Membrane adsorbers are thin, synthetic, microporous or macroporous membranes that are derivatized with functional groups akin to those on the equivalent resins. The membranes are stacked 10-15 layers deep in a comparatively small cartridge, generating a much smaller footprint than columns with a similar output, This reduced buffer consumption but increases teh flow rate even though the bed height is much lower and there is a reduced pressure drop. Despite the increased flow rate, adsorption is efficient becasue the transport of solutes to their binding sites in a membrane adsorber occurs mianly be convection, while pore diffusion (the predominant mechanisms in resins) is minimal. These benefits reduce process tiems to less than 10% of those associated with traditional stainless-stell columns. Antoher important advantage is the linear scale-up for parameters such as frontal surface area, bed volume, flow rate and static bidning capacity, while normalized DBC reamins fairly constant at 10% or complete breakthrough. Scaling up processs scale oeprations therefore is a lot less troublesome than would be i the case for standard resins, making the cpressing of up to 100 kg batches of antibodies a realistic proposal. Giovannoni (“Antibody Purificaiton using Membrane Adsorbers” Downstream Processing, BioPharm International, October 2009)

Some of the uses of membrane separation include separation of particulates from the process stream in order to get the stream ready for further processing (e.g., guard filters for chromatogrpahy columns), spearation of impurites form the process stream (e.g., viral clearance and sterile filtration, concentration of the feed stream to reduce total volume of the process stream or to reach a targeted product concentraiton (e.g., ultrafiltration) and finally, buffer exchange to deliver the product in the desired buffer system (e.g., dafiltration). (Rathore and Shirke “preparative Biochemistry & Biotechnology, 41: 398-421, 2011). 

The use of membrane chromatography or normal flow membrane based adsorbers is well known. All of these devices are basically formed of a housing having an inlet and an outlet and one or more layers of an adsorptive membrane located between the inlet and outlet. The membranes are typically rendered adsorptive by surface modification, in situ copolymerization or grafting, direct formation from adsorptive materials or by the inclusion of adsorptive particles in the membrane matrix during formation of the membrane (Phillips, WO/2003/040166)

Advantages of Membrane adsorbers/chromatography

Membrane chromatography offers a cost effective alternative to traditional packed-bed chromatography in flow-trhough operations, such as polishing for the removal of viruses and contaminants in antibody manufacture. They are particularly attractive at larger scales, where columns suffer form the rising costs of resins and buffers and fall foul of scale related packing issues that reduce column efficiency. Membranes are integral to many bioprocesses because they can be used as disposable modules. Membrane chromatography involves the use of thin, synthetic microporous or macroporous membranes that are stacked 10-15 layers deep in a comparatively small cartyridge and membrane devices in a range of sizes are available from suppliers such as Millipore (Intercept), Pall (Mustang) and Sartorius-Stedim Biotech (Sartobind) with funcitonal groups equilvaent to the corresponding resins (e.g., membranes containing activated quaternary ammonium groups for anion exchange). The use of such membrane devices results in the complete elimination of cleaning and validation, a major expense in downstream antibody processing. Anotehr significant functional advantage over resins is that the transport of solutes to their binding sites occurs mainly by convention, while pore diffusion is minimal which means that they can oeprate at much greater flow rates than columns, considerably reducing buffer consumption and shortening the overal process time by up to 100 fold. (Gottschalk, Biotechnol. Prog. 2008, 24, 496-503).

The use of conventional packed-bed chromatography with FT-AEX requires column of very large diameter to permit high volumetric flow rates, to prevent a process botteneck at the polishing step (2). Proper flow distribution in production columns requires a minimum bed height and thus a large bed volume. There are at least two major reasons for this: non-uniform packing and inadequate header design. The result is that such packed column are dramatically under utilized because they are packed for peed and not optimized for binding capacity. The disadvantages een with AEX columns have led to the developmend and utilization of membrane chromatography or membrane adsorbers. Zou, “Membrane Chromatography as a Robust Purificaiton System for large-scale antibody production”. BioProcess Interantion, pp. 32-37.

Membrane adsorbers used in the purification of mAbs involve affinity and ion exchagne adsorption mechanisms. Affinity is generally used in the initial capturing step, while ion exchange is used for a polishing step. The choice of a ligand is strongly dependent on the target molecule characteristics and on its concentraiton in the feed solution. Protein A binds to the Fc part of many different antibodies. (Boi, J. Chromatography B, 848 (2007) 19-27).

Modes of Operation

Bind and Elute Mode:

Giovannoni (“Antibody Purificaiton using Membrane Adsorbers” Downstream Processing, BioPharm International, October 2009) disclsoes using membrane adsorbers for both flow through and retention steps in antibody polishing. After an initial capture step using a traditional Protein A column, the feed is loaded onto a Srtobind Q AEX membrane adsorbe (Sartorius) which elutes directly into a Sartobind S CEX adsorber. The AEX adsorber is oeprated in flow through mode to retin HCP, DNA and leachate while the CEX adsorber oeprates in retention mode, allowing the pure antibody to be separated from positively charged impurities. Because the eluate form the frist adsorber is laoded directly and automatically onto the second, the process can be regarded as a single, integrated polishing step that achieves up to 90 recoery and 99.9% purities as detereind by SEC, cation exchange HPLC an d SDS-PAGE. The final step is a nonofiltration operation to mechanically remove endogenous or adventitious virus particles <20 nm in diameter using an NFP Millipore cartridge filer. 

Flow through mode: 

Anion exchange membranes operated in flow through mode are effective downstream of Protein A purification (Zhou, (2006) Biotechnol. Prog. 22, 341-349). 

Q and other charged MA devices have been in development for chromatography purposes for more than 15 years. Some limitations for large production scale are distorted or poor inlet flow distribution, nonidentical membrane proe size distribution, uneven membrane thickness and lower binding capacity. The first 3 weaknesses can be improved to some degree when multiple layer configuations are ued. This configuation for the Q membrane is used in viral vaccine production. Q AEX adsorber devices have also been used for endotoxin removal at process scale. Low binding capacity is still a major disadvantage in bind/elute mode but in FT mode, the limitations are no longer considered as major issues, particularly when FT-MA is used as the polishing step for antibody purification. Antibody recovery in the fT mode is comparable to columns, normally about 98-100%. Buffer usage for MA can be reduced to only 5% of that for a conventional packed bed chromatography.  (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

Pompiati (US13/141306) discloses a membrane anion exchange chromatography step in which an immunoglobulin in monomeric form can be obtained from an AEX material in a flow through mode which is perofremd in a narrow pH value range of pH 7.8-8.8.

Overload Mode: 

Brown (Biotechnol. Appl Biochem (2010) 56, 59-70) discloses overloading cation and anion exchange membranes such as the Mustang S and Q and Sartobind S membranes (Pall Corporation) in normal flow mode as a final purificaiton step in the purfication of mAbs resulting in retention of impurities and breakthrough of purified antibody. Membranes are an ideal polishing step because they have a distinct flow rate advantage and sufficient capacity within a relatively small footprint for binding trace impurities and contaminants. 

Membrane Absorber Materials

Van Reis (US 7,001,550) discloses filtration membranes modifed with a charged compound for separating proteins such as antibodies. The membranes may be cellulose, cellulose diacetate and triacetate, cellulose nitrate and cellulose diacetate/cellulose nitrate blends. The membranes are commercially avaialbe from various sources such as Millipore Corp. Preferably the filtration membrane has hydroxyl groups available on the surface for reaction with a derivatizing compound. Preferably the hydroxyl groups are primary alcohol moieties such as those of a cellulose matrix. In one embodimetn the membrane is a cellulose membrane, perferqably a composite regenrated cellulose (CRC) modified to have a net positive or ngetative charge. In another embodimetn, the charged omcpound includes a linker arm between the charged moiety and the moiety covalently linked to a reactive group of the membrane. In one emdoiment, the filtration method is ultrafiltration.

Commerically available membranes are often regenerated cellulose, polyethersulfone and polyvinylidene fluoride in the microfiltration range. They can be modified by chemical activation, coating or frafting. Alternatively, they can be prepared by copolymerisation of two functional monomers or by other methods like cellulose derivative membranes, poly (ether-urethane-urea) membranes, PVC and PTFE microporus composit sheets, macroporous chitin and chitosan membranes. (Chen, J. Chromatography A, 1177 272-281, 2008).

Membrane Ion-exchange Chromatography:

Membrane IEX are available from different companies such as membrane CEX materials (Mustang C, MustangS, Sartobind CM, Sartobind  S) and AEX such as MustangQ, Sartobind Q.

Dwonstream purificaiton processes for monoclonal antibody production typically involves multiple chromatogrpahy steps, including one or more ion-exchange columns. Conventional ion-exchange column chromatogrpahy steps are effective and reliable, but generally have low product throuput (kg processed/h). As monoclonal antibodies become mroe widley used, more efficient process cale product is necessary. membrane chromatogrpahy has increasingly been reported as a potnetially advantageous tool to purify proteins. One advantage of membrnes over conventional preparative beads or gells is the elimination of pores with long diffusive path lenghts. Fro the membranes, binding sites are located along the through pores rather than nestled within log difficusive pores. Accordingly, mass transport of the mAb to the ibnding site relies on convection rather than diffusion. Consistent with convective rather than diffusive mass transport, binding capacities of ion exchange membranes have been found to be independent of flow-rate. In addition, breakthorugh capacities of IEX membranes are often comparable in magnitude to commonly used ion exchange resins. (Due to economic and prcoess restrictions, CEX membranes may not currently be advantageous for process scale antibody purificaiton in a bind and elute mode. however, AEX membranes in a flow thorugh mode may provide a resaonble alternative to columns for the remvoal of lw levels of omputires such as DNA, HCPs and virus. Knudsen, “Membrane ion-exchange chromatogrpahy for process-scale antibody purificaiton” J. Chromatorpahy A, 907 (2001) 145-154). 

Virus filters are known in the art and are supplied by Millipore and Asahi Chemical Industry Co., Ltd. Suitable paravovirus retentive filters include Viresolve Pro (Millipore), which has an asymmetric dual layer structure and is made form polyethersulfone (PES). The membrane is designed to retian viruses greater than 20 nm while allowing proteins of MW less than 180 kDa to permeate throught he membrane. Other filters suitable for removal of small viruses, including parvovirueses from protein solutions include Novasip DV20 and VD50 Virus Removal Filter Capsules (Pall Corp), Virosart CPV, Planova N (Asahi Kasei) and BioEX (Asahi Kasei). (Mehta, WO 2011/031397). 

Cored Anion Beads: Cored Q beads consisting of a rigid core and a thin agarose gel coating have been developed for antibody flow through chromatography. Various rigid cores may be used including ceramic, glass (e.g., borosilicate, alkaline resistant, etc), metal and plastics (e.g., polystyrene, polethylene, etc). Wang, J. Chromatogrpahy A, 1155 (2007) 74-84

Anionic charge modified microporous filter membranes:

Chu (US4,604,208) discloses a hydrophilic anionic charge modified microporous filter membrane.

Anionic (positive charged) UF: 

A positively charged ultrafiltration membrane repels positively charged proteins such as IgG monoclonal antibodies, despite being small enough to pass through the prores. This permits their selective retention and concentraiton while weakly alkaline, neutral, and weakly acidic contaminants pass through the membrane. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70).

HPTFF has been described in which a positive charge is created on the surface of an UF with a size cutoff of about 100-300 kDa. When a sample of crude IgG is introduced wtihin a narrow pH and conductivity, IgG is repelled from the membrane surface and thus prevented from passing through the pores. The majority of contaminant species either binds to the surface or passes through the pores by convective mass transport and is thus eliminated. The method also permits concentration of the antibody. (Gagnon, US 14/555060, published as US Patent No: 9,890,205)

Gagnon (US 15/120,111, published as US 2017/0057992) discloses a method of purifying IgG using a porous electropositive membrane where the pores have a hydrodynamic diameter between about 10-15 nm or have an average hydrodynamic diameter of about 3-6 nm or a pore size corresponding to a globular protein having a MW of about 10-50 kDa, wherein during at least a portion of the contacting step the preparation includes a salt such as sodium, potassim or ammonium chloride, at a concentration greater than about 50 mM and pH from about 3-9 and where the process includes a final buffer exchange step with buffer having a salt concentration 20 mM or less and pH from about 5 to within about 0.5 pH units of the isolectric point of the most aklaine glycoform of the IgG antibody. In one embodiment, the electropositive memtrane retains at least 60-99% of the antibody. In one embodiment the electropositive membrane incldues a plurality of positvely charged nitrogen containing moeities such as primary, secondary, tertialy, quaternary amines such as tris(2-aminoethyl)amine (TREN), diethylenetriamine, triethylenetriamine, tetraethylenepentamine, polypropylenimine tetramine, poly(amidoamine). 

Lebreton (Biotechnology and Bioengineering, 100(5), 2008) discloses using positvely charged cellulosic membranes of 100 kDa MWCO and operating under a aselected range of buffer pH and ionic strenght for purificaiotn of Fab’2 from  HCP. 

Shirataki (US 2010/0228010) discloses purifying a protein such as an antibody using a porous membrane having a graft chain on a pore surface and an AEX group such as a trimethylamino group fixed to the graft chain. Impuriites such as HCP, DNA nd endotoxins are absorbed by the AEX of the membrane. The maximum pore size of the porous membrane is preferably 0.1-0.8 um. 

Van Reis (WO 01/08792) discloses filtration membrane possessing a net charge, either positive or negative and methods of making the charged membranes as well as using them in protein purificaiton. The charged membranes repel proteins having the same charge as the membrane, thereby retaining such proteins on the upstream side of the membrane. Proteins pass through the membrane pores if they have a net neutral charge or a polarity opposite that of the membrane and are smaller than the average pore diamter. In one mebodiment RhuMab HER2 (pI 8.9) was separated form BSA (pI 4.8). The proteins were mixed in a pH 4.5 buffer cuasing the rhuMab HER2 to be positively charged and BSA to be neutral. Only BSA passed through the membrane into the filtrate because it was not repelled by the positive surface or pores of the membrane. 

Immobilized Metal Ion Affinity Groups

–CU(II) chelated to Tris(2-aminoethyl)amine (TREN)

TREN is a tetradentate chelating ligand that was first reported in immobilized metal ion affintiy chromatogrtaphy (IMAc). It has been used along with IDA with Ni(II) immobized on a poley(ethylene vinyl alcohol) (PEVA) hollow fiber membrane for the pufication of IgG from huma plasma. The adsorption of Cu(II) chelated to IDA and TREN immobilzed on PEVA hollow fibers and agarose gels was explored by Borsoi-Ribeiro (J. Mol. Recognit. 2013, 26; 514-520). The TREN was covalently coupled via amine groups present in the chelating ligad to the reactive epoxy groups introduced into PEVA hollow fibers using epichlorohydrin or bisoxirane. 

TREN is a quadridentate chelating ligand used in IMAC with four nitrogen atoms. TREN chelated with copper and nickel ions has been used in protein purificaiton. Due to its high amine residue content, TREN can serve as an anion exchanger. At a pH lower than 10.0, TREN is positvely charged and can adsorb negatively charged molecules and thus can be used to purify IgG from serum proteins. It costs less and is more stable than ligands traditionally used in affintiy chromatographies and thus is advantageous in this respect. 

While AEX has been used for clearance of impurities such as virsues and HCP and DNA, anion exchange adsorbents based on the traditional quateranyr amine (Q) ligand are sensitive to salt concentraiton, leading to reduced clearance levels of impurites at moderate solt concetnraitons (50-150 mM). For this reason, alternative salt tolerant AEX ligands like trsi-2-aminoethyl amine have been used. Riordan “Salt tolerant membrane adsorbers for robust impurity clearance” published online 2009 disclose that such membrane adsorbers that incorproate such salt tolerant AEX lgiands provide a robust approach to impurity clearance during the purificaiton of mAbs. 

Ganon (US 9,975,919) discloses a method of reducing impurities such as aggregates that includes contacting a protein preparation that includes an antibody with a solid surface such as a membrane that includes at least one surface bound ligand such as tris(2-aminoethyl) amine (TREN) cpable of binding a metal. In one embodiment, the preparation is first contacted with allantoin followed by contacting with solid surface with TREN wherein the operating conditions are selected to prevent bidnign of the antibody followed by spearating the protein preparation from the solid surface. 

Etzel (WO 2008/0088720) discloses a disposable micro-porous filter membrane and an immobilzied ligand such as tris(2-aminoethyl) aminethat irreversibly and selectively binds virsues under a range of conductivities and pH values but do not bind mAbs. The lignd is dimensioned and configured to irreversibly and slectivly bind virsues and simultaneously to have a pKa sufficiently high to repel basic proteins such as mAbs via electrostatic charge resuplsion.

HIC Membranes:

–Hydrophilic, regenerated, stabilized cellulose with hydrophobic phenyl groups covalently attached to the base matrix: Examples are Sartobind HIC membrane adsorbers (Fraud et al. “Hydrophobic-Interaction Membrane Chromatography for Large-Scale purification of biopharmaceuticals, June 1, 2009,

Microfiltration Membranes

(Ghosh (J Membrane Science 237 (2004) 109-117) disclsoes seapration of human plasma proteins HSA and HIgG by using a PVDF microfiltraiton membrane which retained HIgG while the HSA passed through. In the presence of binding buffer (1.75 M ammonium sulfate in 20 mM sodium phsphate, pH 6.5) the membrane bound HIgG while HSA passed through. The bound HIgG was eluted using 20 mM soidum phosphate, pH 6.5. 

Ligands

Membrane adsorbers used in the purificaiton of monoclonal antibodies involve affinity and ion exchange adsorption mechanisms. (Chen, J. Chromatography A, 1177 272-281, 2008).

Bhut (biotechnlogy and bioengineering, 2011, 108(1), pp. 2645-2653) developed a surface initiated polymerization protocol designed to provide uniform and variable chain spacing to anion exchange membrane adsorbers . The dynamic binding capacity of IgG increased nearly linerarly with increasing polymer chain desnity. The high DBC of IgG (greater than 130 mg/ml) was independent of linear flow velocity. 

In Conjunction with Other Steps

IEX Membrane — Parvovirus filter:

Prefiltration using IEX adsorbers can improve parvovirus filter throughput of mAbs. The membranes work by binding trace foulant, and although some antibody product also binds, yields of greater than 99% are easily acheived by overloading. (Brown, Biotechnolgy and Bioengineering, 106(4), 2010). 

Mehta (US 2011/0034674; EP2462158; see also WO 2011/031397) discloses that the fouling of parvovirus filters can be improved by subjecting a composition comprising a antibody/protein to a CEX membrane absorber step and an endotoxin removal step in either order prior to passing through the virus fitler. Mehta also discloses that Mustang S, a strong negatively charged ion-exchanger, when used as a prefilter is known to increase the cpacity of parvovirus retentive filter by several fold. 

Depth Filtration –Parvovirus Filtrer:

Although depth fitlers have traditionally been used successfully for clarificaiton of cell culture fluid, there are limitations when used dowsntream of capture steps such as a prefilter to parvovrius retentive filter. Depth fitlers are not base stable which prevents the sanitizaiton of process train after installation, resulting in open processing and potential for bioburden grwoth. The composition of Dfs includes diatomaceous earth as a key component which is typically food grade and presents quality concerns. DFs also tend to leach metals, betaglycans and other impurities. (Mehta, US 2011/0034674). 

AEX Membrane -CEX Membrane:

Giovannoni (“Antibody Purificaiton using Membrane Adsorbers” Downstream Processing, BioPharm International, October 2009) disclsoes using membrane adsorbers for both flow through and retention steps in antibody polishing. After an initial capture step using a traditional Protein A column, the feed is loaded onto a Srtobind Q AEX membrane adsorbe (Sartorius) which elutes directly into a Sartobind S CEX adsorber. The AEX adsorber is oeprated in flow through mode to retin HCP, DNA and leachate while the CEX adsorber oeprates in retention mode, allowing the pure antibody to be separated from positively charged impurities. Because the eluate form the frist adsorber is laoded directly and automatically onto the second, the process can be regarded as a single, integrated polishing step that achieves up to 90 recoery and 99.9% purities as detereind by SEC, cation exchange HPLC an d SDS-PAGE. The final step is a nonofiltration operation to mechanically remove endogenous or adventitious virus particles <20 nm in diameter using an NFP Millipore cartridge filer.

CEX Membrane – CEX:

Thayer (US Patent Application 16/433,763 published as US 2020/0102346; see also US Patent Applicaion No: 14/365,449, published as US Patent 10/364268) discloses enhancing efficiency of downstream purificaiton steps for mAbs which includes using CEX Membrane in overload mode such that at elast one contaminant remains bound to teh membrane while the polypeptide of interest is primarily in the effluent and subjecting the membrane effluent to a cation excahnge chromatography step. 

See also  Membrane Adsorbers See also filtration in conjunction with Protein A chromatography for the purification of antibodies See also  “Anion exchange” for membrane absorbers with anion exchange ligands.

Companies:  disposable depth filter system: Pall, Cuno, Millipore; Sartorius Stedim Biotech

Introduction:

Although the chromatography steps play the major role in the purificaiton of antibodies, filtraiton steps are integral to any process. These can be in the form of ultrafiltration steps in TFF mode, 0.2 um membrane filtration steps for particle reduction/bioburden control, or virus reduction filtration steps to reduce the level of viruses in the process stream. Bonnerjea (J. Chromatography B, 848 (2007) 64-78)

The first series of operations in a purification train – the harvest step – focuses on the removal of particulate impurities with the goal of delivering a particle-free sueprnatant that can be fed into the subsequent, mostly chromatographyy-based prcoesses. Typically, tangential flow microfiltraiton, depth filtration and particular centrifugation are used to clarify cell culture suspensions. These initial steps remove large particles (greater than 1-2 um diameter) and are frequently followed by the use of a polishing depth filter and one or more absolute filters to remove smaller particles and to reduce the turbidity of the feedstream. (Thommes, Process Scale Purificaiton of Antibodies 2009, pp. 293-308). 

The terms “microfiltraiton” and “ultrafiltration” reference fitlraiton parameters commonly understood in the art. In particular, the term micrfiltraiton refers to the use of a fitlraiton membrane with a proe size betwen 01.-10 um and “ultrafiltraiton” refers to the use of a filtraiton membrane with a pore size between 0.001-0.1 um. Microfiltraiotn is typically used for clarifcaiotn, sterilization, removal of micropartciculates and for cell harvests; ultrfiltraiton is typically used for separating and concentrating dissolved molecules )oe.g., protien, peptides, nucleic acids, carbohydrates and other biomolecules), for exchange buffers and for gross fractionation. (Becker et al. (US 2015/0274773). 

Filter Sizing:

Very few filtration difficulties are observed at laboratory scale and 0.2 um filters, such as syringe filters may be suficient for the small product volumes,. However, filtraiton difficulites are enhanced as processes are scaled-up. Filters should either be sized appropriately for a particular application or in the absence of data, a generic approach may be taken whereby a train of pre-filters of decreasing pore size may be used prior to the final 0.2 um filter. For filter-sizine, an adequate volumne of product is reuqired to that ideally the filtraiton operation can be continued until the filter becomes blocked. Hence, the pcoress may need ot be partially sacled-up before this data can be obtained. One pilot process oepratea t 130L fermentation scale was observed to have fitlraiton issues in 2 out of 4 batches. During a futher pilot batch, filter sizine was performed at two stages in the purificaotn process which had proved to be difficult to filter previously. Bonnerjea (J. Chromatography B, 848 (2007) 64-78)

The use of large proe size pre-filters can be efective at reducing filter area and coses. For example, filter sizing data fo r the neutralised eluate form a Protien A chromatogrpahy column showed the reuqirement for three 30” 0.45/0.2 um Sartopore 2 filters or alternatively one 30′ 0.8/0.45 um pre-filter in line with one 10” 0.45/0.2 um Sartopore 2 filter. With the larger volumes anticipated with tire increases, filter sizing should be a key consideraiton of any scale-up process. Filter performance can vary depending on supplier and material of construction, emphasising the advantages of evluating several ilter types prior to scaling up. Bonnerjea (J. Chromatography B, 848 (2007) 64-78)

Size Porosity that retains Most of Antibody:

Gagnon (US 15/120,111, published as US 2017/0057992) discloses a method of purifying IgG using an electropositive membrane (AEX membrane) having a porosity that retains at least 50% antibodies/non-adsorbed solutes with a hydrodynamic diameter greater than a selected size of about 10 nm but permits passage of non-adsorbed solutes with a hydrodynamic diameters less than the elected size. In addition, at least o portion of the contacting step the sample contains a salt at a concentraiton less than about 50 mM and a pH in the range from about 3- to 0.5 pH units of the pI of the most alkaline glycoform of the IgG or a salt at a concetnration greater than about 50 mM and a pH from about 3-9 and (b) a final contacting step by eitehr (i) an absence of excess salt  or not greater than 20 mM and (ii) a pH value in a range from about 5 to within about 0.5 pH units of the pI of the most alkaline glycoform of the IgG antibody. In one embodiment, IgG is fractionaed by precipitation in 2 M ammonium sulfate, the antibody precipitates, mostly contaminants, are eliminated with the supernatant. The IgG is resolubilized by reducing the ammonium concentration to 1 M (by the addition of water). This high salt sample is then introduced into a FFF with electropositive membranes with an average pore size corresponding to a hypothetical globular protein of 50 kDA. IgG remains retains, but the high salt largely . The high salt buffer is then displaced by a low salt buffer such as 50 mM Tris, pH 8.2 and any remaining acidic contaminants bind to the electropostive membrane surface, but the IgG does not. In anotehr embodiment, mammalina cell culture harvest containing IgG was clarified by adjsutment to pH 5.2, addition fo 1% allantoin, follwoed by 0.4% sodium caprylate acid, incubated 2 hr, 5% BioWorks TREN was added, 4 hours incubation, centrifuggation to remove contaminants and then the preparation was applied directed to a TREN bearing cellulose membrane with a pre size corresponding with a globular protein with a mass of 30 kDA. The preparation was then buffer exchanged into 50 mM Tris, 2 mM EDTA, 200 mM Histidinet, 2 M NaCL, pH to dissociate non-specific interactions, then buffer exchanged into 50 mM Tris, pH 8.2 

Ultrafiltration (UF)

Two types ulfiltration methods are commonly used for ultrafiltration (UF). In direct flow filtration (DFF) the feed is forced direclty toward the membrane. As a result, molecules larger than the pores aggregate at the membrane surface and form a gel that clobks the flow of the smaller molecules through the pores, so that the flux rate decreases rapidly as filtraiton proceeds. The protein solution is often stirred during DFF in order to keep the retained protein from aggregating and blocking the pores of the membrane. The other main UF process is tangential flow filtration (TFF) in which teh sample flows accross the surface of the membrane as pressure on the solution forces smaller molecules in the solution outwards trhough the pores of the membrane. The flow of solution across the membrane during TFF helps prevent a gel of aggregated molecules from forming on the surface of the membrane that blocks the pores and preevents smaller molecules form passing through. As a result, the flux rate for TFF drops off much more slowly as filtration proceeds than occurs during DFF. Yang (WO/2004/001007)

Depth Filtration (See outline)

High Performance Tangential Flow Filtration (HPTFF)

HPTFF has been described in which a positive charge is created on the surface of an UF membrane with a cutoff of about 10-300 kDa. When a sample of crude IgG mAb is introduced within a narrow range of pH and conductivity, IgG is repelled form the membrane surface and thus prevented form passing through the pores. The majority of contaminant speceis either binds to the surface or passes through the pores by convective mass transport and is thereby eliminated. The method also permits concentration of the antibody. (Gagnon, 14/555060)

Flocculation -Filtration

Flocculation is a smilar process to coagulation, wehre suspended partciles clump together becasue the attractive forces between them overcome any repulsive forces caused by like surface charges. Such repulsive forces can be eliminated, for example, through the addition of inorgnaic electrolytes, which shield the surface charges, or by the addition of polyelectrolytes that bind to and neutralize the surface charge. Flocculation has been used mainly for the removal of whole cells from fermentation broth. Particles are often coagulated or flocculated prior to filtration to reduce the passage of small noncoagulated particles through teh filter and to produce a more porous cake which is easier to remove. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Lowering pH + Divalent cation (e.g., Ca2+ or Mg2+ or Cu2+ or Co2+ or Mn2+):

Romero (WO2008/127305) disclose a method of isolating a biomacromolecule such as an antibody by lowering the pH of the composition, adding a divalent cation and separating the antibody from the impurity. The lowering of the pH of the harvest feed prior to filtration causing flocculation of large cells and cellular debris along with precipitation of other impurities like DNA which improves mass transfer of the composition near the surface of the filter (lowering the pH also reducing plugging or filling of the pores of the filter) thus reducing transmembrane pressure across the filter. Because lowering the pH of the composition can result in coprecipitation of the biomacromolecule of interest as well as the impurity, addition of divalent cations to the pH adjusted composition is suitable for increasing the recvoery of the biomacromolecule of interest. 

Conditions:

Water as DF medium: 

Graunhofer (US2009/0291062) discloses aqueous formultations of proteins such as antibodies such as adalimumab in water based on a diafiltration process using water as a diafiltration medium. The resulting aqueous formultaiton has a significant decrease in the overall percentage of excipients and low conductivity. 

Applying gas to permeate side of UF

Bolton (WO2010/111378) discloses a method of generating a highly concentrated antibody/protein solution by circulating the first antibody solution through an UF device and applying a flow of gas to the permeate side of the porous membrane and then collecting the second permeate solution. Exposure of the permeate side of the membrane to a flow of gas such as air allows evaporation of the absorbed protein free solvent into the air so as to remove the solvent which concentrates the antibody solution, while the membrane prevents the antibody from being exposed to air which can damage proteins. In one embodiment, the UF device is connected to a retentate tank in a lopp so that the first protein solution is allowed to return back to the retentate tank which allows for continous reciruclaiton of the solution. When the protein solution inside the apparatus achieves a desired concentraiton or desired feed pressure, the reciruclation may be ramped down and the highly concentrated protein solution may be collected. 

Stabilizing Agents

–Salt of Surfactant: 

A major drawback in the applicaiton of UF membranes in bioseparations is protein fouling. Flux loss occurs over a long term period, and flux does not recover even when filtration is resumed after depressurization. Chen (J. Membrane Sci. 67(2-3): 249-261, 1992) discloses that the small anionic surfactant (AOT) provides a reduction of protein deposition by altering the electrostatic interaction between the protein and membrane surface. When used in conjunction with nonionic surfactants or when polyethyelene oxide segments are added to their backbond, the anionic surfactants showed significant flux improvement and fouling resistance compared with that of the single AOT or the nonionic surfactant. 

In order to inhibit aggregation and loss of biological activity when producing a highly concentration solution of MAbs by UF, a stabilizing additive such as a polyol, and/or a viscoity reducing agent such as a salt or surfactant is typically added to the composition containing the antibodies (US6,171,586, US2002/0045571).

–Addition of Amino Acids

–Glycine

Van Holten (US6,096,872) discloses methods of nanofiltration and ultra filtration of anti-D immunogloulin in high ionic buffer with an excipient such as polysorbate 80. The high ionic strengh buffer can be 150 mM NaCl-glycine. In one embodiment, a human IgG product was suspended in glycine buffer and filtered through an UFG.

 –Additional of basic amino acids: 

Hongo (US 13/260419, now US 9,056,896) teaches removing small viruses from antibody solution with a virus removing membrane using a monoclonal antibody solution supplemented with a basic amino acid such as arginine, histinde, lysine. The basic amino acid is thought to have the effect of decreasing the potential of the antibody surface and thus suppressing electrostatic interaction with the negative charge of the virus removing membrane. The basic amino acid also has an effect of suppressing antibody-antibody hydrophobic interaction at a pH range near the isolelectirc point of antibodies where they tend to associate with each other through hydrophobic interaction since the electrostatic repulsion between antibodies decreases.f

–Acetate or Histidine: Yang (WO/2004/001007) discloses that antibodies and histidine or acetate at a concentration in the range of 3-48 mM can be concentrated efficiently by UF to a high concentration with retention of biological activity and relatively little aggregation, even in the absence of a stabilizing or viscoscity reducing additive such as a surfactant, a polyol, a saccharide, a salt or high buffer concentration:

Temperature:

Bolton “Acheiving high mass throughput of therapeutic proteins through parvovirus retentive filters” Biotech. Progress 26(6) 1671-1677 (2010) discloses an optimum temperature of 35C for maximizing throughput through the Virosart CPV and Virsesolve Pro filters in methods of using parvovirus retentive filters . 

–Zeta (surface) potential: 

Hongo (US 13/260419) disclose that if the zeta potential Ei1 (mV) of a monoclonal antibody in solution minus the zeta potential Em (mV) of the virus removing membrane is more than 20mV, the electrostatic interaction between antibody and membrane is strong, which id disadvantageous for filtration. Yet if the zeta pootential Ei0 (mV) of the monoclonal antibody in the solution (pH=4 and ionic strenght of 0.1 mM) is less than 10 mV, the soothing effect of the antibody on base potential is weak, and an improvement of filtration rate is not achieved. Thus a most beneficial zeta potential Ei1 (mv) of a monoclonal antibody in solution satisfies the following conditions: (a) 0 mV≤Ei1-Em≤20mv, with respect to the zeta potential (Em (mV) of the virus removing membrane and (b) 10mV≤Ei0-Ei1≤40mV, with respect to the zeta potential Ei0 (mV) of the monoclonal antibody in the solution (pH=4 and ionic strenght of 0.1 mM) containing the monoclonal antibody. 

Particular Contaminants which are Removed

DNA: 

Charlton, (Bioseparation, 8, 1999, 281-291) discloses using positively charged filters (anion exchangers) for removal of DNA from solutions containing antibodies at various ionic strenght (salt concentrations). If the filter is operating as a true anion exchangers, then increasing the ionic strenght of the buffer results in reduced adsorption of the DNA. The ooposite effect is observed at low ionic strenght where hydrophobic interactions are playing a role in adsorption. Similarly, the enhanced DNA adsorption observed at high salt concentration is partially reversed by teh addition of a non-ionic surfactant such as polysorbate 80. 

Viruses:

–Parvovirus filtration:

–—Prefiltration:

——Depth filters or IEX membrane adsorptive pre-filters or mixed mode 

Brown (“increasing parvovirus filter throughput of monoclonal antibodies using ion exchange membrane adsorptive pre-filtration” Biotechnolgy and Bioengineering, 106(4), 2010) dscloses pre-filtraiton using Ion exchange membrane adsorbers can imporve parvovirus filter throput of mAbs. 

Olsen (US 2018/0072769) discloses flowing a fluid including a recombinant antibody through a pre-filer before the fluid is flowed thorugh a virus filter.  Examples of pre-filters include a Sartorius Virosart Mas pre-filter, a Millipore pre-filter, a Sartopore 2 pre-filter, a Sartobind STIC pre-filter, a Sartobind Q pre-filter, a Sartobind HIC Phenyl pre-filter, a Sartobind S pre-filter, Millipore Viresolve Pro Shield pre-filter, CUNO delipid pre-filter and Millipore XOHC pre-filter. In some embodimetns, a virus filter includes a polyamide membrane, a cation exchange based based membrane, an anion exchange based membe or.a HIC based membrane. Additional examples of pre-filters are known in the art. Olsen also disclos eusing a depth filter having anionic and hydrophobic propters (used CUNO delipid filter) to purify Alexion 1210 (also called BNJ441). Significant improvement in aggregate removal and particulate content was observed when depth filtration was performed immediately prior to virus filtration. A depth filter having anionic and hydrophobic properties resulted in significant removal of host cell protein and soluble protein aggregates. 

—-additives:

—–non-ionic surfactant:

Brown, (US 14/007,610, published as US 2014/0309403) discloses a method of reducign fouling of an ultrafiltration membrane by the addition of a non-ionic surfactn such as plysorbate 20 during the filtration of a solution that contains an antibody and parvovirus particles.   

Multiple UF/DF: 

Konstantinov (US2006/0149042) discloses a process of subjecting a supernatant to an initial UF, then adjusting the conductivity of the retentate, such as by diafiltration with water for injection (WFI), diluent or buffer, and then subjecting the solution to a second UF. The process increases the concentration of cell culture supernatant containing macromolecules and an orgnaic polymer with higher yields.

Pore Size (MWCO)

–Second Mebrane smaller pore size:

Chtourou (US 7,186,410) discloses a process for preparing human immunoglobulin concnetrates form plasma using an AEX at alklaine pH and further comprising concentrating the immunoglobulin by UF and sterilization by filtering it through nanometric filters of a porosity decreasing fromm 100 to 15 nanometers. 

—-In final Filtration step:

Falkenstein (WO2011039274) discloses antibody solutions with a concetnraiton of more than 100 g/l are prone to difficulties such as blocking of the employed filter by aggregates formed during the formulation or concentraiton process during the final filtartion step.. Falkenstein discloses a method for filtration of antibodies which have been concentrated to at least 100 g/l as by diafiltration by combination of two immediately consecutive double membrane filtration steps wtih a first filter of 3.0 um (“pre-filter) and 0.8 um (main filter) pore size and a second filter of 0.45 um (pre-filter) and 0.22 um (main filter) pore size. Such filters such as Sartoclean CA (3.0 um + 0.8 um filter cartridge) and Sartobrain P (0.45 um + 0.2 um filter cartride) were employed. Sartoclean CA mini cartiridges are available with 3.0 um/0.8 um cellulose acetate double membranes and Sartobran Sartorius is available as doulbe filtraiton unit 0.45/0.2um. 

–Second membrane larger MWCO

Becker et al. (US 2015/0274773) discloses processing a crude feed stream to thereby eliminte the need for time consuming impurity precipitation by ombining at elast two TFF unit oeprations. In one embodiment, the first TFF unit was conducted with an UF membrane having a 50 kD cut-off value and a second TFF unit having a 300 kD cut-off. A third unit with a a 50 kD cut-off can also be incorporated. In certain emobdiments, the dual stage TFF methods may also include the membranes with even larger pore sizes such as with sterilization prior to the initial/first TFF unit oepration. Sterizization of load fluids, e.g., cell culture sueprnatants containing proteins of interest vial filtration methods are well known in the art and typically include filtratioon of the sueprnatant through filter membranes having pire sizes ranging form about 0.1-45 um. Accordingly dual stage TFF membhos comprising sterilizaiton of cell culture supernatant prior to the TF unit operations by filtration through a suitable filter membrane having a pore size between 0.1-.45 um is contemplated also. 

Gonzalez (US8,772,461 and WO/2009/129226; see also US14/294460) discloses a method for concentrating a protein such as IgG by ultrafiltering the solution using a first membrane to form a first retentate solution comprising the protein, diafiltering this first retentate with an aqueous solution using the first membrane to form a second retentate solution comprising the protein at about the first concentration, formulating the second retentate having the diafiltered protein with glycine and adjusting the pH and then ultrafiltering the second retentate solution using a second membrane to form a final retentate solution. In one embodiment, the second membrane has a molecular weight cutoff of about twice the MWCO of the first membrane. 

Smider US2006/0088883) discloses that based on a calculated MW, a polypeptide of greate and lesser size can be isolated using UF through membranes of different pore sizes. As a first step, the protein mixture is UF through a membrane with a pore size that has a lower MWCO than the MW of the recombinant catlytic polypeptide or the proteolytic antibody light chain. The retentate of the UF is then UF against a membrane with a MWCO greater than the MW of the recombinatn catalytic polypeptide or the proteolytic antibody light china. The polypeptide will pass through the membrane into the filtrate which can then be processed in a next step of column chromatography.

Wang (“cascade ultrafiltration bioreactor-separator system for continous production of F(ab’2) fragment from immunoglobulin G, J. Membrane Science 2010, 351(1-2), 96-103) discloses a continous two stage UF system for fragmentation of IgG by pepsin and purification of the F(ab’)2 fragment thus generated. In the first stage, a 10 kDa MWCA membrane retains pepsin, IgG and F(ab’)2 while allowing degraded Fc sub fragments through. In the second stage, a 70 kDa mebrane retains both IgG and (F(ab’)2 while allowing pepsin through. Under this method, the pepsin concentration within the first stage is significantly higher than in the feed. The first st6age thus carrys out IgG digestion while the second stage serves to separate pepsin and (F(ab’)2.

Surface Area of Membrane

–Second Membrane Smaller Surface Area; Recirculating Wash buffer:

Teschner (US 12/789345 (now US  8,546,548 and US 13949565, now US 9,175,068 and US 14/855,686, published as US 2016-0244512; see also US/188839, published as US 2019/0085064) disclose a method for concentrating IgG by concentrating a first solution compirsing IgG to 2-10% (w/V) by UF using a first UF, diafiltering the first IgG concentrate comprising the same first UF/DF membrane. concentrating the first IgG diafiltrate using the first UF membrane, collecting this IgG concentrate, washing the first UF by recircualting a wash buffer through the UF, transfering this IgG post wash solution into a second UF/DF comprising a 2nd UF membrane and concentrating it by UF, collecting this IgG concentrate and combining it with the prior IgG concentrate. In one embodiment, the second membrane has a surface area which is less than the frist surface area such as no more than a tenth of the surface area of the first UF membrane. 

First Tank for dilution of Precipitation – First and Second Filtration Units adapted to return first retentate and second retentation or second permeate to frist tank having the precipitate for a final dilution factor

(Menyawi, US 17/054,018, published as US 2021/0246162) disclsoes a clsoed system for extrating a prtoein of interest which includes a first tank adapted to dilute a precipitate to a first dilution factor to form a suspension, receiving the suspension into a first filtraiton unit to produce a first permeate enriched with the protien of itnerest and a first rententate depleted of the protien of interest wherein the first filtraiotn unit si adapted to retun the first retentate to the first tank and a second tank in connected with the first filtraiton unit for recovering the first permeate enriched iwth the protein of interest as well as a second filtraiton unit for concentrating the first permeate in the second tnak which is adapted to produce a seecond retentate enriched with the protien of itnerest and a second permeate depleted of the protien of itnerest wherien the seocnd unit is optionally adapted to return the second retentate and/or the the second permeate to teh frist tank. The clsoed sytem reduces cost of goods such as water, buffers and chemicals needed for the spearation of the protein of interest. 

Temperature

–Elevated Temeprature:

Winter (US 2006/0051347 also published as US 2007/0237762) teaches processes for concentrating proteins such as antibodies by first ultrafiltering and then diafiltering and then a second ultrafiltering whereby one or more of the first ultrafiltering, the second ultrafiltering and the diafiltering are accomplished at elevated termpatures such as 30-50C.

In conjunction with Non-Affinity A Purification Schemes          

UF/DF – AEX:

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

ultrafiltration/TFF: 

While diverse methods for preparing and purifying therapeutic MAbs have been developed, they typically have in common a final step of concentration by ultrafiltration that precedes formulation of the final product. TFF is also commonly used for diafiltration and concentration of MAb preparation in the final steps of preparing a highly concentrated MAb solution (WO 2004/001007).

Carbon filtration-AEX-Mixed Mode-Carbon filtration: 

Ishihara (US 13/826195) disoses mAb purification using activated carbon in a non-adsorption mode where a clarified solution is passed through an activated carbon filter (e.g., CUNO Lte., Zeta carbon filter). The resulting activated carbon eluate is applied to an AEX and the eluate is passed through a multimodal chromatography column with the eluate then being passed through an activated carbon filter. 

Carbon filtration-CEX-Activated C-AEX: see Ishihara (US 13/826195)

Non-affinity chromatography/HPTFF:

Fahrner (US 2003/0229212A1) teaches a method for purifying a target protein such as an antibody using an ion exchange chromatography step such as cation or anion exchange and/or mixed mode ion exchange chroatography followed by HPTFF.

Design of the membrane Housing

Design of the membrane housing has been knwon to have significant impact on the efficiency of a membrane based processing step. Conventional membrane housing are in the form of flat sheet cassettes and of hollow fiber and spiral bound capsules. An optimal design would maximize the membrane surface area and the flow rate, keeping the size of the moedule to a minimum so as to reduce holdup and dead spaces and make sanitization and validation easier to perform, resulting in significant savings of coset and process time. Pleated membrane configuration is an approach that allws more surface area in a relatively small membrane moedule. A deep pleat membrane confirugation provides more surface area as compared to the traditional fan pleated membrane contruction. The deep pleated design can provide as much as double surface area and hence can be oeprated at double the flow rate of conventiaonl fan pleated design. Keleenpack capsules from Pall Life Sciences are avialbe with Ultipleat (deep pleat) membrane configuration and have been shown to give good recoveries and high flux rates during clarificaiton of mAbs from CHO and hybridoma cell cultures. (Rathore & Shirke) “Recent Devleopments in Membrane-Based Separation in Biotechnology Processes: Review” Prepative Biochemistry and Biotechnolgy 41: 4, 398-421). 

See also Immunoaffinity generally under Affinity Chromatography

Particular Types of Antibodies Used

VHH antibodies:

VHH which are naturally devoid of light chains can be used to generate immunosorbent materials for the purification of human IgG antibodies. Advantages of VHH fragments are that they are single domain peptides, which are exceptionally stable even at higher temepratures. They are also small and easily produced in cost-efficient host organisms such as Saccharomyces cerevisiae. In addition, due to the sequence similarity between these VHH fragments and the human VH3 domain family, immunogenecity is expected to be very low compared to bacterial surface proteins like Protein A and G (WO2006/059904; EP-A-656946).(Low, J Chromatography B, 848 (2007) 48-63)

VHH are basically a single variable heavy chain fragment. They are very stable, low molecular weight (12 kDa) sequences. Antibodies are raised in camels or LLamas and the VHH genes are then cloned and expressed in yeast. The VHH fragmetns contain three CDRs which allows for both high selectivity and customization. The resulting molecules show good stability in caustic (0.1N NaOH). Because of their small size they are able to penetrate deeply into molecules and reognize unique conformational epitopes, alhough this capability may be limited when immobilized on a chromatography resin. Unlike Protein A, however, VHH fragments are monomeric and do not have the capability to bind multiple IgG molecules, which may impose a limitation on capacity. (Low, J Chromatography B, 848 (2007) 48-63)

BAC (the Bio Affinity Company) has developed a proprietary technology (CaptureSelect) based on camelid VHH domains. In the case of the CaputreSelect LC-kappa, the matrix is based on a VHH that recognizes huamn kappa light chains through high affinity binding at the light chain constant domain. Unlike protein L which binds the variable domain and ignores a significant fraction of the kappa light chain population, this product binds 100% of human kappa light chains. In the case of Capture Select LC lambda, the matrix is based on a proprietary ligand that reognizes human lambda light cahins through high affinity binding at the light chain constant domain. BAC also has a CaptureSelect IgG Fc that is designed to purify all human IgGs (including IgG3) and fusion proteins. The amtrix is based on a VHH that recognizes all four subclasses of human IgG through high affinity binding at the Fc region of the heavy chain. (“Novel Affinity Ligands for Bioprocessing” Innovations in Pharmaceutical Technology.

Frenken (US 2016/0207986) disclsoes immobilisation of wngle domain antigen bidning fratments of antibodies naturally devoid of light chains onto a solid surface. Ffater coupling of both tagged an untagged antimouse Fc VHH, complete mouse serum was loaded onto the columns. An elution buffer of Ha2HPO4/NaH2PO4 150 mM NaCl with addition of 12 mM HCL, final pH 2 was used. Immobilised tagged VHH and untaggged VHH has essentially the same results. 

Hermans (US 2015/037627) discloses immobilization of IgG-Fc domain binding VHH fragments onto sepharose, loading human IgG in PBS pH 7.4 on the column with a lienar flow of 150 cm/h, washing with PBS pH 7.4 and eluting with 0.1 M glycine buffer pH 3.0. 

FAB antibodies

—using MgCL elution buffer:

Moorenweis (US 15/166761, published as US 2016/0347826) discloses using the HuCAL Platinum library that includes the CysDisplay selection technology in order to generate Fab antibodies against human IgM, IgA and IgE that bind at neutral pH but can be eluted from the antigen under mild condition (e.g., pH 4-5). Selection of the anitgens using elution under mild conditions resulted in 14 anitobdies against IgM, IgA and 17 antibodies against IgE. Recombinant antiboides were isolated from the HuCAL Platinum library of human antibody genes by three rounds of panning with the antigens. For pannings, some antigens were passively adsorbed to microtiter plates and other antigens were coupled to beads. The pahge antibody library was incubated with the antigens and specific antibody pahge were eluted by elution with 1-2 MgCL. 

Single-chain Antibodies (scFv)

Kuman (US 15/774,398, published as US 2018/0327803) discloses immobilization of a single-chain antibody as a separation ligand for a human serum-derived IgG polyclonal antibody. In one embodiment, the single-chain antibodies are produced by phage display of a produced antibody library to serum-derived IgG or IgA polyclonal antibody coupled to multilamellar liposomes. Several clones were identified which bound to not only a human IgG antibody but also a human IgA antibody coupled to the multilamellar liposomes, suggesting that the single-chain antibodies bind to the L chain, specifically to the kappa chain, of a human antibody which is a region common to both a human IgG antibody and a IgA antibody. The single chain antibody was coupled to a carrier (the carboxyl group of Sepharose/agarose carreir in the form of beads, was esterified with NHS and the purified single-chain antibody was contained with the coolumn so that an amino group of the single-chain antibody forms an amide bond with teh caroxyl group). To the column, a human serum-derived IgG polyclonal antibody was supplied, the column washed and the human serum-derived IgG polyclonal antibody eluted with 0.5 M arginine (pH 1.5). 

pH Dependent antibodies (binding affinity higher at higher verus lower pH):

The purification of an antigen by affinity chromatography using serum antibodies, produced by a host animal’s response to the antigen which are bound to a solid support as an immunoadsorbent has been used for many years. Two serious shortcomings are that if antibodies of high affinity are used to extract the antigen from a sample, harsh conditions are requried to dissociate the antigen form the antibodies after non-adsrobed impurities have been washed away. The required conditions for this include for example a pH of less than 3 or greater than 11 or a concentrated chaotrope such as guanidine or urea solution which can dentautre the antigen and the antibodies. To avoid this, it has become common practice to use immobilized antiobdies of low affinity as an immunoadsorbent which permits elution of the antigen from the body of the immunoadsorbent using mild, non-denaturing conditions. However, the requisite step of washing the column to elute impurities also elutes some of the antigensuch that efficiency of separation is reduced. In addition, low affinity antibodies cannot efficiently bind antigens which are present in the media at relatively low concentrations (e.g., less than 10 ng/ml). Bartholomew WO 83/03678). 

Bartholomew (WO 83/03678) describes a different approach which is using antibodies having a high affinity for the antigen at a first pH, usually at or near pH 7, but with a much lower affinity iat a second lower pH, such as in the range of pH 4.5-4.0. It is believed that the change in binding affinity is likely due to protonation of histidine residues or deprotonation of lysine or possibly tyrosine or aginine residues in either the antibody, the antigen or both which alters the aility of the antigen and antibody to complex with each other. 

Ito (FEBS 11483, August 1992) “The His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values”) discloses that Hisitdine residues can be introduced into various sites in the amino acdi sequences of antibodies by site-directed mutagenesis so as to alter the binding constants of the antibodies with antigen (e.g., lysozime) at various pH values and at two ionic strenghts. Binding constants at low ionic strengh were higher than those at high ionic strenght at all pH values tested. Binding constants at higher pH were higher than those at lower pH. Differences between the binding constants at pH 5.2 nad those at pH 7.8 at low ionic strenght are large than those at higher ionic strenght. 

Domains/Regions on the Antibody bound

Binding to light chains: 

The amino acid sequences as described in WO2006/059904 relate to VHH fragments that bind to the light chain of human antibodies of either the kappa or lambda isotype.

A CaptureSelect biotin anti-labda binds to constant domain of human lambda light chains. 

A Capture Select anti-LC-kappa binds to constant domain of human kappa light chains. 

Binding to Fc part of IgG: 

The amino acid sequences as described in WO2009/011572 relate to VHH fragments that bind to the Fc part of human IgG. Thus they do not allow purification of fragments of human IgG that do not comprise the Fc domain, such as Fab of F(ab’)2 fragments of human IgG.

Several mouse monoclonal antibodies have been described that are capable of binding to the Fc domain of human IgG antibodies (Nelson PN, “Characterisation of anti-IgG monoclonal antibody A57H by epitope mapping. Biochem Soc Trans 1997; 25: 373). A problem associated with the use of mouse Mabs or antibodies from non-human species is the release of Mabs form the matrix which causes contaimination in the purified preparations that is difficult to remove. (Hermans US13/982970)

–Binding to CH3 domain of Human IgG-Fc:

A captureSelect biotin conjugate from TheroScientific binds to CH3 domain of human IgG-Fc. 

Binding to CH1 domain: 

–CaptureSelect™ Biotein Anti-IgG-CH1 Conjugate: (BAC, which was aquired by ThermoFisher Scientific)

Hermans (US13/982970) discloses antigen-binidng proteins which comprise one or more single binding domains which do not comprise a light chain but has full antigen-binding capacity. Examples of such ABP are are VHHs obtained by immunisation of camelids (e.g., llama, camels, dromedaries, Bacterian camles, alpacas, vicunas and guanacos) or sharks). The ABPs bind to an epitope of the CH1 domain that involves one or more of the amino acids: a phenylalamine at position 122, none or a single cysteine at either one of positions 127 and 128, a serine or a lysine at position 156 and/or an asparagine or a serine at position 216. Hermans discloses that the molecules are useful for purifying a target molecule having a human IgG-CH1 domain such as a Fab fragment. The ABPs are advantageous in that they bind the CH1 domain of each of human IgG1, IgG2s, IgG3 and IgG4. The ABPs can be used to purify any human IgG dervied Fab fragment independent of its IgG subclass, light chain isotype and VH subclass. The ABPs do not bind to human non-IgG related antibody isotypes or to the Fc or Fv domain or to free light chainsof a human IgG (accordingly there is no copurification of free light chains).  

Particular types of Proteins Purified

Glycan-targeting antibodies: 

Glycan targetting antibodies recognizing a specific carbohydrate structure have been used, such as antibodies specific for the Lewis x antigen (Cho, “use of glycan targeting antibodies to identify cancer-associated glycoproteins in plasma of breast cancer patients, Anal Chem. 80:5286-5292 (2008)

Commonly Used Affinity Resins for Antibody Purification  (see also Protein L under Affinity Chromatography)

KappFabSelect: is a highly corss-linked agarose with a recombinant protein/ligand produced in S. crevisiae with affinity for the constant domain of the immunoblogulin kappa light chain (See GE Heealthcare Life Scince “KappaSelect, 25 ml”

LambdaFabSelect: is a highly corss-linked agarose with a recombinant protein/ligand produced in S. crevisiae with affinity for the constant domain of the immunoblogulin lambda light chain (See GE Heealthcare Life Scince “LambdaFabSelect Data file 28-9448-22AA”

Capto L: is an affinity chromatography medium (resin) for the purificaiton of antibodies and antibody fragments. It combines a rigid, high-flow agarose matrix with the immunoglobulin binding recombinant protein L ligand, which has strong affintiy to the variable region of antibody’s kappa light chain. (GE Healtchare Life Sciences “Capto L, HiTrap Protein L Hiscreen Capto L”). Native Protein L is a 76-106k protein containing 4-5 highly homologous consecutive extracellular domains responsible for the protein’s interaction with Ig Kappa light chains. Given that is target is the akppa light chain, Protein L will bind to representaitves of most antibody classes, including IgG, IgM, IgA, IgE, and IgD. However, it does not recognize antibodies for related fragments from certain animal species. As the binding site for Protein L is located in the framework region 1 (a less variable region than the CDRs of the variable domain) of the kappa light chain, fragments derived form antibodies that have the kappa light chain can be purified using Protein L. About 60% of mammalian IgG light chains are kappa chains, with the remaining 40% being lambda chains that lack binding sites for Protein L. (GE Healthcare “a platform approach to purificaiton of antibody fragments” 

Protein L is able to bind to some kappa light chain variable domains without interfering with the antigne binding site. This characteristic has the great advantage of allowing purificiaton not only of IgGs, but also antibody fragments, such as single-chain variable fragments (scFv) which are devoid of constant comains. However, PpL is only effective in binidng certain subtypes of kappa light cahins. For example, it binds human VkI, VkIII and VkIV subtypes, but does not interact with VkKK subtype. (Lakhrift, MABS 2016, 8(2), 379-388). 

HIC is a well studied major polishing step in the purificaiton process of IgG based products and known for its capability to remove aggregated from of antibody. (Chen, J. Chromatography A, 1177 (2008) 272-281

Modes of operation:

Bind & elute mode: 

When used in bind-elution mode, there are two main limitation to HIC. HIC resins have relatively lower binding capacity and lower step yield compared to the other chromatography steps used in mAb purificaiton. Second, sufficient binding of mAb proteins to HIC resins is usually achieved with increasing salt concentrations in the binding buffers and the elution product may thus contain a fair amount of salt. However, optimization of resin pore size can significantly improve the binding capacity of standard HIC resins to increase HIC unit process efficiency. (Chen, J. Chromatography A, 1177 (2008) 272-281).

There are two main limitations when using bind elution mode. In general, HIC resins have relatively lower binding capacity and lower step yield compared to other chromatography steps used in mAb purificaiton. Secondly, sufficient binding of mAb proteins to HIC resins is usually acheived with increasing salt concentrations in the binding buffers, and the elution product pool from HI step purificaiton may still contain fair amount of salt, which often complicates sample manipulations and process flow transitions during large scale manufacture. In this respect, efforts have been made to optimize resin pore size to facilitate mass transport of mAb molecuels into ligand binding sites to increase binding capacity and hydrophobic charge induction (HCIC) mied mode chromatography design to allow mAb molecules to bind tot he resin at elatively low salt conditions. (Chen J Chromatography A, 1177 (2008) 272-281). 

Hunter (US2016/0083453) discloses a method from removing multimers from a sample of polyclonal antibodies which includes HIC operated under bind and elute conditions, where monomers are multimers are both bound to the column and then monoerms are selectively eluted with a change in salt concentration and/or pH. The loading buffer is substantially the same as the equilibration buffer and the resin may be washed in a buffer that is substantially the same as the loading buffer. For elution, the buffer may be a lower ionic strengt phosphate buffer. In some embodiments, the buffer comprises a step wise or linear gradient of decreasing salt. 

–Sequential HIC

(Mccue (US 2011/0129468) discloses a method of purifying an Ig (constant part) fusion protein using HIC in bind and elute mode and then contacting the eluate with a second HIC in bind and elute mode.

Flow through mode:

For flow through, process related impurities such as HCPs and aggregates will bind to the HIC media while product flows through the column Herigstad ((US13831181, published as US 9,249,182)

Ghose (US 9,994,609) discloses purification of antibodies by HIC under no-salt conditions with a pH of about 5-7 in flow through mode. 

Herigstad (US 14/077574, published as US 8,946,395) discloses purification of an antibody using HIC that combines flow through and bind-elute tehniques (also known as weak-partitioning mode). A porition of the antibody binds to the HIC and a substantial protion of a contaminant also binds. The flow through of the antibody is collected and by washing the HIC meida another fraction of the antibody is collected. 

Pan (US2008/0167450) teaches that while HIC conducted in bind and elution fashion is an efficient mode to remove dimers and aggregates, this mode has realtively low yield and separation resolution for other product related isomers and has to contend with high salt concentration in the eltuion pools. Thus, HIC in bind and elution mode is becoming less popular in antiboyd production. Instead, HIC chromatotraphy in a flow through mode is gaining interest as a way to remov a large percentage of aggregates.

(Nti-Gyabaah, US 14/355014, published as US 2014/0288278) discloses hydrophobic interaction chromatography (HIC) for the purification of monoclonal antibodies from heterogeneous aggregates. n. In one embodiment, the HIC is conducted in flowthrough mode with operating codnitions of pH 7, conductivity of 110 mS/cm, protein loading of at least 425 gm of protein per liter of resin. 

Shukla (US7,427,659; see also US 2005/0136521) discloses a method for separating a target protein such as an antibody using HIC with branched hdyrocarbon functional groups in an aqueous salt solution and collecting the unbound flow through fraction containing the target protein.

–No salt conditions:

HIC has two primary challenges: In general, binding capacity has been limited, especially in comparison to IEX. To circumvent this issue, HIC is sometimes used in flowthrough mode in which the product of interest flows through wile the more hydrophobic impurities remain bound to the column. This strategy is popular as a polishing step in antibody processes since aggregates are usually more highy retained on HIC. Second, the use of high concentraitons of salts is highly undersirable in any manufacturing prcoess because it can cause correosion of stainless stell tanks. Efforts to operate HIC under reduced or no-salt conditions have those been atempted. (Ghose, “purification of monocloanl antibodies by hydrophobic interaction chromatography under no-salt conditions”, Monoclonal antibodies, 5(5), 2013, pp. 795-800). 

Ghose, “purification of monocloanl antibodies by hydrophobic interaction chromatography under no-salt conditions”, Monoclonal antibodies, 5(5), 2013, pp. 795-800) discloses operating HIC in flwothrough mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin was selected and the pH of the mobile phase was modulated to acheive the required selectively. Under the pH 6 and below, antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product flowed through while impurities such as aggreagates and HCPs bound to the column. 

Rinderknecht (WO/1996/033208) discloses purifying antibody using HIC at low sat concentration. The antibody is eluted from the column in the fraction which does not bind to it.

Ghose (US14/775,774, US 2016/0024145; see also US 16/005642, published as US2019/0144495) discloses a method of purifying an antibody using HIC in flow through mode where the solution does not contain a salt and has a pH of about 5-7.

 Hybrid mode:

A hybrid mode can involved the use of an HIC media that allows for the product to be immobilized but then removed by successive washes of buffer identical to or substantially similar to the loading buffer such as where the salt concentration is adjusted within about 20% of the concentration of the loading buffer. 

Herigstad ((US13831181, published as US 9,249,182; see aslo 14/978553, published as US 2016/0115193)

Overload Mode:  See HIC conditions generally

Conditions:

A large numbr of factors are known to affect HIC performance such as resin matrix, hydrophobic ligand type and density, resin pore size, antibody concentration, amount of laoding, type and concentration of the salting-out salt, pH and operation temperature. In addition, detergents, alcohols, polyols, amino acids and chaotropic agents can be added to the wash and/or elution to improve resolution and/or recovery. While epxerience chromatographers amy be able to cut through the factors with a limited set of experiments, developing the HIC step for optimal yield and purity can still be time consuming due to the large number of actors involved. (Lu, Current Pharmaceutical Biotechnology, 2009, 10, 427-433). 

Bind and Elute Mode:

–Binding:

—-High salt concentration: 

Geenrally, the protein preparation in a high salt buffer is loaded on the HIC column. The salt in the buffer interats with water molecules to reduce the solvation of the proteins in solution, thereby exposing hydrophobic regions in the protein which are then adsorbed by hydrophobic groups on the matrix. The more hydrophobic the molecule, the less salt is needed to promote binding. Senczuk (US 8,273,707)

Hydrophobic interactions are strongest at high salt concentration but the actual concentraitons can vary over a wide range depending on the nature of the protein of interest, salt type and particular HIC ligand chosen. and thus HIC is conventionally performed folloiwng salt elution step such as one used in connection with ion exchange chromatography. For example the salt concentrations shown to be effective in aggregate reduction are in the range of 80mM-1000mM depending on the salt type and HIC adsorbent. Herigstad ((US13831181)

—-Low salt concentration: 

Rinderknecht (US5,641,870) teaches that in contrast to performing HIC at neutral pH in the presence of high salt concentration to elute the antibody, a low pH HIC at low salt concetnration such as 0-0.25M salt can be used. Preferably, no salt gradient is used to elute the antibody. Rinderknecht teaches that buffers that will control the pH within this range include phosphate, acetate, citrate or ammonium buffers.

Sanchayita (US14/775,774, US 2016/0024145) discloses a method of purifying an antibody using HIC in flow through mode where the solution does not contain a salt and has a pH of about 5-7.

–Wash: 

—-Isocratic conditions

Herigstad ((US13831181, now US 9,249,182; see also 14/978553, published as US 2016/0115193) disclsoes a method for reducing a preparation comprising a protein of interest such as an antibody by contacting the antibody/misture with HIC miedia in the presence of a load buffer and collecting a flow through fraction and then contacting the HIC with a wash buffer that is substantially similar to the load buffer and collecting the wash fraction. In one embodiment, the protein of inters binds to the HIC media at a Kp of greater than 90.  and the antibody comprses adalimumab. In one embodiment the Kd for the binding of adlimumab is at least 0.47 and the Kd for the binding of the at least one impourity is 0.01 and the Qmax is at least 6, In one embodiment, the load and/or wash buffer is in the range of 80 mM-1000 mM. 

–Elution: 

—-Decreasing Salt Gradients:

Usually, a dcreasing salt gradient is used to elute proteins from a HIC. As the ionic strenght decreases, the exposure of the hydrophobilic regions of the protein increases and proteins elute from the column in order of increasing hydrophobicity.Senczuk (US 8,273,707)

—-Low pH and Low salt:

Rinderknecht (US5,641,870) teaches elution of an antibody from HIC with a buffer having a pH of about 2.5-4.5 (see also binding above using low pH).  that in contrast to performing HIC at neutral pH in the presence of high salt concentration to elute the antibody, a low pH HIC at low salt concetnration such as 0-0.25M salt can be used. Preferably, no salt gradient is used to elute the antibody.

Flow through Mode:

Sanchayita (US14/775,774, US 2016/0024145) discloses a method of purifying an antibody using HIC in flow through mode where the solution does not contain a salt and has a pH of about 5-7. 

In conjunction with other Types of Chromatography

Bioprrocess engineers often try to develop processes using chromatographyc steps arranged in order to reduce the number of buffer changes. One well known example is using the high conductivity elution buffer from an ion exchange chromatogrpahy (IEC) step as a basis for formulating a high conductivity adsorption buffer in a following HIC step. The latter typically involves adding even more salt to the IEC elution buffer to form  a suitable HIC adsorption buffer (Van Alstine, WO2011/0352382). 

Herigstad ((US13831181, now US 9,249,182) discloses using HIC in combination with other types of chromatography for the purificaiton of an antibody such as Affinity – HIC, HI-Affinity, IEX-HIC, Mixed Mode -HIC, HIC-filtration

Nti-glyabaah (US2014/0288278) disclsoes methods of purifying an antibody such as adalimumab by binding the antibody to Protein A then to AEX in flow through mode and a polishing step which can be mixed mode resin or HIC. 

Protein A – HIC:

Cunnigham (US 2022/0306727) disclsoes prparing masked antibodies with reduced aggregation which includes Protein A chromatography followed by HIC.

See also HIC and HCIC under “affinity chromatography” 

Hydrophobic charge induction chromatography (HCIC) is a type of mixed mode chromatography which as advatnages of mild elution conditionk high capacity and salt-toelrance. The binding of HCIC resin and target proteins can be acheived vai hydrophobic interactions at neutral pH, and then prtoeins can be eluted via electrostatic repulsion between proteins and charge dligands under acidic conditions. (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

Advantages of HCIC for the purification of antibodies: 

A sorbent that employes HCIC does not require feedstock preconditioning, such as concentration, dilution, pH adjustment or addition of specific sals, to bind antibodies optimally. Proteins are absorbed directly from a variety of sources under physiological conditions and elution is achieved by simply lowering the pH.(Guerrier, 755 (2001) 37-46).

CIC shows specific advantages over traditional methods for antibody purification. Sample preparation is primarily limited to the clarificaiton of feedstocks, which can be loaded onto the column without adjustment of ionic strength or pH. IgG dynamic binding capacity ranging from 25-35 mg/ml-1 of sorbent at pH708 and a 10% breakthorugh, are routinely acheived even for murine IgG1. Concomitant purification and concentration effects are acheived in a single step. Several parameters contribute to acheive the sorbent proerties for IgG capture, such as the structure of the ligand as a whole and the ligand density attached to the porous matrix. Although most of the investigations to date have been focused on the separation of antibodies of IgG class, preliminary trials have also been performed to separate IgA. Specificity was not extensively investigated, but binding capacity determinations showed significantly lower numbers than for IgG.  (Boschetti “Antibody spearation by hydrophobic charge induction chromatography” Trends in Biotechnology 20(8) 2002).

Conditions: 

Over a pH range 6-9, the ligand is uncharged. Under these conditions, the ligand and the spacer arm behave much like a hydrophobic site and bind the protein by hydrophobic association. When pH of the mobile phase is modified, the ligand takes a net ionic charge. Under these conditions, the antibody also carries a similar net positive charge, therefore desorption occurs on the basis of electrostatic repulsion between the solid phase sorbent and the protein. In contrast to hydrophobic interaction chromatography or thiophilic chromatography, hydrophobic charge induction chromatography is controlled on the basis of pH rather than salt concentration (Guerrier, Bisoseparation, 9, 211-221, 2000). 

Hydrophobic charge induction chromatography provides for antibody capture without need for adjustment of pH or ionic strengh of typical feedstocks. Like bioaffinity chromatography on Protein A/G, capture is accomplished under near physiological conditions. HICIC is based on the pH dependent behavior of dual-mode, ionizalbe ligands (e.g., pyridine derivative). Over a pH range 6-9, the ligand is uncharged. Under these conditions, the ligand the spacer arm behave much like a hydrophobic stie and bind the protein by hydrophobic association. The sorbent is designed so that binding occurs without need for addition of lyotropic salt. In contrast to HIC or thiophilic chromatography, hydrophogic charge induction chromatography is controlled on the basis of pH rather than salt concentraiton. When pH is modified, the ligand takes a net ionic charge and the antibodies also carries a similar net positive charge and thus desorption occurs. (Guerrier Bioseparation 9: 211-221, 2000)

Washing:

–Caprylic acid is a saturated fatty acid which is commonly used to precipitate protein impurities and separate IgG from plasma, serum and ascites. Tong used caprylate as the albumin selective additive to enhance adsorption selectivity of MEP Hypercell. The reustls showed that the addition of 50 mM adoium caprylate in the laoding ufer and 75 mM in teh washing buffer could greatly improve IgG purity when separating form BSA/IgG mixtures. (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

—ethylene glycol + Inorganic salt: 

Falkenstein (US13/883243, published as US Paent No: 9422329; see also US Patent Application No: 15/216099, published as US Patent 10377794 and US Patent Application 16/778,128, published as US 20200165297) discloses washing an antibody from a multimodal weak CEX such as Capto MMC or Streamline CST using ethylene glycol and an inorganic salt such as sodium chloride, potassium chloride and ammonium chloride. In some embodiments, a binding buffer comprising an inorganic salt and denaturant such as gaunidinium hydrochloride or urea is applied to the multimodal weak CEX.

Elution:

—ethylene glycol + Inorganic salt: 

Falkenstein (US13/883243, published as US Paent No: 9422329; see also US Patent Application No: 15/216099, published as US Patent 10377794 and US Patent Application 16/778,128, published as US 20200165297) discloses eluting an antibody from a multimodal weak CEX such as Capto MMC or Streamline CST using ethylene glycol and an inorganic salt such as sodium chloride, potassium chloride and ammonium chloride. In some embodiments, a binding buffer comprising an inorganic salt and denaturant such as gaunidinium hydrochloride or urea is applied to the multimodal weak CEX.

Particular Resins Used

MEP HyperCell: 

MEP HyperCel is the most widely used HCIC resin whcih was commercialied by Pall. The funcitonal ligand of MEP HyperCel is 4-mercaptoethyl-pyridine (MEP) which contains a pyridine structure and a hydrophobic long chain with a sulfur atom. It has a pKa of 4.8 that enables adsorption happened at neutral pH.  (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

Guerrier (Bioseparation 9: 211-221, 2000) discloses using MEP-HyperCell for the purificaiton of antibodies from ascite fluid, cell culture and cell culture supernatant. The column was initially equilibrated with a phosphate buffered slaid (25 mM phosphate containing 150 mM sodium chloride, pH 7.4), the colunm then loaded, washing tih 50 mM Tris-HCl buffer pH 8 to wash out protein impuriteis. 4-mecaptoethyl pyridine or 4-MEP has enhanced specificity for immunogloublins compared to a simple phenyl group. The pKa of the ligand is 4.8. At pH<4,7, the ligand takes on a predominant postivie charge. As a result the pH of the mobile phase needs only be reduced to 4.0-4.8 for desorption to ccur. 

HCIC in combination with other purification techniques: 

(As to other forms of chromatography followed by HCIC, see respective sections. For example, CEX-HCIC, see cation exchange for purification of antibodies)

HCIC – (Ion affinity or ion exchange or HIC or RPC): 

Rossi (WO/2005/049649) describes HIC used for the purification of IL-18 binding protein and further a step slected from immobilized metal ion affinity chromatography, ion exchange chromatrogaphy, HIC and RPC.

HCIC-AEX:

Boschetti (TRENDS in Biotechnology, 20(3) 2002) teaches that it is known to combine HCIC with AEX.

HCIC-CEX

Arunakumari (US13/410,740, now US8,697,847) teaches a method of purifying an antibody using CEX followed by HIC where there is no in -process tangential flow filtration step. 

Rossi (WO2005/049649) teaches purifcation of IL-18 binding protein using HCIC in combination with IEX such as CEX.

—HCIC-CEX-HIC-UF/DF: 

Boyle (US2004/0033535) discloses purification of alphaOPGL-1 antibody by HCIC with MEP HyperCel to remove a majority of host cell proteins and DNA. The MEP HypercerCel resin is washed with equilibration buffer (20 mM Tris pH 7.2) and the antibody eluted form the resin using a low pH buffer (20 mM Sodium Acetate, pH5). The MEP pool is titrated to pH 3.7 and help for about 60 minutes to inactivate virus (viral inactication), then filered and then the antibody is further purified by CEX using SP Sepharose HP which removed additional CHO cell proteins, DNA, lower weight MW proteins and aggregated forms of alphaOPGL-1. The column is washed and eluted with a linear gradient of increasing salt. The antibody is further purified by HIC and then concentration and fiafiltrated by TFF UF.

(HCIC -Hyrdroxyapatite (HT):  

Guerrier (J. Chromatogr. B. 755 2001, 37-46) teaches isolation of antibodies using HCIC in combination with hydroxyapatite as a second step. The optimal adsorption on the MEP HyperCel column was 7-9 at physiological ionic strengh.

As to Fc receptors generally see Fc Receptors in Signal Transduction

Fc receptor is a general term that refers to any one of several proteins that bind to the Fc region of an immunoglobulin. Fc receptors can be soluble or membrane bound. An  example of a membrane bound Fc receptor is the FcRn (Gastinel (US5,623,053).

Godavarti (US2007/0072307) discloses methods of purifying polypeptides haivng a Fc region such as antibodies using Protein A/G. In one embodiment an Fc receptor can be used.

In General

Inactivation of contaminating virus and removal of this virus is an important concern in the medical industry for the production of recombinant protein. (Zou, WO/2008/036899)

Low Ph Hold: A Protein A/Virus invactivation step: is included in almost all downstream monoclonal antibody processes. This step is placed after the Protein A step because the product at this stage already is in low pH. The virus inactivation is performed during a time somewhere between 30-60 minutes, at a pH of 3.6-3.8 (IPC0m000183319D, anonymous publication dated 3/18/2009 from the IP.com electronic publication website).

Filtration: 

–Nanometer filtration: At present, sized baed nanometer filtration technology is perhaps the most robust viral removal unit operation. Based on the sizes of the mammalian virues commonly occurred in mAb production, these nanometer filters used can be divided into two classes: 50 and 20 nm pore sizes although there are also filters with a nominal pore size of 15 and 35 nm. Large pore filters are efficient in retaining large particle viruses like x-MuLV and pseudorabies virus (PRV). On the other hand, filters with a small pore sizes (20 nm) remove both large viruses and small virus particles such as MMV and respiratory enteric orphan III. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

–Aeptic filtration: For more than 30 years, aseptic filtraiton with 0.2 um membranes has remained virtually unchanged becasue of the quality, predictability and flexibility of the technology. Producign mAb at high concentraiton (up to 150 g/L) showed that aseptic filtraiton can be challenging since a limited volume of antibody therapeutics at such concentrations will readily clog most 0.2 um membranes. However, dual layer filters has been shown to correct this problem. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

–Membrane Chromatography (MC) or memrane adsorber (MA):

Q and other charged MA devices have been in development for chromatography purposes for more than 15 years. Some limitations for large production scale are distorted or poor inlet flow distribution, nonidentical membrane proe size distribution, uneven membrane thickness and lower binding capacity. The first 3 weaknesses can be improved to some degree when multiple layer configuations are ued. This configuation for the Q membrane is used in viral vaccine production. Q AEX adsorber devices have also been used for endotoxin removal at process scale. Low binding capacity is still a major disadvantage in bind/elute mode but in FT mode, the limitations are no longer considered as major issues, particularly when FT-MA is used as the polishing step for antibody purification. FT-MA chmatography is useful to remove impurities below 1% concentraiton including viruses. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

Purification Schemes which include Protein A chromatography and Virus Inactivation/Reduction

Low “future of antibody purificaition” J. Chromatography B, 848 (2007) 48-63)) teaches that although many variations exist, the vast majority of antibody purification process follow a process flow of 1. cell culture harvest, 2. protein A chromatogrpahy, 3. viral inactivation, 4. polishing steps, 5, viral filtration and 6. UF/DF.

Cell culture-Depth Filtration (DF) – Protein A-Viral inactivation-Polishing-Viral filtration-UF/DF: 

Shukla, (J. Chromatogr. B 848 (2007) 28-39) disclose an antibody purification scheme (1) cell culture harvest  as by centrifugation followed by debth filtration, (2) Protein A chromatography (3) low pH viral inactivation, (3) two polishing chromatographic steps chosen from CEX, AEX, HIC and hydroxyapatite (4) viral filtration (5) UF/DF.

(Zou, WO/2008/036899) discloses methods for remvoing viral contaminatns from a therpaeutic protein solution which includes the step of passing the solution through a protein A chromatography column before being passed through a depth filter at a pH within 1 pH unit of the isoelectric point of the virus. In one embodiment the pH is within the range of 4-6 and the virus is less than about 30 nm. 

Protein A – virus inactivation -AEX – CEX – UF/DF

Large scale purification of mAb typically consists of four steps: protein A chromatography, virus-inactivation – AEX (Q sepharose),  – CEX (CM sepharose) and UF/DF. The affinity column is generally the first step after harvest and clarification. In order to inactivate potential viruses, the eluate is typically subjected to a virus inactivaiton process followed by an AEX to reduce host cell proteins, DNA, protein A and potential viruses. Next, a CEX is typically used to further reduce the residual amounts of host cell proteins and antibody aggregates. A hydrophobic interaction column step may be used in place of the CEX. The next step is likely to be a nanofiltraiton virus removal step, using a DV20 or Planova filter. Finally, the pool is then diafiltered and further concentration (Sabbadini, WO2008/070344A2). 

Ishihara (J. Chromatography A, 1176 (2007) 149-156) disclose purification of antibodies which includes 3 sequential chromatography steps (Protein A/anion/cation and also includes a low pH virus inactivaiton step after the Protein A step.

Protein A – Viral Inactivaiton -CEX – DF

Loetscher (WO2007/068429) discloses an antibody purificaiton schemed with the steps of Protein A chromatography – Viral inactivation – CEX – flow through chromatography using Q-Sepharose FF – Diafiltration – 

Protein A – Viral Inactivation – CEX – AEX – UF/DF

Yao (US13/266860) also discloses a scheme of (1) protein A chromatography, (2) inactivating virus (3) CEX (4) AEX, (5) filtering virus particles (6)UF/DF and (7) fine filtering. 

Protein A – viral inactivation – HIC – UF/DF -AEX – NF

Ransohoff (US2013/0260419) teaches a continous process for the purification of antibody which includes clarification, Protein A, low pH viral inactivation, HIC, UF/DF, AEX, NF.

–Protein A – CEX – HIC – Viral inactivation – DF

Shadle (US 5,429,746) discloses a method for separating IgG monomers from aggregates by contacting the mixture with a hydrophobic interaction chromatographic support and selectively eluting the monomer from the support.  3 purification steps are involved (Protein A affinity, cation exchange, and hydrophobic interaction chromatograph), 2 viral inactivation steps, and a diafiltration step to exchange the product into a final buffer of choice.

Conditions for Viral Inactivation

Low pH

Brorson (Biotechnology and Bioengineering, 82(3), 2003) discloses that a 30 minute incubation time of ≥30 min, at room temperature in acetate or citrate is a robust viral inactivation step. 

Detection of Viruses

Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell based infectivity assay has been the standard virus quantification method. However, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantificaiton. It is about 100 fold more sensitive than the cell based infectivity assay. (Shi, Biotechnology and Bioengineering, 87: 884-96, 2004). 

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. 

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 hydroxyapatite chromatography in flow through mode to enhance removal of protein A and other contaminants. 

—-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:

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 mixed mode such as a Capto adhere column which may be conducted in flow through mode.

–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 (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 mixed mode such as a Capto adhere column which may be conducted in flow through mode. 

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)

Protein A chromatography exploits the fact that murine IgG binds to Protein A Sepharose at pH>8 but does not bind at pH<3.0. Typically, the pH of the MAb-containing solution is adjusted to 8.5 and passed over a column of Protein A. With the MAb bound to the protein A, contaminants are washed from the column with a pH 8.5 buffer, Finally, the purified MAb is eluted by passing a pH 3.0 buffer over the column. (Profy, EP0282308A2)

The interaction between IgG and Protein A has been studied in detail and the interaction has been shown to primarily consist of hydrophobic interactions along with some hydrogen bonding and two salt bridges. The primary binding site for Protein A on the Fc region is at the juncture of Cgamma2 and Cgamma3 domains. Variations in affinity amongst IgGs of different species and subclasses for Protein A have been reported. Antibodies belonging to the same sublcass have greater than 95% homologus Fc regions and are expected to have similar binding affinity. Studies have revealed that a highly conserved histidyl residue is present in the center of the Protein A binding site of IgG which aligns facing a complimentary and similarly conserved histidine residue on Protein A itself. At alkaline or neutral pH, these residues are unchared and there are no restrictions on interfacial contact. At low pHs, the complementary histidine groups take on a positive charge resulting in electrostatic repulsion between the two proteins and a concomitant reduction in hydrophobic contact area between them. This electrostatic repulsion is strong enough to elute the antibody off the Protein A column. Elution pH is a critical parameter during Protein A process development. It has been observed that the eltuion pH can vary significantly even among antibodies which have homologous Fc region which is surprising based on conventioanl wisdom that the itneraction with Protein A is primarily dictated by the Fc region of the antibody. In addition to the classical binding stie, some immunoglobulins have been shown to have an “alternate binding” site for Protein A on their heavy chain variable domain. In particular, IgMs as well as some IgGs and IgAs that contain heavy chains from the human VH3 gene family have been shown to exhibit this behavior. Binding studies between Protein A and antibodies have shown that while all 5 domains of Sp1 (E, D, A, B, C) binding IgG via their Fc region, only domains D and E exhibit significant Fab bidning.  The Z domain, a functional analog of the B domain was shown to have negligible bidning to the antibody variable region.   (Ghose, Biotechnol Bioneg. 2005, 92(6), 665-73). See also Starovasnik, Protein Sci, 1999, 8(7), 1423-31) showing that immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc porition of the antibody.

Cell Culture Adjustment

Additiona of Benzoate:

Beigie (US 16/228291, published as US 2019/0233468) discloses that adjustment of a harvest to 0.5 M soidum benzoate at pH 7.2 or 0.5 M sodium sodium benzoate and pH 9 prior to protein A purificaiton leg to improved removal of PLBL2 and HCP impurities. 

Binding

The interaction of immobilized Protein A or Protein G with immunoglobulins is pH dependent. The binding capacity for Protein A is optimal at pH 8.9, whereas the binding capacity of Protein G is high over a broader pH range. “Protein A Antibody Purification Handbook” Pro-Chem Inc. 2005.  Protein A column typically bind antibodies at neutral pH and can be eluted at low pH (typically between pH 3 and 4). It has been shown that a highly conserved histidyl residue in the center of the protein A binding region of IgG faces a complementary histidyl residue on protein A. Those residues take on a positive charge at low pH, thus repelling each other and weakening the protein A-IgG hydrophobic association. That results in elution of IgG from the affinity column. (Shukla, “strategies to address aggregation during protein A chormotography” 36 BioProcess Technical. May 2005).

The basic protocol of protein A affinity column is to bind at neutral pH and elute at acid pH (Fahrner, Biotec. Genetic Eng. Reviews, 18, 2001, p. 309, last ¶). See Affinity chromatography.

Antibody concentration

By using the Beer-Lambert law, the concentration of IgG (mg/ml) in the sample can be measured by multiplying the adsorbance at 280 nm by 0.72. If IgM or IgA are purified, multiply the absorbance at 280 nm by 0.84 or 0.94 respectively. These antibody concentrations are only estimates but can provide a reliable and quantiative method for determining the concentrations of pure antiboyd solutions. Most researchers use a sandwich ELISA assay to accurately measure antibody concentyrations within a range of 1-20 mg/ml sample. Antibodies can also be monitored for purifyt by SDS-PAGE. IgG appears in a reducing SDS-PAGE as 25 kDa and 50-55 kDa bands and IgM appears as 25 kDa and 70-80 kDa bands. Recovery of immunoglobuilins can be quantified by a standard protein assay, scanning densitometry of reducing or non-reducing SDS polyacrylamide gels or ELISA. Proteus “Protein A Antibody Purificaiton Handbook, 2005).

Hober (J. Chromatogr B 848 (2007) 40-47) discloses that the capacity amoung several commercial SPA medi resins ranged from 0.5 to 20 mg/ml and that for large scale applications, optimisation incdes dynamic capacity, antibody concentration in the load, the number of column volumes for elution/equilibration/wash, the load flor rate and the elution/equilibration/wash flow rate.

–Polycarboxylic acid

Ngo (US4,933,435) teaches purifciation of immunoglobulins on Protein A adsorbent at pH of 6-10 and containing at least one polycarboxylic acid which is any organic acid having more than one carboxyl group, including salft forms thereof. Among the numerous carboxylic acids listed are acetate, glycine, aspartate, gluatamine, malate, glutarate, succinate, Tertrate, EDTA.

–Salts

      –—-NaCL: Millipore “Affinity Chromatography Media” 2004, teaches that optimal binding for monoclonal antibodies is with 50 mM phosphate and 0.15M NaCl pH 7.5.

It is assumed that binding between IgG1 and protein A is at least partly based on hydrophobic interactions so that conditions which favour these interaciton should also enhance the binding of IgG1 to protein A. Accordingly it has been disclosed that increasing the pH and/or the ionic strenght of the binding buffer enhances the affinity of these mabs for protein A. (Van Sommeren (“Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to prtoein A sepharose 4 fast flow” Preparative Biochemistry, 22(2), 135-149 (1992), showing high dynamic binding capacities even at neutral pH with 0.1 M tris 1.5M(NH4)2SO4 and 0.1 M tris 1.0M Na2SO4 buffers (pH7.5). 

      —–Kosmotropes (Katropic Salts):

The term “kosmotrope”(order-maker)  denotes solutes that stabilize proteins. An alternative name for kosmotrope is “compensatory solute” as they ahve been found to compensate for the deleterious effects of high salt contents (which destroy the natural hydrogen bonded network of water) in osmaotically stressed cells. They should be distinguished from “chaotropes” (disorder-maker) which denote solutes that destablize proteins. (Jungbauer (US8,058,410).

Ngo (J. of Immunoassay & Immunochemistry, 29(1), 2008, 105-115) disclose that kosmotropes (kosmotropic salts) such as ammonium phosphate, ammonium sulfate and sodium sulfate, enhance the yield of antibody purfied by Protein A chromatography. Ngo disclsoes purification of immunoglobulin on protein A using a buffer having a pH 7.5-10 and containing a combination of monovalent cations such potassium ions and polybasic anions such as phosphate ions. These monovalent cations and polybasic anions  ions can be provided by the use of potassium phosphate or ammonium phosphates and ammonium sulfates and sodium sulfates.

Wang (13/898929, 13/898984 and 14/085503) teaches using a kosmotropic salt such as ammonium sulfate, sodium sulfate, sodium citrate, potassium sulfate, potassium phosphate, sodium phosphate, to improve antibody binding onto a Protein A resin. The kosmotropic slat contributes to teh stability and structure of water-water interactions and causes water molecules to favorably interact with proteins and stabilize the intermolecular interaction, thereby increasing the retention of the antibody of interst on the protein A resin. 

–Effect of Temperature: Yityoong (J Immunological Methods, 109 (1988) 43-47) disclose that certain mouse IgG subclasses that bound weakly to protein A at ambient temperature would bind more firmly if the temperature of the system was lowered to 4C. At this temperature, irrellevant proteins could be washed off easily.

When adsoprtion buffers of relatively low ionic strenght are used, improvement of the binding of IgG1 antibodies to protein A can also be obtained by lowering the temperature (Van Sommeren (“Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to prtoein A sepharose 4 fast flow” Preparative Biochemistry, 22(2), 135-149 (1992). 

Wash

Non-specific binding of mAbs to protein A affinity media is usually due to either ionic or hydrophobic interaction with the base matrix or immobilization chemistry. Glass based matrices, for instance ProSep®-A, may exhibit relatively higher HCP levels compared to agarose based matrices. This is htought to be due to higher non-specific binding inherent to the glass matrix ompared to agarose (IP.com Number : IPCOM000127319D, August 22, 2005). 

The aim is to modify the post load wash buffer in such a way as to disrupt these interactions, which will elute the non-specifically bound contaminants without prematurely eluting the mAb. Several approaches have proven effective which include selecting a pH for the intermediate wash buffer that is between the loading and the elution buffers, and/or inclusion of salt, detergents or amino acids such as arginine (Millipore Technical Brief “Increasing Purity of ProSep®-vA affinity chromatography media using an intermediate wash step” 2006. 

Millipore “Affinity Chromatography Media” 2004, teaches that wash buffers usually have the same buffer components as the loading buffer (see above) to ensure the captured product remains bound to the column while non-binindg contaminatns are washed off. However, in the case of non-specific binding due to ionic interactions, adding salt (1M NaCl) has been found to be effective. 

Multiple washing

–-Using differing pH and/or conductivity:

Mendiratta (WO 2014/207763) discloses a method for the purification of mAb from cell culture which includes Protein A with several wash buffers. The second wash is conductited at a pH and/or conductivity higher than the first wash and the third wash at a pH and/or conductivity lower than the second wash. 

Buffer additives: (amino acids):

—Arginine:

Barron ( http://www.priorartdatabase.com/IPCOM/000127319 ) describes an intermediate wash solution for Protein A chromatography containing 0.5 to 2.0 M arginine in a phosphate/acetate buffer at pH 5-7.5 which is used to remove HCP contaminants. They also tested an intermediate wash which contained sodium chloride at 0.5-2 M at pH 5.0-7.5 but reported that NaCL wash showed no significant decrease in HCP.

PCOM000127319D, August 22, 2005 also discloses that introducing an intermediate arginine wash step before elution of ProSep-vA high capacity column can significanlty decrease the aount of HCP non-specific binding caused by a model CHO feedstock. 

Eckermann (US13/522030, now US 9,284,347) disclose a method of depleting impurities from a solution that contains a protein which comprises the Fc domain of an immunoglobuilin by protein A chromatography using a wash buffer fwhich contains 1) arginine, 2) sodium chloride 3) an alcohol such as isopropanol, n-propanol and ethanol and 4) polyvinylpyrrolidone and/or a detergent.

Ritzen “J. Chromatography B, 856 (2007) 343-347 reports that washing of a mAb bound to an affinity resin with 0.5M arginine is an effective way for specific removal of protein bound endotoxin. 

Sun (US20080064860 and 20080064861 see also WO2008/031020) also describe washing that conntains arginine at a concentration of 0.1-2.0 M and pH 4.5-8.0. Bing (US2011/015730) also discloses an intermediate washing of an affinity matrix where the wash comprises and equilibrating buffer having similar pH and sale concentrations as the feed and further compirsing an elevated concentration of salts (e.g., sodium chloride). In some embodiments the wash may contain one or more charged amino acids such as arginine. 

—-Arginine + High pH

Frauenschuh (US 13/516960, now US 9505803; see also 15/336878, published as US 20170044211; see also US 16/363,170, published as US 2019/0233469) disclose a wash solution for affinity chromatography which uses arginine and a nonbuffering salt such as a halogen salt such as sodium chloride (NaCL), magensium chloride (MgCl2) and potassium chloride (KCl), preferably at high pH above 8.0. The combination of arginine and a nonbuffering salt is reported to remove more impurities than wash solutions containing either arginine or salt alone and results in a sharper elution peak correlating with a high concentration of the protein of interest. The term “nonbuffering salt” does not include buffering salts such as sodium acetate, sodium phosphate and Tris, which contribute to retaining the pH of a wash solution udner the applied conditions. However, Frauenschuh (US 14/122,707, published as US 9,663,552) discloses a wash buffer peromred without the presence of a nonbuffer salts where the wash buffer includes arginine at a pH at least 8.5. 

—-Arginine + guanidine:

Holstein (“Protein A Intermediate wash strategies” BioProcess International, 13920, 2015) disclsoes that increasing arginine in a Protein A intermediate wash buffer dramatically reduced the level of HCP. Increasing GuHCL concentration also eld to less HCP in the elution pool. In addition, an intermediate wash consisting of 0.05M Tris, 2 M GuHCL, pH8.5 led to the loest HCP in the elution pool. 

Wang (US 15/659093, published as US 2018/0021696) disclsoes  method for preparaing a protein of interest using a wash buffer for Protein A that includes a basic amino acid such as arginine and guanidine. 

–Caprylate:

Gruber (WO0214186350) discloses a method for separating a recombinantly produced polypeptide such as an antibody from HCP using a Protein Chromatography column which includes a washing buffer that has between about 50-100 mM sodium caprylate at pH 8-9. In one embodiment, the wash buffer includes about 100 mM sodium capyrlate in 100 mM Tris at a pH of about 9 and about 2.5M sodium chloride. 

—-Arginine + caprylate

Aboulaich “A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies” Biotechnol. Prog. 2014, 30(5)) discloses identifying HCPs that assocaite with mAbs by imobilizing mAb onto chromatography resinfollowed by incubation with HCPs obtained from supernatant of non-mAb producer cells The dissociation of the HCPs form the mAb using various washes were identified using mass spectometry. A combination of sodium capryalte and arginine as a wah buffer was more beneficial than with arginine alone and reduced the levels of a alrge number of HCPs to undetectable levesl (0%).  Aboulaich disclsoes that this approach could potentially facilitate rapid development of wash condtions to improve HCP clearance during the Protein A capture step for mAb mnufacturing. 

Dumetz (US 16/330,588, published as US 2021/0284686) discloses a method of purifying an antibody from HCPs such as Phospholipase B-like 2 protien and cathepsin L using a superantigen chromatography solid support such as Protein A with a wash buffer that includes about 50 mM caprylate and more than about 0.5 M arginine. 

–Histidine

Ishihara (EP 2583973, published 8/20/2011) discloses purificaiton of mAbs using Protein A chromatography such as MabSelect SuRe where the column was washed with an equislibration buffer that inlcudes histidine pH 7.0. 

—-Histidine + Tris

Falkenstein (US 15/900,443, published as 2018/0186865) dicloses a wash buffer for use in Protein A chromatography for the purificaiton of IgG4 or IgG1 isotype antibodies that incldues both histidine and tris.  anda.a pH of about 6.5 or higher. In one embodiment, the was step includes a low conductivity aqueous solution. Falkenstein (US 15/900,449, published as 2018/0186866) also discloses a method for purifying a bispecific antibody using Protein A with a wash buffer of low conducitvity. In one embodiment, the buffer aslo includes Tris. In one embodiment, the method further includes an additional wash using a high conductivity which can also include histidine. McDonald (US 15/900, 461, published as 2018/0186832) also discloses a method of purifying a human IgG1 or IgG4 antibody with a Protein A that includes using a wash buffer with low conductivity of 0.5 mS/cm or less. In one embodimetn the low conductivity aqeuous solution has a pH of about 7 or more and includes Tris. 

Adjustment of pH

–Intermediate pH:

Non-specific binding is usually due to eitehr ionic or hydrophobic interaction with the base matrix or ommobilization chemistry. The aim is to modify the post-load wash buffer in wuch a way as to disrupt these interactions, which will elute the non-specifically bound contaminants without prematurely eluting teh MAb. Selecting a pH for teh ntermedaite wash buffer that is between the loading and the elution buffers is one approach to do this. For example, if the loading pH is 7.5 and the lution pH is 4.0, then an intermediate wash buffer pH of 5.0 might be optimal. In teh case where an intermediate pH wash is not practical or is insufficient alone to attain required HCP levels, then the second step is to evaluate the addition of a salt, amino acid, detergent or solvent in the intermedaite wash buffer (see below). Millipore Corporiation (2006) “Increasing purity on Prose. 

—High pH:

Shukla “Host cell protein clearance during Protein A chromatography: development of an improved column wash step” Biotechnol. Prog 2008) discloses a Protein A wash buffer with high pH such as 9.0. A wash buffer consisting of 1 M urea and 10% isopropanol was partciularly effective. 

Wang (US 15/558,033, published as 2018/0078876, published as US 10695693; see also US Patent Application No: 16/881,102, published as US 20200282332) discloses a method of purifying an antibody using an affinity chromatography such as Protein A and a high alkaline pH such as one at least 9.0. 

Addition of Caprylic Acid

–Caprylic Acid

It has been shown that by adding 25 mM carpylic acid to the wash buffer during purificaiton of polyclonal IgG from ovine serum using Protein A mimetics the level of non-specifically bound albumin could be reduced. The purity of IgG could be increased form about 80 to 95% by introducing this intermediate wash after laoding of serum to the column. Monie, “Evaluation of the 96-well format for screening of chromatographic buffer condtions” Master’s Degree thesis, Uppsala, University School of Engineering, October 2006.

—–Caprylic Acid with salt

Gruber (US 14/890,823, published as US 2016/0108084) discloses a method of reducing HCP by loading a cell culture sueprnatant onto a Protein A chromatography column and washing with a buffer that includes 25 -200 mM fatty acid and sodium chloride at a concentraiton of about 1-2.5M. 

—–Caprylic Acid without salt

Goklen (US 14/775868, published as US 2016/0024146) discloses a method for purifying an antibody using Protein A chromatography that includes a first wash buffer that includes 10-125 mM sodium caprylate without the addition of NaCL. 

Jensen (US 2017/0044210) discloses a method for purifying a target protein such as an antibody by loading a sample with the protein onto an affinity chromatography and using 2-10 mM caprylic acid so as to inactivate and way away viruses followed by eluting the target protein. 

—-Caprylic Acid + NaCL:

Gruber (US 14/890823, published as 2016-0108084) discloses that wash buffer at pH 9, with 2.5M sodium chloride and 100 mM sodium cpayrlate improved HCP clearance. 

Hydrophobic electrolyte solvent

Blank (6,127,526) discloses a method for purifying an antibody by Protein A chromatography by adsorbing it onto a solid phase comprising silica or glass such as controlled pore glass column or silicic acid column, removing contaminants by washing with a hydrophobic electrolyte solvent such as TMAC or TEAC and recvoering the antibody.

Salts:

–Salts + detergents, solvents and polymers

Protein A chromaotgraphy wash buffers containing salt (such as sodium chloride) alone or in combination with either a detergent (e.g., tween 20), a solvent (e.g., hexylene glycol) or a polymer (e.g., polyethylene glycol) have been described (Breece, 6,870,034). 

Benzoate Salt + benzyl alchol: Beigie (US 16/228,291, published as US 2019/0233468) discloes that using 0.5 M sodium benzoate pH 7.0 and benzyl alcohol in an intermeiate wash was very effective at removing HCPs such as PLBL2 during Portien A purification. 

tegramethylammonium chloride(TMAC): One way to decrease the amount of host cell proteins in the elution pool when using Prosep media is by using an intermediate wash with tegramethylammonium chloride(TMAC) (Fahrner, Biotech. Genet. Eng. Rev. 18, 2001, p. 311, 2nd ¶).

Urea

–Urea + Isopropanol

Shukla (WO 2007/1009163; see also Biotechnology Progress, 24(5), 2008, pp.1115-1121) disclose that a combination of 1M urea and 10% isopropanol in the wash buffer minimized HCP levels during Protein A chromatography for the purificaiton of antibodies.

–Urea + NaCL

Woo (US 15/924,163, published as US 2018/0265543) discloses a method of purifying an antibody using protein A chromatography wehre a wash solution that includes urea is used. Results showed that a wash solution that includes arginine, guanidine or a comination of urea with sodium chloride, resulted in a reduction of HCP. 

–Urea + Dextran Sulfate

Zhengjian (WO 2016/153978) discloses use of dextran to enhance Protein A chromatography wash. In one emboodiment other wash buffer components such as salt which is sodium chloride and a chaotropic reagent which is urea was combined with dextran sulfate. 

Elution

The most common elution conditions for Protein A or G affinity and immuno-affinity separations involve a reduction in pH to between pH 2.5 and 5.0. “Protein A Antibody Purification Handbook” Pro-Chem Inc. 2005.

Millipore “Affinity Chromatography Media” 2004, teaches teaches that the highest pH which gives acceptable recovery of the target antibody should be used which for IgG is usually pH3.0-4.0. 

A variety of strategies have evolved to address the issue of aggregation/precipitation during Protein A elution such as modification of the elution buffer to make the buffer conditions more conducive to product stability. Stabilizers such as arginine have been added to the elution buffer to reduce aggregation of eluting antibodies. Low temperature operation of the Protein A step can in some cases reduce product aggregation. (Shukla, J. Chromatogr. B 848 (2007) 28-39)

–Addition of amino acids:

——Arginine, histidine and proline:  

Arginine is an effective agent for elution of antibodies from Protein A and G columns (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555). Arakawa (“protein Expression and Purification, 36: 244-248, 2004) disclose eluting antibodies from a Protein A column using an elution buffer containing 0.5-2.0 M arginine at pH 4.1-5.0. Recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 30% recovery (Arakawa, “Elution of antiboides form a Protein-A column by aqueous arginine solutions, Protein Expression and Purificaiton 36 (2004) 244-248). 

(Chmielowski (US13/058301) discloses the amino acids histidine, proline or arginine in elution buffers as stabilization agents for Protein A affinity.

–Acetate: 

Chmielowski (US13/058301) discloses a method for purifying a monoclonal antibody by contacting the sample with Protein A affinity chromatography at a temperature from about 15-27C and eluting with acetate.

Shukla (US 2009/0306351) teaches a method for purifying a monoclonal antibody with an elution buffer of 100 m acetate, pH 3.6. 

–Citrate (citric acid/sodium citrate): 

Chmielowski (US13/058301) discloses a method for purifying a monoclonal antibody by contacting the sample with Protein A affinity chromatography at a temeprature from about 15-27C and eluting with citrate.

Hickman (WO 2010/048192 A2) teaches equilibration with a suitable buffer such as Tris/NaCL, pH around 7.2. Following loading of the column, the column can be washed as for example with 20 mM citric acid/sodium citrate, 0.5 M NaCl at pH of about 6.0 and then eluted with a suitable elution buffer such as  acetic acid/NaCl buffer, pH around 3.5.

Shukla (BioProcess International, May 2005, pp.36-44) teaches various combinations of elution buffers during protein A chromatography of a monoclonal antibody including 50 mM citrate, pH 3.6. Yumioka (EP1563710) teaches a method purifying monoclonal antiboidies on a protein A affinity chromatography which involves eluting in a citrate buffer of pH3.5. The buffer can further comprise arginine.

–pH: Since most antibodies retain the native structure above pH 4.0, various attempts have been made to elute the bound antibodies above this pH (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555).

—-Reducing pH or Temperature or adding Protease inhibitors: 

Fahner disloses a method for reducing leacing of protein A during protein A affinity chromatography by reducing temperature or pH or by adding one or more protease inhibitors to a composition that is subjected to protein A affinity chromatography. In one emboidment the termpature is in the range form about 3C -20C.

Nielsen discloses a method for reducing leaching of protein A by reducing temperature or pH or by adding protease inhibitor(s) to a composition that is subjected to protein A affinity chromatography (WO 2005/016968).

For effects of temperature on protein A chromatography see also Van Sommeren “Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antiobides to protein A Sepharose Fast Flow” Preparative Biochemistry, 22, 135-149, 1992  and Tu “Temperature effects binding of murine monoclonal IgG antibodies to protein A. J. Immunological methods, 109, 43-47, 1988. 

Shukla (BioProcess International, may 2005, pp. 36-44) teaches conducing protein A chromatography at room temperatures (20-25C) but that colder termpatures gave a lower rate constant for aggregation (2-8°C).

—-Neutral or Alkaline pH  or Magnesium chloride

Deepak (IN 183939) discloses prapring pure monospecific polyclonal antibodies to malarial lactate dehydrogenose useful for the diagnosis of mario by adsorbing serum collected from animals having antibodies against LDH on Protein/A separhose column and eluting with a buffer having a pH of 2-5 or 10-11.8 or with a sale such as Magneisu or lithium chlorida or potassium thiocynate (3-5M). 

Tustian (US 2016/0024147) disclose that employing a chaotropic agent and pH gradient or pH step elution buffers results in imporved peak resolution between closely related molecular species. Bi specific antibodies containing aprotein A binding ablating substitution CH3 domain paired with a protein A binding CH3 domain are separated with high peak resultion from monosepcific antibodies containing aprotein A binidng ablating substituted CH3 domain wpaired with a protein A binding ablating substitutedCH3 domain and monospecific antibodies containing a protein A binding CH3 domain paired with a protein A binidng CH3 domain. Useful chatropic agents include magesium chloride and calcium chlroide. In one embodiment the VH binding bsAb was loaded on a Mabsapture A resin and following a series of washes the antibodies were eluted from pH 6 to 3 with magnesium chloride. 

—-Ph Gradients:

Bian (US 14/768254, published as US 2016/0122305) discloses a method of purifying an antibody using a MAbSelect SuRE column using a pH gradient that used a high and low pH buffer. In particular, Bian showed that using two types of modified C domain resins for the Protein A ligan was particularly effective effetive in removing agregates with the pH gradient whereas a large aggregate peak was observed in the fractions eluting towards the end of the profile for MabSelect SurE, a large agreggate peak was observed in botht eh early and late eluting fractions for the modified C domains. 

Brown (13/393525, published as US 2013/0041139) teaches purification of polypeptides having a CH2/CH3 region such as antibodies by binding the polypeptide to Protein A and leuting with a pH gradient starting at a pH of 5.0 using an elution buffer that contains a high and low pH buffer such as acetate and formate.

Davis (US2010/0331527) discloses islation of a bispectific antibody on a solid support comprising Protein A by employing a pH gradient, such as a linear gradient from pH 4.2 to pH 2.8. 

Duhamel, J. Immunological Methods 31: 211-217 (1979) discloses using a pH gradient to elute column bound IgG into at least two major overlapping peaks centered 0.4 pH units apart in the gradient with the first peak centered at pH 4.7 and the seccond at pH 4.3. Upon second passage of each component, the high pH eluting IgG conatined less than 1% IgG1, 95% IgG2 and 5% IgG4 and the low pH eluting IgG contained 90% IgG1, 6% IgG2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. 

Better (US5,576,184) discloses loading of anytibody onto a Protein A colum and elution with a pH gradient (pH2-9). The antibody was found to elute between pH 3.5 and 4.0. 

Ishihara (US 2006/0257972) discloses applying an antibody mixture to a Protein A column and performing a gradient elution where a buffer (pH 2.5) onctianing 0.10 M sodium acetate, 0.10 M glycine and 0.15 M sodium chloride is gradually added to a buffer (pH 8.0), which contains 0.10 M disodium phosphate, 0.10 M sodium acetate, 0.10 M glycine and 0.15 M sodium chloride to gradually lower the pH level (o.e., pH gradient elution). 

Martin discloses that guinea pig serum was chromatography using protein A-Sepharose chromatography in citrate-phosphate buffer at pH 7.3. The bulk of serum proteins eluted in the starting buffer. Two peaks of protein A bound serum proteins were eluted by a decreasing pH gradient, a small peak centered at pH 4.7 and a larger peak centered at pH 4.3. IgG contained in the peak eluted at pH 4.7 has fast gamma immunoelectrophoretic mobility and IgG in the peak eluted at pH 4.3 had slow gamma mobility. 

Pan (US2008/0167450 and WO2008085988) teaches methods of purifying Fc domain containing polypeptides comprising binding said polypeptide to protein A and eluting with a pH gradient such as one where the top of the gradient starts above pH7 and the bottom is at or below 4.0. 

Pan Analytical Biochemistry 388: 273-278 (2009) also disclose that under the typical step gradient mode, protein A tightly binds the Fc portion of teh antibody under neutral pH conditions (pH 7.0-8.0) while impurities are washed away after which the acidic pH buffer (pH 3-4) is used to release the antibody from the protein A column. A pH gradient instead of a typical step gradient mode can also be used. 

–Salt Gradients:

Van Alstine (US 2007/0213513) discloses in a multi step chromatography procedure with affinity chromatography uisng salt gradient elution will require a subsequent deilution step before the next step of ion exchange chromatograph. 

 Cleaning

Millipore “Affinity Chromatography Media” 2004, teaches that the use of a low pH 9pH 1.5) regeneration with phosphoric or hydrochloric acid solution after ever cycle is very effective at removing strongly bound material from ProSep-A media which is protein A immobized on porous glass. 

Staphylococcus aureus Protein-A (SpA):

Protein A affinity chromatography has become well established as the preferred capture step for purification of human monoclonal IgG for in vivo applicaitons, so much so that it has become largely regarded as generic (Gagnon, “Polishing Methods ofr Monoclonal IgG Purification” chapter 17 in “process Scale Bioseparations for the Biopharmaceutical Industry”, p. 491). There are, however, some problems with Protein A chromatography. In order to elute the antibody, acidic conditions are required due to the high affinity of the antibodies to the resin. Explosure to these acidic conditions can result in the formation of protein aggregates. Some strategies to address aggregation include a additional chromatography steps or to use stabilizing agents (this has a problem itself in that these stabilizing agents must now be purified) (Chmielowski (US13/058301).  

It is well established that immunoglobulin binding proteins interact with a number of distinct epitopes on teh full lenght antibody and this adds to their functioanl flexiblity. Protein L binds to the Vl of Kappa light chains, Protein M interacts through conserved framework regions on the VL and Vk and binds both isotpyes with high affinity (low nM). Proteins A and G are multi-specific prtoeins possessing binding affinity to the Fc as well as the Fab protion of teh antibody. Each binds to the hinge region connecting CH2 and CH3 on teh Fc protion of teh IgG with high affinity. Additionally, Protein binds to the VH3 subset of VH domains which includes about 30-50% of the circulating IgGs. Protein G binds to the constant domain of teh Fab porition of IgG through its interction with teh CH1 domain, a highly conserved domain across many isotypes and species. (Baily, J of Immunological Methods, 415 (2014) 24-30). 

Multiple Binding Domains of SpA: (see multimers of SpA domains under “Affinity Chromatography”)