Method for recovering product from perfusion culture

Abstract

The present disclosure provides methods for purifying target proteins (e.g., antibodies) from host cells, such as mammalian cells, using a purification protocol that incorporates a harvest recovery operation that includes a continuous solids-discharging disk stack centrifugation step followed by flocculation and depth filtration steps. The protein recovery methods of the present disclosure recover high yields of purified target protein from perfusion cultures with packed cell volumes of 16% or greater using a non-conventional but effective process.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of biomolecule processing, and more particularly to the field of recovering biomolecules from perfusion cultures. [Background technology]

[0002] Modern medicine faces many challenges in its efforts to address the wide variety of diseases and conditions that afflict humans and other animal species. The development of new and better treatments has expanded beyond efforts to develop new and better pharmaceuticals, including macromolecular biological compounds or biologics, such as monoclonal antibodies (mAbs), antibody fragments, antibody-like modalities, multispecific antibodies, engineered proteins, and biosimilars. In recent years, these efforts have yielded results at a steady pace, resulting in an ever-growing group of therapeutically active biologics that are approved for human use and fulfill unmet needs in the medical community. A natural companion to the effort to develop novel therapeutics is the effort to improve manufacturing processes to maintain product quality, reduce production costs, increase efficiency and sustainability, and improve the yield of intact, functional therapeutic biologics while streamlining manufacturing at pilot- and commercial-scale manufacturing levels.

[0003] The manufacture of therapeutic biologics is a multistep process involving production, recovery, and purification / polishing operations. Biologics are typically produced by expression from host cells in cell culture (typically mammalian cell culture for expression of a desired recombinant protein from the host cells). Cell culture is followed by a harvest or recovery operation, in which the culture medium containing the recombinant protein is removed from the bioreactor and an initial clarification is performed to prepare for the separation and purification of the target therapeutic protein from other components of the cell culture broth. Such components include intact cells, cellular debris, process- and product-related impurities, e.g., nucleic acids such as DNA, proteins such as non-target host cell proteins, as well as high- and low-molecular-weight variants of the desired therapeutic protein, lipids, carbohydrates, viral particles, etc.

[0004] As cell culture practices improve and pack cell volumes, cell densities, titers, and process- and product-related impurities increase, pressure is placed on downstream processes to effectively separate and purify target proteins. This is particularly true for harvest operations of perfusion cultures (e.g., continuous perfusion cultures), which can be overwhelmed by increased pack cell volumes, rising impurity levels, and increased titers. Summary of the Invention [Problem to be solved by the invention]

[0005] For the foregoing reasons, there remains a need in the art for a biomolecule (e.g., biologic) purification process that includes a harvest recovery step that efficiently and cost-effectively provides high-yield separation of biologics from other cell culture components resulting from turbid perfusion cultures that have a packed cell volume (PCV) of at least 16% at harvest. [Means for solving the problem]

[0006] The present disclosure provides a method for separating a recombinant protein produced in a perfusion culture from at least one other perfusion culture component, the method comprising: (a) recovering a pool or eluate stream from a perfusion culture comprising the recombinant protein and at least one other perfusion culture component, wherein the perfusion culture has a packed cell volume (PCV) at harvest of at least about 16%; (b) introducing the recovered pool or eluate stream into at least one continuous solids discharge disc stack centrifuge; (c) operating the continuous solids discharge disc stack centrifuge, thereby separating the liquid component into a centrate and a high-density composition; (d) collecting the centrate; (e) adding a flocculant to the centrate at a temperature between 8°C and 12°C; and (f) subjecting the centrate to a filtration step.

[0007] In some embodiments, the perfusion culture of any of the disclosed methods is harvested from a single-use bioreactor, for example, a single-use 500 L bioreactor, or a single-use bioreactor that is 2,000 L or larger.

[0008] In some embodiments, the perfusion culture harvest pool or eluate stream of any of the disclosed methods had a turbidity of at least 180 NTU at harvest. In some embodiments, the perfusion culture harvest pool or eluate stream of any of the disclosed methods had a viable cell density of at least 2 x 10 at harvest. 7 viable cells / ml or a viable cell density of at least 3 x 10 at the time of harvest 7 viable cells / ml or a viable cell density of at least 5 x 10 at the time of harvest 7 viable cells / ml.

[0009] In some embodiments, the perfusion cultures of any of the disclosed methods had a PCV at harvest of at least about 20%, or a PCV at harvest of at least about 24%, or a PCV at harvest of at least about 26%, or a PCV at harvest of at least about 30%.

[0010] In some embodiments, the temperature of the perfusion culture at the time of harvest in any of the methods of the disclosure is about 8 to about 12° C. For example, the temperature of the perfusion culture at the time of harvest in any of the methods of the disclosure is about 10° C.

[0011] In some embodiments, the continuous harvest eluate stream from the perfusion culture of any of the methods of the present disclosure is introduced into a continuous solids discharge disk stack centrifuge.

[0012] In other embodiments, the perfusion harvest pool of any of the discontinuous batches of the disclosed methods is introduced into a continuous solids discharge disk stack centrifuge.

[0013] In some embodiments, the turbidity of the centrate of any of the disclosed methods is greater than about 160 NTU, e.g., greater than about 180 NTU, or greater than about 200 NTU, or greater than about 250 NTU, or greater than about 300 NTU, or greater than about 500 NTU, or greater than about 600 NTU, or greater than about 750 NTU.

[0014] In some embodiments, a flocculant is added to the centrate of any of the disclosed methods at a temperature of about 10° C. In exemplary embodiments, the flocculant is poly(diallyldimethylammonium chloride) (pDADMAC) or its monomer diallyldimethylammonium chloride (DADMAC), and pDADMAC or DADMAC is added to at least 0.04% (w / w), or to 0.04-0.15% (w / w), or to 0.05% (w / w).

[0015] In any of the disclosed methods, the recombinant protein is a eukaryotic protein, such as a mammalian protein. In some embodiments, the mammalian protein is an antigen-binding protein.

[0016] In any of the methods of the disclosure, the recombinant protein is a mammalian antigen-binding protein, which is a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a bispecific T cell engager molecule (BiTE®).

[0017] In other embodiments, the protein of any of the methods of the present disclosure is a granulocyte colony stimulating factor, an erythropoiesis stimulating agent, a HER receptor, a cell adhesion molecule, a growth factor, an osteoinductive factor, insulin, a coagulation protein, a colony stimulating factor, a blood group antigen; a growth hormone, a growth hormone receptor, a T cell receptor; a neurotrophic factor, a neurotrophin, a relaxin, an interferon, an interleukin, a viral antigen, a lipoprotein, an integrin, a rheumatoid factor, an immunotoxin, a surface membrane protein, a transport protein, a homing receptor, an addressin, a regulatory protein, or an immunoadhesin.

[0018] In some embodiments, the filtration step in any of the disclosed methods comprises depth filtration. Exemplary depth filters that may be used in any of the disclosed methods include a MILLISTAK+® COHC filter, a MILLISTAK+® COSP filter, a SARTOCLEAR® DL60 filter, or a SARTOCLEAR® DL75 filter. In one embodiment, the depth filter is a MILLISTAK+® COHC filter or a MILLISTAK+® COSP filter, and the centrate is passed through the depth filter at a flux rate of 150 LMH or less or a pressure of 10 psi or less. In some embodiments, the pressure is 2 psi or less. In some embodiments, the flux rate is between 90 and 150 LMH.

[0019] The present disclosure also provides any of the methods of the present disclosure, further comprising at least one additional chromatography step. Exemplary additional chromatography steps include ion exchange chromatography, hydrophobic interaction chromatography, or multimodal chromatography.

[0020] Additionally, the present disclosure provides any of the methods of the present disclosure, further comprising at least one or more viral filtration steps, viral inactivation steps, and / or UFDF steps.

[0021] The present disclosure provides a target protein produced by any of the methods disclosed herein.

[0022] In another embodiment, the present disclosure provides a method for maintaining a low differential pressure during depth filtration of a load feed from a perfusion culture, the method comprising the steps of obtaining a flocculated centrate from the perfusion culture having a packed cell volume (PCV) at harvest of at least 16%; passing the centrate through a depth filter at a flux rate of 150 LMH or less and a differential filter pressure of 10 psi or less; and recovering an eluate.

[0023] The present disclosure also provides a method for producing isolated and purified recombinant protein from a perfusion culture, comprising: (a) initiating a perfusion culture in a disposable bioreactor; (b.) inoculating the bioreactor with cells engineered to recombinantly express a protein of interest; (c.) culturing the cells until the perfusion culture has a packed cell volume of at least 16%; (d.) reducing the temperature of the culture to between 8°C and 12°C and collecting the perfusion culture from the bioreactor as a harvest pool or eluate stream; and (e.) introducing the harvest pool or eluate stream into at least one continuous solids discharge disk stack centrifuge. (f.) collecting the centrate from the centrifuge; (g.) adding a flocculating agent to the centrate at a temperature of 8°C to 12°C; (h.) passing the flocculated centrate through a depth filter at a flux rate of 90 to 150 LMH and a pressure of 10 psi or less, wherein the flocculated centrate is at a temperature of 4°C to 12°C; (i.) subjecting the filtered centrate to one or more chromatography, filtration, and / or UFDF unit operations; and (j.) obtaining an isolated and purified recombinant protein.

[0024] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated and purified recombinant protein produced by any of the methods disclosed herein. [Brief explanation of the drawings]

[0025] [Figure 1]Effect of flocculation on small-scale normalized depth filter loading levels. Figure 1 shows normalized small-scale depth filtration performance using untreated and flocculated effluent from a large-scale, disposable, continuous solids-discharging disk stack centrifuge. In all conditions tested except one, flocculation of the centrate increased the normalized depth filter throughput compared to the control. Normalized throughput was calculated by dividing the small-scale depth filtration final throughput (L / m²) of the pDADMAC-treated (dark gray) and untreated (light gray) conditions by the depth filtration final throughput (L / m²) of the control (black) condition. The control (black) condition was operated using untreated effluent combined with an X0HC depth filter. The pDADMAC condition was operated using 0.05% (w / w; weight pDADMAC / weight centrate) pDADMAC-treated centrate combined with a COHC, COSP, X0HC, X0SP, DL60, or DL75 depth filter. Untreated conditions were run using untreated centrate with C0SP, C0HC, or X0SP depth filters. The following runs were terminated early during small-scale depth filtration due to time limitations: xmAb run 1, xmAb run 2, and bispecific 2. Higher throughputs would have been achieved for these runs if the run time had been extended. Error bars represent ±1 standard deviation from the mean. Bsp = bispecific. [Figure 2]Effect of aggregation and small-scale depth filtration on percent yield. Figure 2 shows the yields of the small-scale aggregation and depth filtration steps of the experiment shown in Figure 1. Aggregation, followed by depth filtration conditions (dark gray) had similar or improved process yields when compared to the control condition (black) or untreated condition (light gray). Percent yield was calculated by dividing the measured total protein mass (g) in the depth filtrate pool by the measured total protein mass (g) in the centrate pool. pDADMAC conditions were operated using 0.05% w / w pDADMAC-treated centrate in combination with COHC, COSP, XOCC, XOSP, DL60, or DL75 depth filters. Untreated conditions were operated using untreated centrate with COSP, COHC, or XOSP depth filters. Error bars represent ±1 standard deviation from the mean. Bsp = bispecific. [Figure 3] Effect of small-scale aggregated host cell protein (HCP) levels. Figure 3 shows the small-scale aggregation and depth filtration HCP log reduction values ​​(LRV) for the experiment shown in Figure 1. The aggregation followed by depth filtration condition (dark gray) demonstrated similar HCP clearance as the control condition (black) and untreated condition (light gray) after accounting for HCP assay variability. HCP LRV was calculated by applying a base-10 logarithmic transformation to the ratio of measured HCP levels in the centrate to those in the depth filtrate pool. The control condition (black) was operated using untreated in combination with an X0HC depth filter. The pDADMAC condition was operated using 0.05% (w / w) pDADMAC-treated centrate in combination with a COHC, COSP, X0HC, X0SP, DL60, or DL75 depth filter. The untreated condition was operated using untreated centrate with a COSP, COHC, or X0SP depth filter. Bsp=bispecificity. [Figure 4]Effect of small-scale aggregation and depth filtration DNA levels. Figure 4 shows the small-scale aggregation and depth filtration DNA LRVs for the experiment shown in Figure 1. Aggregation followed by depth filtration conditions (dark gray) showed similar or improved DNA clearance compared to the control (black) and untreated conditions (light gray). DNA LRV was calculated by applying a base-10 logarithmic transformation to the ratio of measured DNA levels in the centrate to the depth filtrate pool. The control condition (black) was run using untreated in combination with an X0HC depth filter. The pDADMAC condition was run using 0.05% (w / w) pDADMAC-treated centrate in combination with a COHC, COSP, X0HC, X0SP, DL60, or DL75 depth filter. The untreated condition was run using untreated centrate with a COSP, COHC, or X0SP depth filter. Bsp = dual specificity. [Figure 5A] Effect of pDADMAC on centrate turbidity. Figure 5 shows the effect of centrifugation operating conditions and pDADMAC concentration on supernatant turbidity. Supernatant turbidity values ​​were measured after incubation with pDADMAC and laboratory-scale centrifugation. Variation in centrifuge feed flow rate and bowl speed was used to generate variation in initial centrate turbidity. pDADMAc concentrations above 0.04 (w / w%) reduced centrate turbidity for the two model molecules tested (i.e., mAb1 Run 2 (A) and xmAb Run 1 (B)). [Figure 5B] Effect of pDADMAC on centrate turbidity. Figure 5 shows the effect of centrifugation operating conditions and pDADMAC concentration on supernatant turbidity. Supernatant turbidity values ​​were measured after incubation with pDADMAC and laboratory-scale centrifugation. Variation in centrifuge feed flow rate and bowl speed was used to generate variation in initial centrate turbidity. pDADMAc concentrations above 0.04 (w / w%) reduced centrate turbidity for the two model molecules tested (i.e., mAb1 Run 2 (A) and xmAb Run 1 (B)). [Figure 6A]Effect of pDADMAC concentration on small-scale depth filtration performance. Figure 6 shows the effect of pDADMAC concentration on small-scale normalized depth filtration throughput using large-scale centrate from (A) mAb1 (Run 1) and (B) mAb2. Compared to the control (black), increased depth filtration throughput was observed using 0.05 w / w% to 0.1 w / w% pDADMAC for mAb1 and 0.035 w / w% to 0.1 w / w% pDADMAC for mAb2. Untreated was combined with an X0HC depth filter. pDADMAC conditions were operated using 0.02 w / w%, 0.035 w / w%, 0.05 w / w%, or 0.1 w / w% pDADMAC-treated centrate combined with a COHC depth filter. Normalized throughput was calculated by dividing the small-scale depth filtration final throughput (L / m2) of the pDADMAC-treated condition by the small-scale depth filtration final throughput (L / m2) of the control untreated condition. [Figure 6B] Effect of pDADMAC concentration on small-scale depth filtration performance. Figure 6 shows the effect of pDADMAC concentration on small-scale normalized depth filtration throughput using large-scale centrate from (A) mAb1 (Run 1) and (B) mAb2. Compared to the control (black), increased depth filtration throughput was observed using 0.05 w / w% to 0.1 w / w% pDADMAC for mAb1 and 0.035 w / w% to 0.1 w / w% pDADMAC for mAb2. Untreated was combined with an X0HC depth filter. pDADMAC conditions were operated using 0.02 w / w%, 0.035 w / w%, 0.05 w / w%, or 0.1 w / w% pDADMAC-treated centrate combined with a COHC depth filter. Normalized throughput was calculated by dividing the small-scale depth filtration final throughput (L / m2) of the pDADMAC-treated condition by the small-scale depth filtration final throughput (L / m2) of the control untreated condition. [Figure 7]Effect of pDADMAC and PEG3000 treatment on depth filtration performance. Figure 7 shows a comparison of small-scale depth filtration performance between pDADMAC and pDADMAC + PEG3000 treatment conditions using a representative large-scale centrate from mAb1 Run 3. The large-scale run of mAb1 Run 3 was performed using a stainless steel continuous discharge centrifuge. The initial untreated centrate turbidity values ​​were similar to those observed using a disposable continuous solids discharge centrifuge and were considered representative of depth filtration testing. An increase in normalized depth filtration throughput was observed for both pDADMAC and pDADMAC + PEG3000-treated centrates compared to the control. The untreated material was combined with an X0HC depth filter. pDADMAC at 0.05 w / w% was combined with a COHC depth filter. pDADMAc (0.05 w / w%) + PEG3000 was combined with COHC and COSP depth filters. Normalized throughput was calculated by dividing the small-scale depth filtration final throughput (L / m2) for the pDADMAC and / or pDADMAC+PEG-treated conditions by the small-scale depth filtration final throughput (L / m2) for the control untreated condition. For the large-scale run of mAb1 run 3, small-scale depth filtration was performed the day after the end of the process (day 19). Normalized throughput was calculated by dividing the small-scale depth filtration final throughput (L / m2) for the pDADMAC-treated condition by the small-scale depth filtration final throughput (L / m2) for the control untreated condition. [Figure 8]Effect of flocculation on the sequential pressure trend of depth filtration. Figure 8 shows the differential pressure profile of a large-scale depth filtration train performed using 0.05 w / w% pDADMAC and a COHC filter (dark gray) compared to a control small-scale depth filtration experiment performed with untreated centrate and an XHC filter (black). Representative large-scale runs used were: mAb1, Run 1; xmAb1, Run 1; xmAb1, Run 2; and Bispecific 2, Run 1. The use of flocculation and COHC depth filtration resulted in a low and stable differential pressure profile when compared to the XHC control, where a rapid increase in differential pressure was observed. Delta A pressure represents the differential pressure across the first-stage depth filter. Delta B pressure represents the differential pressure across the second-stage bioburden reduction filter. Note that the data plotted with the permeability trend on the y-axis showed no difference compared to the absolute pressure trend in this figure. [Figure 9] Comparison of product quality between pDADMAC-treated and untreated material. Figure 9 shows a comparison of product quality between pDADMAC-treated and untreated material from xmAb1 run 1. The aggregation followed by depth filtration conditions (light gray) showed similar product quality to the control conditions (black) and the pDADMAC-treated centrate (dark gray). Normalized product quality values ​​were calculated by dividing the product quality values ​​for the pDADMAC-treated centrate conditions (dark gray) and the pDADMAC-treated depth filtrate pool conditions (light gray) by the product quality value for the untreated control condition (black). Abbreviations: SE = size exclusion chromatography; CEX = cation exchange chromatography; rCE = reduced capillary electrophoresis with sodium dodecyl sulfate; nrCE = nonreduced capillary electrophoresis; Main = median molecular weight range; Main peak = main or median peak obtained from chromatographic fractionation; LMW = low molecular weight; MMW = medium molecular weight. DETAILED DESCRIPTION OF THE INVENTION

[0026] "Cell density" refers to the number of cells in a given volume of culture medium. "Viable cell density" (VCD) refers to the number of viable cells in a given volume of culture medium as measured by a standard viability assay (such as trypan blue exclusion).

[0027] As used herein, "packed cell volume" (PCV), also referred to as "packed cell volume fraction" (PCV%), is the ratio of the volume occupied by cells to the total volume of the cell culture, expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). Changes in packed cell volume can result from changes in cell diameter. Packed cell volume is a measure of the solids content in a cell culture. Solids are removed during harvesting. More solids mean more effort to separate the solids from the desired product during harvesting. Also, the desired product may become entrapped in the solids and be lost during the harvesting process, resulting in a lower product yield. Packed cell volume is a more accurate way to represent the solids content in a cell culture than cell density or viable cell density because host cells vary in size and cell cultures also contain dead and dying cells and other cell debris. In addition, under growth arrest, some cells increase in size, and the packed cell volume before and after growth arrest may differ due to the increase in biomass as a result of the increase in cell size.

[0028] "Centrate," as used herein, refers to the low-density composition that is formed or is forming when cell culture broth is subjected to a continuous solids discharge disk stack centrifuge (e.g., a disposable continuous solids discharge disk stack centrifuge). In operation, the centrifuge fractionates the cell culture broth into a low-density composition and a high-density composition that includes at least a portion of the solids originally found in the cell culture broth.

[0029] A "target protein" is any naturally occurring, synthetically produced, or recombinantly expressed protein, peptide, or fragment thereof that is recovered according to the methods of the present disclosure.

[0030] "Low density composition" or "light phase" is a synonym for "centrate" which refers to the liquid exiting a disc stack centrifuge after removal of at least some solids from the input fluid.

[0031] "Dense composition" or "heavy phase" refers to material enriched in particulate matter such as cells and cell debris that results from a continuous solids discharge disk stack centrifuge.

[0032] A "bioreactor" is any vessel suitable for culturing cells capable of expressing a target protein. Bioreactors can range in size from laboratory to commercial scale and can have a variety of enhancements, including pump-controlled inlets and / or outlets, thermometers, CO and / or O gauges, means for agitating the culture, etc. A disposable bioreactor is a bioreactor that has a disposable bag instead of a culture vessel.

[0033] "Depth filtration" refers to the filtration of a particulate-laden liquid through a medium of sufficient depth to effectively separate particles of various sizes from the liquid.

[0034] "Tangential flow filtration" refers to the filtration of a particulate-containing liquid in which the liquid is directed tangentially across the surface of a filtration medium, e.g., by gravity-induced transmembrane pressure, such that a portion of the liquid enters the filtration medium and is thereby filtered.

[0035] "Q / Sigma" is the ratio of the centrifuge feed flow rate to the theoretically equivalent settling area required to achieve the same degree of solid-liquid separation in the settling pan. In the case of a disc stack centrifuge, it is calculated as follows:

number

[0036] "Load factor" is the ratio of centrifugal feed flow rate to separation area (KQ) based on experimental results. KQ is calculated as follows:

number

[0037] "Turbidity" is given its ordinary and accustomed meaning of the degree to which a liquid lacks transparency. As used herein, turbidity is measured in conventional nephelometric turbidity units (NTU) unless otherwise specified.

[0038] Samples from perfusion cultures with PCVs of at least 16% at harvest were found to have higher turbidity levels. While turbidity decreases during harvest, the greater the turbidity at harvest, the more effort required to separate the turbid material from the desired product. Increased turbidity in perfusion cultures increases the risk of harvest equipment failure and / or poor performance, resulting in reduced product yield. Turbid perfusion cultures with PCVs of at least 16% present a challenge for conventional harvest operations. Turbidity is not completely reduced by centrifugation; turbidity values ​​as high as 1500 NTU were detected in light-phase centrate samples collected from disposable, continuous solids-discharging disc-stack centrifuges of perfusion cultures with PCVs of at least 16% at harvest.

[0039] "Unit operation" refers to a functional step performed as part of a process to purify a recombinant protein of interest. For example, a unit operation can include, but is not limited to, steps in an operation such as recovery, capture, purification, polishing, viral inactivation, viral filtration, concentration, and / or formulation of a recombinant protein of interest. A unit operation can be designed to achieve a single objective or multiple objectives, such as a combination of capture and viral inactivation steps. A unit operation can also include holding or storage steps between processing steps.

[0040] "Perfusion culture" refers to a cell culture method in which fresh medium is supplied to a bioreactor throughout the course of the cell culture and the culture broth is passed through a cell retention device in the bioreactor that selectively retains certain cell culture components (e.g., cells and, optionally, recombinant proteins), while removing spent medium, by-products, impurities, and, optionally, recombinant proteins in the permeate.

[0041] Fed-batch culture is the workhorse for producing therapeutic proteins. The fed-batch strategy is versatile and flexible and can be used with any bioreactor volume. In a fed-batch process, the culture is initiated at a low cell density in a portion of the bioreactor's total working volume (typically 50-75% volume) and is replenished with fresh nutrient medium throughout the culture, up to 100% of the bioreactor's total working volume. The feed medium replenishes nutrients throughout the cell culture to extend cell life and improve productivity. Spent medium and waste products are not removed from the bioreactor. Cells, protein, spent medium, waste products, etc. are all retained in the bioreactor and accumulate over time. Therefore, fed-batch culture is limited by the volume of the bioreactor; therefore, larger bioreactors (e.g., production bioreactors) operating at tens of thousands of liters are preferred for increased production. Over time, fed-batch cultures will decline due to the accumulation of spent medium, waste products, etc., which negatively impact cell mass, productivity, and titer. Harvesting is typically initiated when productivity drops, typically when the packed cell volume (PCV) is 10% or less at harvest.

[0042] In recent years, the use of perfusion has gained popularity in mammalian cell culture processes. Unlike fed-batch culture, perfusion culture is initiated at a high cell density at or near the full working volume of the bioreactor. Like fed-batch culture, perfusion culture systems supply fresh medium to the bioreactor throughout the course of the cell culture. Unlike fed-batch culture, perfusion culture systems utilize pumps to direct the contents of the bioreactor through a retention device (e.g., an ultrafilter, a microfilter, or a hollow fiber filter) that selectively retains or returns certain cell culture components (cells and, optionally, recombinant proteins) to the bioreactor. As a result, perfusion cultures can be maintained for longer periods than fed-batch cultures, for example, for 15 days or more. Throughout the course of perfusion culture, cell mass accumulation is higher, resulting in a higher number of cells at harvest, larger cell size, increased cell debris, by-products, and impurities compared to fed-batch culture. Spent medium (or nutrient-depleted medium), waste products, by-products, product- and process-related impurities, recombinant proteins, etc. are removed as waste in the filter permeate or collected for recovery. The perfusion culture is harvested through a retention device, collected from the permeate, or bulk harvested directly from the reactor. Due to the limitations of these retention devices, perfusion processes are performed in small-volume bioreactors (typically 500 L to 2,000 L). Such bioreactors include single-use bioreactors.

[0043] Cell culture broth from perfusion cultures with a PCV of at least 16% at harvest contains high concentrations of cell debris, product- and process-related impurities, and by-products, which affect protein filter passage, strain the capacity of the pumping system, reduce harvest yields, and increase the levels of impurities and by-products in the harvest pool. In contrast to the 10% PCV values ​​often seen in fed-batch cultures, PCV levels of 30% or more are not uncommon in perfusion cultures. This high solids content increases the burden on downstream harvest, purification, and polishing operations. Filters are prone to blockage and clogging as the high-solids perfusion culture passes through, resulting in the need to replace filters during harvest, which is time-consuming, reduces efficiency, reduces yield, and increases harvest costs. Pump capacity can also be limited by solution viscosity and high PCV, which further affect the cost / efficiency of harvest. The connections between the perfusion system and the retention device (tubing length, diameter, etc.) can complicate matters and increase the density of the culture broth entering the filter, resulting in cross-flow obstructions, reducing productivity in the bioreactor, and negatively affecting cell culture broth harvest. The harvest capacity of perfusion cultures with PCVs greater than 16% at harvest typically reaches only about 70%, leaving a significant amount of product behind.

[0044] The increase in solids content in continuous perfusion cultures and the shortcomings of current retention systems used in perfusion cultures highlight the need for improvements in the perfusion culture harvest process.

[0045] Harvest recovery processes for fed-batch cultures have traditionally relied on centrifugation, filtration, or a combination of these techniques to separate target proteins from culture particles, including cells, cell debris, and, to a lesser extent, process- and product-related impurities such as host cell proteins (HCPs) and nucleic acids such as DNA. Intermittent solids discharge disk stack centrifugation is well suited to the clarification of these low-solids fed-batch cultures and is widely used for fed-batch culture harvests. However, while intermittent solids discharge disk stack centrifugation is suitable for the clarification of low-solids cell culture broths, it has limitations in its solids transport capacity. Furthermore, as cell culture solids content increases, the limitations of intermittent solids discharge disk stack centrifugation become apparent as the centrifuge bowl fills more quickly with cells and debris, thus necessitating more frequent solids discharge. Frequent solids discharge reduces product recovery and increases centrate turbidity, thereby making downstream filtration more difficult. Due to its limited solids transport capacity, intermittent solids discharge disk stack centrifugation is not a realistic or viable option for high-PCV perfusion cultures. Therefore, intermittent solids discharge disk stack centrifugation is often combined with another separation technique, such as filtration, or resizing of impurity particles, to accommodate the limitations of disk stack centrifugation, for example, by generating larger particles by flocculation or sedimentation of the cell culture broth prior to centrifugation.

[0046] The aggregates have a larger average particle size than the natural product, resulting in more efficient clarification of the fed-batch culture broth in subsequent disc stack centrifugation operations, which improves the performance of the fed-batch culture broth for separation by disc stack centrifugation and also promotes more efficient clarification by depth filtration after such centrifugation due to reduced levels of small particles that would otherwise contribute to clogging of the filter pores.

[0047] Filtration (particularly depth filtration) has been used both as a primary recovery separation technique and as a partner to centrifugation, such as intermittent solids-discharging disk stack centrifugation, to separate target biologics from fed-batch culture broth. Depth filters have been established as one of the primary technologies routinely used in fed-batch culture recovery processes. Depth filters utilize various porous filtration media (e.g., diatomaceous earth, cellulose, and / or synthetic fibers) to create a three-dimensional network of pores that collectively establish a tortuous path through which materials must pass to pass through the filter. Larger particulate matter (e.g., cells and cell debris) from the cell culture broth can rapidly plug the filter by clogging its pores or surface.

[0048] In the case of perfusion culture, the primary means of recovery is the employment of tangential flow microfiltration, in which the direction of cell culture broth flow is tangential to the direction of migration through the filter. During perfusion culture, as the cell culture broth passes through the filter, larger particles (e.g., cells and, optionally, recombinant proteins) migrate across the surface of the filter and are returned to the bioreactor rather than settling and clogging the filter pores. Further improvements have been achieved through the use of alternating tangential flow microfiltration, in which the direction of cell culture broth across the membrane surface is periodically reversed, thereby creating a flushing action and improving filter performance. The motivation for developing alternating tangential flow microfiltration emphasizes the fact that particulate matter in the perfusion culture broth tends to easily clog filters, yet the use of alternating tangential flow microfiltration for perfusion cultures with PCVs of at least 16% at harvest has been an ineffective solution to the filter clogging problem, especially when used for such perfusion cultures. The increasing solids content of perfusion cultures has led to the search for more effective recovery methods. There has been a focus on improving filter technology to increase the surface area of ​​the filter to accommodate increased solids loads. The results of this effort have not yet provided a solution for such cultures, and may not be able to solve the problem without increasing material and labor costs and impacting efficiency and productivity.

[0049] The use of perfusion culture as a component of commercial-scale protein manufacturing allows for reduced equipment size and facility footprint, reduced material and environmental impact, incorporation of disposable components, and efficient, flexible production at all scales and with many therapeutic modalities. This provides a strong incentive to develop more effective recovery processes capable of handling these higher-solids perfusion cultures. The disclosure herein relates to the recovery of recombinant proteins produced by perfusion cultures with PCVs of at least 16% at harvest using a process previously considered inapplicable or impractical for perfusion cultures (i.e., centrifugation), specifically the use of a continuous solids-discharge disk-stack centrifuge. As described herein, centrifugation of perfusion cultures with PCVs of at least 16% results in acceptable clearance of cellular material, but centrate turbidity can be as high or higher than the harvested material and must be reduced before the centrate can be used with depth filtration.

[0050] Continuous perfusion cultures can achieve PCVs of 16% to 45% or more at low culture volumes (up to 2000 L), compared with fed-batch cultures, which have PCVs of less than 10% at cell culture volumes more than 10 times larger than perfusion cultures. This makes these high-solids perfusion cultures less suitable for the filter harvest methods typically used for most perfusion culture harvests. While the PCVs of these continuous perfusion cultures are also not comparable to fed-batch cultures, which have PCVs of less than 10%, the higher culture volumes and higher yields make them more suitable for larger-scale intermittent or continuous solids-discharge disk stack centrifugation and other centrifuge harvest methods. For commercial-scale perfusion cultures, harvest methods such as intermittent solids-discharge centrifugation are not an option because, at higher PCVs, the centrifuge bowl must be constantly opened to discharge solids, resulting in relatively ineffective separation of the centrate from debris. Additionally, conventional stainless steel intermittent or continuous discharge centrifuges are not suitable for use in modern single-use manufacturing facilities (e.g., open floor plan designs with minimal utility) because the equipment does not have a closed process design and requires clean-in-place and steam-in-place for product exchange.

[0051] The introduction of continuous solids discharge disk stack centrifugation technology into the process is revolutionary in that the state of the art held the view that continuous solids discharge disk stack centrifugation cannot distinguish between the density of target proteins in a cell culture broth and the density of biological particulates, such as cells, cell debris, host cell proteins (HCPs), or nucleic acids (e.g., DNA), in that liquid. An exemplary continuous solids discharge disk stack centrifuge system is the disposable continuous solids discharge disk stack centrifuge, which is expected to be too costly for long-term use. An intermittent solids discharge disk stack centrifuge is a disk stack centrifuge that continuously produces cell paste, accumulates the cell paste in the centrifuge bowl, and periodically or intermittently discharges the cell paste from the centrifuge. The solids discharge interval is based on the PCV of the cell culture, the cell culture harvest flow rate, and the centrifuge bowl solids retention space. This type of centrifuge is typically used for clarification of lower-density, lower-PCV cell cultures. Continuous solids-discharge disk stack centrifugation continuously generates cell paste in the centrifuge bowl and continuously transports the paste out of the centrifuge at a predetermined cell paste concentration. This type of centrifuge can be used for PCV of a much wider range of cell cultures than harvest methods based on intermittent solids-discharge centrifugation. The development of disk stack centrifugation provides a technology for separating materials based on density differences with relatively low or non-existent shear forces, which is suitable for incorporation into harvest processes involving higher PCV perfusion cultures. Disclosed herein is the surprising discovery that continuous solids-discharge disk stack centrifugation effectively separates target proteins, such as antibodies, from biological particulates found in actual perfusion culture broths (especially those with high recovery turbidity) with PCVs of at least 16%, with process yields of approximately 85% to 90% for the target protein, compared to approximately 70% for the target protein using standard harvest techniques (e.g., microfiltration) for perfusion cultures. An exemplary system is a disposable continuous solids-discharge disk stack centrifuge.The benefits of increased PCV with approximately 85%-90% process yield, combined with the cost savings from reduced downtime and labor required to clear centrifuge clogs, more than offset the cost of disposable centrifuges, resulting in an economically attractive target protein recovery method. The incorporation of continuous solids-discharging disk-stack centrifugation, followed by centrate clarification using flocculation or sedimentation, provides a scalable means by which proteins can be recovered in a continuous flow over time, even in perfusion cultures with higher solids content, while still maintaining a clear flow path and minimizing contamination concerns for any subsequent filtration process steps.

[0052] A disc-stack continuous solids discharge centrifuge was chosen because, similar to typical microfiltration harvests, it effectively removes intact cells and larger cell debris fragments without increasing recovery volume, and its closed-device design allows it to be operated using open-floor-plan manufacturing facilities. When used in conjunction with a perfusion culture process, the centrate does not contain intact cells but still contains high concentrations of particles too small to be separated by centrifugation. Turbidity levels of 1500 NTU have been observed in the centrifugal harvest of these higher PCV perfusion cultures. These small particles clog depth and sterile filters at low loading capacities, resulting in the need for larger membrane areas for filtration. Flocculation is used to aggregate small particles into larger ones, increasing the average particle size in solution and reducing the total particle concentration. When combined with a depth filter pore size that effectively removes aggregated particles (e.g., COHCs), this improves the loading capacity of depth and sterile filters and reduces the total membrane area required to filter the centrate at large scale.

[0053] In addition to improved process yields, reduced pool volumes and more consistent recovery performance between lots were observed. Smaller pool volumes with consistent product amounts improve the compatibility of purification equipment and facilitate optimization of manufacturing plant production schedules, raw material usage, and workload allocation. Furthermore, the incorporation of a disc stack continuous discharge centrifugation step reduces subsequent depth filtration load levels to a point where the depth filtration media has sufficient solids retention space to be effective in further purifying the target protein without immediate, unproductive clogging.

[0054] Using continuous solids discharge disk stack centrifugation technology, cell culture broth is separated into a heavy phase stream (solids) and a light phase stream (centrate). The disclosed methods described herein are applicable to several modes of centrifugation operation, specifically: (1) continuous separation of proteins from cellular material in the harvest stream of a perfusion culture manufacturing process with a higher solids content; (2) continuous separation of proteins and impurities from the cellular material, followed by reintroduction of the cellular material into a bioreactor; and (3) efficient batch harvest of product from a bioreactor, for example, continuous solids discharge disk stack centrifugation of a perfusion culture with a higher solids content, where the bioreactor effluent is held in a surge or holding tank for periodic delivery to the centrifuge.

[0055] Additionally, the continuous solids discharge disk stack centrifuge provides a closed system that is scalable and can be used to manufacture therapeutic target proteins without the need for isolation rooms and other requirements to avoid contamination and ensure product quality. This method is compatible with bioreactors preferred for perfusion culture and the open ballroom configuration adopted by many who use continuous perfusion culture in bioreactors. For example, a disposable continuous solids discharge disk stack centrifuge is compatible with a disposable bioreactor. Commercial-scale stainless steel intermittent solids discharge disk stack centrifuges or continuous solids discharge disk stack centrifuges, even if they were available, would not be compatible with commercial-scale disposable bioreactors and perfusion systems.

[0056] The suitable harvest flow rate from a cell culture bioreactor depends on several process design decisions. To size the continuous solids discharge disk stack centrifuge device to match the desired harvest flow rate, operation of the unit can be continuous or operated in a periodic (batch) mode, as described above. Typically, the flow rate is adjusted to maintain a nearly constant load or Q / Sigma value.

[0057] In the methods described herein, the harvest operation begins by collecting the cell culture fluid directly from the bioreactor or from a surge or holding tank and subjecting it to centrifugation using a continuous solids discharge disk stack centrifuge. The centrate may also be collected in a collection holding vessel or may be connected in-line to a surge vessel prior to flocculation and loading onto one or more depth filters.

[0058] The continuous solids discharge disk stack centrifuge device itself can be constructed of stainless steel, plastic, or another material. When the device is a disposable continuous solids discharge disk stack centrifuge, it is combined with a disposable insert that contains the centrifuge bowl and other product-contacting surfaces or flow paths. This disposable insert can be functionally closed with a disposable sterile connector and can be sterilized by irradiation before use. The disposable insert sterile connector allows for attachment to a bioreactor or surge tank and a centrate collection tank to functionally close the centrifugation operation.

[0059] The method described herein recovers a recombinant protein of interest from a perfusion culture having a PCV of at least 16% by subjecting the culture broth to continuous solids discharge disk stack centrifugation in the first step of the harvest operation. More specifically, the target protein (e.g., recombinant protein) harvest operation can be understood to have two parts: (1) removal of cells, cellular debris, etc., and (2) clarification of the centrate containing the protein of interest. To achieve this result, the disclosed method employs a continuous solids discharge disk stack centrifugation step. Any continuous solids discharge disk stack centrifuge known in the art is contemplated for use in the disclosed method. Because the system can be a disposable continuous solids discharge disk stack centrifuge, the components that contact the culture are disposable components that do not necessarily include materials and construction methods designed to withstand repeated use. Nevertheless, the disposable centrifuges contemplated for the disclosed method can be used in continuous harvest methods for various periods of time, and thus the centrifuge can be made to remain operational for extended periods of use.

[0060] The present continuous solids discharge disc stack centrifuge can be used to perform an initial separation of the culture broth into a heavy phase (solids containing cells) and a centrate (containing secreted target protein (e.g., recombinant protein and solids that did not migrate to the heavy phase)) before reaching the depth filtration step. The centrate is further clarified using flocculation before the depth filtration step. Continuous solids discharge disc stack centrifuges, such as disposable continuous solids discharge disc stack centrifuges, can be based on commercially available units from, for example, Alfa Laval Corporate AB (Lund, Sweden), GEA Westfalia (Oelde, Germany). All forms of continuous solids discharge disc stack centrifuges are suitable for use in disposable form according to the methods of the present disclosure.

[0061] The continuous solids discharge disk stack centrifuge technology can be applied to the recovery of culture broth from disposable or stainless steel bioreactors. Preferably, disposable bioreactors for use in such methods have a volume of 5,000 L or less.

[0062] In some embodiments, the theoretical set area (Sigma value) normalized flow rate of the centrifuge, Q / Sigma, is in the range of 4.6E-9 to 2.2E-8 m / s disclosed herein as useful for target protein recovery. The harvest flow rate from a bioreactor depends on several process design decisions. As a result, there is a wide range of harvest flow rates when compared across mammalian cell culture processes. For single-batch harvests, higher flow rates are preferred. For continuous harvest strategies, such as cell-blood product separation, lower flow rates are preferred. Continuous solids-discharge disk stack centrifuges are typically operated continuously as cell culture batches are fed into the centrifuge.

[0063] Successful continuous solids discharge centrifugation is characterized by high process yield and clearance of cells and cell debris (Table 1). Variability in suspended particle levels is observed in perfusion culture processes with PCVs of at least 16%. Therefore, the ability of a continuous solids discharge centrifuge to remove cells and cell debris was quantified by comparing light-phase turbidity with established baseline turbidity values. Baseline turbidity values ​​were measured from cell culture supernatants produced after centrifugation of 50 mL cell culture samples for 17 minutes at approximately 2094 relative centrifugal force (rcf). As disclosed herein, purification was successful when approximately 100% of the cells were removed from the light phase and there was a high yield of the target protein. As shown in Table 1, effective cell and cell debris separation was observed; although the light-phase turbidity was less than twice the baseline turbidity value, this degree of turbidity is sufficient to prevent subsequent purification by depth filtration. The centrate is generally maintained at 10° C., although the centrifuge may warm the light phase to 16-25° C. The centrifuge is held at 10° C. before and during flocculation. In the experiments disclosed herein, the centrate was not sparged, but it is expected that the centrate could be sparged, especially if the centrate is stored for a period of time.

[0064] The levels of submicron cell debris, product- and process-related impurities, and other culture by-products collected in the centrate produced by the high-solids perfusion culture described herein were found to be significantly higher than those found in fed-batch processes at any scale. The high level of impurities in the centrate more easily and quickly clogs depth and sterile filters, necessitating additional filtration area to prepare the centrate for further processing. Therefore, the highly turbid centrate must be further clarified before depth filtration. Centrifugation is followed by a flocculation or sedimentation operation to clarify the centrate in a fast, efficient, and cost-effective process. Clarifying the centrate before depth filtration, rather than before centrifugation as is common in fed-batch cultures, (a) avoids flocculation-related cell lysis, which can adversely affect product quality; (b) potentially avoids or minimizes flocculation-related changes in the rheological behavior of the cell culture or cell precipitate that affect centrifugation; and (c) reduces any depth filtration membrane area required to produce a clarified centrate that can be loaded onto a chromatography column. Unlike fed-batch culture harvest methods, where flocculation is typically performed as a first step, followed by centrifugation to remove flocculated material, adding flocculants directly to perfusion culture broth with a PCV of at least 16% before centrifugation does not make commercial-scale harvest operations viable. Flocculated material would interfere with continuous solids-discharge centrifugation.

[0065] The flocculant may be added to the centrate in a batch mode or in an in-line continuous mode. A predetermined amount of flocculant solution is added to the centrate pool to achieve a target flocculant concentration. Alternatively, the flocculant solution may be added at a predetermined ratio of flocculant flow rate to centrate flow rate to achieve a target flocculant concentration.

[0066] An exemplary flocculant suitable for use in the methods herein is a cationic flocculant. Many process- and product-related impurities are negatively charged in cell culture media, and cationic flocculants are used to reduce / remove them from the centrate. Exemplary cationic flocculants include polymers of diallyldimethylammonium chloride, such as polydiallyldimethylammonium chloride (pDADMAC) and its monomer, diallyldimethylammonium chloride (DADMAC), as well as polyethyleneimine (PEI), polyacrylamide (PAA), and chitosan (U.S. Pat. No. 9,371,554 and McNerney et al., mAbs 7:2 413-427 (2015)). Further exemplary flocculating agents include simple acids, e.g., organic acids such as caprylic acid or octanoic acid (WO 2010 / 151632, incorporated herein by reference), divalent cations, polycationic polymers such as polyethyleneimine (PEI), non-ionic polymers such as polyethylene glycol (PEG) (U.S. Pat. No. 9,371,554 and McNerney et al., mAbs 7:2 413-427 (2015), incorporated herein by reference), and polyalkylene glycols (and transition metals such as zinc, U.S. Pat. App. Pub. No. 2009 / 0292109, incorporated herein by reference), non-ionic surfactants, and stimuli-responsive polymers such as benzylated poly(allylamine) (Kang et al., Biotechnol. Bioeng. 110:2928-2937 (2013), incorporated herein by reference). A "stimuli-responsive polymer" or "smart polymer" is a compound containing multiple chemical subunits (monomers) that is sensitive to at least one trigger from the external environment, including temperature, light, electric or magnetic fields, and chemicals, resulting in a detectable change in the properties of the polymer, such as solubility and the ability to induce aggregation.Such agents are useful in the methods of the present disclosure as aggregating compounds or attached to an insoluble matrix, such as silica beads or eXtreme-Density (XD) cell culture processes, to aid in centrifugal clearance (Schirmer et al., Bioproc. Intl. 8:32-39 (2010), incorporated herein by reference).

[0067] In some embodiments, the flocculating agent is poly(diallyldimethylammonium chloride) (pDADMAC) or its monomer, diallyldimethylammonium chloride (DADMAC). In some embodiments, pDADMAC is added to the centrate at 0.04-0.15% (w / w). In some embodiments, pDADMAC or DADMAC is added to the centrate at 0.05% (w / w). Flocculation is carried out at 8-12°C, with flocculation typically carried out at 10°C. In some embodiments, flocculation is carried out at about 8°C, 8.5°C, 9°C, 9.5°C, 10°C, 10.5°C, 11°C, 11.5°C, or 12°C. In some embodiments, flocculation is carried out at about 10°C. After addition of the flocculating agent, the mixture is mixed for 30 minutes before beginning depth filtration. In some embodiments, the flocculated mixture is stored with mixing at 8-12°C (e.g., 10°C) for up to 4 hours. In some embodiments, the entire recovery process, from cooling the contents of the bioreactor to depth filtration of the centrate, is carried out at 10° C. or with the liquid at 10° C. In some embodiments, centrifugation, flocculation, and depth filtration are carried out with the liquid at 10° C.

[0068] Depth filters utilize various porous filtration media (e.g., diatomaceous earth, cellulose, and / or synthetic fibers) to create a three-dimensional network of pores that collectively establish a tortuous path through which materials must pass to pass through the filter. Particulate matter from cell culture broth (e.g., cells, cellular debris, by-products, and both product- and process-related impurities) can rapidly plug the filter by clogging the filter's pores or surface. As should be known in the art, the rate and extent of filter blockage or clogging is affected by the particle amount and size distribution of the cellular debris, as well as the chemistry and pore size of the depth filter media. Disclosed herein is data confirming that when unprocessed centrate was combined with larger pore sizes such as X0HC, or even COHC, there was almost immediate clogging (see Figure 8). Flocculation ensures the formation of larger molecular aggregates, which reduces the amount of cellular debris particles and increases particle size, reducing the incidence of blockage or clogging, especially when combined with a suitable filter type (e.g., COHC, COSP, DL75, DL60). The selection of an appropriate depth filter type is based on empirical optimization of flocculation, the chemistry of the depth filter media, and the pore size range of the depth filter media. In particular, the pore size must match the size of the cellular debris present in the solution. Without flocculation, particles were found in the size range of less than 1-2 μm. With flocculation, particles were found in the 50-100 μm range. Overall, fewer particles were found after flocculation. Small and large particles can clog the filter. The worst performance was observed when the selected pore size of the depth filter did not match the size of the particles in the solution, which explains why COHC filters using untreated centrate performed worse than X0HC filter types. As shown in Figure 1, a wide range of depth filter types exhibit increased depth filtration loads compared to the X0HC control. The X0HC control was selected for untreated centrate because it had previously been shown to provide the best depth filtration capacity for untreated centrate. Increased depth filtration capacity reduces the amount of depth filter required to filter a given centrate volume, reducing the filter area footprint in manufacturing facilities.

[0069] Suitable depth filters, as should be known in the art, are composed of a porous matrix of synthetic or cellulose fibers optionally attached to a charged resin used to attract and retain oppositely charged filter aids. Some exemplary depth filters incorporate diatomaceous earth as an inorganic filter aid. Depth filters may also include a binder. For all depth filters, including cellulosic depth filters, the thicker the material (e.g., the cellulose fiber mat or material), the more sinuous and complex the pathways through the filter material, resulting in particles of various sizes being trapped and removed from the liquid being clarified. Cellulosic depth filters typically have a net positive charge, which also provides the basis for the adsorptive removal of negatively charged impurities.

[0070] The disclosed method includes subjecting the clarified centrate from the continuous solids-discharging disc stack centrate to depth filtration to remove impurities after the flocculation step disclosed herein. The depth filtration operation can include one or more depth filters of the same or different types. Depth filters suitable for use in the disclosed method include, but are not limited to, cellulose, synthetic fiber mesh, or a combination of both, as well as depth filters made from the pre-treated filtration matrix described in Singh et al., Biotechnol. Bioeng. Biol 110:1964-1972 (2013), which is incorporated herein by reference. Based on the current small-scale depth filtration data disclosed herein, exemplary depth filtration types suitable for use with the flocculated centrate in the recovery methods of the present disclosure include COHC (nominal pore size distribution of 0.2-2 um), COSP (nominal pore size distribution of 0.2-2 um), DL75 (nominal pore size distribution of 1-14 um), and DL60 (pore size distribution of 0.6-10 um). Those skilled in the art may identify other filter media suitable for use in the methods of the present disclosure using only ordinary skill. Expanding on the foregoing observations, exemplary depth filters include cellulosic depth filters, MILLISTAK+® DOHC filters, MILLISTAK+® COHC filters, MILLISTAK+® COSP filters, SARTOCLEAR® DL60 filters, SARTOCLEAR® DL75 filters, Clarisolve 20MS filters, Clarisolve 40MS filters, Clarisolve 60HX filters, 3M™ Zeta Plus™ filters, diatomaceous earth, and 3M™ Emphaze™ AEX Hybrid Purifiers. All of these filter media provide useful purification without unacceptable losses in yield, but, as described herein, filter media loading is reduced if the pore size of the filter media is not properly selected based on the particle size in the centrate being filtered.Based on the data disclosed herein, it is anticipated that multi-layer filters will be advantageous for purifying target proteins with high yields, but multi-layer filters are not a requirement of the disclosed methods. Experiments on the depth filtration component of the purification method are designed to obtain data useful for achieving high loading levels to minimize the number of filters required to clarify the centrate sufficiently to pass through any subsequent downstream filters, such as Protein A guard filters (typically 0.2 μm pore size), while maintaining high process yields.

[0071] Additionally, depth filtration may be followed by another sterile or bioburden reducing filter (e.g., Millipore SHC filter, Sartopore 2 filter (Sartorius), and Pall filter) with a pore size of 0.2 μm or less to ensure bioburden reduction. Upon completion of depth filtration, the purified centrate is typically held at 2-8° C. for up to 72 hours. This holding may be reduced depending on molecular stability.

[0072] The present disclosure further contemplates methods of combining the present recovery process with post-recovery recovery steps known in the art (e.g., target biomolecule recovery, e.g., affinity-based recovery, which for immunoglobulin and immunoglobulin-like targets may be based on binding of target affinity chromatography materials utilizing Staphylococcus proteins such as Protein A), as well as chromatographic fractionation and polishing steps, in which the chromatographic medium may be in any form, including beads in column form, and the fractionation and polishing steps may rely on any discriminatory characteristic of the target, such as size (e.g., size exclusion chromatography), affinity, charge (e.g., anionic or cation exchange chromatography), hydrophobicity (e.g., hydrophobic interaction chromatography), multimodal or mixed-modal (combining different modes), or any characteristic known to be useful for distinguishing between molecules using chromatographic media. Briefly, the present disclosure provides any method of purifying a target biomolecule (e.g., a biologic such as an antibody) that has been recovered using the methods disclosed herein.

[0073] The present disclosure provides a method for separating a recombinant protein from cell culture broth in a batch harvest stream from a cell culture bioreactor. The culture is harvested from the bioreactor when a predetermined parameter (e.g., culture duration, titer, viable cell density, or packed cell volume) is met. The bioreactor is cooled to a temperature of 12°C or below. The cell culture broth is then either harvested directly from the bioreactor or collected in a holding tank and held at 10-12°C. The harvest cell viability, viable cell density (VCD), packed cell volume, and / or baseline turbidity of the culture are determined using conventional techniques known in the art.

[0074] The present disclosure provides for the separation of recombinant proteins from cell culture broth in a periodic harvest stream from a perfusion culture process, where the cell culture broth passes through a surge tank / vessel before or after a continuous solids-discharging disk-stack centrifuge separates the solids (heavy phase) from the liquid supernatant (light phase or centrifuge bed). The centrate is then subjected to a flocculation / precipitation step before being loaded onto one or more depth filters and, optionally, onto one or more sterilizing filters. The eluate is then collected in a holding tank and either stored at 4°C or subjected directly to a purification chromatography operation.

[0075] The present disclosure provides for the continuous separation of recombinant proteins from continuous cell cultures, in which batches of cell culture broth are passed through a continuous solids-discharging disk-stack centrifuge to separate solids from the cell culture supernatant (centrate). In some embodiments, the solids may be recycled or reintroduced into the bioreactor. The centrate is collected in a harvest pool and subjected to the remaining steps of the harvest method described herein. In some embodiments, the method includes continuous harvest directly from the working bioreactor, and some embodiments provide in-line cooling.

[0076] The recovery operations described herein can be combined with additional recovery strategies, such as additional centrifugation, such as disk stack centrifugation; filtration, including tangential flow filtration, microfiltration, ultrafiltration, and depth filtration; sedimentation, and chromatography media-based separations, typically using columns, including anion and / or cation exchange, affinity-based exchange (e.g., immunoaffinity exchange), molecular sieve, and / or polishing chromatography. Any purification or clarification protocol known in the art to be applicable to cell culture materials, including cell culture supernatants and liquids containing cellular material, is understood by the methods disclosed herein. In a typical operation, separation by continuous solids-discharging disk stack centrifugation is the first step after removal of the cell culture from the bioreactor (or a surge tank or holding tank useful for batch-mode separations). Following the centrifugation, flocculation, and filtration recovery steps described herein, the purification or clarification protocol can include any combination of downstream filters, columns, and / or other forms of centrifugation useful for separating biomolecules such as proteins.

[0077] Beyond the aforementioned techniques, the methods of the present disclosure contemplate a wide variety of chromatographic steps involved in the separation or clarification of biomolecules of interest (e.g., proteins), including size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, multimodal or mixed-modal chromatography, and affinity chromatography such as immunoaffinity chromatography. Various media may be used in various configurations designed to accommodate biomolecules, e.g., proteins, batch separation, and continuous purification modes.

[0078] The disclosed methods contemplate manufacturing processes in which cells are cultured in three or more distinct phases. Each phase may occur in its own bioreactor vessel or other vessel suitable for cell culture. Alternatively, multiple phases may occur in a common vessel. In commercial processes for protein production using mammalian cells, there are generally multiple (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10) growth phases / stages occurring in different culture vessels preceding a final N production phase. For example, cells may be cultured in one or more growth phases prior to an N-1 seed stage, which may be cultured in one or more N-1 bioreactors. The duration of the N-1 stage may range, for example, from 7 to 14 days and may be designed to maintain cells in exponential growth prior to inoculation of the production (N) bioreactor. In some embodiments, cells from the N-1 bioreactor are transferred to the (N) production bioreactor and grown under conditions that maximize protein production. In other embodiments, there is no transfer, ie, the N-1 production stage and the N production stage occur in the same bioreactor.

[0079] A "cell" or "cells" includes any prokaryotic or eukaryotic cell. Cells can exist either ex vivo, in vitro, or in vivo, either separately or as part of a higher-order structure such as a tissue or organ. Cells include "host cells" that can be genetically engineered to express proteins of commercial or scientific interest. Host cells are typically derived from lines arising from primary cultures that can be maintained in culture indefinitely. Host cells can be prokaryotic (e.g., E. coli) or eukaryotic (e.g., yeast, insect, or animal cells (e.g., CHO cells)). Host cells cultured under appropriate conditions will express the protein of interest, which can then be collected from the culture medium (if the host cells secrete the protein into the medium), directly from the host cells that produce the protein (if the protein is not secreted), or from both sources. The selection of an appropriate host cell will depend on various factors, including the desired expression level, protein modifications (such as glycosylation or phosphorylation) that are desirable or necessary for activity, and the ease of folding into a biologically active molecule.

[0080] Genetic engineering of host cells involves transfecting, transforming, or transducing with a recombinant polynucleotide molecule and / or otherwise modifying the cell (e.g., by homologous recombination and gene activation, or fusion of recombinant cells with non-recombinant cells) to cause the host cell to express a desired recombinant protein. Methods and vectors for genetically engineering cells and / or cell lines to express a biomolecule (e.g., a protein) of interest are well known to those of skill in the art. Expression systems and constructs in the form of expression vectors, such as plasmids or transcription or expression cassettes, containing one or more polynucleotides encoding a biomolecule (e.g., a protein) of interest, such as those identified herein, are provided, as well as host cells containing such expression systems or constructs. As used herein, "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, naked DNA, DNA complex, etc.) suitable for use to transfer and / or transport protein-encoding information to a host cell and / or a specific location and / or compartment within a host cell. Vectors may include viral and non-viral vectors, as well as non-episomal mammalian vectors. Vectors may be expression vectors, such as recombinant expression vectors or cloning vectors. A vector can be introduced into a host cell to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors may contain sequence components, generally including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a transposon / transposase, and a selectable marker. These elements may be selected as needed by those skilled in the art, and one or more of each selected component may be included in the cloning vector.

[0081] Preferably, the host cell is a eukaryotic cell, such as a mammalian cell. Any mammalian cell suitable for recombinant protein expression is suitable for use in connection with the present disclosure. Suitable mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, mouse myeloma (NS0, Sp2 / 0) cells, baby hamster kidney (BHK) cells, human embryonic kidney (293) cells, fibrosarcoma (HT-1080) cells, human embryonic retina (PER.C6) cells, hybrid kidney and B cell (HKB-11), amniotic cell production of CEVEC (CAP) cells, human liver (HuH-7) cells, and any other cells used or suitable for use in clinical and / or commercial manufacturing. The most commonly used cell lines are derived from CHO cells. CHO cells are widely used to produce complex recombinant proteins. Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplifiable gene expression systems enable high-level recombinant protein expression in these cells (Kaufman RJ (1990), Meth Enzymol 185:537-566). The glutamine synthetase (GS) knockout CHOK1SV cell line, which utilizes GS-based methionine sulfoximine (MSX) selection, is also widely used. CHOK1 cells (ATCC CCL61) are also included.

[0082] Key attributes and performance parameters may be measured to better inform decisions regarding the performance of each step during production. These key attributes and parameters may be monitored in real time, near real time, and / or after the fact. Key parameters such as consumed medium components (e.g., glucose), metabolic levels of by-products accumulating in the medium (e.g., lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content, may be measured during cell culture. Key attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, clumping, cell number, packed cell volume, percent yield, and titer may be monitored during appropriate stages in the manufacturing process. Process and product impurities may also be monitored throughout the manufacturing process.

[0083] "Culturing" or "culturing" refers to the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. Cell culture medium and tissue culture medium are used interchangeably to refer to a medium suitable for the growth of host cells in in vitro cell culture. Typically, cell culture media contain buffers, salts, an energy source, amino acids, vitamins, and essential trace elements. Any medium capable of supporting the growth of suitable host cells in culture may be used. Cell culture media, which can be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells (e.g., components effective in controlling the timing of recombinant protein production), are commercially available and include, among others, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media, such as the EX-CELL™ 300 series, which can be obtained from American Type Culture Collection or SAFC Biosciences, as well as other vendors. Cell cultures can also be supplemented with individual concentrated feeds for specific nutrients that may be difficult to formulate or rapidly depleted in cell culture media. Such nutrients may be amino acids such as tyrosine, cysteine, and / or cystine (see, e.g., WO 2012 / 145682). The cell culture medium may be serum-free, protein-free, growth factor-free, and / or peptone-free. The cell culture may also be enriched by the addition of nutrients at higher than normally recommended concentrations. In a preferred embodiment, the cell culture medium is enriched with 10 6 Cell densities greater than 10 cells / ml (i.e., 10 8 The concentration is adjusted for growth of host cells to a cell density approaching, reaching, or exceeding 100 cells / ml.

[0084] The perfusion feed medium may be formulated or supplemented to achieve a concentration of at least 5 g / L of non-ionic block copolymer when the cell culture is passed through hollow fiber filters with a pore size or molecular weight cut-off (MWCO) that will not retain the recombinant protein in the bioreactor (WO 2015 / 188009).

[0085] Various media formulations may be used during the culture period, for example, to initiate the culture, to facilitate the transition from one stage (e.g., growth stage or phase) to another stage (e.g., production stage or phase), and / or to optimize conditions in the cell culture (e.g., concentrated media provided during perfusion culture). A basal media formulation containing essential media components is typically used to initiate the cell culture. A growth media formulation may be used to promote cell growth and minimize protein expression. A production media formulation may be used to promote the production of a biomolecule of interest (e.g., protein) and cell maintenance while minimizing the growth of new cells. A feed medium consumed during the production phase of the cell culture, typically a medium containing more concentrated components such as nutrients and amino acids, may be used to supplement and maintain the active culture, particularly a culture operated in perfusion mode. Such concentrated feed medium may contain most of the components of the cell culture medium, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or even about 1000x their normal amounts or concentrations.

[0086] Cell growth can be limited or stopped during a cell culture run, particularly during the production phase. Such methods include, either alone or in combination, temperature shift, pH shift, the use of chemical inducers of protein production and cell cycle inhibitors, and nutrient limitation or starvation. For example, a temperature shift can be used to transition from the growth phase to the production phase. The growth phase can occur at a first temperature of about 35°C to about 38°C, and the production phase can occur at a second temperature of about 29°C to about 35°C, optionally about 30°C to about 35°C or about 30°C to about 34°C. Optionally, about 32°C to about 33°C. In addition, the growth phase can occur at a higher pH than the production phase. pH shift can be used separately or in combination with temperature shift and / or addition of chemical inducers.

[0087] Another method for maintaining cells in a desired physiological state is to induce cell growth arrest by exposing the cell culture to low L-asparagine conditions and / or asparagine starvation (see, for example, WO 2013 / 006479). Cell growth arrest can be achieved and maintained through a culture medium containing a limiting concentration of L-asparagine and maintaining a low concentration of L-asparagine in the cell culture. Maintaining a concentration of L-asparagine at 5 mM or less can be used to induce and maintain cells in a growth-arrested state, thereby increasing productivity.

[0088] Additionally, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added before, simultaneously with, and / or after the temperature shift. If the inducer is added after the temperature shift, it can be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift. Cell cycle inhibitors, compounds known or suspected to regulate cell cycle progression and the associated processes of transcription, DNA repair, differentiation, senescence, and apoptosis, are also useful for inducing cell growth arrest. Cell cycle inhibitors that interact with the cycle machinery, such as cyclin-dependent kinases (CDKs), are useful because they interact with proteins from other pathways, such as AKT, mTOR, and other pathways that directly or indirectly affect the cell cycle.

[0089] The disclosed method can be used as part of a large-scale production process in which cells are cultured in three or more different phases. Each phase can be carried out in its own bioreactor vessel or other vessel suitable for cell culture. Alternatively, multiple phases can be carried out in a common vessel. Suitable vessels include fluidized bed bioreactors, with or without microcarriers, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors. As used herein, the general term bioreactor refers to any vessel used for cell culture.

[0090] For use in the methods described herein, cell cultures are operated in perfusion mode. Mammalian cells, such as CHO cells, can be cultured in bioreactors on a small scale, from less than 100 ml to less than 1000 ml. Alternatively, large scale bioreactors, from 1000 ml to over 2,000 liters, can be used. In one embodiment, bioreactors between 1 liter and 2000 liters are used. In one embodiment, the bioreactor is between 10 liters and 2000 liters. In one embodiment, the bioreactor is between 10 liters and 100 liters. In one embodiment, the bioreactor is between 30 liters and 50 liters. In one embodiment, the bioreactor is between 30 liters and 2000 liters. In one embodiment, the bioreactor is between 100 liters and 2000 liters. In one embodiment, the bioreactor is between 500 liters and 2000 liters. In one embodiment, the bioreactor is between 1000 liters and 2000 liters. Larger scale cell cultures, such as those for clinical and / or commercial scale biomanufacturing of protein therapeutics, can be maintained for weeks or months while the cells produce the desired protein.

[0091] In some embodiments, the methods of the present disclosure are carried out using single-use bioreactors, also known as disposable bioreactors, which utilize disposable bags instead of traditional stainless steel culture vessels. The use of single-use technology minimizes infrastructure requirements associated with traditional cell culture, such as steel / glass commercial-scale vessels and related equipment. Single-use bioreactors offer flexibility in the manufacturing process, and on-site assembly, reconfiguration, sterilization, and validation are faster, easier, and less costly than traditional, built-in stainless steel cell culture plants. Single-use bioreactors include a disposable plastic sterile bag supported by a non-disposable support structure. The culture is agitated within the bag by agitation or rocking, and air and oxygen spargers are also provided, as well as sensors for measuring and adjusting various parameters of the culture (e.g., pH, temperature, oxygen, cell density, etc.). Disposable bioreactors are commercially available, e.g., Bio STR®, Satorius, Goettingen Germany; MOBIUS®, Millipore, Burlington, Mass.; XCELLEREX®, Cytiva, Marlborough, Mass. In some embodiments, it is expected that cultures of 3 kL or more will be grown in a bioreactor of suitable size.

[0092] A bioreactor system maintains conditions within the bioreactor to support cell culture. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). "Running" a bioreactor system means maintaining conditions within the bioreactor system to support cell culture. A bioreactor "run" typically involves inoculating a prepared bioreactor with a seed culture, subjecting the cells to one or more growth and / or production phases until predetermined parameters (time, viable cell density, packed cell volume) are met, and then harvesting the contents of the bioreactor.

[0093] "Culturing" or "culturing" refers to maintaining cells in a culture medium under conditions suitable for the survival and / or growth of cells separate from a multicellular organism or tissue, and for the production of a protein product. Cell cultures are typically operated in batch, fed-batch, or perfusion mode. In batch mode, a fixed amount of culture medium and cells is placed in a bioreactor at the beginning of a run. During cultivation, the medium volume in the reactor remains constant, while the nutrient content of the medium is reduced and the waste and by-product content increases. The cell concentration increases during the run, reaches a steady state as the nutrient content is depleted and waste increases, and then decreases. Fed-batch culture begins with an inoculation of cells and a fixed amount of culture medium. Unlike batch culture, the volume of medium in the bioreactor increases as concentrated nutrients are added during cultivation. As with batch culture, the waste and by-product content increases during the run, reaches a steady state as the nutrient content is depleted and waste increases, and then decreases.

[0094] Perfusion culture, like batch and fed-batch culture, begins with a constant inoculation of cells and culture medium. Unlike fed-batch culture, the culture begins at or near the final working volume of the reactor. Also unlike fed-batch culture, fresh feed medium is added to the bioreactor while an equal volume of spent medium is perfused or removed from the bioreactor through a retention device. In the case of the method described herein, the perfusion is continuous perfusion. A retention device such as a tangential flow filtration (TFF) system or an alternating tangential flow (ATF) system may be used to remove spent medium and unwanted products from the bioreactor. Alternatively, sonication or cell sedimentation techniques may be used to remove spent medium and unwanted products.

[0095] RTF relies on the use of a recirculation means (most typically a diaphragm pump) to shuttle the cell culture through a module (e.g., a hollow fiber filter module) to allow removal of spent medium while retaining the cells in the bioreactor. See, e.g., U.S. Pat. No. 6,544,424; Furey (2002) Gen. Eng. News. 22(7), 62-63. An advantage of ATF is the cleaning effect on the filter induced by the alternating flow. An exemplary method of the present disclosure includes operating a (N) production bioreactor using an ATF perfusion system.

[0096] Typically, hollow fiber filters are used in ATF systems (although this is not required). When cell culture, including cell culture medium, cells (whole and lysed), soluble expressed recombinant proteins, host cell proteins, waste products, etc., is introduced into the filter depending on the pore size or molecular weight cutoff (MWCO), the hollow fiber material retains certain cell culture components (in addition to the cells themselves) on the lumen side (inside) and allows certain components to pass through the filter (permeate) based on the pore size or molecular weight cutoff of the hollow fiber material. The retained material (retentate) is returned to the bioreactor. Fresh perfusion cell culture medium is added to the bioreactor, and permeate is withdrawn from the filter at predetermined intervals or continuously to maintain a desired or constant bioreactor volume. The permeate can be discarded, stored in a holding tank, bag, or tote, or transferred directly to another unit operation, such as a recovery operation.

[0097] Exemplary filters (e.g., ultrafilters and precision filters) include, but are not limited to, Millipore Prostrac, Cytiva hollow fiber filters in many pore size ranges, Repligen filters in various pore sizes, Spectrum hollow fiber filters in various pore sizes, and Ashai Kasei hollow fiber filters.

[0098] Typically, hollow fiber filters are used in perfusion culture retention systems. In various embodiments, hollow fibers have an inner diameter of about 0.5 mm to about 1 mm and can be any suitable length (e.g., about 30 cm to about 110 cm). Hollow fibers for microfiltration typically have pore sizes in the range of 0.1 μm to 10 μm or a molecular weight cutoff of 500 kDa to 750 kDa or more and can be used to pass proteins (e.g., monoclonal antibodies or engineered antibody-like proteins) into the permeate. Ultrafiltration hollow fibers typically have pore sizes in the range of 0.01 μm to 0.1 μm or a molecular weight cutoff of 300 kDa or less and can be used to retain the desired protein in the retentate and return it to the bioreactor. This can be used, for example, to concentrate recombinant protein products for recovery. Such filters are commercially available and include Xampler (Cytiva, Marborough, MA), Midikros (Spectrum Laboratories, Inc., Dominguez, CA), and XCell ATF®, Repligen, Waltham, MA. The temperature of the culture broth exiting the bioreactor is controlled, and this temperature is typically maintained at 10-12°C. The culture broth is removed from the bioreactor using a pump to a continuous solids discharge disk stack centrifuge or surge tank. As should be understood, the centrifuge itself also has a pump.

[0099] The cell culture medium can be drawn from the bioreactor into the filter module by a pumping system that passes the cell culture through or along the filter (e.g., through the lumen side of the hollow fibers). Examples of cell pumping systems include peristaltic pumps, double diaphragm pumps, low shear pumps (Levitronix™ pumps, Zurich, Switzerland), and alternating tangential flow systems (ATF™, Repligen, Waltham, MA). Permeate can be drawn from the filter by using a peristaltic pump. In the examples provided herein, perfusion is achieved by using an alternating tangential flow system.

[0100] Medium exchange in bioreactors is expressed as vessel volumes per day (VVD) or bioreactor volumes per day (BV / d). The exchange rate can be minimized, for example, by setting it at about 0.25 BV / d, but more frequently it is set at a value of at least 1 BV / d, and typically this rate is set at a value of at least 2 to 3 BV / d.

[0101] After expression in the perfusion culture, the target protein of interest is then harvested according to the methods of the present disclosure. This harvesting process clarifies or purifies, or partially clarifies or purifies, the target protein from at least one impurity co-found in the culture broth, such as residual cell culture medium, cell extract, cells, cell debris, undesirable cell or medium components, product-related and / or process-related impurities. The harvested recombinant protein of interest may be stored in a surge tank, holding tank, bag, or other container adapted for feeding a chromatography column skid. The harvested recombinant protein of interest may also be fed directly to a chromatography column skid as an eluate stream. At harvest, the PCV of the perfusion culture is at least 16%. In one embodiment, the PCV of the perfusion culture is at least 18%. In one embodiment, the PCV of the perfusion culture is about 20 to about 30%. In one embodiment, the PCV of the perfusion culture is about 16%. In one embodiment, the PCV of the perfusion culture is about 18%. In one embodiment, the PCV of the perfusion culture is about 19%. In one embodiment, the PCV of the perfusion culture is about 20%. In one embodiment, the PCV of the perfusion culture is about 21%. In one embodiment, the PCV of the perfusion culture is about 22%. In one embodiment, the PCV of the perfusion culture is about 23%. In one embodiment, the PCV of the perfusion culture is about 24%. In one embodiment, the PCV of the perfusion culture is about 25%. In one embodiment, the PCV of the perfusion culture is about 26%. In one embodiment, the PCV of the perfusion culture is about 27%. In one embodiment, the PCV of the perfusion culture is about 28%. In one embodiment, the PCV of the perfusion culture is about 29%. In one embodiment, the PCV of the perfusion culture is about 30%. In one embodiment, the PCV of the perfusion culture is about 30% to about 35%. In one embodiment, the PCV of the perfusion culture is about 16% to 45%. Recovery and further purification of the target protein of interest can be achieved by downstream unit operations.

[0102] Downstream operations purify and polish the target protein. These downstream operations utilize capture chromatography, which includes resins and / or membranes containing agents that will bind and / or interact with at least one desired protein, impurities, or contaminants. Examples of capture chromatography include affinity chromatography, size exclusion chromatography, ion exchange chromatography (IEX), such as cation exchange (CEX) and anion exchange (AEX) chromatography, hydrophobic interaction chromatography (HIC), multimodal or mixed-mode chromatography (MMC), immobilized metal affinity chromatography (IMAC), and the like. Such materials are known in the art and commercially available.

[0103] Affinity chromatography is commonly used in biomanufacturing processes as a first, supplemental step to isolate and concentrate target proteins of interest (e.g., recombinant proteins) from crude or clarified materials. Examples of such affinity chromatography materials include those utilizing Staphylococcus proteins such as Protein A, Protein G, Protein A / G, and Protein L; substrate-binding capture mechanisms; antibody- or antibody-fragment-binding capture mechanisms; aptamer-binding capture mechanisms; and cofactor-binding capture mechanisms. Immobilized metal affinity chromatography can be used to capture proteins that have, or have been engineered to have, affinity for metal ions. Protein A is highly selective for a wide range of antibodies and antibody-like proteins, and its robust removal of process-related impurities and high yields make it the bulk purification process of choice. Protein A materials are commercially available from a number of suppliers. For example, MABSELECT™ SURE Protein A, Protein A Sepharose FAST FLOW™ (Cytiva, Marborough, MA), PROSEP-A™ (Merck Millipore, UK), and TOYOPEARL™ 650M Protein A (TosoHass Co., Philadelphia, PA).

[0104] Intermediate and / or polishing unit operations utilize various chromatographic methods for the continuous purification of the protein of interest and for the clarification of contaminants and impurities such as DNA, host cell proteins, product-related impurities, variant products and aggregates, viruses (e.g., by viral adsorption), etc. These chromatographic unit operations utilize agent-containing resins and / or membranes that can be operated in a variety of configurations, such as a flow-through mode, in which the protein of interest is contained in the eluate and the contaminants and impurities bind to the chromatographic medium; a frontal or overload chromatography mode, in which a solution containing the protein of interest is loaded onto the column until the adsorption sites are occupied, and the species with the least affinity for the stationary phase (protein of interest) begin to elute; and a bind-and-elute mode, in which the protein of interest binds to the chromatographic medium and is eluted after the contaminants and impurities have passed through or been washed off the chromatographic medium, or any other method that achieves some degree of purification of the target protein by chromatography. As noted above, examples of such chromatographic methods include, among others, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and gel filtration.

[0105] Multiple chromatography unit operations (usually one, two, or three, each typically performing a different function or operating in a different mode) are combined depending on the requirements of the manufacturing process. Ion exchange chromatography, based on electrostatic interactions between charged surfaces, separates a protein of interest from impurities based on different adsorption and desorption rates, which reflect the level of electrostatic attraction of the protein (or impurities) to the chromatography medium. Cation exchange chromatography refers to chromatography performed on a solid-phase medium that is negatively charged and has cations available for exchange with cations in an aqueous solution passed over or through the solid phase. The charge can be imparted by attaching one or more charged ligands to the solid phase, e.g., by covalent bonding. Alternatively, or in addition, the charge can be an inherent property of the solid phase (e.g., as in the case of silica, which has an overall negative charge). Cation exchange chromatography is typically performed in bind-and-elute mode, allowing many high-pi proteins of interest to bind to the chromatography material. Cation exchange chromatography can also be performed in flow-through mode. CEX chromatography is typically used for the removal of high molecular weight (HMW) contaminants, the removal of process-related impurities, and / or viral clearance.Commercially available cation exchange media are available, including, but not limited to, sulfopropyl (SP) functional groups immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™, or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (Cytiva), FRACTOGEL-SO™, FRACTOGEL-SE HICAP™, and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL® XS, TOYOPEARL® HS (Tosh Bioscience, King of Prussia, PA), UNOsphere™ (BioRad, Hercules, CA), and S Ceramic Hyper D™ F (Pall, Portland, FL). Washington, NY), POROS(TM) (ThermoFisher, Waltham, MA).

[0106] Anion exchange chromatography refers to chromatography performed on a solid phase medium that is positively charged and has anions available for exchange with anions in an aqueous solution passed over or through the solid phase. Anion exchange chromatography is typically performed in flow-through mode. Due to the high pI of many therapeutic proteins, these therapeutic proteins do not readily bind to AEX chromatography materials. AEX chromatography is used, for example, for viral clearance and impurity removal. Commercially available anion exchange media are available, including, but not limited to, quaternary amines (Q) immobilized on agarose (e.g., Source 15 Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Cytiva), FRACTOGEL EDM TMAE™, FRACTOGEL EDM DEAE™ (EMD Merck), TOYOPEARL Super Q® (Tosh Bioscience), POROS HQ™, POROS XQ™ (ThermoFisher).

[0107] Mixed-mode or multimode chromatography (MMC) refers to chromatography performed on solid-phase media that utilize a combination of interaction mechanisms, such as ion exchange (CEX or AEX) and hydrophobic interactions. Commercially available multimodal chromatography media are available, including Capto™ Adhere (Cytiva).

[0108] Hydrophobic interaction chromatography refers to chromatography performed on a solid phase medium that utilizes interactions between hydrophobic ligands and hydrophobic residues on the surface of the protein of interest. Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sepharose™ (Cytiva), Tosoh hexyl (Tosoh Bioscience), and Capto™ phenyl (Cytiva).

[0109] Hydroxyapatite chromatography refers to chromatography performed on a solid phase medium that utilizes positively charged calcium and negatively charged phosphate. Depending on the pI of the protein and the pH of the buffer, the hydroxyapatite medium can function as a cation or anion exchanger.

[0110] Unit operations involved in the inactivation, reduction, and / or elimination of viral contaminants may involve processes that manipulate the environment and / or rely on filtration. Viral mitigation measures are important to ensure the safety of protein therapeutics and may be implemented one or more times throughout downstream purification stages of recovery operations. Viral contaminants can arise from a variety of sources, including the use of reagents of animal origin, adventitious viral contaminants in host cell lines, or system failures in GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped viruses. For enveloped viruses, the envelope allows the virus to identify, bind, invade, and infect target host cells. Therefore, enveloped viruses are susceptible to inactivation methods. Various methods can be employed to inactivate viruses, including heat inactivation / pasteurization, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, chemical inactivators, the addition of detergents, and solvent / detergent treatments. Surfactants, such as detergents, can be very effective at solubilizing membranes and therefore specifically inactivating enveloped viruses. One method for achieving viral inactivation is incubation at low pH (e.g., a pH below 4). Low pH viral inactivation can be followed by a neutralization unit operation that readjusts the virally inactivated solution to a pH more compatible with the requirements of the next unit operation. Low pH viral inactivation is typically performed after purification of the harvest solution using affinity chromatography (particularly affinity chromatography utilizing substrate-binding ligands derived from Staphylococcus aureus, such as Protein A chromatography), because elution from such chromatographic materials is usually performed at low pH. Exemplary low pH viral inactivation methods are described in U.S. Patent Application Nos. 63 / 168,608 and 63 / 159,217. Exemplary detergent inactivation is described in International Patent Organization Publication No. WO 2020 / 190985. Low pH viral inactivation may also be followed by filtration, such as depth filtration, to remove any resulting turbidity or precipitate.

[0111] Non-enveloped viruses are more difficult to inactivate without risk to proteins during production and are removed by filtration methods. An exemplary process is described in International Patent Organization Publication No. WO 2020 / 159838. Virus filtration can be performed using microfilters or nanofilters such as those available from PLAVONA® (Asahi Kasei, Chicago, IL), VIROSART® (Sartorius, Goettingen, Germany), VIRESOLVE® Pro (MilliporeSigma, Burlington, MA), Pegasus™ Prime (Pall Biotech, Port Washington, NY), and CUNO Zeta Plus VR (3M, St. Paul, MN). Virus filtration can occur at one or more steps in downstream operations of the biomanufacturing process. Typically, virus filtration precedes any ultrafiltration or diafiltration (i.e., UF / DF) operations, but can also occur after UF / DF.

[0112] Unit operations may also include product concentration and buffer exchange of the protein of interest into a desired formulation buffer for bulk storage of the drug substance. Buffer exchange and product concentration may be achieved using known methods for ultrafiltration and diafiltration. Unit operations to achieve drug product fill / finish may also follow. The disclosed method further optionally includes concentrating the protein product using ultrafiltration and diafiltration (UF / DF). The purified protein is subjected to ultrafiltration and diafiltration operations, including concentrating or diluting the purified protein by ultrafiltration; buffer exchanging the purified, concentrated / diluted protein into the desired formulation by diafiltration; and further diluting or concentrating the formulated purified protein by a second ultrafiltration until the target protein concentration is reached. One or more stability-enhancing excipients may optionally be added directly to the UF / DF retentate feed tank containing the formulated purified protein to obtain the formulated drug substance, or may be added to the UF / DF eluate pool. Filters for use in UF / DF operations are well known and common in the art and are commercially available from a variety of sources. There are many types of materials available, such as regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), polyethersulfone (PES) membrane, and Omega (Pall Corporation, Port Washington, NY). Multiple filters can be used up to the capacity that the holder, skid, or physical configuration of the UF / DF system will allow or that is necessary to achieve the desired objectives of the production process.

[0113] As is known in the art, key attributes and performance parameters of purified proteins of interest can be measured to better inform decisions regarding the performance of each step during production. These key attributes and parameters can be monitored in real time, near real time, and / or after the fact. Key parameters such as consumed media components (e.g., glucose), levels of accumulating metabolic by-products (e.g., lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content, can be measured during cell culture. Key attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, yield, and titer can be monitored during appropriate stages in the production process. Monitoring and measurements can be performed using known techniques and commercially available equipment.

[0114] Pharmaceutical compositions (e.g., solutions, suspensions, etc.) may contain one or more of the following: buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives; sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that may function as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0115] Target protein (e.g., biomolecule of interest) The terms "polypeptide" and "protein" are used interchangeably throughout this disclosure and refer to molecules comprising two or more amino acid residues linked by peptide bonds. Polypeptides and proteins also include polymers (i.e., naturally occurring polypeptides or proteins produced by non-recombinant cells) having one or more deletions from, insertions into, and / or substitutions of amino acid residues in the native sequence, or molecules produced by genetically engineered or recombinant cells having one or more deletions from, insertions into, and / or substitutions of amino acid residues in the amino acid sequence of the native protein. Polypeptides and proteins also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. Polypeptides and proteins also include modifications, including, but not limited to, glycosylation, lipid conjugation, sulfation, derivatization such as gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0116] The terms "polynucleotide," "nucleic acid molecule," or "engineered nucleic acid molecule" are used interchangeably throughout this disclosure and include both single-stranded and double-stranded nucleic acids, including genomic DNA, RNA, mRNA, cDNA, nucleic acids of synthetic origin, or some combination thereof not related to a sequence normally found in nature. The term "isolated polynucleotide," "isolated nucleic acid molecule," or "isolated engineered nucleic acid molecule" specifically refers to a sequence not normally found in nature, such as a sequence of synthetic origin. An isolated nucleic acid molecule comprising a specific sequence may, in addition to the specific sequence, also include sequences encoding up to 10 or even up to 20 other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences. The nucleotides comprising the nucleic acid molecule may be ribonucleotides or deoxyribonucleotides, or may be modified forms of either type of nucleotide. The modifications include base modifications such as bromouridine derivatives and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoroamidates.

[0117] Biomolecules of interest (e.g., polypeptides and proteins) may be of scientific or commercial interest, such as protein-based therapeutics. Proteins of interest (e.g., proteins) may include secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins, among others. Biomolecules of interest may be produced by recombinant animal cell lines using cell culture methods, and may be referred to as "recombinant proteins" to indicate the source of the protein biomolecule, but not its structure. Proteins of the present disclosure may have the same structure as a naturally occurring protein, may be fragments thereof (e.g., fragments that specifically bind to a binding partner or enzymatically active fragments), and may also be chimeras or fusions of at least a portion of two or more proteins. Expressed proteins may be produced intracellularly or secreted into the culture medium, from which they may be recovered and / or collected according to the methods of the present disclosure. The term "isolated protein" or "isolated recombinant protein" refers to a polypeptide or protein of interest that is purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. Biomolecules of interest include proteins that exert a therapeutic effect by binding to a target (including, inter alia, the targets listed below, targets derived therefrom, targets related to them, and modifications thereof).

[0118] "Purifying" means increasing the purity of a protein in a composition by removing (partially or completely) at least one impurity from the composition. Protein recovery and purification is achieved by the disclosed methods, resulting in a more "homogeneous" protein composition that meets yield and product quality criteria (e.g., reduced product-related impurities and increased product quality).

[0119] As used herein, the term "isolated" means (i) free from at least some other proteins or polynucleotides with which it is normally found; (ii) essentially free from other proteins or polynucleotides derived from the same source (e.g., the same species or the same cell); (iii) separated from at least about 50 percent of the polypeptides, polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (iv) in operative association (by covalent or non-covalent interactions) with polypeptides or polynucleotides with which it is not naturally associated; or (v) not occurring in nature.

[0120] Biomolecules (e.g., proteins) of interest include "antigen-binding proteins." Antigen-binding proteins refer to proteins, polypeptides, or fragments thereof that contain an antigen-binding region or portion that has affinity for another molecule (antigen) to which it binds. Antigen-binding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFv) and two-chain (bivalent) scFv), muteins, multispecific proteins, and bispecific proteins.

[0121] An scFv is a single-chain antibody fragment that contains the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136, all of which are incorporated herein by reference in their relevant portions. An scFv retains the ability of the parent antibody to specifically interact with a target antigen.

[0122] The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or antigen-binding regions thereof that compete with the intact antibody for specific binding. Unless otherwise specified, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, hetero-IgG, bispecific, and oligomers or antigen-binding fragments thereof. Antibodies include IgG1, IgG2, IgG3, or IgG4 types. Also included are proteins having antigen-binding fragments or regions, e.g., Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single-chain antibody molecules, single-domain V H H, complementarity determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a target polypeptide.

[0123] Also included are human proteins, humanized proteins, and other antigen-binding proteins (eg, human and humanized antibodies) that do not produce a significant adverse immune response when administered to humans.

[0124] Also included are modified proteins, such as proteins that are chemically modified non-covalently, covalently, or both covalently and non-covalently. Also included are proteins that further include one or more post-translational modifications that may be produced by cell engineering systems or that are introduced ex vivo by enzymatic and / or chemical methods or by other methods known in the art.

[0125] The terms "multispecific construct," "multispecific protein," and "multispecific antibody" are used herein to refer to proteins that have been engineered to simultaneously bind to and neutralize at least two different antigens or at least two different epitopes on the same antigen. For example, multispecific proteins can be engineered to target immune effectors to tumors or infectious agents in combination with targeted cytotoxic agents. Multispecific proteins include triabodies, tetravalent bispecific antibodies, diabodies, triabodies, or tetrabodies capable of binding to multiple targets, multispecific proteins without antibody components such as minibodies, and single-chain proteins. Coloma, MJ, et al., Nature Biotech. 15:159-163 (1997) (relevant portions are incorporated herein by reference).

[0126] The most common and diverse group of multispecific proteins are those that bind two antigens and are referred to herein as "bispecifics," "bispecific constructs," "bispecific proteins," and "bispecific antibodies." Bispecific proteins can be classified into two broad categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like molecules retain Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). The Fc region helps improve solubility and stability and facilitates some purification procedures. Non-IgG-like molecules are smaller and have enhanced tissue penetration (see Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol & Oncology 8:130-143, 2015; Spiess et al., Mol Immunol 67, 95-106, 2015; Williams et al., Chapter 41 Process Design for Bispecific Antibodies in Biopharmaceutical Processing Development, Design and Implementation of Manufacturing Processes, Jagschies et al., eds., 2018, pages 837-855, all of which are incorporated herein by reference in relevant parts). Bispecific proteins may have binding specificities for different antigens or several epitopes and be used as a framework for additional components that increase the binding specificity of the molecule.

[0127] Forms of bispecific proteins, including diabodies, are constantly evolving and include, but are not limited to, single chain antibodies, quadromas, knob-in-holes, cross mAbs, dual variable domain IgG (DVD-IgG), IgG single chain Fv (scFv), scFv-CH3 KIH, dual acting Fab (DAF), half molecule exchange, kappa lambda bodies, tandem scFv, scFv-Fc, diabodies, single chain diabodies (scDiabodies), scDiabody-CH3, triple bodies, miniantibodies, minibodies, TriBi minibodies, tandem diabodies, scDiabody-HAS, tandem scFv-toxins, dual affinity retargeting molecules (DARTs), nanobodies, nanobodies-HSA, dock and lock ( DNL), strand exchange engineered domain SEEDbody, Triomab, Leucine Zipper (LUZ-Y), antibodies generated using XmAb® technology; Fab-arm exchange, DutaMab, DT-IgG, charge pair, Fcab, orthogonal Fab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-VV(L)-IgG, KIH Bispecific T cell engagers including IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-Ig4 (four-in-one), Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, intrabody, ImmTAC, HSABody, IgG-IgG, CoV-X-Body, scFv1-PEG-scFv2, BiTE® molecules (Fan, supra; Spiess, supra; Sedykh, supra; Seimetz et al., Cancer Treat Rev 36(6)458-67, 2010; Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottschalk editor, pp. 559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011 (relevant portions of each are incorporated herein by reference). Biomolecules of interest (e.g., proteins) can also include recombinant fusion proteins containing multimerization domains, such as leucine zippers, coiled coils, or the Fc portion of immunoglobulins. Also included are proteins containing all or part of the amino acid sequence of differentiation antigens (referred to as CD proteins), or their ligands, or proteins substantially similar to any of these.

[0128] Biomolecules (e.g., proteins) of interest can also include engineered receptors, such as chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs), as well as other proteins containing antigen-binding molecules that interact with target antigens. CARs can be engineered to bind to antigens (such as cell surface antigens) by incorporating antigen-binding molecules that interact with the target antigen. CARs typically incorporate an antigen-binding domain (such as an scFv) in conjunction with one or more costimulatory ("signaling") domains and one or more intracellular activation domains.

[0129] In some embodiments, the biomolecule (e.g., protein) of interest can include a colony-stimulating factor, such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are erythropoiesis-stimulating agents (ESAs), such as Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methoxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), and Binocrit®. (epoetin alfa), epoetin alfa Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, and molecules or variants or analogs thereof, and biosimilars of any of the foregoing.

[0130] In some embodiments, the biomolecule (e.g., protein) of interest may include a protein that specifically binds to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factors (CSFs), other blood and serum proteins, blood group antigens, receptors, receptor-related proteins, growth hormones, growth hormone receptors, T-cell receptors, neurotrophic factors, neurotrophins, relaxin, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0131] In some embodiments, the biomolecule (e.g., protein) of interest binds to one or more of the following, alone or in any combination: CD proteins, including but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, such as HER2, HER3, HER4, and EGF receptor, EGFRvIII; cell adhesion molecules, such as LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin, growth factors, such as, but not limited to, vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factors, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, such as For example, aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), such as TGF-alpha and TGF-beta, e.g., TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, and TGF-beta5, among others, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors, insulin and insulin-related proteins, such as, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin -like growth factor binding proteins; (clotting and coagulation-related proteins, e.g., Factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, e.g., urokinase and tissue plasminogen activator (“t-PA”), bombadin, thrombin, thrombopoietin, and thrombopoietin receptors, colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF, among others, other blood and serum proteins, e.g., but not limited to, albumin, IgE,and blood group antigens, receptors and receptor-associated proteins, such as flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, T cell receptor, neurotrophic factors, such as, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), relaxin A chain, relaxin B chain, and prorelaxin, interferons, such as interferon-alpha, -beta, and -gamma, interleukins (IL) and their receptors, such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2, or IL-17 receptor, IL-1RAP , IL1-α, IL-1β, viral antigens, including, but not limited to, AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, Ig kappa, ROR-1, ERBB2, mesothelin, RANTES (regulated upon activation, normally expressed and secreted by T cells), mouse gonadotropin-related peptide, DNase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, PSA, addressin, regulatory protein, immunoadhesin, antigen-binding protein, somatotropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-Met, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, P-cadherin, NKG2D-1,Programmed cell death protein 1 and its ligands PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin, SS1(dsFv)PE38 conjugate, Legionella pneumophila pneumophila (lly), gpA33, B7H3, interferon (IFN) gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7 integrin, platelet-specific (platelet glycoprotein Iib / IIIb (PAC-1), transforming growth factor beta (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRα), sclerostin, and biologically active fragments or variants of any of the foregoing.

[0132] In some embodiments, the biomolecule (e.g., protein) of interest includes abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosuzumab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, Ximab, ipilimumab, lerdelimumab, rumiliximab, ixekizumab (lxdkizumab), mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, palivizumab, panitumumab, pembu Lorisumab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargramostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, zalutumumab, and biosimilars of any of the foregoing.

[0133] In some embodiments, the biomolecule (e.g., protein) of interest may include: blinatumomab, catumaxomab, ertumaxomab, solitomab, targomiR, rutikizumab (ABT981), vanucizumab (RG7221), memtolumab (ABT122), ozoralizumab (ATN103), flotetuzumab (MGD006), pasotuxizumab (AMG112, MT112), lym phomun(FBTA05), (ATN-103), AMG211(MT111, Medi-1565), AMG330, AMG420(B1836909), AMG-110(MT110), MDX-44 7, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, Injiu Labeled IMP-205, xm734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013 (ACE910), RG7597 (MEDH7945A), RG7802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM- 111, MM141, MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352, and modified molecules or variants or analogs thereof, and biosimilars of any of the foregoing.

[0134] Biomolecules (e.g., proteins) of interest according to the present disclosure encompass all of the foregoing, and further include antibodies comprising one, two, three, four, five, or six of the complementarity-determining regions (CDRs) of any of the foregoing antibodies. Also included are variants comprising regions whose amino acid sequence is 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identical to the reference amino acid sequence of the biomolecule of interest in protein form. Identity in this regard can be determined using a variety of well-known and readily available forms of amino acid sequence analysis software. Preferred software includes those implementing the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of sequence searching and alignment. Other algorithms may also be used, particularly where speed is an important consideration. Commonly used programs for DNA, RNA, and polypeptide alignment and homology matching that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter of which is an implementation of the Smith-Waterman algorithm for execution on massively parallel processors manufactured by MasPar.

[0135] Chimeric antigen receptors (CARs) incorporate one or more costimulatory (signaling) domains known in the art to increase the potency of CAR-T cell responses. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (see above), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016) (relevant portions of each are incorporated herein by reference).Exemplary costimulatory domains for incorporation into a CAR can be derived from, among other sources: CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LF A-1), lymphocyte function-associated antigen-1 (LF A-1), lymphocyte function-associated antigen-1 (LDA ... la / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signaling lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8beta, IL-2Rbeta, IL-2Rgamma, IL-7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDL-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CD l-lb, ITGAX, CDl-lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, 41-BB, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof.A costimulatory domain can include one or more of the extracellular portion, the transmembrane portion, and the intracellular portion of the protein from which it is derived.

[0136] The following examples, both demonstrative and prophetic, are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope. [Example]

[0137] Example 1: Disposable continuous solids discharge disc stack centrifugation, flocculation, and depth filtration of high density cell cultures. This example provides a harvesting method for purifying target proteins from high-density, continuously perfused mammalian cell cultures using a purification protocol that utilizes a disposable, continuous solids-discharging, disk-stack centrifugation step, followed by flocculation and depth filtration. This harvesting method produces high yields of purified protein from high-density cultures using an unconventional yet effective process.

[0138] Nine different perfusion cultures were run at 500 L scale. The cultures contained one of four monoclonal antibodies (mAbs) (i.e., mAb1 (runs 1, 2, and 3), mAb2, mAb3, or mAb4), xmAb (runs 1 and 2), or the cultures contained one of two bispecific antibodies (Bsp) (i.e., Bsp1 and Bsp2).

[0139] On day 0, CHO cells expressing the monoclonal or bispecific antibody were seeded into a 500 L disposable bioreactor with a working volume of 450-500 L of serum-free, chemically defined batch medium. The culture was initiated in batch mode at 36 °C with stirring. The culture was then grown in a 500 L disposable bioreactor with a nominal surface area of ​​2.1 m. 2Cultures were continuously perfused until harvest using an X-CELL ATF-6® alternating tangential flow (ATF) filtration system (Repligen, Waltham, MA) equipped with an ATF-6 filter and a serum-free, chemically defined perfusion medium. The cultures were subjected to a low temperature shift (32-33°C) to increase production and maintained at this low temperature until harvest.

[0140] On day 15 or 18, the contents of the bioreactor were bulk harvested. First, the culture was cooled to 10°C. The cell culture broth was then either harvested directly at 10°C or collected in a holding tank and held at 4°C. The viability, viable cell density (VCD), percent packed cell volume (PCV%), and baseline turbidity of the harvested cells were determined using conventional techniques, and the results are shown in Table 1.

[0141] Undiluted cell culture was subjected to continuous centrifugation using a 500 L disposable continuous solids discharge disk stack centrifuge (Alfa Laval Primo™, Fresno, CA). The cell culture was continuously loaded into the centrifuge by an in-line mechanism at a specific flow rate (Table 1). The centrifuge was operated at an equivalent sedimentation velocity (Q / Sigma) of 5.25 E-9 m / s to 1.5 × E-8 m / s or 0.6 to 1.8 (L / h / KQ), with PCV% values ​​ranging from 16 to 30%.

[0142] The feed flow rate and heavy phase pump flow rate were adjusted to maintain the desired heavy phase PCV%. The bowl speed and feed flow rate were controlled to maintain the desired light phase turbidity value. The reported values ​​for centrifuge feed flow rate, centrate flow rate, heavy phase flow rate, and recovery yield are average values ​​at various sampling points over the course of the centrifugation process for a given sample (see Table 1).

[0143] As shown in Table 1, the average bowl speed varied depending on the molecule being recovered and ranged from 4713 to 5200 rpm using an Alfa Laval Primo™ bowl. The average centrifuge feed flow rate was 0.7 to 2.0 L / min (LPM). The centrate was continuously collected in a collection tank and held at 10°C with mixing. The concentrate (heavy phase) was discharged to waste. The average centrate flow rate was 0.79 to 1.51 L / min. The average concentrate (heavy phase) flow rate was 0.29 to 0.61 L / min. The average concentrate (heavy phase) PCV% was 77 to 96%. In-line centrate measurements were performed periodically, and the average centrate turbidity was 165 to 940 nephelometric turbidity units (NTU). The average theoretical yield of the centrifugation was 88 to 99%. In implementing the disposable, continuous solids discharge disk stack centrifuge according to the disclosed method, attention is paid to the feed flow rate, bowl speed, and heavy-phase PCV % target, which are affected by the feed cell density and the heavy-phase pump flow rate. These parameters are summarized using the Q / Sigma value, as described herein, which is positively correlated with turbidity, since an increase in feed flow rate (Q) for a given equivalent settling area (Sigma) increases turbidity. Important centrifugation operating parameters include centrifuge bowl speed, feed flow rate, cell culture solids (PCV), and heavy-phase (cell paste) solids (PCV). Exemplary values ​​for these parameters are disclosed throughout this application. Adjusting these parameters controls the normalized settling velocity (Q / Sigma) and theoretical process yield. As known in the art, Q / Sigma provides a measure of normalized settling velocity, where Q is the feed flow rate and Sigma is the normalized settling area of ​​the spinning bowl. To achieve a relatively low Q / Sigma, the centrifuge is operated at a lower feed flow rate, allowing for a longer residence time for the cells and capture of larger debris, resulting in less debris in the centrate.

[0144] Centrate turbidity is generally proportional to particle (especially particulate) concentration. As the data herein demonstrate, harvest and centrate turbidity vary to some extent due to differences in cell culture, cell density, packed cell volume, and percent viability of various cultures to be harvested according to the present disclosure. It is noteworthy that existing techniques using intermittent solids discharge centrifugation are only suitable for low cell density cultures with PCV values ​​of 3-12% at cell culture harvest. Other approaches to obtaining proteins of interest from cell cultures using fed-batch cell cultures result in lower turbidity, which is attributed to lower cell densities and lower PCV%, consistent with reduced particle load. Some approaches operate harvest operations in which cell cultures with relatively low PCV% are mixed with cell paste. This results in a mixed cell-containing solution with an artificially elevated PCV% in an environment with artificially high cell viability and correspondingly low cell debris, allowing for high cell culture feed or flow rates that would be unsuitable for actual cell culture harvest operations.

[0145] As demonstrated by the experiments disclosed herein, a disposable, continuous solids-discharge, disc-stack centrifuge was effective in removing high yields of whole cells from cell culture broth. The observed mean centrate turbidity ranged from 165 to 941 NTU, indicating high levels of cell debris remaining in solution. The primary contributor to centrate turbidity is accumulated cell debris, which is primarily due to cell death mechanisms within the bioreactor or cell culture vessel, but may also be influenced by the centrifuge's operating method. Lower feed flow rates and higher bowl speeds, for example, reduce turbidity. Turbidity fluctuations complicate depth filter sizing. This complexity was overcome by adding a flocculation step after centrifugation. The addition of a flocculation step improves process robustness against turbidity fluctuations in actual target protein recovery. The benefits of adding a flocculation step were demonstrated in a comparative study comparing centrate clarification by flocculation followed by depth filtration with clarification by depth filtration alone (see Example 2). First, the effect of flocculation concentration on turbidity was tested by adding pDADMAC at various concentrations to the centrate, followed by laboratory-scale centrifugation and supernatant turbidity measurements. Low turbidity was observed at concentrations of 0.04%–0.10% (w / w) pDADMAc (Figure 5). A concentration of 0.05% (w / v) pDADMAC, followed by depth filtration, was tested using various depth filters, and the results were compared to those obtained using depth filtration alone. More specifically, the results of these experiments showed a 0.85–16.2-fold increase in depth filtration loading capacity after pDADMAC treatment compared to depth filtration alone (Figure 1). In addition to increased depth filter loading levels, similar or improved DNA and host cell protein (HCP) clearance was observed with flocculation and depth filtration compared to depth filtration alone (Figures 3 and 4). Improved filter loading and impurity clearance did not adversely affect process yield when compared to depth filtration alone (Figure 2). In general, similar or improved process yields were observed for flocculation and depth filtration when compared to depth filtration alone.The level of product loss is acceptable given the increased clarity of the centrate, which was suitable for direct application to chromatographic fractionation (e.g., affinity binding of antibody biopharmaceuticals to a Protein A column).

[0146] [Table 1]

[0147] Example 2. Depth filtration of flocculated centrate In the disclosed recovery method, after disposable, continuous solids-discharging disc stack centrifugation and flocculation / sedimentation, the centrate material is further purified using depth filtration. Therefore, the liquid fraction of the flocculated centrate material obtained from the large-scale recovery described in Example 1 was used in flocculation and small-scale depth filtration experiments, as briefly described in Example 1 above, to compare depth filtration performance between untreated centrate and pDADMAc-flocculated centrate. To 1 L centrate samples, pDADMAc (polydiallyldimethylammonium chloride, Sigma Aldrich, St. Louis, MO) stock solution was added to a final concentration of 0.025-0.01% (w / w) pDADMAc. A 1 L sample from each centrate was left untreated to serve as a treatment control. The pDADMAC solution was added continuously to the centrate over approximately 1 minute, and stirring was continued for at least 30 minutes at 10°C. Agitation was achieved using a stir plate and stir bar, and the stirring speed was varied to maintain constant mixing of the centrate and flocculated material. A total of 56 conditions were tested across various modalities and various depth filters. Results indicated that 0.035-0.10% (w / w) pDADMAC defined the optimal concentration range for flocculation of various centrates containing target proteins (see Figures 5 and 6). The data indicated optimal and suboptimal, yet still effective, flocculant concentrations, and consistent with this data, the present disclosure contemplates a range of 0.025-0.15% flocculant for use in the disclosed recovery methods.

[0148] The particle size distribution in unprocessed centrates of various molecules was measured using dynamic light scattering (DLS) to be in the 1-60 μm range, with most particles in the 1-40 μm range. Therefore, filters with larger pore size distributions, such as COHC and COSP, may not be suitable for unprocessed material. This particle size range is consistent with the nominal pore size distribution of these filters. Additionally, the number of finer particles in the unprocessed material increases, potentially causing internal filter clogging and further compromising depth filtration throughput. In contrast, pDADMAC flocculation works by agglomerating finer molecules together into larger particles. DLS data showed that upon pDADMAC treatment, particle number decreased and average particle size increased. This may explain the increased throughput of depth filters used after pDADMAC flocculation; larger particles are retained by the filter, preventing internal clogging compared to unprocessed material, which contains a larger number of finer particles, constituting a higher percentage of the total particles filtered. Selecting the correct treatment type, treatment concentration, and nominal pore size rating of the depth filter is relevant to optimizing depth filter loading, process yield, and impurity removal.

[0149] Six small-scale micropod depth filters were tested: MILLISTAK+® X0HC, MILLISTAK+® X0SP, MILLISTAK+® COHC, MILLISTAK+® COSP (MilliporeSigma, Burlington, Mass.), SARTOCLEAR® DL60, and SARTOCLEAR® DL75 (Sartorius, Bohemia, NY). Millipore and Sartorius depth filters each had a 23 cm 2 and 25cm 2 It was.

[0150] The depth filters used in the small-scale experiments varied in pore size distribution and material composition. Millipore X0HC and COHC depth filters are composed of cellulose and inorganic filter aids. X0HC pore sizes ranged from 0.1 to 0.5 μm, while COHC depth filter pore sizes ranged from 0.3 to 1.0 μm. Millipore X0SP and COSP depth filters are synthetic with the same pore size distribution as the X0HC and COHC depth filters. Sartorius DL60 and DL75 filters have a wider pore size distribution than Millipore depth filters, with nominal pore size distributions of 0.6 to 10 μm and 2 to 14 μm, respectively. The DL60 depth filter has a pure cellulose prefilter combined with a coarse secondary filter. The DL75 depth filter contains a coarse primary-grade filter and a fine secondary filtration-grade filter. The variety of pore size distributions and material properties in the depth filters used in this development ensured that a large depth filtration dataset was available to support appropriate depth filter selection for the recovery methods of the present disclosure.

[0151] Prior to loading the treated and untreated samples, the depth filters were flushed with purified water or buffer solution at a temperature of approximately 10°C and a flow rate of less than 400 liters per square meter per hour (LMH) flux to ensure a minimum of 100 L / m 2 After flushing the primary depth filter, a secondary 0.2 μm Optiscale Capsule SHC filter was attached to the primary filter (MilliporeSigma, Burlington, Mass.) and this secondary filter was flushed at the same flux rate, achieving a target throughput of at least 50 L / m. 2 The surface of the secondary filter was 3.6 cm 2 and the first-stage to second-stage depth filtration surface area ratios were 6.38 to 7.14 for Millipore and Sartorius depth filters, respectively.

[0152] After flushing with water, the inlet of the primary depth filter was drained to remove excess water. The depth filter was then filled with the respective load (untreated centrate or flocculated centrate) to ensure the removal of air bubbles. The filter was then loaded with either 1 L of untreated centrate or 1 L of flocculated centrate. The inlet flux during the experiment was maintained between 90 LMH and 150 LMH. The final depth filtration throughput varied depending on the depth filtration performance. At the end of the experiment, the turbidity of the depth filtrate pool was measured.

[0153] Depth filtration experiments were terminated when the inlet pressure of the first stage of the filter reached at least 20 psig or when the desired throughput was achieved. In some cases, due to time limitations, depth filtration experiments were terminated before the desired throughput was achieved. Based on data from both depth filtration and flocculation studies, a range of 0.02 to 0.1% (w / w) pDADMAC was shown to be the optimal concentration range for inducing beneficial flocculation in the disclosed recovery method.

[0154] To evaluate the performance of depth filtration at small scale, the following parameters were included: depth filtration final throughput, depth filtration yield, host cell protein (HCP) levels (otherwise known as CHOP levels) in the depth filtrate pool, DNA levels in the depth filtrate pool, normalized depth filtration throughput, HCP log reduction value (LRV), and DNA LRV. Normalized values ​​were calculated by dividing the final values ​​of the various parameters by the control condition (X0HC + untreated), using untreated X0HC depth filters as the control condition. Normalized values ​​indicate the magnitude of increase or decrease in the various parameters tested and thus help better assess the degree of improvement of the novel recovery technology. LRV was calculated by dividing the measured load value by the measured pool value and applying a base-10 logarithmic transformation.

[0155] Improved performance in depth filtration was characterized by an increase in depth filtration throughput value or normalized depth filtration throughput value, an increased or equivalent percent yield, and an increased or equivalent HCP LRV and / or DNA LRV.

[0156] Figure 1 shows normalized depth filtration throughput values ​​for various molecules, treatment types, and depth filters. The normalized depth filtration magnitude for the flocculated centrate conditions ranged from 0.85 to 16.2 times that of the untreated centrate. Three main treatment types were tested: control (X0HC + untreated), pDADMAC treatment (various depth filters + 0.05% (w / w) pDADMAC flocculation), and untreated (various depth filters other than X0HC + untreated). All conditions were run at 150 LMH flux or less. Overall, the data demonstrate a much higher depth filtration throughput using 0.05% (w / w) pDADMAC flocculation compared to the control condition using X0HC depth filters and untreated centrate, regardless of the material tested and the final PCV% of the sample. Specifically, with the exception of mAb1 and X0HC, all depth filters performed better with 0.05% (w / w) pDADMAC aggregation. While the control and untreated conditions reached a maximum pressure of 20 psig or less within a short period of time, the pDADMAC-treated conditions could be maintained at a differential pressure of less than 10 psig and operated for a longer period of time. This is advantageous because a larger volume of material could be filtered before peak pressure was reached. A peak pressure of 20 psig was considered a hazard and safety risk, and therefore depth filtration operations were limited.

[0157] Figure 2 shows that the percent yield for the 0.05% (w / w) pDADMAC-treated condition was comparable to or higher than both the control and untreated conditions. The exceptions were depth filters XOSP and XOCC in combination with pDADMAC treatment. As a result, the use of XOSP or XOCC filtration with pDADMAC-treated centrate is not preferred due to the lower yields. The control and untreated conditions exhibited lower yields, which is explained by clogging of the depth filter as it reached maximum pressure. No recovery buffer flush was performed in these experiments.

[0158] Small-scale flocculation and depth filtration pools, shown in Figure 1, were evaluated for impurity levels. Impurity levels in the depth filtrate pool were measured and compared to the centrate pool using log reduction value (LRV) calculations. When high LRV values ​​are observed, high impurity clearance is observed. HCP and DNA values ​​for the pDADMAC-treated (dark gray), untreated (light gray), and control (black) conditions are shown in Figures 3 and 4. Variability in impurity LRV was observed between centrate lots, but overall, the impurity LRV of the pDADMAC-treated centrate was similar to or better than the untreated centrate and control conditions.

[0159] The present disclosure provides a harvest method utilizing disposable continuous solids discharge disk stack centrifugation combined with centrate flocculation, e.g., using pDADMAC treatment for flocculation, and depth filtration, resulting in higher harvest yields compared to conventional perfusion culture harvest methods using microfiltration. Molecules produced in continuous perfusion cultures, such as those disclosed in Example 1, have high PCVs ranging from 16% to 30%. High PCV cell cultures pose challenges to existing microfiltration harvest technologies, resulting in variable harvest yields and high costs due to increased filter use. In addition, some molecules have demonstrated low recovery yields (approximately 70%) resulting from product losses due to cell culture variability, filter variability, and equipment variability, which together lead to variable amounts of product lost during harvest operations. Current intermittent solids discharge centrifuge technology cannot handle such high PCV cell culture harvests due to clogging of the centrifuge. It is recognized that continuous solids discharge disk stack centrifugation may not provide sufficient clarification of high solids cell culture harvests, as seen in low throughput and, in some cases, low percent yield, HCP clearance, and DNA clearance in examples of unprocessed centrate, and may not offer a robust harvest process option by itself or even in combination with depth filtration. It is the combination of continuous solids discharge disk stack centrifugation followed by flocculation that results in a robust process for recovering a wide variety of target proteins from high solids perfused cell cultures, which process also makes it feasible to incorporate a depth filtration step to further purify the target protein while maintaining high yield in a cost-effective series of steps that is compatible with further conventional downstream purification operations.

[0160] Treatment of the centrate with 0.05% (w / w) pDADMAc resulted in aggregation of smaller particles, reducing the total particle mass while effectively increasing the particle size distribution. As a result, when combined with an appropriate depth filter (e.g., Millistak+® COHC, Millistak+® COSP, Sartoclear® DL75, or Sartoclear® DL60), the centrifugation and aggregation steps increased depth filtration throughput while simultaneously recovering most of the target protein with comparable or improved recovery yield, HCP clearance, and DNA clearance. In addition, pDADMAC aggregation was able to maintain low pressure differentials throughout the run, an important aspect both in terms of safety and the ability to filter the desired amount of material while meeting depth filtration throughput targets (see Figure 8).

[0161] Example 3 Effect of pDADMAC concentration on turbidity and small-scale depth filtration performance A pDADMAC dosing study was conducted to understand the effect of pDADMAC concentration on the turbidity of the flocculated samples. Dosing studies were repeated using centrates with varying turbidities. Variation in centrate turbidity was induced by changing the modality and disposable continuous solids discharge centrifuge operating conditions as described in Table 2. Dosing studies were performed by adding pDADMAC from a stock solution to 40 mL of centrate to the final pDADMAC concentrations shown in Table 2. Control (0% pDADMAC w / w) and pDADMAC-treated centrates were mixed for at least 15 minutes. Samples were centrifuged at 3000 rpm for 17 minutes using a JS4.2 centrifuge (Beckman Coulter, Indianapolis, IN). After centrifugation, the supernatant was placed in a new conical tube, and the turbidity of the supernatant was measured using a Hach 2100P Portable Turbidimeter (Hach Company, Loveland, CO).

[0162] [Table 2]

[0163] Figure 5 shows the results of the pDADMAC dosing study described in Table 2. Figure 5A shows the results of the pDADMAC dosing study using mAb 1 (Run 2) from Table 1 and the conditions described in Table 2. This study evaluated the effect of pDADMAC concentration on the turbidity levels of various centrate sources subjected to various centrifugation flow rates and bowl speeds. Note that Table 1 shows only the average values ​​for the feed flow rates and bowl speeds. The test results revealed low supernatant turbidity values ​​in the pDADMAC concentration range of 0.04 to 0.10% (w / w). pDADMAC concentration levels below 0.04% were undesirable because they resulted in high turbidity levels.

[0164] Figure 5B shows the results of a pDADMAC dosing study from xmAb1 (Run 1). This study evaluated the effect of pDADMAC concentration on centrate sources with various initial turbidity levels. Various initial turbidities in the centrate were generated by varying the inlet flow rate of the disposable centrifuge. High- and low-turbidity centrates exhibited initial turbidity values ​​of 1500 NTU and 290 NTU, respectively. The results showed that regardless of the initial centrate turbidity level, the supernatant turbidity values ​​were low over the pDADMAC concentration range of 0.02–0.10% (w / w). A pDADMAC concentration level below 0.02% was undesirable because turbidity levels were not reduced after pDADMAC treatment.

[0165] After determining the optimal pDADMAC concentration that resulted in the lowest turbidity values, small-scale depth filtration studies were conducted using pDADMAC at concentrations of 0.02% (w / w), 0.05% (w / w), and 0.1% (w / w) to determine the concentration range that resulted in the highest depth filtration throughput. Small-scale depth filter operation was carried out as described in Example 2.

[0166] Monoclonal antibodies mAb1 (Run 1) and mAb2 were harvested from cell culture broth using a disposable, continuous solids discharge, disk-stack centrifuge as described in Example 1, Table 1. pDADMAC aggregation was performed at small scale using a 2% pDADMAC stock solution. For mAb1 (Run 1), the pDADMAC concentration range tested was 0.01%, 0.02%, and 0.05%. For mAb2, the pDADMAC concentration range tested was 0.035%, 0.05%, 0.1%, and 0.15%. All pDADMAC concentrations used in this experiment were (w / w). COHC depth filters were used for pDADMAC-treated samples. For each experiment, untreated centrate was filtered through an X0HC depth filter as a control.

[0167] The normalized depth filtration throughput values ​​from these experiments are shown in Figure 6. Normalized depth filtration throughput is calculated as the final depth filtration throughput (L / m) at small scale for pDADMAC treatment conditions. 2 The % filtration throughput was calculated by dividing the % filtration throughput (%) by the final depth filtration throughput of the control untreated condition. A normalized throughput greater than 1 indicates improved depth filtration performance compared to the control. pDADMAC concentrations between 0.035% and 0.1% (w / w) pDADMAC showed improved depth filtration performance when compared to the control.

[0168] Example 4 Effect of pDADMAC and PEG on small-scale depth filtration performance Centrate from mAb1 Run 3 was collected. This large-scale run was not included in Example 1 because it did not use a disposable continuous solids discharge centrifuge device but instead was performed using a stainless steel continuous solids discharge centrifuge. The average centrate turbidity was 182 NTU, which was similar to the average centrate turbidity for mAb1 Runs 1 and 3 and was therefore considered representative material for pDADMAC-treated depth filtration studies. Centrate obtained from the large-scale run was subjected to flocculation with 0.05% (w / w) pDADMAC alone or in combination with 3% PEG3000, as described in Example 2, followed by depth filtration. The pDADMAC and PEG3000 solutions were added to the centrate simultaneously. The flocculated centrate was mixed for at least 30 minutes before being loaded onto a COHC or COSP depth filter. Untreated centrate was loaded onto an X0HC depth filter as a control because it had previously been determined to provide the highest depth filter loading capacity for untreated centrate. The X0HC filter was used for the untreated control centrate because it is the best filter type in terms of particle size in solution for the untreated condition (see Figure 1). We used this as a control to understand how similar / different each molecule is to each other.

[0169] Figure 7 shows that the pDADMAC + PEG 3000 condition achieved increased depth filtration throughput compared to 0.05% w / w pDADMAC alone. Thus, the present disclosure provides an efficient and effective method for target protein recovery in which a continuous solids discharge disk stack centrifugation precedes a flocculation step, preferably using 0.02% to 0.15% pDADMAC with or without about 3% PEG (e.g., 3% PEG 3000).

[0170] Example 5 Comparison of large-scale and small-scale depth filtration performance mAb1 Run 1, xmAb Run 1, xmAb1 Run 2, and Bsp2, as shown in Table 1, were subjected to both large-scale and small-scale depth filtration. At large scale, centrate was collected from the disposable continuous solids discharge centrifuge described in Table 1 for mAb1 Run 1, xmAb1 Run 1, xmAb1 Run 2, and Bsp2. This centrate was subjected to large-scale flocculation and depth filtration. Large-scale depth filtration was performed using a COHC depth filter. The filter size of the COHC used for the large-scale run was 1.1 m. 2 Parallel small-scale depth filtration tests were conducted for each centrate lot, using untreated centrate and X0HC depth filters as controls. Both the small-scale and large-scale depth filters were flushed with DI water as described in Example 2. Flocculation was performed using a 2% (w / w) pDADMAC stock solution to achieve a final pDADMAC concentration of 0.05% (w / w) in the centrate. Each small-scale depth filtration condition was performed using at least 1 L of flocculated material. For each of three representative runs, small-scale depth filtration performance was evaluated using a flocculated centrate depth filtration flow rate of 150 LMH. Large-scale depth filtration performance for mAb1 Run 1, xmAb1 Run 1, xmAb1 Run 2, and BSP2 was evaluated using flocculated centrate flow rates of 65, 95, 155 LMH, and 120 LMH, respectively.

[0171] Figure 8 compares the depth filtration performance of large-scale flocculated centrate run using a COHC depth filter and 0.05% (w / w) pDADMAC treatment with the small-scale depth filtration performance using an XHC depth filter. The data demonstrates improved differential pressure trends for flocculation and XHC depth filtration (gray) compared to the control condition (black) using untreated centrate and COHC depth filtration. pDADMAC treatment and COHC filtration demonstrated stable differential pressures below 10 psig. Maintaining low differential pressures during production is an important goal, as a maximum allowable pressure of approximately 20 psig is desired in order to maintain safety and reduce risks associated with high pressure. Being able to reliably maintain low differential pressures for longer periods of time results in more material being filtered, thereby reducing the impact on downstream processes in the recovery operation. A summary of the recovery process yields for these large-scale runs is shown in Table 3. Process yields ranged from 86% to 92%, representing an improvement over the average yield of approximately 70% for microfiltration recovery processes.

[0172] [Table 3]

[0173] Example 6 Product Quality Considerations The deep filtrate pool containing the target protein was subjected to preparative Protein A purification, followed by analytical testing by chromatographic fractionation or electrophoresis to assess product quality. The types of analytical chromatography used included size exclusion chromatography (SE) and cation exchange chromatography (CEX). Electrophoretic assessment of product quality was determined using reduced capillary electrophoresis (rCE) with sodium dodecyl sulfate and non-reduced capillary electrophoresis (nrCE). These different types of analysis were performed to rigorously assess product quality.

[0174] Figure 9 shows a comparison of product quality for xmAb1 Run 1 for the pDADMAC-treated COHC filtrate pool (dark gray), the untreated centrate COHC pool (light gray), and the untreated XHC filtrate pool (black). The COHC conditions were performed using 0.05% (w / w) pDADMAC as described in Example 2. As shown in Figure 9, product quality assessment using SE, CEX, rCE, and nrCE, respectively, showed comparable results among the three materials subjected to quality assessment (i.e., untreated centrate, pDADMAC-treated centrate, and COHC depth filtration pool). These results demonstrate that treatment of cell culture centrifugation centrate with a flocculant such as pDADMAC does not adversely affect the product quality profile.

Claims

1. 1. A method for separating a recombinant protein produced in a perfusion culture from at least one other perfusion culture component, comprising: (a) harvesting a pool or effluent stream from a perfusion culture comprising the recombinant protein and at least one other perfusion culture component, wherein the perfusion culture has a packed cell volume (PCV) at the time of harvest of at least about 16%; (b) introducing the collected pool or eluate stream into at least one continuous solids discharge disk stack centrifuge; (c) operating said continuous solids discharge disk stack centrifuge, thereby separating the liquid components into a centrate and a high density composition; (d) collecting the centrate; (e) adding a flocculant to the centrate; (f) subjecting the centrifuged solution to a filtration step; A method comprising:

2. 10. The method of claim 1, wherein the continuous solids discharge disc stack centrifuge is a disposable continuous solids discharge disc stack centrifuge.

3. 3. The method of claim 1 or 2, wherein the flocculant is added to the centrate at a temperature of between 8°C and 12°C.

4. 4. The method of claim 3, wherein the flocculant is added to the centrate at a temperature of about 10°C.

5. 5. The method of any one of claims 1 to 4, wherein the perfusion culture is harvested from a disposable bioreactor.

6. 6. The method of claim 5, wherein the disposable bioreactor is at least 500 L.

7. 6. The method of claim 5, wherein the disposable bioreactor is 2,000 L or larger.

8. 8. The method of any one of claims 1 to 7, wherein the perfusion culture harvest pool or effluent stream had a turbidity of at least 180 NTU at the time of harvest.

9. The perfusion culture harvest pool or effluent stream has a viable cell density of at least 2 x 10 cells at harvest. ７ The method according to any one of claims 1 to 8, wherein the concentration of viable cells was 1000 or more per ml.

10. The perfusion culture harvest pool or effluent stream has a viable cell density of at least 3 x 10 cells at harvest. ７ The method of claim 9, wherein the total number of viable cells per ml was 100.

11. The perfusion culture harvest pool or effluent stream has a viable cell density of at least 5×10 at harvest. ７ The method of claim 9, wherein the total number of viable cells per ml was 100.

12. 12. The method of any one of claims 1 to 11, wherein the perfusion culture harvest pool or effluent stream had a packed cell volume of at least about 18% at the time of harvest.

13. 13. The method of claim 12, wherein the perfusion culture had a packed cell volume of at least about 20% at harvest.

14. 13. The method of claim 12, wherein the perfusion culture had a packed cell volume of at least about 24% at harvest.

15. 13. The method of claim 12, wherein the perfusion culture had a packed cell volume of at least about 26% at harvest.

16. 13. The method of claim 12, wherein the perfusion culture had a packed cell volume of at least about 30% at harvest.

17. 17. The method of any one of claims 1 to 16, wherein the temperature of the perfusion culture at the time of harvest is from about 8°C to about 12°C.

18. 18. The method of claim 17, wherein the temperature of the perfusion culture at the time of harvest is about 10°C.

19. 19. The method of any one of claims 1 to 18, wherein a continuous harvest eluate stream from the perfusion culture is introduced into the continuous solids discharge disc stack centrifuge.

20. 20. The method of any one of claims 1 to 19, wherein discrete batches of perfusion harvest pool are introduced into said continuous solids discharge disk stack centrifuge.

21. 21. The method of any one of claims 1 to 20, wherein the turbidity of the centrate is greater than about 160 NTU.

22. 22. The method of any one of claims 1 to 21, wherein the flocculating agent is poly(diallyldimethylammonium chloride) (pDADMAC).

23. 23. The method of claim 22, wherein poly(diallyldimethylammonium chloride) (pDADMAC) as a flocculant is added to at least 0.04% (w / w).

24. 23. The method of claim 22, wherein poly(diallyldimethylammonium chloride) (pDADMAC) as a flocculant is added to 0.04-0.15% (w / w).

25. 23. The method of claim 22, wherein poly(diallyldimethylammonium chloride) (pDADMAC) as a flocculant is added up to 0.05% (w / w).

26. The method of any one of claims 1 to 25, wherein the recombinant protein is a eukaryotic protein.

27. 27. The method of claim 26, wherein the eukaryotic protein is a mammalian protein.

28. 28. The method of claim 27, wherein the mammalian protein is an antigen-binding protein.

29. 29. The method of claim 28, wherein the antigen binding protein is a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a bispecific T cell engager molecule (BiTE®).

30. 30. The method of any one of claims 1 to 29, wherein the protein is a granulocyte colony stimulating factor, an erythropoiesis stimulating agent, a HER receptor, a cell adhesion molecule, a growth factor, an osteoinductive factor, insulin, a coagulation protein, a colony stimulating factor, a blood group antigen; growth hormone, a growth hormone receptor, a T cell receptor; a neurotrophic factor, a neurotrophin, relaxin, an interferon, an interleukin, a viral antigen, a lipoprotein, an integrin, a rheumatoid factor, an immunotoxin, a surface membrane protein, a transport protein, a homing receptor, an addressin, a regulatory protein, or an immunoadhesin.

31. The method of any one of claims 1 to 30, wherein the filtration comprises a depth filter.

32. 32. The method of claim 31, wherein the depth filter is a MILLISTAK+® COHC filter, a MILLISTAK+® COSP filter, a SARTOCLEAR® DL60 filter, or a SARTOCLEAR® DL75 filter.

33. 32. The method of claim 31, wherein the depth filter is a MILLISTAK+® COHC filter or a MILLISTAK+® COSP filter.

34. 34. The method of any one of claims 31 to 33, wherein the centrate is passed through the depth filter at a flux rate of no more than 150 LMH and a pressure of no more than 10 psi.

35. 35. The method of claim 34, wherein the pressure is 2 psi or less.

36. 35. The method of claim 34, wherein the flux rate is 90 to 150 LMH.

37. 37. The method of any one of claims 1 to 36, further comprising at least one additional chromatography step.

38. 36. The method of claim 35, wherein the chromatography step is selected from ion exchange chromatography, hydrophobic interaction chromatography, or multimodal chromatography.

39. 39. The method of any one of claims 1 to 38, further comprising at least one or more virus filtration, virus inactivation, and / or UFDF steps.

40. A target protein produced by the method of any one of claims 1 to 39.

41. 1. A method for maintaining a low differential pressure during depth filtration of a load feed from a perfusion culture, comprising: (a) obtaining a flocculated centrate from a perfusion culture having a packed cell volume (PCV) at harvest of at least 16%; (b) passing the centrate through a depth filter at a flux rate of 150 LMH or less and a pressure of 10 psi or less; (c) collecting the eluate; A method comprising:

42. 1. A method for producing isolated, purified recombinant proteins from a perfusion culture initiated in a single-use bioreactor, comprising: a. inoculating the bioreactor with cells engineered to recombinantly express a protein of interest; b. culturing the cells until the perfusion culture has a packed cell volume of at least 16%; c. reducing the temperature of the culture to 8°C to 12°C and collecting the perfusion culture from the bioreactor as a harvest pool or effluent stream; d. introducing said collection pool or eluate stream into at least one continuous solids discharge disk stack centrifuge, wherein said collection pool or eluate stream is at a temperature between 4°C and 12°C; e. collecting centrate from said centrifuge; f. adding a flocculant to the centrate at a temperature of 8°C to 12°C; g. passing the flocculated centrate through a depth filter at a flux rate of 90-150 LMH and a pressure of 10 psi or less, wherein the flocculated centrate is at 4°C to 12°C. h. subjecting the filtered centrate to one or more chromatography, filtration, and / or UFDF unit operations; i. Obtaining an isolated and purified recombinant protein; A method comprising:

43. 41. A pharmaceutical composition comprising an isolated, purified recombinant protein produced by the method of claim 40.

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