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.