Methods of harvesting products from perfusion cultures

Abstract

The present disclosure provides methods for purifying a target protein, e.g., an antibody, from a host cell, such as a mammalian cell using a purification protocol that incorporates harvest and recovery operations including a continuous solids discharge disc stacked centrifugation step followed by a flocculation and deep filtration step. Protein harvesting methods of the present disclosure use an unconventional but effective process to recover high yields of purified target proteins from perfusion cultures having a cell hematocrit greater than or equal to 16%.

Description

Technical Field

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

[0002] Modern medicine faces many challenges in its efforts to address the various diseases and conditions that plague humans and other animal species. The development of new and better treatments goes beyond the efforts to develop new and better drugs, which include large molecule biological compounds or biologics, such as monoclonal antibodies (mAbs), antibody fragments, antibody-like formats, multispecific antibodies, engineered proteins, biosimilars, etc. In recent years, these efforts have steadily yielded results, with an increasing number of therapeutically active biologics approved for use in humans and meeting unmet needs in the medical community. A natural adjunct to the efforts to develop new therapeutic agents is the effort to improve manufacturing processes to increase the yield of complete functional therapeutic biologics while maintaining product quality, reducing production costs, improving efficiency and sustainability, and simplifying production at pilot and commercial scale production levels.

[0003] The manufacture of therapeutic biologics is a multi-step process involving production, recovery, and purification / refining operations. Biologics are typically produced by host cell expression in cell culture, which is typically a mammalian cell culture used to express the desired recombinant protein from the host cells. The 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 initially clarified in preparation for the separation and purification of the target therapeutic protein from other components of the cell culture fluid. Such components include intact cells, cell debris, process and product related impurities, including nucleic acids such as DNA, proteins such as non-target host cell proteins and high and low molecular weight variants of the desired therapeutic protein, lipids, carbohydrates, viral particles, etc.

[0004] As cell culture practices improve and cell packed volumes, cell densities, titers, process and product related impurities increase, downstream processes are under pressure to effectively isolate and purify target proteins. This is especially true for harvesting operations from perfusion cultures (including continuous perfusion cultures), which can be overwhelmed by increasing cell packed volumes, increasing impurity levels and increasing titers.

[0005] For the reasons stated above, there is a continuing need in the art for a biomolecule (e.g., biologic) purification process involving a harvest recovery step that efficiently and cost-effectively results in high yield separation of the biologic from other cell culture components produced by a turbid perfusion culture having a packed cell volume (PCV) of at least 16% at harvest. Summary of the invention

[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) harvesting a pool or eluate stream from a perfusion culture comprising the recombinant protein and at least one other perfusion culture component, the perfusion culture having a packed cell volume (PCV) of at least about 16% at the time of harvest; (b) introducing the harvested pool or eluate stream into at least one continuous solids discharge disc stack centrifuge; (c) operating the continuous solids discharge disc stack centrifuge to separate the fluid components into a centrate and a high density composition; (c) collecting the centrate; (d) adding a flocculant to the centrate at a temperature of 8°C to 12°C; and (e) 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, such as a single use 500 L bioreactor or a 2,000 L or larger single use bioreactor.

[0008] In some embodiments, the perfusion culture harvest pool or eluate stream of any of the disclosed methods has a turbidity of at least 180 NTU at the time of harvest. In some embodiments, the perfusion culture harvest pool or eluate stream of any of the disclosed methods has a turbidity of at least 2×10 7 viable cells / ml, or at least 3 × 10 7 viable cells / ml, or at least 5 × 10 7 The viable cell density was 1 viable cell / ml.

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

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

[0011] In some embodiments, a continuously harvested eluate stream from a perfusion culture of any of the disclosed methods is introduced into a continuous solids discharge disc stack centrifuge.

[0012] In other embodiments, discrete batches of perfusion harvest cells of any of the disclosed methods are introduced into a continuous solids discharge disc stack centrifuge.

[0013] In some embodiments, the centrate of any of the disclosed methods has a turbidity greater than about 160 NTU, such as a turbidity 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 monomeric 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 disclosed methods, the recombinant protein is a mammalian antigen binding protein, and the antigen binding protein is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a bispecific T cell engager molecule

[0017] In other embodiments, the protein of any of the disclosed methods 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 transporter, a homing receptor, an addressin, a regulatory protein, or an immunoadhesin.

[0018] In some embodiments, the filtering step in any of the disclosed methods comprises depth filtering. Exemplary depth filters that can be used in any of the disclosed methods include C0HC filter, C0SP filter, DL60 filter or DL75 filter. In one embodiment, the depth filter is C0HC filter or COSP filter, and wherein centrifuge liquid passes through this depth filter with 150LMH or less flux rate and 10psi or less pressure. In certain embodiments, this pressure is 2psi or less. In certain embodiments, this flux rate is 90 to 150LMH.

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

[0020] In addition, the present disclosure provides any of the disclosed methods, further comprising at least one or more virus filtration steps, virus 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 loaded feed derived from a perfusion culture, the method comprising the steps of: obtaining a flocculated centrate derived from a perfusion culture having a packed cell volume (PCV) of at least 16% at harvest; passing the centrate through the depth filter at a flux rate of 150 LMH or less and a filtration differential pressure of 10 psi or less; and recovering the eluate.

[0023] The present disclosure also provides methods for producing isolated, purified recombinant proteins from perfusion cultures, the methods comprising the steps of: (a) initiating a perfusion culture in a single-use 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; (e.) introducing the harvest pool or eluate stream into at least one continuous solids discharge circle; in a disc stack centrifuge, wherein the harvest pool or eluate stream is between 4°C and 12°C; (f.) collecting centrate from the centrifuge; (g.) adding a flocculant to the centrate at a temperature between 8°C and 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 between 4°C and 12°C; (i.) subjecting the filtered centrate to one or more chromatography, filtration and / or UFDF unit operations; and (j.) obtaining an isolated, purified recombinant protein.

[0024] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated, purified recombinant protein produced by any of the methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 . Effect of flocculation on small-scale normalized depth filter loading levels. Figure 1 Normalized small-scale depth filtration performance using untreated and flocculated material from large-scale single-use continuous solids discharge disk stack centrifugation is shown. Centrate flocculation increased normalized depth filter throughput relative to control under all but one of the conditions tested. Small-scale depth filtration final throughput (L / m) of pDADMAC treated (dark grey) and untreated (light grey) conditions was plotted. 2 ) divided by the final throughput of the control condition (black) (L / m 2 ) was used to calculate normalized throughput. Control conditions (black) were run using untreated material paired with a XOHC depth filter. pDADMAC conditions were run using 0.05% (w / w; weight pDADMAC / weight centrate) pDADMAC treated centrate paired with a COHC, COSP, XOHC, XOSP, DL60 or DL75 depth filter. Untreated conditions were run using untreated centrate paired with a COSP, COHC or XOSP depth filter. Due to time constraints, the following runs were terminated early during the small scale depth filtration process: xmAb Run 1, xmAb Run 2 and Bispecific 2. Higher throughput would have been achieved for these runs if the run time had been extended. Error bars represent standard deviations from the mean ± 1. Bsp = bispecific.

[0026] Figure 2 . Effect of flocculation and small-scale depth filtration on percent yield. Figure 2 Shows Figure 1 Small scale flocculation and depth filtration step yields for the experiments shown. Flocculation followed by depth filtration conditions (dark grey) had similar or improved step yields when compared to control conditions (black) or untreated conditions (light grey). Percent yields were calculated by dividing the total protein mass (g) measured in the depth filtrate pool by the total protein mass (g) measured in the centrate pool. pDADMAC conditions were run using 0.05% w / w pDADMAC treated centrate paired with COHC, COSP, XOHC, XOSP, DL60 or DL75 depth filters. Untreated conditions were run using untreated centrate paired with COSP, COHC or XOSP depth filters. Error bars represent standard deviations from the mean ± 1. Bsp = bispecific.

[0027] Figure 3 . Effect of host cell protein (HCP) levels on small-scale flocculation. Figure 3 Shows Figure 1 Small-scale flocculation and depth filtration HCP log reduction values ​​(LRVs) for the experiments shown. After accounting for HCP assay variability, the post-flocculation depth filtration condition (dark grey) showed similar HCP clearance to the control condition (black) and the untreated condition (light grey). The HCP LRV was calculated by applying a logarithmic transformation to the ratio of the HCP levels measured in the centrate pool to the HCP levels measured in the depth filtrate pool. The control condition (black) was operated using untreated material paired with a X0HC depth filter. The pDADMAC condition was operated using 0.05% (w / w) pDADMAC treated centrate paired 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 = bispecific.

[0028] Figure 4 . Effects of small-scale flocculation and depth filtration on DNA levels. Figure 4 Shows Figure 1 Small scale flocculation and depth filtration DNA LRVs for the experiments shown. Post-flocculation depth filtration conditions (dark grey) showed similar or improved DNA clearance relative to control conditions (black) and untreated conditions (light grey). DNA LRVs were calculated by applying a base 10 logarithmic transformation to the ratio of DNA levels measured in the centrate pool to those measured in the depth filtrate pool. Control conditions (black) were run using untreated material paired with a X0HC depth filter. pDADMAC conditions were run using 0.05% (w / w) pDADMAC treated centrate paired with a COHC, COSP, X0HC, X0SP, DL60 or DL75 depth filter. Untreated conditions were run using untreated centrate paired with a COSP, COHC or X0SP depth filter. Bsp = bispecific.

[0029] Figure 5. 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 bench-scale centrifugation. Changes in centrifuge feed flow rate and centrifuge bowl speed were used to generate changes in initial centrate turbidity. pDADMAC concentrations greater than 0.04 (% w / w) reduced the centrate turbidity of the two model molecules tested, namely mAb 1 Run 2 (A) and xmAb Run 1 (B).

[0030] Figure 6. 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 centrates from (A) mAb1 (Run 1) and (B) mAb 2. Increased depth filtration throughput was observed using 0.05% to 0.1% w / w pDADMAC (for mAb1) and 0.035% to 0.1% w / w pDADMAC (for mAb 2) compared to the control (black). Untreated material was paired with a X0HC depth filter. pDADMAC conditions were operated using 0.02%, 0.035%, 0.05% or 0.1% w / w pDADMAC treated centrates paired with a COHC depth filter. The small-scale depth filtration final throughput (L / m 2 ) divided by the final throughput of the small-scale depth filtration under untreated conditions (L / m 2 ) to calculate the normalized throughput.

[0031] Figure 7 Effects of pDADMAC and PEG3000 treatment on depth filtration performance. Figure 7 A comparison of small-scale depth filtration performance between pDADMAC and pDADMAC+PEG3000 treatment conditions using representative large-scale centrifuges from run mAb1 run 3 is shown. The mAb1 run 3 large-scale run was performed using a stainless steel continuous discharge centrifuge. The initial untreated centrifuge turbidity values ​​were similar to those observed using a single-use continuous solid discharge centrifuge and are considered representative of the depth filtration study. When compared to the control, an increase in normalized depth filter throughput was observed for the centrifuges treated with pDADMAC and pDADMAC+PEG3000. Untreated material was paired with a X0HC depth filter. 0.05% w / w pDADMAC was paired with a COHC depth filter. pDADMAC (0.05% w / w)+PEG3000 was paired with COHC and COSP depth filters. The final throughput (L / m2) of the small-scale depth filtration of the pDADMAC and / or pDADMAC+PEG treatment conditions was compared to the control. 2 ) divided by the final throughput of the depth filtration under untreated conditions (L / m 2 ) was used to calculate the normalized throughput. For the mAb1 run 3 large-scale run, a small-scale depth filtration was performed one day after the end of the process (day 19). The final throughput (L / m) of the small-scale depth filtration for the pDADMAC treatment condition was calculated by 2 ) divided by the final throughput of the small-scale depth filtration under untreated conditions (L / m 2 ) to calculate the normalized throughput.

[0032] Figure 8 .The effect of flocculation on the continuous pressure trend of depth filtration. Figure 8 The pressure differential curves of a large-scale depth filtration series using 0.05% w / w pDADMAC and COHC filters (dark grey) are shown compared to a control small-scale depth filtration experiment using untreated centrate and XOHC filters (black). Representative large-scale runs used are: mAb1 run 1, xmAb 1 run 1, xmAb 1 run 2, and bispecific 2, run 1. Compared to the XOHC control where a rapid increase in pressure differential was observed, the use of flocculation and COHC depth filtration produced a low and stable pressure differential curve. ΔA pressure represents the pressure differential of the 1st stage depth filter. ΔB pressure represents the pressure differential of the 2nd stage bioburden reduction filter. Note that the data plotted by the permeability trend on the y-axis is not different from the absolute pressure trend in this figure.

[0033] Fig. 9 Comparison of product quality between .pDADMAC treated and untreated material. Fig. 9 A comparison of product mass between pDADMAC-treated and untreated material from xmAb1 Run 1 is shown. The post-flocculation depth filtration condition (light grey) showed similar product mass to the control condition (black) and the pDADMAC-treated centrate (dark grey). Normalized product mass values ​​were calculated by dividing the product mass values ​​of the pDADMAC-treated centrate (dark grey) and pDADMAC-treated depth filtrate pool (light grey) conditions by the product mass value of the untreated control condition (black). Abbreviations: SE = size exclusion chromatography, CEX = cation exchange chromatography, rCE = reduced sodium dodecyl sulfate capillary electrophoresis, nrCE = non-reduced capillary electrophoresis; major = median molecular weight range; major peak = major or median peak resulting from chromatographic fractionation; LMW = low molecular weight; MMW = mid molecular weight. DETAILED DESCRIPTION

[0034] "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 determined by a standard viability assay, such as trypan blue dye exclusion.

[0035] As used herein, "packed cell volume" (PCV), also known as "percent packed cell volume" (%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. [Biotechnology and Bioengineering] December 20: 95 (6): 1228-33). Changes in packed cell volume may be caused by changes in cell diameter. Packed cell volume is a measure of the solid content in the cell culture. Solids are removed during harvest. More solids means that more effort is required to separate the solid material from the desired product during the harvest process. In addition, the desired product can be trapped in the solids and lost during the harvest process, resulting in a reduced product yield. Because host cells vary in size and cell cultures also contain dead and dying cells and other cell debris, packed cell volume is a more accurate way to describe the solid content within a cell culture than cell density or viable cell density. Furthermore, some cells increase in size while in a growth-arrested state, and therefore, the packed cell volume before and after the growth arrest will likely be different due to an increase in biomass resulting from an increase in cell size.

[0036] As used herein, "centrate" is a low density composition that is or is being formed when subjecting a cell culture fluid to continuous solids discharge disc stack centrifugation (such as single use continuous solids discharge disc stack centrifugation). In operation, the centrifuge fractionates the cell culture fluid into a low density composition and a high density composition that contains at least some of the solids originally found in the cell culture fluid.

[0037] A "target protein" is any naturally occurring, synthetically produced, or recombinantly expressed protein, peptide, or fragment thereof that is harvested according to the methods of the present disclosure.

[0038] "Low density composition" or "light phase" is synonymous with "centrate," which refers to the fluid exiting a disc stack centrifuge after at least some solids have been removed from the input fluid.

[0039] The "high density composition" or "heavy phase" is material rich in particulate matter, such as cells and cell debris resulting from continuous solids discharge disk stack centrifugation.

[0040] A "bioreactor" is any container suitable for culturing cells capable of expressing a target protein. Bioreactors can range in size from laboratory to commercial scale and can have various modifications, including pump-controlled inlets and / or outlets, thermometers, CO2 and / or O2 meters, means for stirring the culture, etc. A single-use bioreactor is one that has a disposable bag instead of a culture vessel.

[0041] "Depth filtration" is the filtration of a fluid containing particles through a medium of sufficient depth to achieve separation of particles of various sizes from the fluid.

[0042] "Tangential flow filtration" is the filtration of a fluid containing particles in which the fluid is directed tangentially across the surface of a filter medium using transmembrane pressure caused, for example, by gravity, causing some of the fluid to enter the filter medium and thereby be filtered.

[0043] "Q / ∑" is the ratio of the centrifuge feed flow rate to the theoretical equivalent settling area required to achieve an equivalent degree of solid-liquid separation using settling discs. For a disc stack centrifuge it is calculated as: where ω is the angular velocity; N is the number of disks; r2 is the maximum radius of the disk; r1 is the minimum radius of the disk; and α is the semi-cone angle of the disk.

[0044] The "load factor" is the ratio of the centrifuge feed flow rate to the separation area KQ based on empirical results. KQ is calculated as: Where n is the drum speed in RPM; N is the number of disks; r2 is the maximum radius of the disk; r1 is the minimum radius of the disk; α is the semi-cone angle of the disk.

[0045] "Turbidity" has its ordinary and customary meaning, namely the degree to which a fluid lacks clarity. As used herein, turbidity is measured in conventional nephelometric turbidity units (NTU), unless otherwise specified.

[0046] Samples of perfusion cultures having a PCV of at least 16% at harvest were found to have elevated turbidity levels. The turbidity decreased during the harvest process, however, the greater the turbidity at harvest, the more effort required to separate turbid material from the desired product. Increased turbidity in perfusion cultures increases the risk of failure and / or reduced performance of harvesting equipment, resulting in reduced product yield. Turbid perfusion cultures having a PCV of at least 16% present a challenge to conventional harvesting operations. Turbidity was not completely reduced by centrifugation, with turbidity values ​​as high as 1500 NTU detected in light phase centrate samples collected at harvest from single use continuous solids discharge disc stack centrifugation of perfusion cultures having a PCV of at least 16%.

[0047] "Unit operation" refers to a functional step performed as part of a process for purifying a recombinant protein of interest. For example, a unit operation may include steps in an operation such as, but not limited to, harvesting, capturing, purifying, refining, viral inactivation, viral filtration, concentrating, and / or formulating a recombinant protein of interest. A unit operation may be designed to achieve a single objective or multiple objectives, such as a combination of capture and viral inactivation steps. A unit operation may also include storage or storage steps between processing steps.

[0048] "Perfusion culture" refers to a cell culture method in which fresh culture medium is fed into a bioreactor during the cell culture process and the culture fluid is passed through a cell retention device that selectively retains certain cell culture components (e.g., cells and optionally recombinant proteins) in the bioreactor while removing spent culture medium, byproducts, impurities, and optionally recombinant proteins in the permeate.

[0049] Fed-batch culture is an important means of producing therapeutic proteins. The fed-batch strategy is widely used and flexible and can be used for all bioreactor volumes. In the fed-batch process, the culture starts with a low cell density, a portion of the total working volume of the bioreactor (usually 50%-75% volume), and fresh nutrient medium is supplemented during the culture process until 100% of the total working volume of the bioreactor. The feed medium supplements nutrition during the cell culture to extend the life of the cells and improve productivity. The spent culture medium and waste products are not removed from the bioreactor. Cells, proteins, spent culture medium, waste, etc. are all retained in the bioreactor and accumulate over time. Therefore, fed-batch culture is limited by the volume of the bioreactor, so larger bioreactors such as production bioreactors operating at tens of thousands of liters are conducive to increasing production. Over time, the fed-batch culture will decline due to the accumulation of spent culture medium, waste, etc., which will adversely affect cell quality, productivity and titer. Harvest usually starts when productivity decreases. Usually, the cell pressure volume (PCV) is less than or equal to 10% when harvested.

[0050] In recent years, the use of perfusion is more and more common in mammalian cell culture technology.Different from fed-batch culture, perfusion culture starts with a high cell density equal to or close to the total working volume of the bioreactor.As with fed-batch culture, the perfusion culture system feeds fresh culture medium into the bioreactor during the cell culture process.Different from fed-batch culture, the perfusion culture system utilizes a pump to guide the contents of the bioreactor through a retaining device (filter, such as an ultrafilter, a microfilter, a hollow fiber filter), which selectively retains certain cell culture components (cells and optional recombinant proteins) or returns them to the bioreactor.Therefore, perfusion culture can maintain a longer time than fed-batch culture, for example 15 days or longer.In the process of perfusion culture, compared with fed-batch culture, more cell mass is accumulated, which results in a larger cell number, a larger cell size, cell debris, by-products and impurities increase during harvest.Spent culture medium (or culture medium exhausted of nutrition), waste, by-products, products and process-related impurities, recombinant proteins, etc. are removed or collected as waste in the filter permeate for harvest. Perfusion cultures are harvested through retention devices and collected from the permeate, or harvested in bulk 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.

[0051] The cell culture fluid from the perfusion culture has a PCV of at least 16% at harvest, which contains high concentrations of cell debris, product and process-related impurities, by-products, etc., which can affect the ability of the protein to pass through the filter and classify the pumping system, thereby reducing the harvest yield and increasing the level of impurities and by-products in the harvest pool. It is not uncommon to reach PCV levels of 30% and higher for perfusion culture, compared to PCV values ​​of 10% commonly observed in fed-batch culture. This high solid content increases the burden on downstream harvesting, purification and refining operations. When the perfusion culture material with a high solid content passes through, the filter is more easily blocked and blocked, resulting in the need to replace the filter during harvest, which takes time, reduces efficiency, reduces yield and increases harvest costs. Pump capacity may also be limited by solution viscosity and high PCV, which further affects the cost / efficiency of harvesting. The connection of the perfusion system and the retention device (tubing length, diameter, etc.) can complicate the problem and increase the density of the culture fluid entering the filter, resulting in cross-flow failures, reducing productivity in the bioreactor and negatively affecting the harvest of cell culture fluid. Perfusion cultures with a PCV greater than 16% at harvest typically only reach about 70% harvest capacity, leaving a large amount of product.

[0052] The increased solids content in continuous perfusion cultures and the shortcomings of current retention systems for perfusion cultures highlight the need for improved perfusion culture harvesting processes.

[0053] Harvest recovery process for fed-batch culture traditionally relies on centrifugation, filtration or a combination of these technologies to separate target protein from culture particles, which include cells, cell debris, and to a lesser extent, separation of impurities associated with process and product, such as host cell protein (HCP) and nucleic acid such as DNA. Intermittent solid discharge disc stacking centrifugation is very suitable for clarifying these low solid content fed-batch cultures, and is widely used in fed-batch culture harvesting. However, although intermittent disc stacking centrifugation is suitable for clarifying cell culture fluids with low solid content, its solid transport capacity is limited. In addition, as the cell culture solid content increases, the limitation of intermittent solid discharge disc stacking centrifugation becomes obvious, because the centrifuge drum is filled with cells and debris faster, and therefore requires more frequent solid discharge. Frequent solid discharge reduces product recovery and increases the turbidity of the centrifuge, making downstream filtration more difficult. Due to limited solid transport capacity, intermittent solid discharge disc stacking centrifugation is not a realistic and feasible option for high PCV perfusion culture. Therefore, intermittent solids discharge disc stack centrifugation is often combined with another separation technique, such as filtration, or the size of the impurity particles is changed to fit the limitations of disc stack centrifugation, for example by flocculating or sedimenting the cell culture fluid to produce larger particles prior to centrifugation.

[0054] The flocculated material has a larger average particle size than the native material, resulting in more efficient clarification of the fed-batch broth in a subsequent disc stack centrifugation run. This results in improved performance of disc stack centrifugation of fed-batch broth and also facilitates more efficient clarification by depth filtration following such centrifugation due to reduced levels of small particles that would otherwise cause clogging of the filter pores.

[0055] Filtration, particularly depth filtration, is used both as a primary harvest separation technique and as a partner to centrifugation, such as intermittent solid discharge disc stack centrifugation, to separate target biological agents from fed-batch culture broth. Depth filters have become one of the main technologies routinely employed in fed-batch culture harvest recovery processes. Depth filters utilize various porous filter media, such as diatomaceous earth, cellulose, and / or synthetic fibers, to form a three-dimensional network of pores that together form a tortuous path that the material must take to pass through the filter. Larger particulate matter (e.g., cells and cell debris) from the cell culture broth can quickly contaminate the filter by plugging the pores or surface of the filter.

[0056] For perfusion cultures, the primary means of harvesting is by tangential flow microfiltration, in which the direction of cell culture fluid flow is tangential to the direction of migration through the filter. During perfusion culture, as the cell culture fluid passes through the filter, larger particles (such as cells and optionally recombinant proteins) migrate across the surface of the filter and return to the bioreactor, rather than settling and plugging the pores of the filter. Further improvements have been achieved by using alternate tangential flow microfiltration, in which the direction of cell culture fluid passing through the membrane surface is periodically reversed, thereby providing a flushing action and improving filter performance. The motivation for developing alternating tangential flow microfiltration highlighted the fact that particulate matter in the perfusion culture fluid can easily plug filters; however, the use of alternating tangential flow microfiltration for perfusion cultures with a PCV of at least 16% at harvest has been an ineffective solution to the filter plugging problem, particularly when used for such perfusion cultures. The increased solids content of perfusion cultures has led to a search for more efficient harvesting methods. The focus has been on improving filtration technology to increase the surface area of ​​the filter to accommodate the increased solids loading. The results of this effort have not yet proposed a solution for such cultures and may not be able to solve the problem without increasing material and labor costs and affecting efficiency and productivity.

[0057] The use of perfusion cultures as a component of commercial-scale protein production allows for reduced equipment size and facility footprint, reduced material and environmental impacts, the use of single-use components, and efficient and flexible production at all scales and in multiple therapeutic formats. This provides a strong impetus for the development of more efficient harvesting processes for perfusion cultures that can handle these higher solids contents. The disclosures herein relate to harvesting recombinant proteins produced by perfusion cultures having a PCV of at least 16% using methods previously thought to be unsuitable or feasible for perfusion cultures, namely centrifugation, particularly using a continuous solids discharge disc stack centrifuge. As described herein, centrifugation of perfusion cultures having a PCV of at least 16% results in acceptable clearance of cellular material, however, the centrate turbidity can be as high or higher than that of the harvested material and needs to be reduced before the centrate can be used for depth filtration.

[0058] Compared to fed-batch cultures, which have PCVs below 10% in the cell culture volume, continuous perfusion cultures can achieve PCVs of 16% to 45% or higher in low culture volumes (2000 L or less), which is 10 times higher than perfusion cultures. This makes these high solids content perfusion cultures less suitable for filtration harvesting methods commonly used for most perfusion culture harvests. The PCVs of these continuous perfusion cultures are also not comparable to fed-batch cultures, which have PCVs below 10%, higher culture volumes and higher yields, making them more suitable for larger scale intermittent or continuous solids discharge disc stack centrifugation and other centrifugation harvesting methods. For commercial-scale perfusion cultures, harvesting methods such as intermittent solids discharge centrifugation are not an option because at higher PCVs, the centrifuge bowl will constantly open to discharge solids, resulting in relatively inefficient separation of the centrate from the debris. Additionally, conventional stainless steel intermittent or continuous discharge centrifuges are not suitable for use in modern single-use manufacturing facilities (eg, open floor plan designs with minimal utility) because the equipment does not have a closed process design and requires in-place cleaning and in-place steaming for product conversion.

[0059] The introduction of continuous solid discharge disc stack centrifuge technology into the process is innovative because the prior art believes that continuous solid discharge disc stack centrifuges cannot distinguish the density of target proteins in cell culture fluids and biological particles (such as cells, cell debris, host cell proteins (HCPs) or nucleic acids (e.g., DNA)) in the fluid. An exemplary continuous solid discharge disc stack centrifuge system is a single-use continuous solid discharge disc stack centrifuge, and it is expected that single-use equipment is too expensive for long-term use. Intermittent solid discharge disc stack centrifuges are disc stack centrifuges that continuously produce cell paste, accumulate cell paste in the centrifuge bowl, but periodically or intermittently discharge cell paste from the centrifuge. The solid discharge interval is based on the cell culture PCV, the cell culture harvest flow rate, and the centrifuge bowl solid retention space. This type of centrifuge is generally used for lower density, lower PCV cell culture clarification. Continuous solid discharge disc stack centrifuges continuously produce cell paste in the centrifuge bowl and continuously transport the paste out of the centrifuge at a predetermined cell paste concentration. This type of centrifuge can be used for a wider range of cell culture PCVs than harvesting methods based on intermittent solid discharge centrifugation. The development of disc stack centrifugation provides a technology for separating materials based on density differences, which involves relatively low or non-existent shear forces, suitable for incorporation into harvesting processes involving higher PCV perfusion cultures. The surprising discovery disclosed herein is that continuous solid discharge disc stack centrifugation effectively separates target proteins (such as antibodies), particularly those with high harvest turbidity, from biological particles found in actual perfusion culture fluids with a PCV of at least 16%, with a step yield of about 85% to 90% for the target protein compared to about 70% step yield for the target protein using standard harvesting techniques for perfusion cultures (e.g., microfiltration). An exemplary system is a single-use continuous solid discharge disc stack centrifuge. The benefits of an increased PCV of about 85% to 90% step yield, combined with the cost savings of reduced downtime and labor required to unclog the centrifuge, more than offset the cost of the single-use centrifuge, thereby achieving an economically attractive target protein harvesting method. Combining continuous solids discharge disc stack centrifugation with subsequent clarification of the centrate using flocculation or sedimentation provides a scalable method by which proteins can be harvested in a continuous flow over time while still maintaining an unobstructed flow path and minimizing contamination issues with any subsequent filtration process steps, even with perfusion cultures at higher solids contents.

[0060] Disc stack continuous solid discharge centrifugation was selected because it can effectively remove intact cells and larger cell debris without expanding the harvest volume as in typical microfiltration harvests, and because of its closed equipment design, it can be operated using open plan manufacturing equipment. When used in conjunction with a perfusion culture process, the centrate does not contain intact cells, but still contains a high concentration of particles that are too small to be separated by centrifugation. Turbidity levels of 1500NTU were observed from the centrifugal harvests of these higher PCV perfusion cultures. These small particles clog depth filters and sterile filters at low loadings, requiring large membrane areas for filtration. Flocculation is used to aggregate small particles into large particles. This increases the average particle size in the solution and reduces the total particle concentration. When used in conjunction with a depth filter pore size that effectively removes flocculated particles (e.g., COHC), the loading capacity of the depth filter and sterile filter is increased, thereby reducing the total membrane area required for large-scale filtration of the centrate.

[0061] In addition to improved step yields, reduced pool volumes and more consistent harvest performance from batch to batch were observed. Smaller pool volumes and consistent product amounts improve assembly of purification equipment and help optimize production planning, raw material usage, and labor allocation at manufacturing plants. Furthermore, the incorporation of a disc stack continuous discharge centrifugation step reduces the subsequent depth filtration load level to the point where the depth filtration media has sufficient solids retention space to effectively further purify the target protein without immediate and unproductive clogging.

[0062] The cell culture fluid is separated into a heavy phase (solids) and a light phase (centrate) stream by using continuous solids discharge disc stack centrifuge technology. The disclosed methods described herein are applicable to a variety of centrifuge operation modes, specifically: (1) continuous separation of protein from cell material in the harvest stream of a higher solids content perfusion culture manufacturing process; (2) continuous separation of protein and impurities from cell material with subsequent reintroduction of the cell material back into a bioreactor; and (3) efficient batch harvesting of product from a bioreactor, such as continuous solids discharge disc stack centrifugation of a higher solids content perfusion culture, where the output from the bioreactor is held in a buffer tank or holding tank for periodic delivery to a centrifuge.

[0063] In addition, the continuous solids discharge disc stack centrifuge provides a closed system that is scalable and can be used to produce therapeutic target proteins without isolation chambers and other requirements to avoid contamination and ensure product quality. The method is compatible with bioreactors that favor perfusion culture and the open ball chamber setup adopted by many who use continuous perfusion culture in bioreactors. For example, a single-use continuous solids discharge disc stack centrifuge is compatible with single-use bioreactors. Commercial-scale stainless steel intermittent solids discharge or continuous solids discharge disc stack centrifuges, even if available, are not compatible with commercial-scale single-use bioreactors and perfusion systems.

[0064] The appropriate harvest stream flow rate from a cell culture bioreactor depends on several process design decisions. To adjust the size of the continuous solids discharge disc stack centrifuge device to accommodate the desired harvest flow rate, the operation of the unit can be continuous, or in a periodic (batch) mode, as described above. Typically, the flow rate is adjusted to maintain a near constant load or Q / ∑ value.

[0065] In the methods described herein, the harvest operation begins with collecting the cell culture fluid directly from the bioreactor or from a buffer tank or holding tank and centrifuging it using a continuous solids discharge disc stack centrifuge. The centrate can also be collected in a harvest holding container or connected in series to a buffer container before flocculation and loading onto one or more depth filters.

[0066] The continuous solids discharge disc stack centrifuge device itself can be constructed of stainless steel, plastic or other materials. When the device is a single-use continuous solids discharge disc stack centrifuge, it is combined with a single-use insert that includes the centrifuge bowl and other product contact surfaces or flow paths. The single-use insert can be functionally closed with a single-use sterile connector and sterilized by radiation prior to use. The single-use insert sterile connector can be connected to a bioreactor or buffer tank and a centrate collection tank to functionally close the centrifugation operation.

[0067] The method described herein subjects the culture fluid to continuous solid discharge disc stack centrifugation in the first step of the harvesting operation to recover the target recombinant protein from the perfusion culture having a PCV of at least 16%. In more detail, it will be appreciated that the harvesting operation of the target protein (e.g., recombinant protein) has two parts: (1) removing cells, cell debris, etc., and (2) clarifying the centrifuge containing the target protein. In order to achieve this result, the disclosed method employs a continuous solid discharge disc stack centrifugation step. Any continuous solid discharge disc stack centrifuge known in the art is intended to be used in the method disclosed herein. Since the system can be a single-use continuous solid discharge disc stack centrifuge, the components that contact the culture are single-use components that do not need to include materials and construction methods designed to withstand repeated use. However, it is contemplated that the single-use centrifuge used in the disclosed method can be used in continuous harvesting methods of varying durations, and therefore, the centrifuge can be configured to remain operable over an extended period of use.

[0068] A continuous solids discharge disc stack centrifuge can be used to perform an initial separation of the culture broth into a heavy phase (solids, including cells) and a centrate phase (including secreted target protein (e.g., recombinant protein and solids that did not enter the heavy phase)) before reaching a depth filtration step. Flocculation is used to further clarify the centrate before the depth filtration step. Continuous solids discharge disc stack centrifuges, including single-use 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 single-use form according to the methods of the present disclosure.

[0069] Continuous solids discharge disc stack centrifuge technology can be applied to harvest culture broth from single use or stainless steel bioreactors.Preferably, the single use bioreactor used in such methods has a volume of no greater than 5,000L.

[0070] In some embodiments, the centrifuge theoretical setting area (∑ value) normalized flow rate Q / ∑ range for recovering target protein disclosed herein is 4.6E-9 to 2.2E-8 m / s. The harvest flow from the bioreactor depends on several process design decisions. Therefore, when compared with the mammalian cell culture process, the flow rate range of the harvest flow is very wide. For single batch harvesting, higher flow rates are preferred. For continuous harvesting strategies, such as cell exudate product separation, lower flow rates are preferred. Continuous solid discharge disc stack centrifuges are typically operated in a continuous manner while cell culture batches are fed into them.

[0071] Successful continuous solids discharge centrifugation is characterized by high step yields and removal of cells and cell debris (Table 1). For perfusion culture processes with a PCV of at least 16%, changes in suspended particle levels were observed. Therefore, the ability of the continuous solids discharge centrifuge to remove cells and cell debris was quantified by comparing the light phase turbidity to an established baseline turbidity value. The baseline turbidity value was measured by the cell culture supernatant produced after centrifuging a 50 mL cell culture sample at about 2094 relative centrifugal force (rcf) for 17 minutes. As disclosed herein, when about 100% of the cells were removed from the light phase, the yield of the target protein was high and successful purification was achieved. As shown in Table 1, effective cell and cell debris separation was observed; the light phase turbidity was less than 2 times the baseline turbidity value, however, this degree of turbidity was sufficient to prevent subsequent purification by depth filtration. The centrate is typically maintained at 10°C, but the centrifuge may warm the light phase to 16°C-25°C. The centrate was maintained at 10°C before and during flocculation. In the experiments disclosed herein, the centrate was not sparged, but it is contemplated that the centrate could be sparged, particularly if the centrate is stored for a period of time.

[0072] It is found that the level of submicron cell debris, product and process-related impurities and other culture byproducts produced by the high solid content perfusion culture collected in the centrate described herein is significantly higher than that observed in any scale of fed-batch process. High concentrations of impurities in the centrate are more likely and faster to contaminate depth filters and sterile filters, thereby requiring additional filtration area to prepare the centrate for further processing. Therefore, it is necessary to further clarify the high turbidity centrate before depth filtration. After centrifugation, flocculation or precipitation operations are carried out to clarify the centrate with a fast, efficient and cost-effective process. Clarify the centrate before depth filtration, rather than before centrifugation as is common in fed-batch culture (a) to avoid cell lysis related to flocculation that may adversely affect product quality, (b) potentially avoid or minimize changes in the rheological behavior of cell culture or cell sediment related to flocculation that affects centrifugation, and (c) reduce any depth filtration membrane area required for the clarified centrate that can be loaded onto the chromatographic column. Unlike fed-batch culture harvesting methods, where flocculation is typically performed as a first step followed by centrifugation to remove the flocculated material, adding flocculants directly to a perfusion broth having a PCV of at least 16% prior to centrifugation does not result in a viable commercial-scale harvesting operation. The flocculated material prevents continuous solids discharge centrifugation.

[0073] The flocculant can be added to the centrate in batch mode or in series continuous mode. A predetermined amount of flocculant solution is added to the centrate pool to achieve a target flocculant concentration. Alternatively, the flocculant solution can be added at a predetermined ratio of flocculant flow rate to centrate flow rate to achieve the target flocculant concentration.

[0074] Exemplary flocculants suitable for the methods herein are cationic flocculants. Since many impurities associated with the process and product in the cell culture medium are negatively charged, cationic flocculants are used to reduce / remove them from the centrifuge. 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 (US 9,371,554 and McNerney et al., mAbs [monoclonal antibodies] 7: 2 413-427 (2015), incorporated herein by reference). Additional exemplary flocculants include simple acids, including organic acids such as octanoic acid (WO 2010 / 151632, incorporated herein by reference), divalent cations, polycationic polymers such as polyethyleneimine (PEI), nonionic polymers such as polyethylene glycol (PEG) (US 9,371,554 and McNerney et al., mAbs [monoclonal antibodies] 7:2 413-427 (2015), incorporated herein by reference) and polyalkylene glycols (and transition metals such as zinc, U.S. Patent Publication No. 2009 / 0292109, incorporated herein by reference), nonionic surfactants, and stimuli-responsive polymers (Kang et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 110:2928-2937 (2013), incorporated herein by reference) such as benzylated poly(allylamine). "Stimuli-responsive polymers" or "smart polymers" are compounds containing multiple chemical subunits (monomers) that are sensitive to at least one trigger from the external environment (including temperature, light, electric or magnetic fields) and chemicals that results in detectable changes in polymer properties (such as solubility and the ability to induce aggregation). Such agents can be used in the methods according to the present disclosure as flocculating compounds or attached to insoluble matrices such as silica beads or limiting density (XD) cell culture processes (Schirmer et al., Bioproc. Intl. [International Journal of Biological Pharmaceuticals] 8:32-39 (2010), incorporated herein by reference) to aid in centrifuge clarification.

[0075] In some embodiments, the flocculant is poly(diallyldimethylammonium chloride) (pDADMAC) or its monomeric 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 performed at 8-12°C, wherein flocculation is typically performed at 10°C. In some embodiments, flocculation is performed 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 performed at about 10°C. After adding the flocculant, the mixture is mixed for 30 minutes and then depth filtration is initiated. In some embodiments, the flocculated mixture is stored at 8-12°C, e.g., 10°C, for up to 4 hours under mixing. In some embodiments, the entire harvesting process from cooling of the bioreactor contents to depth filtration of the centrate is performed at or with fluids at 10° C. In some embodiments, centrifugation, flocculation, and depth filtration are performed with fluids at 10° C.

[0076] Depth filters utilize various porous filter media, such as diatomaceous earth, cellulose, and / or synthetic fibers, to form a three-dimensional network of pores that together form a tortuous path that material must take to pass through the filter. Particulate matter from the cell culture fluid, such as cells, cell debris, byproducts, and product and process-related impurities can quickly contaminate the filter by clogging the pores or surfaces of the filter. As is known in the art, the rate and extent of filter fouling or clogging is affected by the number and size distribution of cell debris particles and the chemistry and pore size of the depth filter media. The data disclosed herein show that when untreated centrate is paired with XOHC or even larger pore sizes such as COHC, clogging occurs almost immediately (see Figure 8 ). Flocculation ensures the formation of larger molecular aggregates, which reduces the number of cell debris particles and increases the particle size, thereby reducing the incidence of fouling or plugging, especially when paired with an appropriate filter type (e.g., COHC, COSP, DL75, DL60). Selection of an appropriate depth filter type is based on empirical optimization of flocculation, depth filter media chemistry, and depth filter media pore size range. Specifically, the pore size is matched to the size of the cell debris in the solution. In the absence of flocculation, particles were found to range in size from less than 1-2 μm. In the presence of flocculation, particles were found to be in the 50-100 μm range. Overall, there were also fewer particles after flocculation. Both small and large particles can plug the filter. The worst performance is observed when the selected pore size of the depth filter dose does not match the particle size in the solution, which is why the performance of the COHC filter using untreated centrate is worse than that of the X0HC filter type. As Figure 1As shown, various depth filter types showed an increase in depth filtration load relative to the X0HC control. The X0HC control of untreated centrate was selected because it has been previously demonstrated to provide the best depth filtration capacity for untreated centrate. The increased depth filtration capacity reduces the number of depth filters required to filter a fixed centrate volume, thereby reducing the filtration area in the manufacturing equipment.

[0077] Suitable depth filters are composed of a porous matrix of synthetic or cellulose fibers, optionally connected to a charged resin for attracting and retaining oppositely charged filter aids, as known in the art. Some exemplary depth filters incorporate diatomaceous earth as an inorganic filter aid. The depth filter may also include a binder. For all depth filters, including cellulose depth filters, the thicker the material (e.g., cellulose fiber pad or material), the more tortuous and complex the path through the filter material, which results in particles of various sizes being captured and removed from the clarified fluid. Cellulose depth filters typically have a net positive charge, which also provides a basis for the adsorption removal of negatively charged impurities.

[0078] The method of the present disclosure includes exposing the clarified centrate from the continuous solid discharge disc stack centrate to depth filtration to remove impurities after the flocculation step as disclosed herein. The depth filtration operation may include one or more depth filters of the same or different types. Depth filters suitable for the method of the present disclosure include, but are not limited to, cellulose, synthetic fiber nets, or a combination of the two, and depth filters made of pretreated filter matrices as described in Singh et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 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 of centrates suitable for flocculation in the harvesting method of the present disclosure include COHC (0.2-2 μm nominal pore size distribution), COSP (0.2-2 μm nominal pore size distribution), DL75 (1-14 μm nominal pore size distribution) and DL60 (0.6-10 μm pore size distribution). Those skilled in the art will be able to identify other filter media suitable for use in the disclosed methods using only routine techniques. Expanding on the foregoing observations, exemplary depth filters include cellulose depth filters, D0HC filter, C0HC filter, C0SP filter, DL60 filter, DL75 filter, Clarisolve 20MS filter, Clarisolve 40MS filter, Clarisolve 60HX filter, 3MTM Zeta Plus TM Filters, Diatomaceous Earth and 3M TM Emphaze TM AEX hybrid purifier. All of these filter media will provide useful purification without causing unacceptable yield loss, but as pointed out herein, if the filter media pore size is not appropriately selected based on the particle size in the filtered centrate, the filter media load will be reduced. Based on the data disclosed herein, it is expected that multilayer filters will be beneficial for purifying target proteins obtained in high yields, but multilayer filters are not a requirement according to the methods disclosed herein. Experiments related to the depth filtration component of the purification method are designed to generate data that can be used to achieve high loading levels, so as to minimize the number of filters required to clarify the centrate enough to pass through any subsequent downstream filters (such as protein A protection filters (usually 0.2 μm pore size)) while maintaining high step yields.

[0079] In addition, depth filtration may be followed by more than one sterile or bioburden reduction filter (e.g., Millipore SHC filter, Sartopore 2 filter (Sartorius), and Pall filter) with a pore size no greater than 0.2 μm to ensure bioburden reduction. After depth filtration is complete, the purified centrate is held at 2-8°C, typically for no more than 72 hours. Depending on the stability of the molecule, retention may be reduced.

[0080] The present disclosure further contemplates methods that combine the harvesting process with post-harvest recovery steps known in the art, such as target biomolecule recovery, e.g., affinity-based recovery, which for immunoglobulin and immunoglobulin-like targets can be based on binding of a target affinity chromatography material utilizing Staphylococcus proteins such as Protein A, and chromatographic fractionation and polishing steps, wherein the chromatographic medium can be in any form, including beads in column form, and the fractionation and polishing steps can rely on any discriminating property of the target, such as size (e.g., size exclusion chromatography), affinity, charge (e.g., anion or cation exchange chromatography, hydrophobicity (e.g., hydrophobic interaction chromatography), multimodal or mixed-mode (a combination of different modes), or any property known to be useful for distinguishing molecules using a chromatographic medium. In short, the present disclosure provides any method for purifying a target biomolecule (e.g., a biologic, such as an antibody) that has been harvested using the methods disclosed herein.

[0081] The present disclosure provides the separation of recombinant proteins from the cell culture fluid in the batch harvest flow from the cell culture bioreactor.When the predetermined parameters (e.g., culture duration, titer, viable cell density or cell packing volume) are met, the culture is harvested from the bioreactor.The bioreactor is cooled to a temperature of 12 ° C or lower.Then the cell culture fluid is harvested directly from the bioreactor or collected in a storage tank and maintained at 10-12 ° C.The harvested cell viability, viable cell density (VCD), cell packing volume and / or baseline turbidity of the culture are measured using conventional techniques known in the art.

[0082] The present disclosure provides separation of recombinant proteins from cell culture fluid in a periodic harvest stream from a perfusion culture process, wherein the cell culture fluid is passed through a surge tank / container before or after a continuous solids discharge disc stack centrifuge, thereby separating solid material (heavy phase) from a liquid supernatant (light phase or centrate). The centrate is then subjected to a flocculation / precipitation step and then loaded onto one or more depth filters, and optionally also onto one or more sterile filters. The eluate is then collected into a storage tank and stored at 4°C or directly subjected to a purification chromatography operation.

[0083] The present disclosure provides continuous separation of recombinant proteins from continuous cell cultures, wherein multiple batches of cell culture fluids are passed through a continuous solids discharge disc stack centrifuge to separate solid materials from the cell culture supernatant (centrifuge). In some embodiments, the solids can be recycled or reintroduced into the bioreactor. The centrifuge is collected in a harvesting tank and the remaining steps of the harvesting method described herein are performed. In some embodiments, these methods relate to continuous harvesting directly from a working bioreactor, and some embodiments provide series cooling.

[0084] The harvesting operations described herein can be combined with additional harvesting strategies, including additional centrifugation, such as disc stack centrifugation; filtration, including tangential flow filtration, microfiltration, ultrafiltration and depth filtration; sedimentation methods and separation based on chromatographic media, typically using columns including anion and / or cation exchange, affinity-based exchange (e.g., immunoaffinity exchange), molecular sieves and / or polishing chromatography. The methods disclosed herein include any purification or clarification scheme known in the art that is suitable for cell culture materials (including cell culture supernatants and fluids containing cell materials). In typical operations, separation by continuous solid discharge disc stack centrifugation is the first step after removing the cell culture from the bioreactor (or a buffer tank or storage tank useful in batch mode separation). After the centrifugation, flocculation and filtration harvesting steps described herein, the purification or clarification scheme can include any combination of downstream filters and / or columns and / or other centrifugation forms that can be used to separate biomolecules such as proteins.

[0085] In addition to the above techniques, the methods of the present disclosure contemplate a variety of chromatographic steps involved in separating or clarifying a biomolecule of interest (e.g., a protein), including size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, multimodal or mixed mode chromatography, and affinity chromatography, such as immunoaffinity chromatography. Various media can be used in various configurations designed to facilitate separation of biomolecules (e.g., proteins) in batch and continuous purification modes.

[0086] The method disclosed herein contemplates a production method for culturing cells in three or more different stages. Each stage can be carried out in its own bioreactor container or other container suitable for cell culture. Alternatively, more than one stage can be carried out in a shared container. For commercial methods using mammalian cells to produce proteins, there are usually multiple, such as at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10 growth phases / stages, which occur in different culture containers before the final N production stage. For example, cells can be cultured in one or more growth phases before the N-1 seed stage and cultured in one or more N-1 bioreactors. The duration of the N-1 stage can be in the range of, for example, 7 to 14 days, and can be designed to maintain exponential growth of cells before inoculating the production (N) bioreactor. In some embodiments, cells from N-1 bioreactors are transferred to (N) production bioreactors and grown under conditions that maximize protein production. In other embodiments, there is no transfer, i.e., the N-1 and N production stages occur in the same bioreactor.

[0087] "One or more cells" include any prokaryotic or eukaryotic cells. Cells can be in vitro cells, in vitro cells or in vivo cells, and can be isolated or as part of a higher structure (such as a tissue or organ). Cells include "host cells", which can be genetically engineered to express proteins with commercial or scientific significance. Host cells are typically derived from pedigrees from primary cultures, which can be maintained in culture for an unlimited time. Host cells can be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast, insect or animal cells (e.g., CHO)). Host cells cultured under appropriate conditions express target proteins, which can then be collected from the culture medium (if the host cell secretes it into the culture medium), directly from the host cells that produce it (if the protein is not secreted), or from two sources. The selection of suitable host cells will depend on various factors, such as the required expression level, protein modifications (e.g., glycosylation or phosphorylation) required or necessary for activity, and easy folding into biologically active molecules.

[0088] Genetically engineered host cells involve transfection, transformation or transduction of cells with recombinant polynucleotide molecules, and / or otherwise altered (e.g., by homologous recombination and gene activation or fusion of recombinant cells with non-recombinant cells) to cause host cells to express desired recombinant proteins. Methods and vectors for genetically engineered cells and / or cell lines to express target biomolecules (e.g., proteins) are well known to those skilled in the art. Expression systems and constructs in the form of expression vectors are provided, such as plasmids or transcription or expression cassettes, which contain one or more polynucleotides encoding target biomolecules (e.g., proteins), such as those identified herein, and host cells containing such expression systems or constructs. As used herein, "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, naked DNA, composite DNA, etc.) suitable for transferring and / or transporting information encoding proteins to a specific location and / or compartment in a host cell and / or a host cell. Vectors can include viral and non-viral vectors, as well as non-additive mammalian vectors. Vectors can be expression vectors, such as recombinant expression vectors or cloning vectors. The vector can be introduced into a host cell to allow replication of the vector itself, and thereby increase copies of the polynucleotides contained therein. The cloning vector can contain sequence components, which generally include but are not limited to an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a transposon / transposase, and a selective marker. One of ordinary skill in the art can appropriately select these elements, and one or more of each selected component can be included in the cloning vector.

[0089] 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 the context of 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 cells (HKB-11), CEVEC amniotic fluid cells (CAP) cells, human liver (HuH-7) cells, and any other cells used for or suitable for clinical and / or commercial manufacturing. The most commonly used cell line is from CHO cells. CHO cells are widely used in the production of composite recombinant proteins. Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 77: 4216-4220) DXB11 and DG-44 are ideal CHO host cell lines because the efficient DHFR selectable and amplifiable gene expression system allows high levels of recombinant protein expression in these cells (Kaufman R.J. (1990), Meth Enzymol [Enzyme Methodology] 185: 537-566). Glutamine synthetase (GS)-knockout CHOK1SV cell line is also widely used, which utilizes methionine sulfoximine (MSX) selection based on glutamine synthetase (GS). CHOK1 cells (ATCC CCL61) are also included.

[0090] Key attributes and performance parameters can be measured to better guide the decision-making of each step performance during the manufacturing period. These key attributes and parameters can be monitored in real time, near real time and / or monitored afterwards. During cell culture, main key parameters can be measured, such as the level of the culture medium components (such as glucose) consumed, the metabolic byproducts (such as lactic acid and ammonia) that may be accumulated in the culture, and the parameters related to cell maintenance and survival, such as dissolved oxygen content. Key attributes such as specific productivity, viable cell density, pH, osmotic pressure, appearance, color, aggregation, cell count, cell packing, yield percentage and titer can be monitored at the appropriate stage of the manufacturing process. Process and product impurities can also be monitored in the whole manufacturing process.

[0091] "Culture" or "culturing" refers to the growth and propagation of cells outside 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 culture medium suitable for the growth of host cells during in vitro cell culture. Typically, a cell culture medium contains buffers, salts, energy sources, amino acids, vitamins, and trace essential elements. Any culture medium capable of supporting the growth of appropriate host cells in culture can be used. Cell culture media that can be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in a specific cultured host cell (including components that effectively control the production time of the recombinant protein) are commercially available and include RPMI-1640 medium, RPMI-1641 medium, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium, F-12K medium, Ham's F12 medium, Iscov's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz L-15 medium, and serum-free medium such as EX-CELL TM 300 series, etc., the culture medium can be obtained from the American Type Culture Collection or SAFC Biosciences and other suppliers. Cell culture can also be supplemented with independent concentrated feeds of specific nutrients, which may be difficult to be formulated in cell culture or quickly exhausted in cell culture. Such nutrients can be amino acids such as tyrosine, cysteine ​​and / or cystine (see, for example, WIPO Publication No. 2012 / 145682). Cell culture medium can be a serum-free, protein-free, growth factor-free and / or peptone-free culture medium. Cell culture can also be enriched by adding nutrients higher than its usual recommended concentration. In a preferred embodiment, the cell culture medium is adjusted so that the host cell grows to more than 10 6 The cell density is close to, reaches or exceeds 10 cells / ml. 8 The cell density was 10 cells / ml.

[0092] The perfusion feed medium can be formulated or supplemented to achieve a concentration of at least 5 g / L of the non-ionic block copolymer when the cell culture is passed through a hollow fiber filter having a pore size or molecular weight cutoff (MWCO) that does not retain recombinant proteins in the bioreactor (WO 2015 / 188009).

[0093] A variety of media formulations can be used during the culture process, for example, to initiate the culture, promote the transition from one stage (e.g., the growth stage or growth period) to another stage (e.g., the production stage or production period) and / or optimize the conditions during the cell culture (e.g., concentrated culture media provided during the perfusion culture process). Basal culture media formulations containing basic culture media components are generally used to initiate cell culture. Growth medium formulations can be used to promote cell growth and minimize protein expression. Production medium formulations can be used to promote the production of target biomolecules (e.g., proteins) and the maintenance of cells while minimizing the growth of new cells. Feed media, typically a culture medium containing more concentrated components such as nutrients and amino acids, which are consumed during the production phase of the cell culture, can be used to supplement and maintain active cultures, particularly cultures operated in perfusion mode. Such concentrated feed medium can contain most of the components of the cell culture medium, for example, about 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, or even about 1000-fold their normal amounts or concentrations.

[0094] During cell culture, especially during the production phase, cell growth may be restricted or stagnant. Such methods include temperature changes, pH changes, chemical inducers and cell cycle inhibitors using protein production, nutrient restriction or starvation, alone or in combination. For example, temperature changes can be used to transition from the growth phase to the production phase. For example, the growth phase can be carried out at a first temperature of about 35°C to about 38°C, and the production phase can be carried out 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 be carried out at a higher pH than production. pH changes can be used alone or in combination with temperature changes and / or the addition of chemical inducers.

[0095] Another method of 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, e.g., WIPO Publication No. WO 2013 / 006479). Cell growth arrest can be achieved and maintained by a culture medium containing a limiting concentration of L-asparagine and maintaining a low concentration of L-asparagine in the cell culture. Maintaining the concentration of L-asparagine at 5 mM or less can be used to induce and maintain cells in a growth arrest state, thereby increasing productivity.

[0096] In addition, chemical inducers of protein production, such as caffeine, butyrate and hexamethylenebisacetamide (HMBA) can be added before, simultaneously and / or after the temperature change. If the inducer is added after the temperature change, the inducer can be added one hour to five days after the temperature change, optionally one to two days after the temperature change. Cell cycle inhibitors, compounds known or suspected to regulate cell cycle progression and the related processes of transcription, DNA repair, differentiation, aging and apoptosis can also be used to induce cell growth arrest. Cell cycle inhibitors such as cyclin-dependent kinases (CDKs) that interact with the cycle mechanism are useful, as are those molecules that interact with proteins from other pathways such as AKT, mTOR and other pathways that directly or indirectly affect the cell cycle.

[0097] The method disclosed herein can be used as a part of a larger production process, whereby cells are cultured in three or more different stages. Each stage can be carried out in its own bioreactor container or other containers suitable for cell culture. Alternatively, more than one stage can be carried out in a shared container. Suitable containers include fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake bottles or stirred tank bioreactors with or without microcarriers. In general terms as used herein, a bioreactor is any container for cell culture.

[0098] For use in the methods described herein, cell cultures are operated in perfusion mode. Mammalian cells (such as CHO cells) can be cultured in a bioreactor at a smaller scale of less than 100 ml to less than 1000 ml. Alternatively, a larger bioreactor of 1000 ml to more than 2,000 liters can be used. In one embodiment, 1 liter to 2000 liters is used. In one embodiment, the bioreactor is 10 liters to 2000 liters. In one embodiment, the bioreactor is 10 liters to 100 liters. In one embodiment, the bioreactor is 30 liters to 50 liters. In one embodiment, the bioreactor is 30 liters to 2000 liters. In one embodiment, the bioreactor is 100 liters to 2000 liters. In one embodiment, the bioreactor is 500 liters to 2000 liters. In one embodiment, the bioreactor is 1000 liters to 2000 liters. Larger scale cell culture, such as for clinical and / or commercial scale biomanufacturing of protein therapeutics, can be maintained for weeks or months, during which the cells produce the desired one or more proteins.

[0099] In some embodiments, the methods of the present disclosure are performed using single-use bioreactors (also referred to as disposable bioreactors) that use disposable bags instead of traditional stainless steel culture containers. The use of single-use technology minimizes the infrastructure requirements associated with traditional cell culture, such as steel / glass commercial-scale containers and related machinery. Single-use bioreactors provide 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 equipment. Single-use bioreactors include disposable plastic sterile bags supported by non-disposable support structures. The culture is stirred by an agitator in the bag or by shaking, and air and oxygen sprayers and sensors are also provided to measure and adjust various parameters of the culture, such as pH, temperature, oxygen, cell density, etc. Single-use bioreactors are commercially available, for example, from Satorius, Göttingen, Germany. Bio Millipore, Burlington, MA Cytiva, Marlborough, MA In some embodiments, it is expected that cultures of 3 kL or more will be grown in a bioreactor of appropriate size.

[0100] Bioreactor systems maintain conditions within the bioreactor to support cell culture. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, edited by D. Rickwood, Oxford University Press, New York (1992). "Operation" of a bioreactor system refers to maintaining conditions in the bioreactor system to support cell culture. A bioreactor "operation" typically includes the following steps: inoculating a prepared bioreactor with a seed culture, subjecting the cells to one or more growth phases and / or production phases until predetermined parameters (time, viable cell density, cell packing) are met, and then harvesting the contents of the bioreactor.

[0101] "Cultivation" refers to maintaining cells in a culture medium under conditions suitable for the survival and / or proliferation of cells other than multicellular organisms or tissues and suitable for producing protein products. Cell culture is usually operated in batch, fed-batch or perfusion mode. In batch mode, a fixed amount of culture medium and cells are added to the bioreactor at the beginning of the operation. During the cultivation process, the volume of the culture medium in the reactor remains constant, while the nutrient content of the culture medium decreases, and the waste and by-product content increases. The cell concentration continues to increase during operation and tends to stabilize and decline as the nutrient content is exhausted and the waste increases. Fed-batch culture starts with inoculated cells and a fixed amount of culture medium. Unlike batch culture, the volume of the culture medium in the bioreactor increases as concentrated nutrients are added during the cultivation process. Similar to batch culture, the waste and by-product content increases during operation and will tend to stabilize and decline as the nutrient content is exhausted and the waste products increase.

[0102] Perfusion culture, such as batch culture and fed-batch culture, starts with fixed inoculation of cells and culture medium. Unlike fed-batch culture, culture starts at the final working volume of the reactor or close to the final working volume. Also different from fed-batch culture, when fresh feed medium is added to the bioreactor, an equivalent amount of spent culture medium is perfused or taken out from the bioreactor by a retaining device. In the case of methods described herein, perfusion is continuous perfusion. Retaining devices, such as tangential flow filtration (TFF) systems or alternating tangential flow (ATF) systems can be used to remove spent culture medium and unwanted products from bioreactors. Alternatively, sonic technology or cell sedimentation techniques can be used to remove spent media and unwanted products.

[0103] ATF relies on the use of a recirculation device (most typically a diaphragm pump) to move the cell culture back and forth through the module (e.g., through a hollow fiber filter module) to allow the spent culture medium to be removed 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. The benefit of ATF is the cleaning effect on the filter caused by the alternating flow. An exemplary method of the present disclosure includes operating (N) production bioreactors using an ATF perfusion system.

[0104] Typically, hollow fiber filters are used in ATF systems (although this is not required). When cell culture (including cell culture medium, cells (complete and lysed), soluble expressed recombinant proteins, host cell proteins, waste, etc.) are introduced into the filter, depending on the pore size or molecular weight cutoff (MWCO), the hollow fiber material can retain certain cell culture components (except the cells themselves) on the lumen side (inside), and allow 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 culture medium is added to the bioreactor, and the permeate is taken out from the filter at predetermined time intervals or continuously to maintain the required or constant bioreactor volume. The permeate can be discarded, stored in a storage tank, bag or tote or directly transferred to another unit operation, such as a harvesting operation.

[0105] Exemplary filters (eg, ultrafilters and microfilters) include, but are not limited to, Millipore prostrack, Cytiva hollow fiber filters of various pore size ranges, Repligen filters of various pore sizes, Spectrum hollow fiber filters of various pore sizes, and Ashai Kasei hollow fiber filters.

[0106] Typically, hollow fiber filters are used for perfusion culture retention systems. In various aspects, the hollow fiber has an inner diameter of about 0.5 mm to about 1 mm, and can have any suitable length (e.g., about 30 cm to about 110 cm). The hollow fiber used for microfiltration typically has a pore size of 0.1 μm to 10 μm or a molecular weight cutoff of 500 to 750 kDa or more, and can be used to allow proteins (e.g., monoclonal antibodies or engineered antibody-like proteins) to enter the permeate. Ultrafiltration hollow fibers typically have a pore size 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 to return to the bioreactor. This can be used, for example, to concentrate recombinant protein products for harvesting. Such filters are commercially available, such as Xampler (Cytiva, Marborough, MA), Midikros (Spectrum Laboratories, Inc, Dominguez, CA, California), XCell (Repligen, Waltham, MA). The temperature of the culture broth leaving the bioreactor is controlled and is typically maintained at 10-12°C. A pump is used to move the culture broth from the bioreactor to a continuous solids discharge disc stack centrifuge or surge tank. It should be understood that the centrifuge itself also has a pump.

[0107] The cell culture fluid can be pumped out of the bioreactor and into the filter module by a pumping system that moves the cell culture through or along the filter (e.g., through the lumen side of the hollow fiber). Examples of cell pumping systems include peristaltic pumps, double diaphragm pumps, low shear pumps (Levitronix, Zurich, Switzerland), TM pump) and alternating tangential flow system (ATF from Repligen, Waltham, MA) TM ). The 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.

[0108] The medium exchange in the bioreactor is expressed as the volume of the vessel per day (VVD) or the volume of the bioreactor per day (BV / d). The exchange rate can be minimized, such as by setting it to about 0.25 BV / d, but more frequently it is set to a value of at least 1 BV / d; typically, the exchange rate is set to a value of at least 2 BV / d to 3 BV / d.

[0109] After expression in the perfusion culture, the target protein of interest is then harvested according to the methods disclosed herein. The harvesting process clarifies or purifies, or partially clarifies or purifies the target protein, away from at least one impurity found in the culture fluid, such as remaining cell culture medium, cell extracts, cells, cell debris, unwanted cell or culture medium components, products and / or process-related impurities. The harvested target recombinant protein can be stored in a buffer tank, storage tank, bag or other container suitable for providing feed to a chromatographic column skid. The harvested target recombinant protein can also be directly provided to the chromatographic column skid as an eluent stream. At the time of harvesting, 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%-about 30%. In one embodiment, 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%-about 35%. In one embodiment, the PCV of the perfusion culture is about 16%-45%. Recovery and further purification of the target protein of interest can be accomplished by downstream unit operations.

[0110] Downstream operation purifies and refines the target protein. Downstream operation utilizes capture chromatography including resin and / or membrane, and these resins and / or membrane contain reagents that will be combined and / or interacted with at least one desired protein, impurity or pollutant. The example of capture chromatography includes affinity chromatography, size exclusion chromatography, ion exchange chromatography (IEX) such as cation exchange (CEX) and anion exchange (AEX) chromatography, hydrophobic interaction chromatography (HIC), multimode or mixed mode (MMC), immobilized metal affinity chromatography (IMAC) etc. Such materials are known in the art and can be commercially available.

[0111] Affinity chromatography is often used as an initial capture step in biomanufacturing processes to separate and concentrate a target protein of interest, such as a recombinant protein, from crude or clarified material. Examples of such affinity chromatography materials include those utilizing: Staphylococcal 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; cofactor binding capture mechanisms; and the like. Immobilized metal affinity chromatography can be used to capture proteins that have an affinity for metal ions or have been engineered to have an affinity for metal ions. Protein A is highly selective for a variety of antibodies and antibody-like proteins, and a large number of purification methods as first-line methods rely on strong removal of process-related impurities and high yields. Protein A materials are commercially available from a number of suppliers. For example, MABSELECT TM SURE Protein A, Protein A Sepharose FAST FLOW TM (Cytiva, Marborough, MA), PROSEP-A TM (Merck Millipore, UK) and TOYOPEARL TM 650M Protein A (TosoHass Co., Philadelphia, PA).

[0112] Intermediate and / or polishing unit operations utilize various chromatography methods to continuously purify the protein of interest and remove contaminants and impurities, such as DNA, host cell proteins, product-related impurities, variant products and aggregates, viruses (such as by viral adsorption), etc. These chromatography unit operations utilize resins and / or membranes containing reagents, which can be operated in a variety of configurations, such as flow-through mode, in which the protein of interest is contained in the eluent and the contaminants and impurities bind to the chromatography medium; 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 material with minimal affinity for the stationary phase (the protein of interest) begins to elute; and bind and elute mode, in which the protein of interest binds to the chromatography medium and elutes after the contaminants and impurities flow through or are washed off the chromatography medium, or any other method used to achieve a degree of purification of the target protein by chromatography. As mentioned above, examples of such chromatographic methods include 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); reverse phase chromatography and gel filtration, etc.

[0113] Multiple chromatography unit operations, usually one, two or three, each usually performing a different function or operating in a different mode, are combined according to the requirements of the manufacturing process. Ion exchange chromatography based on electrostatic interactions between charged surfaces separates the target protein from impurities based on different rates of adsorption and desorption that reflect the electrostatic attraction level of the protein (or impurity) to the chromatographic medium. Cation exchange chromatography refers to chromatography performed on a negatively charged solid phase medium, which has cations that can be used to exchange cations in an aqueous solution that passes through or passes through the solid phase. The charge can be provided by connecting one or more charged ligands to the solid phase, such as by covalent attachment. Alternatively or additionally, the charge can be an inherent property of the solid phase (e.g., as in the case of silica with an overall negative charge). Cation exchange chromatography is usually operated in a binding and elution mode, and the high pI of many target proteins can be bound to the chromatographic material. Cation exchange chromatography can also be operated in a flow-through mode. CEX chromatography is commonly used to remove high molecular weight (HMW) contaminants, process-related impurities and / or virus removal. Commercially available cation exchange media include, but are not limited to, sulfopropyl (SP) functional groups immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW TM 、SP-SEPHAROSE FAST FLOW XL TM Or SP-SEPHAROSEHIGH PERFORMANCE TM CAPTO S TM 、CAPTO SPImpRes TM CAPTO S ImpAct TM (Cytiva), FRACTOGEL-SO3 TM , FRACTOGEL-SE HICAP TM and FRACTOPREP TM (EMD Merck, Darmstadt, Germany), XS, HS (Tosh Bioscience, King of Prussia, PA), UNOsphere TM (BioRad, Hercules, CA) and SCeramic Hyper D TM F (Pall, Port Washington, NY), POROS TM(Thermo Fisher, Waltham, MA)).

[0114] 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 aqueous solution that is passed through or through the solid phase. Anion exchange chromatography is typically run in flow-through mode. Due to the high pI of many therapeutic proteins, they do not readily bind to AEX chromatography materials. For example, AEX chromatography is used for viral clearance and impurity removal. Commercially available anion exchange media are available and include, but are not limited to, quaternary amines (Q) immobilized on agarose (e.g., Source 15Q, Capto TM Q, Q-SEPHAROSE FAST FLOW TM (Cytiva), FRACTOGEL EDM TMAE TM FRACTOGEL EDM DEAE TM (EMD Merck), TOYOPEARLSuper (Tosh Bioscience), POROS HQ TM 、POROS XQ TM ,(ThermoFisher).

[0115] Mixed-mode or multimodal chromatography (MMC) refers to chromatography performed on a solid phase medium that exploits a combination of interaction mechanisms, such as ion exchange (CEX or AEX) and hydrophobic interactions. Commercially available multimodal chromatography media are available and include Capto TM Adhere (Cytiva).

[0116] Hydrophobic interaction chromatography refers to chromatography performed on a solid phase medium that exploits the interaction between a hydrophobic ligand 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 TM (Cytiva), Tosoh Bioscience, and Capto TM Phenyl (Cytiva).

[0117] Hydroxyapatite chromatography refers to chromatography performed on a solid phase medium using positively charged calcium and negatively charged phosphate. Depending on the pI of the protein and the pH of the buffer, the hydroxyapatite medium can act as a cation or anion exchanger.

[0118] Unit operations involved in inactivating, reducing and / or eliminating viral contaminants may include processes that manipulate the environment and / or rely on filtration. Viral mitigation measures are essential to ensure the safety of protein therapeutics and may be performed once or multiple times in the downstream purification stage of the harvest operation. Viral contaminants may come from a variety of sources, including the use of animal-derived reagents, adventitious viral contaminants in host cell lines, or system failures at GMP production sites. Viruses are divided into enveloped viruses and non-enveloped viruses. For enveloped viruses, the envelope allows the virus to recognize, bind, enter and infect target host cells. Therefore, enveloped viruses are susceptible to inactivation methods. Various methods can be used to inactivate viruses and these methods include heat inactivation / pasteurization, UV and gamma ray irradiation, use of high-intensity broad-spectrum white light, addition of chemical inactivators, surfactants, and solvent / detergent treatments. Surfactants (such as detergents) can dissolve a variety of membranes and may therefore be very effective in specifically inactivating enveloped viruses. One method of achieving viral inactivation is incubation at low pH (e.g., pH less than 4). After low pH virus inactivation, a neutralization unit operation can be performed, which will readjust the virus-inactivated solution to a pH value that is more in line with the requirements of subsequent unit operations. Low pH virus inactivation is typically performed after purification of the harvested fluid by affinity chromatography, particularly affinity chromatography using a substrate-binding ligand from Staphylococcus aureus, such as protein A chromatography, because elution from such chromatographic materials is typically performed at low pH. Exemplary low pH virus inactivation methods are described in U.S. Patent Application No. 63 / 168,608 and U.S. Patent Application No. 63 / 159,217. Exemplary detergent inactivation is described in World International Patent Organization Publication No. WO 2020 / 190985. Filtration, such as depth filtration, can also be performed after low pH virus inactivation to remove any resulting turbidity or precipitation.

[0119] Non-enveloped viruses are more difficult to inactivate without risk to the protein being produced and can be removed by filtration methods. An exemplary process is described in World Patent Organization Publication No. WO 2020 / 159838. Virus filtration can be performed using micro or nano filters (e.g., (Asahi Kasei, Chicago, IL), (Sartorius, Goettingen, Germany), Pro (MilliporeSigma, Burlington, MA), Pegasus TMPrime (Pall Biotech, Port Washington, NY) and CUNO Zeta Plus VR, (3M Company, St. Paul, MN)). Virus filtration may occur at one or more steps in the downstream operations of the biomanufacturing process. Typically, virus filtration is performed before any ultrafiltration or diafiltration (i.e., UF / DF) operation, but may also be performed after UF / DF.

[0120] The unit operation may also include product concentration and buffer exchange of the target protein into the desired formulation buffer for bulk storage of the drug. Buffer exchange and product concentration may be accomplished using known ultrafiltration and diafiltration methods. Unit operations for achieving drug product filling / completion may be followed. The method according to the present disclosure further optionally includes using ultrafiltration and diafiltration (UF / DF) to concentrate the protein product. The purified protein is subjected to ultrafiltration and diafiltration operations, including concentrating or diluting the purified protein by ultrafiltration; buffer exchange of the purified concentrated / diluted protein by diafiltration into the desired formulation; and further diluting or concentrating the formulated purified protein by a second round of ultrafiltration until the target protein concentration is reached. Optionally, one or more stability-enhancing excipients may be added directly to the UF / DF retentate feed tank containing the formulated purified protein to produce a formulated drug substance, or added to a UF / DF eluate pool. Filters for 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, including regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Slice, ECO (Sartorius, Goettingen, Germany), polyethersulfone (PES) membranes, and Omega (Pall Corporation, Port Washington, NY). Multiple filters can be used depending on the capacity allowed by the actual setup of the rack, skid or UF / DF system or the capacity required to achieve the desired goals of the production process.

[0121] As known in the art, the key attributes and performance parameters of the target protein of purification can be measured to provide information for the decision-making of the performance of each step in the relevant production process better. These key attributes and parameters can be monitored in real time, near real time and / or monitored afterwards. During cell culture, main key parameters can be measured, such as the level of the medium components (such as glucose) consumed, the metabolic byproducts (such as lactic acid and ammonia) accumulated and the parameters related to cell maintenance and survival, such as dissolved oxygen content. Key attributes such as specific productivity, viable cell density, pH, osmotic pressure, appearance, color, aggregation, percentage yield and titer can be monitored at the appropriate stage of the production process. Known techniques and commercially available equipment can be used to monitor and measure.

[0122] Pharmaceutical compositions (e.g., solutions, suspensions, etc.) may include 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 solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methyl parabens; 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 tonicity, such as sodium chloride or glucose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0123] Target protein (e.g., biological molecule of interest)

[0124] The terms "polypeptide" or "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 macromolecules having one or more deletions, insertions and / or substitutions of amino acid residues of the native sequence, i.e., polypeptides or proteins produced by naturally occurring cells and non-recombinant cells; or produced by genetically engineered cells or recombinant cells, and including molecules having one or more deletions, insertions and / or substitutions of amino acid residues of the amino acid sequence of a native protein. Polypeptides and proteins also include amino acid polymers in which one or more amino acids are chemical analogs of corresponding naturally occurring amino acids and polymers. Polypeptides and proteins also include modifications, including but not limited to derivatizations, such as glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0125] The terms "polynucleotide", "nucleic acid molecule" or "engineered nucleic acid molecule" are used interchangeably throughout this disclosure and include single-stranded and double-stranded nucleic acids, including genomic DNA, RNA, mRNA, cDNA, nucleic acids of synthetic origin, or some combination that is unrelated to sequences that are normally found in nature. The terms "isolated polynucleotide", "isolated nucleic acid molecule" or "isolated engineered nucleic acid molecule" specifically refer to sequences that are not normally found in nature, such as sequences of synthetic origin. An isolated nucleic acid molecule comprising a specified sequence may include coding sequences for up to ten or even up to twenty other proteins or portions thereof in addition to these specified sequences, or may include operably linked regulatory sequences that control the expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences. The nucleotides comprising the nucleic acid molecule may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Modifications include base modifications such as bromouridine and inosine derivatives; ribose modifications such as 2',3'-dideoxyribose; and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, anilinothioate, anilinophosphoramidate, and phosphoramidate.

[0126] Target biomolecules (e.g., polypeptides and proteins) may have scientific or commercial significance, including protein-based therapeutic agents. Target biomolecules (e.g., proteins) include, in particular, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Target biomolecules can be produced by recombinant animal cell lines using cell culture methods, and can be referred to as "recombinant proteins" to indicate the source of the protein biomolecule rather than the structure. The protein disclosed herein may have the same structure as a naturally occurring protein, may be a fragment thereof, such as a fragment or an enzymatically active fragment that specifically binds to a binding partner, and the protein may also be a chimera or fusion of at least part of two or more proteins. The expressed one or more proteins may be produced intracellularly or secreted into the culture medium, and may be recovered and / or collected from the culture medium according to the methods disclosed herein. The term "isolated protein" or "isolated recombinant protein" refers to a target polypeptide or protein that is purified from proteins or polypeptides or other contaminants that interfere with its treatment, diagnosis, prevention, research, or other uses. Biomolecules of interest include proteins that exert therapeutic effects by binding to targets, particularly those listed below, including targets derived therefrom, targets associated therewith, and modifications thereof.

[0127] "Purification" means increasing the purity of a protein in a composition by removing (partially or completely) at least one impurity from the composition. Recovery and purification of proteins are achieved by the methods disclosed herein, thereby producing a more "homogeneous" protein composition that meets yield and product quality benchmarks (such as reduced product-related impurities and increased product quality).

[0128] As used herein, the term "isolated" means (i) free from at least some proteins or polynucleotides with which it is normally found, (ii) substantially free from other proteins or polynucleotides from the same source, such as from the same species or from the same cell, (iii) separated from at least about 50% of the polynucleotides, lipids, carbohydrates or other materials with which it is associated in nature, (iv) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not associated in nature, or (v) not found in nature.

[0129] Biological molecules (e.g., proteins) of interest include "antigen binding proteins". "Antigen binding protein" refers to a protein, polypeptide, or fragment thereof that includes an antigen binding region or antigen binding 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 double-chain (bivalent) scFv, mutant proteins, multispecific proteins, and bispecific proteins).

[0130] scFv is a single-chain antibody fragment that has 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 relevant parts. scFv retains the ability of the parent antibody to specifically interact with the target antigen.

[0131] The term "antibody" includes glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or an antigen binding region thereof that competes with an intact antibody for specific binding. Unless otherwise indicated, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, heteroIgG, bispecific antibodies, and oligomers or antigen binding fragments thereof. Antibodies include IgG1, IgG2, IgG3, or IgG4. Also included are proteins with antigen binding fragments or antigen binding regions, such as 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 bind a specific antigen to a target polypeptide.

[0132] Also included are human, humanized, and other antigen binding proteins, such as human antibodies and humanized antibodies, which do not produce a significant deleterious immune response when administered to humans.

[0133] Also included are modified proteins, such as proteins chemically modified by non-covalent bonds, covalent bonds, or both covalent and non-covalent bonds. Also included are proteins further comprising one or more post-translational modifications, which can be obtained by cell modification systems or by enzymes and / or chemical methods introduced in vitro or by any other means known in the art.

[0134] "Multispecific constructs", "multispecific proteins" and "multispecific antibodies" are used herein to refer to proteins that are recombinantly engineered to simultaneously bind 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 in combination with targeted cytotoxic agents or infectious agents against tumors. Bispecific proteins include trispecific antibodies, tetravalent bispecific antibodies, multispecific proteins that do not contain antibody components (such as diabodies, triabodies or tetrabodies, minibodies), and single-chain proteins that can bind to multiple targets. Coloma, MJ et al., Nature Biotech. [Nature-Biotechnology] 15: 159-163 (1997), incorporated herein by reference in relevant parts.

[0135] The most common and diverse multispecific proteins are proteins that bind two antigens, referred to herein as "bispecifics," "bispecific constructs," "bispecific proteins," and "bispecific antibodies." Bispecific proteins can be divided into two major 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 aids in some purification operations. Non-IgG-like molecules are smaller, which may enhance 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, pp. 837-855, all of which are incorporated herein by reference in relevant parts). Bispecific proteins are sometimes used as a framework for additional components with binding specificities for different antigens or epitopes, increasing the binding specificity of the molecule.

[0136] The formats of bispecific proteins including bispecific antibodies are constantly evolving and include, but are not limited to, single chain antibodies, quadromas, knobs-in-hole structures, crossover monoclonal antibodies, dual variable domain IgG (DVD-IgG), IgG single chain Fv (scFv), scFv-CH3 KIH, bifunctional Fab (DAF), half-molecule exchange, κλ-bodies, tandem scFv, scFv-Fc, diabodies, single chain diabodies (sc diabodies), sc diabodies-CH3, triabodies, minibodies, minibodies, TriBi minibodies, tandem diabodies, sc diabodies-HAS, tandem scFv-toxin, dual affinity redirected molecules (DARTs), nanobodies, nanobodies-HSA, docking and locking (DNL), chain exchange engineered domain SEED bodies (SEEDbody), trifunctional antibodies (Triomabs), leucine zippers (LUZ-Y), Fab-arm exchange, DutaMab, DT-IgG, charged 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 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, intracellular antibody, ImmTAC, HSAbody (HSABody), IgG-IgG, Cov-X-body, scFv1-PEG-scFv2, bispecific T cell engagers (including 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, 2nd ed., Uwe Gottschalk, ed., pp. 559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011, each of which is incorporated herein by reference in relevant parts)). The biological molecules of interest (e.g., proteins) may also include recombinant fusion proteins including, for example, multimerization domains such as leucine zippers, coiled coils, Fc portions of immunoglobulins, etc. Also included are proteins comprising all or part of the amino acid sequence of a differentiation antigen (referred to as CD proteins) or its ligands or proteins substantially similar to any of these.

[0137] Purpose biomolecules (e.g., proteins) can also include genetically engineered receptors, such as chimeric antigen receptors (CAR or CAR-T) and T cell receptors (TCR), and other proteins comprising antigen binding molecules that interact with the target antigen. By comprising antigen binding molecules that interact with the target antigen, CAR can be engineered to bind antigens (such as cell surface antigens). CAR generally connects antigen binding domains (such as scFv) with one or more costimulatory ("signaling") domains and one or more intracellular activation domains in series.

[0138] In some embodiments, the biomolecule of interest (e.g., protein) may include a colony stimulating factor, such as granulocyte colony stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to (filgrastim) and (Pegfilgrastim). Also included are erythropoiesis stimulating agents (ESAs), such as (Epoetin α), (darbepoetin alfa), (Epoetin delta), (methoxypolyethylene glycol-epoetin beta), MRK-2578, INS-22, (Epoetin ζ), (Epoetin β), (Epoetin ζ), (epoetin alfa), epoetin alfa Hexal, (Epoetin α), (Epoetin theta), (Epoetin theta), (Epoetin θ), Epoetin α, Epoetin β, Epoetin ζ, Epoetin θ, Epoetin δ, Epoetin ω, Epoetin ι, tissue plasminogen activator, GLP-1 receptor agonist, and molecules or variants or analogs and biosimilars of any of the foregoing.

[0139] In some embodiments, the biomolecules of interest (e.g., proteins) can include proteins that specifically bind to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony stimulating factors (CSF), other blood and serum proteins, blood group antigens, receptors, receptor-associated proteins, growth hormone, growth hormone receptors, T cell receptors, neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transporters, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0140] In some embodiments, the biomolecule of interest (e.g., protein) 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 (including, for example, HER2, HER3, HER4, and EGF receptor), EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin), growth factors (including 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-α), erythropoietin (EPO), nerve growth factor (e.g., NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4 and TGF-β5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des (1-3)-IGF-I (brain IGF-I) and osteoinductive factor, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein); (coagulation and coagulation-related proteins such as factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, thrombopoietin and thrombopoietin receptors, colony stimulating factors (CSF), including the following: M-CSF, GM-CSF and G-CSF, other blood and serum proteins, including but not limited to albumin, IgE and blood group antigens, receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors, T cell receptors, neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophins-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), relaxin A-chain, relaxin B-chain and prorelaxin, interferons,Including, for example, interferon-α, -β and -γ, 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 enveloped virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surface Surfactants, tumor necrosis factor-α and -β, enkephalinase, BCMA, Igκ, ROR-1, ERBB2, mesothelin, RANTES (regulator of activation of normal T cells expressed and secreted), mouse gonadotropin-related peptide, DNA enzyme, FR-α, 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, transporter, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, PSA, addressin, regulatory protein, immunoadhesin, antigen binding protein, growth hormone, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, tight junction protein (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 ligands PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin, SS1(dsFv)PE38 conjugate, Legionella pneumophila (lly), gpA33, B7H3, interferon (IFN)γ, interferon gamma-induced protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin 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 β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin,and biologically active fragments or variants of any of the foregoing.

[0141] In some embodiments, the biomolecule of interest (e.g., protein) includes abciximab, adalimumab, adelimumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, brentuximab, brodalumab, mocantuzumab, canakinumab, cetuximab, certolizumab, canakinumab, daclizumab, monoclonal antibody, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitazumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, leridizumab, ruximab, lxdkizumab, mapatumumab, motesanib phosphate diphosphate), muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelleukin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rituximab, rituximab, romiplostim, lomosozumab, sargramostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volocizumab, zalumab, zalutumumab, and biosimilars of any of the foregoing.

[0142] In some embodiments, the biomolecules of interest (e.g., proteins) can include blinatumomab, catumaxomab, ertuinomab, solituzumab, targomiRs, rugizumab (ABT981), vanucizumab (RG7221), nonatolumab (ABT122), ozoralixumab (ATN103), floteuzmab (MGD006), pertuximab (AMG112, MT112), lymphomab (lymphomab), omun (FBTA05), (ATN-103), AMG211 (MT111, Medi-1565), AMG330, AMG420 (B1836909), AMG-110 (MT110), MDX-4 47. TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011(JNJ64052781), IMCgp100, Indium 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 their modified molecules or variants or analogs and biosimilars of any of the above.

[0143] The biomolecules of interest (e.g., proteins) according to the present disclosure encompass all of the foregoing, and further include antibodies comprising 1, 2, 3, 4, 5, or 6 complementary determining regions (CDRs) of any of the above antibodies. Also included are variants comprising regions of amino acid sequences that are 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identical to a reference amino acid sequence of a 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 that implement the Smith-Waterman algorithm, which is considered to be a satisfactory solution to the problem of searching and aligning sequences. Other algorithms may also be employed, particularly where speed is an important consideration. Common programs for alignment and homology matching of DNA, RNA, and polypeptides that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waterman algorithm for execution on massively parallel processors manufactured by MasPar.

[0144] Chimeric antigen receptor (CAR) comprises one or more costimulatory (signaling) domains known in the art to increase the efficacy of CAR-T cell responses. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (supra), Song et al., Blood [blood] 119: 696-706 (2012); Kalos et al., Sci Transl. Med. [Science Translational Medicine] 3: 95 (2011); Porter et al., N. Engl. J. Med. [New England Journal of Medicine] 365: 725-33 (2011) and Gross et al., Annu. Rev. Pharmacol. Toxicol. [Annual Review of Pharmacology and Toxicology] 56: 59–83 (2016), each of which is incorporated herein by reference in relevant parts.Exemplary co-stimulatory domains for incorporation into CARs can be derived from, among other sources, CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ξ), CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNF, TNFr, integrin, signaling lymphocyte activation molecules, 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, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, C D49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-lc, ITGBl, CD29, IT GB2, 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 a fragment or combination thereof. The costimulatory domain may comprise one or more of the extracellular portion, transmembrane portion, and intracellular portion of the protein from which the costimulatory domain is derived.

[0145] The following examples (both actual and predicted) are provided to illustrate specific embodiments or features of the invention and are not intended to limit the scope thereof.

[0146] Examples

[0147] Example 1 Single Use Continuous Solids Discharge Disc Stack Centrifugation, Flocculation and Depth Filtration of High Density Cell Cultures.

[0148] This example provides a harvesting method for purifying a target protein from a high-density continuous perfusion mammalian cell culture using a purification scheme that utilizes a single-use continuous solids discharge disk stack centrifugation step followed by flocculation and depth filtration. The harvesting method uses an unconventional but effective approach to produce high-yield, purified protein from high-density cultures.

[0149] Nine different perfusion cultures were performed at 500 L scale. The cultures contained one of four monoclonal antibodies (mAbs), mAb 1 (runs 1, 2, and 3), mAb 2, mAb 3, or mAb 4, xmAb (runs 1 and 2), or the cultures contained one of two bispecific antibodies (Bsp), Bsp 1 and Bsp 2.

[0150] On day 0, CHO cells expressing monoclonal or bispecific antibodies were inoculated into a 500 L single-use bioreactor at a working volume of 450-500 L serum-free, chemically defined batch medium. Cultivation was started in batch mode at 36 °C with stirring. X-CELL An alternating tangential flow (ATF) filtration system (Repligen, Waltham, MA) was used with a nominal surface area of ​​2.1 m 2 The cultures were continuously perfused with ATF-6 filters and serum-free, chemically defined perfusion medium until harvest. The cultures were cold-shifted (32-33°C) to increase yield and maintained at lower temperatures until harvest.

[0151] On day 15 or day 18, the contents of the bioreactor were harvested in bulk. Initially, the culture was cooled to 10° C. The cell culture fluid was then harvested directly at 10° C. or collected into a storage tank and maintained at 4° C. Harvest cell viability, viable cell density (VCD), percent packed cell volume (% PCV), and baseline turbidity of the culture were determined using conventional techniques and the results are shown in Table 1.

[0152] A 500 L single-use continuous solids discharge disc stack centrifuge (Alfa Laval Primo, Fresno, CA) was used. TM) The undiluted cell culture was centrifuged continuously. The cell culture was continuously loaded into the centrifuge at a specific flow rate through a series mechanism (Table 1). The centrifuge was operated at an equivalent sedimentation rate (Q / ∑) of 5.25E-9 m / s to 1.5×E-8 m / s or 0.6 to 1.8 (L / h / KQ), and the % PCV value was 16%-30%.

[0153] 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 harvest yield are averages for different sample time points during centrifugation for a given sample (see Table 1).

[0154] As shown in Table 1, using Alfa Laval Primo TM The drum was rotated with the average drum speed varying depending on the molecules being harvested and was 4713-5200 rpm. The average centrifuge feed flow rate was between 0.7-2.0 L / min (LPM). The centrate was continuously collected into a harvest tank and maintained at 10°C with mixing. The concentrate (heavy phase) was discharged to waste. The average centrate flow rate was between 0.79-1.51 L / min. The average concentrate (heavy phase) flow rate was between 0.29-0.61 L / min. The average concentrate (heavy phase) % PCV was 77%-96%. Online centrate measurements were performed regularly with an average centrate turbidity between 165-940 nephelometric turbidity units (NTU). The average theoretical centrifuge yield was between 88%-99%. When performing single-use continuous solid discharge disc stack centrifugation according to the method disclosed herein, pay attention to the feed flow rate, drum speed and heavy phase % PCV target, which is affected by the cell density of the feed and the flow rate of the heavy phase pump. As described herein, these parameters are summarized using Q / ∑ values, and Q / ∑ values ​​are positively correlated with turbidity, because for a given equivalent sedimentation area (∑), an increase in feed flow rate (Q) will increase turbidity. Important centrifugal operating parameters include centrifuge drum speed, feed flow rate, cell culture solids content (PCV) and heavy phase (cell paste) solids content (PCV). Exemplary values ​​of these parameters are disclosed throughout the application. These parameters are adjusted to control normalized sedimentation rate (Q / ∑) and theoretical stepwise yield. As known in the art, Q / ∑ provides a measure of standardized sedimentation rate, where Q is the feed flow rate, and ∑ is the standardized sedimentation area of ​​the rotating drum. To achieve relatively low Q / ∑, the centrifuge is operated at a lower feed flow rate to allow more residence time to capture cells and larger debris, thereby producing less debris in the centrate.

[0155] Centrate turbidity is usually directly proportional to the concentration of particles (especially fine particles). As shown in the data herein, harvest and centrate turbidity vary to a certain extent due to differences in the percentage of viability of various cultures harvested according to the present disclosure, cell culture, cell density, cell packed volume, and the present invention. It is worth noting that the prior art using intermittent solid discharge centrifugation is only applicable to lower cell density cultures with PCV values ​​of 3%-12% when cell culture is harvested. Other methods of obtaining target protein from cell culture using fed-batch cell culture result in lower turbidity, but this is due to lower cell density and lower %PCV, consistent with the reduced particle load. In some methods, a harvest operation is designed in which a cell culture with a relatively low %PCV is mixed with a cell paste. The result is a mixed cell-containing fluid in which %PCV is artificially increased in an environment with artificially high cell viability and correspondingly low cell debris, thereby allowing high cell culture feed or flow rate that is not suitable for real-world cell culture harvest operations.

[0156] As demonstrated by the experiments disclosed herein, single-use continuous solid discharge disc stack centrifugation can effectively remove whole cells from cell culture fluids at high yields. However, the average centrate turbidity observed was 165 to 941 NTU, indicating that a large amount of cell debris was retained in the solution. One of the main causes of centrate turbidity is accumulated cell debris, which is mainly caused by cell death mechanisms in bioreactors or cell culture containers, but may also be affected by the centrifuge operation mode. For example, lower feed flow rates and higher drum speeds will reduce turbidity. Changes in turbidity complicate the sizing of depth filters. This problem is overcome by increasing the post-centrifugation flocculation step. Increasing the flocculation step improves the robustness of the process to cope with the effects of turbidity changes in the actual target protein harvest. The benefits of adding a flocculation step are revealed by comparative studies, in which the centrate clarification by flocculation followed by depth filtration is compared with the clarification by depth filtration alone (see Example 2). First, the effect of flocculation concentration on turbidity is studied by adding different concentrations of pDADMAC to the centrate, and then centrifugation and supernatant turbidity measurements are performed on a laboratory scale. Low turbidity was observed at concentrations between 0.04% and 0.10% (w / w) pDADMAC (Figure 5). A concentration of 0.05% (w / v) pDADMAC was tested using different depth filters followed by depth filtration and the results were compared to those obtained using depth filtration alone. More specifically, the results of these experiments showed that depth filtration loading increased by 0.85-16.2-fold ( 0.05% (w / v) pDADMAC) after pDADMAC treatment compared to depth filtration alone. Figure 1 ). In addition to increasing 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 ( Figure 3 and 4 Improved filter loading and impurity removal do not adversely affect step yields when compared to depth filtration alone ( Figure 2 ). In general, similar or improved step yields were observed for flocculation and depth filtration when compared to depth filtration alone. The level of product loss was acceptable given the increased clarity of the centrate, which is suitable for direct application in chromatographic fractionation, such as affinity binding of antibody biologics to a Protein A column.

[0157]

[0158] Example 2 Depth Filtration of Flocculated Centrifuge Liquid

[0159] In the harvesting method disclosed herein, after a single use of continuous solid discharge disc stack centrifugation and flocculation / precipitation, the centrate material is further purified using depth filtration. Therefore, the fluid fraction of the flocculated centrate material obtained from the large-scale harvest described in Example 1 was used for flocculation and small-scale depth filtration experiments to compare the depth filtration performance between untreated and pDADMAC flocculated centrates, as briefly described in Example 1 above. pDADMAC (polydiallyldimethylammonium chloride, Sigma Aldrich, St. Louis, MO) stock solution was added to a 1L centrate sample to a final concentration of 0.025%-0.01% (w / w) pDADMAC. A 1L sample from each centrate was untreated to serve as a treatment control. The pDADMAC solution was continuously added to the centrate for about one minute and stirred at 10°C for at least 30 minutes. Stirring was achieved using a stirring plate and a stirring bar; the stirring rate was changed to maintain constant mixing of the centrate with the flocculated material. A total of 56 conditions were tested in different formats and different depth filters. The results showed that 0.035%-0.10% (w / w) pDADMAC defined the optimal concentration range for flocculation of various centrates containing target protein (see Figures 5 and 6). The data showed optimal and suboptimal but still effective flocculant concentrations, and consistent with the data, the present disclosure contemplates a range of 0.025%-0.15% flocculant for use in the harvesting methods of the present disclosure.

[0160] Filters with larger pore size distributions (such as COHC and COSP) may not be suitable for untreated materials, as the particle size distribution in the untreated centrates of various molecules measured using dynamic light scattering (DLS) ranged from 1 μm to 60 μm, with most particles in the 1-40 μm range. This size range is consistent with the nominal pore size distribution of these filters. In addition, the number of finer particles increased in the untreated material, which could cause internal filter clogging and further exacerbate the depth filter throughput. In contrast, pDADMAC flocculation works by aggregating finer molecules together to form larger particles. DLS data showed that after pDADMAC treatment, the number of particles decreased and the average particle size increased. This may explain the increase in throughput of the depth filter used after pDADMAC flocculation; compared to untreated materials containing a large number of finer particles (a higher proportion of the total particles filtered), the larger particles are retained by the filter and prevent internal clogging. Selecting the correct treatment type, treatment concentration, and depth filter nominal pore size rating is related to optimizing depth filter loading, step yield, and impurity removal.

[0161] Six small micro-pod depth filters were tested. X0HC, X0SP, C0HC, COSP (MilliporeSigma, Burlington, Mass.), DL60 and DL75 (Sartorius, Bohemia, NY). Millipore and Sartorius depth filters are 23 cm 2 and 25cm 2 .

[0162] The depth filters used for the small-scale experiments differed in their pore size distribution and material composition. The Millipore X0HC and COHC depth filters are composed of cellulose and an inorganic filter aid. The X0HC pore size range is 0.1-0.5 μm, and the COHC depth filters have a pore size range of 0.3-1.0 μm. The Millipore X0SP and COSP depth filters are synthetic and have the same pore size distribution as the X0HC and COHC depth filters. The Sartorius DL60 and DL75 have a wider pore size distribution than the Millipore depth filters, with nominal pore size distributions of 0.6-10 μm and 2-14 μm, respectively. The DL60 depth filter has a combination of a pure cellulose pre-filter and a coarse secondary filter. The DL75 depth filter contains a coarse primary filter and a secondary fine filter. The variety of pore size distributions and material properties in the depth filters used for development ensures that a large depth filtration data set is available to support the selection of appropriate depth filters for the disclosed harvesting methods.

[0163] Prior to loading the treated and untreated samples, the depth filter was flushed with purified water or a buffer solution at a temperature of about 10°C at a flow rate of less than 400 liters per square meter per hour (LMH) to achieve a flow rate of at least 100 L / m 2 After flushing the primary depth filter, a secondary 0.2 μm Optiscale Capsule SHC filter was connected to the primary filter (MilliporeSigma, Burlington, Mass.) and the secondary filter was flushed at the same flux rate with a target throughput of at least 50 L / m 2 The surface area of ​​the secondary filter is 3.6 cm 2 , the depth filtration surface ratios of stage 1 to stage 2 are 6.38 and 7.14 for Millipore and Sartorius depth filters, respectively.

[0164] After water flushing, the inlet of primary depth filter is drained, to remove unnecessary water. Then the depth filter is perfused with their respective loading (untreated centrate or flocculated centrate), to ensure removal of bubbles. Then 1L untreated centrate or 1L flocculated centrate is loaded into the filter. The inlet flux during the experiment remains between 90LMH and 150LMH. Final depth filtration throughput varies according to the performance of depth filtration. The turbidity of the depth filtrate pool is measured at the end of the experiment.

[0165] The depth filtration experiment was terminated when the stage 1 inlet pressure in the filter reached at least 20 psig or when the desired throughput was reached. In some cases, due to time constraints, the depth filtration experiment was terminated before the desired throughput was reached. Based on data from both the depth filtration and flocculation studies, a pDADMAC range of 0.02%-0.1% (w / w) was demonstrated to be the optimal concentration range for inducing beneficial flocculation in the harvesting method of the present disclosure.

[0166] The following parameters are included to evaluate the performance of small-scale depth filtration: depth filtration final throughput, depth filtration yield, depth filtrate pool host cell protein (HCP) level (also referred to as CHOP level), depth filtrate pool DNA level, normalized depth filtration throughput, HCP logarithm reduction value (LRV) and DNA LRV. Untreated XOHC depth filter is used as control condition and the final value of various parameters is divided by control condition (XOHC+ untreated) to calculate normalized value. Normalized value shows the increase and decrease amplitude of the various parameters tested, therefore helps to better evaluate the improvement of new harvest technology. LRV is calculated by applying a logarithmic transformation to the base of ten to the measured load value divided by the measured pool value.

[0167] Improved performance in depth filtration is characterized by increased depth filtration throughput values ​​or normalized depth filtration throughput values, increased or comparable percent yield, and increased or comparable HCP LRV and / or DNA LRV.

[0168] Figure 1Depth filtration normalized through values ​​of various molecules, treatment types and depth filters are shown. Compared with the values ​​of untreated centrate, the normalized depth filtration values ​​of the centrate conditions of flocculation are in the range of 0.85-16.2 times. 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 except X0HC+ untreated). All conditions were operated at less than or equal to 150LMH flux. Generally, data show that the depth filtration through amount using 0.05% (w / w) pDADMAC flocculation is much higher than the control conditions using X0HC depth filter and untreated centrate, regardless of the final % PCV of material and test sample. Specifically, except mAb1 and X0HC, all depth filters perform better when 0.05% (w / w) pDADMAC flocculation. The control and untreated conditions reached a maximum pressure of no more than 20 psig in a short period of time, while the pDADMAC treated conditions maintained a pressure differential of less than 10 psig and could be operated for a longer period of time. This is advantageous because it indicates that a greater volume of material can be filtered before reaching the peak pressure. A peak pressure of 20 psig is considered a hazard and safety risk, thus limiting deep filtration operations.

[0169] Figure 2 The percent yields for the 0.05% (w / w) pDADMAC treated conditions are shown to be comparable or higher than the control and untreated conditions. The exceptions to the depth filters are XOSP and XOHC paired with pDADMAC treatment. Therefore, due to the lower yields, it is not preferred to use XOSP or XOHC to filter the centrate treated with pDADMAC. The control and untreated conditions showed lower yields due to the maximum pressure being reached due to clogging of the depth filter. In these experiments, no recovery buffer rinse was performed.

[0170] Evaluate Figure 1 Impurity levels for small scale flocculation and depth filtration pools shown in . The depth filtrate pool impurity levels were measured and compared to the centrifuge pool using a log reduction value (LRV) calculation. When high LRV values ​​were observed, high impurity removal was observed. Figure 3 and Figure 4 HCP and DNA values ​​are shown for pDADMAC treated (dark grey), untreated (light grey) and control conditions (black). Variation in the impurity LRV was observed between centrate batches, but in general, the impurity LRV of pDADMAC treated centrate was similar or better than that of untreated centrate and control conditions.

[0171] The present disclosure provides a harvesting method that utilizes single-use continuous solid discharge disc stack centrifugation combined with flocculation of the centrate, such as flocculation using pDADMAC treatment, and depth filtration that results in higher harvest yields than traditional perfusion culture harvesting 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 present challenges to existing microfiltration harvesting technologies due to increased filter usage resulting in variable harvest yields and high costs. In addition, the harvest yields of some molecules are low (about 70%), which is caused by product losses attributable to cell culture variability, filter variability, and equipment variations, which together result in varying amounts of product losses during the harvesting operation. Current intermittent solid discharge centrifuge technology is unable to handle such high PCV cell culture harvests due to centrifuge plugging. It is recognized that continuous solids discharge disc stack centrifugation may not provide adequate clarification of high solids content cell culture harvests and may not provide a robust harvesting process option on its own or even in combination with depth filtration, as seen in low throughput and in some cases low percent yield, HCP clearance, and DNA clearance in the untreated centrate example. The combination of continuous solids discharge disc stack centrifugation and subsequent flocculation results in a robust process for harvesting a variety of target proteins from high solids content perfusion cell cultures, and the process also makes it possible to incorporate a depth filtration step to further purify the target protein while maintaining high yield in a series of cost-effective steps that are compatible with further conventional downstream purification operations.

[0172] Treatment of the centrate with 0.05% (w / w) pDADMAC resulted in the aggregation of smaller particles, reducing the total amount of particles while effectively increasing the particle size distribution, making it possible to obtain a more uniform distribution of particles when used with an appropriate depth filter (e.g., C0HC, C0SP, DL75 or DL60), the centrifugation and coagulation steps resulted in an increase in depth filtration throughput while providing similar or improved harvest yields, HCP clearance, and DNA clearance when harvesting most target proteins. In addition, pDADMAC flocculation was able to maintain a low differential pressure throughout the run, which is an important aspect in terms of safety and the ability to filter the desired amount of material while meeting the target depth filtration throughput goals (see Figure 8 ).

[0173] Example 3 Effect of pDADMAC concentration on turbidity and small-scale depth filtration performance

[0174] The pDADMAC dosage study was conducted to understand the effect of pDADMAC concentration on the turbidity of flocculated samples. The dosage study was repeated using centrates with different turbidities. The changes in centrate turbidity were produced by changing the mode and single-use continuous solid discharge centrifuge operating conditions, as described in Table 2. The dosage study was conducted by adding pDADMAC from the stock solution to 40 mL centrates to the final pDADMAC concentration shown in Table 2. The control (0% w / w pDADMAC) and pDADMAC-treated centrates were mixed for at least 15 minutes. The 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 turbidity meter (Hach Company, Loveland, CO, Colorado).

[0175] Table 2. pDADMAC dosing study of mAb 1 and xmAb 1

[0176]

[0177] FIG5 shows the results of the pDADMAC dosing study as described in Table 2. Figure 5A Results of a pDADMAC dosage study using mAb 1 (Run 2) of Table 1 and the conditions described in Table 2 are shown. This study evaluated the effect of pDADMAC concentration on turbidity levels of various centrate sources subjected to different centrifuge flow rates and bowl speeds. Note that Table 1 only shows the average values ​​for feed flow rates and bowl speeds. The results of the study revealed low supernatant turbidity values ​​within a pDADMAC concentration range of 0.04 to 0.10% (w / w). pDADMAC concentration levels below 0.04% are undesirable as they result in high turbidity levels.

[0178] Figure 5BDescribed are the results of a pDADMAC dosage study from xmAb1 (Run 1). This study evaluated the effect of pDADMAC concentration on a source of centrates with different initial turbidity levels. Different initial turbidities in the centrates were produced by varying the inlet flow rate of a single-use centrifuge. High turbidity and low turbidity centrates produced initial turbidity values ​​of 1500 NTU and 290 NTU, respectively. The results of the study showed low supernatant turbidity values ​​within the pDADMAC concentration range of 0.02 to 0.10% (w / w), regardless of the initial centrate turbidity level. Concentration levels of pDADMAC below 0.02% are undesirable because they do not reduce the turbidity level after pDADMAC treatment.

[0179] After determining the optimal pDADMAC concentration that resulted in the lowest turbidity values, a small-scale depth filtration study was performed 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. The small-scale depth filtration run was performed as described in Example 2.

[0180] Monoclonal antibodies mAb 1 (run 1) and mAb 2 were harvested from cell culture fluid using a single-use continuous solid discharge disc stack centrifuge as described in Example 1 Table 1. 2% pDADMAC stock solution was used to perform pDADMAC flocculation on a small scale. For mAb 1 (run 1), the pDADMAC concentration range tested was 0.01%, 0.02% and 0.05%. For mAb 2, 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 samples processed with pDADMAC. For each experiment, untreated centrifuge was filtered with a XOHC depth filter as a control.

[0181] The normalized depth filter throughput values ​​from these experiments are shown in Figure 6. The final throughput (L / m 2 ) divided by the final depth filtration throughput of the control untreated condition. A normalized throughput greater than one indicates improved depth filtration performance relative to the control. pDADMAC concentrations between 0.035% and 0.1% (w / w) pDADMAC showed improved depth performance when compared to the control.

[0182] Example 4 Effects of pDADMAC and PEG on small-scale depth filtration performance

[0183] The centrate of mAb 1 run 3 was collected. This large-scale run is not included in Example 1 because it was not produced using a single-use continuous solid discharge centrifuge device, but was performed using a stainless steel continuous solid discharge centrifuge. The average centrate turbidity was 182 NTU, which is similar to the average centrate turbidity of mAb 1 runs 1 and runs 3, so it is considered to be a representative material for the depth filtration study of pDADMAC processing. As described in Example 2, the centrate obtained from the large-scale was flocculated with 0.05% (w / w) pDADMAC alone or in combination with 3% PEG 3000, followed by depth filtration. pDADMAC and PEG 3000 solutions were added to the centrate simultaneously. The flocculated centrate was mixed for at least 30 minutes and then loaded onto a COHC or COSP depth filter. Untreated centrate was loaded onto a XOHC depth filter as a control because it had previously been determined that this provided the highest depth filter loading for untreated centrate. X0HC filters were used for the untreated control centrate because it is the optimal filter type for particle size in solution for the untreated condition (see Figure 1 ). We use this as a control to understand how each molecule is similar / different from each other.

[0184] Figure 7 The pDADMAC+PEG3000 condition was shown to achieve 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 harvesting, wherein continuous solid discharge disk stack centrifugation precedes a flocculation step, preferably using 0.02%-0.15% pDADMAC, with or without about 3% PEG, such as 3% PEG3000.

[0185] Example 5 Comparison of large-scale and small-scale depth filtration performance

[0186] As shown in Table 1, large-scale and small-scale depth filtration was performed for mAb 1 Run 1, xmAb 1 Run 1, xmAb 1 Run 2, and Bsp 2. At large-scale, centrate was collected from a single-use continuous solids discharge centrifugation as described in Table 1 for mAb 1 Run 1, xmAb 1 Run 1, xmAb 1 Run 2, and Bsp 2. The centrate was flocculated and deep filtered at large scale. Large-scale depth filtration was performed using a COHC depth filter. The filter size of the COHC used for large-scale runs was greater than or equal to 1.1 μm. 2. Small-scale depth filtration studies were performed in parallel for each centrate batch using untreated centrate and X0HC depth filters as controls. Depth filters in small-scale and large-scale depth filters were rinsed 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 the 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 of mAb 1 Run 1, xmAb1 Run 1, xmAb1 Run 2, and Bsp 2 was evaluated using flocculated centrate flow rates of 65, 95, 155 LHM, and 120 LMH, respectively.

[0187] Figure 8 The depth filtration performance of large-scale flocculated centrate using COHC depth filters and 0.05% (w / w) pDADMAC treatment is shown compared to the small-scale depth filtration performance using XOHC depth filters. The data show that flocculation and COHC depth filtration performance (grey) are improved in terms of differential pressure trends when compared to control conditions using untreated centrate and XOHC depth filtration (black). pDADMAC treatment and COHC filtration show stable differential pressures below 10 psig. Maintaining low differential pressures during manufacturing is an important goal because a maximum allowable pressure of about 20 psig is desired during the process to maintain safety and reduce risks associated with high pressure. Ensuring that the low differential pressure can be maintained for a longer duration results in more material being filtered, and this reduces any impact on downstream steps in the harvesting operation. The harvesting step yields for these large-scale runs are summarized in Table 3. The step yields ranged from 86% to 92%, which is an improvement over the average microfiltration harvesting step yield of about 70%.

[0188] Table 3. Summary of large-scale single-use continuous solids discharge disc stack centrifuge, flocculation, and depth filtration process performance.

[0189]

[0190] Example 6 Product Quality Considerations

[0191] Product quality is assessed by subjecting the deep filtrate pool containing the target protein to preparative protein A purification followed by analytical testing by chromatographic fractionation or electrophoresis. The types of analytical chromatography used include size exclusion chromatography (SE) and cation exchange chromatography (CEX). Electrophoretic assessment of product quality is performed using reduced sodium dodecyl sulfate capillary electrophoresis (rCE) and non-reduced capillary electrophoresis (nrCE). These different types of analysis are performed to provide a rigorous assessment of product quality.

[0192] Fig. 9 Comparison of xmAb 1 Run 1 product quality for the pDADMAC treated COHC filtrate pool (dark grey), untreated centrate COHC pool (light grey), and untreated XOHC filtrate pool (black) is shown. COHC conditions were performed as described in Example 2 using 0.05% (w / w) pDADMAC. Fig. 9 As shown in, product quality evaluation using SE, CEX, rCE and nrCE, respectively, showed comparable results in the three types of materials subjected to quality evaluation (i.e., untreated centrate, pDADMAC treated centrate and COHC depth filter pool). These results confirm that treatment of cell culture centrate with a flocculant such as pDADMAC does not negatively affect product quality characteristics.

Claims

1. A method for separating a recombinant protein produced in a perfusion culture from at least one other perfusion culture component, the method comprising: (a) harvesting a pool or eluate stream from a perfusion culture comprising the recombinant protein and at least one other perfusion culture component, the perfusion culture having a packed cell volume (PCV) of at least about 16% at the time of harvest; (b) introducing the harvest pool or eluate stream into at least one continuous solids discharge disc stack centrifuge; (b) operating the continuous solids discharge disc stack centrifuge to separate the fluid component into a centrate and a high density composition; (c) collecting the centrifuge liquid; (d) adding a flocculant to the centrifuged liquid; and (e) filtering the centrifuged liquid.

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

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

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

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

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

7. The method of claim 5, wherein the single-use bioreactor is 2,000 L or larger.

8. The method of any one of claims 1-7, wherein the perfusion culture harvest reservoir or eluate stream has a turbidity of at least 180 NTU at the time of harvest.

9. The method of any one of claims 1 to 8, wherein the perfusion culture harvest pool or eluate stream has at least 2×10 7 The viable cell density was 1 viable cell / ml.

10. The method of claim 9, wherein the perfusion culture harvest pool or eluate stream has at least 3×10 7 The viable cell density was 1 viable cell / ml.

11. The method of claim 9, wherein the perfusion culture harvest pool or eluate stream has at least 5×10 7 The viable cell density was 1 viable cell / ml.

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

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

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

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

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

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

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

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

20. The method of any one of claims 1-19, wherein discrete batches of perfusion harvest tanks are introduced into the continuous solids discharge disc stack centrifuge.

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

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

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

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

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

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

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

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

29. The method of claim 28, wherein the antigen binding protein is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a bispecific T cell engager molecule 30. The method of any one of claims 1-29, wherein the protein is granulocyte colony stimulating factor, erythropoiesis stimulating agent, HER receptor, cell adhesion molecule, growth factor, osteoinductive factor, insulin, coagulation protein, colony stimulating factor, blood group antigen; growth hormone, growth hormone receptor, T cell receptor; neurotrophic factor, neurotrophin, relaxin, interferon, interleukin, viral antigen, lipoprotein, integrin, rheumatoid factor, immunotoxin, surface membrane protein, transporter, homing receptor, addressin, regulatory protein or immunoadhesin.

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

32. The method of claim 31, wherein the depth filter is C0HC filter, C0SP filter, DL60 filter or DL75 filter.

33. The method of claim 31, wherein the depth filter is C0HC filter or C0SP filter.

34. The method of any one of claims 31-33, wherein the centrate is passed through the depth filter at a flux rate of 150 LMH or less and a pressure of 10 psi or less.

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

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

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

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

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

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

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

42. A method for producing an isolated, purified recombinant protein from a perfusion culture initiated in a single-use bioreactor, the method comprising the steps of a. inoculating the bioreactor with engineered cells to recombinantly express the target protein; 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 between 8°C and 12°C and collecting the perfusion culture from the bioreactor as a harvest pool or eluate stream; d. introducing the harvest pool or eluate stream into at least one continuous solids discharge disc stack centrifuge, wherein the harvest pool or eluate stream is between 4°C and 12°C; e. collecting the centrate from the centrifuge; f. adding a flocculant to the centrifuge at a temperature of 8°C and 12°C; g. 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 between 4°C and 12°C; h. subjecting the filtered centrate to one or more chromatography, filtration and / or UFDF unit operations; and i. Obtaining isolated and purified recombinant protein.

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

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