange chromatography is typically used. Protein A and cation exchange are fun in bind-and-elute modes, while the anion exchange is run in flow through mode (for antibodies with pI greater than about 8). Running in these modes in this order produces a high yield process capable of meeting purity requirements (Fahrner, Biotec. Genetic Eng. Reviews, 18, 2001, p. 302, last ¶). see also Tugcu (“Maximizing productivity of chromatography steps for purificaiton of monoclonal antibodies, Biotechnology and Bioengineering, 99(3), 2008) which discloses that CEX placed after Protein A will reduce the extent of titration and resulting conductivity going to CEX.

The protein A column captures the antibody of interest while the bulk of the impurities pass through the column. The protein then is recovered by elution. Since most of the proteins of interest have isoelectric points (PI) in the basic range (8-9) and therefore being positivley charged under normal processing conditions (pH below the PI of the protein), they are bound to the cation exchange resin in the second column. Other positively charged impurities are also bound. The protein of interest is then recovered by elution. The anion exchange column is typically operated in a flow through mode, such that any negatively charged impurities are bound to the resin while the positively charged protein of interest is recovered in the flow through stream. (WO2008/079280).

Hickman (US2016/0083452) discloses a method of antibody purificaiton using primary recovery such as pH reduction/centrifugation/filtration, Protein A affinity chromatography such as a MabSelect.TM. from GE Healthcare, CEX and then subjecting this eluate to AEX. In certain embodiments, the CEX or AEX, dpending on which is used first, is next filtered using a delipid filter that can be followed by a bi-layer filter cartridge.

Kozlov (EP2027921) teaches a method for purifying monoclonal antibodies via Protein A/G affinity – CEX – AEX having a membrane coated with a polymer such as a polyallylamine.

Kramer (US13/881201) discloses purficiation of antibodies using serially connected in line CEX followed by ZEX, both in flow through mode and operating as one single unit.

Liu (J of Chromatography A 1218 (2011) 6943-6952 teaches a MAB purificaiton process employing protein A affinity chromatgoraphy, isocratic overloaded CEX and AEX. See overlood chromatography under conditions for CEX.

Nielsen (WO/2009/138484) discloses an antibody purificaiton process using Protein A, virus inactivation, CEX, which can be performed at a temperature below room temperature, virus inactivation, AEX which can be performed below room temperature. .

Moya (US2009/0232737) discloses that an example of a chromatography process for the purification of an antibody which involves use of protein-A affinity followed by CEX, followed by AEX. The protein A column captures the antibody while the builk of the impurities pass through the column. The protein is then recovered by elution from the column. Since most of the antiboides have isoelectric points (PI) in the basic range and thus are positively charged under normal processing conditions (pH below the PI of the prtoein), they are bound to the CEX resin (other positively charged impurities are also bound). The antibody is then recovered by eluction under conditions (pH, salt concentration) in which the protein elutes while impurities remain bound to the resin. The ZEX column is typically perated in a flow through mode such that any negatively charged impurities are bound to the resin while the positivley charge antibody is recovered in the flow through stream.

Kulkarni (13/518701) teaches a process for purifying antibodies with protein A chromatography, then CEX and then AEX. The eluate from the CEX is loaded onto the AEX resin without substantial adjustment of pH.

Soice (US8,536,316) discloses a method for purifying an Fc region containing target property in a sample by contacting the same with affinity chromatography, contacting the eluate with CEX, contacting the eluate with AEX where the method eliminates the need for a buffer exchange step between the affinity chromatography and cation exchange chromatography, as well as the cation exchange step and anion exchange step.

–Protein A-CEX-AEX-HA:

Eon-Duval (US2010/0267932A1; see also US2010/090961) discloses a process for purification of an Fc fusion protein using Protein A affinity chromatography, cation exchange, anion exchange and hydroxyapatite chromatography. In a preferred embodiment the flow through from the anion exchange is loaded onto the HA resin without previous dilution or dialysis.

Mazzola (WO2009/017491) teaches a method for purifying a monoclonal antibody by contacting the sample/antibody solution with Protein A chromatography, eluting the antibody at about pH 3.5, inactivating viral contaminats by adjusting eluting to pH 2.5-4.5, adjusting the antibody thereof eluate to pH 3.5 to 7.5 and binding to CEX, eluting and filtering through AEX mediaum and then binding to ceramic hydroxyapatite.

Protein A – CEX – AEX- HIC:

Hickman (WO 2010/048192; US2010/0135987) teaches ion Protein A, ion exchange (either cation or anion) followed by ion (AEX or CEX) followed by hydrophobic interactive chromatography (HIC). The hydrophobic chromatographic step facilitates elimination of aggregations. The procedure uses a high salt buffer which promotes interaction of the antiobdies with the hydrophobic column. The column is eluted using lower concentrations of salt.

Protein A – CEX – HIC:

The mixture applied to the HIC column following affinity and/or CEX may contain immunoglobulin aggregates, misfolded specie s and residue material form the affinity step. Shadle, (US 5,429,746; see also WO95/22389) teaches Protein A affinity chromatography followed by an optional viral inactivation step, CEX, viral inactivation and then HIC.

Protein A-CEX-HIC-AEX:

Bacac (US2014/0242079) discloses purification by Protein A affinity chromatography, then CEX. The product containing fraction were pooled and then subjected to HIC in bind-elute mode. The eluate therefrom was then subjected to AEX in flow through mode.

Blaisdell (WO2009/058769) discloses purifying a protein in sample comprising Protein A, IEX which can be CEX, eluting the sample from the IEX and determining if the sample contains hydrophobic variants. If so, the sample is loaded onto a HIC which is conducted in flow through mode.

Protein A -CEX -NF – Mixed Mode:

–Protein A – CEX -Mixed Mode with AEX/Hydrophobic interaction (i.e., Capto Adhere) –DF (in PBS):

(Meh (US 14/809,211, published as US 2016/0185841 and US14/995278, published as US2016/0185841; see also US 16/447,439, published as US 2019/0330269; see also US Patent Applicaiton 17/154678, published as 20210139535) discloses a method for purifying amAb using affinity chromatography such as Protein A, CEX, NF, followed by a mixed mode chromatography such as Capto Adhere which has AEX and Hydrophobic interaction, then diafiltering the composition into PBS such that the mAb is substantiall free of Bis-Tris.

Protein A – CEX – UF/DF – AEX:

Kokke (WO/2013/189544) discloses subjecting an antibody cell culture harvest to Protein A, usbjecting the antibody to at least one polishing step such as CEX, subjecting the partially purified antibody to a UF/DF and then to AEX.

Affinity Chromatography in conjunction with Filtration

Prefiltration (activated Carbon) – Protein A:

Martin (EP1577391A1) discloses a prefilter for purification systems such as affinity chromatography and UF. The prefilter, positioned upstream of the system inlet, reduces the presence of non-specific binding species that enter the system. Suitable agents for the prefilter are activated carbon, charged cation or anion entities, fumed silica, glass, etc.

Depth Filtration-Protein A:

Paglia (WO/2008/051448) discloses a method for reducing protein A contamination in an antibody preparation using a charge modified depth filter.

Singh (US 2013/0012689) discloses a process for the primary clarification of feeds, including chemically treated flocculated feeds containing target biomolecuels such as mAbs using a primary clarification depth filtration device without the use of a primary clarification centrifugation step or TF microfiltration step. The depth filtration device contains a porous depth filter having graded porous layers of varying pore ratings. In one embodiment, such primary clarification DF is used prior to Protein A chromatography. In one embodiment, the clarified cell culture continuosly flow onto the next step of the purification process such as the Protein A affinnity chromatography as well as several other steps (activated carbon , AEX, CEX and virus filtration) performed in a flow through mode.

Siwak teaches a prefilter for purification systems such as affinity chromatography columns and UF systems which reduces the presence of non-specific binding species that enter the system, thereby extending the yield, pcacity and lifetime of the system. Suitable agents for the filter includes hydrophobic entities, lipophilic entities, activated carbon, charged cation or anion entities, fumed silica, glass, CPG.

Yigzaw (Biotechnol. Prog. 2006, 22, 288-296) discloses the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant mAbs process stream.

Protein A -Dual-layer Filtration:

Bonnerjea (J. Chromatography B, 848 (2007) 64-78) discloses filtration of an eluate from a Protein A chomatography column under pressure through a dual-layer 300 cm2 Sartopore 2 (0.45/0.2 um) sterile filter capsule until flow temrinated. The 300 cm2 Sartopore 2 filter blocked after 6.05 L had passed through the filter. Based on these findings, the estmination was made that one 20” filter owuld be reuqired for the in-process filtraiton of the eluate of the Protein A affinity column at the 2000 L fermenter scale.

Protein A — UF/DF — AEX

Kooke (US 14/410562) discloses a method of purifying an antibody composition using UF/DF and then AEX.

Protein A and Depth Filtration

Pagliaa (WO2008/051448) discloses reducing prtoein A contaimination in a binding molecule preparation by cotacting a charge modified epth filter with a olution having a pH greater than about 8 to obtain a pre-treated charge modified ddepth filter and contacting this depth filter with the binding molecule preparation and collection the preparation that flow through the filter. In one embodiment, the charged depth filter is an anion exchange filter.

–Protein A – Depth Filter –IEX

discloses a method for purifying an antibody using Protein A affinity chromatography, incactivating virues in the first eluate and processing the incactivated eluate through at least one depth filter and then processing the filtered eluate through at least one IEX.

Wang (US 2012/0264920) discloses a method for purifying an antibody using a capture chromatogrphy resin such as Protein A and then prcoessing the eluate witha acombination step that includes one or mroe depth filters and and ion exchange followed by an intermediate/final polishing step that may include HIC.

–Protein A -DF -HIC:

–Protein A -DF –MM:

Althouse (US 15/035,091, published as 2016/0272674) discloses a method for producing an impurity reduced antibody preperation using affinity chromatogrpahy, filtering the sample through a DF and contacting the filtered sample to a resin haing both IEX and hydrophobic interaction functionalities such as Capto MMC (weak CEX), Capto MMC ImpRes and NuviaCPrime (hydrophobic cEX) as well as capto adhere (MM, AEX).

Suenaga (EP2360183) discloses a method of purifying an antibody using a mixed mode resin having both an ion exchagne and hydrophobic functional group which includes Protein A affinity chromatographyvirus filtration followed by passing the filtrate through a depth filter two days latter, buffer exchange and then capto Adhere mixed mode column and then cation exchange chromatography.

Wang (WO 2011/146179) teaches a method of purifying an antibodys using a cpature chromatography resin such as Protein A, a depth filter or series of DP arranged after the capture chromatography resin and a mixed mode chromatogrpahy resin such as Capto adhere arranged after the depth filter.

Protein A – Anionic Filter membranes:

Gagnon teaches protein A affinity chromatography has become well established as the preferred capture step for purification of human monoclonal IgG for in vivo applications, so much so that it has become largely regarded as generic and that anion exchange is a polishing method to complete purification after Protein A (p. 491, lines 1-3). Gagnon also teaches that anion exchange is nearly as universal as protein A in the purification of monoclonal IgG and is employed frequently as the last chromatography step because of its ability to scavenge endotoxins that may have entered the process via contaminated manufacturing materials or inappropriate sample handling. (p. 492, lines 1-3). Gagnon further teaches that while the majority of established anion exchange applications are still performed on conventional ion exchanges, charged membrane filtration is becoming increasingly popular (Pete Gagnon, “Process Scale Bioseparations for the Biopharmaceutical Industry”, chapter 17 from “Process Scale Bioseparations for the biopharmaceutical Industry”, CRC Press 2006, ISBN: 973-1-57444-517-6, pp. 491Biotechnology and Bioprocessing, published July 2, 2006).

Affinity in combination with Viral inactivation See outline

Affinity A chromatography and HCIC

Protein A -HCIC -CHT/AEX/CEX: Ley (US 2009/0149638) discloses systems and methods for purifying proteins such as antibodies. In one aspect, Ley discloses Pro-A followed by hydrophobic charge induction chromatography followed by either ceramic hydroxyapatite or AEX. Optionally, the elution of the first elute (i.e., Pro A) can be performed under acidic conditions and maintained in holding tank for 1-2 hours to inactivate viral load. Advantageously, many of the processes enable one chromatography step to follow another step without an intermediate UF/DF step and without a holding tank.

Affinity A chromatography and Mixed Mode

Affinity — Hydroxyapatite

–Protein A – Ion exchange – hydroxyapatite:

Sun discuses purifcation of antibodies using a scheme of Protein A affinity chromatography, viral inactivation, followed by AEX, an optional virus filtration and then finally hydroxyapatite (US2005/0107594A1)

Mazzola (WO2009/017491A10 discloses methods ofr purifying antibodies using ceramic hydroxyapatite. The method involves caputring an IgG on Protein A affinity chromatography, followed by at least one ion exchange technique prior to adsorbing the IgG to hydroxyapatite and selectively eluintg the product.

Affinity — Mixed Mode

–Protein A- Mixed mode (Capto Adhere)

GE Healthcare’s Capto adhere anion exchanger offers multimodal functionality designed for post protein A polishing in MAb processes. Contaminants left in a product pool after protein A capture are mreoved by operating Capto adhere resin in flow through mode so that they bind to the resin. The Capto adhere ligand is N-benzyl-N-methyl ethanolamine which displays several different modes of interaction, the most dominant being ionic. (Eriksson “MAb contaminant Removal with a multimodal anion exchanger” BioProcess International 52, February 2009.

Engstrand (WO2006/043895) discloses methods of separating antibodies using a resin having multi-modal-anion-exchange ligands such as N-benzyl-N-methyl ethanolamine conducted in flow through mode. It can be used as an intermediate purification or polishing step such as after Protein A.

Duthe (US 2014/0323698 and WO2013/075849; see also Duthe US 14/889,397, published as US 2016/0083454) discloses a two step chromatogrpahy process for purificaiton of mAbs using a same mother buffer solution that include Bis Tris in combination for example NaCL, acetic acid and water. The first column can be a Protein A column and the second a multi-modal resin column such as Capto Adhere which is a multimodal anion exchanger with a highly cross linked agrose base matrix. . In one embodiment, the method further includes UF and DF against a histidine buffer.

Gagnon (WO2010/030222) discloses non-aggregated antibody purification using Protein A followed by by multimodal anion exchange such as Capto Adhere in the presence of zwitterions.

Sanofi (WO 2013/075740) discloses a method for purifying antibodies using only two chromatographic steps which are affinity protein A and one multi-modal resin chromatography such as Capto Adhere (GE Healthcare) where all buffers used during the steps can be prepared starting from the same moother solution Bis Tris in combination with NaCL, acetic acid and water.

Wang, (US20120264920) disclosees Protein A followed by a combination processing step which can include viral inactivationand ion-exchange and then an intermediate/final polishing step such as a mixed-mode chromatography such as Capto adhere conducted in flow through.

Yonan (US 15/545271, published as US 2018/009876) disclsoes purification of adalimumab using Protein A followed by a Capto adhere resin (SEX/HIX mixed mode resin; the ligand is N-Benyzl-N-methyl emthanol) in flow through mode followed by CEX.

—-Protein A -Mixed mode — AEX:

Duthe US 14/889,397, published as US 2016/0083454) teaches a three chromatophy step process for purifying an antibody from a solution by Protein A, then mixed mode and then AEX. The elution buffers for the Protein A and MM each are Bis Tris, acetic acid, NaCL and water and the AEX is performed in flow through mode.

–Protein A – Virus Inactivation – MM – CEX:

Suenaga (US2011/0251374) disclsoes antibody purification by Protein A, virus inactivation, MM such as Capto adhere and then CEX.

Affinity Chromatography in combination with HIC (hydrophobic interaction chromatography)

ProA-IEX (CEX/AEX)-HIC: Blaisell (WO2009/058769) discloses a method of purifying an antibody from hydrophobic varients in a sampel using Protein A affinity chromatography, loading onto an IEX such as a CEX or AEX followed by HIC which is conducted in flow through mode.

Defrees (US2010/0075375) discloses purifcation of polypeptide conjugates using AEX conducted in flow through mode and then HIC that includes a sufficient salt conceetration to affect binding of the polypeptide conjugate to the MIC medium which is usbsequently eluted from the HIC.

Herigstad (US14/077,574, now US 9,249,182) teaches HIC in combination with affinity chromatography such as as Affinity – HIC, Affinity – AEX – HIC, Affinity-Mixed mode- HI.

Ishihara (US2006/0257972; see also EP1614693) teaches a method for purifying antibodies by Protein A affinity, AEX, CEX, and then purification by HIC such as a Phenyl Sepharose HP column.

Kremer (US 13/578679) discloses a method for purifying antibodies using Protein A followed by in-line AEX in flow through mode followed by HIC also in flow through mode and one additional purification step.

Ramachandra (US2011/0097340) teaches purification of anti-VEGF antibodies using Protein A followed by AEX in flow through mode and then HIC which removes aggregates, DNA and host cell proteins followed by NF.DF.

Shadle (WO/1995/022389) teaches a method for purifying an antibody using Protein A chromatography (bind and elute), then adsorbing the antibody onto an ion exchange and eluting it onto an HIC, eluting the antibody anre recovering it.

(Wang, US20120264920) discloses antibody purification using Protein A capture chromatography, viral inactivation, HIC, nanofiltration.

Affinity Chromatography in combination with HCIC (Hydrophobic Charge Induction Chromatography)

PRoA-MEP-CHT/AEX: Chen (WO2009/045897) discloses Protein A followed by MEP which is a hydrophobic charge induction chromatography resin and then CHT (ceramic Hydroxyapatite resin) or AEX for purification of antibodies. The steps follow each other without intermediate diafiltration or UF steps (i.e., no adding salt or diluting or complicated manipulation between the column pairs).

Affinity in combination with or followed by Carbonaceous purification step

Bian (US 13/565463, US 2013/1097200) discloses that carbonaceous material such as activated carbon can be incorporated in chromatography column based protein purifications processes in a flow through mode. In some embodiments, the sample is contacted with a carbonaceous materials after a Protein A affinity capture step. Alternatively, Protein A may be used after contacting the sample with a carbonaceous material.

Affinity in Combination with Caprylic Acid Precipitation (see precipitation of antibodies)