System and method for end-to-end continuous downstream processing

Abstract

Provided herein are methods for purifying a protein of interest using continuous countercurrent downstream processing. Also provided herein are methods of purifying a protein of interest using continuous countercurrent affinity nanoparticle dialysis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 366,159, filed on June 10, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0003] Materials Submitted Electronically Are Incorporated by Reference

[0004] Incorporated herein by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently with this document and identified as follows: a 50,990-byte xml file named "CMTFF-100-WO-PCT.xml" created on June 7, 2023. Background Art

[0005] As biology moves to the forefront of drug development, the need for improved manufacturing processes is growing. With the expected increase in demand for recombinant protein therapeutics, more cost-effective and flexible manufacturing processes are needed. In fact, various economic analyses estimate that process development and clinical manufacturing costs can account for 40% to 60% of drug development costs. Together with commercial manufacturing, which is primarily driven by consumable downstream processing costs, this can amount to up to 25% of sales revenues for biologics. Therefore, more efficient downstream processing is needed. Summary of the invention

[0006] The present disclosure relates to a method for purifying a product of interest using countercurrent flow filtration, the method comprising: (a) contacting a first solution containing the product of interest and impurities with a binding molecule to form a complex, wherein the complex contains the product of interest bound to the binding molecule; (b) contacting the first flowing solution containing the complex with a first side of a semipermeable membrane, wherein the complex has a molecular weight exceeding the molecular weight cutoff of the semipermeable membrane, such that the complex is retained on the first side of the membrane; (c) passing the impurities through the semipermeable membrane, wherein the impurities have a molecular weight below the molecular weight cutoff of the semipermeable membrane and are retained on the second side of the semipermeable membrane in a second flowing solution countercurrent to the first flowing solution; and (d) dissociating the complex to form free product of interest and free binding molecule.

[0007] In one aspect, the method further comprises (e) regenerating the binding molecule, wherein the regenerated binding molecule is capable of forming a complex upon contact with the product of interest in the first solution or the second solution.

[0008] The present disclosure also provides a method for purifying a product of interest using countercurrent flow filtration, the method comprising: (a) contacting a first mobile solution containing a product of interest and impurities with a first side of a semipermeable membrane, wherein the product of interest passes through the semipermeable membrane to form a complex with a binding molecule on the second side of the semipermeable membrane, wherein the complex has a molecular weight exceeding the molecular weight cutoff of the semipermeable membrane, so that the complex is retained on the second side of the semipermeable membrane; (b) optionally retaining impurities on the first side of the semipermeable membrane, or wherein impurities flow through the semipermeable membrane, wherein the impurities have a molecular weight lower than the molecular weight cutoff of the semipermeable membrane, and either remain on the first side of the semipermeable membrane in a first mobile solution countercurrent with a second mobile solution, or pass through the semipermeable membrane to reach the second side of the semipermeable membrane; wherein the second mobile solution flow rate is lower than the first mobile solution flow rate, and (c) dissociating the complex to form free products of interest and free binding molecules. The method also includes (e) regenerating binding molecules, wherein the regenerated binding molecules are capable of forming complexes when contacting the product of interest in the first mobile solution or the second mobile solution. In some aspects, the regenerated binding molecules pass through the second side of the semipermeable membrane again in the second mobile solution.

[0009] In one aspect, the second mobile solution comprises second binding molecules that can bind to impurities in the first solution and / or the second mobile solution. In another aspect, unbound binding molecules diffuse across the semipermeable membrane into the second mobile solution.

[0010] In some aspects, the product of interest is a protein. In one aspect, the binding molecule comprises protein A, protein G, a cation exchange resin, or an anion exchange resin. In another aspect, the binding molecule comprises protein A. In one aspect, a first solution comprising the product of interest is obtained from a bioreactor. In one aspect, the second mobile solution comprises a positively charged polymer. In another aspect, the positively charged polymer is DEAE dextran.

[0011] In one aspect, the impurities include low molecular weight species. In another aspect, the positively charged polymer binds low molecular weight species that have diffused across the semipermeable membrane.

[0012] In one aspect, steps (a) to (c) are repeated, and wherein the binding molecule comprises protein A. In another aspect, steps (a) to (c) are repeated, and wherein the binding molecule comprises a cation exchange resin. In another aspect, steps (a) to (c) are repeated, and wherein the binding molecule comprises an anion exchange resin.

[0013] In some aspects, the binding molecule includes an assembled nanoparticle. In some aspects, the assembled nanoparticle is an assembled ferritin nanoparticle comprising 24 fusion protein monomers. In some aspects, each fusion protein monomer comprises i) a self-assembling nanoparticle monomer; ii) a linker; and iii) an immunoglobulin binding domain; in some aspects, the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NO: 1-3. In some aspects, the immunoglobulin binding domain is a protein A Z-domain. In some aspects, the protein A Z-domain comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NO: 4-7. In some aspects, the linker comprises an amino acid sequence selected from one of SEQ ID NO: 8-27. In some aspects, uncomplexed and / or unassembled fusion protein monomers diffuse into a second mobile solution through a semipermeable membrane. In some aspects, the fusion protein monomer also comprises a purification tag at one end of the fusion protein monomer. In some aspects, the purification tag comprises 6, 8 or 10 repeated histidines. In some aspects, the fusion protein monomer further comprises a protease site between the purification tag and the rest of the fusion protein monomer. In some aspects, the fusion protein monomer comprises a protease site, i.e., a HRV-3C protease site. In some aspects, the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 28-36. In some aspects, the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 32, or is encoded by a nucleic acid comprising SEQ ID NO: 52. In some aspects, the fusion protein monomer comprises 2 to 5 immunoglobulin binding domains. In some aspects, 2 to 5 immunoglobulin binding domains are protein A Z-domains. In some aspects, 2 to 5 immunoglobulin binding domains are separated from each other by a linker. In some aspects, the fusion protein monomers are capable of assembling into assembled nanoparticles comprising 24 fusion protein monomers.

[0014] In some aspects, steps (b) and (c) are repeated. In some aspects, steps (b) and (c) are repeated 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0015] In some aspects, the permeate from step (b) and / or step (c) is recycled to the first mobile solution and / or the upstream mobile solution. In some aspects, the permeate from step (b) and / or step (c) is recycled to the first mobile solution and / or the second mobile solution and / or the upstream solution. In one aspect, the protein dissociated is diafiltered. In some aspects, the semipermeable membrane is a dialysis membrane.

[0016] In one aspect, the first mobile solution and / or the second mobile solution has a flow rate of about 30 ml / min to about 60 ml / min. In another aspect, the first mobile solution and the second mobile solution have the same flow rate. In another aspect, the first mobile solution and the second mobile solution have different flow rates. In another aspect, the first mobile solution and / or the second solution are pulsed. In another aspect, the pulse enhances mass transfer across the semipermeable membrane. In another aspect, the pulse volume is less than the volume of the pores of the semipermeable membrane. In another aspect, the pulse volume is about half the volume of the pores of the semipermeable membrane. In another aspect, the pulse volume is greater than the volume of the pores of the semipermeable membrane.

[0017] In one aspect, the product of interest of about 0.1 kg / day, about 0.5 kg / day, about 1 kg / day, about 2 kg / day, about 3 kg / day, about 4 kg / day, about 5 kg / day, about 6 kg / day, about 7 kg / day, about 8 kg / day, about 9 kg / day or about 10 kg / day is purified. On the other hand, the product of interest comprises antibody, Fab, fusion protein, naturally occurring protein, chimeric protein or any combination thereof. On the other hand, the protein comprises the antibody selected from IgM, IgA, IgE, IgD and IgG, and wherein optionally about 10mM to about 1M NaCl or more preferably about 150mM NaCl are added in the first mobile solution. On the other hand, the protein comprises antibody and the antibody is the IgG antibody selected from IgG1, IgG2, IgG3 and IgG4. On the other hand, the antibody is a monoclonal antibody.

[0018] In one aspect, the first mobile solution is concentrated to about 50 g / L to about 100 g / L of the product of interest. In one aspect, the product of interest is a monoclonal antibody. In one aspect, the free product of interest is concentrated to about 50 g / L to about 100 g / L.

[0019] In one aspect, wherein the molecular weight of the complex is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times the size of the molecular weight cutoff of the semipermeable membrane. In some aspects, steps (a) to (c) are performed a second time, and wherein the second mobile solution of the second time is added to the first mobile solution of the first time, and wherein the MWCO of the filter used for the second time has a MWCO of the same size or greater than the first step (a)-(c). In some aspects, steps (a) to (c) are performed a second time, and wherein the first mobile solution of the second time is added to the first mobile solution of the first time, and wherein the MWCO of the filter used for the second time has a MWCO of the same size or greater than the first step (a)-(c). In some aspects, steps (a) to (c) are performed a third time, and wherein the second mobile solution of the third time is added to the first mobile solution of the second time, and wherein the MWCO of the filter used for the third time has a MWCO of the same size or greater than the first or second step (a)-(c). In some aspects, steps (a) to (c) are performed a third time, and wherein the third first mobile solution is added to the second first mobile solution, and wherein the MWCO of the filter used the third time has a MWCO that is the same size or greater than the MWCO of the first or second performance of steps (a)-(c). In some aspects, the second and third steps (a) to (c) are performed consecutively. In some aspects, the third first mobile solution or the second mobile solution flows into the second first mobile solution, and / or wherein the second first mobile solution or the second mobile solution flows into the first first mobile solution.

[0020] In one aspect, the present disclosure provides a method of using a solution effluent of filtrate or dialysate from a sequential downstream step as a wash solution for an upstream step, wherein the upstream step has a filter having a MWCO of the same size or greater than the downstream step generating the effluent.

[0021] In one aspect, the present disclosure provides a method of using a solution effluent from a retentate of a sequential downstream step as a wash liquid for an upstream step, wherein the upstream step has a filter having a MWCO of the same size or smaller than the downstream step generating the effluent, and the filtrate is directed downstream. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram showing the sequential countercurrent affinity colloid capture steps.

[0023] Figure 2 Critical flux data for the dehydration stage are shown.

[0024] Figure 3A and Figure 3B. Figure 3A A schematic diagram showing the three-stage CM dehydration is shown, and Figure 3B A schematic diagram of the fully continuous downstream process is shown.

[0025] Figure 4 Shown are the sizes of complexes formed at different molar ratios of sProA / mAbl.

[0026] Figure 5A and Figure 5B Schematic diagram showing the three stages of dewatering and countercurrent washing. Figure 5C Affinity colloid separation using soluble protein A for capture antibody is shown. Figure 5D A soluble Protein A purification cycle using dialysis-based Protein A recycle is shown.

[0027] Figure 6 Critical flux data for the wash phase are shown.

[0028] Figure 7 The effect of salt concentration on the partition coefficients of sProA and mAb during the first elution step is shown.

[0029] Figure 8 Shown are data generated during the first elution of the CEX polishing step, where the partition coefficients of sProA and mAbl were measured at pH 3 and varying concentrations of NaCl.

[0030] Fig. 9 A schematic diagram of a one-stage system is shown. A peristaltic pump (P1) is used to pump the feed containing mAb, impurities and resin slurry into the TFF module. The permeate flux is regulated by a peristaltic pump (P2).

[0031] Fig.10 A schematic diagram of a single-pass tangential flow filtration ultrafiltration system is shown. A peristaltic pump (P1) is used to pump the feed containing mAb, impurities and resin slurry into the TFF module. The permeate flux is regulated by a peristaltic pump (P2).

[0032] Fig.11 Shown are the HCP partition coefficients and Kp impurities for 5% Fractogel TMAE resin at various supplied HCP concentrations in a 10 g / L mAb pool.

[0033] Fig.12 A to Fig.12 B. Fig.12 A shows a preparative SEC chromatogram indicating the fractions that were pooled based on the analytical SEC column results. Fig.12B shows an A280 HPLC-SEC trace (TSKgel SuperSW mAb) of fractions from each of the characteristic peaks (A3, B3 and C3). Fractions containing only peaks 1 and 2 on HPLC-SEC were pooled. Fig.12 C shows the SEC chromatogram of 7.8 mL of 2.2 mg / ml dialyzed ferritin (50 mM tris pH 7.4) loaded onto a XK26 / 70 prepacked Superose 6 column using a 50 mM Tris pH 7.4 mobile phase at a flow rate of 40 cm / h (isocratic elution phase), and the corresponding SDS PAGE gel of mock pool samples (L1-L4) representing each peak (1-4), showing that all peaks contain primarily ferritin (31 kDa band).

[0034] Fig.13 Results of a method for making antibody and nanoparticle samples that do not produce precipitation are shown.

[0035] FIG. 14A to FIG. 14D It shows ( Fig.14A ) soluble protein A (cycle 0), ( Fig. 14B ) purified nanoparticles (cycle 1), ( Fig. 14C ) Nanoparticle permeation from cycle 1 (cycle 1b), ( Fig.14D ) Affinity chromatogram of the neutralized eluate (cycle 2) from cycle 1. Based on peak area rather than mass balance, all soluble protein A (sProA) was found in the eluate. The nanoparticles also showed binding, elution, and even rebinding after elution.

[0036] Fig.15 Shown is an overlay of preparative SEC chromatograms showing isocratic elution for three neutral pH runs: antibody and nanoparticles together, antibody alone, and nanoparticles alone.

[0037] Fig.16 Shown are SDS-PAGE results of fractions of a neutral pH antibody and nanoparticle mixture run on preparative SEC.

[0038] Fig.17 Preparative SEC chromatogram overlay showing isocratic elution of three separate low pH loads: antibody and nanoparticles together, antibody alone, and nanoparticles alone

[0039] Fig.18 Shown are the results of SDS-PAGE of fractions of the low pH antibody and nanoparticle mixture run on SEC.

[0040] Fig.19 Shown is a comparison of the antibody and nanoparticle mixture on a SEC column at neutral pH and low pH.

[0041] Fig. 20 SDS PAGE gels of load, retentate and filtrate samples at neutral and low pH are shown with ladder bands. Annotated images of the ladder bands are included next to the indication of heavy chain (HC), light chain (LC) and ferritin monomer (NP384). The dashed box highlights the lanes demonstrating filter-based separation.

[0042] Fig.21 Next to the volumes of retentate and filtrate after centrifugation at 2000 g for 15 s at room temperature, a list of sieving coefficients for each separation is shown.

[0043] Fig. 22 Shown are normalized heat curves from isothermal titration calorimetry; 0.5 g / L nanoparticles were titrated with mAb, showing an average of 14 binding sites per nanoparticle with an equilibrium dissociation constant of 25 nM.

[0044] Fig.23 A schematic diagram of a one-stage system is shown. A peristaltic pump (P1) is used to pump the feed containing mAb and CM into the TFF module. The permeate flux is regulated by a peristaltic pump (P2) on the retentate outlet.

[0045] Fig.24 Size exclusion chromatogram showing size enriched feed, 0.5 mg / mL nanoparticles, retentate after 2 dialysis volumes, and 300 kDa TFF size enriched final product

[0046] Fig.25 Schematic diagram showing multi-stage countercurrent single-pass TFF

[0047] Fig.26 Shown is the effect of pre-filling the membrane with conditioned medium on the operation of the TMP

[0048] Fig. 27 SDS-PAGE gel for TFF elution steps is shown. Purified mAb 0.5 g / L and 0.05 g / L, in wells 1 and 2, respectively; pure nanoparticles 0.23 g / L and 0.02 g / L, in wells 3 and 4, respectively; well 5 - Bio-Rad Precision Plus unstained protein marker; well 6 - TFF feed; TFF permeate sample wells P1 to P5; TFF retentate sample wells R1 to R5.

[0049] Fig.28 Schematic diagram showing the continuous countercurrent affinity nanoparticle dialysis procedure (C3ANDo)

[0050] Fig.29A and Fig.29B . Fig.29AThe effective diffusion coefficient of mAb in a ~500 kDa membrane and the effect on mAb recovery at various feed fluxes are shown ( Fig.29B ).

[0051] Fig.30 A scheme of the C3ANDo capture step is shown.

[0052] Fig.31 The scheme for the C3ANDo elution study is shown.

[0053] Fig.32 Shown is an SDS-PAGE gel of the C3ANDo elution steps, including the recycle phase (40 min-640 min) and single pass (well 4-well 9) samples, and a Bio-Rad Precision Plus unstained protein marker as a molecular weight ladder.

[0054] Fig.33 Shown is an SDS-PAGE gel of the C3ANDo elution steps, including recycle phase (well 10-well 12) and single pass (well 4-well 9) samples, and a Bio-Rad Precision Plus unstained protein marker (well 3) as a molecular weight ladder. DETAILED DESCRIPTION

[0055] The present disclosure provides an efficient method for removing contaminants using countercurrent filtration during protein purification without the need for chromatography. Thus, the present disclosure provides a method for purifying a product of interest that uses about 1 / 10 the amount of water and solvent used in a chromatographic process.

[0056] I. Definitions

[0057] In order to more easily understand the present disclosure, some terms are first defined. As used in this specification, unless otherwise explicitly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0058] It should be noted that the term "a" or "an" refers to one or more of the entity; for example, "a feed medium" should be understood to mean one or more feed media. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0059] As used herein, the term "and / or" should be understood as specifically disclosing each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0060] It should be understood that wherever herein aspects are described with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for the skilled person.

[0062] Units, prefixes and symbols are expressed in their SI recognized form. Numerical ranges include numerical values ​​that limit the range. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below can be more fully defined by reference to the specification in its entirety.

[0063] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated components.

[0064] The term "about" or "substantially comprising" means that a value or composition is within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, these terms may mean values ​​of up to an order of magnitude or up to 5 times. When a specific value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value or composition.

[0065] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range, and where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0066] The terms "polypeptide" or "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention; the intervention is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modification operation, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically encompass antibodies and polypeptides containing an Fc domain (e.g., immunoadhesins).

[0067] "Anion exchange resin" or "anion exchange polymer" refers to a solid or liquid phase that is positively charged and therefore has one or more positively charged ligands attached thereto. Any positively charged ligand attached to a solid phase suitable for forming an anion exchange resin may be used, such as a quaternary amino group.

[0068] "Cation exchange resin" or "cation exchange polymer" refers to a negatively charged solid or liquid phase and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Any negatively charged ligand attached to a solid phase suitable for forming a cation exchange resin may be used, such as carboxylates, sulfonates, and other ligands as described herein.

[0069] As used herein, the term "product of interest" is used in its broadest sense, including any product (natural or recombinant) that needs to be purified, including proteins, nucleic acids, macromolecules or lipids present in a mixture. Such products of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies) and / or any fusion proteins. Such products of interest may also include DNA, RNA, cDNA and antisense oligonucleotides. In some aspects, the product of interest refers to any protein that can be produced by the methods described herein. In some aspects, the product of interest refers to any DNA or RNA sequence that can be produced by the methods described herein. In some aspects, the product of interest is a protein of interest.

[0070] As used herein, the term "protein of interest" is used in its broadest sense and includes any protein (natural or recombinant) present in a mixture that needs to be purified. Such proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies) and / or any fusion proteins. In some aspects, a protein of interest refers to any protein that can be produced by the methods described herein. In some aspects, a protein of interest is an antibody. In some aspects, a protein of interest is a recombinant protein.

[0071] The terms "purification", "separation" or "isolation" as used interchangeably herein refer to increasing the purity of a product of interest from a composition or sample comprising the product of interest and one or more impurities. Typically, the purity of a product of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0072] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or base that must be added to cause a unit pH change. Buffer solutions resist changes in pH under the action of their acid-base conjugate components. Buffer solutions used with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0073] As used herein, the term "continuous countercurrent modular tangential flow filtration" or "CMTFF" refers to the general technology described herein in which tangential flow filtration is used in one or more separate embodiments, optionally in series or in parallel, wherein the flow on the permeate side of the membrane flows in a direction countercurrent to the retentate side of the membrane.

[0074] As used herein, the term "C3ANDo" or "continuous countercurrent affinity nanoparticle dialysis" refers to the general dialysis method used herein in which one or more separate embodiments are optionally connected in series or in parallel, wherein the flow on the lumen side of the dialysis module flows in a direction countercurrent to the shell side of the module.

[0075] As used herein, the term "sProA" or "soluble protein A" refers to any protein A that is soluble in solution. Typically, protein A is therefore not bound to a chromatographic resin or is not bound to a membrane. In some aspects, protein A is derived from Staphylococcus. Soluble protein A will remain bound to the Fc region of an IgG immunoglobulin.

[0076] As used herein, the term "contaminant" is used in its broadest sense, covering any undesirable component or compound in a mixture. In cell culture, cell lysate or clarified body (for example, clarified cell culture supernatant), contaminant includes, for example, host cell nucleic acid (for example, DNA) and host cell protein present in cell culture medium. Host cell contaminant protein includes but is not limited to those produced naturally or recombinantly by host cells, and protein (for example, proteolytic fragments) and other process-related contaminants associated with or derived from a product of interest. In certain embodiments, another means such as centrifugation, sterile filtration, deep filtration and tangential flow filtration are used to separate the contaminant precipitate from cell culture.

[0077] The term "HMW species" refers to any one or more unwanted proteins present in a mixture. High molecular weight species may include dimers, trimers, tetramers or other polymers. These species are generally considered to be product-related impurities and may be covalently or non-covalently attached and may also, for example, consist of misfolded monomers in which hydrophobic amino acid residues are exposed to polar solvents and may cause aggregation.

[0078] The term "LMW species" refers to any one or more unwanted species present in a mixture. Low molecular weight species are generally considered product-related impurities and may include truncated species, charge variants, or half molecules of compounds that are expected to dimerize (such as monoclonal antibodies).

[0079] The term "host cell protein" or HCP refers to an undesirable protein produced by the host cell that is unrelated to the production of the desired product of interest. Undesirable host cell proteins can be secreted into the upstream cell culture supernatant. Undesirable host cell proteins can also be released during cell lysis. Cells used for upstream cell culture require proteins for growth, transcription, and protein synthesis, and these unrelated proteins are undesirable in the final drug product.

[0080] As used herein, the term "fed-batch culture" or "fed-batch culture process" refers to a method of culturing cells in which additional components are provided to the culture at a certain time after the start of the culture process. A fed-batch culture can be started with a basal medium. A culture medium in which additional components are provided to the culture at a certain time after the start of the culture process is a feed medium. The fed-batch culture is usually stopped at a certain time point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0081] The term "chromatography" refers to any kind of technique for separating a protein of interest (e.g., an antibody) from other molecules present in a mixture (e.g., contaminants). Typically, the protein of interest is separated from other molecules (e.g., contaminants) due to the different rates at which the individual molecules of the mixture migrate in a fixed medium under the influence of a mobile phase or during binding and elution. The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to any kind of adsorbent, resin, or solid phase that separates a protein of interest (e.g., a protein containing an Fc region, such as an immunoglobulin) from other molecules present in a mixture during separation. Non-limiting examples include particles, monolithic or fiber resins, and membranes that can be placed in a column or filter cartridge. Examples of materials for forming a matrix include polysaccharides (e.g., agarose and cellulose); and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl) benzene, polyacrylamide, ceramic particles, and derivatives of any of the above substances. Examples of typical matrix types suitable for use in the disclosed methods are cation exchange resins, affinity resins, anion exchange resins, or mixed mode resins.

[0082] "Part " is a functional group that is attached to the chromatographic matrix and determines the binding properties of the matrix. The example of "part " includes but is not limited to ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, sulfophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups and mixed mode groups (combinations of the aforementioned groups). Some preferred ligands that can be used herein include but are not limited to strong cation exchange groups, such as sulfopropyl, sulfonic acid; strong anion exchange groups, such as trimethylammonium chloride; weak cation exchange groups, such as carboxylic acid; weak anion exchange groups, such as N5N diethylamino or DEAE; hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl; and affinity groups, such as protein A, protein G and protein L. In order to be able to more easily understand the present disclosure, some terms are first defined. As used in the present application, unless otherwise explicitly provided in this article, each term in the following terms should have the meaning set forth below. Additional definitions are set forth throughout the present application.

[0083] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., an Fc region containing a protein of interest or an antibody) specifically binds to a ligand that is specific to the protein of interest. Such ligands are commonly referred to as biospecific ligands. In some aspects, a biospecific ligand (e.g., protein A or its functional variant) is covalently attached to a chromatographic matrix material, and when a solution contacts a chromatographic matrix, the protein of interest in the solution can approach the biospecific ligand. During the chromatographic step, the protein of interest generally retains its specific binding affinity to the biospecific ligand, while other solutes and / or proteins in the mixture do not bind the ligand significantly or specifically. The binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatographic matrix, while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. Then under suitable conditions (e.g., low pH, high pH, ​​high salt, competitive ligands, etc.), the specifically bound protein of interest is removed from the immobilized ligand in an active form, and passed through the chromatographic column together with the elution buffer, without the contaminating proteins or protein impurities that were previously allowed to pass through the column. Any component can be used as a ligand for purifying its corresponding specific binding protein (e.g., antibody). However, in various methods according to the present disclosure, protein A is used as a ligand containing the Fc region of the target protein. One of ordinary skill in the art can easily determine the conditions under which the target protein (e.g., protein containing the Fc region) is eluted from the biospecific ligand (e.g., protein A). In some aspects, protein G or protein L or functional variants thereof can be used as biospecific ligands. In some aspects, a biospecific ligand (such as protein A) is used to bind to the protein containing the Fc region at a pH range of 5-9, the biospecific ligand / target protein conjugate is washed or rebalanced, and then eluted with a buffer solution containing at least one salt having a pH higher than or lower than 4. In some aspects, protein A is not bound to the chromatographic column resin and is therefore a soluble protein A or "sProA".

[0084] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or base that must be added to cause a unit pH change. Buffer solutions resist changes in pH under the action of their acid-base conjugate components. Buffer solutions used with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0085] As used herein, the term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, electric current flows by the transport of ions. Therefore, as the amount of ions present in an aqueous solution increases, the solution will have a higher conductivity. Conductivity is measured in millisiemens per centimeter (mS / cm) and can be measured using a conductivity meter.

[0086] As used herein, the term "mobile phase" refers to a liquid or gas that flows through a chromatography system, causing the materials to be separated to move at different rates on the stationary phase. Mobile phases can be polar or non-polar. Polar mobile phases are often used in conjunction with non-polar stationary phases, and these chromatographic separations are known as reversed phase chromatography. In contrast, non-polar mobile phases are often used in conjunction with polar stationary phases, and are often referred to as normal phase chromatography.

[0087] As used herein in the context of chromatography, the term "stationary phase" refers to a solid or liquid phase in a chromatography system that selectively adsorbs the materials to be separated. Typically, silica is used as the stationary phase.

[0088] As used herein, the term "chromatography column" or "column" in relation to chromatography refers to a container, usually in the form of a cylinder or hollow column, filled with a chromatography matrix or resin. A chromatography matrix or resin is a material that provides physical and / or chemical properties for purification.

[0089] The terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic process in which an ionizable solute of interest (e.g., a protein of interest in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate conditions of pH and conductivity so that the solute of interest interacts more or less nonspecifically with the charged compound than with solute impurities or contaminants in the mixture. Contaminating solutes in the mixture may be washed out of the column of ion exchange material faster or slower than the solute of interest, or may bind to or be excluded from the resin. "Ion exchange chromatography" specifically includes cation exchange (CEX), anion exchange (AEX), and mixed mode chromatography.

[0090] As used herein, "perfusion" or "perfusion culture" or "perfusion culture process" refers to the continuous flow of a physiological nutrient solution through or through a cell population at a steady rate. Because perfusion systems typically involve retaining cells within a culture unit, perfusion cultures characteristically have relatively high cell densities, but culture conditions are difficult to maintain and control. In addition, because cells are grown at high density and retained within the culture unit, the growth rate typically decreases over time, causing the cell growth to enter a late exponential phase or even a stationary phase. This continuous culture strategy typically involves culturing mammalian cells, such as non-fixation-dependent cells, expressing a polypeptide and / or virus of interest during a production phase in a continuous cell culture system.

[0091] "Antibodies" (Ab) shall include, but are not limited to, glycoprotein immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. In some aspects, the antibody is a full-length antibody.

[0092] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, IgD, IgE and IgM. IgG subclasses are also well known to those skilled in the art, and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. The term "antibody" may include multivalent antibodies (e.g., trivalent antibodies) that can bind more than two antigens. A trivalent antibody is an IgG-type bispecific antibody composed of two regular Fab arms fused to an asymmetric third Fab-sized binding module via a flexible linker peptide. This third module replaces the IgG Fc region and consists of a heavy chain variable region fused to a CH3 with a "knob" mutation and a light chain variable region fused to a CH3 with a matching "hole". The hinge region does not contain a disulfide bond to facilitate antigen access to the third binding site. In the absence of explicit indication, the term "antibody" includes monospecific, bispecific or multispecific antibodies, as well as single-chain antibodies, unless the context indicates otherwise.

[0093] As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of VL, VH, LC and CH1 domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs), and (vii) a combination of two or more isolated CDRs optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by a synthetic linker using recombinant methods, which enables them to become a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for usefulness in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0094] An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-L1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity with other antigens (such as PD-L1 molecules from different species). In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0095] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody that has two different heavy / light chain pairs, thereby generating two antigen-binding sites that are specific for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0096] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are substantially identical and which exhibit a single binding specificity and affinity for a particular epitope. A monoclonal antibody is an example of an isolated antibody. Monoclonal antibodies can be produced by hybridoma technology, recombinant technology, transgenic technology, or other techniques known to those skilled in the art.

[0097] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence, wherein the first amino acid sequence is not naturally linked to the second amino acid sequence in nature. Amino acid sequences that are normally found in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally found in the same protein can be placed in a fusion polypeptide in a new arrangement, for example, the fusion of the Factor VIII domain of the present disclosure with the Ig Fc domain. For example, a fusion protein is formed by chemical synthesis, or by forming and translating a polynucleotide in which the peptide regions are encoded in a desired relationship. A chimeric protein can also include a second amino acid sequence linked to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0098] In all aspects of the fusion protein monomer, the self-assembled nanoparticle monomer is a ferritin monomer. Ferritin is an intracellular protein that stores iron and releases iron as needed. Ferritin is widely conserved and found in almost all living organisms. In its native state, ferritin is a globulin of 24 subunits that self-assembles to form hollow nanoparticles. Vertebrates have two types of ferritin, a light subunit (L) with a molecular weight of about 19 kDa and a heavy subunit (H) with a molecular weight of about 21 kDa. Amphibians also express another type of ferritin, called M ferritin. Most organisms express ferritin similar to vertebrate H-type ferritin.

[0099] Ferritin is a hollow globular protein with a mass of 474 kDa and comprises 24 ferritin monomer subunits. Therefore, as used herein, "assembled ferritin nanoparticles" refer to assembled proteins comprising 24 ferritin monomer subunits. In some aspects, the ferritin monomer subunit is a "fusion protein monomer" containing at least one self-assembly (sa) ferritin monomer sequence and one or more affinity ligands, which are separated by any number of homologous or heterologous linker sequences. A histidine tag (e.g., 8 histidines) may also be included to facilitate purification by immobilized metal affinity chromatography (IMAC). When a histidine tag is included, a cleavable sequence may be included to optionally enzymatically cleave the histidine tag with a protease after expression.

[0100] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range, and where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0101] As used herein, "culturing" refers to growing one or more cells in vitro under defined or controlled conditions. Examples of definable culture conditions include temperature, gas mixture, time, and medium formulation.

[0102] As used herein, the term "seeding" refers to adding cells to a culture medium to initiate a culture.

[0103] As used herein, the term "induction" or "induction phase" or "growth phase" of cell culture refers to the initial inoculation of a bioreactor (e.g., a seed bioreactor) at the start of upstream cell culture, and includes an exponential cell growth period (e.g., logarithmic phase) during which cells primarily divide rapidly. During this period, the rate of increase in viable cell density is higher than at any other time point.

[0104] As used herein, the term "production phase" of cell culture refers to the period of time during which cell growth is static or maintained at a near constant level. The density of viable cells remains approximately constant over a given period of time. Logarithmic cell growth has ended, and protein production is the main activity during the production phase. At this point, culture medium is usually supplemented to support continued protein production and obtain the desired glycoprotein product.

[0105] As used herein, the term "expression" is used to refer to transcription and translation occurring within a cell. The expression level of a product gene in a host cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell, or both.

[0106] As used herein, the terms "culture medium" and "cell culture medium" as well as "feed medium" and "fermentation medium" refer to nutrient solutions used to grow and / or maintain cells, particularly mammalian cells. Without limitation, these solutions typically provide at least one component from one or more of the following categories: (1) an energy source, typically in the form of a carbohydrate such as glucose; (2) all essential amino acids, typically a basic set of twenty amino acids plus cysteine; (3) vitamins and / or other organic compounds at lower required concentrations; (4) free fatty acids or lipids, such as linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are required at typically very low concentrations (often in the micromolar range). The nutrient solution may be optionally supplemented with one or more components from any of the following categories: (1) hormones and other growth factors, such as serum, insulin, transferrin, and epidermal growth factor; (2) salts, such as magnesium salts, calcium salts, and phosphates; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; (5) protein and tissue hydrolysates, such as peptone or peptone mixtures obtained from purified gelatin, plant materials, or animal by-products; (6) antibiotics, such as gentamicin; (7) cell protectants, such as pluronic polyols; and (8) galactose. Commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) are suitable for culturing host cells. In addition, any medium described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980) can be used as a culture medium for the host cells. Any other necessary supplements may also be included at appropriate concentrations.

[0107] Various aspects of the disclosure are described in more detail in the following subsections.

[0108] II. Countercurrent Filtration

[0109] The present disclosure provides an efficient method for removing contaminants using countercurrent filtration during protein purification without the need for chromatography. Thus, the present disclosure provides a method for purifying a product of interest that uses about 1 / 10 the amount of water and solvent used in a chromatographic process.

[0110] The present disclosure provides a method for purifying a product of interest using countercurrent flow filtration, the method comprising: (a) contacting a first solution containing the product of interest and impurities with a binding molecule to form a complex, wherein the complex contains the product of interest bound to the binding molecule; (b) contacting the first flowing solution containing the complex with a first side of a semipermeable membrane, wherein the complex has a molecular weight exceeding the molecular weight cutoff of the semipermeable membrane, so that the complex is retained on the first side of the membrane; (c) passing the impurities through the semipermeable membrane, wherein the impurities have a molecular weight lower than the molecular weight cutoff of the semipermeable membrane and are retained on the second side of the semipermeable membrane in a second flowing solution countercurrent to the first flowing solution; and (d) dissociating the complex to form a free product of interest and a free binding molecule, and (e) passing the product of interest or the binding molecule through the semipermeable membrane, wherein the smaller of the product of interest or the binding molecule has a molecular weight lower than the molecular weight cutoff of the semipermeable membrane and is retained on the second side of the semipermeable membrane in a second flowing solution countercurrent to the first flowing solution, while the larger of the product of interest or the binding molecule is retained on the first side of the membrane.

[0111] The present disclosure provides a method for purifying a product of interest using modular countercurrent flow filtration, the method comprising: (a) contacting a first solution containing the product of interest and impurities with a binding molecule to form a complex, wherein the complex contains the product of interest bound to the binding molecule; (b) contacting the first flowing solution containing the complex with a first side of a semipermeable membrane under pressure, wherein the complex has a molecular weight exceeding the molecular weight cutoff of the semipermeable membrane, so that the complex is retained on the first side of the membrane; (c) passing the impurities through the semipermeable membrane by convection, wherein the impurities have a molecular weight below the molecular weight cutoff of the semipermeable membrane, thereby becoming a filtrate; and (d) dissociating the complex to form a free product of interest and a free binding molecule, and (e) passing the product of interest or the binding molecule through the semipermeable membrane, wherein the smaller of the product of interest or the binding molecule has a molecular weight below the molecular weight cutoff of the semipermeable membrane and is retained on the second side of the semipermeable membrane in a second flowing solution countercurrent to the first flowing solution, while the larger of the product of interest or the binding molecule is retained on the first side of the membrane.

[0112] In some cases, the filtrate from one module is used to dilute another solution upstream of the semipermeable membrane so that the filtrate is used in a countercurrent manner.

[0113] In some aspects, the method further comprises: (e) regenerating the binding molecule by contacting a solution comprising the binding molecule with a first side of a semipermeable membrane, wherein the binding molecule has a molecular weight exceeding a molecular weight cut-off of the semipermeable membrane, such that the binding molecule is retained on the first side of the membrane, wherein the regenerated binding molecule is capable of forming a complex upon contact with the product of interest in the first solution or the second solution. Fig. 9In some aspects, the second mobile solution comprises a second binding molecule that can bind to impurities in the first solution and / or the second mobile solution. In some aspects, unbound binding molecules diffuse across the semipermeable membrane into the second mobile solution.

[0114] In some aspects, the binding molecule comprises Protein A, Protein G, a cation exchange resin, or an anion exchange resin. In some aspects, the binding molecule comprises Protein A.

[0115] "Cation exchange resin" or "cation exchange membrane" refers to a negatively charged solid phase and has free anions for exchanging cations in an aqueous solution passing through or through the solid phase. Any negatively charged ligand attached to a solid phase suitable for forming a cation exchange resin can be used, such as carboxylates, sulfonates, and other ligands as described below. Commercially available cation exchange resins include, but are not limited to, for example, those with sulfonate-based groups. In some aspects, the cation exchange resin is selected from those with the following groups: sulfonate-based groups (e.g., MonoS, Minis, Source 15S and 30S, SP from GE Healthcare). Fast Flow, SP HighPerformance, Capto S, Capto SPImpRes; available from Tosoh SP-650S and SP-650M; available from BioRad High S; Ceramic HyperDS from Pall Technologies, M and LS SP and Spherodex LS SP); sulfoethyl-based groups (e.g., SE; obtained from Applied Biosystems S-10 and S-20); sulfopropyl-based groups (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh; HS-20, HS 50 and XS); sulfoisobutyl-based groups (e.g., EMD SO3 - ); sulfoethyl-based groups (e.g., SE52, SE53, and Express-Ion S from Whatman); carboxymethyl-based groups (e.g., CM FastFlow; Hydrocell CM from Biochrom Labs Inc.; MACRO- CM; Ceramic HyperD CM from Pall Technologies, M CM, LS CM; Matrx from Millipore C500 and C200; CM52, CM32, CM23 and Express-Ion C from Whatman; CM-650S, CM-650M and CM-650C); sulfonic acid and carboxylic acid based groups (e.g., Carboxy-Sulfon); carboxylic acid-based groups (e.g., WP CBX from JT Baker; MAC-3; obtained from Sigma-Aldrich Weak cation exchanger, Weak cation exchangers and Weak cation exchanger; and EMD COO-); sulfonic acid-based groups (e.g., Hydrocell SP from Biochrom Labs Inc.; Fine mesh strong acid cationic resin; UNOsphere S, WP Sulfonic from JTBaker; S membrane; obtained from Sigma-Aldrich Strong cation exchanger, Strong cations and or orthophosphate-based groups (e.g., P11 from Whatman). Other cation exchange resins include carboxymethyl cellulose, BAKERBOND ABX™, CeramicHyperD Z, Matrex Cellufine C500, Matrex Cellufine C200, or any combination thereof.

[0116] "Anion exchange resin" or "anion exchange membrane" refers to a solid phase that is positively charged and therefore has one or more positively charged ligands attached thereto. Any positively charged ligand attached to a solid phase suitable for forming an anion exchange resin can be used, such as a quaternary amino group. Commercially available anion exchange resins include DEAE cellulose. In some aspects, the anion exchange resin is selected from DEAE cellulose, available from Applied Biosystems, PI 20, PI 50, HQ 10, HQ20, HQ 50, D 50; from Sartorius Q; MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Fast Flow, Q High Performance, QAE and FAST Q (GE Healthcare); WP PEI, WPDEAM, WP QUAT from JTBaker; Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc.; UNOsphere Q, MACRO- DEAE and MACRO- High Q; Ceramic HyperD Q, ceramic HyperD DEAE, available from Pall Technologies M and LSDEAE, Spherodex LSDEAE, QMA LS, QMA M and Q; obtained from Dow LiquidSeparations Fine mesh strong base type I and type II anion resins and MONOSPHER E 77 weak base anion; obtained from Millipore Q film, Matrex A200, A500, Q500 and Q800; available from EMD EMD TMAE, EMD DEAE and EMD DMAE; obtained from Sigma-Aldrich Weak and strong anion exchangers type I and II, Weak and strong anion exchangers type I and II, Weak and strong anion exchangers type I and II, TSK gel Q and DEAE 5PW and 5PW-HR from Tosoh, SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C; QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q from Whatman; and Q (Sartorius Corporation, New York, USA). Other anion exchange resins include POROS XQ, Q, Q Sepharose TM XL, Q Sepharose TM Big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q Sepharose TM High performance, QSepharose TM XL, Resource Q, Capto Q, Capto DEAE, Toyopearl GigaCap Q, Fractogel EMDTMAE HiCap, Nuvia Q or PORGSPI or any combination thereof.

[0117] In some aspects, a first solution comprising a product of interest can be obtained from a bioreactor. In some aspects, a first solution can be obtained from a bioreactor without any further downstream processing steps prior to performing the methods described herein.

[0118] In some aspects, the second mobile solution comprises a positively charged polymer. In some aspects, the positively charged polymer may include DEAE dextran. In some aspects, the positively charged polymer may bind low molecular weight substances that have diffused across the semipermeable membrane.

[0119] In some aspects, steps (a) to (c) of the methods described herein are repeated, and wherein the binding molecule comprises protein A. In some aspects, steps (a) to (c) of the methods described herein are repeated, and wherein the binding molecule comprises a cation exchange resin. In some aspects, steps (a) to (c) of the methods described herein are repeated, and wherein the binding molecule comprises an anion exchange resin.

[0120] In some aspects, the dissociated protein is diafiltered.

[0121] In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of at least about 10 ml / min to about 1,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of about 10 ml / min to about 9,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of about 10 ml / min to about 8,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of about 10 ml / min to about 7,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of about 10 ml / min to about 6,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of about 10 ml / min to about 5,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution may have a flow rate of about 10 ml / min to about 4,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution can have a flow rate of about 10 ml / min to about 3,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution can have a flow rate of about 10 ml / min to about 2,000 ml / min. In some aspects, the first mobile solution and / or the second mobile solution can have a flow rate of about 30 ml / min to about 60 ml / min, about 31 ml / min to about 60 ml / min, about 32 ml / min to about 60 ml / min, about 33 ml / min to about 60 ml / min, about 34 ml / min to about 60 ml / min, about 35 ml / min to about 60 ml / min, about 36 ml / min to about 60 ml / min, about 37 ml / min to about 60 ml / min, about 38 ml / min to about 60 ml / min, about 39 ml / min to about 60 ml / min, about 40 ml / min to about 60 ml / min, about 41 ml / min to about 60 ml / min, about 42 ml / min to about 60 ml / min, about 43 ml / min to about 60 ml / min, about 44 ml / min to about 60 ml / min, The invention can be applied to a flow rate of about 40 ml / min to about 60 ml / min, about 45 ml / min to about 60 ml / min, about 46 ml / min to about 60 ml / min, about 47 ml / min to about 60 ml / min, about 48 ml / min to about 60 ml / min, about 49 ml / min to about 60 ml / min, about 50 ml / min to about 60 ml / min, about 51 ml / min to about 60 ml / min, about 52 ml / min to about 60 ml / min, about 53 ml / min to about 60 ml / min, about 54 ml / min to about 60 ml / min, about 55 ml / min to about 60 ml / min, about 56 ml / min to about 60 ml / min, about 57 ml / min to about 60 ml / min, about 58 ml / min to about 60 ml / min, or about 59 ml / min to about 60 ml / min.In some aspects, the first flowing solution and the second flowing solution have the same flow rate. In some aspects, the first flowing solution and the second flowing solution have different flow rates.

[0122] In some aspects, the first flow rate and / or the second flow rate may include about 30 ml / min, about 31 ml / min, about 32 ml / min, about 33 ml / min, about 34 ml / min, about 35 ml / min, about 36 ml / min, about 37 ml / min, about 38 ml / min, about 39 ml / min, about 40 ml / min, about 41 ml / min, about 42 ml / min, about 43 ml / min, about 44 ml / min, about 45 ml / min, about 46 ml / min, about 47 ml / min, about 48 ml / min, about 49 ml / min, about 50 ml / min, about 51 ml / min, about 52 ml / min, about 53 ml / min, about 54 ml / min, about 55 ml / min, about 56 ml / min, about 57 ml / min, about 58 ml / min, about 59 ml / min, or about 60 ml / min.

[0123] In some aspects, the first flow rate is higher than the second flow rate. In some aspects, the second flow rate is higher than the first flow rate.

[0124] In some aspects, the first mobile solution and / or the second mobile solution are pulsed. In some aspects, the pulse enhances the mass transfer across the semipermeable membrane. In some aspects, the pulse volume is less than the total volume sum of the holes of the semipermeable membrane. In some aspects, the pulse volume is about half of the total volume sum of the holes of the semipermeable membrane. In some aspects, the pulse volume is greater than the total volume sum of the holes of the semipermeable membrane. In some aspects, a peristaltic pump can be used to generate pulses in the first mobile solution and / or the second mobile solution. In some aspects, a piston pump can be used to generate pulses in the first mobile solution and / or the second mobile solution. In some aspects, a piezoelectric actuator can be used to generate pulses in the first mobile solution and / or the second mobile solution. In some aspects, the pulse flow is not completely reversible, resulting in diffusion mass transfer.

[0125] In some aspects, the semipermeable membrane has a molecular weight cut-off of about 1,000 kDa. In some aspects, protein A has a molecular weight exceeding 1,000 kDa, wherein protein A cannot diffuse through the semipermeable membrane. In some aspects, the product of interest is a monoclonal antibody with a molecular weight of about 150 kDa. In some aspects, the complex comprising the monoclonal antibody and the protein has a molecular weight exceeding 1,000 kDa. In some aspects, the semipermeable membrane is a ceramic membrane.

[0126] In some aspects, the semipermeable membrane has a molecular weight cutoff of about 500 kDa to about 1000 kDa. In some aspects, the complex comprising the monoclonal antibody and the protein A has a molecular weight of more than 1,000 kDa. In some aspects, the host cell protein (HCP) has a molecular weight of less than 150 kDa.

[0127] In some aspects, the complex comprising a product of interest (e.g., a monoclonal antibody) and a binding molecule (e.g., protein A) is dissociated to form a free product of interest (e.g., a monoclonal antibody) and a free binding molecule (e.g., protein A). In some aspects, the product of interest (e.g., a monoclonal antibody) has a molecular weight exceeding the molecular weight cut-off of a semipermeable membrane, and the free binding molecule (e.g., protein A) has a molecular weight less than the molecular weight cut-off of a semipermeable membrane, wherein the free binding molecule (e.g., protein A) is removed by diffusing through the semipermeable membrane. In some aspects, the product of interest (e.g., a monoclonal antibody) has a molecular weight less than the molecular weight cut-off of a semipermeable membrane, and the free binding molecule (e.g., protein A) has a molecular weight exceeding the molecular weight cut-off of a semipermeable membrane, wherein the product of interest (e.g., a monoclonal antibody) is removed by diffusing through the semipermeable membrane.

[0128] In some aspects, the molecular weight cutoff of the semipermeable membrane is about 1kDa to about 10kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 100kDa to 200kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 300kDa to 400kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 400kDa to 500kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 500kDa to 600kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 600kDa to 700kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 700kDa to 800kDa. In some aspects, the molecular weight cutoff of the semipermeable membrane is about 900kDa to 1,000kDa.

[0129] In some aspects, the binding molecule may include a protein A mimetic peptide. In some aspects, the protein A mimetic peptide may include proteinogenic amino acids and / or non-natural amino acids and / or synthetic amino acids arranged in a non-cyclic form without disulfide bonds or in a branched or cyclic form with one or more covalent bonds (such as disulfide bonds).

[0130] In some aspects, the methods described herein are capable of purifying about 0.1 kg / day, about 0.5 kg / day, about 1 kg / day, about 2 kg / day, about 3 kg / day, about 4 kg / day, about 5 kg / day, about 6 kg / day, about 7 kg / day, about 8 kg / day, about 9 kg / day, or about 10 kg / day of product of interest.

[0131] In some aspects, the purified product of interest is free of residual Protein A.

[0132] In some aspects, the method described herein may include a first countercurrent filtration, wherein the binding molecule includes protein A. In some aspects, the method also includes a second countercurrent filtration, wherein the binding molecule includes a cation exchange resin or an anion exchange resin. In some aspects, the method also includes a third countercurrent filtration, wherein the binding molecule includes a cation exchange resin or an anion exchange resin. In some aspects, the ion exchange resin is different between the second countercurrent filtration step and the third countercurrent filtration step. In some aspects, the method also includes a diafiltration step.

[0133] In some aspects, the methods described herein can purify products of interest from about 0.1 kg / day to about 0.5 kg / day. In some aspects, the methods described herein can purify products of interest from about 0.5 kg / day to about 1 kg / day. In some aspects, the methods described herein can purify products of interest from about 1 kg / day to about 2 kg / day. In some aspects, the methods described herein can purify products of interest from about 2 kg / day to about 3 kg / day. In some aspects, the methods described herein can purify products of interest from about 3 kg / day to about 4 kg / day. In some aspects, the methods described herein can purify products of interest from about 4 kg / day to about 5 kg / day. In some aspects, the methods described herein can purify products of interest from about 5 kg / day to about 6 kg / day. In some aspects, the methods described herein can purify products of interest from about 6 kg / day to about 7 kg / day. In some aspects, the methods described herein can purify products of interest from about 7 kg / day to about 8 kg / day. In some aspects, the methods described herein can purify products of interest from about 8 kg / day to about 9 kg / day. In some aspects, the methods described herein are capable of purifying about 9 kg / day to about 10 kg / day of product of interest.

[0134] Without wishing to be bound by theory, in some aspects, the methods described herein can be separated using affinity colloids. In some aspects, the methods described herein can be separated using anion exchange polymer affinity colloids. In some aspects, the methods described herein can be separated using protein A by diffusing through a semipermeable membrane to capture a product of interest (e.g., a monoclonal antibody). In some aspects, the methods described herein can be separated using protein A by diffusing through a semipermeable membrane to capture a product of interest (e.g., a monoclonal antibody), and impurities (e.g., HCP) can be diffused through a semipermeable membrane. In some aspects, a product of interest (e.g., a monoclonal antibody combined with protein A) compounded with a binding molecule can be dissociated at low pH. In some aspects, protein A can be diffused through a semipermeable membrane.

[0135] In some aspects, the purification methods of the products of interest can be modularly combined. In some aspects, the products of interest (e.g., monoclonal antibodies) obtained from the bioreactor can first be subjected to protein A affinity colloid separation as described herein. The products of interest (e.g., monoclonal antibodies) can then be further subjected to cation exchange polymer affinity colloid separation, followed by anion exchange polymer affinity colloid separation, followed by a diafiltration step.

[0136] In some aspects, a dirty product (e.g., a product of interest) is obtained from a bioreactor, and the product (e.g., a product of interest) is concentrated and purified using one of the separation methods described herein (e.g., affinity colloid separation, anion exchange polymer affinity colloid separation, etc.). The concentrated product is then subjected to a second separation method. The dirty permeate from the second separation is sent back to the dirty product stream. The concentrated product (e.g., a product of interest) from the second separation method is then diafiltered. The slightly dirty permeate from the diafiltration step is sent back to the concentrated product (e.g., a product of interest) from the first separation method.

[0137] In some aspects, the methods disclosed herein can be performed at elevated temperatures due to increased diffusion rates and decreased viscosity. In some aspects, the methods described herein can be performed at a temperature of about 37° C., about 38° C., about 39° C., or about 40° C. In some aspects, a semipermeable ceramic membrane is used in the methods described herein when performed at a temperature of about 37° C., about 38° C., about 39° C., or about 40° C.

[0138] In some aspects, the semipermeable ceramic membranes can be used in dialysis or diafiltration / ultrafiltration modes. These semipermeable ceramic membranes have the advantage of being able to be cleaned and used multiple times compared to polymer-based semipermeable membranes.

[0139] In some aspects, semipermeable membrane fouling can be reduced by a variety of methods including, but not limited to, backwashing, forward flushing, pulse operation, ultrasonic fouling reduction, gas entrainment, and / or electrophoretic fouling reduction.

[0140] In some aspects, high performance tangential flow filtration (HPTFF) can also be used for diafiltration / ultrafiltration. HPTFF is an ultrafiltration operation known to those skilled in the art in which the permeate is recirculated simultaneously with the retentate to maintain a more consistent transmembrane pressure.

[0141] In some aspects, the methods herein can be performed with a first solution comprising a product of interest having a concentration of about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, about 200 g / L, or more than 200 g / L of the product of interest.

[0142] dialysis effluent

[0143] In some aspects, it is acceptable for the dialysate solution to be used upstream of the previous step. In some aspects, the dialysate solution is titrated to a new pH and / or mixed with a high concentration of salt solution to achieve the desired ionic strength for the upstream step. In some aspects, other solutions are added to the dialysate solution, including but not limited to arginine, urea, guanidine, ethanol, isopropanol, other alcohols, octanoic acid and other solutions suitable for washing the product of interest. In some aspects, the system is operated continuously. In some aspects, the dialysate solution is stored in a small buffer tank. In some aspects, the buffer tank is less than the volume of solution in one day. In some aspects, the solution is recycled from the downstream step to the upstream step and needs to use a buffer tank.

[0144] In some aspects, the permeate from the ultrafiltration step can also be used in a countercurrent manner from one step to another step (e.g., as described herein). In some aspects, the permeate from the single-pass tangential flow filtration step can also be used in a countercurrent manner from one step to another step (e.g., as described herein). In some aspects, the dialysate from the dialysis step can be used in a countercurrent manner from one step to another step. In some aspects, the retentate from the dialysis step can also be used in a countercurrent manner from one step to another step.

[0145] In some aspects, the previous upstream step or the step after the receiving step that receives the countercurrent solution must have an effluent directed to waste in order to remove any impurities that may be in the recirculating solution. In some aspects, if the recirculating solution comes from the permeate of a step that employs a semipermeable membrane with a molecular weight cutoff (MWCO), the receiving step of the subsequent step must employ a membrane with the same MWCO or greater that is directed to waste. Otherwise, even a small amount of material larger than the MWCO of the upstream step will accumulate in the system over time. In some aspects, the upstream step may have a semipermeable membrane with a MWCO greater than that of the downstream step, so that any large material that passes through the downstream filter can easily pass through the upstream filter to the waste stream.

[0146] In some aspects, if the recirculating solution is from the retentate portion of a semipermeable membrane, the receiving step must have a semipermeable membrane of the same or smaller size than the MWCO of the downstream step, and the retentate of the upstream step must be diverted to waste.

[0147] In some aspects, all combinations of recycling from the retentate or permeate portion of a downstream step to an upstream step are contemplated, where the upstream step (or a step after the upstream step but before the downstream step) has a waste stream on the retentate portion of the permeate portion of the filter.

[0148] In some aspects, accumulation of trace materials in the recycle loop can be handled by diverting a portion of the recycle line to waste or by periodically emptying the recycle loop.

[0149] In some respects, if this step is an anion exchange step, then receiving step or subsequent step must have the effluent of removing anionic compounds. In some respects, if this step is a cation exchange step, then receiving step must have the effluent of removing cationic compounds. For example, if the downstream step relates to anion exchange nanoparticles or polymers, then the cationic component will flow through the permeate or dialysate of membrane. If this material is used in the upstream, then the upstream step must allow a part of cationic component flow to be discharged. This can be completed with cationic exchange nanoparticles or polymers, which are combined with retaining cationic compounds on the retentate side, and the effluent flowing to the downstream step comes from the permeate. Or, the upstream step can be an affinity step, which is orthogonal to ion exchange, and the cationic component will flow through the affinity step and arrive at the waste line.

[0150] In some aspects, even trace levels of impurities can accumulate in the recycle loop without such a removal step upstream. In some aspects, this accumulation can be managed by splitting, where a portion of the recycle loop is diverted to waste. This split is a less efficient method because the product is also directed to waste.

[0151] In some aspects, the disclosure relates to a method of using a solution effluent from a successive downstream step as a wash solution for an upstream step. In some aspects, the solution effluent is mixed with a solution stream to adjust the pH or salt concentration or other excipient concentration to a desired level.

[0152] In some aspects, the present disclosure relates to a method of using a solution effluent of filtrate or dialysate from a consecutive downstream step as a wash solution for an upstream step, wherein the upstream step has a filter having a MWCO of the same size or greater than that of the downstream step generating the effluent.

[0153] In some aspects, the present disclosure is directed to a method of using a solution effluent from a retentate of a sequential downstream step as a wash liquid for an upstream step, wherein the upstream step has a filter having a MWCO of the same size or smaller than that of the downstream step generating the effluent, and the filtrate is directed downstream.

[0154] In some aspects, the present disclosure relates to a method of using solution effluent from successive downstream steps as a wash solution for an upstream step, wherein the downstream step retains anions and the upstream step allows cations to be diverted to waste.

[0155] In some aspects, the present disclosure relates to a method of using solution effluent from successive downstream steps as a wash solution for an upstream step, wherein the downstream step retains cations and the upstream step allows anions to be diverted to waste.

[0156] In some aspects, the present disclosure relates to a method of using a solution effluent from successive downstream steps as a wash solution for an upstream step, wherein the downstream step retains certain classes of impurities and the upstream step allows these impurities to be diverted to waste.

[0157] In some aspects, the upstream step is an Fc protein-containing concentration step and the upstream step is a sequential Protein A wash step.

[0158] In some aspects, the disclosure provides the effluent stream from the continuous concentration step in the binding molecule recirculation, to wash the complex of the binding molecule and the product of interest. In some aspects, titrant, salt or other excipients are added to the effluent stream. In some aspects, dialysis or ultrafiltration steps are continuous. In some aspects, the effluent enters a small tank, wherein the tank size is less than the effluent storage capacity of one day. In some aspects, the effluent enters a small tank, wherein the tank size is less than the effluent storage capacity of three hours.

[0159] In some aspects, the recycling binding molecule is a Protein A affinity ligand.

[0160] In some respects, the present disclosure relates to a method for using the solution effluent from a dialysis step or an ultrafiltration concentration step in a step upstream of a dialysis or ultrafiltration step. In some respects, titrants, salts or other excipients are added to the effluent. In some respects, the dialysis or ultrafiltration step is continuous. In some respects, the effluent enters a canister, wherein the canister size is less than the effluent storage capacity of one day. In some respects, the effluent enters a canister, wherein the canister size is less than the effluent storage capacity of three hours. In some respects, the product of interest is a part for a protein, a DNA, an RNA, a virus or a virus-like particle, a synthetic molecule or an affinity complex.

[0161] ProA recycles SPTFF effluent

[0162] Typically, the affinity fraction stream extracted from the step of separating the affinity ligand from the product of interest is dilute. A semipermeable membrane with a MWCO lower than the MW of the affinity ligand can be used to concentrate the affinity ligand and then recirculate it. The permeate or dialysate from this concentration step can be used as part of the wash solution for the affinity step in countercurrent mode. This solution can be titrated or mixed with other solutions that may have high concentrations of salts or other related compounds that help remove impurities in the wash solution.

[0163] In some aspects, the disclosure relates to a method for washing a complex of an affinity moiety and a product of interest using an effluent stream from a continuous concentration step in the recirculation of an affinity moiety. In some aspects, the method also includes adding a titrant, a salt or other excipient to the effluent stream. In some aspects, the dialysis or ultrafiltration step is continuous. In some aspects, the effluent enters a canister, wherein the canister size is less than the effluent storage capacity of a day. In some aspects, the effluent enters a canister, wherein the canister size is less than the effluent storage capacity of three hours. In some aspects, the binding molecule is protein A. In some aspects, the product of interest has a HIS tag. In some aspects, protein A is greater than 500kDa. In some aspects, protein A is less than 50kDa.

[0164] Binding molecules

[0165] In some respects, when binding molecule (for example, also referred to as affinity moiety) is a recombinant protein, and when product of interest is a recombinant protein, binding molecule can be prepared in the cell expression system identical with product of interest. In some respects, the growth medium for generating binding molecule is identical with the growth medium for generating product of interest. This method of generating binding molecule reduces the total cost of binding molecule, because the impurity in binding molecule is identical with the impurity in product of interest. This similarity of impurity means that binding molecule (for example, affinity moiety) can be purified, so that it is cleaner than the step of mixing binding molecule (for example, affinity moiety) and product of interest. When this step is a capture step, and product of interest has a large amount of impurities, binding molecule can be dirtier than expected, because this step will purify binding molecule / product of interest complex.

[0166] For example, the product of interest has far more than 100,000ppm of host cell proteins (HCP) after capture. The binding molecule can have 1,000ppm or even 10,000ppm HCP, and still purify the product of interest with minimal impact. Generally, it is expected that the binding molecule has a purity of less than 100ppm or even less than 10ppm of HCP. The higher the allowed impurity level of the binding molecule, the lower its manufacturing cost.

[0167] The initial purification of the binding molecule in this system is relatively easy. If the binding molecule is larger than the product of interest, the binding molecule can be passed through a series of identical filters used with the product of interest. First, the binding molecule is concentrated by asymmetric dialysis or a single pass TFF step, and then the binding molecule is washed in a dialysis or CM-TFF step. Finally, the material is filtered by a CM-TFF step or a single pass TFF step with a MWCO or aperture greater than the affinity portion.

[0168] If the binding molecule is smaller than the product of interest, it can be purified by a system that uses an "elution" step similar to the affinity / target cycle.

[0169] Washing solution

[0170] In some aspects, suitable solutions for washing the product of interest during one or more wash steps include solutions made from arginine, urea, guanidine, ethanol, isopropanol, other alcohols, octanoic acid, and other suitable solutions.

[0171] Design of binding molecules (e.g., affinity moieties)

[0172] In some aspects, binding molecule has two unique features. First, it is designed to be easily separated from the product of interest under non-binding conditions. Second, under binding conditions, the complex formed between the binding molecule and the product of interest contains two or more products of interest (e.g., monoclonal antibodies). In some aspects, the quantity of the product of interest of each complex is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20. In some aspects, the molar ratio of the binding molecule to the product of interest is about 1:2 or 1:3 or higher. In some aspects, the binding molecule can bind to the product of interest of twice or more times its own weight. In some aspects, the mass ratio of the binding molecule to the product of interest is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10.

[0173] In some aspects, the binding molecule can be easily separated from the product of interest under "elution" or non-binding conditions. For example, when the pH is reduced to about 3.5, the Fc-containing portion will elute or no longer bind to Protein A.

[0174] In some aspects, protein A (e.g., binding molecule) can be constructed to be significantly smaller than containing Fc compounds (e.g., products of interest). In some aspects, for ultrafiltration separation, this means that protein A (e.g., binding molecule) must be 1 / 5 to 1 / 10 of the size of containing Fc compounds (e.g., products of interest). In some aspects, for dialysis separation, if protein A is less than 1 / 3 of the size of containing Fc compounds, acceptable purification can be performed. In some aspects, if the product of interest is a typical antibody (150kDa MW), the protein A portion should be less than 30kDa. 100kDa MWCO membrane is sufficient to retain antibodies and allow most of the affinity ligands to pass through. The MW of native protein A is between 40-60kDa, which is about 1 / 3 of an antibody. However, native protein A cannot be separated with ultrafiltration or dialysis equipment. Protein A is either retained together with the antibody, or they all pass through the membrane.

[0175] For example, a single domain of Protein A may bind antibodies in solution but does not produce a complex of two or more antibodies, and ultrafiltration or diafiltration of a crude feed stream does not remove much more impurities than the same system and only unbound antibodies.

[0176] In some aspects, a protein A construct having two binding domains can bind two antibodies (e.g., a product of interest), which can allow the formation of a filterable complex. Under elution conditions, some protein A constructs can pass through an ultrafilter or diafilter with a 100 kDa MWCO or 75 kDa MWCO while retaining the antibodies.

[0177] In some aspects, protein A can be constructed to be significantly larger than containing Fc compounds (e.g., products of interest). For example, a protein A portion greater than 500kDa can be produced. A 500kDa membrane is sufficient to allow antibodies to flow through the membrane by convection while retaining affinity ligands. A 300kDa membrane also may allow antibodies to pass through, but with lower efficiency. In dialysis, a 300kDa membrane is sufficient to allow antibodies to diffuse through it. Larger MWCO membranes are also effective.

[0178] In some aspects, the binding molecules prepared from peptides or proteins may have a his tag (usually a tag with six histidines at the amino or carboxyl terminus of the protein). If the elution conditions are above pH 7, divalent cations (such as copper or cobalt) may be added to aggregate the binding molecules, thereby allowing filtration. At pH 4 or 5, the his tag is positively charged, and a cation exchange method may be used to separate the binding molecules from the product of interest.

[0179] In some aspects, the product of interest comprising DNA may have a sequence that specifically binds to large nanoparticles or microparticles, thereby allowing separation to occur.

[0180] In some aspects, the binding molecules comprise an elastin-like peptide sequence that allows spontaneous aggregation under certain conditions.

[0181] In some aspects, the binding molecules have multiple possible intrinsic modes that allow for separation under elution / non-binding conditions.

[0182] In some aspects, for small molecular weight synthetic or naturally occurring molecules, binding molecules comprising small peptides can be found, for example, by phage display screening. In some aspects, if the peptide is significantly larger than the small molecular weight compound under non-binding conditions, the peptide can be separated from the compound. Most peptides will be larger than the above-mentioned small molecule targets.

[0183] In some aspects, the binding molecule must have an affinity constant that is much smaller than the concentration of the product of interest in solution.

[0184] In some aspects, polymers can be used as binding molecules. In some aspects, polymers are ionic and / or hydrophobic.

[0185] In some aspects, the complex formed by the binding molecule and the product of interest contains at least two products of interest per complex or at least three products of interest per complex. In some aspects, the size of the complex is significantly larger than the product of interest itself to facilitate separation across a semipermeable membrane.

[0186] In some aspects, a protein A molecule (eg, a binding molecule) can bind to three antibodies (eg, a product of interest) and be purified on a 300 kDa membrane or a 500 kDa membrane. The complex can pass through a 700 kDa membrane.

[0187] In some aspects, very large protein A (e.g., binding molecule) can bind to an antibody (e.g., product of interest) and be retained by a semipermeable membrane, but the cost of protein A is too high. In addition, the separation of large MW protein A from antibodies becomes more challenging because the concentration of protein A is very high and is retained by the membrane. The retention of high concentration protein A causes the performance of the semipermeable membrane to decrease. Flux decreases, resistance increases, and antibodies can be more highly retained. Therefore, compared with antibodies, low concentrations of protein A are preferred. Therefore, in some aspects, the molar ratio of antibody to protein A is 2: 1 or more preferably 3: 1. In some aspects, for very large protein A parts, such as those higher than 500kDa, the molar ratio will be 4: 1 or 8: 1 or even 20: 1. The 8: 1 molar ratio is a 2.4: 1 antibody: protein A mass ratio. Due to cost considerations and filtration efficiency, a higher mass ratio is preferred.

[0188] In some aspects, very small protein A molecules (e.g., binding molecules) should effectively bind at least two antibodies (e.g., products of interest) so that a chain reaction can occur and a complex of three or more antibodies can be formed. In some aspects, the size of the complex can be controlled in the following manner: adding a protein A molecule that only binds to one antibody, thereby producing a "dead end" in the chain reaction. The size of the complex should not be too high because the complex can become difficult to filter. In some aspects, for protein A / mAb complex, the size of the complex can be 300kDa, 500kDa, 700kDa, 1MDa. In some aspects, the size can be as small as 70nm and as large as 1 μm, and the complex is filtered out by a 0.2 μm filter. In some aspects, the size can be 10 μm or 100 μm. The cost of a microfilter is usually cheaper than a nanofilter, so the largest filterable particles are preferred. 300kDa particles allow economical purification, even if the 300kDa membrane is more expensive than the 0.2 μm membrane.

[0189] In some aspects, very small protein A molecules (e.g., binding molecules) can bind to an antibody (e.g., a product of interest), and they can contain another affinity domain that binds to another protein A molecule, which itself binds to the antibody. Therefore, in some aspects, a large complex can be formed. For example, in some aspects, a single domain protein A can have a his tag, which can bind the protein A domains to each other in the presence of chelated metal ions (such as cobalt, copper, nickel or other) so as to form a complex of protein A and the antibody. His-tagged proteins are known to those skilled in the art of protein expression and purification. Without limitation, binding molecules (in some aspects, protein A domains) can be tagged with 3-6 histidines. In some aspects, the molar ratio of protein A to mAb is about 2:1, but the mass ratio is quite high, about 1:10 or 1:15 or even 1:30. In some aspects, the size of the complex can be controlled by adding a protein A domain without a his tag. In some aspects, the ratio of non-his-tagged protein A to his-tagged protein A can be 1:2 (it produces relatively small particles), 1:3, 1:5, 1:10 or higher. In some aspects, the more his-tagged protein A is relative to non-his-tagged protein A, the larger particles will be generated. In some aspects, larger particles produce particles that are difficult to filter. In some aspects, the preferred ratio is 1:3 to 1:5. Single domain protein A (containing or not histidine) can be separated by ultrafiltration or dialysis with a 50kDa membrane, more preferably a 75kDa membrane and most preferably a 100kDa membrane. The his-tagged protein A passes through the membrane, and the antibody is retained. In some aspects, a chelated metal is added to the protein A before the antibody, and in some aspects, the antibody is combined with the protein A and then the chelated metal is added.

[0190] The method of the present disclosure may also relate to the use of self-assembled nanoparticles. Such nanoparticles will have the ability to assemble and bind multiple antibodies simultaneously, thereby producing large complexes. In some aspects, the binding molecule is a nanoparticle. The self-assembled nanoparticle is formed by the self-association of monomers encoded in each plasmid. The number of monomers assembled into the nanoparticle is different (e.g., about 2, about 5, about 12, about 24-60 or more). The MW of the fused nanoparticle is the product of the monomer size and the number of monomers in the nanoparticle, while the MW of the mAb-bound substance is the product of the monomer MW, mAb MW, the number of monomers in the nanoparticle, and the capacity % (molmAb / mol ProA Z binding domain). While the nanoparticles present on the retentate side of the filter membrane after elution should have an apparent MW greater than the cutoff of the elution membrane filter (e.g., 300 or 500 kDa MWCO), the affinity-bound mAb-nanoparticle complexes should have a MW greater than the cutoff of the wash and binding membrane filters (e.g., 500, 750, 1000 kDa MWCO or 0.22 μm). The optional additional protein AZ domains increase the mass of the nanoparticles and also increase the mass of the mAb-nanoparticle complexes. Similarly, the mass ratio of bound mAb to nanoparticles will increase with the number of Z domains only if the additional mAb can bind.

[0191] Self-assembled nanoparticles span a variety of sizes, homomonomer component numbers and sequences. Ferritin nanoparticles form hollow spheres containing 12 nm pores, which are composed of 24 monomers, each containing 5 helical domains. This choice is made because of its pH resistance <4, which is necessary to maintain its structure under conditions used to dissociate mAbs from affinity ligands. The above-mentioned Helicobacter pylori ferritin was modified from the WT form to include amino acids at its N-terminus, thereby allowing favorable spatial separation of the N-terminus modified with affinity ligands. Stable protein 1 (SP1) exhibits high temperature stability and pH stability and is composed of 12 subunits, which have been shown to be reliable supports for enzymes and carbon nanotube-bound proteins that form biomaterials. SP1 also has the characteristic that it can reversibly form nanorods, which may be beneficial for size retention of mAbs (and nanoparticles) on large pores.

[0192] In various aspects of the method, the fusion protein monomer comprises: i) a self-assembling nanoparticle monomer; ii) a linker; and iii) an immunoglobulin binding domain. In various aspects, the linker connects the self-assembling nanoparticle monomer and the immunoglobulin binding domain.

[0193] In various aspects, the method further comprises regenerating the fusion protein monomers, wherein the regenerated fusion protein monomers are again capable of forming assembled nanoparticles and complexes when contacted with the protein of interest in the first flowing solution or the second flowing solution.

[0194] In various aspects of the method, the self-assembled nanoparticle monomer is a ferritin monomer. In various aspects of the fusion protein monomer, the ferritin monomer is a Helicobacter pylori ferritin monomer or a Pyrococcus furiosus ferritin monomer. In various aspects, the ferritin monomer is a Helicobacter pylori ferritin monomer. In various aspects, the ferritin monomer is a Pyrococcus furiosus ferritin monomer. In various aspects, the ferritin monomer is a human ferritin monomer. In various aspects, the ferritin monomer is a human H-chain ferritin monomer. In various aspects, the ferritin monomer is a human L-chain ferritin monomer. In various aspects, the ferritin monomer is a ferritin monomer disclosed in Table 1 below.

[0195] In various aspects of the method, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 1. In various aspects, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 1. In various aspects, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 2. In various aspects, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 2.

[0196] In various aspects, the assembled nanoparticle is an assembled ferritin nanoparticle. In various aspects, the assembled ferritin nanoparticle comprises any of the ferritin monomers described above. In various aspects, the assembled ferritin nanoparticle comprises more than one type of ferritin monomers described above.

[0197] In some respects, the bispecific property of small molecule binding molecules need not be combined with the same site on the product of interest. The complex of two or more products of interest can be produced by combining with two different sites on the product of interest. In some respects, for molecules without two binding sites thereon, this heterogeneous bispecific affinity is preferred. Because the antibody is a dimer, the antibody can combine two protein A molecules, so that the complex can become larger. Most molecules will not have two affinity binding sites, so the bispecific property of affinity ligands requires two different affinity parts on the same site.

[0198] In some aspects, bispecific peptides can be produced by linking two affinity peptides together so that the combined peptide binds to two different epitopes on a small synthetic or natural target, allowing for the discovery and purification of larger complexes by nanofiltration systems such as those described. In some aspects, the MWCO is expected to be much lower than that of proteins, DNA, RNA, or viruses or virus-like particles. MWCOs approaching 500Da, 1kDa, 2kDa, 4kDa, 8kDa, 16kDa, 32kDa can be used for such systems.

[0199] In some respects, for small molecular weight synthetic molecules, affinity moieties consisting of small DNA or RNA chains can be found, for example, by conventional screening methods. Bispecific moieties can be produced by two different affinity moieties, which bind to two different epitopes on a small synthetic target, thereby allowing for the discovery and purification of larger complexes by nanofiltration systems (such as those described). In some respects, it is expected that the MWCO is much lower than that of proteins, DNA, RNA or viruses or virus-like particles. MWCOs approaching 500Da, 1kDa, 2kDa, 4kDa, 8kDa, 16kDa, 32kDa can be used for such systems.

[0200] In some aspects, for small molecular weight synthetic or naturally occurring molecules, binding molecules consisting of small peptides can be found, for example, by phage display screening. Some targets may include aspirin or metabolites, such as those discussed in Mulukutla et al., Biotech Bioeng, Vol. 114, 2017, which inhibit CHO cell growth. These affinity ligands must have an affinity constant much smaller than the concentration of the product in solution. In some aspects, if the peptide is significantly larger than the small molecular weight compound under non-binding conditions, the peptide can be separated from the compound.

[0201] In some aspects, the present disclosure provides a binding molecule with two or more binding sites, wherein each of the binding sites is combined with an epitope on a product of interest, and the complex comprising the binding molecule and the product of interest is greater than the product of interest. In some aspects, the complex is about 25%, about 50%, about 100%, about 200% or about 400% greater than the product of interest. In some aspects, the complex comprises a binding molecule and about two or more products of interest. In some aspects, the complex loses affinity for the product of interest under specific elution conditions. In some aspects, the binding molecule has 2, 3, 4, 5, 6, 7, 8, 9 or 10 binding sites for the product of interest. In some aspects, the binding molecule comprises two or more protein A binding domains with a molecular weight of about 300kDa, about 400kDa, about 600kDa or about 1,000kDa. In some aspects, two or more protein A binding domains can bind to a target molecule containing Fc twice the mass of protein A. In some aspects, the complex comprises three or more fab fragments with a molecular weight of about 100 kDa, about 300 kDa, about 600 kDa, or about 1,000 kDa. In some aspects, the complex comprises a DNA with a molecular weight of about 300 kDa, about 400 kDa, about 600 kDa, or about 1,000 kDa with two or more affinity sites. In some aspects, the complex comprises a peptide with a molecular weight of about 1 kDa, about 2 kDa, about 4 kDa, about 8 kDa, or about 16 kDa with two or more binding sites. In some aspects, the complex comprises an RNA with a molecular weight of about 300 kDa, about 400 kDa, about 600 kDa, or about 1,000 kDa with two or more affinity sites.

[0202] In some aspects, filtration is continuous dialysis. In some aspects, filtration is continuous modular countercurrent tangential flow filtration. In some aspects, by filtering under specific elution conditions, complex is separated into product of interest and binding molecule, and the MWCO of membrane or the pore size of membrane is less than binding molecule and greater than product of interest.

[0203] In some aspects, the binding molecule has two or more binding sites for the product of interest, wherein one of these sites is combined with the first target of interest, and another site is combined with the second product of interest. In some aspects, the binding molecule has a molecular weight less than the product of interest. In some aspects, the second binding molecule has only one binding site in these binding sites. In some aspects, the mixture of binding molecules and the product of interest produces a complex with a weight ratio of at least one unit binding molecule and about two or more units of the product of interest. In some aspects, the ratio of binding molecules to complexes is controlled by the size of the complex. In some aspects, the ratio of binding molecules to the product of interest is about 1:3, about 1:5 or about 1:10. In some aspects, the filtration of the complex is used to purify the product of interest. In some aspects, filtration is continuous dialysis. In some aspects, filtration is continuous modular countercurrent tangential flow filtration. In some aspects, by filtering under elution conditions to separate the binding molecule and the product of interest, wherein the binding molecule passes through the filter, and the product of interest is retained. In some aspects, the filter area is greater than 1m 2 In some aspects, filtration requires the addition of polymers or particles that are larger than the molecular weight cutoff of the filter used, which are bound to the affinity moiety.

[0204] In some aspects, the binding molecule has two or more binding sites, wherein one of these sites is combined with an epitope in two similar epitopes on the product of interest, and another site is combined with an epitope in two similar epitopes on the independent product of interest. In some aspects, the independent binding molecule has only one binding site in these binding sites. In some aspects, the complex comprises at least one binding molecule and two or more products of interest. In some aspects, the ratio is controlled by the size of the complex. In some aspects, the ratio is about 1:3, about 1:5 or about 1:10. In some aspects, the complex is filtered to purify the product of interest. In some aspects, filtering is continuous dialysis. In some aspects, filtering is modular countercurrent tangential flow filtration. In some aspects, the binding molecule and the product of interest are separated by filtering under elution conditions. In some aspects, filtering requires adding a polymer or particle greater than the MWCO of the filter used, which is combined with the binding molecule. In some aspects, the binding molecule has a molecular weight three times that of the product of interest.

[0205] Dialysis-based affinity separation

[0206] In some aspects, if the size of the binding molecule is greater than the MWCO of the dialysis membrane, and the product of interest is less than the MWCO of the membrane, the binding molecule can be placed in a dialysate solution. In some aspects, the product of interest flows into the dialysis equipment on the retentate side of the filter, and the larger binding molecule is suspended in the dialysis solution on the other side of the membrane, flowing in countercurrent with the product of interest. Unbound large binding molecules cannot diffuse across the membrane to the retentate side because it is greater than the MWCO. The product of interest diffuses through the membrane and combines with the large binding molecule. After combining, because the complex is too large, it cannot diffuse or convect back to the retentate side.

[0207] In some aspects, the dialysis process uses a higher flux of solution on the clean dialysis side of the membrane compared to the retentate side of the membrane to allow diffusion. The ratio of α or dialysis flow rate to retentate flow rate is typically 2, 4, 8, or 16 times the retentate flow rate.

[0208] Adding a large binding molecule to the dialysis solution allows the flow rate of the dialysis solution to be much lower than that without adding a large binding molecule. The effective flow rate ratio or α is proportional to the affinity constant and inversely proportional to the concentration of the affinity ligand. Some equilibrium models can be applied, such as the Langmuir isotherm. For example, in some aspects, in a system where MWCO is 500kDa or 700kDa or 1000kDa, a large protein A portion can be used to keep the concentration of the target equal to the concentration in the retentate (α=1), or more preferably, α is reduced to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64 or 1 / 128, and therefore the concentration of the antibody from the retentate to the dialysate is increased to 2, 4, 8, 16, 32, 64 or 128 times.

[0209] Under flow conditions, the amount of the product of interest passing through the semipermeable membrane is related to the diffusion rate of the product of interest in the membrane, the affinity and concentration of the binding molecules on the dialysis side of the membrane, and the area of ​​the membrane, and is inversely proportional to the thickness of the membrane, etc. In some aspects, the mass transfer of the product of interest in the lumen can affect the amount of the product of interest passing through the membrane, especially when the thickness of the retentate flow field is much greater than the thickness of the membrane (for example, in the case of hollow fibers, that is, when the hollow fiber lumen is much greater than the membrane). The method of inducing radial flow in the lumen of the membrane or the method of inducing orthogonal flow in the case of a flat plate is known. Similarly, mass transfer may be affected by the flow distribution of the dialysate. In some aspects, a method of providing radial flow to allow the dialysate material to reach the inside of a bundle of hollow fiber membranes is used. In some aspects, these methods include spacing hollow fibers at the top and bottom of the filter cartridge to allow radial flow, using Dean vortex, using pulse flow, using bubbles to provide radial mixing. In some aspects, kinked membranes or twisted membranes can also provide radial mixing. In some aspects, in the case of a flat plate, many methods in these same methods can be used. In some aspects, screens can also be used to create mixing orthogonal to the membrane.

[0210] III. Products of Interest

[0211] In some aspects, the methods disclosed herein can be applied to any protein product (e.g., a product of interest). In some aspects, the protein product is a therapeutic protein. In some aspects, the therapeutic protein is selected from antibodies or their antigen binding fragments, Fc fusion proteins, anticoagulants, coagulation factors, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, receptors, and thrombolytic agents. In some aspects, the protein product is an antibody or its antigen binding fragment. In some aspects, the protein is a recombinant protein.

[0212] In some aspects, the protein product is an antibody or an antigen-binding fragment thereof. In some aspects, the protein product is a chimeric polypeptide comprising an antigen-binding fragment of an antibody. In certain embodiments, the protein product is a monoclonal antibody or an antigen-binding fragment thereof ("mAb"). The antibody can be a human antibody, a humanized antibody, or a chimeric antibody. In certain embodiments, the protein product is a bispecific antibody.

[0213] In some aspects, the mixture comprising protein product and pollutant comprises the product of the previous purification step. In some aspects, the mixture is the crude product of the previous purification step. In some aspects, the mixture is a solution comprising the crude product of the previous purification step and buffer (e.g., starting buffer). In some aspects, the mixture is included in the crude product of the previous purification step reconstructed in the starting buffer.

[0214] In some aspects, the source of the protein product is bulk protein. In some aspects, the source of the protein product is a composition comprising the protein product and non-protein components. The non-protein components may include DNA and other contaminants.

[0215] In some aspects, the source of the protein product is from an animal. In some aspects, the animal is a mammal, such as a non-primate (e.g., a dairy cow, a pig, a horse, a cat, a dog, a rat, etc.) or a primate (e.g., a monkey or a human). In some aspects, the source is a tissue or a cell from a human. In some aspects, such terms refer to non-human animals (e.g., non-human animals such as pigs, horses, dairy cows, cats or dogs). In some aspects, such terms refer to pets or farm animals. In some aspects, such terms refer to people.

[0216] In some aspects, the protein product purified by the methods described herein is a fusion protein. "Fusion" or "fusion protein" comprises a first amino acid sequence connected in frame with a second amino acid sequence, and the first amino acid sequence is not naturally connected to the second amino acid sequence in nature. The amino acid sequences that are usually present in a single protein can be gathered together in a fusion polypeptide, or the amino acid sequences that are usually present in the same protein can be placed in a fusion polypeptide in a new arrangement. For example, by chemical synthesis, or by forming and translating a polynucleotide (wherein the peptide region is encoded with a desired relationship) to form a fusion protein. The fusion protein may also include a second amino acid sequence that is connected to the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond. After transcription / translation, a single protein is produced. In this way, a variety of proteins or fragments thereof can be incorporated into a single polypeptide. "Operably connected" is intended to represent the functional connection between two or more elements. For example, the operable connection between two polypeptides fuses two polypeptides in frame together to produce a single polypeptide fusion protein. In a specific aspect, the fusion protein also includes a third polypeptide, as discussed in further detail below, and the third polypeptide may include a linker sequence.

[0217] In some aspects, the protein purified by the method described herein is an antibody.Antibody may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heterologous conjugated antibodies, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F (ab') 2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies) and the antigen-binding fragments of any of the above.In some aspects, antibody as described herein refers to polyclonal antibody groups. The antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some aspects, the antibody described herein is an IgG antibody or its class (e.g., human IgG1 or IgG4) or subclass. In one aspect, the antibody is a humanized monoclonal antibody. In some aspects, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In some aspects, the antibody described herein is an IgG1 or IgG4 antibody.

[0218] The present disclosure relates to methods disclosed herein, wherein the product of interest is an antibody, an antigen-binding antibody fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof. In some aspects, the product of interest is a full-length IgG antibody. In some aspects, the antibody is an IgG1, IgG2, IgG3, and / or IgG4, or a hybrid thereof. In some aspects, the antibody is a monoclonal antibody.

[0219] In some aspects, the method disclosed herein is accomplished using bacterial cells, yeast cells, insect cells or mammalian cells. In some aspects, the mammalian cell is a Chinese hamster ovary cell. In some aspects, the product of interest is prepared by the method disclosed herein.

[0220] sequence

[0221]

[0222]

[0223]

[0224] Example

[0225] Example 1: Systems and methods for end-to-end continuous downstream purification

[0226] Capture Steps

[0227] Experiments were performed using a Chinese hamster ovary (CHO) cell line expressing the monoclonal antibody IgG 1. Studies were performed in a 3-liter glass stirred tank reactor (Chemglass Life Sciences, USA) with a 2-liter working volume or in a 500-liter perfusion bioreactor. In a tangential flow filtration (TFF) perfusion system, a 0.2 μm pore size and a membrane area of ​​980 cm were used. 2 The cell cultures were continuously harvested from day 0 to day 14 using a polyethersulfone (PES) hollow fiber membrane (Repligen Corporation, USA). In the TFF system, a low shear magnetically suspended centrifugal pump was used as a recirculation device (Levitronix, Zurich, Switzerland).

[0228] Affinity colloid capture step

[0229] The continuous countercurrent affinity colloid capture procedure includes 4 steps, such as Figure 1First, dehydration of conditioned medium (CM); second, addition of soluble protein A (sProA); third, dehydration of the CM and sProA complex; and fourth, 2 stages of countercurrent washing.

[0230] During the dehydration stage, the use area is 0.5m 2 30kDa Ultracel Pellicon capsule filter (Catalog No. PCC030C05 Millipore-Sigma, USA). Two peristaltic pumps were used to control the feed and retentate flow rates (Watson-Marlow Fluid Technology Group, USA). Transmembrane pressure (TMP) was monitored by placing PendoTech pressure sensors (Cole-Parmer, USA) on the feed, retentate and permeate lines. Critical flux studies were performed using the flux step procedure of Li and Zydney (Li Z, Zydney AL. Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates. Biotechnology Prog. 2017; 33(6): 1561-1567). TMP was evaluated as a function of time during constant flux operation, with a stepwise increase in filtrate flux to determine the onset of fouling.

[0231] To determine the optimal conditions for mixing sProA and mAb, a series of studies were performed in batch mode using a well-performing mAb ("mAb1"). The mAb was aliquoted into 100 mL glass beakers and placed on a magnetic stirring plate at a constant mixing rate of 300 RPM at room temperature. sProA (Cat. No. 10-2001-1M Repligen Corporation, USA) was added to the beaker at a flow rate of 0.05 mL / min using a programmable syringe pump. The optimal binding conditions were determined by varying the molar ratio of sProA:mAb, and the data were evaluated by dynamic light scattering (DLS), size exclusion chromatography (SEC), and protein A binding.

[0232] To concentrate the CM and sProA mixture, a 0.1 m column chromatography column with a Biomax 300 kDa membrane, C sieve was used. 2Pellicon capsule filters. Two peristaltic pumps were used to control the feed and retentate flow rates (Watson-Marlow Fluid Technology Group, Wilmington, MA), while TMP was monitored by placing PendoTech pressure sensors on the feed, retentate, and permeate lines. The retentate from this stage was sent to a 2-stage countercurrent wash step to further remove impurities. The area of ​​each filter during the wash was 0.1 m 2 , and the feed and retentate fluxes for each filter were selected based on critical flux studies using a flux ladder procedure. The continuous countercurrent affinity colloid capture steps are shown in Figure 1 middle.

[0233] analyze

[0234] mAb concentration was determined by analytical protein A affinity chromatography using a 20×2.1 cm inner diameter POROS A / 20 protein A column (Thermo Fisher Scientific). Antibody concentration was calculated using a standard curve established for the molecule of interest according to Beer's law. mAb purity was assessed by a size exclusion chromatography (SEC) column using a 30×7.8 cm inner diameter TSKgel G2000SWxl column (Tosoh Bioscience LLC). Product purity was calculated as the ratio of the monomer peak area to the sum of all peak areas according to the SEC chromatogram. The size of mAb and sProA complex was assessed by DLS using a Zetasizer Nano (Malvern Panalytical, UK). HCP levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cygnus Technologies, USA). Samples were diluted using sample dilution buffer (Cygnus Technologies) until the HCP concentration in the sample was in the range of 1 ng / mL-100 ng / mL. The ELISA procedure followed the kit manufacturer's protocol.

[0235] CM dehydration

[0236] Figure 2 Critical flux studies of the dehydration stage at feed fluxes of 5, 10, and 15 LMH are shown, where the concentration of the feed material (CM mAb1) was 1 mg / mL. Figure 2As shown, when the TMP was below 1 psi, a maximum concentration factor of 7 was achieved at a feed flux of 5 LMH. The data generated here can be used to design a dehydration process based on the flow rate out of the perfusion bioreactor. The permeate flow rate can also be adjusted based on the desired final concentration factor, taking into account the initial concentration of the CM to be used. Based on the critical flux data generated, a 3-stage TFF for dehydration was designed, in which 670 L CM of mAb1 generated during days 11-13 of the 500 L perfusion bioreactor was concentrated. As shown in Figure 3, two peristaltic pumps were used to control the feed and retentate fluxes on each filter. The average concentration of CM was 1 mg / mL, and it was concentrated 8.3× during the process, with the flux and pressure of each filter described in Table 1. Nearly 1-log HCP reduction was achieved during this step, with almost no measurable HCP retention on the membrane. In addition, by reducing 0.5-1 log HCP during this step, the final purity of the captured product will also be improved, mainly due to the interaction of HCP with sProA and mAb complexes.

[0237] Table 1: Flux and concentration factors during the 3-stage CM dehydration

[0238] Filter 1 Filter 2 Filter 3 Feed flux (LMH) 16.8 5.8 2.9 Retentate flux (LMH) 5.8 2.9 2.1 Concentration Factor 2.9 2 1.38 TMP(opsi) 2.1 1.8 1.9

[0239] mAb and sProA mixed

[0240] The optimal binding conditions of sProA and mAb were determined by varying the molar ratio of sProA:mAb. Figure 4 The size of the complexes formed by mAb1 at different sProA:mAb molar ratios is shown. Refined mAb at a concentration of 4 mg / mL and pH 7.4 was used for each study, with 40 mg / mL of sProA at pH 7.2 added to increase the molar ratio. The size of the complexes was studied using DLS and SEC, and based on the data, a soluble complex of approximately 700 kDa was formed at a 0.3 sProA:mAb molar ratio. In addition to the size of the complexes, the binding yield was also studied using ProA-bound HPLC. For mAb1, 100% yield was achieved at a molar ratio of 0.3 and higher; where 100% yield means that all mAb has formed a complex and there is no free mAb in solution. Based on the data generated in this study, a 0.3 sProA:mAb molar ratio was used for all remaining studies reported here.

[0241] 3-stage dehydration and CC washing

[0242] As shown in Figure 5, a 3-stage dehydration and CC wash was used, where the mAb and sProA complex was concentrated first. The retentate from this stage was then diluted with the permeate from the second wash stage and concentrated again in the first wash stage. Finally, the retentate from the first wash stage was diluted again with fresh buffer of 50 mM Tris, pH 7.4, and further concentrated during the second wash stage. A critical flux study was performed on the wash stage to determine the optimal flow rate for this step. Figure 6 As shown, with a feed flux of 5 LMH, a concentration factor of 5 can be achieved. Therefore, based on this data, the feed and retentate fluxes for each stage were controlled at 5 and 1 LMH, respectively, in order to achieve a 5x concentration factor during each stage. At the end of the capture step, a concentration factor of 40 was achieved with a final HCP level of 590 ppm.

[0243] These data show an HCP level of 590 ppm and an antibody yield of 90%, which operates most efficiently at high protein concentrations, thereby reducing the amount of wash solution per kg of product. The final purity of the product is comparable to that of a protein A column; this has the advantage of reducing the process quality index (PMI) by at least 2 / 3. In addition, the final cost of the purification process is further reduced by replacing the expensive protein A chromatography resin with a soluble form of protein A that is recycled and reused at least 10-20 times. It has been demonstrated that the process is economically viable using commercially available sProA. In addition, sProA can be prepared by any manufacturer, including in-house, thus eliminating supply chain constraints.

[0244] The extent of removal (R) of each substance on TFF is given as follows:

[0245] R = C0 / C, where C0 is the concentration of the substance in the feed entering the filter and C is the concentration of the substance in the final retentate leaving the filter. Figure 7 Shown are the removal folds for mAb, sProA, and a complex of mAb and sProA at a molar ratio of 3; where mAb and sProA passed through the filter, while 100% of the complex of mAb and sProA was retained on the filter.

[0246] CEX Refining

[0247] In order to separate sProA and mAb after the capture step, a CEX polishing step was developed. Studies were conducted in batch contact mode using 96-well plates (Cat. No. 136101, Fisher Scientific) to determine the optimal conditions for separating mAb and sProA. UNOsphere cation exchange resin (Cat. No. 1560111, BioRad) was prepared and washed with a buffer (pH 3) containing 50mM glycine, then settled and centrifuged at 1000×g for 10 minutes to determine the volume concentration. The mAb and sProA complex from the capture step was titrated to pH 3 using glycine / HCl and mixed with 20% resin at 20mg / mL binding capacity. After incubating the sample for 5 minutes, it was centrifuged at 1000×g for 10 minutes to collect the filtrate containing unbound protein. During the next step, in order to elute sProA, the first elution buffer was mixed with the sample, which was then centrifuged to collect the filtrate containing sProA. During the last step, the second elution buffer was mixed with the sample and centrifuged to collect the mAb as the filtrate in this step. The volume of the buffer during both elution steps was 20x the volume of the settling resin, and the incubation times for the first and second elution steps were 10 and 20 minutes before centrifuging the samples.

[0248] Figure 8 Data generated during the first elution of the CEX polishing step are shown, where the partition coefficients of sProA and mAb1 were measured at pH 3 and different concentrations of NaCl. Based on the data, 0.8 M NaCl was able to elute 91% of sProA at pH 3, while no mAb was eluted in this step. During the second elution step, 50 mM Tris and 1 M NaCl at pH 7.8 were used, whereby 70% of the mAb was eluted.

[0249] AEX Refining Materials and Methods

[0250] The synthetic feed (simulated neutralized protein A elution pool) containing the monoclonal antibody mAb1 and impurities (i.e. HCP) was adjusted to a salinity of pH 8.5 and 10mM NaCl (conductivity, 2.5 ± 0.3mS / cm). The synthetic feed was prepared by adding HCP enriched from the conditioned medium (mAb1) to the mAb. The enriched HCP was prepared by loading the mAb1 conditioned medium (adjusted to pH 8.5, conductivity 2.5mS / cm) onto a Capto Q column (20cm bed height, 1.1cm diameter) pre-equilibrated with buffer A (10mM Tris pH 8.5, 10mM NaCl). The column was washed with buffer A after loading, and the bound HCP was then recovered by step elution with 10mM Tris pH 8.5, 1M NaCl. The eluted HCP pool was treated with 10 μL Benzonase endonuclease (EMD Merck, Germany) for 2 h to digest host cell DNA and RNA. TM Buffer A was dialyzed using a dialysis bottle (3.5K MWCO) (Thermo Scientific, USA) and the resulting HCP pool was buffer exchanged. mAb concentrations were determined using Solo-VPE (Repligen, USA). Chromatographic separation was performed using Fractogel TMAE (M) anion exchange resin (EMDMerck, Germany), which has a particle size range of 40 μm-90 μm as described by the manufacturer. The resin was washed and resuspended in binding buffer (10 mM Tris HCl pH 8.5, 10 mM NaCl). Adsorbent and binding buffer conditions are empirical and described elsewhere (Kelley et al., 2008). After centrifugation at 1000 × g for 5 minutes, the resin slurry concentration (% v / v) was determined by the sedimented resin volume.

[0251] Batch Binding Experiment

[0252] Batch binding experiments were used to screen the optimal binding conditions for adsorbent and impurity removal. This method allows for more convenient and rapid screening while maintaining functional similarity to the expected continuous operation. Anion exchange resin (e.g., Fractogel TMAE) was pre-balanced and resuspended in buffer A (as described above) to prepare a 50% (v / v) resin slurry. The required amount of resin slurry was moved into a 1.5 mL low protein binding tube (catalog number 90411, Thermo Scientific) containing a mAb pool with or without impurities to produce a 1% or 5% final resin suspension. The mAb pool was pretreated with buffer A. The resulting mixture was incubated on a rotary mixer for 20 minutes at room temperature (22 ± 2 ° C). After incubation, the sample containing the resin was filtered through a 0.45 μm rotary filter to recover the refined mAb. mAb concentrations were measured on Solo-VPE (Repligen, USA). In-house assays of HCP ELISA and real-time PCR were used to quantify HCP and host cell (CHO) DNA in unpolished and polished mAbs, respectively.

[0253] It should be noted that some screening studies were performed in 1 mL 0.45 μm 96-well filter plates (Cat. No. 8129, Pall Corporation, USA). The operating conditions (pH and conductivity) were selected based on the weak partitioning mode (WPC) of operation (reference). In WPC, the differential interactions of the product of interest and impurities with the chromatographic resin are determined by the partition coefficient K p For proteins (mAb or impurities), K p It is the ratio of the concentration of solute bound to the resin divided by the concentration in solution at equilibrium (citation).

[0254]

[0255] Continuous single-pass TFF anion exchange chromatography

[0256] like Fig. 9 As shown, the continuous single-pass TFF anion exchange chromatography system includes a surface area of ​​980 cm 2Hollow fiber module (catalog number S04-P20U-10-N, Repligen, USA) and peristaltic pump.Peristaltic pump is used to control feed and permeate flow rate.Considering the high normalized water permeability (400LMH / psi) of the hollow fiber module used in this study, the arrangement allows to adjust the permeate flux.Pre-balance resin slurry (40%v / v) with binding buffer (10mM Tris HCl pH 8.5, 10mM NaCl).Slurry is manually mixed in batches with feed (10g / L mAb and impurities) to produce a load material with a desired final resin slurry concentration of 1% to 5%.The mixture is incubated for 5 minutes, and feed pump P1 is used to apply to the hollow fiber module with a feed flux of 50LMH.The flow velocity between coordination pumps P1 and P2 is used to obtain the permeate flux and slurry concentration factor along the desired hollow fiber membrane, wherein P1>P2 and slurry concentration factor are the ratio between feed flow rate and retentate flow rate. The selection of the slurry concentration factor depends on the percentage of slurry in the load material and the expected product recovery in a single pass operation. For 1% and 5% initial slurries, the selected slurry concentration factors were targeted at 20x and 5x, respectively (Table 2), which resulted in a nominal final slurry concentration of 20% and 25% after SPTFF. The purified mAb product was continuously collected in the permeate, while the impure concentrated resin was recovered from the retentate.

[0257] Table 2: Comparison of HCP reduction in synthesis feed at different mAb concentrations .

[0258]

[0259] *No Benzonase treatment

[0260] Water Recycling and PMI

[0261] Serum Concentrating Factor

[0262] The product recovery or step yield percentage in a single pass operation is directly proportional to the slurry concentration factor and the hollow fiber module. The slurry concentration factors successfully evaluated for 5% resin slurry are 5x, 10x, 15x; for concentration factors exceeding 15, a sharp increase in transmembrane pressure of up to 25 psi may be due to blockage of the hollow fibers.

[0263] Batch Binding Experiment

[0264] Fig.11The calculated “Kp impurity” values obtained at various HCP concentrations are shown, while maintaining the mAb concentration constant at 10 g / L with 5% Fractogel TMAE resin. In the case of HCP challenges of 600 PPM or less, the residual HCP levels in the purified mAb material were below the detection limit of the assay. At higher (>600 ppm) HCP challenges, a 2.3 log reduction of HCP was observed, where the Kp impurity value of HCP > 3500. Similarly, the partition coefficient of the purified mAb product was determined and found to be <1.0 at a salinity of 10 mM (NaCl) (data not shown). From the principle of WPC, a lower product Kp value is preferred as this reduces the likelihood of product loss, especially at higher mAb loads, and allows maximum utilization of the effective binding capacity of the adsorbent to remove HCP and DNA.

[0265] Continuous single-pass TFF anion exchange chromatography

[0266] The functional similarity of the batch binding study to the TFF system simplifies the transition to the TFF operating mode while leveraging the results from the batch binding. Table 1 shows the HCP reduction observed when operating in the TFF mode. For 1% and 5% resin slurries at a 10 mg / mL mAb feed, a 1.9 log reduction of HCP was observed, which is comparable to the batch binding results. Selecting the 1% resin slurry provided a 91% product recovery, compared to 74% product recovery observed when using the 5% slurry in the TFF mode. It should be noted that the product discussed here is the result of the slurry concentration factor, rather than the result of the product Kp (Kp < 1). Interestingly, a significant reduction in the HCP log reduction was observed at an 85 mg / mL mAb feed.

[0267] Water during the recovery of the purified product from the permeate will offset the PMI of this step. As Fig. 9 shown, a 35 g / L mAb feed was concentrated to 80 g / L using a 30 kDa single-pass TFF module to recover 56% of the water feed, with no detectable HCP breakthrough in the recovered water (Table 3).

[0268] Table 3: Water recovery and host cell protein breakthrough in water recovered from mAb feed

[0269]

[0270] Additional recovery strategies include, but are not limited to: a permeate concentrated to 150 g / L containing 10 g / L mAb will recover 93% of the water used in the load material; a permeate concentrated to 150 g / L containing 25 g / L mAb will recover 83% of the water used in the load material; a permeate concentrated to 150 g / L containing 50 g / L mAb will recover 66% of the water used in the load material; and a permeate concentrated to 150 g / L containing 100 g / L mAb will recover 33% of the water used in the load material.

[0271] As outlined above, 1% and 5% resin slurries in the loaded material were evaluated for slurry concentration factors of 20 and 5, respectively. The HCP loadings on 1% and 5% resin were 0.6 mg / mL resin and 0.12 mg / mL resin, respectively. A 2-log HCP removal was observed at a feed mAb concentration of 10 g / L.

[0272] Example 2: Design of ferritin nanoparticles and expression in E. coli

[0273] Particle Design

[0274] The protein nanoparticles may comprise monomers self-assembled into 24-mer particles via a self-assembly domain derived from Helicobacter pylori ferritin (Hpftn). An N-terminally modified form, Bf-HpFtn, was used. The design was based on the N-terminus of Bf-HpFtn, with the addition of a linker domain, an antibody binding domain, and an affinity tag. The particle design was optimized primarily by changing the linker region, and nanoparticle 384 was ultimately selected.

[0275] The self-assembly domain (Bf-HpFtn) of nanoparticle 384 comprises a peptide according to SEQ ID NO: 1. It is connected to the antibody binding domain via a linker, and an example (incarnation) of the linker comprises SEQ ID NO: 26. The antibody binding domain (Z-domain) comprises SEQ ID NO: 4. The affinity tag comprises a polyhistidine tag and an enterokinase cleavage site as described in SEQ ID NO: 50. Therefore, the entire protein monomer composed of the above parts in nanoparticle 384 is SEQ ID NO: 30.

[0276] Construct design and generation

[0277] The protein sequence of the protein monomer is in Geneious The software is reverse translated into nucleotide sequence, and the software selects to realize E. coli optimization by random codon usage with rare codons removed. The obtained sequence of the designed coding gene can be found in SEQ ID NO:52. Synthetic nucleotides are ordered from commercial suppliers, and the synthetic nucleotides have the vector overlap designed for cloning into the internal vector derived from pET28, but have the beta-lactamase resistance gene replaced into the vector. The insert fragment is cloned in the following manner: overlap assembly is performed using a commercial cloning kit based on the Gibson reaction, and then transformed into the E. coli cloning strain. The plasmid obtained from the single colony isolate is confirmed by Sanger sequencing, and then the plasmid is transformed into E. coli BL21 (De3) for expression to form strain Bl_NP0384.

[0278] Nanoparticle expression

[0279] Protein from strain B1_NP0384 is produced by fed-batch growth and IPTG induction of the expression strain. Growth medium comprises 20.3g / kg yeast extract, 10.1g / kg sodium sulfate and 7g / kg potassium dihydrogen phosphate, and is supplemented to 100 μg / mL carbenicillin after autoclaving. The strain is grown overnight in a shake flask at 37°C, then inoculated into the 30L same medium in the bioreactor at 37°C, and the feed cycle is started after initial oxygen rebound. The feed comprises 550g / kg glucose supplemented with 5.4g / kg magnesium sulfate and 333g / kg yeast extract. When the optical density 550nm is 80, induction is started by adding the IPTG that final concentration is 0.3mM, and all available precautions are taken to maintain oxygen supply. The culture is cooled before harvesting after 3 hours of induction. Cell paste is harvested by centrifugation and stored in -80°C.

[0280] Nanoparticle purification of the insoluble fraction (inclusion bodies)

[0281] For protein purification, cells were suspended in phosphate buffered saline (PBS) at pH 7.4 to six volumes (ml / g cell paste). After being completely resuspended by agitation, the cell suspension was kept stirred by a magnetic stirring bar, and protease inhibitors (2.3 tablets / L of Roche Complete04693132001) and DNA enzyme (Pierce Universal Nuclease 2656968, 100 μL / L; DNA enzyme I Roche 10104159001, 33 mg / L) were added. The cell suspension was stirred, and lysozyme (Thermo89833) was then added to 40 mg / L, and the cells were stirred at room temperature for about 40 minutes. The cell suspension was cooled on ice, and then the cells were passed through a LM20 microfluidizer twice at 12,000 psi, while the reaction chamber and cooling loop were kept immersed in an ice-water bath. The lysed cells were centrifuged at 3000 × g in a 1L centrifuge bottle, and the supernatant was decanted. The inclusion body precipitate was then suspended in PBS containing 0.5x B-PER II (Thermo 78260) extraction reagent at a ratio of 5 mL / g precipitate, and an additional 33 mg / L DNase I was added. The suspension was completed by stirring, and the suspension was then centrifuged at 10,000×g for 20 minutes in a 500 mL centrifuge bottle. The supernatant was decanted and the precipitate was then washed in PBS (5 mL / g precipitate). The suspension was centrifuged at 10,000×g for 20 minutes and decanted.

[0282] Urea solubilization of inclusion bodies

[0283] The washed inclusion body precipitate was then suspended in PBS at 5 ml / g precipitate by stirring until completely suspended, and then three volumes of 8 M / kg urea, 50 mM Tris pH 7.8 were added to the stirred solution of the inclusion body suspension (final concentration was 6 M urea / kg). The solution was kept at 4°C overnight, then warmed to room temperature and centrifuged at 15,000 × g for 20 minutes. The supernatant containing the dissolved protein monomer was further purified by immobilized metal affinity chromatography (IMAC).

[0284] Immobilized metal affinity chromatography (IMAC) purification (2.1×19 cm)

[0285] The supernatant containing urea-dissolved protein is filtered through a 0.45 μm filter and loaded onto a 77 mL Ni-NTA post (2.1 × 19 cm), which was pre-equilibrated with 50 mM Tris-HCl, 6 M urea (pH 7.8) containing 5 mM imidazole for 5 column volumes (CV), with a flow rate of 15.2 mL / min. After sample loading, the post was washed with 50 mM Tris-HCl, 6 M urea (pH 7.8) containing 5 mM imidazole for several CVs to remove weakly bound impurities. After post washing, the protein bound was eluted with a step gradient of 50 mM Tris-HCl, 6 M urea (pH 7.8) containing 500 mM imidazole. After elution, the post was cleaned with 0.5 M sodium hydroxide for 5 CVs, followed by 5 CVs of deionized (DI) water and 20% ethanol storage solution. To avoid loss of chelated nickel on the Ni-NTA resin, the column was refilled with 3 CV of 0.1 M NiSO4 before each run, followed by 5 CV washes with DI water and equilibration buffer. The eluted protein was collected in an AKTA Avant fraction collector and kept at 6°C until the refolding step.

[0286] Dialysis for urea removal and refolding

[0287] Batch dialysis was performed primarily to remove urea, allowing for refolding and self-assembly of the nanoparticles while adjusting to a favorable buffer. The IMAC eluate equilibrated to ambient temperature was loaded onto a 10 kDa MWCO Slide-A-Lyzer TM The precipitate was dialyzed into a dialysis cassette (Thermo Fisher) and washed overnight with agitation in 200 dialysis volumes (DV) of 50 mM Tris buffer (pH 7.8), filtered through a 0.22 μm filter, and then stored at 4°C.

[0288] Preparative SEC

[0289] use Pure (Cytiva) loaded 3 mL of 0.22 μm syringe-filtered, dialyzed nanoparticle 384 (ferritin) into a HiPrep equilibrated with 50 mM Tris pH 7.8. TM 16 / 60 The column was eluted isocratically with equilibration buffer at 0.5 ml / min (15 cm / h) for at least 2 CVs to collect fraction A in a 96-well deep-well plate. 280≥5mAU, with a maximum fraction volume of 1mL. The column was washed with 0.5CV of 0.5M NaOH, 1.5CV of equilibration buffer, 1CV of water, and 1.2CV of 20% ethanol at the same flow rate. All buffers were filtered through 0.22μm before use. Fractions were analyzed for protein concentration (A280, EC1%=7.7) on a Stumner (Unchained Labs, Pleasanton CA) and for purity using the TSKgel SuperSW mAb method described herein. Fractions were pooled to include only those fractions with peaks 1 and 2, as Fig.12 A to Fig.12 Shown in B. Peak 3 contained smaller fragments suspected to be incompletely assembled ferritin species and was therefore excluded.

[0290] Nanoparticle binding, dissociation and characterization

[0291] When the sample containing both nano-particle and antibody is prepared, precipitation is visible immediately. In order to solve this problem, the precipitation sample containing excessive antibody for nano-particle is added in multiple saline solution, and visual assessment is redissolved by outward appearance and by the absorbance reading at 600nm (measurement of light scattering of particles in solution). In addition, independent nano-particle and independent antibody are also added in these salts as a control, and determine whether salt affects the precipitation of any one itself in these components. The precipitation sample of antibody and nano-particle is put into 150mM NaCl solution, and it is found that it has best redissolved. In addition, this condition can not make any one component itself precipitate. However, adding salt in the precipitated sample does not make the precipitation sample fully redissolve.

[0292] Based on the assumption that the precipitation interaction might be difficult to completely reverse but could be prevented, salt was added to the antibody before the nanoparticles were added. A method was designed to produce bound nanoparticle and antibody samples in which a stock solution of 5 M NaCl was added to a solution containing the antibody and then the nanoparticles were added to this solution. The amount of NaCl stock solution added to the antibody was calculated so that the final concentration of the solution was equal to 150 mM NaCl. When this method was tested, clear samples with no visible precipitation were obtained, as shown in Figure 2. Fig.13 Visible in.

[0293] IgG Sepharose 6 affinity column ("Reverse Protein A, mAb column")

[0294] One set of samples containing nanoparticles was loaded onto a 3.9 mL Tricorn 5 / 20 column (Cytiva, Marlborough, MA) packed with IgG Sepharose 6 Fast Flow affinity resin (Cytiva) to a bed height of 19.7 cm and connected to Pure 25 (Cytiva) FPLC system. For all runs, the method followed the sequence of Table 4, setting the linear speed to 300 cm / h.

[0295] Table 4: Chromatographic method used for IgG 6 Sepharose Fast Flow affinity chromatography

[0296]

[0297] 1 Column volume. 2 Cleaning-in-place disinfection method.

[0298] Four chromatographic runs were performed as in Table 5 and the results are presented in FIG. 14A to FIG. 14D In. An engineering run (cycle 0) was first performed to confirm the binding capacity of the resin (i.e., the resin can indeed bind protein A). Recombinant native protein A ligand (rPA, Repligen, Waltham, MA) from Staphylococcus aureus (S. aureus) diluted to pH 7.4 with 50mM tris (pH 7.4) was loaded, and although 25% was lost on the column according to mass balance, all chromatogram areas and 75% of the quantitative mass were attributed to the eluent, indicating that the column was functional. When loaded with less than half the mass of soluble protein A load (cycle 1), approximately half of the purified nanoparticle load was bound and appeared in the eluent according to the chromatogram peak area, while half appeared in the flow-through. Cycle 1b shows that the population in the flow-through was also able to bind to the column, indicating that it was overloaded in cycle 1 and therefore had a lower capacity than soluble protein A, which may be due to its much larger size and therefore inaccessibility to some ligands. Cycle 2 shows that it can bind again when the eluate from cycle 1 is titrated to neutral pH with 0.5M Tris (pH 11) and reloaded. In summary, these data indicate that the nanoparticles can bind mAb, dissociate from mAb (enabling elution), and regenerate by pH titration to bind mAb at least once again.

[0299] Table 5: Affinity chromatography experiments and performance (recovery) metrics .

[0300]

[0301] 1 Not recorded, 2 Undetermined

[0302] Combined SEC and SDS-PAGE for complex formation and resolution

[0303] In order to prove that complex is formed between antibody and nanoparticle, SEC and SDS-PAGE are used to carry out experiment.First, 4.2mL antibody with a concentration of 9.6mg / mL in the pH 7.8 buffer containing 50mM Tris and 215mM NaCl is stirred, and 1.8mL nanoparticle with a concentration of 5.1mg / mL in 50mM tris (pH 7.8) is continuously added at a rate of 0.1mL / min. The resulting mixture contains 6.7mg / mL antibody and 1.5mg / mL nanoparticle in the pH 7.8 buffer containing 50mM tris and 150mM NaCl.

[0304] The material was then analyzed using a HiPrep 16 / 60 Sephacryl S-500HR column (Cytiva). The column was connected to The column was washed with 1 CV of water and then balanced with 2 CV of 50mM tris (pH 7.8). The 50mL Superloop produced by Cytiva was loaded with 3mL volume or 2.5% column volume for antibody and nanoparticle mixture, and 2 CV of the same equilibrium buffer was passed through the column to elute the mixture. The elution peak measured with 280nm absorbance was fractionated with 2mL increments. The column was then sterilized with 0.75 CV of 0.5M NaOH, washed with 1 CV of water, and then put back into the 20% ethanol aqueous solution storage buffer by applying 1.2 CV. The same method was repeated twice with the 17.4mg / mL antibody alone of 3mL load and the 5.0mg / mL concentration of 3mL load.

[0305] Fractions from the antibody and nanoparticle mixture runs were then analyzed using SDS-PAGE according to the protocol described below.

[0306] SDS-PAGE results ( Fig.16 ) shows that the front of the peak contains nanoparticles and antibodies, while the back of the peak contains only antibodies. This can be seen by comparing fractions B2 and B8 on the gel, where the line showing the nanoparticle monomer is visible in B2 but not in B8. In addition, Fig.15 Evaluation of the results showed that the antibody alone did not elute before about 100 mL of elution volume corresponding to fraction B6 on the gel, but when the antibody and nanoparticles were combined, the antibody could be seen as early as 80 mL. Because the antibody appeared at an earlier elution volume than when it was alone, indicating a larger size, we inferred that the antibody was binding to the nanoparticles.

[0307] In order to prove the low pH dissociation of antibody and nanoparticle, repeat the use of this SEC and SDS-PAGE combined with each other, but carry out under low pH.As carried out above, produce the mixture containing 6.7mg / mL antibody and 1.5mg / mL nanoparticle in the pH 7.8 buffer containing 50mM tris and 150mM NaCl.Then, use the 1M glycine of pH 3.5 this solution is titrated to pH 3.5.1mL low pH buffer is added to every 2mL original mixture, and the sample is diluted to the antibody concentration of 4.5mg / mL and the nanoparticle concentration of 1.0mg / mL, all at pH 3.5.

[0308] For the SEC procedure, the same HiPrep 16 / 60 Sephacryl S-500HR column (Cytiva) was connected to pure, and use the same operation method, but there are two major differences. First, the equilibrium buffer for this operation is 100mM glycine pH 3.5, so that the column environment can be maintained under low pH conditions. Second, an additional washing step with 0.5 CV of water is added before disinfection with 0.5M NaOH to prevent any interaction between acid and base. The loading amount of the mixture of antibody and nanoparticles is 5.5mL or 4.5% column volume, and this increase is to take into account the dilution caused by the titration procedure. Repeat this same procedure for the control, wherein 5.5mL of single antibody is injected at 4.5mg / mL and 5.5mL of single nanoparticles are injected at 1.0mg / mL.

[0309] The fractions from the low pH SEC run with the antibody and nanoparticle mixture were then run on an SDS-PAGE gel according to the method given below. The antibody and nanoparticle mixture produced two peaks ( Fig.17 and Fig.18 ); the first peak is entirely nanoparticles and elutes where the nanoparticle peak alone would normally elute, and the second peak is the antibody peak, which elutes where the antibody peak alone would normally elute. Unlike the neutral pH experiment, where the antibody eluted earlier than it would normally elute when injected alone (due to the larger size), the antibody here does not show an increase in size based on the time it eluted from the SEC column, but elutes at the same residence time as when there were no nanoparticles mixed in the load. Therefore, this indicates that at low pH, the antibody and nanoparticles are completely dissociated.

[0310] exist Fig.19In Figure 1, SEC traces of a mixture of antibodies and nanoparticles loaded at neutral pH and low pH are compared. The disinfection peak is located at a different point on the x-axis due to the additional water wash in the low pH method. However, it is obvious that the peak after disinfection is much larger in the neutral pH run than in the low pH run. This suggests that more material is bound or retained on the column for the neutral pH run compared to the low pH run, which we speculate may be due to the formation of larger protein complexes under neutral pH conditions.

[0311] Nanoparticle filter performance

[0312] To understand the complex filtration performance on a small scale under elution conditions, a 500 μL volume, 300 kDa MWCOPES centrifugal filter (VS0152; Satorius Stedim, PA) was used.

[0313] First, after adding 5M NaCl (aqueous solution) to a final concentration of 150mM, two mixtures of antibody and nanoparticle 384 were combined in 50mM Tris pH 7.4 and 7.8, respectively, to reach a concentration of 0.86mg / ml and 0.40mg / ml for antibody and nanoparticle, respectively. The mixture was further diluted to pH 3.5 with 100mM glycine pH 3.0 or 50mM Tris pH 7.4, 150mM NaCl, to reach a final concentration of 0.20 and 0.43 for nanoparticle 384 and mAb, respectively, at pH 7.4 or pH 3.5. Each of the two pH mixtures was supplemented by a set of two controls, which contained either mAb or nanoparticle alone, with the same protein, NaCl concentration and pH as in the mixture, but with buffer to replace the absent components. A total of six spin filters were first equilibrated in buffer matching the subsequent load (500 μL, 1 spin, 2000 g, 1 min), and then the samples were loaded and spun at 2000 g for 15 s at ambient temperature (RT).

[0314] The volumes of the load, filtrate, and retentate were recorded and the absorbance at 280 nm (A280) was measured on a Nanodrop 2000 spectrophotometer (Thermo-Fisher, Waltham MA) blanked with the corresponding sample buffer. 0.5 mL of sample was titrated to neutral pH units by adding 2% v / v of 0.5 M Tris base solution, the volume addition ratio of this Tris base solution was applied to lower volumes of load, filtrate, and retentate samples as needed, and SDS-PAGE was performed as follows.

[0315] Fig. 20SDS-PAGE gels of mixtures separated at neutral and low pH are shown. At neutral pH, nanoparticles, when present alone or in complex, appear in both the filtrate and the retentate, with fewer nanoparticles passing through the filter when in complex. When present alone at neutral pH, mAb is evenly distributed across the filter, but when in complex, mAb is more highly retained. At pH 3.5, under elution conditions, separation is confirmed, with only mAb passing through the filter when in complex, while nanoparticles are found only in the retentate.

[0316] Only a small volume of the load was passed through the filter to avoid gel fouling and to generate a realistic scaled-down model of what might occur during TFF ( Fig.10 Negligible volume loss was observed. The sieving coefficients of both mAb and nanoparticles decreased with pH, ​​while comparable sieving coefficients of mAb complexes were observed at each pH.

[0317] Analytical methods

[0318] Purity was determined using an Agilent (Santa Clara, CA) 1260 Infinity II bio-inert LC system equipped with TSKgel SuperSW mAb HR; 4 μm; 7.8 mm×30 cm (Tosoh, King of Prussia, PA). Samples were injected and eluted isocratically at 0.5 mL / min for 30 minutes while recording the absorbance at 280 nm. An arginine-rich mobile phase (100 mM anhydrous disodium hydrogen phosphate, 100 mM sodium sulfate, 1 M arginine, pH 6.8) was used and a guard column was not included to avoid material loss.

[0319] SDS PAGE

[0320] By adding Novex with 5% bME TM 2X TrisGlycine SDS (LC2676, Thermo-Fisher) was used to prepare the sample buffer, which was added to the sample at a 1:1 volume ratio, heated to 95°C for 2 minutes, and cooled to room temperature. All samples and SeeBlue TM Plus 2,3kDa-198kDa ladder (LC5925; Thermo-Fisher; no heating / cooling) was electrophoresed on 4%-12% bis-tris PAGE with 1xMES running buffer at 200V for 35 minutes or at 150V for 90 minutes, followed by SimplyBlue TMSafestain (LC6060; Thermo-Fisher) staining and imaging. Low pH samples were supplemented with 2.5% v / v of pure β-mercaptoethanol after cooling and before loading.

[0321] Example 2: Materials and methods for TFF and C3ANDo

[0322] Isothermal calorimetry (ITC)

[0323] The titrant material was 28 mg / mL monoclonal antibody (mAb) purified by bind-elute protein A and passed through anion exchange chromatography to remove impurities. The ITC cell material was 0.5 g / L-1.0 g / L nanoparticles and was produced by the methods described in this section. Both resulting materials were buffer exchanged using a Slide-A-Lyzer TM Dialysis cassette, 3.5KMWCO (Thermo Scientific, USA) dialyzed to obtain a final formulation of 50mM Tris (pH 7.8), 150mM NaCl (Tris saline). The material was degassed using a vacuum degasser. mAb and nanoparticle concentrations were determined using Solo-VPE (Repligen, USA). The ITC chamber material (nanoparticles) was titrated with a titrant (mab) using a MicroCai PEAQ-ITC automatic device (Malvern Panalytical, USA), while measuring the energy released or absorbed by the chamber during the entire titration process and compared with a reference chamber filled with water for injection (WFI). Each titrant was injected 12 times at 2μL / s for titration, and mixed into the ITC sample chamber of 200μL ITC chamber material at 1000RPM at 20°C. Control titrations were performed using tris saline, nanoparticles, and tris saline as ITC chamber materials and mAb, tris saline, and tris saline as titrant materials. After entering the concentrations of the titrant and ITC chamber materials, the energy released or absorbed by the chamber during the entire titration was converted into thermodynamic properties (KD, enthalpy, entropy, free energy) and molar ratios using the PE SQ-ITC automatic software. The three control titrations performed were also uploaded to the software to minimize the energy that was not directly related to the combination of nanoparticles and mAb.

[0324] Conditioned medium (CM) dehydration

[0325] Dehydration of conditioned media from perfused cell cultures can significantly reduce membrane area and the water required for subsequent product capture and polishing steps. Dehydration was demonstrated using a single-pass TFF operation evaluated in different membrane units / geometries with MWCO ranging from 30 kDa to 50 kDa. mAb-conditioned media (CM) (obtained from perfused cell cultures) containing mAb or mAb-fortified CM (simulating higher titers) was filtered through a 0.22 μm PES filter. mAb-fortified conditioned media was prepared by adding purified mAb to mAb-depleted CM. Fig.23 As shown, the continuous single-pass TFF system includes a cassette-type membrane or hollow fiber module and a peristaltic pump.

[0326] Peristaltic pumps are used to control feed and retentate flow rates. This arrangement allows for regulation of permeate flux and volume concentration factor (VCF=Q feed / Q retentate). Phosphate buffered saline, 10mM sodium phosphate buffer, 150mM NaCl pH 7.4 are used to pre-balance the membrane unit by flushing the membrane with a feed flux of 40LMH to 20 times the hold-up volume of the membrane unit. In a typical study, CM filtered by 0.22 μm is applied to the TFF module with a feed flux of 10LMH using feed pump P1. The flow rate between pumps P1 and P2 is coordinated to obtain the desired permeate flux and VCF along the membrane module, wherein P1>P2. The selection of VCF depends on the mAb concentration in the load material and the dehydration expected in the single-pass mode. Regarding the process performance measured by the transmembrane pressure of the membrane in the 30kDa-50kDa MWCO range, several offsets of feed flux and mAb concentration are evaluated. Continuously collect concentrated CM at the retentate, and collect mAb-depleted CM at the permeate. mAb concentrations were determined using an analytical protein A column. mAb samples were loaded onto a 0.1 mL POROSTM pre-filled protein A affinity column (ThermoFisher, Waltham, MA) filled to a bed height of 3.0 cm and connected to an Agilent Series Gradient 1200 (Agilent) HPLC system. For all runs, the method had a set linear velocity of 6000 cm / h, the binding buffer was 1X PBS, pH 7.2, and the elution buffer was 1X PBS, pH 7.2+0.1% H3PO4.

[0327] Table 6: Overview of flux and feed concentration deviations

[0328]

[0329] Nanoparticle size enrichment

[0330] A feed containing about 0.5 mg / mL of purified nanoparticles (previously described nanoparticle purification method) was used in the ultrafiltration / diafiltration (UF / DF) step for buffer exchange and removal of small size (<300 kDa) impurities, especially monomers and partially formed nanoparticles. 0.11 m2 of 300 kDa PES 2 (Millipore, USA) membrane for UF / DF.Before starting UF / DF, the membrane storage solution was rinsed with 0.25M NaOH, then rinsed with water (WFI), and then balanced with 50mM Tris (pH7.8), 150mM NaCl (Tris saline).Enriched under a feed flux of 260LMH, ultrafiltered to a concentration of about 2mg / mL, then diafiltered 6 DV with tris saline buffer, and followed by tris saline membrane tracking, reaching the final enriched nanoparticles of 1.0mg / mL.The Solo-VPE (Repligen, USA) with an extinction coefficient of 0.77M-1cm-1 was used to determine the nanoparticle concentration.The used membrane was cleaned with 0.25M NaOH and stored in 0.1M NaOH for later use.

[0331] Table 7: Overview of Nanoparticle Size Enrichment

[0332] Concentration (g / L) Yield (%) Size purity by SEC (%) Enriched nanoparticles 1.0 71 94％

[0333] Continuous Countercurrent TFF - Capture, HCP Removal and Elution

[0334] The partitioning of solutes during membrane filtration is expressed as the sieving coefficient (S). S is a measure of the ability of a solute to pass through a membrane and is calculated by the following formula, where C P is the concentration of the solute in the permeate, and C f is the concentration of solute in the retentate.

[0335]

[0336] A sieving coefficient of 1 indicates unrestricted transport of the solute across the membrane, while a sieving coefficient of 0 indicates complete retention of the solute by the membrane. Fig.25 As shown, Experiment S determined the ability to allow predictable separation, recovery, or elimination of molecules of interest in the retentate or permeate using a reasonable number of stages in a multi-stage single-pass TFF (SPTFF) apparatus.

[0337] Sieving coefficient of pure mAb

[0338] For various MWCO membranes, use Fig.23 The experimental setup shown determined the sieving coefficients of pure mAbs in tris saline buffer and are shown in Table 8.

[0339] Table 8: Sieving coefficient S of pure mAb

[0340] Flux, LMH mAb concentration, g / L VCF Membrane MWCO Sieving coefficient, S 10 5 10 300 1 10 5 10 500 1 10 5 10 750 1 10 5 10 1000 1

[0341] Sieving coefficient of pure nanoparticles

[0342] For 300 kDa MWCO membranes, use Fig.23 The experimental setup shown determined the sieving coefficients of pure nanoparticles at pH 7.4 and 3.5 and are shown in Table 9.

[0343] Table 9: Sieving coefficient S of pure nanoparticles

[0344] Flux, LMH Nanoparticles, g / L VCF PH Sieving coefficient, S 10 0.5 10 7.8 0 10 0.5 10 3.5 0.3

[0345] Single-pass TFF-binding and retention of Mab-nanoparticle complexes

[0346] In a typical conjugation step, a purified mAb at a concentration of about 1 mg / mL was extemporaneously mixed with an equal volume of 0.5 g / L nanoparticles to achieve a (mAb: nanoparticle) molar ratio of about 10. Prior to mixing, the mAb and nanoparticles were exchanged with a pH 7.4 tris saline buffer to avoid any pH / salinity drift during mixing. The resulting mAb-nanoparticle mixture was then used as feed to a one-stage SPTFF device with a 300 kDa MWCO membrane module, as shown in FIG. Fig.23 The retentate flow rate was adjusted throughout the run to achieve a VCF of 3 while monitoring the cartridge TMP and outlet flow rate. Free or unbound mAb in the permeate was quantified using ProA-bound HPLC (Table 10).

[0347] Table 10: Summary of nanoparticle complex retention on TFF

[0348] Flux, LMH VCF MWCO mAb%, permeate mAb%, retentate TMP, psi 10 3 300 15 85 9

[0349] As shown in Table 10, 15% of mAb was detected in the permeate, while the remaining mAb was retained on the retentate site on the 300 kDa membrane. This observation is consistent with the mAb and nanoparticle sieving coefficient results discussed earlier. At pH 7.4, pure mAb showed S = 1, indicating unrestricted distribution across the membrane, while pure nanoparticle transport was completely restricted, i.e., S = 0. Upon binding to nanoparticles, the resulting affinity complex (> 900 kDa) resulted in 85% of mAb being retained on the retentate side of the membrane, while allowing free mAb to be distributed into the permeate.

[0350] Single-pass TFF HCP removal

[0351] HCP removal is another aspect of any mAb capture process. After successfully demonstrating the formation and retention of mAb-nanoparticle complexes in pure buffer systems, the study was repeated with conditioned medium (CM). Typically, perfusion CM with about 1 mg / mL mAb titer is temporarily mixed with an equal volume of 0.5 g / L nanoparticles in tris saline pH 7.8 to reach a (mAb: nanoparticle) molar ratio of about 10. Pre-fill CM in a one-stage SPTFF device with 300 kDa and 1000 kDa MWCO membrane modules by flushing the membrane at a 40 LMH feed rate up to 20x system hold-up volume. After pre-filling, mAb-nanoparticle mixture is used as the feed for the SPTFF device, as Fig.23 The retentate flow rate was adjusted throughout the run to achieve a VCF of 3 while monitoring the cartridge TMP and outlet flow rate. Free or unbound mAb in the permeate was quantified using ProA-bound HPLC (Table 11).

[0352] Table 11: HCP removal in TFF with 300 kDa and 1000 kDa MWCO membranes

[0353]

[0354] Similar to the previous results, 11% of mAb was detected in the permeate, while the remaining mAb was retained on the retentate side of the 300 kDa membrane. The observed degree of HCP removal can be further improved by a multi-stage SPTFF device using appropriate wash buffers. For the 300 kDa membrane, a significantly lower operating TMP of 5 psi was observed when compared to 9 psi for mAb-nanoparticles in the pure buffer system ( Fig.26 ).

[0355] Single pass TFF-elution

[0356] To evaluate the recovery of mAb during elution, we used Fig.23 The apparatus shown. The procedure consisted of gradually adjusting the pH of the solution containing the mAb-nanoparticle complexes from 7.8 to 3.5 using 2 M glycine HCl pH 3.0 buffer. The acidified solution was used as the feed to a one-stage single-pass TFF apparatus using a 300 kDa MWCO membrane module. Throughout the operation, the retentate flow rate was adjusted to achieve a VCF of 3 while closely monitoring the TMP and outlet flow rate of the cartridge. The samples collected at the retentate and permeate were reduced and denatured at 70 °C for 5 min and then loaded onto an SDS-PAGE gel using 1x NuPAGE supplemented with 1 mM sodium metabisulfite. TMElectrophoresis was performed in MESSDS buffer. After electrophoresis, the gel was transferred to a fixing solution and stained with Coomassie blue followed by destaining with deionized water. The mAb recovery in the permeate relative to the feed was calculated using image densitometry using ImageJ software.

[0357] SDS-PAGE gel image ( Fig. 27 ) showed that the mAb recovery in the permeate was 64%, which is comparable to the maximum theoretical recovery of a molecule with S = 1 on a 300 kDa membrane of a one-stage device at a VCF of 3 (66%). However, a multi-stage device has the potential to increase the mAb recovery to 90%. On the other hand, in the case of nanoparticles, about 50% of the nanoparticles were found to be partitioned into the permeate, which is higher than the 20% partitioning observed for pure nanoparticles at pH 3.5.

[0358] Table 12: mAb elution results in TFF by image densitometry

[0359]

[0360] Example 4: Continuous Countercurrent Affinity Nanoparticle Dialysis Operation (C3ANDo)

[0361] Diffusion coefficient of mAb (De)

[0362] Convection and diffusion are two mechanisms that drive molecules to move across semipermeable membranes. Unlike convection, diffusion is driven by the difference in concentration gradient across the membrane. The effect of membrane-facilitated diffusion depends on the properties of the membrane and the size and shape of the protein. Therefore, the measurement of the real-world mAb diffusion rate or diffusion coefficient allows estimation of mass transfer in diffusion-dominated systems. A typical C3ANDo device using a hollow fiber membrane operated in a countercurrent manner is described below. The C3ANDo device discussed here is used to evaluate the diffusion rate of mAb and other model proteins using multiple membranes (as shown in Table 13 below), and then mAb capture and elution are demonstrated.

[0363] The hollow fiber modules are mounted in a vertical orientation, with the pump P1 ( Fig.28 )Introduce the feed from the bottom port on the lumen side. The shell side port (shell outlet) near the feed port is attached to pump P2. The distal shell side port (shell inlet) is attached to pump P3. Shell side buffer is introduced into the module using pump P3, while pump P2 regulates the flow rate at the shell outlet. The exhausted flow is collected at the lumen outlet. Peristaltic pumps P1, P2 and P3 are equipped with appropriate pump heads and tubing. Pressure is monitored using pressure sensors placed just before and after the inlet port / outlet port. The shell compartment and lumen compartment of the hollow fiber module are rinsed with buffer before the experiment. The relationship between the feed flow rate and the shell side flow rate is represented by α, where:

[0364]

[0365] In a typical experimental setup with a feed flow (P1) of 0.05 ml / min (throughput 0.12 LMH), both shell side pumps were adjusted to 0.112 ml / min to obtain an α value of 2.25. For some experiments, values ​​in the range of 0.2 to 2.25 were evaluated. The device was operated in a single pass or feed recirculation mode. Small samples were collected regularly from the lumen outlet and the shell outlet for offline analysis.

[0366] Table 13: Experimentally measured protein diffusion coefficients (De)

[0367] Membrane MWCO; Wall thickness Protein / Condition <![CDATA[De(m 2 / s)]]> About 500 kDa; 40 μm mAbs <![CDATA[1.0 to 1.4×10 -11 > About 500 kDa; 40 μm Myoglobin <![CDATA[6.6×10 -11 ]]> About 500 kDa; 40 μm mAb in conditioned media <![CDATA[0.5×10 -11 ]]> 300 kDa; 200 μm Myoglobin <![CDATA[2.5×10 -11 ]]> 300 kDa; 200 μm Ovalbumin <![CDATA[1.6×10 -11 ]]>

[0368] C3ANDo Capture

[0369] In a typical mAb capture study, 1 g / L of purified mAb in tris saline buffer pH 7.8 was used as feed while tris saline buffer pH 7.8 was introduced into the shell side inlet. The feed flow rate and shell side flow rate were maintained at 0.05 ml / min and 0.01 mL / min, respectively, such that α was 0.2. To evaluate the effect of affinity nanoparticles on mAb recovery, similar experiments were performed in which nanoparticles in tris saline buffer pH 7.8 were provided to the shell side inlet, as shown in Figure 2. Fig.29A and Fig.29B The mAb recovery on the shell side was calculated by quantifying the mAb in the lumen outlet using Solo-VPE (Repligen, USA).

[0370] As shown in Table 14 below, the presence of nanoparticles on the shell side increased mAb recovery compared to buffer alone. The mAb-nanoparticle complexes formed were collected at the shell outlet and then used to assess mAb recovery during the C3ANDo elution step.

[0371] Table 14: Effect of shell-side nanoparticles on mAb recovery during capture

[0372] Shell side compartment mAb recovery on the shell side Increase in recovery rate % Tris saline pH 7.8 25％ N / A Nanoparticles + Tris saline pH 7.8 40％ 60％

[0373] C3ANDo elution

[0374] use Fig.31The scheme shown evaluates the feasibility of the diffusion-driven elution method. To demonstrate C3ANDo elution, the mAb-nanoparticle complex (pH 7.8) obtained from the capture step was dialyzed against 0.1M glycine HCl pH 3.5 buffer. As the feed solution travels along the membrane, the solution pH changes from 7.8 to 3.5 due to buffer exchange, resulting in the dissociation of mAb (150 kDa) from the nanoparticles (750 kDa). Due to its relatively small size, the dissociated mAb preferentially diffuses across the membrane to the shell side compartment.

[0375] The lumen outlet flow was recirculated across the membrane. During feed recirculation, the eluted mAb was collected at the shell side outlet. After 1000 min run time, feed recirculation was stopped, operation continued in single pass mode, and the lumen outlet flow was collected. Whenever applicable, samples were neutralized to pH 7.4 using 0.5 M Tris base. These samples were reduced and denatured at 70 °C for 5 min and then loaded onto SDS-PAGE gels using 1x NuPAGE supplemented with 1 mM sodium metabisulfite. TM Electrophoresis was performed in MES SDS buffer. After electrophoresis, the gel was transferred to a fixing solution and stained with Coomassie blue followed by destaining with deionized water. Image densitometry was used to estimate mAb recovery in the permeate relative to the feed using ImageJ software.

[0376] As described above, the C3ANDo elution study was divided into two operation modes, i.e., recirculation mode (0 min-1000 min; Fig.32 ) and one-way mode (1000 min-1600 min; Fig.33 ).from Fig.32 It can be observed that an increased amount of mAb diffused into the shell compartment of the hollow fiber (40 min - 640 min) without any detectable nanoparticles when compared to the feed. The presence of low pH on the shell side caused the mAb to dissociate / elute from the nanoparticles and subsequently diffuse across the membrane to the shell side compartment. A significant decrease in mAb concentration can be seen in the recycled feed sample compared to the feed ( Fig.32 ).exist Fig.33 In the figure, wells 4-9 correspond to the lumen outlet samples collected at time points 1040, 1120, 1240, 1320, 1440 and 1520 minutes after switching to the single-pass mode at 1000 minutes. These samples show the relative enrichment of nanoparticles when the system reaches steady state. The mAbs eluted at 600 and 640 minutes of the recycling phase were loaded into wells 10 and 11, respectively, together with the single-pass samples ( Fig.33 ) for comparison.

[0377] Preferred aspects of the present disclosure are described herein. For those of ordinary skill in the art, after reading the above description, the changes in those preferred aspects may become apparent. Therefore, the present disclosure includes any combination of the above-mentioned elements in all possible variations encompassed by the present invention, unless otherwise indicated herein or clearly contradictory to the context.

Claims

1. A method for purifying a product of interest using countercurrent flow filtration, the method comprising: (a) contacting a first solution comprising the product of interest and impurities with a binding molecule to form a complex, wherein the complex comprises the product of interest bound to the binding molecule; (b) contacting a first flowing solution comprising the complex with a first side of a semipermeable membrane, wherein the complex has a molecular weight exceeding a molecular weight cutoff of the semipermeable membrane, such that the complex is retained on the first side of the membrane; (c) passing the impurities through the semipermeable membrane, wherein the impurities have a molecular weight below the molecular weight cutoff of the semipermeable membrane and are retained on a second side of the semipermeable membrane in a second flowing solution countercurrent to the first flowing solution; as well as (d) dissociating the complex to form free product of interest and free binding molecule.

2. The method according to claim 1, further comprising: (e) regenerating the binding molecule, wherein the regenerated binding molecule is capable of forming a complex when contacted with the product of interest in the first solution or the second solution.

3. A method for purifying a product of interest using countercurrent flow filtration, the method comprising: (a) contacting a first mobile solution comprising the product of interest and impurities with a first side of a semipermeable membrane, wherein the product of interest passes through the semipermeable membrane to form a complex with a binding molecule on a second side of the semipermeable membrane, wherein the complex has a molecular weight exceeding the molecular weight cutoff of the semipermeable membrane, such that the complex is retained on the second side of the semipermeable membrane; (b) optionally retaining the impurity on the first side of the semipermeable membrane, or wherein the impurity flows through the semipermeable membrane, wherein the impurity has a molecular weight below the molecular weight cut-off of the semipermeable membrane and is either retained on the first side of the semipermeable membrane in the first mobile solution countercurrent to the second mobile solution or passes through the semipermeable membrane to the second side of the semipermeable membrane; wherein the second mobile solution flow rate is lower than the first mobile solution flow rate, and (c) dissociating the complex to form free product of interest and free binding molecule.

4. The method according to claim 3, further comprising: (e) regenerating the binding molecule, wherein the regenerated binding molecule is capable of forming a complex upon contact with the product of interest in the first flowing solution or the second flowing solution.

5. The method of claim 2 or claim 4, wherein the regenerated binding molecules are passed again across the second side of the semipermeable membrane in the second mobile solution.

6. The method according to claims 1 to 5, wherein the second running solution comprises a second binding molecule capable of binding to impurities in the first solution and / or the second running solution.

7. The method according to any one of claims 1 to 6, wherein unbound binding molecules diffuse across the semipermeable membrane into the second mobile solution.

8. The method according to any one of claims 1 to 7, wherein the product of interest is a protein.

9. The method of claims 1 to 8, wherein the binding molecule comprises protein A, protein G, a cation exchange resin or an anion exchange resin.

10. The method of any one of claims 1 to 9, wherein the binding molecule comprises Protein A.

11. The method according to any one of claims 1 to 10, wherein the first solution comprising the product of interest is obtained from a bioreactor.

12. The method of any one of claims 1 to 11, wherein the second mobile solution comprises a positively charged polymer.

13. The method of claim 12, wherein the positively charged polymer is DEAE dextran.

14. The method of any one of claims 1 to 13, wherein the impurities include low molecular weight species.

15. The method of claim 14, wherein the positively charged polymer binds low molecular weight species that have diffused across the semipermeable membrane.

16. The method of any one of claims 1 to 15, wherein steps (a) to (c) are repeated, and wherein the binding molecule comprises protein A.

17. The method of any one of claims 1 to 16, wherein steps (a) to (c) are repeated, and wherein the binding molecule comprises a cation exchange resin.

18. The method of any one of claims 1 to 17, wherein steps (a) to (c) are repeated, and wherein the binding molecule comprises an anion exchange resin.

19. The method of any one of claims 1 to 18, wherein the binding molecules comprise assembled nanoparticles.

20. The method of claim 19, wherein the assembled nanoparticle is an assembled ferritin nanoparticle comprising 24 fusion protein monomers.

21. The method of claim 20, wherein each fusion protein monomer comprises i) a self-assembling nanoparticle monomer; ii) a linker; and iii) an immunoglobulin binding domain.

22. The method of any one of claims 20 or 21, wherein the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 1-3.

23. The method of any one of claims 21 or 22, wherein the immunoglobulin binding domain is a Protein A Z-domain.

24. The method of claim 23, wherein the Protein A Z-domain comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 4-7.

25. The method according to any one of claims 21 to 24, wherein the linker comprises an amino acid sequence selected from one of SEQ ID NOs: 8-27.

26. The method according to any one of claims 20 to 25, wherein uncomplexed and / or unassembled monomers of the fusion protein diffuse across the semipermeable membrane into the second mobile solution.

27. The method according to any one of claims 20 to 26, wherein the fusion protein monomer further comprises a purification tag at one end of the fusion protein monomer.

28. The method of claim 27, wherein the purification tag comprises 6, 8 or 10 repeats of histidine.

29. The method of any one of claims 20 to 28, wherein the fusion protein monomer further comprises a protease site between the purification tag and the remainder of the fusion protein monomer.

30. The method of claim 29, wherein the fusion protein monomer comprises a protease site, namely, a HRV-3C protease site.

31. The method of any one of claims 20 to 30, wherein the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 28-36.

32. The method of any one of claims 20 to 31, wherein the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:32, or is encoded by a nucleic acid comprising SEQ ID NO:

52.

33. The method of any one of claims 20 to 32, wherein the fusion protein monomer comprises 2 to 5 immunoglobulin binding domains.

34. The method of claim 33, wherein the 2 to 5 immunoglobulin binding domains are protein A Z-domains.

35. The method of claim 34, wherein the 2 to 5 immunoglobulin binding domains are separated from each other by a linker.

36. The method of any one of claims 20 to 35, wherein the fusion protein monomers are capable of assembling into assembled nanoparticles comprising 24 fusion protein monomers.

37. The method of any one of claims 1 to 36, wherein steps (b) and (c) are repeated, optionally wherein steps (b) and (c) are repeated 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than 10 times.

38. A method according to any one of the preceding claims, wherein the permeate from step (b) and / or step (c) is recycled to the first flowing solution and / or the upstream flowing solution.

39. A method according to any one of the preceding claims, wherein the permeate from step (b) and / or step (c) is recycled to the first flowing solution and / or the second flowing solution and / or the upstream flowing solution.

40. The method according to any one of claims 1 to 39, wherein the dissociated protein is subjected to diafiltration.

41. The method of any one of the preceding claims, wherein the semipermeable membrane is a dialysis membrane.

42. The method of any one of claims 1 to 41, wherein the first flowing solution and / or the second flowing solution has a flow rate of about 30 ml / min to about 60 ml / min.

43. The method of claim 42, wherein the first flowing solution and the second flowing solution have the same flow rate.

44. The method of claim 42, wherein the first flowing solution and the second flowing solution have different flow rates.

45. The method of any one of claims 1 to 44, wherein the first mobile solution and / or the second solution is pulsed.

46. ​​The method of claim 45, wherein pulsing enhances mass transfer across the semipermeable membrane.

47. A method according to claim 45 or 46, wherein the pulse volume is smaller than the volume of the pores of the semipermeable membrane.

48. The method of claim 45 or 46, wherein the pulse volume is about half the volume of the pores of the semipermeable membrane.

49. The method of claim 45 or 46, wherein the pulse volume is greater than the volume of the pores of the semipermeable membrane.

50. The method of any one of claims 1 to 49, wherein about 0.1 kg / day, about 0.5 kg / day, about 1 kg / day, about 2 kg / day, about 3 kg / day, about 4 kg / day, about 5 kg / day, about 6 kg / day, about 7 kg / day, about 8 kg / day, about 9 kg / day, or about 10 kg / day of the product of interest is purified.

51. The method of any one of claims 1 to 50, wherein the product of interest is a protein and comprises an antibody, an antigen binding fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof.

52. The method of claim 51, wherein the protein comprises an antibody selected from the group consisting of IgM, IgA, IgE, IgD and IgG, and wherein about 10 mM to about 1 M NaCl or more preferably about 150 mM NaCl is optionally added to the first mobile solution.

53. The method of claim 52, wherein the protein comprises an antibody and the antibody is an IgG antibody selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

54. The method of claim 53, wherein the antibody is a monoclonal antibody.

55. The method of any of the preceding claims, wherein the first mobile solution is concentrated to about 50 g / L to about 100 g / L of the product of interest.

56. The method of claim 55, wherein the product of interest is a monoclonal antibody.

57. The method of any of the preceding claims, wherein the free product of interest is concentrated to about 50 g / L to about 100 g / L.

58. A method according to any of the preceding claims, wherein the molecular weight of the complex is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times the size of the molecular weight cutoff of the semipermeable membrane.

59. The method of any one of claims 1 to 2 or 5 to 58, wherein steps (a) to (c) are performed a second time, and wherein the second mobile solution of the second time is added to the first mobile solution of the first time, and wherein the MWCO of the filter used the second time has a MWCO that is the same size or greater than the MWCO of the first time steps (a)-(c).

60. The method of any one of claims 3 to 58, wherein steps (a) to (c) are performed a second time, and wherein the first mobile solution of the second time is added to the first mobile solution of the first time, and wherein the MWCO of the filter used the second time has a MWCO that is the same size or greater than that of steps (a)-(c) performed the first time.

61. The method of claim 59, wherein steps (a) to (c) are performed a third time, and wherein the second mobile solution of the third time is added to the first mobile solution of the second time, and wherein the MWCO of the filter used the third time has a MWCO that is the same size or greater than that of steps (a)-(c) performed the first or the second time.

62. The method of claim 60, wherein steps (a) to (c) are performed a third time, and wherein the first mobile solution of the third time is added to the first mobile solution of the second time, and wherein the MWCO of the filter used the third time has a MWCO that is the same size or greater than that of steps (a)-(c) performed the first or second time.

63. The method of claim 61 or 62, wherein the second and third steps (a) to (c) are performed consecutively.

64. The method according to any one of claims 59 to 63, wherein the first mobile solution or the second mobile solution of the third time flows into the first mobile solution of the second time, and / or wherein the first mobile solution or the second mobile solution of the second time flows into the first mobile solution of the first time.

65. A method of using a solution effluent of filtrate or dialysate from a continuous downstream step as a wash solution for an upstream step, wherein the upstream step has a filter having a MWCO of the same size or greater than that of the downstream step generating the effluent.

66. A method of using a solution effluent from the retentate of a successive downstream step as a wash liquid for an upstream step, wherein the upstream step has a filter having a MWCO of the same size or smaller than that of the downstream step generating the effluent, and the filtrate is directed downstream.

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