REMOVAL OF PRO TEIN AGGREGATES FROM

B!OPHARMACEUTICAL PREPARATIONS IN A FLOW-THROUGH

MODE

R a ed p li a <j¾s

[0001 ] The present application claims the benefit of priority of

13. S, Provisional Patent Application No. 61 /609.533, filing dat March 12, 2012, and U.S. Provisional Patent Application No. 61/666,378, filing date June 29, 2012, each of which is incorporated by .reference herein in its entirety.

Field of tbe Invention

[0002 ] This invention relates to methods of removing protein aggregates from biopharraaceotical preparations containing a product of interest in a flow-through mode.

[0003] Protein aggregates are one of the important impurities that need to be removed from hiopharm.aceut.ical preparations containing a product of interest, e. g., a therapeutic protein or an antibod molecule. For example, protein aggregates and other contaminants must be removed from biopharraaee ticai preparations containing a product of interest before the product can be used i diagnostic, therapeutic or other applications. Further, protein aggregates are also often found in antibody preparations harvested from hybrkloma cell lines, and have to be removed prior to the use of the antibod preparation for its intended purpose. This is especially Important in case of therapeutic applications and for obtaining Food and Drug

Adm inistration appr al ,

[0004] Removal of protein aggregates can be challenging as often there are similarities between the physical and chemical properties of protem aggregates and the product of interest in. a hiopharaiacetttical preparation, which is often a monomeric molecule. There are many different methods in the art for the remo val of protein aggregates from biopharmseeutieal preparations including, for example, size exclusion chromatography, ion exchange chromatography and hydrophobic interaction chromatography.

[00051 Several bind and elute chromatography methods are know for separation of protein aggregates from the product of interest For example, hydroxyapatite has been used, in the chromatographic separation of proteins, nucleic acids, as well as antibodies, in hydroxyapati e

chromatography, the column is normall equilibrated, and the sample applied, in a low concentration -of phosphate buffer and the adsorbed proteins are then elated in a concentration gradient of phosphate buffer (see, e.g., Giovannim, Biotechnology and Bioeogmeering 73:522-529 (2000)). However, in several instances, researchers have been unable to selectively elute antibodies from hydroxyapatite or found that hydroxy apati e chromatography did not result in a sufficiently pure product (see, e.g., Junghauer, J. Chromatography 476:257-268 (1989); Giovannini,

Biotechnology and Bioengineering 73:522-529 (2000)),

[0006] Additionally, ceramic hydroxyapatite (CHT), a

commercially available chromatography resin, has been used with some success for the removal of protein aggregates, in a resin format (BIORAD CORP, also see, e.g., U.S. patent publication no, WO 2005/044856), however, it is generally expensive and exhibits a low binding capacity fo protein aggregates,

[0007] A bind and elute cation-exchange chromatography method has also been described* which is sometimes used in the industry for aggregate removal (see, &g, U.S. Patent No. 6,620, 18), however it is often observed that an unfavorable trade-off between monomer yield and aggregate removal needs be made, in a recent review of aggregate removal methods from solutions of monoclonal antibodies, it was noted, concerning a bind and elute chromatography mode that "cation exchange chromatography can be a. useful way to separate aggregate and monomer hut it can be difficult to develop a high yielding step with a high capacity." See, e.g Aldington et aL J. Chrom. B, 848 (2007) 6 -7

[0008] Compared to bind and elute methods and size exclusion chromatography methods known tn the art, protein purification in flow- through .mode is considered more desirable due to better economics, simplicity, time, and buffer savings,

[0009] Attempts have been made in the prior art to implement flow-through aggregate removal based on ^' Hydrophobic- Imeracfcns Chromatography (B1C) media (see, e.g. IIS Patent No, 7.427,659).

However, HIC-based preparative separations have narrow applicability due to generally difficult process development, narrow operating window, and high concentration of salt required in the buffer.

[001 Oj Weak partitioning chromatography (WPC) is another mode of chromatographic operation, in which the product binds weaker than in the case of bind-elute chromatography but stronger than in the ease of flow-through chromatography (See, e.g. U.S Patent No. 8,067,182);

however, WPC also has certain draw back associated with it including, narrow operatin window and lower binding capacity for impurity removal compared to bind and elute methods.

[0011] While, some of the Sow-through methods described in the prior art have been reported to hind aggregates, the specificity for binding aggregates relative to the product of interest appears to be low. Further, there appear to be no known methods in the prior art which exhibit a high specificity for binding lo wer order protein aggregates such as, e.g., dimers,. tfimers and etramers.

.S mmary of the l¾vei¾ t jojS

[0012] The present invention provides novel and improved compositions as well as iSow-throug methods which use such compositions for separating a product of interest, e.g., a therapeutic antibody or a monomelic protein from protein aggregates in a. biopharmaceuticat composition. The compositions and methods described herein are especially useful for separating a monomelic protein of interest from lower order protein aggregates, such as. e. ., dimers, trimers and tetra ers, which are generally difficult to separate from the monoroeric protein.

[0013] The present invention s based, at least in art, on increasing selectivity of binding of protein aggregates compared to a product of interest (i.e., monomeric molecule) to a surface in flow-through mode, thereby to separate the protein aggregates from the product of interest The- present in vent ion is able to accomplish this by the unique design of a surface having a certain densit of cation exchange binding groups, thereby facilitating a greater number of protein aggregates to hind to the surface, as compared to the monomeric molecules.

[0014 j in some embodiments according to the present invention, a flow-through chromatography method of separating a monomeric protein of interest from protein aggregates in a sample is provided, where the method comprises contacting the sample with a solid support comprising one. or more cation exchange binding groups attached thereto, at a density of about 1 to about 30 mM, where the s lid support selectively binds protein aggregates, thereby to separate the monomeric protein of interest from, protein aggregates.

[0015] In some embodiments, the solid suppor used in the methods according to the present, invention is selected from a

chromatographic resin, a membrane, a porous monolith, a woven tabrie and a non- woven fabric.

[0016} In some embodiments, the solid support comprises a chromatographic resin or a porous bead.

[00! 7] In some embodiments, the solid support is a porous poiyvinyiether polymeric head or a porous crosslmked poly.meihacrylate polymer bead. [0 18] In various embodiments, the chromatographic resin or the porous bead comprises a mean particle size of about 50 microns, or between about 0 and about 500 microns, or between about 20 and about 140 microns, or between about 30 and about 70 microns.

[001 ] In some embodiments, a solid support is selected from a ch matographic resin or porous bead where the mean particle size is between 10 micron to 500 microns, or between 20 and 200 microns, o etween 20 and 90 microns. In a particular embodiment the

chromatographic resin or porous bead has a mean particle size of abou t S microns, In general, selectivity may improve with decreasing particle size. One skilled in the art would understand that the mean particle size can be adjusted while maintaining some level selectively for binding protein aggregates based on the needs of a specific application or process,

[0020] In some embodiments, the protein aggregates are lower order protein aggregates such as, for example, diniers, irimers and tetramers, [002 ! ] In other embodiments, the protein aggregates are higher order protein aggregates such as, for example, perstamers and higher order, [0022] In some embodiments, the cation exchange group used in. the methods according to the present, invention is selected irom the group consisting of a sulfonic group, a sulfate group, a phosphonic group, a phosphoric group, and a earboxylic group.

[0023] In some -embodiments, the monomerie protein is an antibody, in a particular embodiment, the antibody is a monoclonal antibody. In other embodiments, the monomerie protein is a recombinant protein, e.g. _* an Fc-fusion protein. In yet other embodiments, the monomerie protein is a non-antibody molecule.

[0024] In some embodiments according, to the present invention, a flow-through chromatography method of separating a monomerie protein of interest from protein aggregates in a sample is provided, where d e method comprising contacting the sample with a solid support comprising one or more cation exchange binding groups attached thereto at a density of abom 1 to about 30 raM, where the solid support birds protein aggregates relative to monomers at a selectivity greater than about 10» thereby to separate the protein of interest from protein aggregates.

[0025] In other embodiments, a tlo -through chromatography method of reducing the concentration of protein aggregates in a sample is provided, the method comprising the steps of: (a) providing a sample comprising a protein of interest and from about I to about 20% of protein aggregates; (b) contacting the sample with a solid support comprising one or more cation exchange binding groups attached thereto, at a density of about 1 to about 30 nlM; and (c) collecting, a flow-through effluent of the sample, where the concentration of protein aggregates in the. effluent is reduced by at least 50% relative to the concentration of the aggregates in (a), thereby reducing the concentration of protei aggregates in the sample.

[0026] h some embodiments according to the methods of the present invention, the concentration of the protein of interest in the effluent is a t least 80% of the concentration of the protein of interest in (a).

[0027] In some embodiments, the ionic conductivity of the sample containing aggregates that, is contacted with the said solid support is within a range of about 0,5 to -about 10 mS/em.

[0028] In some embodiments, a process for purification of a protein of interest (e. ., a monoclo nal anti body} described herein, does not require a bind and elate cation-exchange chromatography step.

Accordingly, such a process eliminates the need for salt addition to elation solution, and use of subsequent dilution steps.

[0029] In some embodiments, a process for purification of a protein of interest ( g,, a monoclonal antibody) is provided, where, the process does not require an increase in. conductivity. Accordingly, such a process does not require dilution after the cation-exchange step in order to reduce conductivity prior to performing the su se uent How-through an ion - exchange step,

[0030] Also encompassed by the present invention are polymers comprising cation exchange groups, where the polymers are attached onto a solid support

[003 i ^' j In some embodiments, such a polymer comprises the following chemical structure;

where R ^{ is a cation-exchange group; ^' ' is any aliphatic or aromatic organic residue that does not contain a charged group: R- i any uncharged aliphatic or aromatic organic linker between any two or more polymeric chains; x. y and z are average molar fractions of each monomer in the polymer, where y>x; and symbol m denotes that a similar polymer chain is attached at the other end of the linker.

[0032 ] i other embodiments, a polymer according _, to the present invention comprises the following chemical structure:

where x, y, and z are average molar fractions of each monomer n the polymer, where x and symbol m denotes that a similar polymer chain is attached at the other end of the linker,

[0033] In yet other embodiments, a polymer according to the present invention comprises the following chemical structure, where the ^• polymer is nkage onto a solid support:

where R. ^! is a cation-exchange group; R ^! is any aliphatic or aromatic organic residue that does not contain a charged group; and x and y are averag molar fractions of each monomer in the polymer, where y>x.

[0034] In some embodiments,, a polymer according to the present invention comprises the .following chemical structure; wherein x and y are average molar fractions of each monomer in the pol mer, where y > χ and wherein the polymer is grafted via linkage onto a chromatography resin.

[0033] in various embodiments, polymers are attached to a solid support.

| 0036j h* various embodiments according to the present invention, the effluent containing the product of interest is subjected to one or more separation methods described herein, where the effluent contains less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 2% protein aggregates,

[0037] n some embodiments according to the present invention, the methods and/or compositions of the present invention may be used in combination with one or more of Protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diatllttation, ultrafiltration,, viral removal filtration, anion exchange chromatography, and/or catio exchange chromatography,

[0038] In some embodiments, the protein aggregates that are selectively removed by the compositions described herein are higher molecular weight aggregates, i.e. protein pen tamers and higher order.

[0039] In some embodiments, the protein aggregates that are selectively removed by the compositions described herein comprise lower order aggregate species, such as protein, doners, trimers, and teiramers. [0040] ϊη some embodiments, the solid supports comprising one or more cation exchange binding groups described herein, are used in a flow- through purification process step in a purification process, where die .flow- through purification process step as well as the entire purification process .may he performed in a continuous manner.

[0041 J In some embodiments, the solid supports described herein are connected to he in fluid conurm caiion with other types of media both upstream and downstream of the solid support. For example, in some embodiments, a solid support comprising one or more cation exchange binding groups, as described herein, is connected to an anion exchange chromatography media upstream and a virus filtration media downstream from the solid support. In a particular embodiment, a sample flows through activated carbon followed by an anion exchange chromatography media followed by a solid support comprising one or more cation exchange binding groups followed by a virus filter. In some embodiments, a static mixer and/or a surge tank is positioned between the anion exchange media and the solid support comprising one or more cation exchange binding groups., in order to perform a pH change.

Brief Descri ion of the Bra iags

[0042] Figure 1 is a schematic depiction, of enhanced aggregate selectivity using a composition having a lower density of cation exchange binding groups as compared to a composition known in the art.

[0043] Figures 2A-2F depict representative chemical structures of various compositions encompassed by the present invention, figures 2A-2D depict cross-linked polymeric structures immobilized on a solid support; Figures 2E-2H depict grafted polymeric structures eovalent!y attached to a solid support. II ^s is a cation-exchange group suc -as g., sulfonic, sulfate, phosphoric, phosphonie or carbox lic group; R ² is any aliphatic or aromatic organic residue that does not contain a charged group; is any uncharged aliphatic or aromatic organic linker between any two or more polymeric chains; x _t y, and. z are average molar fractions of each monomer in the polymer, whereas y>x; symbol m denotes that a similar polymer chain is attached at the other end of the linker; R ⁴ is N l or O; R* is a linear or branched aliphatic or aromatic group, such -Cl¾~ -C^I-L*-, .-(¾¾- _> -£(€%> - CO?-, ~C«R|-; 8* Is a linear or branched aliphatic or aromatic uncharged group containing NH, O, or S linker to the polymer chain; and W and ^¾ are independently selected from a group containing one or more neutral aliphatic a d aromatic organic residues, and may contain heteroaiorns such as (X N, S, P, F, CL and the like,

[0044] Figure 3 A is a graph representing the results of a size exclusion ^' chromatography (SEC) analysis of fractions of a monoclonal antibody (MAb I) passed through three different membrane devices, containing Membrane 7, Membrane 8 or a commercially available ^• membrane (Pali Mustang l ⁾ S membrane), On the x-axts, the total loading of MAb I on the membrane is shown in. g/L and on. the y-axis, the relative concentration of MAb I monomer (represented by % yield) compared to the starting concentration is shown.

[0045] Figure 3B is a graph representing the results of a size exclusion chromatography (SEC) analysis of fractions of MAb 1 passed through three membrane devices, containing Membrane 7, Membrane 8 or Pall Mustang® S membrane. On the x-axis, the total loading of MAb I on the membrane is shown in g L and on the y-asis, the relati ve concentration of MAb I dimer (represented by % yield) compared to the starting concentration is shown. As observed, the dimer break-throug is significantly later for Membrane 7 as compared to both Membrane $ as wel l as the Pali Mustang® S membrane,

[0046] Figure 3C is a graph representing the results of a size exclusive chromatography (SEC) analysis of fractions of MAb 1 passed through three membrane devices, containing Membrane 7, Membrane 8 or Pali Mustang® S membrane. On the x-axis, the total loading of MAb 1. on the membrane s shown In g/L; on the y-axls, the relat v concentration of MAb .1 High Molecular Weight (BMW) aggregate yield compared to d e starting concentration is shown. As observed, the HMW break-through is significantly later for Membrane 7 as compared to both Membrane 8 as well as the Pall Mustang® S membrane.

[0047] Figure 4A is a graph depicting aggregate breakthroughs, as measured by SEC (shown on the right y-axis ^' h for an antibody pool for Membrane 7 (s own by open triangles) and Membrane 8 (shown by open squares) as a function of MAb loading. Also shown is the monomer (i.e., product of interest) yield in the pool for Membrane 7 (closed triangle) and Membrane $ (closed square).

[0048] figure 4B is a graph depicting aggregate breakthroughs

(shown on the right y-axis) for an antibody pool for Membrane 7 for 2 separate runs (run 1 : shows by open circles; run .2: shown by open diamonds) as function of MAb loading. Also shown is the monomer y ield in the pool for Membrane ? (run Ϊ : shown by closed circles: run 2: shown by closed diamonds},:

[0049] Figure 5 is a graph depicting aggregate breakthroughs

(shown on the right y-axis) for an antibody pool for Membrane 7 as a function of MAb ill loading (shown in the x~axis as mg/m.L). Also sho wn Is the monomer yield in the pool tor Membrane 7 (shown in the left y-axi _. s) [0050], Figure 6 A is a graph depicting partition coefficients at pB

5.0 as a function of NaCl concentration for the binding of MAb 1 monomers to Membrane 8 (open squares), and MAb I aggregates to Membrane 8 (open triangles), MAb I monomers to Membrane 7 (closed squares), and MA J aggregates to Membrane ? (closed triangles).

[0051 ] Figure 6FJ is a graph depleting the partitio coefficients at pH S.O as. a function of ^' NaCl concentration for the binding of MAb II monomers to Membrane S (open squares), and MAb 11 aggregates to Membrane 8 (open triangles), MAb 0 monomers to Membrane 7 (closed squares), and MAb II aggregates to Membrane 7 (closed triangles).

[0032] figure 7 is a graph representing selectivity plots tor the binding of MAb I and MAb II to Membranes 7 and 8, respectively, at pH 5.0 Selectivity of Membrane 8 for Ab I is shown by open squares;

selectivity of Membrane 7 for MAb I is shown by closed squares; selectivity of Membrane & for MAb II is shown by open triangles; and selectivity of Membrane 7 for MAb II Is shown by closed triangles.

[0053] Figure 8 depicts a contour plot indkaimg percentage monomer at 1 and 15 g L aggregate loadings.

[0054 ^' J Figure 9 depicts a conto ur plot indicating optimal region of operation (shown in white) at 5, 10 and 15 g/L aggregate loadings. Optimal was defined as >88¾ monomer yield and the aggregate making up < 2% of total protein. The regions in grey do hot meet these criteria.

[0055] Figure .10 is a graph depicting the results of an experiment to investigate effect of flow-rate on throughput of the vims filtration device. The Y-axis denotes pressure drop (psi) and the X-axis denotes throughput of the virus filtration device (kg/nr),

[0056] Figure 11 is a schematic depiction of the connected flow- through purification process, which employs the compositions described herein. An activated carbon containing device is connected directly to an a on-exchange device. The effluent from the amomexehange device passes through a static mixer, where an aqueous acid is added, to reduce pH, and then goes through a cation-exchange flow-through device, according to the present invention, and a vims filter.

[0057] Figure 12 is a graph depicting the results of an experiment to measure HCP breakthrough after an anion exchange chromatography media (ChromaSorh™), The X-axis denotes HCP concentration (ppm) and tbe Y-axis denotes the AFX loading (kg/L) [0058] Figure 13 is a graph depicting the results of an expe iment to measure removal of MAb aggregates as a function of loading of the virus filtration device in the flow-through purification process step. The X-axis denotes the vims filtration loading (kg/nf ) and the Y-axis denotes percentage of MAb aggregates in the sample after virus filtration.

[0059] Figure 1 is a graph depicting the results of art experiment to demonstrate the .removal of MAb aggregates as a function of cumulative protein loading, using a cation-exchange resin, as described herein (Lot #12LPDZ-1 ). The X-axis denotes cumulative protein loading. in mg ml, the left Y-axis denotes the concentration of antibody MAb in mg/ml and the right Y-axis denotes the percentage of MAb aggregates in the sample.

[0060] Figure 15 is a graph depicting the results of art experiment to demonstrate the removal of MAb aggregates as a function of cumulative protein loading, using a cation-exchange resin, as described herein (Lot

12LPDZI 28). The X-axis denotes cumulative protein loading in mg ml, the left Y-axis denotes the concentration of antibody .MAb in mg/ml and the right Y-axis denotes the percentage of MAb aggregates in the sample.

[0061 ] Figure 16 is a graph depicting the results of a experiment to demonstrate the removal of MAb aggregates as a function, of cumulative protein loading, usin the a cation-exchange resin, as described herein (Lot #· 12LPDZ129), The ^' X-axis denotes cumulative protein loading in mg m!, the left Y-axis denotes the concentration of antibody MAb in mg/ml and the right Y-axis denotes the percentage of MAb aggregates in the sample.

10062] Figure i 7 depicts ckomatogram of resin Lot 1712 with

MAb5 at. pFI 5 and 3 minutes residence time.

Pct¾iled escrji tina of the Invention

Several prior art bind and el tc as well a flow-through methods have been described in an attempt to separate protein, aggregates from raonomeric proteins, which are generally the products of interest.

4 [0063] Bind nd eliite methods are generally time consuming, require significant process development and sometimes are not successful in effectively separating the aggregates and in particular, lower order aggregates, .from, a monomelic protein, while maintaining a high yield of monomeric protein. Certain cation-exchange flow-through methods have been described n the art, both with conventional porous resins and membranes; however, they appear to have issues with low capacity of the media tbr binding aggregates, low selectivity for dimers, and often low yield of the product of interest, i.e., monomerie proteins (see, e.g. Li u el al, J, Ckrom. A., 1218 (201 1 ), 6943-6952).

[0064] Additionally, methods have been described in the prior art. which appear to employ cation exchange groups on solid supports to separate proteins. See, e.g., Wu at d> (Effects of stationary phase Hgand density on high-performance ion-exchange chromatography of proteins,. J. Chmm. 598 (1992), 7-13), which discusses having a high density of cation exchange groups on a solid support in. order to have the best

chromatographic resolution of two model proteins. However, recently, the effect of cation exchange binding group density on aggregate removal was specifically investigated (see, e.g., Fogle et αί,. Effects of resin iigand density on. yield and impurity clearance in preparative cation exchange chromatography, L Mechanistic evaluation, ./ Chrom. ,·!. 1225 (2012), 62- 69). It was reported that the resolution of monomelic and high molecular weight antibody forms is largely insensitive to the density of binding groups.

[0065] WPC has also been described for use of impurity removal

(see, e.g., Suda etal _* Comparison of agarose and dextran-gralted agarose strong Ion exchangers for the separation of protein aggregates, J. Chmm, ,4, 1216: pp,52S6~5204, 2009). In case of weak, partitioning chromatography (WPC), the partition, coefficient. p, ranges from 0J to 20; a Kp > 20 is associated with bind-elote chromatography and a Kp<0.i is associated with flow-through chromatography, WPC as at least two serines drawbacks.

5 First, a narrow operating region (0, 1 < p<20k which has to he ^' between flow-through and bind-elute chromatography (see, e.g., U.S. Patent. No. 8,067 J 82). Within this operating region, the selectivity between product and impurities has to be large for efficient separation. Typically for ion- exchange media, the selectivit between product and impurity increases as Kp increases, with highest selectivity under hind-date conditions. Second, the capacity for impurities is. lower than in case of bind-elute mode. Since the Kp values are lower, so will the capacity under typical operating conditions where impurity concentrations are smaller than the that of the product.

[0066] The present invention is able to achieve a superior separation of protei n aggregates and monomenc proteins, as compared to the various methods descri bed in the art. An important distinction of the present invention from those described in the prior art is the use of a low density of cation exchange groups on a solid support as we ^' ll as -a high specificity for the removal of lower order protein aggregates, e.g., dimers, trimers and tetramers, which are typically more diffi ult to remove due to their closeness to monomer in size and surface characteristics,

[0067] In order that the present invention may he more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

L Definitions

[0068] The term "chromatography," as used herein, refers to any kind of technique which separates the product of interest (e.g., a therapeutic protein or antibody) from contaminants and/or protein aggregates in a biopharmaceutical preparation.

[0069] The terms "flow-through process," "flow-through mo e ^* and "flow-through chromatography," as used interchangeably herein, refer to a product separation technique in which a biopharmaceutical. preparation containing the product of interest is intended to flow-through a material. In

US some embodiments, the product of interest flows through the material and the tmdesirable entities bind to the material In a particular embodiment, the material contains a certain density of cation exchange binding groups (i.e., lower than the prior art compositions) and is used for separating a monomerie protein from protein aggregates, where the monomelic protein flows through the material, while the protein aggregates bind to the material.

[0070] ^' The term "affinity chromatography" refers to a protein, separation technique in which a target molecule (e.g., an. Fe region containing protein of interest or antibody) specifically binds to a Hgand which is specific for the target molecule. Such a Hgand is generally referred to as a biospeerfic Hgand. in some embodiments, the biospecific ligand (e.g.. Protein A or a functional variant thereof) is covalently attached to a. suitable chromatography matri material and is accessible t the target molecule in solution as the solution contacts the chromatography matrix. The target molecule generally retains its specific binding affinity for the ^' biospecific hgand during the chromatographic steps, while other solutes and/or proteins in the mixture do not bind appreciably or specifically to the ligand. Binding of the target molecule to the immobilized ligand allows contaminating proteins and impurities to be passed, through the

chromatography matrix, while the target molecule remains specifically bound to the immobilized Hgand on the solid phase material. The- specifically bound target molecule is then removed in its active form from, the immobilized !igand under .suitable conditions (e.g., low H, high V high salt, competing Hgand etc.), and passed through the chromatographic column with the elutlon buffer, substantially free of the contaminating proteins and impurities that were earlier allowed to pass through the column. It. is- understood that any suitable ligand may be used, for purifying its respective specific binding protein, e.g. antibody In some embodiments according to the present invention, Protein A is used as a hgand for an. Fc region, containing target protein. The conditions tor elation from the ^' biospecific ligand (e. .. Protein A) of the target molecule (e.g., an Fc- region containing protein) can be readily deiemuned by one of ordinary skill in the art. in some embodiments _* Protein G or Protein L or a functional variant thereof may be used, as a biospecif c ligand. In some embodiments, a process which, employs a biospecific ligand such as Protein A, uses a pH range of 5-9 for binding to an Fc-region containing protein, followed by washing or re- equilibmt ng the biospecific ligand / target molecule conjugate,. which is then followed by elulion with a buffer having pM about or below 4 which contains at least one salt.

[0071 ] The terms "contaminant/' 'impurity/' and "debr s " as used interchangeably herein, refer to any foreign or objectionable molecule, including a biological macromolecule such as a DNA, an. RNA, one or more host cell proteins, endotoxins, lipids, protein aggregates and one or more additives which may be present in a sample containing the- product of interest that is being separated from one or more of the foreign or objectionable molecules. Additionally, such a contaminant may include any reagent which is used in a step which may occur prior to the separation process. In a particular embodiment, compositions and methods described herein are intended to selectively remove protein aggregates from a sample containing a product of interest.

[0072] The term 'immunoglobulin/' ^* 1g" or "antibody" (used interchangeably herein) refers to a protein having a basic four-pel ypeptide chain structure consisting of two heavy and two light chains, said chains being stabilized, for example, by interchain disulfide bonds, which has the ability to specifically bind antigen. The term "single-chain

immunoglobulin" or "single-chain antibody" (used interchangeably herein) refers to a protei having a two-polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind antigen. The terra " omain" refers to a globular region of a heavy or light chain

I S polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by β-p!eated sheet and/or iatmchaio disulfide bond. Domains are further referred to herein as "constant" or "variable", based on the relative lack of sequence variation within the domains of various class members in the cas of a "constant* domain., or the significant variation within the domains of various c lass members in the ease of "vari able ^" domain. Antibody or polypeptide "d main " are often referred to interchangeably in the art as antibody or polypeptide "regions". The "constant" domains of antibody light chains are referred to interchangeabl as "light chain constant regions", "light chain constant domains", '"CI ^* regions or "CL" domains. The "constant" domains of antibod heavy chains are referred to interchangeably as "heavy chain constant regions", "heavy- chain constant domains' ^* , "CFf ' regions or "ΟΗΓ domains. The "Variable" domains o -antibody light chains axe referred to interchangeably as "light chain variable regions", ^" light chain variable domains", "VL" regions or "Y ^' L" domains. The "variable" domains of antibody heavy chains are referred to interchangeably as "heavy chain variable regions ^" , "heavy chain variable domains ^* ', "V!i" regions or "VH" domains,

|00?3] immunoglobulins or antibodies may be monoclonal or polyclonal and may exist in monomelic or polymeric form, for example, Ig antibodies which, exist in pentameric form and/or IgA antibodies which exist in monomeric, dimeric or mtsltirneric form. The term, "fragment" refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain.

Fragments can be obtained via chemical or enz matic treatment of an intact or complete antibody or antibody chain, fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fah\ F(ah')2 _s Fc and/or Fv fragments.

[0074] The term, "antigen-binding fragment" refers to a polypeptide portion of an immunoglobulin or antibody that binds an antigen or competes with intact antibody (i.e. , with the intact antibody from which they were derived) for antigen binding ( ,* ^> . specific binding). Binding fragments can be produced by recombinant D A techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', (ab¾. Fv, single chains, and single-chain antibodies,

[0075] The term "biophamiaeeuiseai preparation ^* as used herein, refers to any composition containing a product of interest (e.g., a therapeutic protein or an antibody, which is usually a monomer) and. unwanted components, such, as protein aggregates (e.g., lower order protein aggregates and high molecular weigh aggregates of the product of interest).

|O076] As used herein, and unless stated otherwise, the term

"sample" refers to any composition or mixture that contains a target molecule. Samples may be derived from biological or other sources.

Biological sources include eukaryotic and prokaryotic sources, such as plant and animal ceils, tissues and organs. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target molecule. The sample may be "partially purified" (i.e., having been subjected to one or more purification steps, such as filtration, steps) or may be obtained directly from a host cell or organism producing the target molecule (e.g., the sample may comprise harvested cell, culture fluid). In some embodiments, a sample is a cell culture feed. In some embodiments, a sample which is subjected to the flow-through purification processes described herein is an eluate from a bind and elute

chromatography step, e.g., a Protein A affinity chromatography,

[0077] The term "protein aggregate ^" or "protein aggregates/' as used interchangeably herein, refers to an association of at least two molecules of a product of interest, e.g. , a therapeutic protein or antibody. The association of at least two molecules of a product of interest may arise by any mesas including, but not limited to, covalerri, non~eova!eoi, disulfide, or nonreducible crosslinkmg,

[0078] Aggregate concentration can be measured in a protein sample asing Sis?.e Exclusion Chromatography (SEC), a well known and widely accepted, method in the art (sec, e.g. _* Gahrielson et al, J. Ph r , ScL, 96, (2007), 268 -279). Relative concentrations of species of various molecular weights are measured in the effluent using UV absorbance, while the molecular weights of the fractions are determined by performing system calibration following instruction of column manufacturer.

[0079] The term "dimer," "dhnm " "protein diroer" or "protein dinners," as used interchangeably herein, refers to a lower order fraction of protein aggregates, which is predominantly comprised of aggregates containing two rnonorneric molecules, but may also contain some amount of ttimers and tetraraers. This traction is usually observed as the first resolvable peak in a SEC ehromatogram immediately prior to the main monomer peak,

[0080] The term "high molecular weight aggregates," or "HMW." as used interchangeably herein, refers to a higher order fraction of protein aggregates, i. > pentamers and above. This fraction is usually observed as one or more peaks in a SEC chromatograni prior to the dimer peak.

[0081 J The term "binding group/ ^* or 'ligarsd" as used

interchangeably herein, refers to a specific chemical structure immobilized on a solid support (eg., a porous surface), which is capable of attracting a monomelic protein or protein aggregates from a solution. Protein attraction to the binding group can be of any type, including ionic, polar, dispersive, hydrophobic, affinity, metal chelating, or van der Waals.

10082] The term "cation exchange binding group," as used herein, refers to a negatively charged binding group. In a particular embodiment, a binding group is a negatively charged sulfonate -group. [0083] The term "solid support" refers in general to any material

(porous or mm porous ) to which the binding groups are attached. The attachment of binding groups to the solid support can either he through a covalent bond, such as in the case of grafting, or through coating, adhesion, adsorption, and similar mechanisms. Examples of solid, supports used in the methods and compositions described herein include, but are not limited to. membranes, porous beads, winged fibers, monoliths and resins.

[0084] The term "density," a used herein, refers to the

concentration of binding groups or Hgan s on a solid support, which is generally expressed as concentration of ligand in moles per liter of porou media. A widely accepted unit of ion-exchange ligand. density is milfi- equivalent per liter, or meq/L (equivalent to μεο ηιΐ), which corresponds to molar amount of ion-exchangeable groups in a given volume of media. For charged groups with a single ionizabie moiety, the ligand density m meq/L would be equivalent to the density of these groups expressed in m.mofe/L, or nrM.

[0085] The term ^selectivity, as sed herein _* refers to the dimensionless ratio of partition coefficients of two species between a, mobile phase and a stationary phase, A partition coefficient (K _p ) is the ratio Q/C, where Q and C are the bound and free protein concentrations, respectively.

[0086] The term "process step" or "unit operation," as used interchangeably herein, refers to the use of one or more methods or devices to achieve a certain result in a purification process. Examples of process steps or unit operations which may be employed include, but are not limited to, clarification, bind and elute chromatography, virus mactivation, {low- through purification and formulation. It is understood that each of the process steps or unit operations may employ more than one step or method or device to achieve the intended result of thai process step or unit operation. For example, in some embodiments, the clarification step and/or the flow- through purification step may employ more than one step or method or device to achieve that process ste or unit operation. In some embodiments, one or more devices which are used to perform a process step or unit operation are single-use devices and can be removed and/or replaced without having to replace any other devices in the process or even having to stop a process run-.

[0087] As used herein, the term "pool tank" refers to my

container, vessel,, reservoir, tank or bag, which is generally used between process steps and has a size/volume to enable collection of the entire volume of output from, a process step. Pool, tanks may be used for holding or storing or manipulating solution conditions of the entire volume of output from a process step. In various embodiments according to the present invention, the processes obviate the need to use one or more pool tanks.

[0088] In some embodiments, the processes described herein may use one or more surge tanks,

[0089] The term "surge tank" as used herein refers to any container or vessel or bag, which is used between process steps or within a process step (e.g., when a single process step comprises more than one step): where the output from one step flows through the surge tank onto the next- step. Accordingly, a surge tank is different from a pool tank, in that it is noi intended to hold or collect the entire volume of output from a step; but instead enables continuous flow of output from one step to the next, in some embodiments, the volume of a surge tank used between two process steps or within a process step in a process or system described herein, is no more than 25% of the entire volume of the output from the process step. In another embodiment, the volume of a surge tank is no more than 10% of the entire volume of the output from a process step. In some other

embodiments, the volume of a surge tank is less than 35%, or less than 30%, or less than 25%, or less than 20%. or less than 15%, or less than 10% of the entire volume of a cell culture in a bioreactor, which constitutes the starting materia! from which a target molecule is to be purified. [0090] In some embodiments described herein, a surge tank is used upstream of a step which employs the sol d support described herein, [0091 J ^' The terra '"continuous process," as used herein, refers to a process for purifying a target molecule, which includes two or more process steps (or unit operations), such that the output from one process step flows directly into the next process step in the process, without interruption, and where two or more process steps can he performed concurrently for at least a. portion of their duration. To other words, in ease of a continuous process, as described herein, it s not necessary to complete a process ste before the next process step is started, but a portion of the sample is always moving through the process steps. The term "'continuous process" also applies to steps within a process step, in which case, during the performance of a process step including multiple steps, the sample flows continuously through the multiple steps that are necessary to perform the process step, One example of such a process step described herein is the flow-through purification step which includes multiple steps that are performed in a continuous manner, e.g., .flow-throug activated carbon followed by flow- through AEX media followed by flow-through CBX media which, utilizes the solid supports described herein followed by flow-through virus filtration.

[0092] The term "anion exchange matrix' ^* is used herein to refer to a matrix which Is positively charged, e.g. having one or more positively charged ligands, such, as quaternary amino groups, attached thereto.

Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX™ and FAST Q SEPHAROSE™ (GE Healthcare), Other exemplary materials that may be used in the processes and systems described herein are Fraetogel® EMD TMAE, Fraetogei® HMD T AE highcap, Eshmtmo® Q and Fraetogel® EMD DEAE (EMD Mi!lipore).

[0093] The term "active carbon ^* ' or "activated carbon," as used interchangeably herein, refers to a carbonaceous material which has been subjected to a process to enhance its pore structure. Activated carbons are porous solids with very high surface areas. They can be derived from a variety of sources includi ng coal, wood, coconut husk, nutshells, and peat Activated carbon can be produced from these materials using physical, activation involving heating under a controlled atmosphere or chemical activation using strong acids, bases, or oxidants. The activation processes produce a porous structure with high surface areas that give activated carb n high capacities for impurity removal. Activation processes ears be modified to control the acidity of the surface. In some embodiments described herein, activated carbon is used in a flow-through purification step, which typically follows a bind and elute chromatography step or a virus ioaetivalion ste which in turn follows the bind and elute chromatography step. In some embodiments, activated carbon is incorporated within a cellulose media, e.g., in a column or some other suitable device,

[0094] The term "static mixer' refers to a device for mixing two fluid materials, typically liquids. The device .generally consists of mixer elements (non-moving elements) contained in a cylindrical (tube) housing. ^' The overall system design incorporates a method for delivering two streams of fluids into live static mixer. As the streams move through the mixer, the non-moving elements continuously Wend the materials. Complete mixing depends on many variables including the properties of the fluids, inner diameter of the tube, number of mixer elements and their design etc. In some embodiments described herein, one or more static mixers are used in the processes described herein. In a particular embodiment, a static mixer is used for achieving the desired solution change after- an anion exchange chromatography step and before contacting a sample with a cation exchange solid support, as described herein,

I I. Exemplary Solid supports

[0095] The present invention provides solid supports having a certain density of binding groups or hgands attached thereto, which bind protein aggregates more favorably than the monomerie form of a protein which is usually the product of interest Without wishing to be bound by theory, it is contemplated that any suitable solid support- may be used in. case of the present invention. For example, the solid support can be porous or non-porous o -it can fee continuous, such as in the form of a monolith or membrane. The solid support could also be discontinuous, such as in. the form of particles, beads, or fibers . In either case (continuous or

discontinuous), the important features of the solid support are that they have a high surface area, mechanical integrity, integrity i aqueous environment, and ability to provide flow distribution to ensure accessibility of the binding groups,

[0096] Exemplary continuous porous solid supports include mkroporons membranes, i.e. having a pore sizes between about 0.05 micron and 1.0 micron. Porous membranes that may be used In the compositions and methods according to the present Invention may be classified as symmetric or asymmetric in nature, which refers to the uniformity of the pore sixes across the thickness of the membrane, or, for a hollow fiber, across the nrkropotous wall of the fiber. As used herein, the term,

'"symmetric membrane" refers to a membrane that ^' has substantially uniform pore size across the membrane cross-section. As used herein, the term "asymmetric membrane ^** refers to a membrane In which the average pore size is not constant across the membrane cross-section. In some

embodiments, in case of asymmetric membranes, pore sizes can vary evenly or discontinuous!}' a a function of location throughout the membrane cross- section. In some embodiments, asymmetric membranes can have a ratio of pore sizes on one external surface to pore sizes on the opposite external surface, which ratio is substantially greater than one.

[0097] A wide variety of micfoporous membranes made from, a wide variety of materials may be used in the compositions and methods described herein. Examples of such materials include polysaccharides, synthetic and semi-synthetic polymers, metals, metal oxides, ceramics, glass, and combinations thereof

[009S] Exemplary polymers th i can be used to manufacture the mkfoporous membranes that may be used in the compositions and methods described herein include, but are not limited to, substituted or uosubstituted po!yaerylamides, polystyrenes, polymethaery!amides, polyimides, poiyacrylates, polycarbonates, polymethaery1at.es, polyvinyl hydrophilie polymers, polystyrenes, poiysidfbnes, polyeihersulibnes, copolymers or styrene and divi ylbenzene, aromatic polysuifones, poiyietra!luoroet ylenes (PTFE), perfiuo.rinated thermoplastic polymers, poiyoiefins, aromatic polyamides, aliphatic polyamides, ultrahigh molecular weight polyethylenes, polyvhiylidene difluoride ( VDF), polyedieretherketones (PEEK), polyesters, and combinations thereof.

[0099] Exemplary commercially available mieroporous membranes are Durapore and Mil! spore Ex e s® available from EMD Miliipore Corp. (Bilierlca, MA); Supor® available from Pail Corp, (Port Washington, NY) and Sartopore and Sartohran® available from Sartorius Stedim Biotech S.A. (Aubagne Cedex, France),

|00100] Other exemplary continuous solid supports are monoliths, such as Μ monolithic materials available from. BIA Separations

(Viiiaeh, Austria).

[00101 ] Exemplary discontinuous solid supports include porous chromatography beads. As will be readily recog ised by those skilled in the art, chromatography beads can he manufactured from a great variety of polymeric and inorganic materials, such polysaccharides, acrylates, melhaerylaies, polystyrenks, vinyl ethers, controlled pore glass, ceramics and the like.

[00102! Exemplary commercially available chromatography beads are CPG from EMD Miliipore Corp,; Scptarose® from GE Healthcare Life Sciences AB; TOYOPEARL® from Tosoh Bioscience; and P0ROS® from Life Technologies.

[00103] Other exemplar solid supports are woven and ^' non-woven fibrous materials, such as fiber mats and felts, as well as fibers packed into a suitable housing, tor example chromatography column, disposable plastic housing, and the like. Exemplary Solid supports aiso include winged fibers.

!IL Exg pi arv Binding oujs

[00104] A great variety of binding groups or ligands can be attached to solid supports and used tor effective removal of protein, aggregates from a sample, as described herein. In general, the binding group should be capable of attracting and binding to protein aggregates in a solution. Protein attraction to the binding group can be of any type., including ionic (e.g., eatkmic exchange groups), polar, dispersive, hydrophobic, affinity, metal chelating, or van der Waals.

[00105] Exemplary ionic binding groups include, but are not limited to, sulfate, sulfonate, phosphate, phosphorate, carboxylase; primary, secondary, tertiary amine and quaternary ammonium; heterocyclic- amines, such as pyridine, pyrimkiine, pyridiniom, piperazine, and the like.

[00106] Polar groups include a wide variety of chemical entities comprising polarized chemical bonds* such C-CX OO, C- , 0~N,€™N, N- H _> Q~H, C~I% C-CL C ^« 8r, C-S, S~¾ S-O, SO, C-P, P-Cl P-O, P~H< Exemplary ^' polar groups are earbonyi, carboxyL alcohol, thiol, amide, ha! de, amine, ester, ether, thioester, and the like.

[0 107] Hydrophobic binding gro ups are capab le of hydrophobic interactions. Exemplary hydrophobic groups are alky L eycloafky!, haloalkyl, tiuoroaSkyi, aryL and the like.

[00108] At ^" ii nity binding groups are arrangements of several binding functionalities tha in. concert provide a highly specific interaction wit target protein. Exemplary affinity binding groups include Protein A and Protein G and domains and variants thereof,

[00109] In some embodiments, _, a preferred binding group is an ionic group. In a particular embodiment, a binding group is a negatively charged sulfonate group, in general, negatively charged sulfonate groups have several advantages. For example, they exhibit broad applicability io bind positively charged proteins in solution; the chemistry is inexpensive and straightforward with many synthetic manufacturing methods readily available; the interactio between the binding group and proteins is well understood (See, eg.. Stein ef aL J Ckrom, 8, 8 8 (2007) 151 - 158), and the interaction can be easily manipulated by altering solution conditions, and. such interaction can be isolated from other interactions.

IV» Methods of attaching the MacEing graaps to ¾ solid support ami controlling the density of fainditiE groups on the solid support

[001 10) In the compositions and methods described herein, suitable binding groups are attached to a solid support, where the density of the binding groups on the solid support is controlled, such thai to provide a greater capacity to bind protein aggregates versus the product of interest [001 1 1 J The compositions and methods described herein are based o a surprising and unexpected discovery that, a lower density of binding groups on a solid support is more effective in the removal of protein aggregates in a flow-through mode, even though the prior art appears to suggest the desirability to have a high density of binding groups. The compositions and methods described herein are especially effective in the removal of lower order protein aggregates such as, e.g., dhners, trimers and te raniers, in a flow-through mode, which are generally more difficult to separate from the monomeri.c form of proteins, as compared to higher order aggregates such as, e.g., pentamers and higher. [00 I I 2] A variety of methods known in the art and those described herein can be used for attaching binding groups to a solid support for use in the methods described herein. In general, the criteria of successful attachment include achie vement of the desired binding group density and low rate of detachment of binding groups (i.e., low leaching of binding groups), The binding groups could be attached directly to a solid support, or could be Incorporated into a polymeric molecule, which, in turn, can be attached to a solid support. Alternatively, the binding groups can he incorporated into a cross-linked coating applied onto a solid support, with or without ibrming a chernical bond between the coating and the solid support [001 3 j A -number of methods are known in the art for attaching the binding groups to a solid support (see, for example, Ulbricht, M., Advanced Functional Polymer Membranes, Polymer, 47, 2006, 2217-2262). These methods include, but are not limited to, direct modification of the solid support with bindin groups through suitable coupling chemistry; adsorbing, and attaching polymeric molecules. The latter can be

accomplished by either grafting "to" (-when the polymer is pre-made before reaction with the surface) and grafting " ^' from" (when the polymerization is initiated using the surface groups).

[001 14] As described herein, the ability to control the density of binding groups on the surface of the solid support is critically important in order to achieve successful separation of protein aggregates and the product of interest. The density of binding groups, expressed in roo!e L of porous media, or , can be conveniently measured using methods known t those skilled in the art. For example, the density of sulfonic acid groups can be measured using Lithium ion exchange analysis. In this analysis, the hydrogens of the sulfonic acid groups are fully exchanged for Lithium ions, rinsed with water, the lithium ions are subsequently washed off in a concentrated acid solution, and the Lithium concentration in the acid wash solution is measured using on Chmmaiography. [00 ϊ 15] Is protein chromatography, a higher density of Ngands

(binding groups) has usually been desirable since in general it provides a higher binding capacity (see, e. ., Fogle et al. _f X Chram, A _* 1225 (2012) 62- 69). Typical ligami density for most commercially available cation exchange (CEX) resins is at least 1.00 milliequivaients L, or mM for a monovalent ligami For example, SP Sepharose Fast Flow and CM

Sepharose Fast Flow, both available from GE Healthcare, are listed in the product literature to have the concentration of cation-exchange groups 1 SO- 250 mM and 90-1 0 .raM, respectively.

[001 16] A number of methods exist in the art which may be used for controlling the density of binding groups on a solid support When the binding groups are attached directly onto the solid support, the density can be controlled by the length of reaction, type and concentrati on of catalyst, concentration of reagent, temperature, and pressure. When surface pre- treatment (activation) of the solid support is required, for example by partial oxidation or hydrolysis, the extent of the prctrealment will also control the density of binding groups tha will be attached to such, a preheated surface.

[00 17] When the binding groups are incorporated in a polymeric structure, which is either adsorbed, adhered, grafted, or coated onto a solid support, the density of the binding groups can be controlled by the composition of thai polymeric structure. One approach to create the polymeric structure- with controlled density of the binding groups is copolymer! jetton., i.e. polymerization of two or more different monomer types into a single polymeric structure. A binding group can be a part of one of the monomer types used to create the polymeric structure, while other, neutral, monomers can he added to reduce the density of the binding groups, [00118] The choice of monomers used in creating a poly mer comprising binding groups is dictated by reactivity of the monomers, Reactivities of various monomer types and the effects on the polymer composition are well studied and documented (see, lor example. Polymer Handbook 3** e&, John Wiley St Sons, 1 89, p. 11). Well-accepted parameters of monomers that predict the ex osition of the polymer and its structure are the reacti vity ratios of the monomers (see, for example, Qdian, X. Principles of Polymerization, 4 ^th ed. ₉ John Wiley & Sons, 2004,, p. 466).

[001 1 ] A preferred method to attach the binding groups to the solid support is an in -situ polymerization react? on that incorporates the binding group into a cross-linked coating applied onto the solid support. This method is disclosed U.S. Patent Nos. 4,944,879 and 4,618533, as well as published US Patent Publication No, US2 0 208784. This method is facile as well as economical, A charged coating can he created by eopolyraerking a charged acrylic monomer, for example, 2-acrylaraid.o-2- methyJpropanesuifoBic acid (AMPS), with a suitable cross-linker, such as N,N'~metbyiene~l is-acrylamide (MB AM). U.S. Patent. Publication No. US200920 784, incorporated b reference in its entiret herein, discloses a rnieroporons membrane modified with a mixture of AMPS and BAM that can he used for remo val of high molecular weight protein aggregates and for increasing the capacity of nanopo.ro s virus filter. However, the aforementioned patent publication does not discuss contr lling the ligand density, as described herein, to achieve selective removal of protein aggregates and especially lower order protein aggregates, such as, diroers, trimers, tetramcr s etc,

[00120] A neutral monomer that can be used for reducing the density of charged binding Hgands can be selected from a large group of acrylic, methac.rylie and aerylamide monomers such as, for example, aery!arnide, hydroxy propyl aery late, hydroxyethyl acrylate, and

hydroxyethylmetliacrylate. A. _. preferred rnonomer is dlffiethylacrylamtde (DM AM). The reactivity ratio of AMPS and DMA monomers (rp ^:: 0J62, r ₂ -L 108) (see, e.g.. Polymer Handbook 3 ^χά ed., John Wiley and Sons, 1.989, p _> II/l 56) predicts that a polymer including these monomers would have a tendency for short blocks of poly(DMA ) spaced by individual AMPS irnlts, thereby reducing the density of binding groups. Selecting the ratio of DM AM and AMPS in the reaction solution is therefore an

Important method to achieve controlled density of binding groups,

[00121 ] A representative chemical structure of a binding group containing polymer, which is coated onto a solid support, is depicted in. Figure 2A« In order for the polymer to be coated, it is generally cross- linked to other polymers. In Figure 2A, the polymeric structure is shown In which ^! is any aliphatic or aromatic organic residue containing a cation- exchange group, such as e.g., sulfonic, sulfate, phosphoric, phosphonie or carboxylie group; R" is any aliphatic or aromatic organic residue that does not contain a charged group; and R ^* is any uncharged aliphatic or aromatic organic linker between any two or more polymeric chains.

[001 2] In the polymeric structure depicted in Figure 2 A, y>x, which means thai neutral groups (represented by ⁴ⁱ R ^2* ') are present in a greater number than, the charged groups (represented by ⁱ⁴ R ^u '). Here, the x ₅ y, and z are average molar fractions of each monomer in the polymer, and range independently from abotrt 0,001 to 0,999. The symbol m simply denotes that a simi lar polymer chain is attached at the other end of the cross-linker.

[00123] In some embodiments, the polymer containing binding groups is a block copolymer, meaning that it includes a long string or block of one type of monomer {e.g.. containing either neutral or charged binding groups) followed by a long string or block of a different type of monomer (e.g., charged if the first block was neutral, and neutral if the first block was charged).

[00124] hi other embodiments, the polymer containing binding groups contains the monomers in a random order. [00125] In yet other m odim nts, the polymer containing binding groups Is an alternating copolymer _* where each .monomer is always adjacent to two monomers of a different kind on either side.

( ^' 00 ! 26] In some embodiments, a representative chemical st ucture of a binding group containing polymer is depicted in Figure 2.8, in which E ⁴ is NH or O; R ³ is a linear or branched aliphatic or aromatic group,, such - CH . ~C ₂ H , ¾Η ₆ -, -C{CI-I ₃ )rCH: , -Q,¾ ; and R ⁶ is a linear or branched aliphatic or aromatic uncharged group containing H _S O, or S linker to the polymer chain.

[00127] In other embodiments, a representat ve chemical structure of a binding group containing polymer is depicted, in Figure 2C. R' and * are independently selected from a group containing one or more neutral aHphaiic and aromatic organic residues, and may contain heteroaioms such as O, N, S, P, F, CI, and others.

[00128] hi yet other embodiments, a representative structure of binding group containing polymer is depicted in Figure 2D.

[00129] Another representative chemical structure of a binding group containing polymer, which Is grafted to a solid support, is depicted in. figure 2E. The solid support is depicted as a rectangle. In Figure 2E, the polymeric structure is shown in which R* is any aliphatic or aromatic organic residue containing a cation-exchange group, such as &g., sulfonic, sulfate, phosphoric, phosphonic or carboxylie group; R. ² is any aliphatic or aromatic organic residue that does not contain a charged group, in the polymeric structure depicted in Figure 2A, >x, which means that neutral groups (represented by "R ^2w ) are present in a greater number than the charged groups (represented by

[00130] in some embodiments, the graft polymer containing binding groups is a block copolymer, meaning that it includes a long string or block of one type of monomer {e.g., containing either neutral or charged binding groups) following by a lon string or block of a different type of monomer (e.g., charged if the first block was neutral nd neutral if the first block was charged),

[00131 ] In other em ^' bodimenis, the polymer containing binding groups contains the monomers in a random order,

[00.132] In other embodiments, the polymer containing binding groups is an alternating copolymer, whereas each monomer is alwa s adjacent to two monomers of a different kind.

[00133j In some embodiments, a representative chemical structure of a binding group containing polymer is depicted in Figure 21% in which R* is NH or O; R: ^? is a linear or branched aliphatic or aromatic group _* such - C¾- _t -C ₂ H -, -(¼¾-, -C(C¾) ₂ -CHr _f -C ₆ H ; and R ^s is a linear or branched aliphatic or aromatic uncharged group containin NH, (X or S linker to the polymer chain,

[00134] In other embodiments, a representative chemical structure of a binding grou containing polymer is depicted In Figure 20. R' and R ^s are independently selected from a grou containing one or more neutral aliphatic and aromatic organic residues, and may contain, heteroatoms such as O, N, S, 1\ F, CI, and others.

[00135] in yet other embodiments, a representative structure of a binding group containing polymer is depicted in Figure 211.

[00136] The sulfonic asid group in Figures 2B-2D and.2F-2H can be in the protonated form as depicted, as well as in the salt form, containing a suitable eounterion such as sodium, potassium _. , ammonium, and the like, IV. Devices i«eorporat?¾¾ the eeraposit m described herein

{00137] In some embodiments, solid supports having binding groups attached thereto, as described herein, are incorporated into devices. Suitable devices for solid supports, _, such as microporous membranes, include filtration cartridges, capsules, and pods. Exemplary devices also include siacked-plaie filtration cartridges disclosed in the U.S. Publication os. 1)820100288690 Al and JS20O8 257814 Ah incorporated by

33 reference herein. In ease of these devices, a solid support is permanently bonded to the polymeric housing and the devices have a liquid inlet, an outlet and a vent opening, and further minimize the volume of retained liquid. Other exemplary devices include pleated filter cartridges and spiral- wound filter cartridges. Yet other exemplary devices are chromatography columns. Chromatography columns can be produced from a number of suitable materials, such as -glass, metal ceramic, and plastic. These columns can he packed with solid support by the end user, or can also be pre-packed by a manufacturer and shipped to the end user in a packed slate,

V, Methods ¾f f sing the cgm osi lons and deyjees desc ribed herein

[00138] The devices containing solid supports having binding groups attached thereto (e.g., cation exchange binding groups) can he used for removal, of protein aggregates in a flow-through mode. Prior to application tor preparative scale separation, the process must he developed and validated for proper solution conditions such as pH and conductivity. -a»d the range of protein loading on the device must be determined.. The methods for process development and validation are widely known nd routinely practiced in the industry. They usually involve Design of Experiments (DoE) approaches that are illustrated in the Examples herein, [00139] The devices are commonly flushed, sanitized,, and equilibrated with an appropriate buffer solution prior to use. Protein solution is adjusted to a desirable conductivity and pH and is subsequently pumped through a device at. either constant pressure or constant flow. The effluent Is collected and analyzed for the protein yield and aggregate concentration.

[001-40] In some embodiments, a device for aggregate removal, as described herein, is connected directly to a virus filtration device that is designed to ensure size-based removal of viral -particles, for example, as taught in US, Patent No. 7 18,675, incorporated by reference herein, in its entirety.

[001 ] The flow-through aggregate removal step using the compositions and devices described herein can be placed anywhere in a protein purification process, e.g., in an antibody purification process. Table 1 depicts examples of protein purification processes that incorporate flow- through aggregate removal as one or more intermediate steps, which is highlighted in bold. It is understood that many variations of these processes may be used.

[00142] "Protein capture" step, as described herein, refers to the step in a protein purificaiion process which involves isolating the protein of interest from the clarified or unciarified cell culture fluid sample by performing at least the following two steps: i) subjecting the cell culture fluid to a step selected from one or more of: adsorption of the protein of interest on a chromatography resin, a membrane, a monolith, a woven or non-woven media; precipitation, f!oceulation, crystallization, binding to a soluble small molecule or a polymeric iigand, thereby to obtain a protein phase comprising the protein of interest such as, e.g., an antibody: and (ii) reconstituti ng the protein of interest by eluting or dissolution of the protein into a suitable buffer solution,

[00143] Bind elute purification is an optional process step consisting of binding the protein of interest to a suitable chromatography media, optionally washing the bound protein, and eluting it with appropriate buffer solution.

[00144] Flow- through AEX polishing is an optional process step consisting of flowing the solution of protein of interest through a suitable AEX. chromatography media without significantly binding of the protein of interest to the media.

[00145] Activated Carbon I¾w-t¾fo«gh is an optional purification step designed to remove various proces --related impurities, as described in co-pending provisional patent application no. 61/575*349, incorporated by reference herein.

[00146] Virus filtration consists of flo wing the protein solution through a porous membrane, which can. be in the form of flat sheet or hollow fiber that retains he vital particles to high degree of LRV, while passing substantiall all protein of interest

Table 1,

Step I Step 2 Step 3 Step 4 Ste 5

Proem Antibody Bimi/etute Flow- Flow- V has

capture Purification through ihrougb Filtration.

A

AEx" aggregate

polishing removal

Process Antibody Flow- Bisd/elute Flow- Virus

R capture thnmgh Purification through. Filtration aggregate AEX ^"

removal polishing

Process Antibody Bimt/efttte Ffew- Flow- Virus capture Purification throagh through EiltratuM

C

a re ate AE

removal polishing

Process Flow- Antibody Bind/ehae Flow- Virus through capture Purification through Filtration

D

aggregate AE ^"

removal polishing

Process Antibody Fiow- Flow- Virus

capture fhrough through Filtration

E

aggregate EX ^*

removal polishing

Process Antibody Plow- Flow- Bsnd/ehite Virus capture ils r ugh through Purification. Filtration F

aggregate AEX

removal polishing

Process Antibody Flow- Flow- Bimi ehste V sros capture through through Purification Filtration

G

AE aggregate

polishing removal

Process Antibody Flow- Flow- Vims

capture throagh through Filtration

H AEX ^' aggregate

■polishing removal

Process I Aotilxxiv Activated Flow- Flow- Virus capture Carbon through through Filtration

Flow- EX ^" aggregate

through polishing removal [00147] It is understood that in the ^' liable I abo e;, the step of

Antibody Capture, as well as Blud/Elute Purification, can. be operated in any of three modes: (I) batch mode, where the capture media is loaded wi h target protein, loading is stopped, media Is washed and elated, and the pool is collected; (2) senn^ontinuons mode, wherein, the loading is performed continuously and the eiution is intermittent, e.g. , in case of a continuous malncolumn chromatography procedure employing two, three, or more columns; and (3) full continuous mode, where both loading and eiution are performed continuously.

[00148] The optimal flow rate used with the f low-through cation exchange solid support described herein can. sometimes have an effect on the aggregate remo val performance of the solid support and, when the solid support is positioned upstream of a virus filter, it car also affect, the performance of the virus filter. The optima! flow rate can he readily determined in a simple set of experiments using the protein solution of interest. Typical flow rates fall in the range between about 0,05 and 10 CV/rnin.

[00149] Some exemplary processes described In Table 1 , in particular Process E _* Process IT and Process I, do not include a hind and elute cation-exchange chromatography step, while still ensuring aggregate removal using the methods described herein. Elimination of the bind and elate cation-exchange chromatography step offers a .number of significant advantages for the downstream purification process, .e. savings of process time, simplification of process development, elimination of cleaning and cleaning validation, etc. Another strong advantage is the elimination of highrconductivity ehstion, allowing for the entire downstream process to be performed without addition of salt and then without: subsequent dilutions .

[001 0] This invention is furthe illustrated, by the following examples which should not he construed as limiting. The contents of all references, patents and published patent applications died throughout this application, as well as the Figures, are Incorporated herein by reference.

Exam les membrane

100151 J In this experiment, a series of CEX surface-modified membranes were prepared with a variable density of binding groups, which in this case are negatively charged sulfonic acid residues. The density of the cation exchange groups was controlled by formulation of reacti ve solution used for surface modification. In order to achieve a lower density, an uncharged .reactive monomer, N,N-diraeihylacryiamide, was added in d i £ feren t amoun ts ,

[00152] A series of aqueous solutions were prepared containing 2- acrylanui ^} o-2-meth propa«esulfoaic acid (AMPS) ranging from 0 to 4.8%wt. N,N-di.methyl8cryiamid.e ranging from to 4 J%wt, and 0.8% wt. of _f N'-ffiethyleoebisaerykndde. A hydrophilic ultra-high molecular weight polyethylene membrane with pore size rating of 0.65 um and a thickness of 0.125 mm was cut into square pieces of 14 an by 14 em and each piece was submerged in one of solutions for 30 seconds to ensure complete wetting. The excess solution was nipped off,, and the membrane was exposed to 2 MRads of electron ^' beam radiation under inert atmosphere. The membrane was subsequently rinsed with deiomzed water and dried in air.

[Ο 33! Table 2 lists the variants of CEX surface modified membranes that were prepared, it was determined using Lithium ion.

exchange that the anionic group density was 0.13 nrmol/g for Membrane 8 (no DMAM added) and 0.08 mmol/g for Membrane 7 (1 :1 AMPS-.D AM ^' by weight), which correspond to iiganddensiii.es of 34 and 2 ! mM, respectively. Exam le 2. Aiiaivsis f aggregate binding selectivity using sfa k

capacity m asttremeats

[0 154] In this ex eriment. CEX membranes with varied ensity of binding groups prepared in Example I , were tested for their selectivity for binding protein monomers and protein aggregates.

[00155] A 2 g L solution of a partiall purified monoclonal antibody, referred to as M b T containing about J 5% aggregates was prepared in 50 mM Sodium Acetate buffer, pH 5.0, A 14 mm membrane disk was pre-soaked in the acetate buffer and then transferred to 0,5 ml, of antibody solution. The solution, vials were gently shaken for 15 hours, and the molecular weight species left, in solution were analyzed by Sisse- Exclusion Chromatography. The .results are presented in Table 2,

[001.56} While he generally low yields of MAb indicates that the membranes have been loaded at below capacity in this experiment, one of the variants, Membrane 7, demonstrated practically complete removal of dimers and high molecular weight (HMW) aggregates. This indicates that strong aggregate binding selectivity can ^' be- achieved.

n/d - not detected Example 3. Removal of aggrega es in flow-tbrougfa m de for a partially urified iBoaoel aal _. a iib dy (M Abl¾

[00157] la a representative experiment, successful use of the membranes according to the present invention for the removal of protein aggregates from a sample containing a monoclonal antibody in flow-through mode, was demonstrated.

[00158] Five layers of membrane 7 from Example i were sealed into a vented polypropylene device, with a frontal membrane .filtration area of 3 J cm ^" and membrane volume of 0,2 ml-. This type of device i referred to below as the " ICK " device. A purified MAb I at 4.5g/L wa dialyzed into pH 5.0, 50 mM acetate buffer, The resulting material, referred to as partially purified MAb L was diluted to a concentration of I g/l Mab I with 5 % aggregates, in pH 5, 30 mM acetate with a conductivity of -3,0 mS/ern. The material (about 80-100 mL> was then passed through 0.2 ml, devices containing either membrane 7 or 8 from Example T or a 0.18 ml. Aerodi.sk device containing Pal! Mustang® S membrane (commercially available from Thermo Fisher Scientific, Inc., Waltham, MA) at - CV/ ln. Prior to passing the MAb, the membrane devices were wetted with. 18 mil water and equilibrated with 50 column volumes (CV) of 50 mM sodium acetate, pH 5,0, The flow-through of the MAb solution was collected for analysis using analytical size exclusion chromatograph (SEC), The results are shown in Figures 3A-3C. it is clear that Membrane 7 with a lower density of AMPS binding groups compared to Membrane 8, as established in Example 1. offers a superior selectivity lor binding aggregates.

[00159] Table 3 summarizes aggregate binding capacity of the three membranes measured at about 20% aggregate breakthrough. As demonstrated, Membrane 7 exhibits almost a 3-fold increase in aggregate binding capacity as compared to commercially available membranes, e.g., the Pall Mustang® S membrane, as well as Membrane 8.

00160] n another experiment, successful use of membranes according to the present invention in the removal of protein aggregates from a sample containing a different monoclonal antibody, is demonstrated.

[00161] Protein A purified MAb II. was adjusted to pH 5.0 using 1

M T«s base, and to a conductivity of 3,4 mS em using 5 M NaCI solution. The resulting, material referred to partially purified MAb II had a

concentration of 6 g/L with 2.4 % aggregates. The material (about 1 § mL) was then passed through 0,2 mL devices containing either membrane 7 or 8 from. Example ! . Prior to passing the MAb through the membrane, the membrane was wetted with i 8 Q water and equilibrated, with SO column volumes (CV) of 50 mM sodium acetate, pH 5.0. The flow-through of the MAb solution was collected for analysis using analytical ske exclusion chromatography (SEC). The firs 2 fractions were -4.5 mL eac , while the rest, of the 10 fractions were 0.85 mL each. After the MAb treatment, the membrane was washed with 20 CVs of 50 mM sodium acetate, pH 5.0. The bound protein was elated using 50 mM sodium acetate, pH 5,0 - 1 M NaCI in 30 CVs. The total amount of MAb Π loaded on the membranes was 531 mg/hil. The MAb monomer yield was calculated based on amount of monomer in the flow-through versus total monomer passed through the. membrane,

[00162] As shown In Figure 4A, very little aggregate (<Q.1% in. the pool) was seen in the breakthrough pool of Membrane 7 up to -13 g/L aggregate-loading while aggregate breakthrough was observed for

Membrane $ even in the first fiaciion collected indicating superior aggregate capacity for Membrane 7, Th amount of aggregates in the breakthro ugh pool tor Membrane 8 was 0.8 %. At an aggregate-loading of 13 g/L, the .monomer yields were 93 and 86 fo Membrane § and Membrane 7, ^• respectively. Although the yield was slightly lower, no aggregate

breakthrough was observed for Mem brane 7. A later breakthrough of the aggregates is an indication of higher aggregate binding capacity. These breakthrough curves can be used to guide the design of a preparative scale aggregate removal process or device. For example, a device containing Membrane 7 can be loaded to at least 500 g/L to achieve pool purity of <·0,!% aggregates. Generally, higher loading is ver desirable in order to increase the monomer yield and reduce the overall cost associated with the process. In addition, higher yield can be achieved by increasing the loading until aggregate breakthrough is observed or modifying the solution conditions. The flow-through data clearly demonstrates the superior performance of Membrane 7 for the removal of aggregates.

[00163j Figure 4B shows monomer yield and aggregate levels in. the breakthrough pools for two different runs using two different lots of Membrane 7. Run 2 was loaded to 700 mg mL compared, to run 1 at 530 mg/ml. The solution conditions (pH and conductivity) were similar in both, the runs while the MAb concentrations were different (run 1 : 6 g/L; run 2; 4.4 g/L). The amount of aggregates in the pool was similar for both the runs, while there were some differences in the yield (however, it is anticipated that the differences in. yields might be attributed to general variability of analytical techniques used for measuring MAb concentrations).

nterestingly, even at. a MAb loading o 700 g mL the amount of aggregates in the pool is only 0.25% (run2; Figure 4B) which indicates a binding capacity for aggregates to be > 15 g/L. Exam le S. Removal of aggregates in flow-throuEh mode For a partially f urifktt monoclonal . antibody (MAb 01) c ai¾l¾¾ Μ pii-kd¾eed ggregate

[00164] In another experiment, successful use of membranes according to the present invention in the removal of protein aggregates from a. sample containing a yet another .monoclonal antibody, ΜΑ Π, Is demonstrated.

[00165] A solution of partially purified monoclonal. IgG feed (MAb

III) was prepared to 6 g/L in pH 5, 50 mM acetate buffer with a conductivity of 3.5 mS/em. The MAb III had been previously shocked to high pH (1 1) in order to generate 3.7% total aggregates (measured by SEC~HPLC). A Micro .filtration device with a filtration area of 3.1 cm2 and a volume of 0.2 mi- was pre-mokfed using 5 layers of Membrane 7. Prior to passing the MAb, the membrane was wetted with 18 niO water and equilibrated with 50 column volumes (CV) of 50 mM sodium acetate, pH 5.0. The .flow-through of the MAb solution was at 2.5 CV min and tractions were collected for analysis using analytical size exclusion chromatography (SEC). The total amount of MAb III loaded onto the membranes was 930 mg/mi. The monomer yield was calculated based on amount of monomer in the flow- through versus total monomer passed through the membrane.

[00166] Similar to Examples 3 and 4 for MAb I and II, Membran

7 showed a high selectivity and high binding capacity (>10 mg/mL) for MAb III aggregates, as shown in Figure 5.

Example 6; Purification of p o ei monom rs and prutein aggregat s [001 7] l.n this experi ment, preparations of pure antibody monomers and aggregates were generated using preparative SEC tor further characterization of Membrane 7.

[0O16S] Monomers and aggregates were purified from a mixture using the Sephacryl S-300 HR (GE Healthcare) resin. 4.5 raL of the partially purified MAb at. 4.5 - 6 g/L was passed through a 330 mL column. Prior to MAb injection, the column was equilibrated with 1 column volume (CV) of PBS. Two elation fractions were collected: aggregate and monomer fractions. The aggregate fraction was concentrated 1 X using 3000 MW cut-off Amicon U P membrane and analyzed using analytical SEC. The aggregate conten was >70% in these fractions with a concentration of -0,5

Exam le 7;

^" he following experiment was designed to elucidate the mechanism of aggregate binding selectivity observed for the membranes according to the present .invention. Using the. Stersc Mass Action (SMA) .model (see, e.g., Brooks, C, A. and Cramer, S. M,< Sterie mass-faction ion exchange: Displacement profiles and induced salt gradients, AIChE Journal _^ 38: pp. 1969-1 78, 1 92), the number of aggregate interactions with the surface (v) and the affinity constant ( S A) tor the monomer and aggregates were measured, to reflect the affinity of the monomers and the aggregates for the surface,

[00170] The SMA model has been used successfully to explain some of the complexities of nonlinear ion-exc hange interactions. The model lakes into account the sites thai are not available to protein molecules by considering both he sites thai are "lost" (e.g., or not available for binding) due to direct protem-!igand interaction and sites that are slericai!y "lost" due to protein hindrance. The quantity o sites "lost" due to steric hindrance is assumed to he proportional to the bound protein concentration. The SMA equation for a sin le component system is given by:

( i ) where Q and C are the bound and free protein concentrations, respectively, K ts the SMA eqiuHbr m association constant; v is the characteristic charge; o is the steric .factor; C _¾i». is the free salt concentration; is the bound salt concentration available for ion "exchange; and Λ is the total, ionic capacity of the resin. Characteristic charge is the average number of sites on the modified solid support occupied by a protein through ionic interactions, and steric factor is the number of sites siericaM hindered by a protein upon binding to the adsorbent ,

[0 ! 71 ] For linear region of the adsorption isotherm (when Q is very small), Λ » ( H- v and we can approximate the above equation to:

(2)

[00172 j The term Q/C is defined as the partition coefficient (K. _p ), and ratio of partition coefficients is defined as selectivity (S). An alternative method of determining the partition coefficient is by calculating the slope of the adsorption isotherm in the linear region {e.g., as described in U.S. Patent No. 8,067,182 }.

[00173] in one experiment, 0,2 nit membrane Micro devices

(containing either Membrane 7 or 8) were wetted using water, and equilibrated with 20 CV (4 niL) of 50 mM sodium acetate, pH. 5.0 (buffer A). 100 μ) of purified monomer or aggregate (as per the purification process described in Example) at 0.5 g L was infected. The bound, protein was emted using a 20 CV gradient of buffer A and buffer A + 500 mM NaCl The salt concentration for the isocratic rims was chosen based on. the salt concentration (conductivity) range in which the protein eiutes. The isocratic .run was performed by loading 100 μ|. of protein onto the membrane. The running buffer was buffer A salt (different salt concentrations based on protein elution range were used as described above). The elution ehromatograra was monitored using U V280 or UV230 signal, and the retention, volume a the protein peak was noted. The SMA parameters were determined using the isoeratfc elution method as described in Pedersen, et a!, (Pedersen, et. Whey proteins as a model system for chromatographic separation, of prote ns, Chrommogr. B, 790: pp.i6.K173, 2003).

[00174] Table 4 summarises the SMA parameters for MAb ί .for

Membranes 8 and 7. Table 4 siramar zes the SMA parameters for Ab II for Membranes 8 and 1, & A was determined assuming an ionic capacity of 34 and 21 mM. for Membrane § and 7, respectively. Membrane porosity of 78% was used..

Table 5. SMA ram t rs tor MAb 1.1

[001.75] For both MAbs, t was observed thai the difference in the characteristic charge of monomer and aggregates is- higher for Membrane 7 indicating a greater number of interactions for aggregates than monomers. in addition, the affinity (as seen from KSMA) is higher for Membrane 7 than. Membrane 8.

[00176] As depicted in Figures 6A and 6B, it was observed that the partition coefficient i measure of affinity} for aggregates is higher than that observed for monomers in ease of both Membranes 8 and 7, thereby indicating stronger binding to aggregates than monomers. It was also observed thai the effect is more pronounced in case of Membrane 7 relative to Membrane S, demonstrating a superior performance of the former for aggregate remo val.

[0 77} As depicted in Figure 7, Membrane 7 has a higher selectivity (S ~ ra tio of p of aggregate to Rp of monomer} than Membrane 8 for two different MAbs (MAb I and AB II) at pB 5,0 at ail salt concentrations. Notably, the selectivity increases as the sail concentration decreases for both membranes. And, interestingly the increase in selectivity is much higher f or Membrane 7 than Membrane 8, i dicating that higher selectivity could be realized even, at lower density of the binding groups, Also, as depicted in Figure 7. the maximum performance benefits (higher monomer yield and higher aggregate removal) were realised even at lower salt concentrations (or higher selectivity).

Exampl Hi I er ination at operating windo for em rane 7

[00178] in a representative experiment, it was demonstrated using a Design of Experiments approach (DoB), that a practical process window , i.e. combination of solution H, ion conductivity, and protein loading on the membranes according to the present invention, can be achieved, A Design of Experiments (DoE) approach is a wide ly accepted engineering tool for identifying reliable operating conditions (se , for example, Anderson, MX and Whitcomb. FJ, 201 Design of Experiments, Kirk-Othmer

Encyclopedia of Chemical Technology. 1-22 ),

[00179] A central composite surface response, using the DoE approach with 3 factors was performed in batch mode for Membrane 7 in orde to determine its operating window. The parameters that were investigated -were: pH, conductivity and. aggregate loading. Various amounts of partially purified MAb 1 (0.5 - 2.1 niL) at various conditions (Table 5 ) were incuba ted with 13 μ Ι of membrane for 20 hrs. The supernatant was then analyzed for aggregates and yield using analytical SEC.

I able 6. Runs and results of a 3 factor, central composite surface respons

[00180] This DoE study highlighted three key operating parameters

···· loading, pH> and conductivity in determining membrane performance for yield and aggregate removal and suggested a simple procedure to define the desired operating window. The results are plotted in Figures S and 9.

Example 9,

agg egates

[00181] This example demonstrates that the membranes comprising one or more cation exchange groups described herein can. be successfully used to Increase the throughput of a downstream virus filter in a purification process.

[00182] in general, it has been previously reported that a surface- modified membrane to prc-treat the antibody feed can be used before virus

.so filtration (see, for example, U.S. Patent No. 7,1.18,675 and PCX publication no. WO 2010098867, incorporated by reference herein). Because of the high cost of virus filtration, _, increasing the filter throughput has a direct effect on the final cost of the protein product, A number of commercial products are currently marketed specifically to increase the throughput of virus filters, including those available from EMD Mtliipore Corporation, eg., Yireso ve® Preftlter and Viresolve® Pro Shield. However, as demonstrated herein, the membranes according to the present invention are tar superior in protecting downstream virus filters, as compared to the those described in the prior art or presently commercially available.

[00183] A Heat-Shocked polyclonal IgG .feed for testing the protection of a virus filter was prepared at 0.1 g/L, using the IgG from

SeraCare Life Sciences ( ilford MA), Human Gamma Globulin 5% Solution, Catalog # HS-475 L, in either pH 5, 50 mU acetate at S Λ or 1 .0 mS/cm (using NaCi) by the following procedure . One liter of the 0.1 g/L solution (at 8.1 or 16.0 mS/em) was stirred at 170 rprn while heated in a constant water bath set to 65° Celsius for 1 hour after reaching temperature. The solution was removed from the heat and stirring and allowed to cool to room temperature for 3 hours and then refrigerated at 4°C overnight. The next day the Heat- Shocked polyclonal IgG was allowed to warm to room temperature. Fresh solutions of 0.1 g/L polyclonal IgG were prepared in the appropriate sterile filtered pH and conductivity buffers (8.1 or 16.0 mS/cm).

[0 .184] The final solutions for throughput testing were prepared using 9% by volume of the heat-shocked polyclonal IgG stock solutions and. the freshly prepared 0.1 g/L polyclonal IgG solutions. Final pH and conductivity adjustments were made using 1.0 S l HO, aOIl or 4 M NaCL Micro filtration devices with a filtration area of 3.1 en* ⁴ were pre-mokied using 3 layers of . membrane and put in series at a 1.Ί area ratio with

Viresolveii Pro devices (EMD Millipore Corp., Bilieriea, MA). Both devices were prewetted and vented to remove air using the buffer only, pH 5, 50 mM acetate, 8.1 of 16.0 mS/cm. Under constant pressure of 30 psi, an initial flux in mh of buffer/min throughput was measured by mass over a 15 minute period to determine an initial constant flux value. The feed was switched to the heat-shocked polyclonal IgG feed at 30 psi and the volume throughput was measured and plotted versus time until the flux decayed to 25% the initial buffer only flux. The total throughput of heat-shocked polyclonal IgG was measured in Urn ² at V75 and converted to kg of polyclonal lgO/m ^a membrane. As can be seen from Table 6, the Membrane 7 provided superior protection to a vims removal filter (greater volumetric throughput) than Membrane 8,

Table 7

aitt o y ee stream

[001 S3] A solution of a partiall purified monoclonal IgG feed

(MAb 111) was prepared to 6 g/L in pB 5, 50 mM acetate buffer with a conductivity of 8.5 mS/cm (using added NaCl). The MAb III had been previously shocked at a high pH (11) to generate about 4% total aggregates (as measured by SEC-HPLC), Micro filtration devices with a filtration area of 3 J en were pre-moided using 3 layers of membrane and put in series at a 1 :1 area ratio with Viresolve Pro devices (HMD Mi.11i.pore Corporation,

Billeriea, MA). Both devices were prewet and vented to remove air using only the sterile filtered buffer, pH 5, 50 mM acetate, 8.5 mS/cm. The virus membrane was also preconditioned for 10 minutes at a constant flow rate that generated ^' constant back pressure of 30 psi. The MAb ill solution was then fed at a constant flow of 200 L (m ² «h) through, the devices in series and the back pressure vs. time was measured. The total volume throughput was determined at an e.ndpoint where the measured back pressure reached 30 psi The L/ro ² throughput. St the 30 si cut-off was converted to kg of MAb per m ² of membrane.

[00186] As can be seen irons Table S below, the Membrane 7 provided superior protection to a virus removal .filter (greater volumetric throughput) than Membrane

Table 8

time on performance of a virus filtration is investigated, where the virus filter is positioned downstream of Membrane 7 in a .fiow-thraugh purification process, it is observed that a lower flow rate through a device containing Membrane 7 and the vims filtration step results in a higher throughput of the vims filter.

[001 SSj A three-layer device containing Membrane 7, having membrane area 3.1 cm2 and membrane volume 0,12 mL is connected in series to a virus titration device, having a membrane area of 3, 1 em2. About. 3 .mg/niL of a polyclonal human igG (Seracare) in 20 mM sodium acetate, pH 5.0 buffer, is processed through the two connected devices. The experiment is performed at two separate flow-rates, 100 and 200 LM!T A 0.22 μη sterile filter is placed between cation exchange chromatography device and the virus filtration device,

[00189] A pressure sensor is used tor measuring the pressure across the assembly at the different, ^' flow -rates. Normally, a pressure of bout 50 psi is an indication of folding or plugging of the virus filtration membrane. As shown in Figure 10, when the experimeni is performed at a lower flow-rate (i.e., 1 0 LMH), more sample volume can be processed through the virus filtration membrane (Le,, higher throughput} relative to when the sample is processed at a higher flow-rate (le„ 200 LMH). This could be attributed to longer residence time of the sample in the cation exc an e chromatography device, which may result in an improvement hi binding of high molecular weight igG aggregates, thereby preventing early plugging of the virus, filter.

[00 i 90] In this representative experiment, the feasibility of connecting several impurity removal steps into one simple operation, whil meeting parity and yield targets, is demonstrated. This is done b connecting individual devices, na el activated carbo , an anion exchange

chromatography device (e.g., ChromaSorb™}, an in-line static mixer and/or a surge tank for pM change, a cation-exchange iknv-through device for aggregate removal as described herein, and a virus removal device (e.g., Vhesotve® Pro).

[00 i 91 J The set-up, equilibration and procedure are described below,

[001 2] The flow-through purification train consists of fi ve main unit operations; activated carbon (with, optional depth filter in front),, an anion exchange chromatography device (e.g.. ChromaSorb), an in-line static mixer and/or surge tank for in-line pH adjustment, a cation-exchange flow-through device for aggregate removal, and a vims filtration devic (e.g., Viresolve® Pro).

[001 3] Figure .1 1 illustrates the order i which these unit operations are connected.

[001 4] The necessary pumps, pressure, conductivity, and UV sensors are may additionally be included.

[00595 J All devices are individually wetted at a different station, and then assembled. The devices are wetted and pre-ireated according to the manufacturers protocol. Briefly, the depth filter (AIHC grade) is flashed with 100 IJml of water followed by 5 volumes of equilibration buffer 1 (EB1; Protein A elation buffer adjusted to pH 7.5 with 1 M Trls-base, pH 1 1). 2.5 raL of activated carbon n packed into a 2.5 cm Omni fit column as described in co-pending U.S. Provisional Patent Application No. 61/575,349, filing date August 19, 2011 , incorporated by reference he ein, to produce MAb loading of 0.55 kg/L. The column is flushed with 10 CV water, and then equilibrated with EB1 until the p!I is stabilised to pH 7.5. T¾o CbromaSorh devices (0.2 and 0,12 niL) are connected in series to get antibody loading of 4.3 kg/L. The devices are wetted with water at 12.5 CV/min for at least 10 min, followed by 5 DV BBL A disposable helical static mixer (Kof!o Corporation, Cary, JL) with 1.2 elements is used to perform in-line pH adjustments. Two 1.2 niL devices containing Membrane 7 are connected in parallel to remove aggregates, so they can be loaded to about 570 rag/raL of antibody.

[001 6] They are wetted with 10 DV water, followed by 5 DV equilibration buffer 2 (EB2; EB1 Adjusted to pH 5.0 using 1 M acetic acid). The devke$ are farther treated with 5 DV (device volumes) of EB2 + 1 aCL and then equilibrated with 5 DV EB2. A 3.1 cm2 VireSofve# Pro device is wetted with, water pressurized at 30 psl for at least 10 mi.n. The flow rate is then monitored every minute until the flow rate remains constant for 3 consecutive minutes. After all the devices are wetted and equilibrated, they are connected as show in Figure above, EB1 is run through the entire system, until, all pressure readings, and pB readings are stabilised. Following equilibration, the feed (Protein A ei.ut.ion adjusted to pH 7.5) is passed through the flow-through train. During the run, samples are collected before the surge tank and after Viresolve® Pro to monitor IgG concentration and impurity levels (HCP, DMA. leached PrA. and aggregates). After the feed is processed, the system is flushed with 3 dead volumes o EBl to recover protein in. the devices and in the plumbing.

[001 7 J The feed tor the connected, -flow-througfi process Is protein

A eluate of MAb !V, produced in a batch protein A process. The natural level of aggregates in this MAb does not exceed 1 , so a special procedure was de veloped to ncreas the level of aggregates. Solution p!-i was raised to 1 with aqueous NaOH, with gentle stirring, and held for I hour. The pH was then lowered slowly to pH 5 with aqueous HC1 under gentle stirring. The pH cycle was repeated 4 more times. The final level of aggregates is about 5%, mostly consisting of MAb IV d mers and trimers as measured by SEC. The feed is then dialyzed into Tris~HCI buffer, pH 7.5, conductivity about 3

S/crn _*

[001 8} The MAb feed processed for this run is 102 n t, of 13,5 mg/niL M Ab IV at a Sow rate of 0.6 m t/min,

[00199] The HCP breakthrough as a function device loading after

ChroroaSorh is below the upper limit o 10 ppm (Figure 12), The aggregates are reduced from 5% to Ll% by the CEX device ί Figure 13), The MAb IV yield of the connected process is 92%. The throughput on Viresolve# Fro device was >3.7 fcg m2.

[00200] Examples 13-19 demonstrate the feasibility of manufacturing compositions for removing aggregates in a flow-through mode using a cation-exchange resin or winged fibers as solid supports instead of a membrane.

Example 1.3. Preparation of a potymerk ^ (CEX) i-Sllli modified with an AMPS 1)MA sm*f ed copolymer

[00201 J In this representative experiment, a series of cation- exchange (CEX) resins with a grafted AMPS/DMAM copolymer surface (strong CEX) were prepared with a variable density of binding groups, which are negatively charged sulfonic acid residues. The Hgand density and composition, of the strong cation exchange groups was controlled by the composition of reactive solution used for surface modification. In. order to vary ^' the density of the strong cation exchange groups, the charged and uncharged reactive monomers, AMPS and D ^' MAM, were added in various molar ratios. [00202] A .1000 ml. three-necked flask with mechanical stirrer and dropping funnel is marked at a defined volume of 830 mL. In this flask, 8.25g sodium hydroxide is dissolved in 429.34g deionized water. The solution is cooled, to 0°C and 1 .68g AMPS is added slowly in several portions while stirring. Thereafter, 26.22g DM AM Is added. The pH value of the solution is adjusted to 6,0-7,0 by the addition of 65% nitric acid and/or I M sodium, hydroxide. 400 mL sedimented polymeric base head resin with a mean particle size of 50 micron, is added to the solution while stirring gently (120 rpm}> The total volume of the reaction mixture is adjusted to 830 mL

(according to mark) by the addition of deionized water. The p!I value of the mixture is again adjusted to a pH of 6.0 to 7.0 by the addition of 65% nitric acid. The mixture is stirred genily (1.20 rpm} and heated to 40°C. A solution of 6/?5g ammonium cerinm(lV) nitrate and 2,96g 65% nitric acid in I5g deionized water Is added quickly under vigorous stirring (220 rpm). The reaction mixture is then stirred at 120 rpm at 40 ⁸ C for 3 hours,

100203] Thereafter, the reaction mixture is poured onto a glass frit

(porosity P3) and the supernatant is removed by suction. The remaining resin is washed successivel with the following solutions: 3 x 400mL deionized water; 10 x 400raJL 1M sulfuric acid + 0.2 M ascorbic acid; 3 x lOOmL deionized water; H) x 400mL hot deionized water (60°C); 2 x 400mL deionized water; 2 x 400mL 1 M sodium hydroxide; 2 x lOOmL deionked water; during second washing step with deionized water, adjust pH to 6,5-7.0 with 25% hydrochloric acid; 2 x 40QmL 70% ethanol/ 30% deionized water; 2 x. lOOmL de.ioniz.ed water; and 2 x lOOmL 20% ethanol/ 80% deionized water + ISOrn NaCl

[00204] After the above washing procedure is completed, the resin is stored as a I ! (v/v) suspension in a solution of 20% ethanol, 80% deionized water and 150mM NaCl. [00205] Table 9 lists the synthesized sulfonic acid containing sirpBg CEX resins with varying molar ratios of AMPS and DMAM prepared according to this Example.

12LPDZJ 29 were packed in an Oranifit* Chromatography Column with an internal diameter of 6.6 mm to a bed height of 3 cm resulting in about 1 mL packed resin bed. An AKTA Explorer 1 0 (chromatography system) was equipped and equilibrated with bailors appropriate to screen these columns for flow-through chromatography (Table 10), The chromatography columns containing the resin samples I 2LPDZ1 19, 12LPDZ128, and I 2LPDZ129 were loaded onto the chromatography system with equilibration buffet. The feedstock was an IgGl (MAbB) that was purified using ProSep# Ultra Pius Affinity Chromatography Media, and was adjusted to pH 5.0 with 2 M Tris Base. The final MAbB concen ration of th protein A pool was 13 ,8 mg,½L,eontained 2.05% aggregated product, and the conductivity was about 3.5 mS/c-m. The resins were loaded, at a residence time of 3 minutes and to a load density of 414 mg.½L. Table 10: Method for performing chromatography experiments for resin

assayed for total protein concentration on a NanoDrop 2000

spectrophotometer and the aggregate content wa quantified by size exclusion high perfemiance liquid chromatography (SE-HPLC). The aggregate quantification test was performed Tosoh Bioscience TS Gel G30 0SWXL, 7.8 mm x 30 cm, 5 um (Catalog # 08541) column with equilibration buffer of 0.2 M Sodium phosphate pH 7,2, The results show that the mAhB monomelic protein is collected in the flow-through fraction a high concentrations at relatively much lower cumulative protein loadings than the aggregated, product,

[00208] For Lot # I2LPDZ119, 368.1 mg or approximately 88.9 % of protein was recovered at cumulative - protein loading of 414 mg/mL, aggregate level was .reduced from 2.05 % to 0,39 % In the How-through fractions, and the Stri pool contained 21,2 % aggregates suggesting the resins- ability to selectively retain aggregates.

[00209] For Lot # I2LPDZ128, 357.9 mg or approximately 86.4 % of protein was recovered at cumulative protein loading of 414 mg mL, aggregate level was reduced from 2,05 % to Λ 4 % in the flow-through fractions, and the Strip poo! contained 13,4 % aggregates suggesting the resins ability to selective!)-' retain aggregates.

[0021 ] For Lot # 12LPDZ 129, 359.9 rng or approximately 86.9 ¾ of protein was recovered at cumulative protein loading of 414 mg/mL, aggregate level was reduced from 2.05 % to 0,52 % in the flow-through fractions, and the Strip pool contained 16,8 % aggregates suggesting the resins ability to selectively retain aggregates.

[00211] Figure 14 depicts the breakthrough of mAbB monomer and aggregates for Lot # 12LPDZI 19; Figure 15 depicts the breakthrough of MAbB monomer and aggregates for Lot # 1.2LPDZ128; and Figure 16 depicts the breakthrough of MAbB monomer and aggregates for Lot # 12LPDZ129.

[002 ί 2] As demonstrated in Figure 14, with the resin of Lot #

12LPDZ1 19, the MAbB concentration collected in the flow-through -fractions reaches > 90 % its original load concentration with 1 10 nig/mL cumulative protein, loading. Whereas, the aggregate level was only 0,56 % or 27.8 % of the original load concentration at 414 mg/raL cumulative protein loading. This suggests that the resin selectively retains aggregated species to high protein loadings while allowing the protein monomer ( Ab) to be recovered at 88.9 % its total initial mass.

[002131 As demonstrated in Figure 15, with the resin of Lot

#12LPDZ128, the m.AbB concentration collected in the flow-through fractions reaches > 90 % its original load concentration with 138 mg mL cumulative protein loading. Whereas, the aggregate level was only 0.42 % or 20<9 % of the original load concentration at 414 mg mL cumulative protein loading. This suggests that the resin selectively retains aggregated species to high protein loadings while allowing the protein monomer to be recovered at. 86.4 % its total initial mass.

[00214] As demonstrated in Figure 1 (\ with the resin of Lot

#I2LPDZ129, the m.AbB concentration collected in the flow-through fractions reaches > 90 % its original load concentration by 110 mg/niL cumulative protein loading. Whereas, the aggregate level was only 0,99 % or 49,2 % of the original load concentration at 414 mg mL cumulative protein, loading. This suggests thai the resin selectively retains aggregated species to high protein, loadings wh le allowing the protein monomer to be recovered at 86, % its total initial mass,

Exam le IS, FrejMr^^ (CEX)

[0021.51 in a 250 nil... glas jar, 64 nil wet cake of Toyopeari

H 7S-P ^' chromatography resin was added. Next, 1 15g o 5M sodium hydroxide, l S.?5g of sodium sulfate, and 4mL of ally! glyeidyi ether (AGE) were added to the jar containing the resin. The jar was then, placed in a hybridizer at 50¾ overnight, with rotation at medium speed. The next day, the resin was filter drained in a sintered glass filter assembly (EMD Miliiporc

Corporation, Bi!ieriea, MA.) and the wet cake was washed with methanol and then rinsed with deionized water, in a glass vi al, 10 mL wet cake of the AGE activated resin was added. To the glass vial, CL2g of Ammonium persnlfate,

0,3 g AMPS, l >2g DM AM, awl 48g of deio ized water were added and the vial was heated to 6CFC for 1 hours. The next day, the resin was filter drained in a sintered glass .filter assembly (EMD Millipore Corporation,

Bilierica, MA) and the wet cake was washed with a solution of methanol and deionized wafer and the .resin was labeled as Lot. # 1712.

xample 16. Removal of aggregates at va ious r sidence times from a mojij¾ io^

exeha¾ge (CEX) resin modified with an AM ff$ , DMAM

[00.216] ^* The ^ uiiing^si . Lot 1712 from Example 1 was packed in an Omnifit* Chromatography Column with an. internal, diameter of 6.6 mm to a bed height of 3 cm resulting in about 1 m.L packed resin bed. An AK.TA Explorer 100 {chromatography system) was equipped and equilibrated with butlers appropriate to screen these columns for flow-through

chromatography (Similar to Example 1 ). The chromatography columns containing the resin sample were loaded onto the chromatography system with equilibration buffer. The feedstock was m gOl ( AbS) feedstock thai was purified using FroSep® Ultra Plus Affinit Chromatography Media, and was adjusted, to pH 5.0 with 2 M Iris Base, The final concentration of the protein A pool was diluted to 4 mg.'½L,eontained 5.5 % aggregated product, and a conductivity of about 3 ,2 mS/cm. The resin was loaded at a residence time of L 3, or 6 minutes aid to a load density of 1 4 rog/mL. The strip peak fraction for the 3 minute residence timecontatned 95.6 % aggregates indicating a high level of selectivity for aggregated species. The results are depicted in Table 1 1 below.

[00217] Table 1 1 depicts retention of monomer and aggregates for

Lot 1712 with MAb5 at pH 5.0 at 6, 3. or 1. minute residence time. As shown in ^' Table 11, on average, the monomeric species can. be collected at concentrations close to the feed, concentration relatively earl compared to the aggregated species tor ail residence times tested, which suggests that selectivity is relatively insensitive to flow rates.

Table 11

[00218] Figure- 1 ? depicts axhrcjnratograrn of Lot # \ 712 with MAbS a pH 5 and 3 minutes residence time. As depicted in Figure 17, the majority of the product is collected in the ilow-through and this s indicated by the relatively quick breakthrough of protein UV trace. The strip peak size general] y varies based on the conditions and total mass loaded but it is relatively enriched with aggregate species at 95.6 %, compared to the load material which had only 5,5 % aggregates.

Example 17, Purificatio of a moftociunal arifihodv »si»g Proteiti A affmity ehroffiatogra^hv fo towed by the use ef a

[0021 ] In a representative experiment described herein, monoclonal antibody was purified using Protein A affinity chromatography followed by the use of a chromatography resin according to the present invention. The results of this experiment demonstrate an unexpected finding thai the methods did not require art increase in conductivity or the use of dilutions, when, run In a flow-through mode.

[00220] An IgGJ (MAb5) was expressed a cell culture of

Chinese Hamster Ovary (CHO) cells. The ceil culture was clarified by two stage depth filtration followed, by sterile filtration. The clarified cell culture containing 0.5 mg mL mAbS was first purified using ProSep Ultra Plus Aflniiy Chromatography Media (Protein A}. The Protein A chromatography elision, buffer used was 100 is acetic acid. The Protei A elution pool was adjusted to a. pH of 5 ,0 using 2 M Ttis base and the resulting solution had a conductivity of about 3.5 .mS/cm. The Resin Lot # 1.712 was ran. in flow- through mode according to method described herein at 3 minute residence time and flow-through fractions were collected and small aiiqnots were reserved for assaying,

[00221] The flow-through fractions were pooled, adjusted to pE

7.5, and run in flow-through mode using either ChromaSorb, which is a salt- tolerant anion-exchange membrane adsorber, which was loaded to 5 kg L or Fractogel TMAE, which is an anion exchange resin, which was loaded to 1.50

5 ^' ί,ϊ rng/roL. The fractions were assayed for protein concentration, Aggregat level, leached protein A, and Chinese Hamster Ovary Proteins (CHOP). The results are depicted in Table ! 2 below.

[00222] Typically , a chromatographic purification process Involves a traditional bind-aud-eiuie cation exchange chromatography as the step prior to anion, exchange chromatography and further requires a dilution step or a buffer exchange step in order to reduce the conductivity to a level, thai Is suitable tor anion exchange flow-through chromatography. However, as shown in Table 12, the processes described herein using a cation, exchange media according to the present invention do not require an Increase in conductivity n order to operate, and consequentl , do not require a dilution step or a buffer exchange step prior to the parifieation step.

[00223] In this representative experiment, cation-exchange winged fibers were used as the solid support.

[00224] In a I L glass jar, 20g of dry Nylon raulti-lobed, or winged, fibers were combined with 400 g of 4M sodium hydroxide, 24 g. of sodium sulfate, and 160 ml of ally! glyeidyl ether (AGE). The jar was then placed in a hybridizer ai 50°C overnight rotating at medium speed. The following day, the fibers were filtered in a sintered glass filter assembly and the fibers were then washed with methanol and rinsed with Mitli-Q water. A day later, the fibers were washed with water, followed by methanol and then water again, sect ned to a dry cake and dried in vacuum oven at 5CPC for I day. The resulting sample was labeled Sample #1635. In three separate glass vials, 2 grams dr cake of Sample # 1635, AGE activated fibers, were weighed out and added to a glass vial for additional modification by grafting. To the glass vial, ammonium persidfate, AMPS, DMAM, and deionized water were added in amounts specified in Table 13 and the via! was heated to c¾PC for 16 hours with continuous rotation. The following day, the fiber samples were filtered in a sintered glass filter assembly and the wet cake was washed with a solution of deionized water. The vials containing the fibers were labeled as Lot # 1635-1., 16.35-2, and 1635-5.. Next, Lot #1635-5 was titrated for small ion capacity, which was found to be about 28 moi/m.L, It was then assumed that samples #1635-1 and # 1635-2 also had small ion capacity less man 28 μηιοΙ/mL.

Example 19: Removal rrf aggregates fm a jM jjodojtal affiabe y fee

«sj¾»g tr iig cation- xcha ge (CE%) ^i»ge<j,,il|^r|j8¾¾fi§d w¾h AMMjP AM graft d∞ ο*χ®&>

[00225] The resulting modified winged fibers, Lot # 1635 ,

#1 35-2, #1 35-5 from Example 17 were packed in aa Ommfit*

Chromatography Column with an. internal diameter of 6.6 mm to a bed height of 3 em resulting m about 1 mL packed fiber bed. An A TA Explorer 100 (chromatography system) was equipped and equilibrated with buffers appropriate to screen these columns for flow-through chromatography (Similar to Example 13), The chromatography columns containing the winged fiber samples were loaded onto the chromatography system with equilibration buffer. The feedstock was an IgGI (roAhS) feedstock that was purified using protein A affinity chromatography, and was adjusted, to pH 5.0 with 2 M Tris Base, The final concentration of the protein A pool was 4 mg/mL and contained S.5 % aggregated or HMW product. The columns packed with fiber Lot 1.635-1 and Lot # 1 35-2 were loaded to a mass loading, of 64 mg mL aid the column packed with fiber Lot 1635-5 was loaded to a mass loading of 80 mg mL. The results are depicted m Table 14 below.

Table 14

*N/A ^«» Not applicable in : [00226] The specification is most thoroughl understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments in this invention and should not be construed to limit its scope. The skilled artisan readily recognizes that many other embodiments are encompassed by this invention. All publications and inventions are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supercede any such material. The citation of any references herein is not an admission that such references arc prior art to the present invention,

[00227] Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, treatment conditions, and so .forth used in the specification, including claims, are to be understood as being modified in all Instances by the term "about " Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present, invention. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to e very element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims,

[00228] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered b ^¬ way of example only and are not meant to be limiting in any way. It is intended that the specification and examples he considered as exemplary only.

7 with a true scope and spirit ofthe invention being indicated by the following claims.