Improved protein recovery

Abstract

The present disclosure provides a novel wash method using a recycle wash technique that minimizes yield loss due to under-washing and prevents or reduces dilution of the target protein during the recycle wash process.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 718,864, filed August 14, 2018, which is incorporated herein by reference in its entirety. Background Technology

[0003] Subcutaneous delivery of various therapeutic proteins is a rapidly developing field, especially for specific protein subtypes, such as monoclonal antibodies requiring large doses of drug. Proper formulation of these molecules requires concentrations greater than 100 g / L. It is known that manufacturing these molecules at these high concentrations, as well as large-scale recovery of highly concentrated products using tangential flow filtration (TFF) or other filtration methods, is challenging.

[0004] Tangential flow filtration (TFF) is a rapid and efficient method for separating and purifying biomolecules. It can be applied to a wide range of biological fields, such as immunology, protein chemistry, molecular biology, biochemistry, and microbiology. TFF can be used to concentrate and desalt sample solutions ranging from 10 mL to several thousand liters, and can be used for fractionating large biomolecules from small biomolecules, harvesting cell suspensions, and clarifying fermentation broths and cell lysates. Membrane filtration is a widely used separation technique in life science laboratories. Based on membrane porosity, it can be classified as a microfiltration or ultrafiltration process. Ultrafiltration membranes, typically with pore sizes between 0.001 and 0.1 μm, are used for concentrating and desalting dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchanging buffers, and coarse fractionation. Ultrafiltration membranes are usually classified according to molecular weight cutoff (MWCO) rather than pore size.

[0005] It is well known that in high-viscosity ultrafiltration processes intended for high protein concentrations (>150 mg / mL) in the drug substance stage, the burden of achieving the target concentration comes from the ultrafiltration / percolation (UF / DF) process. One problem encountered in manufacturing is the retention of protein in the manufacturing tubing or other "trapped" areas, resulting in poor recovery in the collection container and consequently poor yield. Therefore, typical practice is to employ a recycle rinse to push the retained protein into the collection container to improve the overall yield. If the rinsing behavior follows ideal plug flow conditions, there will be no dilution during the rinse, and instead, the rinse buffer will simply push the protein into the collection container and place it in the "trapped" area, and recovering the protein trapped in the "trapped" area without dilution would not be a problem. However, under real-world conditions, recycle rinses result in product dilution because dilution occurs between the recycle rinse buffer and the protein to be pushed into the collection container. Therefore, it is necessary to reduce or prevent dilution of the target product during the recycle rinse process. Summary of the Invention

[0006] The present disclosure relates to a method of reducing or preventing dilution of a target protein during post-filtration recovery flush. One aspect of the present disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while the filtration is being performed, and (ii) passing a buffer through the filtration assembly during a recovery flush at a flush flow rate of less than 100 liters per square meter per hour (LMH), thereby reducing or preventing dilution of the target protein as compared to dilution of the target protein obtained with a flush flow rate of 300 LMH. In another aspect, the present disclosure relates to a method of improving or increasing concentration of a target protein during post-filtration recovery flush. One aspect of the present disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while the filtration is being performed, and (ii) passing a flush buffer through the filtration assembly during a recovery flush at a flush flow rate of less than 100 liters per square meter per hour (LMH), thereby improving concentration of the target protein as compared to concentration of the target protein obtained with a flush flow rate of 300 LMH.

[0007] In some embodiments, the Reynolds number ("Re") of the flow in the recovery flush is less than 2000, less than 1500, less than 1000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 5, less than 4, or less than 3, wherein Re is calculated using the formula:

[0008] Re = Du p / m, and

[0009] where D is the channel diameter (m) or the equivalent diameter in the case of non-cylindrical flow path geometry, u is the average velocity (m / s) (Q / A c ), p is the density (kg / m 3 ), and m is the viscosity (Pa-s).

[0010] In some embodiments, the Reynolds number (Re) of the flow in the recovery flush is between about 1 and about 50, between about 1 and about 45, between about 1 and about 40, between about 1 and about 35, between about 1 and about 30, between about 1 and about 25, between about 1 and about 20, between about 1 and about 15, between about 1 and about 10, between about 2 and about 40, between about 1 and about 10, between about 2 and about 9, between about 3 and about 8, between about 4 and about 7, between about 4 and about 6, between about 3 and about 7, between about 3 and about 6, between about 3 and about 5, between about 2 and about 8, between about 2 and about 7, between about 2 and about 6, between about 2 and about 5, between about 3 and about 10, or between about 4 and about 6.

[0011] In some embodiments, the Reynolds number (Re) of the flow in the recovery flush is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30. In some embodiments, the Re of the flow in the recovery flush is about 3.8.

[0012] In some embodiments, the flush flow rate is between about 5 LMH and about 100 LMH, between about 10 LMH and about 100 LMH, between about 10 LMH and about 90 LMH, between about 10 LMH and about 80 LMH, between about 10 LMH and about 70 LMH, between about 10 LMH and about 60 LMH, between about 10 LMH and about 50 LMH, between about 10 LMH and about 40 LMH, between about 10 LMH and about 30 LMH, between about 30 LMH and about 50 LMH, between about 20 LMH and about 100 LMH, between about 20 LMH and about 90 LMH, between about 20 LMH and about 80 LMH, between about 20 LMH and about 70 LMH, between about 20 LMH and about 60 LMH, between about 20 LMH and about 50 LMH, between about 20 LMH and about 40 LMH, between about 30 LMH and about 100 LMH, between about 30 LMH and about 90 LMH, between about 30 LMH and about 80 LMH, between about 30 LMH and about 70 LMH, between about 30 LMH and about 60 LMH, between about 30 LMH and about 50 LMH, between about 30 LMH and about 40 LMH, or between about 20 LMH and about 30 LMH.

[0013] In some embodiments, the rinse flow rate is at least about 10 LMH, at least about 20 LMH, at least about 30 LMH, at least about 40 LMH, at least about 50 LMH, at least about 60 LMH, at least about 70 LMH, at least about 80 LMH, at least about 90 LMH, or at least about 100 LMH. In some embodiments, the rinse flow rate is about 30 LMH.

[0014] The present methods can be particularly effective in high viscosity ultrafiltration processes at high protein concentrations (> 150 mg / mL) intended for the drug substance stage. In some embodiments, the viscosity of the target protein is at least about 1 centipoise (cP), at least about 2 cP, at least about 3 cP, at least about 4 cP, at least about 5 cP, at least about 6 cP, at least about 7 cP, at least about 8 cP, at least about 9 cP, at least about 10 cP, at least about 11 cP, at least about 12 cP, at least about 13 cP, at least about 14 cP, at least about 15 cP, at least about 16 cP, at least about 17 cP, at least about 18 cP, at least about 19 cP, at least about 20 cP, at least about 21 cP, at least about 22 cP, at least about 23 cP, at least about 24 cP, at least about 25 cP, at least about 26 cP, at least about 27 cP, at least about 28 cP, at least about 29 cP, or at least about 30 cP.

[0015] In some embodiments, the target protein has a viscosity of at least about 20 centipoise (cP), at least about 21 cP, at least about 22 cP, at least about 23 cP, at least about 24 cP, at least about 25 cP, at least about 26 cP, at least about 27 cP, at least about 28 cP, at least about 29 cP, at least about 30 cP, at least about 31 cP, at least about 32 cP, at least about 33 cP, at least about 34 cP, at least about 35 cP, at least about 36 cP, at least about 37 cP, at least about 38 cP, at least about 39 cP, at least about 40 cP, at least about 41 cP, at least about 42 cP, at least about 43 cP, at least about 44 cP, at least about 45 cP, at least about 46 cP, at least about 47 cP, at least about 48 cP, at least about 49 cP, at least about 50 cP, at least about 51 cP, at least about 52 cP, at least about 53 cP, at least about 54 cP, at least about 55 cP, at least about 56 cP, at least about 57 cP, at least about 58 cP, at least about 59 cP, at least about 60 cP, at least about 61 cP, at least about 62 cP, at least about 63 cP, at least about 64 cP, at least about 65 cP, at least about 66 cP, at least about 67 cP, at least about 68 cP, at least about 69 cP, at least about 70 cP, at least about 71 cP, at least about 72 cP, at least about 73 cP, at least about 74 cP, at least about 75 cP, at least about 76 cP, at least about 77 cP, at least about 78 cP, at least about 79 cP, at least about 80 cP, at least about 81 cP, at least about 82 cP, at least about 83 cP, at least about 84 cP, at least about 85 cP, at least about 86 cP, at least about 87 cP, at least about 88 cP, at least about 89 cP, at least about 90 cP, at least about 91 cP, at least about 92 cP, at least about 93 cP, at least about 94 cP, at least about 95 cP, at least about 96 cP, at least about 97 cP, at least about 98 cP, at least about 99 cP, or at least about 100 cP.

[0016] In some embodiments, the target protein has a viscosity of from about 2 cP to about 10 cP, from about 3 cP to about 10 cP, from about 1 cP to about 9 cP, from about 2 cP to about 8 cP, from about 2 cP to about 7 cP, from about 2 cP to about 6 cP, from about 3 cP to about 6 cP, from about 4 cP to about 5 cP, or from about 2 cP to about 5 cP.

[0017] In some embodiments, the concentration of the target protein after the recovery flush is at least about 100 g / L, at least about 110 g / L, at least about 120 g / L, at least about 130 g / L, at least about 140 g / L, at least about 150 g / L, at least about 160 g / L, at least about 170 g / L, at least about 180 g / L, at least about 190 g / L, at least about 200 g / L, at least about 210 g / L, at least about 220 g / L, at least about 230 g / L, at least about 240 g / L, at least about 250 g / L, at least about 260 g / L, at least about 270 g / L, at least about 280 g / L, at least about 290 g / L, or at least about 300 g / L, prior to the filtration.

[0018] In some embodiments, the concentration of the target protein after the recovery flush is from about 100 g / L to about 300 g / L, from about 110 g / L to about 250 g / L, from about 120 g / L to about 240 g / L, from about 150 g / L to about 250 g / L, from about 130 g / L to about 260 g / L, from about 140 g / L to about 260 g / L, from about 160 g / L to about 220 g / L, or from about 170 g / L to about 230 g / L.

[0019] In some embodiments, the concentration of the target protein after the recovery flush is about 90 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, about 210 g / L, about 220 g / L, about 230 g / L, about 240 g / L, about 250 g / L, about 260 g / L, about 270 g / L, about 280 g / L, about 290 g / L, or about 300 g / L.

[0020] Accordingly, the methods of the present application can result in an increased yield of protein after a recovery flush by preventing or reducing dilution of the target protein during the recovery flush. In some embodiments, the yield of the target protein after the recovery flush, which consists of (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while the filtration is being performed, and (ii) passing a buffer through the filtration assembly at a flush flow rate of less than 100 liters per square meter per hour (LMH) during the recovery flush, is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% compared to the concentration of the target protein prior to the recovery flush.

[0021] In some embodiments, the yield of the target protein after the recovery flush is increased by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, or at least 20% compared to the yield of the target protein obtained after a recovery flush with a flow rate of 300 LMH. In some embodiments, the feed flow rate in (i) is higher than the flush flow rate in (ii).

[0022] In some embodiments, the feed flow rate is at least 200 LMH, at least 210 LMH, at least 220 LMH, at least 230 LMH, at least 240 LMH, at least 250 LMH, at least 260 LMH, at least 270 LMH, at least 280 LMH, at least 290 LMH, at least 300 LMH, at least 310 LMH, at least 320 LMH, at least 330 LMH, at least 340 LMH, at least 350 LMH, at least 360 LMH, at least 370 LMH, at least 380 LMH, at least 390 LMH, at least 400 LMH, at least 410 LMH, at least 420 LMH, at least 430 LMH, at least 440 LMH, at least 450 LMH, at least 460 LMH, at least 470 LMH, at least 480 LMH, at least 490 LMH, or at least 500 LMH.

[0023] In some embodiments, the feed flow rate is between 200 LMH and 500 LMH, between 200 LMH and 450 LMH, between 250 LMH and 450 LMH, 450 LMH, between 300 LMH and 450 LMH, between 300 LMH and 400 LMH, or between 300 LMH and 350 LMH. In some embodiments, the feed flow rate is about 300 LMH.

[0024] In some embodiments, the filtration assembly comprises a membrane. In some embodiments, the filtration membrane useful in the filtration assembly is derived from polyvinylidene fluoride (PVDF), polysulfone, polyethersulfone, polyarylsulfone, regenerated cellulose, polyamide, polypropylene, polyethylene, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile, ethylene copolymer, polyamide (such as “Nylon 6” or Nylon 66”), polycarbonate, PFA, or any combination thereof. In some embodiments, the flush buffer comprises an organic or inorganic acid or a salt thereof.

[0025] In some embodiments, the organic or inorganic acid is citrate (e.g., a mixture of monosodium citrate - disodium citrate, a mixture of citric acid - trisodium citrate, a mixture of citric acid - monosodium citrate, etc.), succinate (e.g., a mixture of succinic acid - monosodium succinate, a mixture of succinic acid - sodium hydroxide, a mixture of succinic acid - disodium succinate, etc.), tartrate (e.g., a mixture of tartaric acid - sodium tartrate, a mixture of tartaric acid - potassium tartrate, a mixture of tartaric acid - sodium hydroxide, etc.), fumarate (e.g., a mixture of fumaric acid - monosodium fumarate, a mixture of fumaric acid - disodium fumarate, a mixture of monosodium fumarate - disodium fumarate, etc.), gluconate (e.g., a mixture of gluconic acid - sodium gluconate, a mixture of gluconic acid - sodium hydroxide, a mixture of gluconic acid - potassium gluconate, etc.), oxalate (e.g., a mixture of oxalic acid - sodium oxalate, a mixture of oxalic acid - sodium hydroxide, a mixture of oxalic acid - potassium oxalate, etc.), lactate (e.g., a mixture of lactic acid - sodium lactate, a mixture of lactic acid - sodium hydroxide, a mixture of lactic acid - potassium lactate, etc.), acetate (e.g., a mixture of acetic acid - sodium acetate, a mixture of acetic acid - sodium hydroxide, etc.), trisamine (e.g., Tris), phosphate, or histidine. In some embodiments, the rinse buffer comprises histidine. In some embodiments, the rinse buffer comprises a sugar. In some embodiments, the sugar is sucrose, trehalose, mannitol, xylitol, erythritol, lactose, glucose, sugar powder, or pullulan. In some embodiments, the rinse buffer comprises 20 mM histidine and 250 mM sucrose.

[0026] In some embodiments, a filtration buffer is used during (i). In some embodiments, the filtration buffer is the same as the rinse buffer. In other embodiments, the filtration buffer is different from the rinse buffer. In some embodiments, the filtration buffer comprises an organic and inorganic acid or a salt thereof.

[0027] In some embodiments, the organic or inorganic acid is citrate (e.g., monosodium citrate-di sodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) acetate (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.), trisamine (e.g., Tris), phosphate, or histidine.

[0028] In some embodiments, the filter buffer comprises a sugar. In some embodiments, the sugar is sucrose, trehalose, mannitol, xylitol, erythritol, lactose, glucose, sugar powder, or pullulan. In some embodiments, the pH of the wash buffer and / or the filter buffer is about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, or about 9.5.

[0029] In some embodiments, the sample is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any combination thereof. In some embodiments, the cell culture sample is derived from a cell culture medium comprising mammalian cells. In some embodiments, the mammalian cells are selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof.

[0030] In some embodiments, the target protein comprises an antibody or a fusion protein. In some embodiments, the target protein comprises an antibody. In some embodiments, the antibody is of an isotype selected from the group consisting of IgM, IgA, IgE, IgD, and IgG. In some embodiments, the IgG antibody is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, an anti-CSF1R antibody, or an anti-IL8 antibody.

[0031] In some embodiments, the target protein comprises an enzyme, a hormone, a cytokine, a cell surface receptor, a protease, a cytokine receptor, or any combination thereof. In some embodiments, the target protein is a fusion protein. In some embodiments, the fusion protein is fused to a heterologous moiety. In some embodiments, the heterologous moiety is a half-life extension moiety. In some embodiments, the half-life extension moiety comprises an Fc. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 (FIG. 1) shows data generated from a scaled down process using a 16 gauge feed tubing (176 cm 2 membrane area. Prior to the recovery flush, many different recovery flush flow rates were tested after the concentration of the TFF retentate was higher than the target concentration. From Figure 1 It can be seen that higher recovery flush flow rates resulted in greater dilution of the concentrated TFF retentate.

[0033] Figure 2 (FIG. 2) shows a comparison of calculated Reynolds numbers using tubing with various cross-sectional areas. The cross-sectional areas are shown in Table 1.

[0034] Figure 3 (FIG. 3) shows a comparison of calculated Reynolds numbers compared to the viscosity of the fluid flowing through tubing with various cross-sectional areas at a fixed velocity. DETAILED DESCRIPTION

[0035] The present disclosure provides an efficient method of reducing or preventing dilution of a target protein during a recovery flush process. Specifically, the method uses a slower flow rate to counteract the dilutive effects of turbulent flow, which occurs when the calculated Reynolds number (Re) is high. In some embodiments, the calculated Re that can be used in the present method is less than 2000.

[0036] As shown in the working examples, the methods are effective in reducing or preventing dilution of the target protein by the recovery flush flow rate during the recovery flush process. In certain embodiments, the present disclosure provides a method of reducing or preventing dilution of a target protein during a recovery flush process using a recovery flush flow rate of 30 LMH, thereby reducing or preventing dilution of the target protein as compared to the dilution of the target protein obtained with a recovery flush flow rate of 100 LMH. Using such a system allows for very little dilution of the target protein during the recovery flush process, which allows for higher final target concentration to be achieved without sacrificing product yield or concentration due to the absence of recovery flush or excessive dilution due to a faster recovery flush rate.

[0037] In certain embodiments, the present disclosure provides a method of purifying a protein of interest from a mixture comprising the protein of interest and one or more contaminants. Possible contaminants include host cell proteins (HCPs), high molecular weight species (HMWs), low molecular weight species (LMWs), or DNA.

[0038] In certain embodiments, the present disclosure provides a method of purifying an antibody. In certain embodiments, the mixture is derived from a harvested cell culture fluid, cell culture supernatant, cell lysate, and clarified bulk.

[0039] I. Terminology

[0040] To enable a clearer understanding of the present disclosure, definitions of certain terms are first provided. As used in this application, unless specifically provided otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0041] The term "and / or," where used herein, is taken to mean either one or both of the entities joined by the term. Thus, use of "and / or" in the phrase, for example, "A and / or B" in this context, is intended to encompass the "A and B," "A or B," "A," (alone), and "B," (alone). Likewise, the term "and / or" as used in the phrase, for example, "A, B, and / or C" is intended to encompass 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).

[0042] It should be understood that wherever aspects are described herein with the language "comprising" or "including" or "consisting of", it will be understood that the other aspects described herein with the language "consisting of" and / or "consisting essentially of" are also provided.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0044] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully

[0045] Use of the alternative (e.g., “or”) should be understood to mean either one, but not both, of the alternatives. As used herein, the indefinite article “a” or “an” should be understood to refer to “one or more” of any listed or enumerated component.

[0046] The term “about” or “substantially” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, depending on the particular context in which the term is used. For example, “about” or “substantially” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” or “substantially” can mean up to 20% of the value. Furthermore, particularly regarding biological systems or processes, the terms can mean up to an order of magnitude or up to five-fold of a value. When a particular value or composition is provided in the application and claims, unless otherwise stated the intent is to adopt “about” or “substantially” meaning unless otherwise indicated in the specific context.

[0047] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the interpretation of values within said range as either a single integer or fraction of the values within said range, as appropriate.

[0048] As used herein, the term "protein of interest" is used in its broadest sense to include any protein (natural or recombinant) that is 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 protein.

[0049] The term "clarification" refers to a process to remove microparticulates. Clarification can reduce the burden on subsequent chromatography (e.g., AEX or CEX) during the purification process. In some examples, clarification is a method to remove colloids, lipids, DNA-RNA, residual cells, and other particulates from a cell culture. Filtration can also be used, and can include a depth filter. "Clarified material" refers to a mixture that has been subjected to a clarification process.

[0050] The term "viral inactivation" refers to a process to remove infectious viral contaminants from a mixture. There are currently many different methods for inactivating infectious pathogenic viruses, including, for example, heat inactivation, solvent / detergent (S / D) inactivation, pH inactivation, chemical inactivation, and / or ultraviolet irradiation inactivation.

[0051] The term "chromatography" refers to any kind of technique that separates a protein of interest (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Typically, the protein of interest is separated from other molecules (e.g., contaminants) due to differences in the rate at which individual molecules of the mixture migrate through a stationary medium under the influence of a mobile phase, or during a binding and elution process. The terms "matrix" or "chromatography 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 a separation process. Non-limiting examples include particulate, monolithic, or fibrous resins that can be placed in a column or cartridge, as well as membranes. Examples of materials used to form the matrix include polysaccharides (such as agarose and cellulose); and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrene divinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above. Examples of typical matrix types suitable for use in the methods of the present disclosure are cation exchange resins, affinity resins, anion exchange resins, or mixed mode resins. A "ligand" is a functional group attached to a chromatography matrix and that determines the binding characteristics of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the above). 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 that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically provided otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0052] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., a protein of interest containing an Fc region or an antibody) is specifically bound by a ligand specific for the protein of interest. Such ligands are often referred to as biospecific ligands. In some embodiments, the biospecific ligand (e.g., protein A or a functional variant thereof) is covalently attached to a chromatography matrix material and is accessible to the protein of interest in solution when the solution contacts the chromatography matrix. During the chromatography step, the protein of interest typically retains its specific binding affinity for the biospecific ligand, while other solutes and / or proteins in the mixture do not bind appreciably or specifically to the ligand. The binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatography matrix while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then removed from the immobilized ligand in an active form under suitable conditions (e.g., low pH, high pH, high salt, competitive ligand, etc.) and passed through the chromatography column with an elution buffer that does not contain the contaminating proteins or protein impurities that were earlier allowed to pass through the column. Any component can be used as a ligand to purify its respective specifically bound protein, such as an antibody. However, in various methods according to the present disclosure, protein A is used as the ligand for target proteins containing an Fc region. Conditions for elution of the biospecific ligand (e.g., protein A) from the target protein (e.g., a protein containing an Fc region) can be readily determined by one of ordinary skill in the art. In some embodiments, protein G or protein L or a functional variant thereof can be used as the biospecific ligand. In some embodiments, the biospecific ligand (e.g., protein A) is used at a pH range of 5-9 to bind to a protein containing an Fc region, to wash or re-equilibrate the biospecific ligand / target protein conjugate, and then to elute with a buffer containing at least one salt having a pH of about equal to or less than 4.

[0053] The terms "purification," "separation," or "isolation," as used interchangeably herein, refer to increasing the purity of a protein of interest from a composition or sample comprising the protein of interest and one or more impurities. Typically, the purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0054] The term "buffer," as used herein, refers to a substance by the presence of which in solution the amount of acid or base that must be added to cause a change in pH units is increased. Buffer solutions resist changes in pH through the action of their acid-base conjugate components. Buffer solutions for use with biological reagents are typically able to maintain a constant hydrogen ion concentration such 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.

[0055] As used herein, the term "chromatography column" or "column" in relation to chromatography is a vessel, typically in the form of a cylinder or hollow column packed with a chromatography matrix or resin. The chromatography matrix or resin is a material that provides physical and / or chemical properties for purification.

[0056] 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 oppositely charged ligands attached (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate pH and conductivity conditions such that the solute of interest interacts more or less non-specifically with the charged compounds than with solute impurities or contaminants in the mixture. Contaminating solutes in the mixture can be washed from the column of ion exchange material, or bind to or be excluded from the resin more quickly or slowly than the solute of interest. "Ion exchange chromatography" specifically includes cation exchange (CEX), anion exchange (AEX), and mixed mode chromatography.

[0057] As used herein, the term "contaminant" is used in its broadest sense to cover any unwanted component or compound within a mixture. In a cell culture, cell lysate, or clarified material (e.g., clarified cell culture supernatant), contaminants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in the cell culture medium. Host cell contaminant proteins include, but are not limited to, those proteins naturally or recombinantly produced by the host cell as well as proteins related to or derived from the protein of interest (e.g., proteolytic fragments) and other process-related contaminants. In certain embodiments, contaminants are separated from the cell culture using means such as centrifugation, sterile filtration, depth filtration, and tangential flow filtration.

[0058] The term "Reynolds number" (Re) is calculated using the following formula:

[0059] Re = Du p / m, and

[0060] where D is the channel diameter (m) or the equivalent diameter in the case of non-cylindrical flow channel geometry, u is the average velocity (m / s) (Q / Ac), p is the density (kg / m 3 ), and m is the viscosity (PA s). The Reynolds number is a dimensionless quantity and describes the ratio of inertial forces to viscous forces in a flowing fluid. It is used in many fluid flow correlations and is used to describe the boundary of fluid flow regimes (laminar, transitional, and turbulent). Laminar flow occurs when a fluid flows in parallel layers that do not break apart. Flow in a tube is usually laminar when the Reynolds number is less than about 2,000. Flow is turbulent when the Reynolds number is greater than about 4,000. Reynolds numbers between 2,000 and 4,000 represent a transition region between laminar and turbulent flow.

[0061] The term "recovery flush" refers to a protein purification step in which a protein sample that has been concentrated beyond its desired target concentration is subjected to a flush to displace other proteins that have not yet reached the collection vessel. The amount of over-concentration in the over-concentration step affects the volume of flush available for the recovery step, as it determines the amount of dilution possible before reaching the final target protein concentration. In many large scale protein purification processes, a significant amount of protein can be retained outside of the collection vessel in tubing, piping, or other "residence" areas, and it is necessary to perform a recovery flush to recover the larger amount of protein product in the collection vessel or other vessel used to collect the purified protein product. Without a recovery flush, the protein product that accumulates in the tubing, piping, or other "residence" areas of a large scale protein purification system can be lost or unrecoverable due to not reaching the protein collection vessel or other vessel used to collect the purified protein product.

[0062] The term "flush buffer" refers to a solution or mixture used in the recovery flush process to increase the overall protein yield, dilute the protein to its final target concentration, or both. The flush buffer is also used to push the protein out of the tubing or other areas of the filtration assembly where the protein has accumulated.

[0063] The term "filtration buffer" refers to a solution or mixture used in the filtration process to pass the feed containing the protein of interest over or through a membrane.

[0064] The term "filtration assembly" refers to the entire system used to perform protein purification. One example of a filtration assembly is a tangential flow filtration (TFF) system, in which the feed containing the protein of interest is subjected to a feed flow perpendicular to the filter membrane. Commercially available TFF systems include Pall Sartorius Sartobind® Flux tangential flow filtration system (GE Life Sciences). Another example of a filtration assembly is a direct flow filtration (DFF) system, in which the feed containing the protein of interest is subjected to a feed flow parallel to the filter membrane.

[0065] The term "feed flow rate" (LMH) or sometimes "cross flow rate" refers to the flow rate in a tangential flow filtration (TFF) protein purification step, in which the feed containing the protein of interest is subjected to a feed flow perpendicular to the filter membrane. The tangential flow of the feed prevents the accumulation of particles trapped at the filter surface and thereby interferes with filtration. During an ultrafiltration concentration step, the feed flow into the ultrafiltration membrane cassette provides the transmembrane pressure (TMP) and cross flow flux necessary for concentration and buffer exchange. In the context of a direct flow filtration (DFF) system, the term "feed flow rate" refers to the flow rate parallel to the filter membrane.

[0066] The term "wash flow rate" (LMH) refers to the flow rate used for a protein purification step in which a protein sample that has been concentrated beyond its desired target concentration is subjected to a wash to displace protein not present in the collection vessel. The wash flow rate represents the rate at which the wash buffer flows through the system during the recovery wash phase of the purification process.

[0067] The term "diafiltration" refers to a process of removing, displacing, or reducing the concentration of a salt or solvent from a solution containing proteins, peptides, nucleic acids, and other biomolecules. The process uses a permeable membrane filter to separate components of a solution or mixture based on molecular size. Molecules smaller than the membrane pores pass freely through the membrane.

[0068] The term "ultrafiltration" refers to a process that uses a filter to separate particles of one size from particles of another size. Ultrafiltration is a pressure-driven membrane process that is widely used for protein concentration and buffer exchange. Ultrafiltration is a size-based separation in which species larger than the membrane pores are retained and smaller species pass through the filter. Ultrafiltration is carried out by differences in filtration rates of different components across the membrane under pressure.

[0069] In some embodiments, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region is comprised of a hinge and three domains, CH1, CH2, and CH3. In some antibodies (e.g., naturally occurring IgG antibodies), each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The heavy chain can or can not have a C-terminal lysine. The term "antibody" can include bispecific antibodies or multispecific antibodies.

[0070] In some embodiments, an "IgG antibody" (e.g., a human IgGl, IgG2, IgG3, and IgG4 antibody) as used herein has the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an IgGl, IgG2, IgG3, or IgG4 antibody can be composed of two heavy chains (HC) and two light chains (LC), wherein the two HC and LC are connected by disulfide bridges in the same number and position as present in naturally occurring IgGl, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bridges).

[0071] Immunoglobulins can be from any of the well-known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into the following subclasses in certain species: IgGl, IgG2, IgG3, and IgG4 in humans, and IgGl, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins (e.g., IgGl) exist in several allotypes, which differ from each other by at most a few amino acids. For example, "antibodies" include naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and wholly synthetic antibodies.

[0072] As used herein, the term "antigen binding portion" 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 shown 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 portion" of an antibody include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the 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 the 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 (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs which can optionally be joined by a synthetic linker. Furthermore, although the two domains of a Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., 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 with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0073] “Fc region” (fragment, crystallizable region), “Fc domain” or “Fc” refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody other than the first constant region immunoglobulin domain (e.g., CHI or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody’s two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. IgG isotypes are divided into the following subclasses in certain species: IgGl, IgG2, IgG3, and IgG4 in humans, and IgGl, IgG2a, IgG2b, and IgG3 in mice. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and the hinge between CHI and CH2 domains. As defined herein, the Fc region of a human IgG heavy chain is defined to extend from amino acid residue D221 for IgGl, V222 for IgG2, L221 for IgG3, and P224 for IgG4, to the carboxy-terminal end of the heavy chain, with numbering according to the EU index as in Kabat, although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain can vary. The CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain in the Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is not present) or 445 (if the C-terminal glycine and lysine residues are not present) of IgG. As used herein, the Fc region can be a native sequence Fc, including any allotypic variant or variant Fc (e.g., non-naturally occurring Fc).

[0074] An "Fc receptor" or "FcR" is a receptor that binds the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include the FcyR family of receptors, including allelic variants and alternatively spliced forms of these receptors. The FcyR family is comprised of three activating receptors (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory receptor (FcyRIIB). Various properties of human FcyRs are known in the art. Most innate effector cell types co-express one or more activating FcyR and the inhibitory FcyRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans), but do not express the inhibitory FcyRIIB in either mice or humans. Human IgGl binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor it binds.

[0075] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.

[0076] As used herein, "isotype" refers to the class of antibody (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by heavy chain constant region genes.

[0077] Amino acids can be referred to herein by either their commonly accepted single-letter codes or by the IUPAC-IUB Biochemical Nomenclature Commission recommended three-letter codes. Likewise, nucleotides are referred to by their generally accepted single-letter codes.

[0078] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not pertain to the specific length of the product. The term "protein," as used herein, is intended to encompass molecules composed of one or more polypeptides, which in some cases can be associated through bonds other than amide bonds. A protein can also be, in another aspect, a single polypeptide chain. In the latter case, the single polypeptide chain can in some cases comprise two or more polypeptide subunits fused together to form the protein. The terms "polypeptide" and "protein" also refer to post-expression modified products of the polypeptide or protein. Post-expression modifications include, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization through known protecting / blocking groups, proteolytic processing, or any combination of the above. A polypeptide or protein can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner including by chemical synthesis.

[0079] The term "polynucleotide" or "nucleotide" as used herein is intended to encompass both single nucleic acids as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). In certain aspects, a polynucleotide comprises conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)).

[0080] The term "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, such as a DNA, cDNA, or RNA segment. The term "isolated" when applied to a nucleic acid or polynucleotide means a nucleic acid molecule, DNA, or RNA that has been removed from its natural environment, e.g., for the purposes of the present disclosure, a recombinant polynucleotide encoding an antigen binding protein contained in a vector is considered isolated. Other examples of an isolated polynucleotide include a recombinant polynucleotide that is maintained in a heterologous host cell or purified (partially or substantially) from other polynucleotides in solution. An isolated RNA molecule includes an in vivo or in vitro RNA transcript of a polynucleotide of the present disclosure. An isolated polynucleotide or nucleic acid according to the present disclosure also includes such a molecule produced synthetically. Additionally, a polynucleotide or nucleic acid can include regulatory elements, such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0081] RANGES: As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the values explicitly recited and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer) as well as fractions of the recited integer values.

[0082] The term "HMW species" refers to any one or more unwanted proteins present in a mixture. High molecular weight species can include dimers, trimers, tetramers, or other multimers. These species are generally considered to be impurities associated with the product; and can be covalently or non-covalently linked; and can also consist, for example, of misfolded monomers with hydrophobic amino acid residues exposed to the polar solvent; and can cause aggregation.

[0083] The term "LMW species" refers to any one or more unwanted species present in a mixture. Low molecular weight species are generally considered to be impurities associated with the product; and can include truncated species or half molecules of compounds intended to be dimers (such as monoclonal antibodies).

[0084] The term "host cell protein" or HCP refers to an unwanted protein produced by a host cell that is not associated with the production of the desired protein of interest. Unwanted host cell proteins can be secreted into the upstream cell culture supernatant. Unwanted 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 not needed in the final drug product.

[0085] Various aspects of the present disclosure are described in further detail in the following subsections.

[0086] II. Method of recovering a rinse

[0087] The present disclosure relates to methods of reducing or preventing dilution of a target protein during post-filtration recovery flush. In one aspect, the present disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while filtration is being performed, and (ii) passing a buffer through the filtration assembly at a flush flow rate of less than 300 liters per square meter per hour (LMH) during a recovery flush, thereby reducing or preventing dilution of the target protein as compared to dilution of the target protein obtained with a flush flow rate of 300 LMH. In another aspect, the present disclosure relates to methods of improving or increasing the concentration of a target protein during post-filtration recovery flush. In one aspect, the present disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while filtration is being performed, and (ii) passing a flush buffer through the filtration assembly at a flush flow rate of less than 300 liters per square meter per hour (LMH) during a recovery flush, thereby improving the concentration of the target protein as compared to the concentration of the target protein obtained with a flush flow rate of 300 LMH. In some embodiments, the desired concentration of the target protein is greater than about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL.

[0088] To accommodate dilution that occurs due to recovery flush, a standard approach for purifying a protein of interest in a downstream manufacturing process is to concentrate the protein solution to a slightly higher concentration (e.g., 20% higher). This strategy works well when the UF / DF operation is performed at a relatively low concentration of 50 mg / mL prior to formulation. For example, the target protein can be concentrated to 55-60 mg / mL during the filtration phase, and diluted to 50 mg / mL prior to formulation. At a protein concentration of 55-60 mg / mL, the viscosity of some monoclonal antibodies is typically in the range of 2-6 cP at room temperature.

[0089] At high protein concentrations, the final concentration and dilution results in a vicious cycle. When the protein concentration is high, in order to allow the recovery flush to dilute the protein to the correct concentration to avoid over-dilution, it is desirable to concentrate the target protein at a very high level (i.e., higher than the desired concentration after filtration). For example, concentrating the protein at a high concentration (e.g., from 150 mg / mL to 170 mg / mL) can take a significant amount of time, result in additional passage through the membrane, and can impact the product quality. In addition, even after reaching a high concentration during / after the filtration step, there is a high risk of over-dilution of the product during the recovery flush. This is because in many large scale protein purification processes, a significant amount of protein can be retained outside the hold tank in the tubing or piping. Thus, because of the retention of a large amount of protein in the tubing or piping, the initial recovery flush can not accumulate the full amount of the recovery product in the collection vessel. This can result in additional recovery flushes, which can further dilute the concentration of the final product. The present method is designed to prevent or reduce the dilution of the target protein during the recovery flush process by slowing the rate of the flush of the flush buffer. Thus, the present method allows for skipping or avoiding additional recovery flush steps during the recovery flush process.

[0090] In some embodiments, the present disclosure relates to methods of reducing or preventing dilution of a target protein during a post-filtration recovery flush. One aspect of the present disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while performing filtration, and (ii) passing a buffer through the filtration assembly at a flush flow rate of less than 300 liters per square meter per hour (LMH), less than about 250 LMH, less than about 200 LMH, less than about 150 LMH, less than about 140 LMH, less than about 130 LMH, less than about 120 LMH, less than about 110 LMH, or less than about 100 LMH during a recovery flush, thereby reducing or preventing dilution of the target protein compared to the dilution of the target protein obtained with a flush flow rate of 300 LMH. In another aspect, the present disclosure relates to methods of improving or increasing the concentration of a target protein during a post-filtration recovery flush. One aspect of the present disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while performing filtration, and (ii) passing a flush buffer through the filtration assembly at a flush flow rate of less than 300 liters per square meter per hour (LMH), less than about 250 LMH, less than about 200 LMH, less than about 150 LMH, less than about 140 LMH, less than about 130 LMH, less than about 120 LMH, less than about 110 LMH, or less than about 100 LMH during a recovery flush, thereby improving the concentration of the target protein compared to the concentration of the target protein obtained with a flush flow rate of 300 LMH.

[0091] To prevent or reduce dilution of the target protein according to the methods of the application, the flow in the filtration assembly is laminar. Laminar flow occurs when a fluid flows in parallel layers with no disruption between the layers. Flow in a tube is generally laminar when the Reynolds number is less than about 2,000. Flow is turbulent when the Reynolds number is greater than about 4,000. Reynolds numbers between 2,000 and 4,000 represent a transition region between laminar and turbulent flow. Thus, in some embodiments, the Reynolds number ("Re") of the flow in the recovery flush is less than 2000, less than 1900, less than 1800, less than 1700, less than 1600, less than 1500, less than 1400, less than 1300, less than 1200, less than 1100, less than 1000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 5, less than 4, less than 3, less than 2, or less than 1, where Re is calculated using the formula:

[0092] Re = Dυρ / μ, and

[0093] where D is the channel diameter (m) or the equivalent diameter in the case of non-cylindrical flow path geometry, υ is the average velocity (m / s) (Q / A c ), p is the density (kg / m 3 ), and μ is the viscosity (Pa-s).

[0094] In some embodiments, the Reynolds number (Re) of the flow in the recovery flush is between about 1 and about 50, between about 1 and about 45, between about 1 and about 40, between about 1 and about 35, between about 1 and about 30, between about 1 and about 25, between about 1 and about 20, between about 1 and about 15, between about 1 and about 10, between about 1 and about 5, between about 1 and about 4, between about 1 and about 3, between about 2 and about 40, between about 2 and about 9, between about 3 and about 8, between about 4 and about 7, between about 4 and about 6, between about 3 and about 7, between about 3 and about 6, between about 3 and about 5, between about 2 and about 8, between about 2 and about 7, between about 2 and about 6, between about 2 and about 5, between about 3 and about 10, between about 4 and about 6, between about 3 and about 4, or between about 2 and about 3.

[0095] In some embodiments, the Reynolds number (Re) of the flow in the recovery flush is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30. In some embodiments, the Reynolds number (Re) of the flow in the recovery flush is about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60. In some embodiments, the Re of the flow in the recovery flush is about 3.8.

[0096] In certain embodiments, the disclosure relates to methods of reducing or preventing dilution of a target protein during post-filtration recovery flushes. One aspect of the disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while filtration is being performed, and (ii) passing a buffer through the filtration assembly at a flush flow rate of less than about 100 liters per square meter per hour (LMH) during a recovery flush, thereby reducing or preventing dilution of the target protein as compared to dilution of the target protein obtained with a flush flow rate of 300 LMH. In another aspect, the disclosure relates to methods of improving or increasing the concentration of a target protein during post-filtration recovery flushes. One aspect of the disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while filtration is being performed, and (ii) passing a flush buffer through the filtration assembly at a flush flow rate of less than about 100 liters per square meter per hour (LMH) during a recovery flush, thereby improving the concentration of the target protein as compared to the concentration of the target protein obtained with a flush flow rate of 300 LMH. In some embodiments, the flush flow rate is between about 5 LMH and about 100 LMH, between about 10 LMH and about 100 LMH, between about 10 LMH and about 90 LMH, between about 10 LMH and about 80 LMH, between about 10 LMH and about 70 LMH, between about 10 LMH and about 60 LMH, between about 10 LMH and about 50 LMH, between about 10 LMH and about 40 LMH, between about 10 LMH and about 30 LMH, between about 30 LMH and about 50 LMH, between about 20 LMH and about 100 LMH, between about 20 LMH and about 90 LMH, between about 20 LMH and about 80 LMH, between about 20 LMH and about 70 LMH, between about 20 LMH and about 60 LMH, between about 20 LMH and about 50 LMH, between about 20 LMH and about 40 LMH, between about 30 LMH and about 100 LMH, between about 30 LMH and about 90 LMH, between about 30 LMH and about 80 LMH, between about 30 LMH and about 70 LMH, between about 30 LMH and about 60 LMH, between about 30 LMH and about 50 LMH, between about 30 LMH and about 40 LMH, or between about 20 LMH and about 30 LMH.

[0097] In some embodiments, the flush flow rate is at least about 10 LMH, at least about 20 LMH, at least about 30 LMH, at least about 40 LMH, at least about 50 LMH, at least about 60 LMH, at least about 70 LMH, at least about 80 LMH, at least about 90 LMH, or at least about 100 LMH. In some embodiments, the flush flow rate is about 30 LMH.

[0098] In certain embodiments, the disclosure relates to methods of reducing or preventing dilution of a target protein during post-filtration recovery flushes. One aspect of the disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while filtration is being performed, and (ii) passing a buffer through the filtration assembly during a recovery flush at a flush flow rate of less than about 30 liters per square meter per hour (LMH), thereby reducing or preventing dilution of the target protein as compared to dilution of the target protein obtained with a flush flow rate of 300 LMH. In another aspect, the disclosure relates to methods of improving or increasing the concentration of a target protein during post-filtration recovery flushes. One aspect of the disclosure relates to (i) passing a sample containing the target protein through a filtration assembly at a feed flow rate while filtration is being performed, and (ii) passing a flush buffer through the filtration assembly during a recovery flush at a flush flow rate of less than about 30 liters per square meter per hour (LMH), thereby improving the concentration of the target protein as compared to the concentration of the target protein obtained with a flush flow rate of 300 LMH.

[0099] In some embodiments, the target protein has a viscosity of at least about 1 centipoise (cP), at least about 2 cP, at least about 3 cP, at least about 4 cP, at least about 5 cP, at least about 6 cP, at least about 7 cP, at least about 8 cP, at least about 9 cP, at least about 10 cP, at least about 11 cP, at least about 12 cP, at least about 13 cP, at least about 14 cP, at least about 15 cP, at least about 16 cP, at least about 17 cP, at least about 18 cP, at least about 19 cP, at least about 20 cP, at least about 21 cP, at least about 22 cP, at least about 23 cP, at least about 24 cP, at least about 25 cP, at least about 26 cP, at least about 27 cP, at least about 28 cP, at least about 29 cP, or at least about 30 cP.

[0100] In some embodiments, the present disclosure can be particularly effective at filtering target proteins having a high viscosity. In some embodiments, the target protein has a high viscosity and is at least about 20 centipoise (cP), at least about 21 cP, at least about 22 cP, at least about 23 cP, at least about 24 cP, at least about 25 cP, at least about 26 cP, at least about 27 cP, at least about 28 cP, at least about 29 cP, at least about 30 cP, at least about 31 cP, at least about 32 cP, at least about 33 cP, at least about 34 cP, at least about 35 cP, at least about 36 cP, at least about 37 cP, at least about 38 cP, at least about 39 cP, at least about 40 cP, at least about 41 cP, at least about 42 cP, at least about 43 cP, at least about 44 cP, at least about 45 cP, at least about 46 cP, at least about 47 cP, at least about 48 cP, at least about 49 cP, at least about 50 cP, at least about 51 cP, at least about 52 cP, at least about 53 cP, at least about 54 cP, at least about 55 cP, at least about 56 cP, at least about 57 cP, at least about 58 cP, at least about 59 cP, at least about 60 cP, at least about 61 cP, at least about 62 cP, at least about 63 cP, at least about 64 cP, at least about 65 cP, at least about 66 cP, at least about 67 cP, at least about 68 cP, at least about 69 cP, at least about 70 cP, at least about 71 cP, at least about 72 cP, at least about 73 cP, at least about 74 cP, at least about 75 cP, at least about 76 cP, at least about 77 cP, at least about 78 cP, at least about 79 cP, at least about 80 cP, at least about 81 cP, at least about 82 cP, at least about 83 cP, at least about 84 cP, at least about 85 cP, at least about 86 cP, at least about 87 cP, at least about 88 cP, at least about 89 cP, at least about 90 cP, at least about 91 cP, at least about 92 cP, at least about 93 cP, at least about 94 cP, at least about 95 cP, at least about 96 cP, at least about 97 cP, at least about 98 cP, at least about 99 cP, or at least about 100 cP. In some embodiments, the target protein has a viscosity of between about 20 cP and about 100 cP, between about 20 cP and about 90 cP, between 25 cP and about 100 cP, between about 25 cP and about 90 cP, between about 30 cP and about 100 cP, between about 30 cP and about 90 cP, between about 30 cP and about 80 cP, between about 30 cP and about 70 cP, between about 40 cP and about 60 cP. In other embodiments, the target protein has a viscosity of about 10 cP, about 20 cP, about 30 cP, about 40 cP, about 50 cP, about 60 cP, about 70 cP, about 80 cP, about 90 cP, or about 100 cP.

[0101] In some embodiments, the target protein has a viscosity of from about 2 cP to about 10 cP, from about 3 cP to about 10 cP, from about 1 cP to about 9 cP, from about 2 cP to about 8 cP, from about 2 cP to about 7 cP, from about 2 cP to about 6 cP, from about 3 cP to about 6 cP, from about 4 cP to about 5 cP, or from about 2 cP to about 5 cP.

[0102] Thus, the methods of the application can improve the concentration of the target protein after recovery wash by preventing or reducing dilution of the target protein during the recovery wash. In some embodiments, the concentration of the target protein after the recovery wash is at least about 100 g / L, at least about 110 g / L, at least about 120 g / L, at least about 130 g / L, at least about 140 g / L, at least about 150 g / L, at least about 160 g / L, at least about 170 g / L, at least about 180 g / L, at least about 190 g / L, at least about 200 g / L, at least about 210 g / L, at least about 220 g / L, at least about 230 g / L, at least about 240 g / L, at least about 250 g / L, at least about 260 g / L, at least about 270 g / L, at least about 280 g / L, at least about 290 g / L, or at least about 300 g / L, at least about 200 g / L, at least about 210 g / L, at least about 220 g / L, at least about 230 g / L, at least about 240 g / L, at least about 250 g / L, at least about 260 g / L, at least about 270 g / L, at least about 280 g / L, at least about 290 g / L, or at least about 300 g / L, before filtration.

[0103] In some embodiments, the concentration of the target protein after the recovery wash is from about 100 g / L to about 300 g / L, from about 110 g / L to about 250 g / L, from about 120 g / L to about 240 g / L, from about 150 g / L to about 250 g / L, from about 130 g / L to about 260 g / L, from about 140 g / L to about 260 g / L, from about 160 g / L to about 220 g / L, or from about 170 g / L to about 230 g / L.

[0104] In some embodiments, the concentration of the target protein after the recovery wash is from about 100 g / L to about 300 g / L, from about 110 g / L to about 300 g / L, from about 110 g / L to about 290 g / L, from about 120 g / L to about 290 g / L, from about 120 g / L to about 280 g / L, from about 130 g / L to about 280 g / L, from about 130 g / L to about 270 g / L, from about 140 g / L to about 270 g / L, from about 140 g / L to about 260 g / L, from about 150 g / L to about 260 g / L, from about 150 g / L to about 250 g / L, from about 160 g / L to about 250 g / L, from about 160 g / L to about 240 g / L, from about 170 g / L to about 240 g / L, from about 170 g / L to about 230 g / L, from about 180 g / L to about 230 g / L, from about 180 g / L to about 220 g / L, from about 190 g / L to about 220 g / L, from about 190 g / L to about 210 g / L, or from about 200 g / L to about 210 g / L.

[0105] In some embodiments, the concentration of the target protein after the recovery wash is about 90 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, about 210 g / L, about 220 g / L, about 230 g / L, about 240 g / L, about 250 g / L, about 260 g / L, about 270 g / L, about 280 g / L, about 290 g / L, or about 300 g / L.

[0106] In some embodiments, the yield of the target protein after the recovery wash according to the method of the application is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% compared to the concentration of the target protein prior to the recovery wash process.

[0107] In some embodiments, the yield of the target protein after recovery flush according to the method of the application is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, or at least about 40% compared to the yield of the target protein obtained after recovery flush with a flow rate of 300 LMH. In some embodiments, the feed flow rate in (i) is higher than the flush flow rate in (ii).

[0108] According to the method of the application, the target protein is passed through the filtration assembly while filtration is taking place. The flow rate in the tangential flow filtration (TFF) step of the feed containing the target protein is referred to herein as the feed flow rate, as opposed to the flush flow rate. Tangential flow of the feed prevents the accumulation of particles trapped at the filter surface and thereby interferes with filtration. During the ultrafiltration concentration step, the feed flow into the ultrafiltration cassette provides the necessary trans-membrane pressure (TMP) and cross-flow flux for concentration and buffer exchange.

[0109] In the present disclosure, the feed flow rate is higher than the flush flow rate. In some embodiments, the feed flow rate is at least 200 LMH, at least 210 LMH, at least 220 LMH, at least 230 LMH, at least 240 LMH, at least 250 LMH, at least 260 LMH, at least 270 LMH, at least 280 LMH, at least 290 LMH, at least 300 LMH, at least 310 LMH, at least 320 LMH, at least 330 LMH, at least 340 LMH, at least 350 LMH, at least 360 LMH, at least 370 LMH, at least 380 LMH, at least 390 LMH, at least 400 LMH, at least 410 LMH, at least 420 LMH, at least 430 LMH, at least 440 LMH, at least 450 LMH, at least 460 LMH, at least 470 LMH, at least 480 LMH, at least 490 LMH, or at least 500 LMH, wherein the flush flow rate is lower than the feed flow rate, for example lower than 100 LMH.

[0110] In some embodiments, the feed flow rate is between 200 LMH and 500 LMH, between 200 LMH and 450 LMH, between 250 LMH and 450 LMH, 450 LMH, between 300 LMH and 450 LMH, between 300 LMH and 400 LMH, or between 300 LMH and 350 LMH. In some embodiments, the feed flow rate is about 300 LMH.

[0111] According to the method of the present application, filtration of the protein of interest is performed using a filtration assembly. The filtration assembly generally comprises elements such as a filter holder, a filter cassette, and ports for flow of filtrate. The filter cassette is the element of the assembly that typically houses the membrane filter used for separation and purification of biomolecules. The filter membrane is used to purify the target protein of interest. In some embodiments, the filter membrane retains cells, cell debris, organelles, and other components while allowing proteins and smaller solutes to pass into the filtrate. In some embodiments, the filtration assembly comprises a membrane. In other embodiments, the membrane that can be used for the filtration assembly membrane is derived from polyvinylidene fluoride (PVDF), polysulfone, polyethersulfone, polyarylsulfone, regenerated cellulose, polyamide, polypropylene, polyethylene, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile, ethylene copolymer, polyamide (such as "Nylon 6" or Nylon 66"), polycarbonate, PFA, or any combination thereof.

[0112] The methods according to the present disclosure include the use of a wash buffer in a recovery wash to recover protein outside of the collection vessel that is retained in tubing, piping, or other "hold-up" areas during the recovery wash. Since not all of the purified protein reaches the collection vessel during the initial purification run, it is necessary to perform a recovery wash to recover the larger amount of protein product in the collection vessel or other vessel used to collect the purified protein product. In some cases, the purification and concentration of the protein to high levels can result in unwanted protein aggregation. It can be necessary to modify the pH, salt content, or other components of the buffer to prevent protein aggregation or other unwanted effects during the purification and concentration process. In some embodiments, the wash buffer comprises an organic or inorganic acid or a salt thereof. In some embodiments, the organic or inorganic acid is a citrate (e.g., a citric acid monosodium-dicitric acid sodium mixture, a citric acid-citric acid trisodium mixture, a citric acid-citric acid monosodium mixture, etc.), a succinate (e.g., a succinic acid-monosuccinic acid sodium mixture, a succinic acid-sodium hydroxide mixture, a succinic acid-disuccinic acid sodium mixture, etc.), a tartrate (e.g., a tartaric acid-sodium tartrate mixture, a tartaric acid-potassium tartrate mixture, a tartaric acid-sodium hydroxide mixture, etc.), a fumarate (e.g., a fumaric acid-monofumaric acid sodium mixture, a fumaric acid-difumaric acid sodium mixture, a monofumaric acid-difumaric acid sodium mixture, etc.), a gluconate (e.g., a gluconic acid-sodium gluconate mixture, a gluconic acid-sodium hydroxide mixture, a gluconic acid-potassium gluconate mixture, etc.), an oxalate (e.g., an oxalic acid-sodium oxalate mixture, an oxalic acid-sodium hydroxide mixture, an oxalic acid-potassium oxalate mixture, etc.), a lactate (e.g., a lactic acid-sodium lactate mixture, a lactic acid-sodium hydroxide mixture, a lactic acid-potassium lactate mixture, etc.) acetate (e.g., an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, etc.), a trisamine salt (e.g., Tris), a phosphate, or a histidine. In some embodiments, the wash buffer comprises histidine. In some embodiments, the wash buffer comprises a sugar. In some embodiments, the sugar is sucrose, trehalose, mannitol, xylitol, erythritol, lactose, glucose, sugar powder, or pullulan. In some embodiments, the wash buffer comprises 20 mM histidine and 250 mM sucrose.

[0113] According to the methods of the present disclosure, a filtration buffer is used while filtering during the process of passing a sample containing the target protein through the filtration assembly at a feed flow rate. The filtration buffer is necessary in protecting downstream chromatography and filtration equipment, and is used in the process of protein purification and concentration. The filtration buffer is different from the rinse buffer, which is used in the rinse recovery process after the process of protein purification and concentration. In some embodiments, the components in the filtration buffer are the same as the components in the rinse buffer. In other embodiments, the components in the filtration buffer are different from the components in the rinse buffer.

[0114] Protein purification and concentration to high levels can result in unwanted protein aggregation. It can be necessary to modify the pH, salt content, or other components of the buffer to prevent protein aggregation or other unwanted effects during the purification and concentration process. In some embodiments, the filtration buffer comprises an organic and inorganic acid or a salt thereof. In some embodiments, the organic or inorganic acid is citrate (e.g., a citric acid monosodium-dicodium citrate mixture, a citric acid-trisodium citrate mixture, a citric acid-monosodium citrate mixture, etc.), succinate (e.g., a succinic acid-monosodium succinate mixture, a succinic acid-sodium hydroxide mixture, a succinic acid-dicodium succinate mixture, etc.), tartrate (e.g., a tartaric acid-sodium tartrate mixture, a tartaric acid-potassium tartrate mixture, a tartaric acid-sodium hydroxide mixture, etc.), fumarate (e.g., a fumaric acid-monosodium fumarate mixture, a fumaric acid-dicodium fumarate mixture, a monosodium fumarate-dicodium fumarate mixture, etc.), gluconate (e.g., a gluconic acid-sodium gluconate mixture, a gluconic acid-sodium hydroxide mixture, a gluconic acid-potassium gluconate mixture, etc.), oxalate (e.g., an oxalic acid-sodium oxalate mixture, an oxalic acid-sodium hydroxide mixture, an oxalic acid-potassium oxalate mixture, etc.), lactate (e.g., a lactic acid-sodium lactate mixture, a lactic acid-sodium hydroxide mixture, a lactic acid-potassium lactate mixture, etc.) acetate (e.g., an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, etc.), trisamine (e.g., Tris), phosphate, or histidine.

[0115] In some embodiments, the filtration buffer comprises a sugar. In some embodiments, the sugar is sucrose, trehalose, mannitol, xylitol, erythritol, lactose, glucose, sugar powder, or pullulan. In some embodiments, the pH of the rinse buffer and / or the filtration buffer is about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, or about 9.5.

[0116] According to the methods of the present application, a sample containing a protein of interest is first passed through a filtration assembly, and then subjected to a recovery rinse. In some embodiments, the sample can have already been subjected to prior purification activities, such as viral filtration, anion exchange chromatography (AEX), cation exchange chromatography (CEX), depth filtration, acid treatment, or protein A chromatography, directly upstream of the filtration and recovery rinse steps. The sample subjected to purification and filtration can be derived from a source such as a cell culture, cell lysate, or clarified material (e.g., clarified cell culture supernatant). In some embodiments, the sample is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any combination thereof. In some embodiments, the cell culture sample is derived from a cell culture medium comprising mammalian cells. In some embodiments, the mammalian cells are selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof.

[0117] Target proteins that can be used in the methods of the present application are any protein, polypeptide, or peptide that is naturally occurring or recombinantly produced. In some embodiments, the target protein has a high viscosity. In some embodiments, the target protein is a protein having a high molecular weight (e.g., 100 kDa to 500 kDa). In other embodiments, the target protein is a protein having a low molecular weight. In other embodiments, the target protein has a molecular weight of at least about 10 kDa, at least about 20 kDa, at least about 30 kDa, at least about 40 kDa, at least about 50 kDa, at least about 60 kDa, at least about 70 kDa, at least about 80 kDa, at least about 90 kDa, at least about 100 kDa, at least about 110 kDa, at least about 120 kDa, at least about 130 kDa, at least about 140 kDa, at least about 150 kDa, at least about 160 kDa, at least about 170 kDa, at least about 180 kDa, at least about 190 kDa, or at least about 200 kDa and up to 400 kDa or 500 kDa.

[0118] In some embodiments, the target protein comprises an antibody or a fusion protein. In some embodiments, the target protein comprises an antibody. In some embodiments, the antibody is of an isotype selected from the group consisting of IgM, IgA, IgE, IgD, and IgG. In some embodiments, the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The methods of the present application can be applicable to any antibody that specifically binds to an antigen. In some embodiments, the antibody is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, an anti-CSF1R antibody, or an anti-IL8 antibody.

[0119] In some embodiments, the target protein comprises an enzyme, hormone, cytokine, cell surface receptor, protease, cytokine receptor, or any combination thereof. In some embodiments, the target protein is a fusion protein. In some embodiments, the fusion protein is fused with a heterologous portion. In some embodiments, the heterologous portion is a half-life extension portion. In some embodiments, the heterologous portion comprises albumin, an immunoglobulin constant region or a portion thereof, an immunoglobulin-binding polypeptide, immunoglobulin G (IgG), albumin-binding polypeptide (ABP), a PAS-modified portion, a HES-modified portion, XTEN, a PEGylated portion, an Fc region, or any combination thereof. In some embodiments, the half-life extension portion comprises a non-peptide portion. In some embodiments, the half-life extension portion comprises a polypeptide. In some embodiments, the half-life extension portion comprises an Fc region. In some embodiments, the fusion protein of this disclosure comprises an amino acid substitution (e.g., an Fc variant) in the immunoglobulin constant region or a portion thereof, said amino acid substitution altering the antigen-independent effector function of the Ig constant region, particularly the protein's circulating half-life.

[0120] Example

[0121] Example 1

[0122] Evaluation of scaled-down designs

[0123] To evaluate potential recycling and flushing processes, a scaled-down model was designed to analyze variations in pipe dimensions. This scaled-down model used 176cm... 2 The membrane area was determined, and tests were initially conducted using 16-gauge tubing. The various tubing types used in the modeling are described in Table 1. An initial protein concentration of 250 g / L was used, and various flushing feed flow rates were tested. This data is plotted in... Figure 1 A higher flushing feed rate results in a lower final protein concentration, and therefore a lower flushing flow rate would be ideal, such as... Figure 1 As shown.

[0124] Table 1. Scale-dependent pipe materials

[0125] Parameters No. 16 CM150 DCM Allegro Inner diameter (mm) 3.1 9.6 12.7 Inner diameter (m) 3.1 x 10 -3 ]] 9.6 x 10 -3 ]] 1.3 x 10 -2 ]]> Cross-sectional area (m 2 )]]> 7.55 x 10 -6 ]]> 7.24 x 10 -5 ]] 1.27x10 -4 ]]>

[0126] The effect of flow rate parameters on Reynolds number was explored using mixtures with fixed viscosity and density, and detailed in... Figure 2 The mixture has a viscosity of 17 cP and a viscosity of 1.08 g / cm³. 3 The density, and used 30 liters / m³ 2A fixed flow rate of 0.03 L / min was used for the CM150 system and a flow rate of 0.04 L / min was used for the DCM due to the cross-sectional differences of the various tubing used. The specific flow rate had to be adjusted for each tubing type.

[0127] The effect of viscosity on Reynolds number was also evaluated using a fixed flow rate of 0.019 m / s. These data are summarized in Table 2. For each tubing type, higher viscosity resulted in a higher calculated Reynolds number. Figure 3

[0128] Example 2

[0129] Evaluation of rinse strategies using multiple monoclonal antibodies

[0130] Three monoclonal antibodies (A, B, and C) were evaluated using the designed rinse process. The data collected from the rinse process for antibody A are detailed in Table 2. These data include the rinse flux (LMH), Reynolds number (Re), retentate concentration, purified drug substance concentration, yield, expected yield, and rinse volume. The process was also validated on a CM150 tangential flow filtration (TFF) scale for monoclonal antibody A after evaluating the scaled down rinse process. In all cases, the flow rate was set to 30 liters / M 2 hour flux. This corresponds to a Reynolds number of 265 in the CM150 system, taking into account the cross-section of the tubing used in the CM150 system. Additional scaled down data and CM150 TFF data were generated for monoclonal antibodies B and C and these data are presented in Tables 2 and 3, respectively.

[0131] Table 2. Data for monoclonal antibody A

[0132]

[0133] Table 3. 30 liter / M 2 hour recovery rinse for monoclonal antibody B

[0134]

[0135]

[0136] Table 3. 30 liter / M 2 hour recovery rinse for monoclonal antibody B

[0137]

[0138] ​It should be appreciated that the specific embodiments detailed in the DETAILED DESCRIPTION section, rather than the SUMMARY and ABSTRACT sections, are intended to explain the claims. The SUMMARY and ABSTRACT sections can set forth one or more, but not all, exemplary aspects of the present application as contemplated by the inventors, and thus, are not intended to limit the present application and the appended claims in any way.

[0139] The present application has been described above by means of functional building blocks which specify the implementation of the specified functions and their relationships. For the purpose of description, the boundaries of these functional building blocks have been defined arbitrarily. Alternate boundaries can be defined so long as the specified functions and their relationships are appropriately performed.

[0140] The foregoing description of specific aspects will so fully reveal the general nature of the application that others can modify and / or adapt for various applications such specific aspects without undue experimentation and without departing from the general concept of the application. Therefore, it is intended that the disclosure and examples set forth herein serve as the best mode for carrying out the application and that other aspects and modifications thereof be included within the scope of the disclosed aspects. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, and that the terms "comprising," "comprises" and / or "comprising" are not intended to exclude the possibility of any

[0141] The breadth and scope of the present application should not be limited by any of the above-described exemplary aspects, but should be defined in accordance with the following claims and their equivalents.

Claims

1. A method for reducing or preventing target protein dilution during a post-filtration recovery rinse, comprising (i) passing a sample containing the target protein through a filter assembly at a feed flow rate while performing the filtration, and (ii) passing a buffer solution through the filter assembly at a rinse flow rate of less than 100 liters / square meter / hour (LMH) during the recovery rinse. The Reynolds number Re flowing in the recovery flush is between 1 and 50, and Re is calculated using the following formula: and Where D is the channel diameter in meters, or the equivalent diameter in the case of a non-cylindrical channel geometry. The average velocity is expressed in m / s, and ρ is expressed in kg / m². 3 The density is expressed in units of ρ, and the viscosity is expressed in Pa·s.

2. A method for improving or increasing the concentration of a target protein during a post-filtration recovery rinse, comprising (i) passing a sample containing the target protein through a filter assembly at a feed flow rate while performing the filtration, and (ii) passing a rinse buffer through the filter assembly at a rinse flow rate of less than 100 liters / square meter / hour (LMH) during the recovery rinse. The Reynolds number Re flowing in the recovery flush is between 1 and 50, and Re is calculated using the following formula: and Where D is the channel diameter in meters, or the equivalent diameter in the case of a non-cylindrical channel geometry. The average velocity is expressed in m / s, and ρ is expressed in kg / m². 3 The density is expressed in units of ρ, and the viscosity is expressed in Pa·s.

3. The method according to claim 1 or 2, wherein the Re flowing in the recovery flush is 1 to 20.

4. The method according to claim 1 or 2, wherein the Re flowing in the recovery flush is 2 to 10.

5. The method according to claim 1 or 2, wherein the Re flowing in the recovery flush is 3.

8.

6. The method according to claim 1 or 2, wherein the flushing flow rate is 5 LMH to 100 LMH.

7. The method according to claim 1 or 2, wherein the flushing flow rate is 20 LMH to 40 LMH.

8. The method according to claim 1 or 2, wherein the flushing flow rate is 30 LMH.

9. The method according to claim 1 or 2, wherein the viscosity of the target protein is from 1 centipoise (cP) to 30 cP.

10. The method according to claim 1 or 2, wherein the viscosity of the target protein is from 20 centipoise (cP) to 100 cP.

11. The method according to claim 1 or 2, wherein the viscosity of the target protein is 2 cP to 5 cP.

12. The method according to claim 1 or 2, wherein the concentration of the target protein is from 100 g / L to 300 g / L before the filtration and after the recovery rinse.

13. The method of claim 12, wherein the concentration of the target protein after the recovery rinse is 170 g / L to 230 g / L.

14. The method of claim 12, wherein the concentration of the target protein after the recovery rinse is from 90 g / L to 300 g / L.

15. The method according to claim 1 or 2, wherein the yield of the target protein after the recovery rinse is 90% to 100% compared to the concentration of the target protein before the recovery rinse process consisting of (i) and (ii).

16. The method of claim 1 or 2, wherein the yield of the target protein is increased by 1% to 20% after the recovery wash compared to the yield of the target protein obtained after recovery washing at a flow rate of 300 LMH.

17. The method according to claim 1 or 2, wherein the feed flow rate in (i) is higher than the flushing flow rate in (ii).

18. The method of claim 17, wherein the feed flow rate is 200 LMH to 500 LMH.

19. The method of claim 17, wherein the feed flow rate is 250 LMH to 450 LMH.

20. The method of claim 17, wherein the feed flow rate is 300 LMH.

21. The method of claim 1 or 2, wherein the filtration assembly comprises a membrane derived from polyvinylidene fluoride (PVDF), polysulfone, polyethersulfone, polyarylsulfone, regenerated cellulose, polyamide, polypropylene, polyethylene, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile, ethylene copolymer, polyamide polycarbonate, PFA, or any combination thereof.

22. The method according to claim 1 or 2, wherein the rinsing buffer comprises organic and inorganic acids or salts thereof.

23. The method of claim 22, wherein the organic or inorganic acid is citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, trimethylamine salt, phosphate, or histidine.

24. The method of claim 22, wherein the rinsing buffer comprises histidine.

25. The method of claim 1 or 2, wherein the rinsing buffer comprises sugar.

26. The method of claim 25, wherein the sugar is sucrose, trehalose, mannitol, xylitol, erythritol, lactose, glucose, powdered sugar, or pullulan.

27. The method of claim 1 or 2, wherein the rinsing buffer comprises 20 mM histidine and 250 mM sucrose.

28. The method according to claim 1 or 2, wherein a filter buffer is used in process (i).

29. The method of claim 28, wherein the filtration buffer is the same as the rinsing buffer.

30. The method of claim 28, wherein the filtration buffer is different from the rinsing buffer.

31. The method of claim 28, wherein the filtration buffer comprises organic and inorganic acids or salts thereof.

32. The method according to claim 31, wherein the organic or inorganic acid is citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, trimethylamine salt, phosphate, or histidine.

33. The method of claim 28, wherein the filtration buffer comprises sugar.

34. The method according to claim 33, wherein the sugar is sucrose, trehalose, mannitol, xylitol, erythritol, lactose, glucose, powdered sugar, or pullulan.

35. The method according to claim 1 or 2, wherein the pH of the rinsing buffer and / or the filtration buffer is 4 to 9.

5.

36. The method according to claim 1 or 2, wherein the sample is selected from pure protein samples, clear bulk protein samples, cell culture samples, and any combination thereof.

37. The method of claim 36, wherein the cell culture sample is derived from a cell culture medium containing mammalian cells.

38. The method according to claim 37, wherein the mammalian cells are selected from Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NSO), young hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), Marshall's bovine kidney cells (MDBK), or any combination thereof.

39. The method of claim 1 or 2, wherein the target protein comprises an antibody or a fusion protein.

40. The method of claim 39, wherein the target protein comprises an antibody.

41. The method of claim 40, wherein the antibody is an isotype selected from IgM, IgA, IgE, IgD and IgG.

42. The method of claim 41, wherein the IgG antibody is selected from IgG1, IgG2, IgG3 and IgG4.

43. The method according to any one of claims 40 to 42, wherein the antibody is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, an anti-CSF1R antibody, or an anti-IL8 antibody.

44. The method according to claim 1 or 2, wherein the target protein comprises an enzyme, a hormone, a cytokine, a cell surface receptor, a cytokine receptor, or any combination thereof.

45. The method of claim 44, wherein the enzyme is a protease.

46. ​​The method of claim 39, wherein the target protein is a fusion protein.

47. The method of claim 44, wherein the target protein is a fusion protein.

48. The method of claim 46 or 47, wherein the fusion protein is fused with a heterologous portion.

49. The method of claim 48, wherein the heterogeneous portion is a half-life extension portion.

50. The method of claim 49, wherein the half-life extension portion comprises Fc.

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