Parif ^' k tk of Biological Molecules ( K ! ) T e present application claims the benefit of priori is of P S, Provisional Patent Application No, 61/6663 I , tiling date Jane 29, 2012, U.S. Provisional Patent Application No. 61/666.561, tiling date Jane 2% 2012, U.S. Provisional Patent Application No.. 1/666320, filing date June 29, 2012. and European Parent

Application E I 20049093 , filing date inly 2, 201 . each of which is incorporated by reference erein In its entirety.

00(52] The present invention provides i ventive and efficient processes and systems for the purification of biological molecules including therapeutic antibodies and Ik- containing proteins.

IkoP gend^^

Efficient and economic large scale production of hiornoleeuks, e.g., therapeutic proteins including antibodies, peptides or hormones, an increasingly important consideration, tor the biotechnology and phamraeeuiicai industries, (knerally, the purification processes are quite elaborate and expensive and Include many different steps.

[0004J Typically, proteins are produced using cell culture methods, e.g., using either mammalian or bacteria! cell lines recombrnantly engineered to produce the protein of interest. In general, following die expression of the target protein. Its separation from one or .more impurities such as, e.g., host cell proteins, media components a d nucleic acids, poses a formidable chal lenge. Such separation and purification, is especially important If the therapeutic proteins are meant lor use in humans and have to be approved by regulatory agencies, such as the Pood and Drug Administration (FDA).

[0005] Conventional processes used today for the purification of proteins often include at least t e folio wing steps: (a) a clarification slop for the removal of ceils and cellular debris,. c,g... using differential centritugation and/or filtration; and (b) one or more downstream chromatography steps to separate the protein of interest front various impurities in the clarified cell culture feed,

I 10006) Wh le the fermentation and cell culture processes can be run either in a ba ch or fed-batch mode or continuously (e.g. in f orm of a continuous perfusion process), the downstream purification processes are typically run as batch processes that are often even physically and logistical ly separated. Between each process step, the sample is typically stored in a holding or pool tank or reservoir in order to chan e solution conditions in order to render it suitable tor ihe next process step.

Consequently, large vessels are required to store the term dia e product. This leads to high costs and very limited manufacturing flexibility and mobility.

|0007] In addition, performing a number of separate batch process steps is labor and cost intensive as well as time consuming.

j ^' 0008 ^' j In ease of monoclonal anti bodies, the industry standard for purification processes typically involves a "tempiated' ^' process, which includes several unit operations. One of the unit operations is a purification step which employs an affinity Isgand called Protein A, isolated irom Staphylococcus} au eus, and which binds the Pc- region of antibodies. Additional unit operations are usuall used in conjunction with, the Protein A unit operation and most, hiopharmaeeutieai companies employ process templates that are quite similar in their use of die nd operations, whereas there may be some variations in the order of the unit operations,

10009] An exemplary tern plated process used in the industry today is shown in Figure 1 , Key aspects of this template Is a cell harvest step, which typically involves use of centrifogation to em e el and cell debris from a cell culture broth, followed, by depth filtration. The cell harvest step is usually followed by a Protein A affinity purification step, which is followed by virus inaetlvadon. Virus hraetivation is typically followed by one or more chromatographic steps, also relerred to as polishing steps, w hich usually rnei ude one or more of cation exchange chromatography and or anion exchange chromatography and/or hydrophobic interaction chromatography and/or mixed mode chroniatography and/or oyd?v>xyapatite ch omatography. The polishing steps are followed by virus filtration and iritrafiliration/diatlltratlon, which completes the templated process. Sec. e.g., Shukia et al, J. Oirornatography 8., 84S (2007) 28-3 ; I . in et al„ MAbs, 2010: Sep-Oet ¾ >}: 480-499,

100 10] General ly, the effluent of the filtration operations and the eluate of the eh.rorn a to graphic operations are col lected in intermediate pool tanks and stored, often overnight, tint.il the next unit operation. 1 he lime needed to complete this process may be as long as 4-7 days. 1 00 ! I J The present invention provides improved templates! processes which, overcome several of die shortcomings of the t em plated processes currently being used by the industry.

I ^' OOO ^' i 2 The present invention provides processes and systems which provide several advantages over the typical templated processes used in the industry today. The templated processes and systems described herein include unit operations that are connected in a continuous or senn-conimaous manner and ob ate ihe need for pool tanks (also called holding tanks) between certain unit operations, where holding tanks are typically used. Alternatively- only surge tanks are employed.,

[0001 3] Dae to a specific combination of certain process steps, the processes and systems described herein re uir fewer steps than typical processes used in the industry and lso significantly reduce the time for the overall purification process, without having an adverse impact on the product yield.

(0O0! 4j In one aspect accord g to the present invention, processes .for purifying a target molecule from a sample are provided. In some embodiments* such a process comprises the steps of: (a) providing a sample comprising the target molecule and one or more impurities; (b) adding at least one precipitant to the sample and removing one or more impurities, thereby to recover a clarified sample; c) subjecting the clarified sample from step (h) to a. hind and elate chromatography step comprising at least two separation units, with each separation unit comprising the same med a, thereby to obtain an eluate comprising the target, molecule; and (d) subjecting the e!uate to flow-through purification comprising use of nvo or more media; w here at least two steps are performed, concurrently lor at. least a duration of their pardon, and wherein the process comprises a single bind and eSole

chromatography step.

[000 51 hi some embodiments, the flo of liquid through the process is continuous, i.e., the process is a continuous process,

[ 0 101 In some embodiments, the process comprises a virus mactfvation step between steps (e l and (d) above, As described herein, the virus inactivatlon step comprises use of a virus naet varion. agent selected from, an acid, a detergent,, a solvent and tem era lure change. [00017 In some mbo im nts, the virus i reactivation ste employs the use of one o more in-line static mixers, in otto mbodimenls. the virus inactivation step comprises die use of one or more surge tanks .

|OO01 fc] In some embodiments, the target molecule is an antibody, e.g.., a .monockmal antibody o a polyclonal antibody.

[000 ] in some embodiments., the recipitant employed n the processes described herein is a stimulus responsive polymer. Λ preferred stimulus responsive polymer is a modified poiyailylamine polymer, which is responsiv to a phosphate stimulus.

100020] Other exemplary precipitants include, but are not limited to, e.g.., an acid, capry lie acid, a iloeculartt and a salt..

[000.21 ) In some embodiments, the removal of Impurities following addition of a precipitant employs the use of one or .more depth filter. In other embodiments, the removal, of one or more impurities employs the use of centrifugation.

[00022] Following precipitation and removal of one or more impurities, the clarified sample s subjected to a. single bind and elate chromatography step, e.g., step (e) mentioned above, which typically employs at least two separation units. In some embodiments, the bind and ehrte chromatography step employs continuous multi- column chromatography (CMC).

100023] in preferred embodiment, the bind and elute chromatography step Is an affinity chromatography step (e.g.. Protein A affinity chromatography). In other enibodiutents, the bind and elute chromatography step comprises the use of a cation exchange (CEX) bind -and elute chromatography step or a mixed mode

chromatography step.

[00024] In some embodiments, the bind and elute chromatograph step (e.g.. Protein Λ affinity chromatography) employs the use of an additive in the loading step, thereby resulting in reducing or eliminating the number of intermediate wash ste s that are used.

[00025] Exemplary additives include salts, detergents, surfactants and polymers. In some embodiments, an additive is a salt (e.g., 0.5M NaCI

[00026] In some erubod intents, the starting sample is a cell culture. Such a sample may be provided in a bioreactor. f 00027] In s me embodiments, the sample is provid d in a essel other than a bioreactor, e.g., ^' ii may ^' be transferred to another vessel from a bioreactor before snbjee mg It to the purification process, as described he ein.

{ ^' 000281 !« _· some embodiments;, a precipitant used in step (b) above is added directly t a bioreactor eor dmng a cell culture. In other embodiments, the precipitant is added to a vessel other than a bioreactor. where the vessel contains a sample comprising a target molecule. In some embodiments, the precipitant is added using a static mixer.

{000291 in some preferred embodiments, the processes described herein include a flow-through purification process operation, which m lo s two or more media selected from activated carbon, anion exchange cinematography med a and cation, exchange chromatography media. In some embodiments, such a flow-through purification operation additionally includes a virus filtration step, which employs the use of a virus filtration membrane.

j 00030] The processes described herein obviate the need to use a pool tank between various process steps. In some embodiments, a process according to the present invention, comprises the use of one or more surge tanks.

[0003 11 The processes described herein may additionally include a formulation step, i ^' n some embodiments., such a formulation step comprises diahltmtion, concentration and sterile.

[00032] As stated above, the processes described herein include a JIow hroirgh purification operation, which typically employs two or more media. In some embodiments, a -now-through purification process operation used in the processes described herein comprises the -following steps, where all steps arc performed in a flow-through mode: fa) contacting the ehtafe .from a Protein A chromatography column with activated carbon; fbj contacting the flow-through sample from step t a) with an anion exchange chromatography media; (c) contacting the flow-through sample from ste (b) with a cation exchange chromatography media; and (d) obtaining the flow-through sample from step (c ^" ) comprising the target molecule, where the level of one or more impurities in. the flow-through sample alter step id) is lower than, the level in the eluatc in step (a?. The steps (a)-tc) described above may be performed in any order. 10003 ] In some embodiments., the flow-through purification step further comprises virus-ishration step, where the flow-through sample from step tej directly flows into a virus filtration step.

100034] In some embodiments,, a solution change is required between, steps (b) and steps (c), where the solution change employs th use f an in- in static mixer and/or a surge tank, so change the pfi

[00035] in some erak>di.ments, the entire How-through purification operation employs a single skid.

f 000 6] In some embodiments, the eluate from a Protein A chromatograph step is subjected to 8 virus inaetivation step prior to contacting the eluate with activated carbon,

[00037] In a particular embodiment described hereto, a process f r purifying a target molecule from a sample is provided, where the process comprises the steps of: (a) providing a bioreactor comprising a eel), culture; (h) adding a precipitant to the b oreactor and removing one or more impurities, thereby resultin in a clarified sample; te) subjecting the clarified sample t a Protein A affinity chromatography step, winch employs at least two separation units, thereby to obtain an eksate; (d) subjecting the duate from step ie) to a virus inactivating agent using an in-line static mix r or a surge tank; (e t contacting the eluate after virus inaetivation to a flow- through puri fication operation comprising contacting the duate in flow-through mode with activated carbon followed by an anion exchange chromatography .media followed by an in dine static mixer and/or a surge tank to change pf i followed b a cation exchange chromatography .media followed by a virus filtration media; and (1) formulating the flow-through sample from step (d) at a desired concentration in a desired buffer, where the process steps arc connected to he in fluid, communicabon with each other, such thai a sample can .flow continuously from one process step to the next, and where at least two process steps (b)~{ft are performed concurrently during at least a portion of their duration.

[00038) in some embodiments described herein, a process for purifying a target molecule f rom a sample is provided, where the process comprises the steps of: (a) providing a bioreactor comprising a cel l culture; (b) adding a precipitant to the bioreactor and removing one or more impurities, thereby resulting in a clarified sample ie) adding one or more addifives selected from, ihe group consisting of a salt a detergent, a surfactant and a polymer to the clarified sample: id) subjecting the

b clarified sam l to a Protein A affinity chromatography step, which employs at least two separation units, thereby to obtain an eluate: (e) subjecting the ehsiue i on) step id) to a virus inactivating agent us ng an in-line static mixer or a surge tank; (i) contacting the eluate after virus inacbvation to a†lovv hrough purification operation comprising contacting the e!uate in Oow-through mode with activated carbon followed by an anion exchange chromatography media followed by an in-line static mixer and/or a sur tank to change pi I followed by a cation exchange

chromatography media followed by a viru filtration media: and (g) formulatin the flow-through sample .from st p (t) at. a desired concentration in a desired buffer, where the process steps are connected to be in Iliad communication with each, other, such tha a sample can flow continuously from one process step u> the n xt, and where at least two process steps (bMg) are perlormed concurrently during at least a portion of their duration,

[{10039] In some embodiments, the additi e in step (c) is 0.5 NaC!.

(00040] A lso provided herein are systems tor use in a purification process, as described herein. In some embodiments, a system includes: (a) a bioreaetor; (b) a Filtration device comprising one or more depth fi lters; (c) one bind and elute chromatography apparatus; and (d) a i!ow-fhrough purification system conjpnsing at least a flow-through anion exchange device, where a liquid Hoses continuously through the devices in {a)~{cj} during a process run, where the devices are connected to be in fluid communication with each other,

[00041] In some embodiments, there is a connecting line between the various devices in the system, lire devices are connected in line such that each device in the system is in fluid communication with devices that precede and follow the device in the syst m.

[00042] In some embodiments, the bioreaetor used in a s tem according to the present invention is a disposable or a single use bioreaetor,

[000431 in some embodiments, the system is enclosed in a sterile envi onment.

[00044] In some embodiments, the bind and elute chromatography apparatus includes at least two separation units, with each unit comprising the same

chromatography media, e.g.. Protein A affinity media. In a particular embodiment, die Protein A media comprises a Protein A ligand coupled to a rigid hydrophilie poly vlnyiether polymer matrix. In other embodiments, the Protein A llgand is coupled to agarose or controlled pore glass. The Protein A llgand may be based o a naturally occurring domain of Protein A from Staphylococcus imr or bo a variant or a fragment of a naturally occurring domain, in a particular embod iment, the Protein A hgand is derived from the C domain of Staphylococcus aureus Protein A.

[00045] In other end.K.!dimeuts, the bind and elate chromatography apparatus includes at least three separation amis. The separation units a e- connected to be m fluid communication with each other, such that a liquid can flow from one separation unit to the next,

[00046] In et other embodiments, the hind and elute chromatography step employs an. additive in the clarified eel! culture during the loading step where the Inclusion of an additive reduces or eliminates the need for one or more wash step before the ehpfo step,

[0004?] in some embodiments, a flow-through purification system additionally comprises an activated carbon device and a cation exchange (CBX) How-through chromatograph device. In some embodiments,, die l!ow-through publica ion system further comprises a virus filtration device,

[00048] In some embodiments, the entire fipw-ihroagh purification s stem employs a single skid.

[00049] F igure ! is a schematic representation of a conventional purification process used in the industry,

100050] Figure 2 is a schematic representation of an exemplary purification process, as described herein. The puniicatkm process shown uses a bioreae or for cell culture followed by the following process steps; clarification; Protein A bind and elute chromatography (capture); virus inactivatiou; .flow-through purification; and.

formulation. As shown, each of the process steps employs one or more devices used to achie ve the intended result of the process step. As shown,, clarification employs precipitation and depth filtration; Protein A bind and elate ch omatograp y is performed using continuous mn!tieoiumn chromatography (CMC); virus inactivation employs two in-line static mixers; fiow-through purifieaiion employs activated, carbon (AO followed by anion exchange AEX) chromatography followed by pH change using an In-line static mixer and a surge tank followed by How-through cation exchange (CEX) chromatography and virus filtration; and formulation employ s a dkfiltatio.ri/con.ce«ira†.k « laugeutial flovv filtration dev c followed by sterile filtration. One or more sterile Hirers are also employed throughout the process.

[000511 Figure- 3 s a graph depicting the results of an experiment to measure pressure of each: depth filter (primary and secondary} and sterile filter used during the clarification step of the process in Figure 2, The X-axes denote filter load (lYnrf with the top X-axis referring to the load of the sterile filter and the bottom X-axis referring to the load of the two depth filters: and the Y-axis denotes the pressure in psL

[00052] i ure 4 is a graph depicting ihe results of an experiment to measure breakthrough of HCP an MAb fol lowing depth filtration prior to loading on the Protein Λ continuous multicoiomn chromatography {CMC) set up. I he X-axis denotes die depth filter load (L/rrT), the left Y-axis denotes MAb concentration

(mg/ il.) and the right Y-axis denotes the .HCP concentration (pg/ L },

[00053] Figure 5 is a schematic depiction of the Oow-through purification process step, as Anther described in Example 3 ,

[00054 J Figure 6 is a graph depicting the results of an experiment to measure pressure profiles after dept filter., activated carhon and virus filtration. The Y-axis denotes pressure (psi) and the X-axis denotes time in. hoars.

[00055] Figure 7 is a graph depleting the results of an experiment to measure HCP breakthrough alter ABX loading. The Y-axis denotes HCP concentration ippm) and the X-axis denotes the AEX loading tkg/L},

[00056] Figure 8 is a graph depicting the results of an experiment to measure removal of MAb aggregates as a function of loading of the virus fi ltration, device during the flow-through purification operation. lire X-axis denotes the varus filtration loading ikg/uY) arid the Y-axis denotes percentage of MAb aggregate in the sample after virus filtration,

[00057] Figure 9 is a graph depicting the results of an experiment to measure pressure profiles alter activated carbon and before virus .filtration during the flow- through purification operation. The X-axis denotes time in hours and the Y-axis denotes pressure in. psi.

[00058] Figure 10 is a graph depicting the results of an experiment to measure pf l and conductivity profiles, where pi 1 is measured before activated carbon and. before CFX llowwhrough device tf the conductivity is measured before CEX flow- through device, The left Y-axis denotes p i the right Y-axis denotes conductivity (mS cm) and the X-axis denotes t me In hours.

I ^' 00059 ^' l Figure 1 1 is a ehromaiograni for Protein A capture of untreated clarified Ab04 using CMC which employs two separation uni s,

[00060] Figure 12 is a chromatogram for Protein A capture of smart -poiytner clarified MAb04 using CMC which employs two separation units.

[000611 Figure 13 s a chromatogram for Protein A capture of caprvhc acid clarified MAbOd using CMC which employs two separation units,.

[00062] Figure 14 is a graph -depicting the results of an. experiment to investigate the effect of residence time on HCP removal using activated carbon and an anion exchange chromatography (AFX) device, as par of the flow-through.

purification operation.. The Y-axis denotes HCP concentration (ppm) and the X-axis denotes AFX load ike 1. >.

| ^' 00063] Figure 15 is a graph depicting the results of an experiment to measure the effect, on pB spike after using a surge tank between the flow-through anion exchange chromatography and cation exchange chromatography step n a flow- through purification operation. The X-axis denotes p! l and the Y-axis denotes time in hours.

[00064] Figure 16 is a schematic depiction of the experimental set up used lo demonstrating that running the ilow-through purification operation in a continuous manner does not have a detrimental -effect on product purity,

[00065] Figure 1 ? is a. graph depleting the result of an experiment to investigate pressure profiles after virus filtration, following use of a virus filtration device in a continuous format and in a batch mode. The Y-axis denotes pressure in psl and the X-axis denotes processing time In hours,

[00066] Figure I S is a graph depicting the results of an experiment to investigate the effect of flow-rate on throughput of the virus filtration device. T he Y- axis denotes pressure drop tpsi) and the X-axis denotes throughput of the virus filtration device (kg/m ^' Y

[00067] Figure 1 depicts a- ehromatogram of Lot # 1 712 with MAbS at pH 5,0 and 3 minute residence time. As depicted in f igure 19, the majority of the product is collected in the How-through and this is indicated by the relatively uick

breakthrough of protein UV trace. The strip peak s ze generally varies based on the cond ion and total mass leaded but a Is relatively enriched with aggregate species at 95,6 %, com pared to the load material which had only 5,S % aggregates;.

[00068] Figure 20 Is a graph, depleting the elation (first peak between 120 to 130 ml) and regeneration (around 1 0 ml) aks from die chromatograni of Protein A.

purification lor cell culture treated with stimulus responsive polymer and/or NaCI, Also shown is the control without any treatment The X-axis represents the v lume passed through the Protein A column. and the Y-axis represents die ahsorbaoce at 280 Km wavelength.

^00069] Figure 21 is a bar graph depletin Ore HCP ^' LRV as a function of NaCI concentration used in the intermediate wash or the loading step daring Protein A chromatography. The X-axis represents the NaCI concentration in Molar ( ) and the Y-axis represents the HCP LRV.

[00070] Figure 22 is a bar graph depicting the product (¾Ab) percentage recovery as a function of the NaCI concentration in either the intermediate wash step or the loading step during Protein. A chromatography. The X-axis represents the NaCI concentration in M and the Y-axis represents the percent MAb recovery.

I ^' OOOTI ] Figure 23 is a bar graph depicting the. HCP concentration in pans per million (ppm) as a. function of the additive included in either the intermediate wash step or the loading ste during Protein A chromatography. T e X-axis represents die additive included and the Y-axis represents the HCP concentration ppm,

[00072] Figure 24 is a bar graph depicting the HCP LRV as a function of the additive included In either the intermediate wash step or the loading step during Protei A chromatography, The X-axis represents the additive Included and the Y-axis represents the HCP TRY,

[00073] Figure 25 is a bar graph depicting the ratio of the additive elution pool volume to the control eiuiion pool volume as a. function of the additive included in either the intermediate wash step or the loading step during Protein A

chromatography. The X-axis represents the additiv included and the Y-asis represents the ratio of the additive e ution pool volume to the control eiution pool volume.

[00074] The present invention provides processes and systems which overcome several, shortcomings associated with the typical tetnplated processes used in the industry for purification or biological molecules such as antibodies.

100075 J As discussed above, typical tenvp ued processes for purification of biobgieal molecules include many d fferent steps, including one or more

chromatographic steps, require use of holding or pool tanks between steps as ed as take several hours to days to complete.

[00076] There have been a lew efforts to .move away front a typical, templated process. For example, PCX Patent Publication No. WO 2012/014183 discusses methods for protein purification in which two or more chromatographic separation, modes are combined in tandem. Additionally, 1 $. Patent Publication No.

2008/0260468 discusses combining a continuous perfusion .fermentation system with a continuous particle removal system and a continuous purification system, where the flow rate of the mixture through the whole process Is kept ubstantially constant. 00077] Furth r, PCI Publication No. WO201 /0$ 1 14? discusses processes tor protein purification but does not appear to describe a continuous or a semi- continuous process,

I00078J Lastly, PCX Publication No. WO2012/078677 describes a continuous process tor nvamdaeture of biological molecules; however, appears to rel on the utilization of multi-valve arrays. Further, the aforementioned PCX publication also does not teach or suggest use of all the process steps described herein. For example, there appears to be no teaching or suggestion of a .(low-through purification operation which includes multiple flow-through steps Including, e.g., use of a flow-through activated carbon device, a flow-through AFX media, a flow-through (/FX media and a flow-through virus filter, lit fact, PCT Publication No, WO201 /078677 does not teach or suggest a cation exchange chromatography step performed in a flow- throu h mode. Lastly, the aforementioned PCX also lads to describe a continuous process that uses a single bind and eliUc chromatography step and can be performed successfully with minimum interventions, as per the processes described herein.

[00079] Therefore, although It appears desirable to have a purification process which: is performed in a continuous mode, tt has been difficult to achieve an efficient continuous process doe to the complexity associated with connecting several Individual unit operations to run in a continuous or even a semi-continuous mode with minimum interventions, eg., iewer solution adjustments (eg,, changes in pfl and/or conductivity). The present invention, however, has been able to achieve exactly thai.

[000801 ^' The present invention also provides ei her advantages over

conventional processes used in the industry today, e,g„ reducing the number of process steps as well as obviating the need to use large pool tanks between process steps for solution adjustments. In case of the processes and. systems described herein, it is noi required to perform large volume dilutions in order to change conductivity, thereby obviating the need to use large pool tanks between r ce s steps.

Additionally, in some embodiments, the processes and sys ems described herein employ iewer control/monitoring equipments (also called "'skids ^" ), which are typically associated with every single process step, compared to conventional processes used in the art.

[000811 I n some embodiments, the present invention also provides processes which employ the inclusion of an additive during the loading step of Protein A chromatography, resulting in the reduction in or el m nation of one or more intermediate wash steps by going straight from the loading step to the eluiion step or by reducing the number of wash steps between the loading step and the elation step., without sacrificing product purity. While U.S. Patent Publication No. 20B0O902S4 discusses inclusion of an amino acid or salt in the sample being loaded onto a Protein A chromatography column, the foregoing publication does not appear to teach or suggest the use of such a Protein A chromatography step in a nu ti -column continuous or a semi-continuous mode., a describee herein. Instead, it discusses the Protien A step to be performed in a batch, single column mode.

[00082] T he present invention demonstrates that even upon the elimination of or reduction in the number of wash steps performed during the Protein A

chromatography step, a reduction in the level, of impurities, e.g.. HCPs, is observed, without sacrificing product purity.

[ 00083 ] hi order thai the present invention ma be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. 100084] Before describing the prese.nl invention in detail, it is to be understood thai this invention is not limited to specific compositions or proces steps, as such .may vary. If must be noted tha as used in this specification and the appended claims, the singular form "a", and ¾ ⁽ⁱ include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a iigand" includes a plurality of ligands and rciereo.ee to "a antibody" includes a plurality of antibodies and the like.

{00085] Unless defined otherwise, all. technical and scientific terms used herein have the same meaning as commonly understood b one of ordinary skill in the art to which this invention is related,

[0008 ^' 6 ^' f The following terms are defined for purposes of the i n vention as described herein,

[00087] A used herein the term "target molecule" or "target com ound" refers to any molecule, substance or compound or mixtures thereof that is isolated, separated or purified from one or more impurities in. a sample using processes and systems, described herein., in various embodiments, th target molecule is a biological molecule such as, e.g., a protein or a mixture of two or more proteins. In a particular embodiment, the target molecule is an. Fe-region containing protein such as an antibody,

00088] The term ^antibody ^** refers to a protein which has the ability to specifically bind to an antigen. Typically, antibodies have a basic four-polypep ide chain structure consisting of two heavy and two light, chains., said chains being stabilized, i r example, by interchain disulfide bonds. Antibodies may be monoclonal or polyclonal and may e ist in mononreric or polymeric form, for example,. g antibodies which exist in pentamerie form and/or IgA antibodies which exist in monomeric,. dirnerie or multimenc form, Antibodies may also include muitispecifk antibodies (e.g., bispeeifie amibodlesp and antibody fragoiems so long as they retain, or are modified to comprise, a Iigand -spec! fie bindin domain. 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 enzymatic treatment of an intact or complete antibody or antibody chain, f ragments can also be obtained by recombinant means. When roduced recomhinaniiy, fragments .may be expressed id one or as part of a larger protein called a fusi n protein. Exemplary fra ments include ah. Fab ¹ , f<ab')2. c and/or Fv fragments.

|0008 ] In s me embodiments, an Fc-region containing protein s a

recombinant protein which includes the Fc region of an immunoglobulin fused to another polypeptide or a fragment thereof. Exemplary polypeptides include, e.g., renin;, a growth hormone, including human growth hormone and bovine growth, hormone: growth hormone releasing factor; parathyroid hormone: thyroid, stimulating hormone; lipoproteins: a- 1 -antitrypsin; insulin o-chaln; insulin, β-chain; proinsuiin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such, as factor V 11FC, factor .IX, tissue factor, and von. Wii!ebraods factor; ami- clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bo besi ; thrombin; hemopoietic growth factor; tumor necrosis factor ·-¾ and ~β; e kephaknasc; ANTES (regulated on activation normally Ύ~ cell exp ssed and secreted); human macrophage inflammatory protein (MIP- 1 -a); a serum albumin such as human serum albumin; MueQerian-inhihiting substance;

relaxin a-chain: reiaxin β-eba!n; prorclaxin; mouse gonadotropm-assoeiated peptide: a microbial protein, such as β-lacfatnase; DNase; IgF: a cytotoxic T-iymphocyte associated antigen (CTI..A) (e.g., CTLA-4): mhibin actixdn vascular endothelial growth factor (VE-GF); receptors or bormorres or growth factors; Protein A or D; rheumatoid factors; a. nenmirophie factor such as bone- derived neurotrophic factor (BDNF), neurotrophin- . -4, ~5. or -6 (ΝΊΜ, NT-4, T-5, or NT- 6)., or a nerve growth, factor such, as HGP- .; platelet-deri ved growth factor (PDGP ; fibroblast growth, factor such as PGP and {$FGF; epidermal growth factor (FGFp transforming growth factor (TOP) such as ί O -alpha and TGP-β, including TGP-pd. TGF-pX ^' KiF-fia. TGF-fM, or TGF-pS; iosu!imiike growth fac or- 1 and -11 (!GP-i and !GF-IJ); des(l--3HGFd (brain fGF-1). .ins-ul.in-l.ike growth factor binding proteins t lGFBPs); CD proteins such s CD3. CD4, CD8, CD I CD20, CD34. and CD40:

erythropoietin; osteoinductive factors; immurtotoxins: a bone morphogenetic protein (BMP); an interferon such as iriter.fero.u-«, -β. and ~γ; colony stimulating factors f t i ^' ··:]. e.g... !vl-CSF. G -CSF, and G-CSP; interieukins (lbs), e ,. I I ^» I to IL IO; superoxide dismutase; T~eell receptors; surface membrane proteins: decay

accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins: homing receptors; addressius; regulatory proteins;

miegrins suc as CD! la. GDI lb, CD! !e, CD l ib an ICAM, VLA-4 and VCA ; a tumor associat d antigen s ch as BER2, !IERJ or HER receptor: and fragments and/or variants of any of the above-listed polypeptides, in addition, an antibody that may be panned using the processes described herein may bind specifically to an of the above-listed polypeptides,

[00090] As used herein, and unless stated otherwise, the terra. ^{^} sam l " refers t any composition or mixture thai contains a target molecule. Samples may be derived from biological or other sources. Biological source include e karyotie and prokaryotie sources, such as plant and animal cells, tissues and organs, I he sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found naked with die target molecule. The sample ma be "partially purified" (i.e., having been subjected to one or more purification steps., such as filtration steps) or ma 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 iced.

[00091 J ^' T he term "impurity" or "contaminant ^" as used herein, refers to any foreign or objectionable molecule, including a biological macromo!eeule such as DM , ENA. one or more host ceil proteins, endotoxins, lipids and one or more additi ves which may be present in a sample containing the targe molecule thai is being separated front one or more of the foreign or objectionable molecules using a process of the present invention. Additionally, such impurity may include any reagent which is used in a ste which may occur prior to the method of the in vention . An impurity may be soluble or insoluble in nature.

j ^' 00O92J The term 'Insoluble impurity,." as used herein, refers to any

undesirable or objectionable entity present in a sample containing a target molecule, where the entity is suspended particle o a solid. Exemplary insoluble impurities include whole ceils, ceil fragments and cell debris,

[000931 The term "solnbte Impurity." as used herein, efers to any undesirable or objectionable entity present in a sample containing a target molecule, where the entity is not an insoluble impurity. Exemplary soluble imparities include host cell proteins fBCPs), DMA. UNA, viruses, endotoxins, cell culture media components, li ids etc. [00094] Th terms Chinese hamper ovary ceil protein ^* and "CHOP" are used interchangeably to refer to a mixture of host cell proteins ΓΉΓΡ") derived ir ns a Chinese hamster ovary 'CHCT) cell culture. The HCP or CHOP is generally present as an impurity in a cell, culture medium or iysate {e. ., a harvested cell culture fluid fl lCCP")} comprising a target molecule such as an antibod or im unoa hesin expressed in a€HO ceil), ^' The amount of CHOP present in a mixture comprising a target molecule provide a measure of the degree of purity for the target molecule, biCP or CHOP Includes., but Is mi limited o _* a protein of interest expressed by the host ceil, such as a CHO host cell Typically, the amount of CHOP in a protein mixture is expressed in parts per .million relative to tire am unt of the target molecule in the mixture. It is understood thai where the host cell is another cell type,. e,g., a mammal an ^' cell besides CI -10, £ com yeast, an insect ceil, or a plant ceil, HCP refers to the proteins, other than target protein, found in a iysate- of that host cell.

[00095] The teen ^m parts pe million ^' " or "pp r are used interchangeably herein to refer to a measure of purity of a target molecule purified using a process described herein. The units ppm refer to the amount of HCP or Cl iOP in nano ramsdnililgram per target molecule In nniligrams/rnilli liter (i.e,, (CHOP ng/ml,)/(ta.rget molecule mg/mL). where the target molecule and the HCPs are in solution).

[00090] The terms "purifying," purification,/ ^* "se a a eC "separating/ ¹

"separation," "IsoIareC "isolating/ ^' or 'isolation," as used herein, refer to increasing the degree of purity of a target molecule from a sample comprising the target molecule and one or more impurities. Typically, the degree of purity of the target molecule is increased by removing (completely or partial ly} at least one impurity from the sample,

fOO097J The terms "bind and elute mode" and "hind and elute process, " as used herein, refer to a separation technique in which at least one target molecule contained in a sample (e.g,, an Fc region containing protein) binds to a suitable resin or media (e.g., an affinity chromatography media or a cation exchange chromatography media) and is subsequently e luted,

f00098J The terms "flow-thrrrugh proeessy "flow-through mode/ ^' and "ftow- through operation," as used interchangeably herein, refer to a separation technique in. which at least one target molecule (e.g., an Fc-region containing, protein or an antibody) contained In a biopharmacemical preparation akmg with one or more impurhies s intended to flow through a material, which usually binds the one or more impurities, where the target molecule usually does not bind ( .e.. Hows through).

[00099] The term "process step" or "unit operation," as u ed interchangeably herein., refers to the use of one or more methods or devices to achieve a certain result in a purification process. Examples of rocess steps or unit operations w hich may be employed in the processes and systems described herein inehsde, but are not limited to, clarification, bind and elute hr mato raphy, virus inactivation, flow-through purification (including use of two or more media selected from activated carbon, anion exchange and cation exchange in a How-through mode) and formulation., !i is understood that each of the process steps or onii operations may employ mor than one step or method or device to achieve the intended result of that process step or unit operation. For example, in some embodiments, the clarification step and/or the ilow- throngh purification operation, as described herein, may employ more than one step or method or device to achieve that process step or unit operation. In some

embodiments, one or more devices which are used to perform a process step or unit operation arc smgboise 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.

f (100 i 00] As used herein, the ter " s em" generally refers So the physical form of the whole purification process, which includes two or more devices to perform, the process steps or unit operations, as described herein. In some

embo iments, the system is enclosed in a sterile environment.

[OtKllOJ 1 As used herein, the term ^separation tmif refers to an equipment or apparatus, winch can be used in a bind and ekuc chromatographic separation or a flow-through ste or a filtration step. For example, separation unit can be chromatography column or a chromatography cartridge which is filled, with a sorbcot matrix or a chromatographic device that contains a media that has appropriate functionality. In some embodiments according to the processes and systems described herein, a single hind and elute chromatograph step is used, in the purification process which employs two or more separation units. In a preferred embodiment, the two or more separation units include the same media.

[000102] In various embodiments, the processes and systems described herein obviate the need to necessarily use poo; tanks, thereby significantly reducing the overall time to run a purification process as well as the overall physical, footprint occupied by the system. Accordingly, in various embodiments according to the present invent on the output from one process step (or unit operation) is the input for the n st step (or unit operation) in the process and flows directly and continuously into the next process step (or unit operation), without ihe need tor collecting the entire output from a pro ess step.

[01)0103] As used herein, ihe tern? "pool tank ^" refers to any 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. Fool tanks may be used for holding or storing or manipulating solution conditions of the entire volume of output from a process step, hi various embodiments according to the present invention, the processes and s stems described herein obviate hie need to use one or more pool tanks.

{ ^' 000 0 1 In some embodiments, the processes and systems described herein may use one or more surge tanks throughout a purification process,

(000105] The term "'sur e t nk" as used herein refers to any container r vessel, or hag, which is used between process steps or within a process step (e.g. , when a single process operation comprises more than one step); where the output, irorn 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 not intended to hold or collect the entire volume of output from 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 t wo process steps or within a process operation (e.g., flow-through purification operation) 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 othe embodiments, the volume of a surge tank is less !han 35%, or less than 30%, or less than 25%, or less than 20%, or less than 1 5%, or less than 10% of the entire volume of a eel! culture in a bioreaeior, which constitutes the starting material from which a target, molec u le is pari l ed,

[000106} The term "continuous process, ^'" as used herein, refers us a. process tor purifying a target molecule, which includes two or more process steps (or unit operations), such that the output front one process step Hows directly into the nex process ste in the process, without interruption and/or without the need to collect h e entire volume of the output from a process step before performing, the next process step. In a preferred etnboiifoienl, iw¾ or more process stops car; he performed concurrently for at least a portion of their duration. In othe words, in c se of a continuous process, s described herein. it is not necessary o complete a process step before lite 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 operation, in which case, during the performance of a process operation Including multiple steps, the sample flows continuously through the multiple steps that are necessar to perform the process operation. One example of such a process operation described herein is the flow through purification operation which includes multiple steps thai are performed in a continuous manner and employs two. or more of How- through activated carbon, flow-through AEX media, flow-throogh€EX media, and flow-through vi u -filtration. In one embodiment, the flow through purification operation is carried out in the order; activated carbon .followed by AEX media followed by CEX media followed by virus filtration. However, it is understood that activated carbon, AEX. media and CEX media may be used in any order.

Accordingly, so some embodiments. AEX is followed by activated carbon followed by CEX media; or alternatively, CEX is followed by activated carton followed by AEX media. In yet other embodiments, activate carbon is followed by ¹ CEX media followed by AEX media. In stOJ other embodiments, AEX media is .followed by CEX media followed by activated carbon: or alternatively, CEX media is followed by AEX media followed by activated carbon.

[000107] Continuous processes, as described herein, also include processes where ^' the input of the fluid materia! in any single process step or the output is discontinuous or intermittent. Such processes may also be referred to as "send- continuous" processes. For example. In some embodiments according to the present invention, the input in a proces step (e.g.. a bind and elate chromatography step) may be loaded continuously; however, the output may be collected intermhtcnt!y, where the other process steps In the purification process are continuous. Accordingly, in some embodiments, the processes and systems described herein include at least one unit operation which is operated In an intermittent matter, whereas the other unit operations in the process or system may be operated in a continuous manner,

OflOI OS] The term "connected process" refers to a process for purifying a target molecule, where the process comprises two or more process steps (or unit operations), which are connected to be in direct fluid communicatio with each other, such that fluid material continuously -flows through the process steps in the process and is in simultaneous contact with two or more rocess steps during the normal o eration of the process, t is understood that at times, at least one process step in die process may be temporarily isolated .from the other process steps by a barrier such as a valve in the closed position. This temporary isolation of individual process steps may be accessary, lor example, during start up or shut down of the process or daring renKwal/rep!aeenien† of i ndividual unit operations. The term "connected process ^' ' also applies to steps within a process operation which are connected to be in fluid cotnmani.cati.on with each other, e.g., when a process operation requires several steps to he performed in order to achieve the intended result of the operation (e.g., the flow- through purification operation used in the methods described herein),

[000109) The term ''fluid comm nic tion/' as used herein, refers to the flow of fluid material between two process steps or f ow of fluid, materia! between process steps of a process operation, where the process steps are connected by any suitable .means (e.g., a connecting line ^' or surge tank), thereby to enable the flow of fluid from one pr cess step to another process step. In some embed invents, a connecting line between two unit operations may be interrupted b one or more valves to control the flow of fluid through the connecting line. A connecting line may be in the form of a tube, a hose, a p e, a channel or some other means that enables flow of liquid between two process steps.

[000 i. 10] The terms "eiarifyT ^Ni elarlifcationT and "clarification siepf ^' as used, herein, refers to a process step for removing suspended particles and or colloids, thereb to reduce mrhidrty, of a target molecule containing solution,, as measured, in NTU (nephelometric turbidity units). Clarification car) be achieved by a variety of means, including centri fugation or filtration. Cemrifugation could, be done in a batch or continuous mode, while filtration could be done in a norma! flow (e.g, depth filtration) or tangential flow mode. In processes used in the industry today, centri fugati on is typically -followed b depth filtration intended, to remove insoluble impurities, which may not have been removed by centrifugatiom Furthermore, methods lot enhancing clarification efficiency can be used, e.g, precipitation.

Precipitation of impurities can. be performed by various means sueh as by

tloceulalion. pbl adjustment (acid precipitation), temperature shifts, phase change due to sfhnul us-responsive polymers or small molecules, or any combinations of these methods. In some embodiments described herein, clarification involves an combinations of two or more of centri fugatlon, filtration, depth filtration and precipitation. In some embodiments _* the processes and systems described herein obviate the need ids; ceniriiugation.

[000 I I I] The term "precipitate," precipitating" or "precipitation" as used herein, refers to process used io clarification, in which the properties of the undesirable impurities are modified such that th y can be m re easily separated from the soluble target molecule. This is typically accomplished by forming large aggregate particles and/or insoluble complexes containing the undesirable Impurities, T hese particles have properties (e.g. density o size) such that they can be more easily separated from the liquid phase that contains the soluble target molecule, such as b filtration or centrifugatioo. In some cases, a phase change is caused, such that the undesirable impurities can be more easily separated from the soluble target molecule.

Precipitation by phase change can be achieved by the addition of a precipitating agent, such as a polymer or a small molecule, in a particular embodiment the precipitant is a stimulus responsive polymer, also referred t as a smart polymer. In some embodiments described herein, the precipitant or precipitating agent is a floeeolant Flocculation. as used herein, is one way of performing precipitation whom the performance typically depends on the fiocculant concentration ^' used ("dose

dependent"). Typical flocculating agents are polyeiectrolytes, such as poiycations. that complex with, oppositely charged impurities.

[0001 i 2] in some embodiments described herein, clarification employs the addition of a precipitant to a sample containing a target molecule and one or more impurities, in some cases, a change In solution conditions (such as temperature. H, salinity) may be used to initiate the precipitation, such as In the case of stimulus responsive polymers. The precipitated material which contains die one or more impurities as well as the precipitating agent is subsequently removed thereby recovering the target molecule in. the liquid phase, where the liquid is then typically subjected to further process steps in order to iurtber purity the target molecule,

[0001 131: Precipitation may be performed directly in a bioreaelor containing a cell culture expressing a target molecule to be purified, where a precipitant is added directl to the bioreaetor. Alternatively, the precipitant may be added to the cell culture, which typically contains the target molecule, in a separate vessel.

[0001 14] In some embodiments, the precipitant is added using a static mixer. In case the precipitant, is a stimulus responsive polymer, both the polymer and the stimulus to which it is responsive, may be added using a static mixer,

a a [0001 15] There art; many wa s known s.o those skilled its the art of removing the precipitated maieriaf such as centnOtgaO sn., filtration or settling or any combinations thereof.

[00 1 10] The term ^Sv setll.ingf ^" as used herein, refers to a sedimentation process in which the precipitated material migrates to the bottom of a vessel under the influence of gravitational, .forces. Settling can be followed by decant ng or tlltermg of the li uid phase or supernatant.

[00 1 17] The term ^{^} stimulus ^'* or "stimuli, ^'*' as used iuterchangeah!y herein, Is meant to refer to a physical or chemical change in the environment which results in. a response by a stimulus responsive polymer. Accordingly, such polymers are responsive to a st mulus and the stimulus results In a change in the solubility of the polymer. Examples of stimuli to which one or more polymers used herein are responsive, include, but arc not limited to, e.g.., changes in temperature, changes in conductivity and/or changes in pj-L In some embodiments a stimulus comprises addition of a completing agent o a complex terming salt to a sample, i various embodiments, a stimulus is generally added after the addition of a polymer to a. sample. Although, the stimulus may also be added during or before addition of a ^• polymer to a sample.

[000 ! The term '"st mulus responsive polymer ' as used herein., refers to a polymer or copolymer which exhibits a change in a physical and/or chemical property after the addition of a stimulus, A. ty pical stimulus response is a change in the polymer's solubility. For example, the polymer poiyi Osoptopyiaerylamide) is water soluble at temperatures below about 35 ^s' C, but become insoluble Irs water ai temperatures of about. 35° CI In a particular -embodiment a stimulus responsive polymer is a modified polyatlylarnine (PAA) polymer which Is responsive to a multivalent ion stimulus (e.g, phosphate stimulu }, Further details regarding this polymer can he found, e.g., in ITS, Publication No. 201 1 1 3000, incorporated by reference herein in its entirety.

[0001 1 ] in some embodiments, a cell, culture is subjected to a depth filter to remove one or more impurities,

[000120] The terms "depth filter" or "depth filtration" as used herein refer to a filter that is capable of retaining particulate matter throughout the filter medium, ather than just on the Olter surface. In some embodiments described herein, one or .more depth filters are used in the clarification process step.

33 [000) 21 ] In some embodiments, clarification results in the removal of -soluble and/or msolnble impurities in a sample which may later on result in the fouling of a filter or device used In a process step in a purification process, thereby making the overall purification process more economical.

| ^' 000122| in various embodiments described, herein, one or more

chromatography steps are included hi a protein purification process..

[000123] The term ''chromatography ^* refers to any kind of technique which separates m anai te of interest (e.g, a target molecule) from other m lecules present in a mixture through differential adsorption onto a media. Usually, the target molecule is separated from other molecules as a result of differences in rales at which the indi vidual molecules of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elutc processes.

[000124] The term "matrix, ^" as used herein, refers to any kind of paniculate sorbeni, bead, resin or other solid phase (e,g,, a membrane, non- oven, monolith, etc J which usually has a functional group or Irgand attached to it. A matrix having a !igand or functional group attached to it is ^' referred to as "m ia,'* which in a separation process, acts as the adsorbent to separate a target molecule (e.g.. an Fe region containing protein such as an hnmisnoglohulin) from other molecule present in a mixture Ce,g., one or more impurities), or alternatively, acts as a sieve to separate molecules based on size (e.g., in case of a virus filtration membrane),

[000125] , Exam-pies of materials for forming the matrix include

polysaccharides (such as agarose and cellulose); and other mechanically -stable substances such as silica ( .g. controlled pore glass), oly{styrenediviuyl H-)enzenc, po! acr i nu.de, ceramic particles and derivatives of any of the above, in a particular embodiment a rigid hydrophllic polyvinylether polymer Is used as a matrix.

(0001 6] Certain media may not contain hgands. Examples of media that may be used in the processes described herein thai do not contain a. li.ga.nd include., but are not limited to, activated carbon, hydroxyapatite, silica, etc.

[0tKi l 7] thi? term "ilgand," as used herein, relets to a functional group ih-ai is attached to a matrix and that determines the binding properties of the media.

Examples of "ligands" include, but are not limited to. ion exchange groups, hydrophobic interaction groups, bydrophille interaction groups, thiophilie Interactions groups, metal affinity groups,, affinity groups, bioaffinil groups, and mixed mode groups (combinations of the aforementioned) ^' . Other exemplary Hgands which may be used Include, but are not lim ted io, strong cation exchange groups, such, as solphoptOpyl, sulfonic acid; strong anion exchange groups, such, as

trimeihylammomum chloride: weak cation exchan groups, such as carhoxylk acid; weak anion exchange groups, such as N dicihylamrno or DBAE; hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl and affinity groups, such as Protein. A, Protein G, and Protein. L, In a. particular eu odirneni, the ligand thai is used in the processes and s stems described herein includes one or more Protein A domains or a l¼tctional variant or fragment thereof, as described in U.S. Patent Publication Nos, 201002218442 and 20130046056, both incorporated by reference herein, which relate to iigands based cither on wild-type moitimerie forms of B, Z or C domains or on mnltimeric variants of Protein A domains (e.g., B. Z or C domain pentaroersi. The iigands described therein also exhibit reduced Fab binding.

[000128 j The term "affinity chromatography" relets to a protein separation technique in which a target molecule (e.g., an Pc region containing protein of interest or antibody) specifically binds io a ligand which is specific ibr the target molecule. Such a ligand is generally eovalently attached to a suitable chromatography matrix material and is accessible to the target molecule in solution as the solution contacts ihc chromatography media. In a particular embodiment,, the ligand is Protein A- or a functional variant thereof, immobilized onto a rigid hydrophille poiyvi&yiether polyme matrix, The target molecule generally retains its specific binding affinity for the ligand 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 io the immobilized ligand allows contaminating proteins and impurities to he passed through the chromatography matrix while the target molecule remains specifically hound to the Immobili ed ligand on the solid phase material. The specifically bound target molecule i then removed in its active form trom the Immobilized ligand under suitable conditions (e.g., lo pB. high pH, high salt, competing ligand etc,), and passed through the chromatographic column with the eUuion buffer, substantially tree of the contaminating proteins and impurities that were earlie allowed to pass through the column, it is understood that an suitable ligand may be used lot' purifying its respective specific binding protein, e.g. an antibody,

f 000129] In some embodiments according to the present invention. Protein A is used as ligand for purifying an Fc region containin target protein, ^" l ite con itions for eiution bom the ligand (e.g.. based on Protein A) of the target molecule (e.g.. an Fc-- region containing protein) can be readily determined by one of ordinary skill in the art. In some embodiments, Protein G or Pmteln I, or a functional variant thereof may be used as a ligand. In om embodiments, a process which employs a ligand such as Protein A, uses 8 pF! range of 5-9 for binding to an Fc-region containing protein, followed b washing. or re-equilibrating the ligand / target molecule conjugate- which is then followed by elation with a buffer having pi I about or below 4 which contains at least one salt.

[000130] The terms "Protein A ^!! and "Prot A" are used Interchangeably herein and encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (e.g.. by peptide synthesis or b recombinant techniques), and variants, thereof which retain the ability to bind proteins which have a€1¾/Qli region, such as an Fc region. Protein A. can be purchased commercially from

Repiigeu. OF. or Permaiech. Protein A is generally Immobilize on a chromatography matrix. A functional derivative, fragment or variant of Protein A used in the methods and systems according to the present invention may be characterized b a binding constant of at least K--W ^' , and preferably K. ^:::: 10 ^' ' M, lor the Fc region of mouse IgG2a or human IgGI . An interaction compliant with such value for the binding constant is termed "high atrioity binding" in the present context. In some

embodiments, such functional derivatives or variants of Protein A comprise at least part of a functional IgO binding domain of wild-type Protein A, selected from the natural domains E, D, A. B, C or engineered mutants thereof which have retained igQ binding functionality.

[0001 311 Also. Protein A deri vatives or variants engineered to allow a single- point attachment to a solid support ma also be used in the affinity chromatography step in the claimed methods.

[00 1. 2 j Single point attachment generally means thai the protein moiety Is attached via a single co valent bond to a chromatographic- support material of the Protein A affinit chromatography. Such single-point at chment may also occur by use of suitably reactive residues which are placed at an exposed amino acid position, namely in a loop, close to the N- or f '-terminus or elsewhere on the outer

eiretmuorenee of the pr te ir¾ fold. Suitable reactive groups are e.g. sniOr dryl or ammo Inaction-;. [000133] In some embodiments, Protein A deri atives of variants are attached via multi o nt attachment to suitable a chromatography matrix.

[ 0001 34] The term ^" affinity chromatography matr x." as used herein, refers to a chromatography matrix which carries hgaods suitable lor affinity chromatography. Typically the ligand (e.g., Proiein A or a junctional var nt or fragment thereof) s covaient!y attached to a chromatography matrix material and is accessible to the target molecule in solution as the solution contacts the chroffiaiography media. Doe example of an affinity chromatography media is a Protein A media. An affinity

chromatography media typicall binds the target molecules with high .specificity based on a lock/key mechanism such as antigen/antibody or en ¾y roe/receptor binding. Examples of affinity media carrying Protein A ligands include Protein A.

SEPHAROSE™ and PRC)SEP«-A. in the ocess and systems described herein, an affinity chromatography step may be used as the single bind and eluie

chromatography ste in the entire purification process. In a particular embodiment, a Protein A based ligand is attached to a rigid hydrophiite poly vinyleiher polymer matrix. In other embodiments, such a ligand. is attached, to agarose or to controlled pore glass.

[000135] The terms "ion-exehange" and "son-exchange chromatography," as used herein., refer to the chromatographic process in which a solute or aoa!yte of interest (e.g., a target molecule being purified) in a mixture, interacts with a charged compound linked (such as by eovalent attachment) to a solid phase ion exchange material, such that the solute or analyte of interest Internets non-speeifteally with the charged compound more of less than solute impurities or contaminants in. the mixture. The contaminating solutes in the mixture eluie from column of the ion exchange material aster or slower than the .solute of interest or are bound to or excluded from the resin relative to the solute of interest.

[0001 6] "Ion-exchange chromatography" specifical ly includes cation exchange, anion exchange, and mixed mode ion exchange chromatography. For example, cation exchange chromatography can hind the target molecule (e.g., an Fc region containing target protein) fallowed by ehstion (e.g,, using cation exchange bind and eluie chromatography or "CBX ^' O or can predominately bind the impurities while the target molecule "Sows through" the column (cation exchange How through chromatography FT - C. TX ) f 0001 7] Anion exchange chromatography can bind the target molecule (e.g., an Fc region containing target protein) followed by e!wiiort or can predominately bind the impurities wh le the target molecule "flows through." the column, als referred to as negative chromatography. In some embodiments and as demonstrated in. the Examples set forth herein, the anion exchang chromatography st p Is performed in a flow through mode,

(000138] The tent* ^" ion. exchange media" refers to a media that is negatively charged (Le„ a cation exchange media) or positively charged (i.e., an anion exchange media). The charge may be provided by attaching one or more charged Uganda to a matrix, e.g., by cova!ertt linkage. Alternatively, or in addition, the charge ma he an inherent property of the matrix (e.g., as is the case of silica, which has an overall negative charge),

1000139} The terra "anion exchange media" is used, herein to refer to a media which is positively -charged, e.g. having one or more positively charged !lgands, such as quaternary amino groups, attached to a matrix. Commercially available anion exchange media Include DEAE cellulose, QA.E SEPBADEX™ and FAST Q

S.EPHAROSE™ (CiE Healthcare). Other exemplary materials that may he used in the processes and systems described heroin are Fraciogel-le- EMD ΊΜΛΕ, Pracrogei® EMD T AE highcap. Eshnnmof ^; Q and Ftactogehlc EMD DEAE (EMD MftHpore).

[000] 40] The term, ^" cation exchange media" refers to a media which is negatively charged, and which has tree cations for exchange with cation in. an aqueous solution contacted with, the solid phase of the media. A negatively charged ligand attached to the solid phase to form the cation exchange media may, for example, he a carboxyiate or sulfonate. Co m rcially available cation exchange media include carboxy-miethyEecllulose. sulphopropyl ISP) immobilized on -agarose i cy.. SP-SEPHA OSE FAST FLOW ^* *** or SfoSEPlIAROSE HIGH

PERFORMANCE ¹ **, from (IE Healthcare) and sulphonyl Immobilized on. agarose (e.g. S-SEPHAROSF EAST FLOW™ from GE Healthcare). Preferred Is Praetogef# EMD SO _. Fractogel® EMD SE Highcap, Eshmtmot? S and Praciogel® i %1l > COO (EMD Milhporo),

[000141 ] The term, "mixed-mode chromatography ^* ' or "muttt-raodal

chromatography ^* as used herein, refers to a process employing a chromatography stationary phase that catties at least two distinct types of functional groups, each capable of interacting with a molecule of interest. Mixed-mode chromatography generally employs a Hgand with more than one mode of interactio with a target protein and/or .impurities. The ligand typically includes at least two dHferent hut co- operative sites Which interact with Ihe substance to he bound. For exam le, one of these sites may ha ve a charge-charge type interaction with the substance of interest, whereas Ore other site may have an electron acceptor-donor type interaction, and/or hydrophobic and/or hydrop ^' hillc interactions with the substance of Interest, Electron donor-acceptor interaction, types include hydrogen-bonding, a t, eadon~«, charge transfer, dipoie-dipoie and induced di.poie interactions. Generally, based on the differen es of the sum of i.nteractio.ns, a target protein and one or more impurities; ma be separated under a range of conditions,

[0001421 The term "mixed mode ion exchange media" or "mixed mode media" refers to a media which is covaiendy modified with cationk and/or anionic and hydrophobic moieties, A commercially available mixed mode Ion exchange media is BAKERBONB ABX ^W (J, T, Baker, Phiilipsbargd J.,) containing weak cation exchange groups, a low concentration of anion exchange groups, an hydrophobic ligands attached to a silica gel. solid phase support matrix. Mixed mode cation exchange materials typically have cation exchange and hydrophobic moieties.

Suitable mixed mode cation exchange materials are CaptxvD MMC (GE Healthcare) and Eshmu.no® I ICX (HMD ilhpote),

[000143 j Mixed .mode anion exchange materials typically have anion exchange and hydrophobic moieties. Suitable mixed mode anion exchange materials are Capto® Adhere (GE Healthcare}.

[000144 J The term ^* iydrophoble interaction chromatography" or ^M H 1C, ^*5 as used herein, refers to a process tor separating molecules based on their

hydrophohicity, i.e., their ability to adsorb to hydrophobic surfaces iron aqueous solutions. Ηϊ€ is usually differentiated .rk>m the Reverse Pha e (H P) chromatography by specially designed HI€ resins that typically have a lower hydrophohlcify, or density of hydrophobic ligands compared to P resins,

[000145] I C ^' : chromatography typically relies on the differences so hydrophobic groups on the surface of solute molecules. These hydrophobic groups lend to hind to hydrophobic groups on the surface of an insoluble matrix, Because llIC employs a more polar, less denaturing environment than reversed phase liquid chromatography, it is becoming increasing popular for protein purification, often in combination with ion exchange or gel Hhration chromatography. [0001 6 } The term ^* %reak hroughd ⁵ as used herein., refers to. the point of time during the loading of sample coritainmg a target molecule on a packed

chromatography column or separation. m\k, when, the target molecule first appears in the output from the column or separation unit. In other words, the term "break- through" s t he point of time when loss of target molecule begins,

[000147 j A. "buffer" s a solution that resists changes in pH by the actio of its acid-base conjugate components. Various buffers which can be employed de end ng, for example, on the desired pli of the buffer, are described in: Buff ts, A Guide for the Preparation and Use of Buffers in Biological Systems, Goefffoy, IX, ed.

Calbfochem Corporation ( 1975), Hon- limiting examples of buffers include ME5 , MOPS, OPSO, ^" iris, HEPHS, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these,

[0001 48] When "lo ding" a sample onto a device or a column or a separation unit containing a suitable media, a buffer is used to load the sample comprising the target molecule and one or more impurities onto the device or column or separation unit, in the bind and. e rte mode, the bufter has a conductivity and/or pB such that the target molecule is hound to media, while ideally all the imparities are not bound and flow through the column. Whereas, in a flow-through mode, a buffer is used to load the sample comprising the target molecule and one or more impurities onto a column or device or separation unit,, wherein the buffer has. a conductivity and/or pB such, that the target molecule is not bound to the media and ows through while Ideall ail or most of the Impurities bind to the media,

[000149] The term "additive" as used herein, refers to any agent which is added to a sample containing a target protein prior to loading of the sample onto a chromatography matrix or dering the loading step, where the addition of the agent eliminates one or more wash steps or reduces the number of wash step which are otherwise designed for impurity removal, to be used subsequent to the loading step and before the elation of the target protein, A single agent may be added to a sample prior to or during the loading or the number of agents may be more than one.

Exemplary addi ives include, but are not limited to, salts, polymers, surfactants or detergents, solvents, chaolropic agents and any combinations thereof in a particular embodiment, such an additive is sodium chloride salt.

1000150] In a particular embodiment, a static mixer Is used for contacting the output from the clarification step with an. additive, where the use of a static miser significantly reduces the time, thus allowing for a simplified connection of he clarification step to the protein A chromatography step.

[00015 J J The term ^" fc-eqnd ibrating' ^' refers to the use of a bullet to recondition the media prior to loading the target molecule. The s me buffer used for loading may be used for rc-eqnilibrating.

[000152] The term ^½i wasfT or "washing" a chromatography media refers to passing an appropriate liquid, e.g., a buffer, through or over the media. Typically washing is used to remove weakly bound contaminants from the media prior to eh.rh.ng the target molecule and/or to remove nou-ho nd or weakly bound target molecule after loading. In some embodiments, the wash buffer s different from the. loading buffer. In other embodiments, the wash bailer and. the loading buffer are the same. In a particular embodiment, a wash step is elimin ted or the number of wash steps is reduced in a purification process by altering the conditions of the sample load.

[000! 53) In some embodiments, the wash steps that are used in the processes described herein employ a. boiler having a conductivity of 20 mS/cm or less, and accordingly, are different from the buffers that are typically used for impurity removal as those typically have a conducti vity greater than 20 mS/cm.

{0001 54) The term ''conductivity ^" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an Increasing amount of Ions present in the aqueous solution, the solution will have a higher conductivity. The nn.lt of measurement for conductivity is miluSenuens per centimeter (mS/em or mS _. and can be measured, using a commercially available conductivity meter (e.g., sold by Orion). The conductivity of a solution .may be altered by changing the concentration of ions therein, f or example, the concentration of a buffering agent and/or concentration of a salt (e.g. NaCl or KG) in the solution .may be altered in order to achieve the desired conductivity, in s m embodiments, the salt concentration of the various buffers is modified, t achieve the desired conductivity, In. some embodiments, in processes where one or more additives are added to a sample load, if one or more wash, steps are subsequently used, such wash steps employ a uffer with a conductivity of about 20 fiiS cm or less.

{0001 55) ^' The term "elate" or '"elating ' or "'elatlotf ^' refers to removal of a molecule (e.g., a polypeptide of interest or an impurity) from a chromatography media by using or altering certain solution conditions, whereb the buffer { referred to as an. "elution buffer ^*" } competes with i.he molecule of interest tor the .!igaruf sites on ihe chromatography resin, A non-lurming example is to eiute a molecule from an ion exchange resin by altering the ionic strength of the buffer surrounding the ion exchange material such thai the butler competes with the .molecule tor the charged sites on the ion exchange material.

[0001561 In some embodiments, the elution buffer a low pH (e.g., having a pi ! in the range of about 2 to about 5, or from about .3 to about 4) and which disrupts Ihe interactions between ligaraf (e.g., Protein A) and die target protein. Exemplary elution buffers include phosphate, acetate, citrate and am.mo.amm ^' buffers, as well as combinations or these. In some embodiments, an elution buffer m y be used which has a high pH (e.g., i I of about 9 or higher). Elution buffers may also concurs additional compounds, e.g., Mg€¾ (2 mM) for facilitating elution.

1000157] In. case virus inacli vation (VI) is desired, a virus inaeti vation buffer may be used to inactivate certain viruses prio to elodng the target molecule. In such instances, typically, the virus inaetivation buffer differs fern loading buffer since it may contain detergent/detergents or have different properties (pfl/cemductivity/salts and their am unts). In some embodiments, virus inaetivation. is performed before the bind and eiute chromatography .step. In some embodiments, virus loaebvation is performed after during or after elution from a bind and elute chromatograph step. In. some embodiments, virus inaeiivatlo i performed in-line using a static mixer, In other embodiments, virus inaetivation employs use of one or more surge tanks.

[000158] The term "bioreaetosy ^* as used herein, refers to an manufacture or engineered device or system, that supports a biologically active environment. In some instances, a h!oreaetor Is a vessel in which a cell culture process is carried out. Such process may either be aerobic or anaerobic. Commonly used biorsaetors arc typicall cylindrical rangin in size from liters to cubic meters, and are often made of stainless steel., in. some embodiments described herein, a hioreaeror is made of a material other than steel and Is disposable or single-use. it is contemplated that the total volume of a bioreaetor may be an vol ume ranging from 100 mi, to up to 10,000 Liters .or more, depending on. a particular process, in some embodiments -according to the processes and systems described herein, the bioreaetor is connected to a unit operation such as a depih hirer. In some embodiments described herein, a bioreaetor is used tor both cell euhnriug as well as for precipitation, where a precipitant may he added directly to a bioreaetor, thereby to preeiphate one or more impurities. (0001591 The e m '"active carbon" or "activated carbon; ^" as used

interchangeably herein, refers to a carbonaceous material which ha been su jected to a process to enhance its po e structure. Activated carbons are porous solids with very high surf ace areas. They can he derived from a variety of sources including coal, w d, coconut husk, nutshells, and peat. Activated carbon can he produced fr m these materials using physical activation involving heating- under a c mrol led atmosphere or chemical activation using strong acids, bases, or oxidants. The activation processes produce a porous structure with high surface areas tha gi ve activated carbon high capacities for impurity removal. Activation processes can be modified to control the acidify of the surface. In some embodiments described herein, activated carbon Is used in a flow through purification step, which wpically follows a bind and elute chromatography step or a virus inaetivation step which in turn follows the bind and elute chromatography step. In some embodiments, activated carbon is incorporated w ithin, a cellulose media. e,g„ in a column or some other suitable device, [000160} The term "static mixer ^* refers to a device for mixing two f uid materials, typically liquids, The device generally consists of mixer elements contained, in a cylindrical (tube) housing. The overall system design incorporates a method for delivering t wo streams of fluids Into the static mixer. As the streams move through the mixer, the non-moving elements continuously blend the materials. Complete mixi n 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 throughou the purification process or system, In a particular embodiment, a static mixer is used tor contacting the output from the bind and elate chromatography step with a virus inactivating agent (e,g,, an acid or any other suitable virus inactivating agent),, where the use of a static mixer significantly reduces the time, which would otherwise be needed to accomplish effective virus inaetivation.

Efpee s^

1000161 ] As discussed above, the present invention provides n vel and

Improved processes for purification of target molecules from a sample fe,g., a cell culture feed) containing a target molecule and one or more Impurities, Tire processes described herein are a vast improvement over existing methods use in the art., in that they reduce the overall time frame required, for a process run ( 12-24 hours relat ve to several days}; include fewer steps relative to most conventional processes: reduce the overall physical footprint of a process by virtue of having fewer unit operations and are easier to execute than a couyersliorsal process. Additionally, some

embodiments, processes according to the present invention m l y devices that may be disposable,

|ί ⁾ 00162 | The processes according to the present invention Include several process steps or unit operations which are intended, to achieve a de ired result and where the process steps (or unit operations) are connected such, that to he in fluid communication with each other and further tha two or more proces steps can be performed concurrently for at least a part of the duration of each process step. In other words, a user does not have to wait for a process step _: to be completed before executing the next process step in tire process, but a user can start a process run such that the liquid sample .containing the target molecule flows through the process steps continuously or semi -continuously , resulting in a purified target .molecule.

Accordingly, the sampl containing die target molecule is typically in contact with mare than one process step or unit operation in the process at any given time,

[ 000163] Each process step (or unit operation) may involve the use of one or more devices or methods io accomplish the process step.

[00016 J The processes described herein are different from conventional processes used in the industry, in that they obviate the need to use pool tanks for holding, diluting, manipulating and sometimes storing the output !fo a process step before the output is subjected to the next process step. In contrast, the processes described herein enable any manipulation of the sample in-line (e.g., using a static mixer) or employ the use of surge tanks (which are usually not more than 10% or 20% or 25% of total volume of the output .from a process step) between process steps or sometimes within a process operation (e,g„ when a process operation employs mote than one method or device), thereby signi ficantly reducing the overall time to perform the process as well as the ph sical footprint of the overall system for performing the process. In a preferred embodiment, processes described herein use no pool tanks hut only surge tanks havin a volume of less than 25%, refe abl les ih&n 10% of the volume of the output from the preceding step.

0 ⁽ 165] The processes described herein include at least three process steps- clarification, bind and ekne chromatography and How-through, purification, Typically, clarification is the first step followed by bind nd el ate chromatography followed by flow-through purification operation. The processes may include additional process steps Including, but not limited to, virus inactivation and formulat on An important aspect of the processes described herein is thai regardless of the number of steps, the process includes only one bind, and elute chromatography step.

[000166] The various process step are performed in a continuous or a. send- continuous manner, as described herein. Following are examples of process steps winch may be used in a continuous or semi-continuous process, as described herein. It i understood that any combinations of the process ste s show n below can. be used., in. other words, any process step under Step 1 in. the Table I below may be combined with any process step under Step 2 and/or any process step under Step 3 and so .forth, it is also understood that additional process steps, w hich, are described elsewhere in the specification may be combined with or used I nstead of one or rnore of the process steps described in Table I below.

Step I Step 2 Siep 3 Step 4 Step 5

Clarification Bind and E ute Virus Flow- Form illation

€ i* r o m ate ra p hs InacttvaiioH thr u

rification

Precipitation Contln nous/sen? i- Vims P low Dialiltrahon in a vessel continuous bind inaetivabon through and

followed by and elute Protein In a surge AEX media concentration depth A tank with or followed by filtration chromatog phy without, sterile

vims filtration nitration

Precipitation Simulated moving Virus blow Concentration in a vessel bed bind and elute maetivation through followed by

.followed by Protein A using a AEX media sterile eentrilugation chromatography static mixer and. CEX intra fon

media with.

or without

virus

filtration

Precipitation Continuous/ Flow DiaOUration in a vessel semi-continuous through followed by followed, by Cation exchange activated sterile settling ana bind and elute carbon filtration rnicr iiltration chromatography media and

O.f d i ABX media

supernatant w h or

without

vires

filtration

Precipitation. Continuous/seini- Flow

in a bioreactor continuous Mixed through

followed by mode bind and activated

depth elate carbon

filtration chromatography media and

ABX media

and CEX

media with

or without

virus

filtration

Precipitation

n a bioreactor

followed by

cmtrifugalio.n

Precipitation

in a bioreactor

followed by

settling and

microti Straiion

o f the

supernatant

[000167] The various process steps (or unit operations} are described in more detail ί α,

[000168] The starting material for th purification process is usually a sample containing a target molecule being purified. Typically, a cell culture producing the target molecule is used, However, samples other than cell cultures may also be used. Exemplary samples include, but are »ot limited to, transgenic mammalian, cell cultures, non-transgen c mammalian cel l cultures, bacterial cell cultures, tissue cultures, microbial termentatiou batches, plant extracts, bioiuels, sea water cultures, freshwater cultures, wastewater cultures, treated sewage, untreated sewag , milk, blood, and combinations thereof Generally, the samples contain various impurities in addition to the target molecule, Such impu.rid.es include media components, cells, cell debris, nucleic acids, host cell proteins, viruses, endotoxins, etc. [00 169] One of the f rst process steps (or unit operations) i the processes and systems described herein is typically clarification. Clarification is ntended t separate one or more soluble and/or insoluble impurities from the target molecule. In some embodiments, insoluble impurities like cells and cellular debris are removed from the sample resulting in a clarified fluid containing the target molecule in solution as well as other soluble Impurities, Clarification is typically peribrmed prior to a step involving capture of the desired target, mo!eeale. Another key aspect of clarification is the removal of soluble and/or insoluble impurities in a sample which may later on result in the fouling of a sterile filter in a purification process, thereby making the overall purification process more economical.

[Ό00 Ι 70] As used in the industry today, clarification generally comprises removal of cells and/or cellular debris a d typically involves cenirifugation as the first step, followed b depth filtration. See, e,g„ S ukla ei aL J, Chromatography B, 848 {200?): 2S-39; Liu et ah. Abs. 2(5): 480-409 (2010) - [ ^' 000.171 ] In some preferred embodiments described herein, clarification obviates the need io use centri.fugat.ion.

[00 1721 for example, in some embodiments, -where the starting volume of the cell culture sample in a bioreactor is less than 2000 liters, or less than 1000 liters or less than 500 liters, tbe eeli culture sample may be subjected to depth filtration alone or to settling and depth Ill.tra0.ori, without the need Ibr eentrifugation..

[0001 3 J In some preferred embodiments, use of precipitation before depth filtration increases throughput and therefore, the amount of sample volume which may he processed without the need f r cenirifjgaiion is also increased. In other words, in some instances, if 1 00 liters of a sample can he processed by depth filtration alone, by combining that with precipitation, a user may e able to process almost twice that amount !,e., 2000 liters,

f00 1 74 j Depth filters are typically used to remove one or mom insoluble Impurities, Depth filters am filters that use a porous nitration medium to retain particles throughout the medi um., rather than just on the surface of the medium,

1 _. 00 1731 hi some preferred ernbodirneuts, a depth fiher Is used, lor clarification, which is capable of filtering cellular debris and particulate .mailer having particle sixe distribution of out 0,5 grn to- about 200 m at a How rate of about !O lUers/mOhr to about 100 bters/m7hr,

[000] ?6| it lias beers fou d that ©specially good results hi the primary removal of particulate impurities can be achieved if the porous depth filt r is anisotropic (he. with a gradual reduction impure size). In some preferred embodiments, the pores have a nominal pore size rating > about 25 μηι. In some preferred embodiments, the depth filter comprises at least 2 graded layers of non-woven libers, wherein, die graded layers have a total thickness of about 0,3 cm to about. 3 cm,

[000177] in s me embodiments _* the depth filters are configured h a deviee which s able to filter high solid feeds containing panicles having a particle sim distribution of approximately 0,5 pro to 200 urn at a. flow rate of about 10 hters/urVhr to about 100 hters /hr until the transmembrane pressure (IMP) reaches 20 psi.

[0001 ?$} In some embodiments, depth fibers comprise a composite of graded layers of non-woven fibers, cellulose, and dlaiomaeeous earth. The non-woven libers comprise polypropylene, polyethylene, polyester, nylon or mixtures thereof.

1000179] Exe.mp.lary depth, filters and methods of use thereof may be found in

U.S. Patent Publication No. 2013001.2689, incorporated by reference herein, which are particularly useful for filtering samples containing particles have a size distribution of about 0, 5 pm to 200 pm. Accordingly, in some embodiments, depth filters used in the clari fication, step include open graded layers, al lowing the larger particles in the feed stream to penetrate Into the depth of the filter, and become captured within the pores of the filter rather than collect on the surface. The open top layers of the graded depth filters enable capturing of larger panicles, while the bottom layers enable capturing the smaller residual aggregated particles. Various advantages of the graded depth filters include a higher throughput retention of larger solids and eliminating th problem of cake formation.

10001 SO] As discussed above, in s me embodiments, clarification includes the use of depth filtration following precipitation. Precipitation may employ acid precipitation, use of a stimulus responsi ve polymer, tioccuiation or settling and any other suitable means/agent for achieving precipitation. Accordingly, in some eumodhnents, a preeiphant, e.g., a stimulus responsive polymer, is added, to a sample to precipitate one or mote soluble and/or insoluble imparities prior to depth filtration, iOOOl 811 Other means of precipitation include, but ate not limited to, use of shortmhain fatty acids such as eapryhc acid, use of fioeookots, changing solution conditions (e.g.. temperature, pH, salinity) and acid precipiiadon.. For example, it has: been reported that m mildly acidic conditions, the addition, of short-chain tatty acids suc as caprylie acid typically precipitates non IgO proteins while IgO is not precipitated.

[000 idoceuktion, as used herein, is one way of perlb.rnt.iog precipitation where the precipitation typically depends on the Oo eukmt concentration used (he,, is "d se dependent ^" ). Typical flocculating agents are polyeleeiro!ytes.. such as poiyeatlons, that complex with oppositely charged impurities,

[000183] Floeeulauts generally precipitate cods, cell debris and proteins because of the interaction between the charges on the eelisO roteins and charges on the olymer (e.g. polyeleetroiytes), thereby creating inso!ob!e complexes,

[000184] t he use of polyelectroiyte polymers in floeculation t purify proteins is wed established .in the art (see, e.g., international PCX Patent Application No. WO2008/091 740, incorporated by reference herein). Precipitation by floeculants can be accomplished w ith a wide range of polymers,, with the only required general characteristic being the polymer must have some level of interaction with a species of interest (e.g., a target molecule or an impurity). Exemplary occn!aots include polymers such as ebitosan and polysaccharides.

000185J F!occuiatiou may also be achieved by chemical treatment resulting, in changes in pH or by addition of a surfactant.

[000186 j There are many ways known to those skilled In the art of removing the precipitated material, such as cetPrifugaiion, depth filtration, filtration or settling or any combinations thereof Settling can be toil owed by decanting or filtering of the liquid phase or supernatant,

[00018?j In some preferred embodiments, stimulus responsive polymers are used for precipitating one or more impurities. Examples of such stimulus responsive polymers can be found, e.g., in U.S. Publication os., 20090036651 , 2010026 /933 and 201 1031 006; each of which is incorporated, by reference herein in its entirety . Stimulus responsive polymers are generally s lu le in. an aqueous based solvent under a certain set of process conditions such as pi L temperature and/or salt concen ati n and are rendered insoluble upon a change in one or more of such conditions ^' and subsequently precipitate out. Exemplary stimulus responsive polymer Include, hut are not limited to, polyaiiylanime, polyal!yiaotine modified with a benz l group or

30 polyvinylamine and polyvmylamine rrs.ods.fied with, a benzyl group, where die stimulus s phosphate or citrate.

[0001881 i some embodiments, a stimulus responsive polymer s continuously added using a stalk raker, in other embod!rnems. both the polymer as well as the stimulus to which it is responsive are added using a static mixer.

[000189] In some embodiments, small molecules are used for precipitating one or more impurities, especially insoluble impurities.

[000190] In some embo ent , small molecules used io the processes described herein are non-polar and caiionk, e.g., as described in PCX Publication No. WO2013028334, incorporated by reference: herein. Exemplary s all molecules that may be used for clarification include, but are not limited to, monoalfcyhrirnethyl ammonium salt Cmm~!imhmg examples include cetyhrimeihylammomum bromide or chloride, teiradecyhrimethyiammoiuum bromide or chloride, alkyitrimethyi ammonium chloride, afkykryltrlmeth l aouiionium chloride,

dodeeyhr.imeth. iao.urion.ium bromide or chloride, dodec f dimeth I -2- phenoxyethy!ammonhuu bromide, hexadecylamine chloride or bromide, dodecy! amine or chloride, and ces idimethyicthyl ammonium bromide or chloride), a moooalkyldimelhylbenxyl ammonium salt (oon-iimiting examples include

alkyldh ediyibeuzyl ammon um chloride and benzethonium chloride), a

dklkykiiureOryi. ammonium salt (non-limiting examples include domi heu hroiui.de, dideeyldimeihyl »mocium lialldes (bromide and chloride salts) and

oetyidodecyldhwthyl ammonium chloride or bromide), a heieroaromatk ammonium salt (non-limiting examples include cetylpyridiurn balides (chloride or bromide salts) and hesariecyipyridinlum bromide or chloride, cis-isomer 1~[3-chioix>aUyi V$,7- triaza~ i~azoniaadamantane, aikyi~Lsoqumoli. m bromide, and

alkyldimethylnapbthylmethyl ammonium chloride), a polysubstltaied quaternary ammonium salt, (non~iun.itmg examples include albyldlmeihylbenzy! ammonium saccharinate and alkyldiu ethykihylbenzyl amraonium eyeiohexylsul amate), and a bis-quaternary ammonium salt (non-limiting examples include I J (bbis(2~mebHyi-4~ an hio ubroli i m. chlorid pdecane, ! ,6-BI,s

eyelohexyl pix pyldiuvetliyl ammonium: chloride hexarse or trklobisookim chloride, and the bis~quat referred to as CDQ by Bucknmn Brochures),

[000191 1 In a particular prerexfed embodiment, the small molecule is beuj thoni m chloride (BZC), [000192] In some embodiments, clarification is performed directly in a bloreaclor. In other words, a precipitant, e.g., a stimulus responsive polymer, may be added directl to a bloreactor containing a culture of cells r ssi g a target molecule, thereby precipitating the ceils and ceil debris, and where the target molecule r mains i the liquid phase obtai ned as a result of the precipitation, in some preferred embodiments, the liquid phase is further subjected to depth filtration. T he liquid phase may also be subjected to cernnfugation, filtration, settling, or

combinations thereof.

[(MM) ! 93] in other embodiments, a stimulus responsi e polymer is added to a vessel which contains the ceil culture and is separate from a bloreactor. Therefore, as used herein, the term ""vessel," refers to a container separate from a bloreactor which is used for cinhuring cells.

|00θ! 94] in some embodiments, a stimulus responsive polymer is added to a sample before eentri&gahon, and eerarilugadon is followed by depth, filtration. In such a process, the size/vokintc of the depth filter which may be required following centritugation is smaller than what is required i the absence of stimulus responsive polymer.

[000195] in some embodiments, a clarified cell culture feed is further subjected to a charged liuoroearbon composition (CFC), to .further remove host cell proteins (!-!CPsp as described in PCX Application No, PCT/US20I 3/3276S (internal ret no, CA- 1303PCTP filed March 18, 2013, which describes a CFC-raodificd membrane for removal of HCPs, CFC-modified membranes can also be used after other process steps In the purification process,, e.g.. following Protein A. bind and elate

chromatography step or following How-through purification process step or following the auiomexeharsge chromatography step, which is part of the flow- through purification process step.

[000 ! %} The clarified sample is typically subjected to a bind and elute chromatography step.

I liad ;md hh - obrmnam r phy

[0001 ?] In various embodiments described herein, the processes and systems include only a single hind and elate chromatography process step for capture, which typically follows clarification. Bind and elute cinematography is intended to bind the iarg t molecule, whereas the one or more impurities Oow through {also referred, to as the "captu e step 1, The bound target. molecule is subse uently e!uted and the eluate or output, itom the bind and elute chromatography s e may be subjected to further purification s e s.

[000198] Bind and elute chromatography may employ a single separation unit or two or three or more separation units.

1:0(101 99] In various embodiments described herein., bind and elute

chromatography that is used s affinity bind and elute chromatography or cation exchange hind and elute chromatography or mixed mode bind and. elate

chromatography. Typical ly, bind and el ute chromatography employs the use of a media which is intended to bind the target molecule.

(000200] In some preferred embodiments, the bind and elute chromatography Is an ailinity chromatography. Suitable chromatography media, that may be used lor affinity chromatography include, but are not limited to, media having Protein A, Protein G or Protein. I. functional groups (e.g.. ProSep® High Capacity (HMD Milllpore), ProSep® Ultra Plus (HMD Millipore). Porost ^: abCapiure A I Liie Technologies), Ab olute® (NovaSep), Protein A Ceramic P!yperD# (Pali

Corporation), Toyopearl AF-rProtein A-6S0F (Tosoh), abSeieet® Sure (0E Healthcare)}. Suitable media are usually packed in a chromatography colum or device,

10002 1 ] hi a particular embodiment, the affinity chromatography media includes a Protein A based ligand coupled to hydropbiiie rigid polyvmyiether polymer matrix.

[000202] In some embodiments according to the present ItiventiotL the bind and elute chromatography process employs continuous muitl-eoiiunn

chromatography, also referred to as CMC.

[000203] in continuous chromatography, several Identical columns are typically connected in an arrangement that allows■columns to be operated In series andmr in parallel, depending on the method requirements. Tims, all columns an: be run simultaneously or may overlap Intermittently in their operation. Each column Is typically loaded, elated, and regenerated several times during a process run.

Compared to conventional chromatography, where a single chromatography cycle is based on several consecutive steps, such as loading,, washing, elation and

regeneration. In ease of continuous chromatography based on multiple identical columns, all these steps ma occur on different columns. Accordingly, continuous hroma raphy operation may result in a bene? utilization of chtoniatogmphy resin and reduced buffer requirements, which benefits process economy.

[000204] Continuous bind and el ute. chromatography also includes simulated moving bed (SMB) chromatography.

0 205J In some preferred embodiments, bind and elate chromatography employs CMC which us s two separation units. In some other preferred

en¾hodin;.ents _:; bind, and el te chromatography employs CMC which uses two or three or more units, in ease of CMC\ the loading of a sample is usually continuous;

however,, the eiution is haermittent or discontinuous i.e., CMC is semi-continuous in nature),

000206] fn some preferred embodiments.. C C ^" employs three separation units, each containing the same chromatography media, and where the separation units are connected such that, liquid can flow from one separation unit to the next separation unit and horn the last to the Of si separation unit, where the sample Is loaded onto the llrst separation unit at a pH and conductivity -which enables binding of the target molecule to the separation unit and where at least part of duration of loading time overlaps with the loading of the consecutive separation unit, where the two separation units are in fluid co man feat ion, such that to enable any target molecules that do not bind io the -first separation, unit being loaded to bind to the next separation unit.

[000207] Different separation units can he at different stages of the process at any given time; i.e., while one separation unit is being loaded, the next separation unit, could fee subjected to washing, dating, re~eqto ibration etc. Also, while the first separation uni Is being subjected to the washing, ebbing, re-equi librating steps, the consecutive separation unit is subjected to the loading step and so forth, such that the sample flows continuously through the separation units and has a velocity above S00 em/h and. hat the chromatography media of the separation units comprises particles with a diameter between 40 and 200 gm and with pore diameters in the range between 50 nm and 200 run.

[000208 J in some embodiments, each separation unit includes an affinity chromatography media suc as. e.g.. Protein A based media. In other embodiments, each separation uni Includes an ion exchange media (e.g., a cation exchange chromatography media) or a mixed-mode chromatography media.

4,y 000209] Exemplary continuous chromatography processes, which may be used in the bind and elute chromatography process step, as described herein, can be found, e.g., in Euro ean Patent Application Nos. E 1 1008021 and EP 12002828,7, both incorporated by reference herein.

[000 10] In sonic embod ments the separation units are connected in a circular manner, also referred to as a simulated moving bed. For example, in certain, instances, at least three separation unit are connected in a circle ami the loading of the sample is shifted sequentially from one separation unit, to the next, e.g., as described in European Patent. No. 204081 1 , incorporated by reference herein.

[00021 I ) I has been found that running hind and elute chromatography in a xenu-conthtuons or continuous mode enables using a reduced volume of an affinity media, by up to 90% of the volume used in a conventional process. Further, separation units with, a reduced diameter, between one third and one tilth compared to a batch, process, can he used. The separation units can be te-osed multiple times within the processing of a particular batch of a target molecule, e.g., during the batch production of a target molecule which is a therapeutic candidate.

[000212] In some embodiments, a separation unit that is being loaded with a sample is in fluid communication with another separation unit over the entire duration, of the loadi ng time.

[000213] In other embodiments, the separation unit that is being loaded is in fluid communication with another separation unit ibr onl part of the duration of the loading time. In some embodiments, two separation units are in fluid communication only tor a second half of the duration of the loading time,

[00021 ] In a batch chromatograph mode, typically loadin of a separation unit (e.g., a chromatography column) is stopped, prior to an excess of target molecule saturating the separation unit. In contrast,, m case of a CMC bind and elute

chromatography process step, as used in the processes and systems described herein, the loading of a separation unit does not have to be stopped as target molecules that do not bind to one separation unit move on to the next separation unit because of fluid communication between, the two separation units, where the outlet of one separation unit is connected with the inlet of a second separation uni t and so forth. It is understood that a person skilled in the art can readily determine when during the loading step., the amount of a target molecule that is not bound, to the separation unit that is being loaded is sufficiently high, such that the outset of the separation unit being loaded needs to be connected to (he inlet of another separation unit. It has been found that this embodiment Is especially effective if ihe separation units comprise media having a particle di meter between 40 and 200 pm and pore diameter ranging from SO to 200 nm. With sue! media, the loading feed am be run continuously at a velocity above 800 om/h. Further details can he found in £1*12002828,7, filed on. April 23. 201 , In some embodiments, ibc outlet of the separation unit or the separation units that are being washed is in a !lnkl communication with the previous separation, unit so that target molecules removed by said, washing are not lost but loaded onto the previous separation unit,

[000215 j I t lias been found that the level of mpuriti s (e.g., HCPs) that end up in the elutioo pool containing the target molecule ean be .significantly reduced with use of certain additi es to the sample load during bind, and elute chromatography. In fact, addition of certain additives io the sample prior to loading or during the loading of ihe sample may obviate the need to use specific wash steps typicall designed to enhance impurity clearance. In other words, the number of wash steps that are typically used is reduced by inclusion of certain additives prior to loading or during the loading of the sample,

[00021 ] In the context of continuous chromatography, a Protein. A column that has completed the loading step and is moved to subsequent z nes is required to complete ail necessary steps within a. time frame expected such thai the column will be ready to accept fresh loading solution. e,g,. as described herein, can be found, e.g.. in European Patent .Application os. EPl 1008021 ,5 and BP 12002828.7, both incorporated by reference herein. The time that is required to complete all necessary steps depends on the number of steps or zones that the column must go through to be ready for loading again. By reducing or eliminating steps, such as intermediate, washing., ihe application of continuous chromatography for higher titers (target protein concentrations) is enabled where the loading phase is expected to be shorter as well as simplifies the timing requited for all titer conditions during continuous chromatography.

[00021 ] Exemplary additives which may be employed to reduce or eliminate one or more intermediate wash steps include, but are not limited to, salts, polymers, surfactants or detergents, solvents, ehaotrople agents and any combinations thereof A "sail ^" is a compound formed by the interaction of an acid and a base. Examples of salts include any and all chloride salts, sulfate salts, _: phosphate salts, acetate and/or citrate salts _* e.g., sod um chloride, ammonium sulfate, ammonium chloride, potassium chiorkle, sodium acetate. In a particular embodiment, the s lt is aCI (e.g., added to a final concentration of 0,5 M aCi), The term "hydrophobi salt' ^' refers to a spec fic salt type with a hydrophobic component suc as, alkylanno.es; telram.ethyiammooium chloride (JMACX ietraethylamt?K>mam chloride (TEAC), ieirapmpylammo um chloride and teirateiy iammoni um chloride. As used herein, & ^polymer" is 8 molecule fanned by covalent linkage oi ^' two or more n-onomeoy where the m nomers are not amino acid residues. Examples of polymers include polyethylene glycol (PEG), propylene glycol, and copolymers of ethylene and propylene glycol (e,g., Piuronics, PP68, etc,}. In a particular embodiment, the polymer is PEG,

[000218] The term ^detergent ^" .refers to surfactants, both ionic and nonionie, sweh as poiysorbates (e.g., poiysorbates 20 or 80); poloxaimers. (e.g., poloxamcr 1 S8); Triton; sodium dodeeyi sulfate (SOS); sodium iauryl sulfate; sodium oolyi glycoside; lauryk myristyk linoieyk or steary nlfbbetaine: (see US6870034B2 for more detergents}. In a particular embodimen the detergent Is a polysorbatc, such as polysorbate 20 (Tween 20).

[000219] The tenn ^solvent" refers to a liquid snbstanee capable of dissolvin or dispensing one or more other substances to provide a solution. ! n some

embodiments, the solvent is an organic, non-polar solvent such as thanol, methanol, isopropanoi aeetonitrlle, hexyiene glycol, propylene glycol and 2,2-ihtodigiycol. The enn "Thaotropic salf ^v refers to a salts that Is known to disrupt the ime r okcu!ar water structure. An example of such a salt is urea and guanidinium. HCL

[000220) In some embodiments, the one or more additives are mixed continuously with a clarified ceil culture using one or more static mixers.

Accordingly. In some embodiments, a clarified ceil culture sample continuously flows to the Protein A chromatography step in a pro ein purification process, where one or more additi es, a described herein:, are continuously mixed, with the clarified- ceil culture prior to loading onto a Protein A chromatography matrix. kj us mackadon

[00022 1 ] hi some embodiments according to the processes and systems described herein, hind and elute chromatography is followed by virus irsaerivaiion (V I ). it is understood (hat. virus inactivafion may not necessarily be performed but is considered optional.

[000222] referably, th output or eh.tate from bind and ehste chromatography is subjected to virus inaetivatkm. Viral inaeiivatkm renders viruses inactive, or unable to infects w ich Is important, especiall in ease he target mokcuie is intended for therapeutic use.

[000223] Many v ruses contain lipid or r tein coats that can be inactivated by chemical alteration, Rather than simply rendering the virus inactive, sosne viral inactivaiion processes are able to denature the virus completely. Methods; to inacti vate viruses are wed known to a person skil led in the art. Some of the snore widely used virus inactivaiion processes .include, ^' e.g., use of one or more of the following: solvent/detergent ina.ciiva.tion (e.g. with Triton X .!00); pasteurisation (heating); acidic pi! inactivaiion; and ultraviolet (UV) ioaeiivation. It Is possible to combine two or more of these processes; e.g., perform acidic pH inactivaiion ai elevated temperature.

[000224] In order to ensure complete and effective virus inactivaiion, virus inactivaiion is often performed over an extended period of time with constant agitation to ensure proper mixing of a virus inact vaiion agent with the sample,. For example, in many large scale processes used in the Industry today, an output or e rate from a capture step is collected in a pool tank and subjected to virus inactivaiion over an extended period of time i e.g,, >! to 2 hours, often followed by overnight storage), [000225] in various embodinieuts described herein, the time required for virus inactivaiion Is significantly reduced by performing virus inactivatlon in-line or by employing a surge tank instead of a pool tank for this step,

000226] Examples of virus inact.lvat.ion techniques that can be used in the processes described herein can be found, e.g., in PCT Patent. Application No

PCT/IJS2013/4567? (internal ref no. P 12/098PCT).

1000227] In some preferred embodiments, virus mactivatton employs use of acidic pi t where the output from the bind and elute chromatography step is subjected to inline exposure to acidic pH for virus inaehvatfon. The pEI used for virus tnaetivation is typically less than 5,0, or preferably between 3,0 and 4.0. In some embodiments, the pi! is about 3.6 or lower. ^' The duration of time used for virus inactivaiion. using an inline method can range from i t) minutes or less, 5 minutes or less, 3 minutes or less, 2 inutes or less, to about I m nute or l ss. In ease of a surge tank, the time required tor inaetivatloo is typically less than 1 hour, or preferably less than 30 min tes,

[000228] la some embodiments described herein., a suitable virus inaetivatlou agent is bUroduced in-line brio a tube or connecting line between bind and. eiute chromatography and the next unit operation in the process ⁽ e.g.. flow through purification), where preferably, the tube or connecting, tine contains a static mixer which ensures proper mixing of the output m en the hind and eiute chromatography process step with the virus inactieation agent before the output goes on to the Bex! unit operation. Typicall , the output from the bind and eiute chromatography flows through the static mixer at a certain flow rate, which ensures a minimum contact time with the virus inactivation agent. The contact ime can he adjusted by using static mixers of a certain length and/or diameter,

(§00229] in some embodiments, a base or a suitable buffer i additionally introduced into the tube or connecting line after exposure to an acid for a duration of time, thereby to bring the pE of the sample to a suitable pi i for the next step, where the pH is not detrimental to lire target molecule. Accordingly, in some preferred embodiments,, both exposure to a low pE as wel as that to a basse buffer is achieved in-line with mixing via a static mixer.

000.230] In some embodiments, instead of an .in-line static mixer, or in addition to an in-line static mixer, a surge tank is used for treating the output from bind and elate chromatography with a virus inaeUvation agent, where the volume of the surge tank is not more than 25% of the total volume of the output from bind, and eiute

chromatography or not more than 15% or not more than 10% of volume of the output iron) hind and eiute chromatography. Because the volume of the surge tank Is significantly less than the volume of a ty pical poo! tank, more efficient m ix ing of the sample with the virus inactivation agent can be achieved,

0002 1 ] in some embodiments _* virus inaetivation can. be achieved by changing the pH of the elutiou buffer during _, bind and eiute chromatography, rather than havin to change pH of the output from bind and eiute chromatography.

00O232| In sonre embodiments described ^' herein, the sample is subjected to a flow-through purification process, following, virus hiaetivatlon. Its some

embodiments, a filtration step may be included after virus inactivaiion and before iiow-through purification. Such a step may be desirable, especially in eases w here turbidity of the sampl s observed following vims i motivation. Such -a filtration stop may emplo a microporoos fitter or a depih Biter.

1000233 Although, .in processes where virus inaeiivatiors ste Is optional, the output Iron? bind and eiute chromatography may be directly su jected to- flow-through purification.

[000234] in various embodiments described herein, the output from bind and ehue chromatography is subjected to a flow-through purification, operation either directly, or following virus inactivation. I some embodiments, flow-through purification operation used m the processes and systems described herein employs two or more process steps or devices or methods or achieving tlow-through purification, which Is intended to remove one or more impurities present in the output from bind and elute chromatography, with or without virus inaetivatkm.

I ^' 00 235 ^' l In some pre errec; emb d ments, flow through purification operation, as described herein, includes one or more of the following steps performed in a flow- through mode: activated carbon; anion exchange chromatography; cation exchange chromatography, mixed mode chromatography, hydrophobic interaction

chromatography and virus til trail on. or combinations thereof In some embodiments, one or more valves, in-line static misers and/or surge tanks may he used between two or more of these steps, in. order to change solution conditions,

1000236] The various steps, one or snore of which ma be used, to achieve flow- !h ough purification, are described in more detail infra.

[000237] As described herein, in some embodiments. In some preferred embodiments, flow-through, purification employs at least one fiow-ihrough anion exchange chromatography (AEX) step, where one or more impurities still remaining in the sample containing the target molecule bind the anion exchange chromatography media, whereas the target molecule flows through,

[000238] In some embodiments, flow-through mixed-mode chromatography or flow-through hydrophobic interaction chromatography may he used instead, or in addition to flow-through anion-exchange chromatography.

000239] Exemplary anion exchange media which may be employed, for AEX chromatography, include, hot are run linnted to, sneh as those based on quaternary ammonium ions, as well as weak ani exchangers, such as those based on primary, secondary _* and tertiary amine. Additional examples of suitable anion exchange media ate Q SepharoseS available from ( E Healthcare BkvSeienees A8 _S P aetogei I ^' M Ah: and Eshmuno Q available Irom HMD Chemicals, Mwstang¾ ^: Q available from Pall. Corp.. Sartohind® Q available from Sarterlus Siedim, and <¾xy«aSorb ^' * ^M devices available from EMD !vlillipote.

[000240] The media can be in the form of particles, mem a es, porous materials or monolithic materials, in preferred embodiments, media are membrane based, matrices, also called membrane adsorbers. The membrane adsorber is preferably a porous membrane sheet made by phase separation methods well known in the art. See, for example _* Zeman L i, Zydney A L, Mieroftltration and IJltrallltrauon:

Principles and Applications, New York: Marcel Dekker, 1 9b, Hollow fiber and tabular membranes are also acceptable matrices. The membrane adsorbers typically have a bed height of 0,5 to 5 mm,

[0002 ! ] Membranes ca be omnuiaet red. from a. broad range of polymeric materials known in the art, including polyoleflns, such as polyethylene and polypropylene, poiyvmylldene fluoride, polyamide. poiytetrailuoroethylene, cellu osics, polysuifone, polyaerylonitrile, etc,

[000242] In order to impart anion exchange properties, the surface of the membranes is usually modified by coating, grafting, adsorption, and plasmadnitiated modification with suitable monomers and/or polymers.

[000243 ] in some embodiments, the anion exchange media that is used lot flow- through anion exchange is a membrane based media having a surface- coated ^" with a cross! inked polymer having attached primary amine groups such as a polyailylamine or a protonated polyailylamine.

[000244] Additional suitable media can. be found in, e.g., U.S. Patent No.

J 37,361. , incorporated by reference herein, which describes porous chromatographic or adsorptive media having a porous, polymeric coating formed on a porous, sell- supporting substrate and anionic exchangers Including such media as well as use methods of parHying a target molecule using such media Such media are particularly suited lor the robust removal of low-level impurities- from oianufaeiured. target molecules, such as monoclonal antibodies, in a manner that integrates well Into existing downstream: purification processes. Typical Impurities include QNA.

endotoxin, H P and viruses. Such media function wed at high salt concentration and high conductivity (high affinity), effectively removing v ruses even under such con itions. High binding capacity without sacrificing device permeability ^' is achieved. Indeed, depending on the coating properties, nucleic acid binding capacities of greater than about 5 mg/mL, or greater than about 25 rng/mL < or greater than, about 35-40 mg/mL, may e achieved. The amou of the anion han e adsorber i much less than that used for a comparable head-based process,

|{M)0243] hi some embodiments, the membranes having art anion exchange functionality are encapsulated to. a suitable muftl-kyer device providing uniform flow through the entire stack of the membrane. The devices cart be disposable or reusable, and can either be preassembied by the membrane manufacturer or assembled by the end user. Device housing materials include thermoplastic- resins, such as

polypropylene, polyethylene, polyxuMbne, poiyvmylidene fluoride, and the like;

thermoset resins such as acrylics, silicones and epoxy rosins; and metals such as stainless steel, ^' The membrane can either be permanently bonded to the device housing, such as by using an adhesive or thermal bonding, or held in place by compression and carefully placed gaskets.

[000246] in some preferred -embodiments, the anlon-exchaoge adsorber device is used at the solution pH value that is at least 0,5- .1 ,0 units below the isoelectric point of the target, protein. The preferred phi range of amoo-exehange adsorber device is from about 6 to about 8, Suitable range of salt concentration is betwee 0 and 500 m.M snore preferably between 10 and 200 mM,

[00024?j ¾ some embodiments, itow-throngh purification may employ additional steps. For example, In a preietTed embodiment, one or more additional flow-through steps are used to addition to anion-exehange chromatography (AEX). The additional flow-through steps include, e,g„ mixed-mode chromatography, cation exchange chromatography, hydrophobic interaction chromatography, activated carbon, size exclusion, or combinations thereof

[00024S] Additional steps which may be included in flow- hrough purification include, e.g.. use of acti ated carbon prior to araon-exchange chromatography or alter anion-exchange chromatography (and/or one or more of mixed mode and HIC), It some embodimerits, activated carbon is incorporated into a cellulose media, e.g„ in a column or a device, Alternatively, activated carbon can be combined with an. anion- exchange media (e.g., in a column er a cartridge), thereby to further remove one or more impurities from a sample containing a target molecule. The column or cartridge may also be disposable:, e.g., MillistakD Pod, The media an be in. the form of particles, membranes, fibrous porous materials or monolithic materials. In case of activated carbon, it can be impregrsated into a porous material, e.g., a. porosis fibrous material.

[000249] It has been .found thai a ll w through activated carbon ste prior to ihe flow-through anion exchange chromatography is especially suitable for the removal of host ceil proteins and leached Protein A, It is also capable of removing a significant amount of potential imparities from cell culture media, such as hormones, surfactants, antibiotics, and amiioam compounds.. In addition, it has been found that an activated carbon containing device reduces the level of turbidity in the sample, for example generated during pB increase of Protein A e!ution tractions,

[000250] Further details about carbonaceous materials, activated carbon, and their use in flow-through purification processes can be found in PCI ^' Publication No.

WO2013/028310, which ½ hereby incorporated by reference,

[000251 } As discussed above, the How-through purification operation used in the processes and systems described herein may include more than one flow-through step.

[000252] In preferred embodiments, .flow-through purification further includes one or more additional flow-through steps, e.g., .for aggregate removal and v irus filtration. In some embodiments, the sample is passed through an adsorpfive depth fi}ter _s or a charged or modified rnieroporous layer or layers in a norma), flow filtration mode of operation, lor aggregate removal Example of flow-through step which may be used lor aggregate removal can be found in, e.g.,, U.S. Patent Nos, 7,1. 1.8,675 and 7,465,397, incorporated by reference herein. Accordingly, In some

embodiments, a two-step filtration process for removing protein aggregates and viral panicles may be used, wherein a sample is first filtered through one or more layers of absorptive depth filters, charged or surface modi fled porous membranes, or a small bed of chromatography media to produce a protein aggregate-fee sample. This may be followed by the use of an ultrafiltration memb ane for virus filtration, as described in more detail below. Ultrafiltration membranes used tor virus filtration are typicall referred to as nano.filfrat.km membranes.

[00025a] In some embodiments, an additional fiow-thr ugh step employs a cation exchange chromatography (CfiX) media. Further details about cation exchange How through devices and their use in flow-through purification processes can. be found In I S, Patent Application No. 13/783,94! (internal rel no. MCA- 1423), incorporated by reference herein. Accordingly, in sonic embodiments, a cation exchange chromatography media thai is used after the anion exchange chromatography step em l s a solid support containing one or more cation exchange binding groups at a density of 1 to 30 mM. Such solid supports are able to bind protein aggregates relative to monomers at a selectively greater than about 10.

[(100254] Its some embodiments., a negatively charged tlhration medium may he used for removal of protein aggregates, e,g., comprisi ng a porous substrate coated with a negatively charged polymerized cross-linked acrylarnidoaJkyl coating, polymerized In situ on the surface of the substrate upon exposure to an electron, beam, and in the absence of a chemical polymerization free- adical initiator, where the coating is formed from a. polymeri¾¾hle acrylamidoalkyi monomer having one or more negatively charged pendant groups and art aer lamido cross-linkin agent. Additional details concerning such filtration media can be found, e.g,, in PCX

Publication No, WO.2010/098867. incorporated by reference herein.

[000255] The use of a flow-through cation-exehange step (CEX) may necess tate a reduction of solution pi l to increase affinity and capacity for imparities, such as antibody aggregates. Such pfl reduction can be performed by a simple in-line addition of suitable solution containing acid, via a three-way valve, a Ί ^" -style connector, a static mixer, or other suitable devices well known in the art, in addition, a small surge vessel can be employed io provide additional mixing and access for sampling, The volume of the surge vessel, which can be in the form of a bag, a container, or a tank, is usually considerably smaller that the volume of the fluid processed with flow-through setup, for example not more than 10% of the volume of the fluid,

[0002561: In some embodiments, the cation exchange media removes protein, aggregates and/or acts as a preo'iher lor a virus-filtration membrane, typically used after cation exchange chromatography.

[00025?] In another embodiment, protein aggregates can be removed using a composite filter material that comprises a calcium phosphate salt. Suitable calcium phosphate salts are dicaleium phosphate anhydrous, dicalcium phosphate dehydrate, tricaktum phosphate and tetraealeium phosphate. In another embodiment, the calcium phosphate salt is hydraxyapaiite. The solution conditions are typically adjusted prior to loading the sample on such device, in particular concentrations of phosphate ion and the ionic strength. Further details about the removal of protein aggregates using a composite filter material that omprises a calcium phosp ate salt in J¾yw-ihro«gh mode can be found in WO201 1 156073 Al , which is Incorporated by reference herein.

£000258] ^' ke entire i!o Ahrougb purification operation (Including the anion exchange chromatography step and one or more additional steps, as described r in), are performed continuously without the use of a pool tank between flow-through process steps,

0002S9] in some embodiments, the fiow-dirough purification process additionall includes virus filtration. However, virus filtration is optional and may not necessarily always be used

H)OO200] In some embodiments, virus filtration involves filtration based on stee exclusion, also referred o as sieving,

£0002611 For virus removal,, the sample is typically passed through an

ultrafiltration fil er thai retains the viruses while the target molecule passes through. According to IUPAC, ultrafiltration is a '"pressure-driven membrane-based separation process in which particles and dissolved maeromoleeules smaller than 0.1 pni and. larger than about 2 nm are rejected/' (IUPAC 'Terminology tor membranes and membrane pr cesse ^*' published in Pure App!. Chern., 1096,- 68. 1479), The ultrafiltration membranes used in this step are usuall specifically designed to remove viruses. In contrast to ultrafiltration membranes used tor protein concentration and. buffer exchange, these membranes are usuall not characterized by the molecular weight eut-ofts, bat rather by typical .retention of viral particles. Viral retention is expressed in log reduction value (L V , which is simply a Lo j;) of the ratio of viral particles in. iced and filtrate in a standardised test. Use of viral filtration in

purification processes can be found in, e.g., Mekzer, T,, and j rnitz. M. eds,, "Filtration and Purification in the Blopharmaeeutieai industry ^" , 2nd eel., Inibrma Healthcare, 2008. Chapter 20,

[000262] Virus-retentive membranes can be rnantitaetured In the form of a fiat sheet, such as VI resolve® Pro from HMD MUlipore Corporation. Idtiporf? VP Grade DV20 from Pali Corporation, Yirosart® CPV ifean Sartoritzs Stedini Biotech, or in the form of hollow fiber, such as Planova ^'m 20N fr m Asahl Kasei Medical Co. They can he single-layer or multi-layer products, and can he manufactured by one of many membrane production processes known in the art. A particularly beneficial combination of throughput retention can be achieved for asymmetric, composite virus-tv eniive membrane, as described in U.S. Publication No, 20120076934 A ^' L incorporated b reference herein,

1 00263] In a particular embodiment, the i!owohrongb purification operation involves at. least an activated carbon ste , an. anion exchange chromatography ste , a cation, exchange chromatography s ep and a vims filtration step,

[000264] Following virus filtration, the sample containing the target molecule .may ^¬ be subjected to one or more additional formulation/eoneentration steps,

.A.d4 i 0. L lW S. cp.§

[000265] As discussed above, in some embodiments, the sam le is subjected to one or more addi ional process steps following virus filtration.

[0002661 in some embodiments, the one or more additional steps include formulation, which may employ diallltration/eoncentration followed by sterile filtration.

[0002671 In some embodiments, following virus filtration, the sample containing target, molecule is subjected to dlailhratiom which typically employs the use of an ultrafiltration membrane in a Tangen ial Flow Filtration fiW} mode, in ease of Tangential Flo Filtration TFF), the fluid is pumped tangentially along the surface of the filter medium. An applied pressure serves to force a portion of the fluid through the filter medium to the filtrate side,

[000268] Diafiliration results in the replacement of the fluid which contains the target molecule with the desired buffer for formulation of the target molecule.

Diaiiltration is typically followed by a step to concentrate the target molecule, performed using the same membrane.

[000260] In another embodiment, single-pass tangential flow filtration (SPI FF) can be used for concentration/diafiltration. A SPIFF module includes multiple ultrafiltration devices connected in scries. The target protein is sufficiently

concentrated/diafiitered after single pass through the SPTf F module without the need lor a retentate loop and pump, enabling continuous operation. More information can be .found in the presentation entitled ""Single pass IFF ^* by Herb Lufe et aL, presented at the American Chem ical Society conference in the spring of 2 1 ! .

[000270] Following diafiltratlon/concentratlon, the sample Is subjected to a sterile ^• filtration step lor storage or any other use. [000271 j Sterile fi tration is typically carried out using Normal Flow Ri.tMi.on ( FPk where the direction of the fluid stream is perpendicular to the filter medium (e.g. a membrane) under an applied pressure. v¾;MsmAs ^

1000272] ^' The present invention also provides systems for purifying a target molecule, wherein the systems include two or more unit operations connected to be in fluid co.rn.municat.io.» w h each other, such thai to perform a process for purifying target molecule in a continuous or scmi«>ntinuoas manner. Each unit operation ma employ one or .more devices to achieve the intended purpose of that unit operation. Accordingly, in some embodiments, th systems described herein, include several devices which are connected to enable the purification process to be run in a continuous or semi-continuous mariner,

[000273} Without wishing to be bound by theory, it is contemplated that a system can be enclosed in a closed sterile environment, thereby to perform the whole purification process in a sterile manner.

1000274] In various embodiments, the very first device in a system is a bioreaetor containing the starting ma e ia , e.g.. cuituring cells expressing a protein to he purified. The hioreactor can he any type of bioreaetor like a batch or a fed batch bioreaetor or a continuous hioreactor like a continuous perfusion ienueutaiion hioreactor. The hioreactor can be made of any suitable material and can be of any ske. Typical materials are stainless steel or plastic. In a preferred embodiment, the bioreaetor is a disposable bioreaetor, e.g. in form of a flexible, collapsible bag, designed for single-use,

b:)00275] Clarification may be performed directly in the bioreaetor, or

ahernatsvely, the bioreaetor can simply he used for cuituring the cells, and clarification is performed in a different vessel.. In yet another embodiment, the cell culture is simply flowed, through a depth filtration device in order to remove one or more impurities. Accordingly, in some preferred emhodirnents, the bioreaetor is in .fluid communication with a device for pe.rfonni.ng depth .filtration.

[000276] The device lor performing clarification (e.g., a depth filtration device} is generally connected to he i fluid communication with a device for performing capture using a bind and elate chromatography (e.g., a continuous multi-column chromatography device comprising two or more separation units). In some ent ilments, the dev ce lor bind and elute chromatography is connected to be In fluid communication with a unit operation for performing flow-through purification, which ma include more than one device/step. In some embodiments, an in-line static mixer or a surge tank is included between the device tor bind and el te

chromatography and the first device used for ilow-th ugh pnrifseatron,

0002?7] In some embodiments, the flow-through purification operation includes more tha one device, e.g., an activated carbon device followed by a AEX

chromatography device followed by an in-line static mixer and/or a serge tank for changing pfl, lb Π owed by a CEX chromatography device followed by a virus Hitration. device. The devices could, generally be in any suitable format, eg., column or a cartridge.

1000278] The last unit operations In the system may include one or more devices for achieving formulation, which includes diafllttatfon/concenttation and sterile filtration,

1000279] Typically, each device includes at least one inlet and at least one outlet, thereby to enable the output from one device to he in fluid communication with the Inlet of a consecutive device in tire system.

|(MK} ^' 280j In most processes and systems used In the industry today, each device used in a purification process employs a process equipment unit, also referred to as a '"skid, ^" which typically includes the necessary pumps, val ves, sensors id device holders. Typically, at least one sbid is associated with each device. In some of the embodiments described herein, the number of skids used throughou the purification process is reduced, for example, in some embodiments, only one skid is used to perform the entire flow-through purification operation, which may Include multiple devices, e.g., activated carbon device, anion exchange chromatography device, cation exchange chromatography device and virus filtration device, along with any ^• equipment needed for solution condition changes. Accordingly, in some

embodiments, a single skid may be used for all of the foregoing steps in .flow-through purification.

101)0281 In. some embodiments, fluid communication between die various devices is continuous; in that the fluid flows directly through all the devices without interruptions. In other embodiments, one or more valves, sensors, detectors, surge tanks and equipment tor any in-line solution changes may be included between, the various devices, ihcreby to temporarily interrupt the iiow of fluid through the system, if necessary, for ex m e, to replace/remove a particular device.

[000282] in some embodiments, one or more surg tanks are included between, the various devices, in some embodiments, not mors than 3 and not more than 2 surge tanks are present in the entire system. The surge tanks located between different devices have no more than 25%, and preferably no snore than 10% of the entire volume of the οι ρι from the first of the two de vices.

[000283) In some preferred embodiments, the s stems described herein include one or more static mixers for buffer exchange and/or in-line dilution,

000284] hi some embodiments, a system further includes one or more sensors and/or probes lor controlling and/or monito.ri.ng one or more process parameters inside the system, for example, temperature, pressure,, p i conductivity, dissolved oxygen (DO), dissolved carbon dioxide IXTb), mixing rate, flow rate, product parameters. The sensor ma also be an optical sensor in some cases.

[000285] In some embodiments, process control may be achieved in ways which do not compromise the sterility of the system,

[000286] in some embodiments, sensors and/or probes may be connected, to a. sensor electronics module, the output of which can be sent to a. terminal board and/or a relay box. The results of the sensing operations may be input into a computer- implemented control system (e.g., a. computers lor calculation and control of various parameters (e.g., temperature and weight/ -oiume measurement , purity:} and for display and user interface. Such a control syst m may also include a combination of electronic, mechanical, and/or pneumatic systems to control, process parameters, h should be appreciated that the control system may perform othe inactions and the invention is not limited to having any particular function or set of functions,

0100287] This invention i further i llustrated by the following examples which should not he construed as limiting. The contents of ail references, patents and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference.

5b Exam l s

Example I t Pr eess for Pynfysng a M o lon l Αηϋ <κΙ 000288] in this representative example, ihe purificatiort of a monoelocal antibody is ^■ achiev d using a purification process in a continuous manner, where various unit operat ns are connected in a manne to operate commnously. An exemplary process is depicted in f gure 2,

[000280] The representative example described below includes the following steps performed in the sequence listed: clarification using depth filtration; use of one or more in-line static mixers to change solution conditions: Protein A bind and e!u e chromatography using continuous ra lttcoiu n chromatography which employs two separation units; pH adjustment of the output using. one or more static mixers; and f!owOhrougli purification which employs depth filtration followed by activated carbon followed by anion exchange chromatography followed by pH adjustment using a static mixer followed by cation exchange chromatography followed by virus filtration.

1000290] In this example a CHO-taaed monoclonal antibody MAb05) is produced in a fed hatch bioreaotor. Approximatel 5.5 I. of cell culture processed with a 0.054 rrfi DOFiC CEMD Miliipore) primary depth biter then further clarified with a 0.054 ob X0HC (HMO Mi!lipore) seeoruiary depth filter where both are processed at a 10 L H flux making the loading app ximately 100 h/tnfi

[0002011 The effluent from the depth filtration is contacted with, a 5 M NaCl solution at a I ; 10 ratio that is then mixed through a static mixer followed by a sterile fiber, ihe ressure is monitored prior to each depth filter and after five sterile filter (Figure 3). Following the static mixer, the solution is passed through a SHC sterile filter (HMD Miliipore) to a final loading of 3200 L/ni2> The effluent from the sterile filter is directed to a surge tank that is monitored with ad ceil to determine the amount filtered. One nth samples arc collected just prior to each load cycle on Protein. A continuous muhi-eolumn chromatography (CMC) (Figure 4). After approximately 70 ml, of cell culture is processed and collected in a surge tank., the clarified solution is simultaneously loaded info the next step for Protein A capture. 1000202] Protein A capture consists of two Protein A columns running on a modified Akta Explorer 100. The Protein A columns have 10 mi, of PfoSep Ultra Plus Protein A media packed into 1 ,6 cm ID Vantage -L (EMD Miliipore) chromatography columns to bed heights of 10.25 and 10.85 cm. The columns are equilibrated with 1 X. PBS, 0.5 M ^' NaCi lor .5 colum volumes (ail column volumes are based on the smallest column). Throughout the run, the loading, flow rate i set so as to have a loading residence time of ! minute. Daring t e .initial loading both columns are placed in series, where the effluent of the primary c lumn Is loaded directly onto the secondary column until a specific load volume is reached. Alter a specific loading volume is passed over the columns, the feed is stopped nd 2 column v lumes (CVs) of the equilibration buffer is passed through the primary column to the secondary column. The primary column is then positioned to undergo washing, elation cleaning and reeqnilibration. while the secondary column is loaded as the primary column. Following the reequilibratioo of the first col mn, the column is then moved to the secondary position to reside in series with the now pr mary column. This serie of events is repeated with each column taking the primary position alter the original primary position column is loaded to a set volume. The first column is loaded a total of three times and the second, column is loaded twice. The e!utions from each column are collected into a beaker with mixing, using a U V trigger to control the start and end time of the elu ion.

[000293] Alter the first two eiuiions arc collected and pooled, the solution is pumped out int a surge tank and. mixed with a 2 solution of iris and. processed through two static mixers to increase the pH to pH 8.0, where the pH of the resulting solution is measured immediately alter the static mixers. The pH adjusteil solution is then processed through m A1 HC deptli filter (EMD illipore) followed by a I em 1.0 Omni fit column packed with activated carbon. The efi!uent from the activated carbon column is then continuously flowed through an anion, exchange chromatography device (e.g., Ch.romaSo.rh ^' ™) (HMD Millipore) to a loading of 4 kg of MAb/L of €hro aSorb ^T , The effluent from the€hrorn.aSorb ^M anion exchanger is then, mixed with 1 M acetic acid, then processed through a static mixer to lower the pH to pH 5.5, The p! l~ad]nsted solution from the static mixer is then flowed through a cation exchange chromatography device used as a prefilter, followed by virus filtration using the Vireso!ve Pro membrane (EMD Millipore), The effluent from the virus filter is directed to a pool tank and sampled.

1000294] This purification process-provides final solution that meets all purification targets, specifically HCP < I ppm, aggregates 1 % with a ouVbOS recovery 60 % fo the overall process. Example 2: Process for Purifying $ Monoclonal Aaiibody

[000295] In this representative example, the purification of a monocloeal. antibody is achieved using a purification process, where various unit operations are onn cted in the se uence described below.

[000296] lilie representative example described below includes the following steps performed in the sequence listed: clarification using stimulus responsive polymer following centri ugation; contacting the supernatant with salt; Protein A. bind and elute chromatograph using continuous muStieolumn chromatography which employs two se aration units; pi 1 adjustment of the output using one or more static mixers; and flow-through purification which employs depth filtration, followed by activated carbon followed by anion exchange chromatography followed by pH adjustment lift ng a static mixer followed by cation exchange chromatography followed b virus filtration.

[000297) In this example, a CHO-hased monoclonal antibody (MAbOS) is produced in a fed-batch bioreactor, .A total of 7 liters of cell culture is contacted with a solution of a stimulus responsive polymer (modified polyailylanfine: responsive to salt addition) to a final polymer concentration of 0,2 % v/v. The cell culture is mixed with the stimulus responsive polymer solution for approximately 10 minutes. About 1.75 nfi. of of 2 M !GHPO,* solution is added and the cell cut tare is mixed for an additional 10 minutes. The pH is then raised to 7.0 by adding 2 bis base id mixing tor 1 5 minutes. The solution is then cent.rifu.ged in 2 L aliquots ai 4.500 g for 10 minutes and the supernatant is decanted and retained. The solids are disposed off. The cell culture supernatant is pooled and then mixed with S M aCi at a 1 : 10 ratio in a batch mode with continuous stirring. The final conductivity of the solution is measured at this point and Is at 55 5 mS/erm The resulting solution is sterile filtered through a 0.22 μη¾ Express SBC filter (HMD Millipore), The steri le filtered solution is the loading materia! for tlie Protein A c romatography,

[000298] The Protein A capture step consists of two Protein A columns running on a modified Akta Explorer 100, The Protein A columns have 1.0 ml, of PtoSep Ultra Plus Protein A media packed into 1 .0 cm I D Vantage- L iE D Mf liipore) chromatography column to bed heights of 1 ,25 and 10,85 em. The columns ore equil ibrated with 1: X T BS. 0.5 M NaCI tor 5 column volumes, CVs (all column volumes are based on the smalks column), Throughout the run, the loading flow mie is set so as to have a loading residence time of about one mi nine.

00 299] During the initial loading, both colum s are placed m series, where the effluent of the primary colu n is loaded directly onto the secondary column until a specific load volume is reached. After a specific loading volume is passed over the c lumns, the feed is stopped and two CVs of the equilibration buffer is passed through the primary column to the secondary column. T he primar column is then positioned to undergo washing, elution, e leaning and reeqmlibration, while the secondary column is loaded as the primary column, hollowing the reeqissiihraooo of the first column, that column is then m ved to the secondary position to reside in series with the now primary column. This series of events is repeated with each column taking the primary position after the original primary position column is loaded to a set volume. Each column is loaded a total of seven times. The ehitions from each column arc collected with a fraction collector, using a !JV trigger to control the start time of the ehstion and collected to a constant volume of approximately 3,5 CVs.

[000300] b low-through purification comprises of six main steps; depth filter; activated carbon;anion exchange chromatography; in-line p adjustment; cation exchange chromatography; and virus filtration.

{ ^' 00030.1 ] Figure 5 illustrates the order in which these steps are connected. The necessary pumps and sensors, e.g., sensors tbrpressure, conductivity and U V are also shown in the schematic,

{000302] Alt devices are individually wetted at a different station, and then assembled as shown in Figure 5. The devices are wotted and pre-treated according to the manufacturer's protocol or as described herein. Briefly, the depth fitter (A! EC) Is fl s ed with 100 L ~ of water followed by 5 volumes of equilibration buffer I (ES I ; Protein A elation buffer adjusted to pi t 7.S with. I fv! Tris-base, phf ! 1 ), 10 nfL of activated carbon is packed into a 2,5 em 11) Omnifi column. The column is flushed with 1 0 CVs water, and then equi librated with EB I until the pH is stabilized, to pi 1 7.5, 1 ,2 mL of anion exchange membrane (7 layers) is stacked into a 4? mm diameter Svvinex device. The device is wetted w ith water at 1 2.5 CV/rnln for at least 10 min, .followed by S device volumes (DVs s of EB I . A disposable helical static mixer (K.oflo Corporation, Cary, lb) with 12 elements is used to perform in-line p! l adjustments. A 3 -layer cation-exchange chmotatogtaphy device (0, 12 ml., membrane volume) is wetted with. 10 DVs water, followed by 5 DVs of equili ration butler 2 (E82: EBI adjusted «,» pB 5.0 using I M acetic acid). The device is further treated with 5 DVs of EB2 + 1 M NaCk and then equilibrated with 5 DV BB2. A 3, 1 cm ³ Viresolve® o virus filtration device is wetted with water pressurized at 30 psi tor at least 10 minutes. The How rate i¾ then monitored every minute until the flow rate remains constant for 3 consecutive minutes. After ail the devices are wetted and equilibrated, they are connected as shown in Figure 5.

1 _. 000303) EBI is run through (he entire system until ah pressure readings and pH readings ate stabilized. Following equilibration,, the teed (i.e.. Protein A e!uate adjusted to pH 7.5) is subjected to Slow-through purification. During the ran, samples are collected before the surge tank ami alter Viresoive% ^: Pro t monitor MAb concentration and impurity levels (e.g.. H€i\ DNA, leached Protein A and

aggregates). Alter the eed is processed, the system I flushed with 3 device volumes of EBI to recover any MAb still remaining in the variou devices as well as the connecting lines between devices.

j 0003041 Figure 6 depicts the pressure readings after depth filter, activated carbon, and Vlrcso!ve¾ ^: Pro in flow- through purification. Generally _y an increase in pressure denotes fouling of filter columns. Notably, the activated catbors column remains fairly protected from any precipitate due the depth filter used before the activated carbon. The Vtresoive€ ^: Pro pressure rises slow ly ith time, but is well below the operating maximum limit (50 psi).

[000305] The HCP breakthrough as a function of time, as measured alter the anion exchange chromatography device is below the target of 10 ppm. The final HCP in the Viresolve® Pro pool is < I ppm (Table I ). T he average leached Protein A in. the elation fractions is 32 ppm. The leached Protein A in the Vhesolve® Pro pool is 4 ppm, The aggregates are reduced from I % to 0.4%»

0OO3Ob| The results from, the experiment are summarized in. fable II below.. Tank- II

E m le 3: hnv-i hrn gh Purification Process Following Batch Protein A

Chromatography

[000307] In this representative experiment, monoclonal antibody solution previously pu.ri.fied by batch protein A is further purified using flow-through purification: to .meet final purity and yield targets. This is done by perfbnrbng the following steps in a flow-through manner: activated carbon: anion exchange chromatography; in-line pH change; cation exchange ehrontatography and virus titration. .

[000308] The set-up, equilibration and run is sim lar to Example 2 except tor some minor modifications. The starting material is a protein A ehrate from a. batch protein A process. Specifically,, the MAb reed processed lor t nm is 102 ml. of 13.5 m.g/niL MAbOS ai a flow rale of 0,6 mL/ in. A depth filter is not used in this study as the feed i filtered through a sterile 0.22 ye filter prior to performing the flow-through, purification.. A. 2.5 mL activated carbon colu n is used which corresponds to a loading of 0,55 kg/l . T wo anion exchange chromatography devices (0.2 and 0,12 mL) are connected in series to get a leading of 4.3 kg/I.,. T wo 1.2 ml, catio exchange chromatography dev ices (7 layers of the mem ran o each de v ice) are connected in parallel to handle aggregates. The MAb loading on the cation, ex han e chromatography devices is about 570 mg/mL. A. 3 J cnf ViresolveTf Fro device is used for virus filtration.

[000309] The f-iCP breakthrough as a function of loadin alter anion exchange chromatography device is belo the targe! of 10 ppm (Figure 7), The final HOP in the Viresolve ^; t ^; Pro pool is < ! ppm fiahle 2). The aggregates are reduced from 5% to 1 J% by the cation exchange chromatography device (Figure S).

[000310] The results from the experiment are summarized so Table III below.

Table II

(00031 1] Figure 9 sho ws the pressure readings before activated carbon nd Viresolve® Pro, Generally, increased p ssur implies the filters are getting fouled. In this case, there is a modest (about 5 psi) increase in pressure in ease of activated carbon. Th Viresolve# Pro pressure rises slowly with time, but is well below the operating maximum limit (50 psi).

(000312] As shown in Figure 10, the pi! after adjustment remains at the target set- point of pi 1 4,9 except during start-up. The pH spikes can be dampened by using a surge tank after the in-line pi I adjustment and before pumping to the cation exchange e h ron ? ato grap hy device,

ExMnp!e 4: Clari ication corrected to Proteitt A chromato rap y

[0003 1 3] In this representative example, clarification is connected to Protein A chromatography io. a continuous manner,

1000 14] I this experiment, the flow-rate that is used for depth filtration Is determined by the residence time used lor Protein. A chromatography, which follows depth nitration. T he flow-rate used in this representative example is slower than thai used in conventional depth filtration., resulting in a higher HCP removal in the output recovered alter Protein A chromatography,

[000315] A monoclonal antohody (MA.b04) cell cuhure teed is obtained and split into three equal portions. The first portion (sample # in Table IV) is clarified using D0HC and X0HC iiHstak-H ) primary and secondary depth filters (EMD illip re) at a filter area ratio of 1 : 1 and a flow rate of 100 tliers:¼¾our (LMITf which Is a typical slux used in. standard clarification processes. The effluent Is tested fo MAb concentration and HC amount The second portion of the cell culture teed (sample #2) is also clarified with the same type and ratio of filters but at a llow rate of 10 L R T his flow rate Is based on a si minute residence time of the Protein A chromatography olumn, which follows clarification, in both cases, the same amount of material. Is processed, corresponding to a throughput of about i (KIL/mT and the two samples are treated in the same mann r,

[000 16] Both clarified cell culture -fluids are subjected to Protein A bind and elute chromatography, resulting in samples #3 and. #4 in Table IV, where the clarification and Protein A chromatography are performed separately (i,e., are not connected). Sample #4, relati ng o slower flow-rate, is lost and. th refore, not able to be nalysed.

[00031 7] The third portion of cell culture feed (sample #$) is processed through an. assembly that has the effluent of the two depth filters continuously loaded onto a Protein A chromatography column {I ., her clarification and Protein A

chromatography are connected). Same chromatographic conditions as above are used. Ail protein A ehiafes are tested for M Ab concentration and HOP amount, in case of sample 5, the six minute residence time of the Protein A chromatography determines the lion -- ate for clariikaOon of about l OLMH.

[0003 1 8] All results are shown in Table IV. T he results indicate that the HCP levels arc 2x lower after slow clarification (i.e., sample #2 relative to sample #1 ). A direct comparison of the corresponding samples following Protein A chromatography is rsof reported due to the sample loss (i.e,, sample #4), A comparison of results relating to sample #5 relative to sample #3 suggests that performing clarification and Protein A chromatography in a continuous and connected manner provides a SX improvement in MAb purity compared to the purity when clarification and Protein A chromatography are run separately. Comparing the estimated LEV values documents the difference.

Table IV

# Sample MAb (g/L HCP HCP (ppin) LEV

(ng/mL)

100 LM H clarified " U t i0%) 250,894 22 1 ,426

.1

1 0 1MB clarified 1.01 (60%) 1 18J I 4 1 17,421

100 LMM clarified and 9.03 7,553 837 2.

3

Protein A purified

10 IMH clarified and not tested.

4

Protein A purified

10 1MB clarified and

5 7.94 808 102 (3.1 ) Protein A purified in a

connected manner

07 Exam le St OartfSeattoa I «g Stimulus esponsive Polymer

[ 000319} in this representat e experiment, clarification is performed using a st mulus responsive polymer using two different processes.

|000a20] In one process,, a stimulus responsive polymer Is added directly to a bioreaetor (which may be a single use or disposable bioreaetor) containing a cell culture expressing a target molecule. In a second process, a cell culture is pumped out of a bioreaetor and contacted with a stimulus responsive polymer using one or more in-line static mixers,

[000321 ] In ease of the process relating to performing clarification directly lo a bioreaetop 60 ml of 10 wt % stimulus responsive polymer is added into a 31, disposable bioreaetor containing a cell, culture and .mixed for at least 5 minutes. 75 rot of 2 Kj!iPOsi is added into the bioreaetor and mixed, lor at least 5 minutes, 2 M I ris base is added inio the bioreaetor while mixing in order to increase pH to between ?»7.3 (approximately 50- 100 ml/}.- The so!n ion is allowed to mix for at least I. minute and then pumped out of the bioreaetor and loaded directly onto a depth film- at a. rate of 100 LMH to remove the precipitate,

[000322] in. case of the process relating to the use of an in-line static mixer, cell culture is pumped out of bioreaetor at a rate of 93,5 LMW to a valve or connector where it is contacted with a stimulus responsive polymer stream flowing a a rate of 1.0 LMH. The combined stream then flows into so inline static mixer sized appropriately to provide efficient mixing. ^' The stream then (lows into a second valve or connector where it Is contacted with a stimulus for the polymer .Rowing at a. rate of 2.3 LMH. The combined stream flows into a second static mixer size appropriatel to provide efficient mixing. The s ream then flows into a third valve or connector where it is contacted with a 2 M Tris base stream flowing at an approximate rate of 2,3 LMH (flow of tris is adjusted to maintain a pH of 7-73 of the combined stream). The combined stream flows into a third static mixer that is sized appropriately to provide efficient mixing. Ibis stream then is loaded directly on one or more depth il Iters in order to remove the precipitate,

[000323] It is noted that different feeds may be more sensitive to pH or may Interact with a stimulus responsive polymer differently. Yields can be maximized by having Ote ability to treat feeds either in bioreaetor, inline or a combination of the two may be us d.

6S [00032 j Use of a stimulus responsive ol mer, as described ^' herein, results in a better performance in the bind and ciute chromatography process step (e.g. Protein A chromatography te , which follows the clarification s e , Add hkrn ally, it s observed thai the method described in this representative e ample results n an increase number of chromatographic cycles of the next bind and elide chromatography step, relative to clarification schemes that do not involve use of a stimulus responsive polymer. Lastly, the eulate obtained subsequent to the bind and eiuie chromatographic step appears to exhibit ess turbidity generation upon phi change, when a stimulus responsive polymer is used upstream of the bind and ciute chromatography step ,

Exam e C*: ffect of Cbinfie iion sing Pre pitation on Et tion Performance of Protein A troRisto raph

[000325] In this representative experiment, the effect of the type of clarification performed on a CHO-hased cell culture producing MAb04 on elution performance of Protein A chromatography is investigated,

[000326] A single hatch of cell culture is split evenly into three aliquots. One aliquot is subjected to clarification using eaprylic acid; another aliquot is subjected to clarification using a stimulus responsive polymer (fe„ modified polyaiiy!amme); and the third aliquot is left untreated.

000327] Following precipitation with the eaprylic acid or stimulus responsive polymer, the solids are removed using eeotrifogation. The untreated cell culture is also cemnfuged alter mixing for the same amount of time as the two treated cultures. Ail are sterile filtered prior to use.

000328] For each clarified sol ution, the conducti vity of the solutions is measured and adjusted with 5 NaCI until the conductivity reaches about 54 mS/cra. The average concentration of added Natl is approximately 0,5 M for all solutions. The higher conductivity ceil culture solutions are sterile filtered prior to loading on to separate Protein. chromatography columns,

[000329] In prder to perform Protein A chromatography for each, eed solution, three columns are packed with ProSep Ultra Plus media with one column having mL of packed media and the other two both having 4,24 ml. of packed media in 10

00 mm inner diameter Omni Fit col mn . T e column bed heights are 5.1 and 5,4 cm for the 4 mL and 4.24 ml, columns, respectively.

[000330] AO chromatography experiments are performed n n Akta Explorer 100 (capryiie and. stimulus responsive polymer treated) or an Akta Avaoi 25 (entreated). rior to the first loading, each column is washed with at least five column voiiimes (CVs) of 0J 5 M phosphoric acid. Ah chromatography runs are performed using the same basic procedure. The columns are equilibrated with 3 CVs of I X I ^' BS ÷ 0,5 NaCf foll wed by loading io ^■ -- 30 g of MA MM per liter of packed media. The loading solution is -flushed out with 2 CVs of equilibration buffer iollow¾d by a 4: CV wash with 25 mM Iris pH 7,0, Following washi g of the colum , the product (K Ab04) is eluted from the column with 5 CVs of 25 mM glycine MCI, 25 mM acetic acid pl:l 2,5, The elation is collected using the s stem's fraction collector with collection starting using a UV trigger and collected for a constant volume of 4 CVs. The column Is cleaned -with 4 CVs of 0, 15 M phosphoric acid followed b a reequiiibratlon step of 1 CVs with equilibration buffer.

[000331 ] The Protein A purification of the dii!erent clarified samples is performed tor twelve (untreated) and nine (capryilic acid and stimulus; responsive polymer treated) successive cycles.

000332| Figures 1 L 1 and 13 depict the overlaid clm natograms for ail experiments, in each case displaying the load, elation and cleaning peaks in sequence. It is evident that the elation peak without stimulus responsive polymer treatment has visible and significant -trailing as compared to the elation peaks from the stimulus responsive polymer treated cell culture, suggesting a less efficient elation when stimulus responsive polymer was not used,

[000533 Additionally, the absorbaoee of the loading solution is noticeably lower for the stimulus responsive polymer treated cell culture compared to the untreated cell culture, where the absorhance is reduced by 0,4 - 0,5 absorbance units (All), suggesting a lower impurity challenge to the column.

[000334] After elation, the pH is raised to 5.0 for both sets of samples, then further raised to pH 7.5. At pH 5.0 there is no visible turbidity of the solutions.

However, at pH ?J al l clution samples exhibit increased levels of turbidity with significantly higher levels (99 - 644 NTH) for the untreated samples, while the stimulus responsive polymer treated clution pool turbidity ranges from 60.5 to 151 Nil! J, which is significantly lower relative to the untreated samples, Esample It Simultaneous Removal «f Soluble and ns luble Impurities from Affinity Cap ure Etnste Us ng Activated Carbon 000335] In this representative experiment, t was demonstrated that activated carbon, when packed -with cellulose media, was capable of remo ing, both insoluble (i.e., particulates) as well as soluble impurities from an eluate from the Protein A bind and ekrte chromatography step fi.e„ capture step).

1000336] In many conventional processes used in the industry today, a depth 11 iter is often used following the Protein A affinity capture step to remove insoluble impurities ( i.e., particulates) before the next step, which typically is a cation exchange bind and eiute chromatography step.

[00O337| in the processes described herein, the use of a depth filter following the Protein A bind and eiute chromatography is obviated. Notably, by using activated carbon following the Protein A bind and el te chromatography step, not only is the need for the cation exchange bind and eiute chromatography ste obviated, but also is the need to use a depth rl et, litis offers many advantages including, e,g., reducing the overall coat, process time as well as the overall physical footprint due to elimination of steps that are typically used,

[0003381 As. demonstrated herein, use of .activated carbon leads to simuitanous removal of both soluble impurities (e.g., HCPsj as well as insoluble impurities (e.g., particulates).

[000339] A cell culture of monoclonal antibody M Ab04 is subjected to Protein A affinity ehromatograpby,. and the pH of the elution pool is adjusted from pll 4,8 to pH 7.5, with dropwise addition of 1.0 M Tris base. In order to change solution conditions suitable 1b? the next step in the process. However, raising the pH of the solution increases the tarhldity, which in this c se is measured to be 28,7 NTU. This solution is .referred to below as the MAb04 Protein A eluate.

[000340] A circular section of a sheet of activated carbon-cellulose media 5/8 inch 1)5 diameter and 5 mm in. thickness is cut and earefally loaded into 1.5 mm diameter OntniOi® Chromatography Columns SKU; Ine, Danhory. C ^' T 06810 USA to result in a column volume of 0.89 ml,. The column is Hushed with 25 niM fris pfi ?, About 40 _m L of the turbid MAb04 Protein. A eluate is passed through the column at a flow rate of 0.20 mL/ntin,. resulting in a residence time of 4,5 minutes. Four 10 m ^' L fmct.io.us ar collected. Bach individual fraction as well as a combined pool composed of all four tractions is evaluated for turbidity and anal ed fo the eoneentra ons of HCP and MAb. HCP anal sis is performed using a commercially available ELISA kit from Cygnus Technologies, Somhport NC USA. catalog .number P550, following kit mamd¾ctuter s protocol. The MAb concentration is measured using an Agilent HPLC system equipped with a Potos® A Protein A analytical column. The results are summarized in Table V.

f 000341 J Ί; he results show that acti vated carbon is unexpectedly effective for the simultaneous removal of both the insoluble impurities (i.e., particulate matter) as well as the soluble impurities (Le, . HCPs) .from the Proiein A ehnde. T he turbidit of the Protein A elua e is reduced from 2H7 NI li to 9.0 HIV, while the concentration of HCP i s red need .from 75 S ppm to 104 ppm.

000342] This result demonstrates that activated carbon, when .packed with cellulose media, can. be used tor removing both soluble as well, as insolubl impurities.

Table V

acti ated

volu e

ear nsi Turbidity MAb HCP HCP

loaded

loading HCP

{NT! ) fmg/mL) (t¾g/wiL) ( m)

(ml,) LMV

(kg/I,)

control 28.7 \ 0,38 7,872 758

1 o. r.i 8,8 9.76 1. 0 1 5 1 ,77

20 0.25 10.3 10.53 Bill S i 0,97

30 0.38 10.3 10.5.5 L660 157 0,68

40 0.50 10,6 10.38 2.333 22S 0.53

40 tpooll 0,50 9.9 10.59 1 ,098 104 0.86

Example S: Effect of Reside ce Time oo Imparity R mo al by Activated

< arbors

1000343] In this representative experiment, it was demonstrated that when activated carbon Is used in a continuous process, as described herein, it results in a greater impurity remo al r lati to when it is not used in a continuous manner.

Notably, whet* activated carbon is employed in a continuous process, as described herein, where it is usually in ϊίιύύ communication with the Protein A bind and date chromatography step upstream and with an anion exchange chromatography media downstream, the sample flows through the activated, carbon at. a (low-rate which s slower Che., having a longer residence time) relative to when activated carbon is used separately as a stand alone operation,

[000344] In this example, Protein A-parlried MAb04 ekrale is further subjected to a flow-through purification step which, employs activated carbon (AC) and an anion exchange chromatography mem rane device (e.g., a CbromaSorb™ device) con.flgu.red in series, at -four different flow rates. Antibody concentration in the feed is determine to be 7,5 g/L; HOP concentration is determined to he 296 ppra. The experiment is performed a pH 7,0, Activated Carbon, grade uehar hID, is obtained from Mead West Vaco, It is packed in a glass Ommfn column 10 bed volume of 0,8 nib. An anion exchange chromatography device with msmhr&rie volume 0,08 mi i connected in series to the AC column. The flo rates are chose such tha the residence time CRT) on. AC is L 2, or 1.0 mins. The MAb loading on AC and the anion exchange chromatography device is held constant at 0,5 kg/L and 5 kg/L, respectively, ibr the 4 difTereni runs (i.e., having the four different residence times stated above).

[000345] Samples from the breakthrough of the anion, exchange chromatography device are collected and analysed for MAb and HC concentrations. The

breakthrough of BCP as a function of MAb loading on the anion exchange

chromatography device at the 4 different residence times on AC mentioned above, is shown in. Figure 1 ,

10003461: As demonstrated in Figure 1.4, lo e flow rates on AC (i.e., longer residence times),, provide better purity t same MAb loadings. Alternatively, the same purity ca he achieved with a smaller volume of AC and the anion exchange chromatogra hy device at a slower flow rate, For example, the larget purity of - I ppm HOP can he achieved using 2 kg/L loading on the anion, exchange chromatography device (and 0.2 kg/L on. AC), operating at 1 minute residence time. Notably, the same purity can be achieved while operating at a. longer residence time of 10 mins (he,, slower How rate) with a igniileand highe loading of 5 kg/L- on the anion exchange chromatography device (and 0,5 kg/L on AO, This finding highlights a potential economic advant ge in using less consumable purification material to achieve the same purity when the flow rate is reduced.

Exam le 9t Advantage nf Using A Surge ^" tank i« the FI«w ^« Thr»ugb

I:½nfkaikj8 Pr cess Step

| ^' 000347| l.o this representative experiment, one or more advantages of using a surge tank in the flow-through purification process step, as described hcreia..are demonstrated,

[000348] Typically, cation exchange flow-through chromatograph requires the sample to be at a pH of about 5. Accordingly, the pH of the sample has to be changed from about neutral. pH to about pH 5.(1, a it flows from the anion exchange chromatography step to die cation exchange chromatography step, when performing flow-thro ugh purl fi eat i on .

000349] While the change In pH can be achieved by using an in-line static raker, tins example demonstrates that it is advantageous to additionall use a surge tank. Accordingly, the . low- of the sample is as follows; anion exchange chromatography step io an in-line static mixer to a surge tank to the cation exchange chromatography step,

[000350| It is observed that if only an in -line static mixer Is used for changing pH conditions between the anion exchange and the cation exchange flow-through chromatography steps, a sudden increase (i.e.. a spike) is seen, as measured using a. pH probe, it is understood that the pH spike is observed due to chemical, differences in the composition of the sample and that of the buffer being added to change the pH, This pH spike is undesirable as it results in the sample being processed lor a duration of time a a higher than is required for optimal results. This example demonstrates thai this pH spike can be reduced or eliminated by the use of a surge tank after the inline static mixer and before the sample contacts the cation exchange chromatography step, as shown .in Figure 1 . 003511 As shown in Figure 15, a pU. spike of about pll 6.5 is observed without the use of a surge took. However., when a surge tank is used., as described herein, the phi fails to below pH 5.3, which is closer to the desirable pH for the su se uent cation exchange chromatography step. xample 10; Running the FI» ~Thr»ugh Purification Process Step in a

Continuous Manner is not Detrimental to Product Purify

[000352] In this representative ^' experiment, it is demonstrated thai running a flow- through purification process in a continuous manner is not. detrimental to product purity. In other words, by comparing the product parity fern a Oow-thmugh purification process step run in a continuous manner to one where the individual steps are performed separately, ii is shown that there is no detrimental effect on the product purity.

[0003.53 j This example demonstrates that by connecting activated carbon and an anion exchange chromatography device (e.g.. ChrornaSorb™} in series to a. cation exchange chromatography device, which acts as a virus preiilier,and a virus filtration device, and operating the entire flow-through purification process step, continuously results in similar capacity of the virus filter, compared to when the acti ated carbon and the anion exchange chromatography device are decoupled ram the cation exchange chromatography device and the virus filtration device,

[000354] The experimental set-up for this experiment is shown in Figure ! 6.

Option I In Figure 16 refers to the continuous process, where the sample from, the surge tank (present after anion exchange chromatography device) is led directl into a cation-exchange chromatography device followed by a. virus filtration device . Option. 2 in f igure 16 refers to a batch process where sample is pooled after activated carbon and the anion exchange chromatography device , and alter a duration of time, it is processed through through a cation exchange chromatography device and the virus d tra d on device.

[000355] In this experiment, the starting s m le is the Protein A hind and. eluie chromatography eloate, which has an HCP concentration of 250 ppm. It is observed that alter activated carbon and anion exchange chromatography device, the HCP levels is reduced to about 4 ppm.

[000356 ] in ease of the batch process (Option 2). the final HCP concentration following virus titration Is observed us he around I ppm; whereas in ease of the c ntinu s rocess (Option 1 ), She BCP concentration lb! lowing virus filtration was about 2 ppm, both of which are towards the lower end of what can be quantified u ing methods known in the art and those described herein, e.g., es ays described in 1 sample 7,

[000357] This result implies that performing all i steps in the fiowohrongh pu ifkabon process step in a continuous manner results in a product purity, which is comparable to when one or more steps arc performed as a stand alone operation.

[000358 J Additionally, it is noted thai the the pressure profiles for the two processes are also very similar, as shown in Figure 17.

Example 11: Effect of RcsMeaee Tit»« on Performanee of Virus Filtration

mbrane

1000359] l.n this representative expernnent. the effect of residence time on performance of the virus filtration is investigated, it is observed thai a lower flow rate through die cation exchange chromatography step and the virus filtration step during: the .flow through purification, process step, results in a higher throughput of the virus filter.

f 000360] Λ three-layer cat on exchange chromatography device, as described in IIS. Patent Application No. 13/783,941 (interna! rel no. MCA- 1423). having membrane area 3, 1 cor and membrane volume 0.12 ml... is connected in a scries to a virus filtration device, ha ving a membrane area of 3 , 1 cm\ About 3 mg/ml of a polyclonal, human IgG (Seracare) in 20 mM sodium acetate, p 5.0 boiler, is processed through the two connected devices. The experiment is performed at two separate iiow-rates, 100 and 200 IMH. A 0,22 μηι sterile filter Is placed between, cation exchange chromatography device and the virus filtration device,

000361 J A pressure sensor is used for measuring th pressure across the assembl at the different iknv rates. Normally, a pressure of about 50 psi is an indication of fouling or plugging of the virus filtration membrane. As ^' s own in Figure 1 8. when die experiment is performed at a lower fiow^raie (i.e., 100 LMH), more sample volume can be processed through the virus filtration membrane t ie,, higher throughput) relative to when the sample is processed at a higher flow-rate (i.e., 200 IMF!}, This could he attribute to longer residence time ot the sample in the cation exchange chromatography device, which may result in an improvement hi binding of high molecular weight igG aggregates, thereby preventing early plugging of t e virus fi iter, xamp e .2. Removal of Aggregates at Various ¾¾klenee tim s f m M&b

f eed askig a Strong€arioa»ex€hi*age (CEX) Reslu Modified with m AMPS 1>M AM Grafted Copolymer 00036.2 _. 1 &* s 250 nil, glass jar, 64 mi wet eake of Toyopead HW75-F

chromatography resin was added. Next, 1 I Sg of $ sodium hydroxide. 18,?5g of s dium sulfate, and 4mL of ally! glycidyl ether (AGE) were added to the jar containing the resin. The jar was then placed in a hybridizer at 50€ overnight, wi th, relation at medium, speed. The next day, the resin was filter drained in a sintered glass filter assembly (HMD Milhpore Corporation. Bilierlcu, MA) and the wet eake was washed wiih methanol and then rinsed with deiooked water. h\ a glass viah 10 nit wet cake of the AGE acti vated resin was added. To the glass vial, 0.2g of Ammonium persuifate, 0,3 g AMPS, 1 ,2g MAM and 48g of delonimi water were added and the vial was heated to 60 ^s' C for 16 hows. The next day, the resin was filter drained in a sintered glass filter assembly (B D MHllpore Corporation, BiHerica, MA) and the wet cake was washed with a solution of methanol and deionized water and the resin was labeled as Lot 1712.

000363] The resin, labeled, as Lot # 1712, was packed in an Goruiih"

Chromatography Column with, an internal diameter of 6.6 mm to a bed height of 3 cm resulting in about I mt packed resin bed. An A TA Explorer 1.00 (chromatography system) was equipped and equilibrated with buffers appropriate to screen these columns for flow-through chromatography. The chromatography columns containing the resin sample were loaded onto the chromatography system with equilibration buffer. The feedstock was an IgGl ( AbS) feedstock that was purified using ProSep¾ ^: Ultra Plus Affinity Chromatography Media, and. was adjusted to pH 5,0 wiih 2 M I ris Base. The final concentration of the Protein A pool was diluted to 4 mg/m.L,comained 5.5 % aggregated product, and a conductivity of about 3,2 niS/cm. The resin was loaded at a residence time of L 3, or 6 minutes and to a load density of 144 mg/nVL. Us strip peak fraction tor the 3 minute residence t necontained 95,6 % aggregates ind cat ng a high level of selectivity for. aggregated species, The results are depicted in Table VI below.

][ 000364] T able Vi depict s retention of monomer and aggregates for .Lot # 1712 with: MAb5 at pH 5.0 at 6, 3, or 1 .minute residence hme. As shown in Table VI, oa average, the monomeric species can be collected m concentrations close to the teed concentration relatively early compared to the aggregated species for ail residence times tessed, which suggests thai selectivity is relatively insensitive to O rates.

Table VI

Average

Flow- m ria iv at 6, 3, or

through e Protein 1 Minute 3 Mift«f¾« I Mf!HJU

C l!eetio Load Rcskfene Resi ence sidence Residence r* e Time Time i e Time

'Hi Oroiclo % % in Flow- Aggregate Aggregate Aggregate

Fraction (mg/ffiL) through s s s

1 .16 13,5 0.0% 0.034 0.0% p 32 94,3 Q..0% 03)% 0,ø%>

3 48 94.4 0.0% 0 0 ^' ' , 0,0%

4 64 95.2 0.0% 0.0% 0.034

5 80 98,3 0.5% 0.0% 0.034

6 96 1003) 0,7% 0,3% 0.0%

? M 2 99.3 1 .1 % 0.9% 2. 1 %

8 128 100.0 2.3% i .6% 2.S%

144 1.00.0 3. 1%: 3.6% 4.8%

£000365} As depicted in Figure 1.9, the majority of the product is collected in the How-through a id this i indicated by the relatively quick breakthrough of protein UV trace. Hie strip peak size generally varies based on the conditions and total mass loaded but it is relatively enriched with aggregate species ai 95,6 %, compared to the load materia! which had only 5.5 % aggregates. Example 13: Removal of Aggregates from a MAt> e d llslag C&iioii-Exchange CEX) Winged fibers adHkd wills an AMPS /!)MA ! Grafted Copolymer.

[000366] fa this representative experiment, cation-exchange winged fibers were used as the solid support,

|000367] In a I I.. glass jar, 20g of dry Ny lon moitb!obed, or winged, libers were combined with 400 g of 4M sodium hydroxide, 24 g of sodium sulfate, and 160 niL of ally! g!yeidyl ether (AGE). Th jar was then placed in a hybridizer at 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 vvith methanol and rinsed with Mdii-Q water, A day later, the fibers were washed with water, followed by methanol, and then water again, suctioned to a dry cake and dried in vacuum oven at 50* ^' C for .1 day. The resulting sample was labeled Sample # 1635, in three separate glass vials, 2 grams dry 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, amm nium persu!faie, AMPS, DMAM, and deiorazed water wure added in amount specified in Table Vii and the vial was heated to 6CLC 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 deionixed water. The vials containing the libers were labeled as Lot 1 635-L 1635-2, and 1635-5. Next, Lot #1635-5 was titrated tor small ion capacity, which was found to be about 8 ptnol/ L. It was then assumed that samples #1635- 1 and #1635-2 also had small ion capacity less than. 28 pinol L.

Table VII

[000368] The resulting modified winged libers. Lot # 1635- L # 1635-2, #1 55-5 were packed la an Omnliit* Chromatography Column with an internal diameter of 6,6 mm to a d heigh! of 3 em resulting in about I ml. packed fiber bed.. An A.KTA Explorer 100 (chromatography s s em) was equipped and equilibrated with buffers appropriate to screen these columns for flow-through chromatography. The cinematography columns containing the winged fiber samples were loaded onto the chromatography system with equilibration buffer. The feedstock was n IgG I (MAbS) feedstock that w s purified using proieiu A affinity elimni tography., and was adjusted t ρΥί 5,0 with 2 M 1 m Base, The final concentration of the protein A pool was 4 mg ml and contained S.5 % aggregated or BMW product. This is the same feedstock as used in Example 1 2, The columns packed with fiber Lot # .1635-1 and Lot 1635-2 were loaded to a mass loading of 64 ntg/mL and the column packed with fiber Lot 1635-5 was loaded to a mass loading of 80 mg/mL. The results are depicted in Table V III below,

Table VI O

*N/A ^:::: Not applicable Example 1 ; The eomhsued effect of stimulus responsive pol mer clarification a»d addi ion, of NaO to he clarified cell cttitare on the Protein A elufkiss pool purity 000369] In this representative experiment a Ab04 cell culture fluid is clarified using either depth filtration or a precipitation step, specifically us ng a stimulus responsive polymer (i,e, _< modified polyall>¾mine}. The resulting clarified solutions are either loaded on to a column containing Pr Sep Ultra Plus resin or have NaCI added to a float concentration of 0.5 M NaCI prior io loadin onto the column, In the absence of the NaCI addition, the column is equilibrated with I X TBS prior to loading, w reas with. NaCI. the equilibration duller Is I X TBS. 0.5 M NaCL All columns are loaded to -approximately 30 g of Ab04 per liter of resin and then washed with equilibration boiler tor 2 CVs followed by a 3 CV wash, with 25 .M Tris pH 7A The bound MAb04 is elated with the 25 rnM glycine HCL 25 mU aeefic acid H 2.5 solution and then cleaned with 5 CVs of 0.15 phosphoric acid pH 1.6 before being reeqiulibrated. with the appropriate equilibration boiler,

[000370] f igure 20 display s an overlay of the eluiion and cleaning peaks, where the elutioo peak generated from the stimulu responsive polymer treated cell culture displays a sharper tail and a reduced cleaning peak, in the presence of NaCL during the load with the stimulus responsive polymer treated cell culture, the cleaning peak is further reduced, thereby indicating a lower level of strongly bound impurities on the resin,

Example 15: Comparison of the effect of different NaCI concentrations during

h t ro d ate a hing, steps it the effect of 0.5 !Vl NaCI present in the loading solution

[000 71 j in this representati e experiment, the impact..of different NaCI concentrations during the intermediate washing steps on the monoclonal antibody ( A ) elation pool purity achieved by Protein A chromatography is directly compared to the impact of ha ving 0.5 M NaCI. in the .sample being loaded onto a Protein A chromatography column,

[000372] A 5 ml, column o P oSe ile Ultra Plu Protein A media, is acquired as a. pre-packed column. Ail chromatography runs are performed using an Akia Explorer chromatography ^' system with a flow rat of 1 .7 mt rmn (- 3 minute residence time) .for ail steps. ^' The same column is used for ah experiments. For the first sot of experiments,, where NaC! is present in the intermediate wash, the chromatography step emplo ed a 5 colum volume equili ration with 1 X TBS followed by loading of 300 mL of clarified cell culture containing a target protein (referred to as MAbOS) at a concentration of approximately 0,57 g/ ^' L, Following the ioading of cell culture, the column is flushed with 10 ml, of equilibration buffer to remove any unbound, product, impurities and other cell culture components, The column is then washed with 5 col umn volumes of 25 mM iris, pH 7.0 that als ncludes 0,5 M NaCl. The column is subsequently washed with. 5 column volumes of 25 rruV! Tris, pH 7.0 without aCl. The product protein (MAbOS) is eluted from the column over 5 column volumes using a holier containing 25 mM Acetic acid and 25 mM Glycine HCI at pH 2.5, The column, is subsequently cleaned with 5 column voiu.rn.es of 0, 15 M Phosphoric acid followed by 10 column volume regeneration step using the equilibration buffer. Equi valent runs are performed where the NaCl conceutrabon during the first wash is varied between 0, 1 ,5 and 2 ,

[000373 ^' ! Th next experiment is performed using the same column with the following changes to the protocol. The cell culture sample being loaded i mixed with a volume of 5 M NaCl such thai the final NaG concentration in the clarified cell culture is 0,5 M NaCl. The equilibration buffer is modified to include 0.5 M NaCl. in the ! X TBS solution. The column is loaded with 333 mL of clarified cell culture with 0.5 M NaCl present to maintain a constant mass loading of the target protein (MAbOS). Intermediate washing is performed as previously described where a total of 10 column volumes of 25 m Tris, pH 73) is used throughout. The elation and cleaning steps and buffers remain identical as described above. ^" Fable IX. provides an abbreviated summary of the steps performed lor each experiment, identi fying the buffers and volumes used for each step where a. potential change is made. Tahfe IX

[000374] Samples of the loading cultures and the elation pools ate measured lor AbOS concentration and Ι Κ.Ύ concentration. Figure 21, shows the HCP LRV as a function of the NaCI. concent ation used in the intermediat wash or used in the loadin step. The Figure illustrates the im o ed level of HOP removal (purification) when 0.5 M NaCI is present in the equilibration buffer and clarified cell culture during the loading phase compa ed to the addition of NaCI. to the intermediate washing steps, at. varying concentrations. The results also provide the measured BCP concentrations in parts per million i pm) based on the ng of BCP per mg of Λ005, shown as number within the corresponding bar. Further.- Figure 22 shows the %

MAbQS recovered in the ehrhon pool as compared to the mass loaded, where it is clearly observed that with 2 NaCI present during the intermediate wash step, a significant loss of product is realized, xamp e 16: Comparison of the roduct purifieaiiea achieved ased «» the Protein A pur ifie k*n step d«rk¾g w h ch sa additive is included

[000375 j In this representative experiment a direct com arison is made etw en Che purification ac ieved by Protein A chromatography with an additi ve present hi the intermediate wash only to the purification a hieve with an additive in the equilibration buffer and cell culture samples thai arc loaded.

{ ^' 000376] A 5 mL column of EroSep ⁾ Ultra Plus Protein A media Is ac uire as a pre-packed column. All chromatography runs are performed using an Akia Avant chromatography system with a flow rale of 1 .7 mL/tnin <-·· 3 minute residence time) for all steps. The same column is used lor all ^' experiments. For all washing experiments, the m thod described in Example 15 above applies where NaO is replaced with a specific additive at a defined concentration. The additives and concentrations used are rovided in Table X.

Table X

} ^' 00iB77J Figure 23 shows the concentration of HCP remaining in the product elation pool for each additive use ! whether it . s present m the fi st intermediate was te or hi the equilibration buffer and cell culture sample. The addition of different sails ( aG, Ammonium Sulfate or TMAC) shows the lowest HCP levels, when the salts are present in the loading phase.

[000378] Figure 24 show the LRV of HCP (relative to the loading HCP concentration) as a function of the additive used and the purification step during which the additive is used. This figure iilustmf.es. again, thai salts are most etleetive at reducing the HCP concentrations, i.e., increasing the HCP FRY, As demonstrated, the presence of the additive in the loading solutions shows improved, impurity clearance when compared to the same additive present only in the intermediate wash. Tables XI and XII summarize the numerical results illustrated m Figures 23 ami 24 with Fable XI showin data when the additiv e is present in the loading step and Table XI I showin data when the additive Is present only during the intermediate wash..

Table XI

Table XI!

[000379] Figure 25 illustrates n example of i - relative elution pool volume depending on the additive identity and the chromatography step during which the additive is included. ^' The Figure shows thai when the ratio of the additive elution pool volume to the control elution pool volume (i.e., where no additives are present) is greater than. 1.90 %„ the additive elution pool, volume exceeds the control elation volume. Conversely, the values less than 100 % indicate a decrease in the additive elation pool volume relative to the control elution poo! volume. This Figure nher demonstrates the impact of the additives on the elution pool volume, A value less than 10 % is preferred. Combining the information provided in Figures 23, 24 and 25 with die numerical data in Tables XI aval X!T di order of the hea performing conditions are TMAC in load > TMAC in wash > Ammonium Sulfate .in load > NaCl. in load > Ammonium snlihte in wash. The order provided here is based on the HOP TRY as of primary importance mllowed by product recovery. UUCP concentration

HO in ppm s of primary im or an e the order chan es slightly to T AC hi load > TMAC n wash > KaCI in load > Ammonium Sulfate in load > Ammon um s«ii¼e In wash,

[000380] The specification s most thoroughly 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. u> limit its scope. The skilled artisan readily recognizes that many other embodiments are encompassed by this invention. All publications and inventions are incorporated b reference m their entirely. 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 are prior art to the present invention.

| ^' K) 381 | Unless otherwise indicated, ail numbers expressing quantities of ingredients., ceil culture, treatment conditions, and so forth used in the specification, including claims, are to he 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 o elements is to be understood to refer to every elemen t in the series. Those skilled in the art will recognize, or be able to ascertain using no more than murine experiment tion, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

| 000382| Many modifications and variations of this in vention can be .made without departing irom its spirit and scope, as will be apparent to ihose skilled in the art. T he specific embodiments described herein are offered by way of example only and are not meant to be limiting i any way. It is Intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.