Tangential flow filtration and nanoresins

Abstract

A method for purifying a biological target. A cell-lysate suspension is mixed with a graphene oxide (GO) nanoresin. A biological target in the suspension binds to the nanoresin. The suspension is washed with a tangential flow filtration system to remove at least some of the unbound cell-lysate components. The biological target is eluted such that the biological target is released from the nanoresin. The eluted biological target is separated from the eluted nanoresin, thereby recovering the nanoresin.

Description

TANGENTIAL FLOW FILTRATION AND NANORESINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a non-provisional of, U.S. PatentApplication 63 / 709,121 (filed October 18, 2024), the entirety of which is incorporated herein by reference. STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number FA9550-16-1-0279 awarded by the Air Force Office of Scientific Research and grant number 1746198 awarded by the National Science Foundation. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form. Thecomputer readable form is incorporated herein by reference. The computer readable file is named Seqeuence.xml and was created on October 17, 2025 (7 kB). BACKGROUND OF THE INVENTION

[0004] Membrane filtration has been used in upstream and downstream applications, such as microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), membrane chromatography (MC), high performance tangential flow filtration and electrophoretic membrane contactor (EMC). A membrane is a heterogeneous interphase barrier between two compartments, enabling selective transport and efficient separation of molecules and ions. Membranes provide ready-to-use solutions in bioprocesses and do not require significant hands-on time. Membranes allow high productivity in a single-batch use. Functionalized membranes enable upscaling and downscaling. More recently proteinpurification / fractionation on the basis of size and / or charge was added to the possible applications. Membranes are also used in the filtration of cell lysates containing, beside proteins, amino acids, salts, organic acids, sugars, vitamins etc.

[0005] Membrane module configurations include hollow fiber, tubular, flat-plate, spiral-wound and rotating devices. There are two standard modes of operation: 1) the dead-end configuration; 2) the cross-flow configuration. In the first mode, the feed approaches the membrane perpendicularly to the surface of the membrane and a particle- free permeate emerge downstream with respect to the membrane. In the second mode the feed stream flows parallel to the surface of the membrane. The component of the feed stream that remains upstream with respect to the membrane is called the retentate, while the flow through is the particle-free permeate. Mass-transfer characteristics are improved by different strategies involving rotating disk filters, cylindrical Taylor vortex devices, conically shaped rotors, and helical coiled Dean vortex systems.

[0006] High performance tangential flow filtration (HPTFF) can separate proteins both in terms of size and charge or both. HPTFF is a 2-dimensional emerging technology for protein purification. Conventional TFF is limited to solutes that differ 10-times in size (e.g. cell-protein, virus-protein, protein-buffer). When using charged membranes, resolving solutes differing 3-times in size is possible. HPTFF compares to UF in terms of mass of product processed per unit area and unit time. HPTFF has been successfully used to separate protein monomers from oligomers, proteins differing only at a single amino acid, and an antigen-binding fragment from a similar size impurity.

[0007] While HPTFF is an exceptionally powerful technique in purifying and concentrating pre-purified proteins and antibodies, it is not suitable to separate proteins and antibodies from cell lysates. It would be advantageous to address its limitations. Unfortunately, no such solution is currently available.

[0008] The discussion above is merely provided for general background information and is not intended to be used as an aid in determining the scope of the claimed subject matter. SUMMARY

[0009] A method for purifying a biological target. A cell-lysate suspension is mixed with a graphene oxide (GO) nanoresin. A biological target in the suspension binds to the nanoresin. The suspension is washed with a tangential flow filtration system to remove at least some of the unbound cell-lysate components. The biological target is eluted such that the nanoresin is separated from the biological target. The eluted biological target is separated from the eluted nanoresin, thereby recovering the nanoresin.

[0010] In a first embodiment, a method for purifying a biological target is provided. The method comprising: mixing a cell-lysate suspension with a graphene oxide (GO) nanoresin, the cell-lysate suspension comprising a biological target; waiting a predetermined time to permit the nanoresin to bind to the biological target, thereby forming a suspension with a resin-bound biological target and unbound cell-lysate components; washing the suspension, with a tangential flow filtration system to remove at least some of the unbound cell-lysate components; eluting the resin-bound biological target with an elution buffer such that the resin-bound biological target is released from the nanoresin, thereby forming a suspension with an eluted biological target and an eluted nanoresin; and separating the eluted biological target from the eluted nanoresin.

[0011] This brief description of the invention is intended only to provide a brief overview of subject matter disclosed herein according to one or more illustrative embodiments and does not serve as a guide to interpreting the claims or to define or limit the scope of the invention, which is defined only by the appended claims. This brief description is provided to introduce an illustrative selection of concepts in a simplified form that are further described below in the detailed description. This brief description is not intended to identify key features or essential features of the claimed subject matter,nor is it intended to be used as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in the background. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the invention encompasses other equally effective embodiments. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. Thus, for further understanding of the invention, reference can be made to the following detailed description, read in connection with the drawings in which:

[0013] FIG.1 is a flow diagram depicting one method for purifying a biological target.

[0014] FIG.2 is a schematic diagram of a tangential flow filtration (TFF) system.

[0015] FIG.3 is a schematic side view of a TFF that uses an applied electric field.

[0016] FIG.4 is a schematic side view of a TFF that applies ultrasonic energy.

[0017] FIG.5 is a schematic side view of a TFF that uses an applied electric field and ultrasonic energy.

[0018] FIG.6 is a schematic depiction of the method of FIG.1.DETAILED DESCRIPTION OF THE INVENTION

[0019] This disclosure provides a method of using high performance tangential flow filtration (HPTFF) used in conjunction with floating chromatographic resins. Two- dimensional (2D) nanoresins are successfully implemented in protein purification processes with HPTFF, as opposed to bulk three-dimensional (3D) resins. 2D nanoresins tend to align with the laminas of the flow of a fluid in conditions of laminar flow. The nanoresins behave as “flying carpets” and not as randomly tumbling spheres. The dynamic stability of the “flight” of 2D nanoresins can be determined by the vectorial sum of different forces: i) the lift force experienced within the flow of the carrier fluid; ii) the drag force caused by fluidodynamic friction; iii) the gravitational force; iv) the buoyancy or upthrust force exerted by the fluid. The motion of the 2D nanoresins can be determined by the flow regime of the fluid. This may be laminar or turbulent depending on the compromise of inertial forces and viscous forces, as predicted by the so-called Reynold number. Also, the structural rigidity and conformation of the 2D nanoresins affect their floating patterns. The strategies in this disclosure provide a new method of protein purification based on 2D immobilized metal affinity chromatography (IMAC) and immunoaffinity chromatography (IAC) nanoresins in combination with TFF.

[0020] Referring to FIG.1, and method 100 depicted therein, the method begins with step 102 wherein a cell-lysate suspension 600 (see FIG.6) is mixed with a two- dimensional nanoresin 602 (see FIG.6) to form a suspension 601 (see FIG.6). The nanoresin 602 is a graphene oxide (GO) nanoresin. In one embodiment, the nanoresin 602 is a GO nanoresin as disclosed in United States Patent 9,339,790 or 9,822,151, the content of which are hereby incorporated by reference. The nanoresin 602 has a two- dimensional surface of nanosheets and does not exhibit the properties of traditional spherical (i.e. three-dimensional) carbohydrate beads that are typically used in protein / antibody separations. The aspect ratios of the nanoresins 602 are not naturally favorable to column-format purifications but are favorable to tangential flow filtration (TFF). 2D nanoresins pack so tightly in a chromatographic column that a high back- pressure arises, unlike the case of conventional resins (spherical-beads). TFF issometimes referred to as cross-flow filtration. As described in U.S. Patent 9,339,790, the two-dimensional nanoresin can be synthesized with oxygen varying between about 8%and about 42%. The term two-dimensional (2D) refers to nanoresins with roughlycircular nanosheets that are one atom thick with various functional groups typical of GO (e.g. OH and COOH groups) and ligands (e.g. NiNTA, bis-NiNTA, tris-NiNTA, Protein A, Protein G, Protein A / G or Protein L) on the surface. The sequences of these proteins are provided elsewhere in this specification. The proteins may be histidine tagged (His- tagged) by the addition of a short (e.g.6-10 amino acids) sequence of histidine amino acids at a terminus (e.g. either the C-terminus or the N-terminus). These nanosheets are corrugated, bend along the surface and may have irregularities such as holes and uneven edges. In some cases, the individual 2D nanoflake is almost rectangular. There areenough COOH groups on the surface that a full coverage on both sides of a GO flake isprovided. The average GO flake is circular and has an average diameter of about 5 m. The surface area of one face is about 20 m2. The total area is about 40 m2. As used in this specification, the term “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0021] In one embodiment, the nanoresin 602 has been surface modified to have atris-nitrilotriacetic acid (tris-NTA) chelating agent as described in United States Patents9,339,790 and 9,822,151: tris-NTA

[0022] The chelating agent may chelate a metal ion, such as Ni2+, Cu2+, Zn2+, Ag+, Fe3+, Ga3+, Zr3+, Ca2+or Co2+.

[0023] The cell-lysate suspension 600 can be produced by conventional means. For example, cells may be prepared by washing with a wash medium (e.g. cold phosphate- buffered saline, PBS) followed by addition of a lysis solution. Lysis suspensions typically contain a detergent (TRITON X-100® , sodium dodecyl sulfate (SDS), N- lauroylsarcosine), salts (e.g. NaCl such as physiological saline), buffering agents (e.g. tris-hydroxymethyl)aminomethane, Tris) and / or chelating agents (e.g. ethylenediaminetetraacetic acid, EDTA). The resulting suspension may be incubated for a predetermined time and / or at a predetermined temperature to promote lysis. Additional disruptions methods (e.g. sonication, freeze-throw cycling, homogenizing, passage through a French press etc.) may also be used. The suspension 600 may also be clarified by, for example, centrifugation. The resulting suspension 600 contains a biological target 604 of interest (see FIG.6) (e.g. a target protein, a target antibody or other biological target) and unbound cell-lysate components 606 (see FIG.6).

[0024] After the suspension 600 is mixed with a binding buffer and a predetermined quantity of the two-dimensional nanoresin 602 to form the suspension 601 (i.e. step 102) step 104 is performed, wherein a predetermined time is allowed to pass to permit the nanoresin to bind to the biological target, thereby forming a suspension 603 with a resin- bound biological target 608 bound to the nanoresin 602 and unbound cell-lysate components 606. Examples of suitable binding buffers include Tris (e.g.100 mM, 1M), aqueous NaCl (e.g.1M or 150 mM at pH=8.0), imidazole (e.g.25 mM). By way of illustration, a predetermined time of at least 1 minute to 1 hour, at least 1 minute to 30 minutes, at least 1 minute to 15 minutes or at least 1 minute to 5 minutes is allowed to pass.

[0025] In step 106 of method 100, the suspension 603 is washed in a tangential flow filtration (TFF) system with a washing buffer. The unbound cell-lysate components 606 pass through a membrane 610 of the TFF system as permeate 212 (see FIG.2) beforebeing collected in the container 214 (see FIG.2). The nanoresin 602 and the resin-bound biological target 608 remain as retentate 210. Due to the size of the nanoresin 602, the nanoresin 602 (and the resin-bound biological target 608) does not pass through the membrane 610. Examples of suitable washing buffers include Tris (e.g.100 mM, 1M), aqueous NaCl (e.g.1M or 150 mM at pH=8.0), imidazole (e.g.25 mM). Step 106 removes at least some of the unbound cell-lysate components 606. In one embodiment, step 106 is repeated at least three times. In one embodiment, two different washing buffers are used in two sequential repeats of step 106. For example, a first washing buffer (10 mM PBS, 150 mM NaCl, pH) is first used, followed by a second washing buffer (1M Tris at pH=8) is then used.

[0026] By way of illustration, and with reference to FIG.2, a system 200 is depicted that includes a feed reservoir 202. The suspension 603 with the resin-bound biological target 608 is placed in the feed reservoir 202. A pump 204 pumps the suspension 600 with the resin-bound biological target 604 from the feed reservoir 202 to provide the feed 206 to a TFF cartridge 208. The TFF cartridge 208 includes the membrane 610. The TFF cartridge 208 may be a variety of commercially available TFF cartridges, for example, a hollow-fiber cartridge, a flat-plate cartridge, a tubular cartridge, a spiral- wound cartridge, etc. The resin-bound biological target 608 does not pass through the membrane 610 and remains upstream of the TFF cartridge 208 as retentate 210 before being returned to the feed reservoir 202. At least some of the unbound cell-lysate components 606 pass through permeate 212 before being collected in container 214. The container 214 is cleaned and / or replaced prior to the step 108. In this manner the retentate 210 that has been returned to the feed reservoir 202 is sent through the TFF cartridge 208 multiple times with impurities being removed and sent to the container 214 on each cycle. In this manner, the liquid component in the suspension functions as a wash buffer. The volume of the retentate in the feed reservoir 202 decreases with each cycle. Once the volume of the retentate in the feed reservoir 202 is reduced to a predetermined volume, the suspension therein may be eluted (step 110).

[0027] In step 108, an elution buffer 216 is pumped into the retentate reservoir 202, and the resin-bound biological target 608 is released from the nanoresin 602 to provide a suspension 607 that comprises an eluted biological target 604a and an eluted nanoresin 602a. In one embodiment, step 108 is repeated at least three times. Examples of suitable elution buffers include Tris (e.g.100 mM), aqueous NaCl (e.g.1M), imidazole (e.g.25 mM or 250-500 mM), glycine (0.1M) with HCl at a pH of 2.5-3.0, a citrate buffer (e.g. 0.1M at pH 3.0) or combination thereof. The selection of elution buffer depends on which ligands were used in the nanoresins 602 and which washing buffer was used in step 106. When using IMAC nanoresins a typical washing buffer (step 106) may be 100 mM Tris, 1 M NaCl, and 25 mM imidazole. The corresponding elution buffer contains 250-500 mM imidazole. For example, the concentration of imidazole in the elution buffer is at least ten times the concentration of imidazole in the washing buffer. When using IAC nanoresins, the washing buffer may be a 1 M NaCl solution at neutral pH. The corresponding elution buffer is 0.1 M glycine-HCl at pH 2.5-3.0. In those embodiments where different washing buffers were used in step 106, the elution buffer may be 0.1 M citrate at pH 3.0.

[0028] In step 110, the suspension 607 (see FIG.6) of eluted nanoresin 602a and eluted biological target 604a is circulated through the system 200 and only the eluted biological target 604a crosses the membrane 610 within the TFF cartridge 208 and into the permeate 212 (downstream of the TFF cartridge 208). In this manner, the eluted nanoresin 602a is separated from the eluted biological target 604a. The eluted biological target 604a is collected in the container 214. The eluted nanoresin 602a remains in the retentate reservoir 202 for subsequent re-use.

[0029] Referring to FIG.3, a system 300 is depicted that is similar to system 200 except in that the TFF cartridge 208 is disposed within an ultrasonic bath that applies ultrasonic energy from a sonic generator 302 to the TFF cartridge 208 during use. The ultrasonic energy increases the rate of permeation across the membrane(s) within the TFF cartridge 208, generally about 25%. The applied frequencies are between 16 Hz and1019 Hz. The flow is either perpendicular (dead end configuration) or tangential (cross- flow configuration).

[0030] Referring to FIG.4, a system 400 is depicted that is similar to system 200 except in that the TFF cartridge 208 is disposed between electrodes 402, 404 such that an electric field is applied across the TFF cartridge 208. In one embodiment, the field is a 240W field. In some embodiments, the electric field is an alternating current (AC) electric field. In other embodiments, the electric field is a direct current (DC) electric field (e.g., >0 to 2400 V / m). In some embodiments, the TFF cartridge 208 comprises a membrane formed from polyvinylidene fluoride (PVDF), a known piezoelectric material is that responsive to applied electric fields. The electric field increases the rate of permeation across the membrane(s) within the TFF cartridge 208, generally about 25%. The flow is either perpendicular (dead end configuration) or tangential (cross-flow configuration).

[0031] Referring to FIG.5 depicts a system 500 that is similar to system 200 except in that both a sonic generator 302 and electrodes 402, 404 are present. System 500 permits the TFF cartridge 208 to be simultaneously or sequentially exposed to ultrasonic energy and / or an applied electric field. The flow is either perpendicular (dead end configuration) or tangential (cross-flow configuration).

[0032] In some embodiments, the pH of the elution buffer is chosen to effect the purification of a given biological target. For example, an acid or a base may be added to alter the pH. The pH may be chosen based on an isoelectric point (pI) of the biological target. pH changes, in some embodiments, affect electroosmotic effects that enhance filtration rates. The viscosity of the cell-lysate suspension may be altered along with the ionic strength of the feed 206.

[0033] The degree of membrane fouling is altered depending on purification conditions. In some embodiments, the use of the disclosed nanoresin decreases proteinfouling (relative to a corresponding system that does not use the nanoresin), as it prevents the formation of a gel / cake on the surface of the membrane.

[0034] As is customary in TFF, pressure sensors and valves may be present in the system 200 that maintain the internal pressure(s) and flow rate(s). In some embodiments, the system 200 has inline flow cells to detect ultraviolet-visible (UV-Vis spectra, pH, conductivity, fluorescence, etc. to detect the target. In some embodiments, the system 200 has an automatic collection system to separate the permeate into fractions. In some embodiments, the system 200 has a pump to add buffer to the permeate side of the cartridge 208 to facilitate separation by removing permeating proteins / antibodies from the membrane. In some embodiments a pre-filter is disposed inline between the feed reservoir 202 and the TFF cartridge 208. In still other embodiments, the system 200 has a sonicating water bath surrounding the TFF cartridge 208 to facilitate separation. A variety of hydraulic pressures (e.g., 0-400 bars), temperatures (e.g., 4°C, 25°C), permeation rates (e.g., >0 to 4 mL / min), numbers of membranes in a cartridge (e.g., 1- 10), and pore sizes (e.g.,100 nm – 1 m) may be used in conjunction with the disclosed systems and methods. Microsieves, as opposed to conventional polymeric membranes, are also contemplated for use with the disclosed system. A stationary configuration may be employed as well as a rotating disk configuration using different rotating speeds. A molecular weight cut-off (MWCO) of 3, 5, and 10 kDa may be used. In some embodiments, the reduction of the transmembrane pressure is obtained by a recirculation pump between the two sides of the cartridge using a co-flow arrangement. High- frequency pulsing of the feed is also contemplated for use with the disclosed systems. Chemical modification of the membrane surface of existing membranes is also contemplated to make them more hydrophilic (e.g. with polyethyleneglycol, PEG) or hydrophobic (e.g. with polydimethylsiloxane). The permeation rate of material across the membrane of the cartridge is generally at least 100 L per square meter per hour.

[0035] The disclosed method can purify a target protein from a cell lysate. This method uses affinity separation techniques to bind the biological target, wash awayunwanted cell lysate, and release the protein / antibody target. The nanoresin is larger than the target protein by 1 or 2 orders of magnitude and remains in the retentate during the purification.

[0036] In those embodiments where the ligand is a protein, the primary sequence of the protein is given below. In select embodiment, the ligand is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical or is identical to one of SEQ ID NO :1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0037] Protein A: MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAA QHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQA PKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAK KLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLL SEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSA NLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDP SVSKEILAEAKKLNDAQAPKEEDNNKPGKEDNNKPGKEDNNKPGKEDNNKPGK EDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDGN KPGKEDGNGVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVV DKKQPANHADANKAQALPETGEENPFIGTTVFGGLSLALGAALLAGRRREL (SEQ ID NO: 1)

[0038] Protein G: MEKEKKVKYFLRKSAFGLASVSAAFLVGSTVFAVDSPIED TPIIRNGGELTNLLGNSETTLALRNEESATADLTAAAVADTVAAAAAENAGAAA WEAAAAADALAKAKADALKEFNKYGVSDYYKNLINNAKTVEGIKDLQAQVVE SAKKARISEATDGLSDFLKSQTPAEDTVKSIELAEAKVLANRELDKYGVSDYHK NLINNAKTVEGVKELIDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKV FKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLK GETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEMVTEVPGDA PTEPEKPEASIPLVPLTPATPIAKDDAKKDDTKKEDAKKPEAKKDDAKKAETLPT TGEGSNPFFTAAALAVMAGAGALAVASKRKED (SEQ ID NO: 2)

[0039] Protein L: CKEETPETPE TDSEEEVTIK ANLIFANGST QTAEFKGTFE KATSEAYAYA DTLKKDNGEY TVDVADKGYT LNIKFAGKEK TPEEPKEEVT IKANLIYADG KTQTAEFKGT FEEATAEAYR YADALKKDNG EYTVDVADKG YTLNIKFAGK EKTPEEPKEE VTIKANLIYA DGKTQTAEFK GTFEEATAEA YRYADLLAKE NGKYTVDVAD KGYTLNIKFA GKEKTPEEPK EEVTIKANLI YADGKTQTAE FKGTFAEATA EAYRYADLLA KENGKYTADL EDGGYTINIR FAGKKVDEKP EEKEQVTIKE NIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYT ADLEDGGYTI NIRFAG (SEQ ID NO: 3)

[0040] Protein A / G: MNAAQHDEAQ QNAFYQVLNM PNLNADQRNG FIQSLKDDPS QSANVLGEAQ KLNDSQAPKA DAQQNNFNKD QQSAFYEILN MPNLNEAQRN GFIQSLKDDP SQSTNVLGEA KKLNESQAPK ADNNFNKEQQ NAFYEILNMP NLNEEQRNGF IQSLKDDPSQ SANLLSEAKK LNESQAPKAD NKFNKEQQNA FYEILHLPNL NEEQRNGFIQ SLKDDPSQSA NLLAEAKKLN DAQAPKADNK FNKEQQNAFY EILHLPNLTE EQRNGFIQSL KDDPSVSKEI LAEAKKLNDA QAPKEEDSLE GSGSGTYKLI LNGKTLKGET TTEAVDAATA EKVFKQYAND NGVDGEWTYD DATKTFTVTE KPEVIDASEL TPAVTTYKLV INGKTLKGET TTEAVDAATA EKVFKQYAND NGVDGEWTYD DATKTFTVTE KPEVIDASEL TPAVTTYKLV INGKTLKGET TTKAVDAETA EKAFKQYAND NGVDGVWTYD DATKTFTVTE KLAAALE (SEQ ID NO: 4)

[0041] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

What is claimed is:

1. A method for purifying a biological target, the method comprising: mixing a cell-lysate suspension with a graphene oxide (GO) nanoresin, the cell- lysate suspension comprising a biological target; waiting a predetermined time to permit the nanoresin to bind to the biological target, thereby forming a suspension with a resin-bound biological target and unbound cell-lysate components; washing the suspension, with a tangential flow filtration system to remove at least some of the unbound cell-lysate components; eluting the resin-bound biological target with an elution buffer such that the resin- bound biological target is released from the nanoresin, thereby forming a suspension with an eluted biological target and an eluted nanoresin; and separating the eluted biological target from the eluted nanoresin.

2. The method as recited in claim 1, wherein the graphene oxide (GO) nanoresin comprises two-dimensional sheets.

3. The method as recited in claim 2, wherein the graphene oxide (GO) nanoresin comprises surface ligands.

4. The method as recited in claim 3, wherein the surface ligands are nitrilotriacetic acid (NTA), bis-nitrilotriacetic acid (bis-NTA), or tris-nitrilotriacetic acid (tris- NTA).

5. The method as recited in claim 3, wherein the surface ligands comprises a protein that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

6. The method as recited in claim 3, wherein the surface ligands comprises a protein that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

7. The method as recited in claim 6, wherein the protein further comprises a histidine-tag at a terminal end of the protein, the histidine-tag having 6-10 sequential histidine amino acids.

8. The method as recited in any one of claims 3-7, wherein the ligands chelate a metal ion selected from Ni2+, Cu2+, Zn2+, Ag+, Fe3+, Ga3+, Zr3+, Ca2+and Co2+.

9. The method as recited in claim 8, wherein the waiting the predetermined time waits for at least 1 minute to 30 minutes.

10. The method as recited in claim 9, wherein the eluting the resin-bound target elutes by eluting with the elution buffer followed by washing with a washing buffer.

11. The method as recited in claim 10, wherein the elution buffer is aqueous imidazole at a first concentration and the washing buffer comprises Tris, NaCl and imidazole at a second concentration, wherein the first concentration is at least ten times the second concentration.

12. The method as recited in claim 10, wherein the elution buffer comprises glycine with a pH of 2.5-3.0 and the washing buffer comprises NaCl with a pH of about 7.

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