Flocculation method

Abstract

To provide a method for harvesting recombinant proteins from a mammalian cell culture solution.SOLUTION: The method comprises culturing mammalian cells expressing a recombinant protein in cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate flocculation, mixing the cell culture medium during the flocculation, allowing the flocculent to settle, and recovering the clarified supernatant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 576,303, filed December 15, 2011, which is incorporated herein by reference.

[0002] The present invention relates to a method for recovering recombinant proteins from mammalian cell culture broth, which method utilizes a cationic polymer, a non-ionic polymer and a non-ionic surfactant. [Background technology]

[0003] Clinical manufacturing of therapeutic proteins is a costly, large-scale endeavor. The demand for ever-larger quantities of therapeutic recombinant proteins has driven advances in cell culture methods, resulting in dramatically improved product titers. High-titer cell culture processes are generally driven by maintaining high viable cell densities throughout longer culture durations. A corresponding increase in biomass solids (viable and nonviable cells) and submicron cell debris particles is also observed. Higher loadings of solids and submicron cell debris particles can pose challenges to mammalian cell culture harvest processes, potentially making harvest methods less efficient at removing debris without substantially reducing product production capacity.

[0004] Cationic polymer flocculants are used in many applications, ranging from drinking water purification, wastewater treatment, use in the petroleum, mining, and paper industries, cosmetics, and medical uses, and have also been used to encapsulate mammalian cells and enzymes and to flocculate microbial cell cultures. However, for use in commercial-scale mammalian cell culture recovery processes, lengthy flocculation settling times can be problematic, leading to a recovery process that is time-consuming and less efficient than standard recovery methods.

[0005] There is a continuing need for improved mammalian cell culture harvest processes, particularly commercial-scale processes. Any improvement that allows for faster harvest times and / or greater recovery can result in cost savings for the production of protein therapeutics. The present invention fulfills this need by providing a fast and efficient cell culture harvest process. Summary of the Invention

[0006] The present invention provides a mammalian cell culture harvest method, the method comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate flocculation, mixing the cell culture medium during flocculation, allowing the flocculent to settle, and recovering the clarified supernatant.

[0007] The present invention also provides a method for harvesting mammalian cell cultures, comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved, adding polydiallyldimethylammonium chloride and PEG 3,000 to the cell culture medium to initiate aggregation, mixing the cell culture medium during aggregation, allowing the aggregates to settle, and recovering the clarified supernatant.

[0008] The present invention also provides a method for harvesting mammalian cell cultures, comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved, adding polydiallyldimethylammonium chloride, PEG 3,000, and Triton X-100 to the cell culture medium to initiate aggregation, mixing the cell culture medium during aggregation, allowing the aggregates to settle, and recovering the clarified supernatant.

[0009] The invention also provides a mammalian cell culture recovery method comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate flocculation, mixing the cell culture medium during flocculation, allowing the flocculants to settle for a first precipitation, collecting the first clarified supernatant, washing the first settled flocculants, allowing the washed flocculants to settle for a second precipitation, and collecting the second clarified supernatant.

[0010] The invention also provides a mammalian cell culture recovery method comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate flocculation, mixing the cell culture medium during flocculation, settling the flocs for a first precipitation, collecting a first clarified supernatant, washing the first precipitated flocs if product recovery in the first clarified supernatant is less than 80%, settling the washed flocs for a second precipitation, and collecting a second clarified supernatant.

[0011] The present invention also provides a mammalian cell culture recovery method comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, adding polydiallyldimethylammonium chloride and PEG 3,000 to the cell culture medium to initiate aggregation, mixing the cell culture medium during aggregation, settling the aggregates for a first precipitation, collecting the first clarified supernatant, washing the first settled aggregates, settling the washed aggregates for a second precipitation, and collecting the second clarified supernatant.

[0012] The present invention also provides a mammalian cell culture harvesting method, comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, adding polydiallyldimethylammonium chloride, PEG 3,000, and Triton X-100 to the cell culture medium to initiate flocculation, mixing the cell culture medium during flocculation, settling the flocculants for a first precipitation, collecting the first clarified supernatant, washing the first sedimented flocculants, settling the washed flocculants for a second precipitation, and collecting the second clarified supernatant.

[0013] In some embodiments, the cationic polymer is polydiallyldimethylammonium chloride.

[0014] In another embodiment, the non-ionic polymer is selected from polyethylene glycol and dextran.

[0015] In another embodiment, the non-ionic polymer is selected from PEG 3,000 and PEG 6,000.

[0016] In another embodiment, the mammalian cell culture recovery method provided above further comprises adding a non-ionic detergent to the cell culture medium. In a related embodiment, the non-ionic detergent is Triton X-100.

[0017] In another embodiment, the cationic polymer and the non-ionic polymer are added simultaneously.

[0018] In another embodiment, the cationic polymer, non-ionic polymer, and non-ionic surfactant are added simultaneously.

[0019] In another embodiment, the cationic polymer is added first and mixed for at least 30 seconds before adding the non-ionic polymer.

[0020] In another embodiment, the cationic polymer is added first and mixed for at least 30 seconds before the non-ionic polymer and non-ionic surfactant are added.

[0021] In another embodiment, the cationic polymer is a polymer of diallyldimethylammonium chloride, polydiallyldimethylammonium chloride, polyethyleneimine, polyacrylamide, or chitosan.

[0022] In another embodiment, the non-ionic detergent is sapoin or Triton X-100.

[0023] In another embodiment, polydiallyldimethylammonium chloride is added at a concentration of at or about 20 to 90 or about 90 pg per total cell density.

[0024] In another embodiment, polydiallyldimethylammonium chloride is added at a concentration of at or about 25 pg per total cell density and the mammalian cells are derived from a diploid cell line.

[0025] In another embodiment, polydiallyldimethylammonium chloride is added at between 43 pg per total cell density and 57 pg per total cell density, and the mammalian cells are derived from a tetraploid cell line.

[0026] In another embodiment, the concentration of PEG 3,000 is 3% or about 3% to 4.5% or about 4.5%.

[0027] In another embodiment, the concentration of PEG 6,000 is 2.5% or about 2.5% to 3.5% or about 3.5%.

[0028] In another embodiment, the concentration of Triton X100 is 0.05% (w / v).

[0029] In another embodiment, the mammalian cell culture broth is between 36°C and 20°C.

[0030] In another embodiment, the mammalian cell culture broth is at or above 20°C.

[0031] In another embodiment, the aggregates from the first precipitation are washed in a 9% sucrose solution. [Brief explanation of the drawings]

[0032] [Figure 1] The structure of PDADMAC is provided. [Figure 2A] The differences in sedimentation times obtained with various molecular weights of PDADMAC are shown. The concentration of each molecular weight of PDADMAC was 57 pg per total cell density. Black diamonds / dashed lines represent PDADMAC with molecular weights of 100,000-200,000. Black squares with dashed lines represent PDADMAC with molecular weights of 200,000-300,000. Black triangles with solid lines represent PDADMAC with molecular weights of 400,000-500,000. [Figure 2B] The clarity of the supernatant obtained when cell culture broth was flocculated with PDADMAC of various molecular weights is shown. From left to right, the bars represent PDADMAC with molecular weights below 100,000; 100,000-200,000; 200,000-350,000; and 400,000-500,000. [Figure 3A] The effect of high molecular weight PDADMAC concentration on the aggregation of 15-20 μm cells is shown. The black dashed diamond represents 11 pg of PDADMAC per total cell density. The black dashed square represents 18 pg of PDADMAC per total cell density. The black solid triangle represents 25 pg of PDADMAC per total cell density. The black dashed circle represents 39 pg of PDADMAC per total cell density. [Figure 3B]Figure 1 shows the effect of high molecular weight PDADMAC concentration on aggregation of 21-24 μm cells. The dotted and dashed black diamonds represent 29 pg of PDADMAC per total cell density. The dashed black square represents 43 pg of PDADMAC per total cell density. The dashed black triangle represents 57 pg of PDADMAC per total cell density. The solid black circle represents 71 pg of PDADMAC per total cell density. The dotted black square represents 86 pg of PDADMAC per total cell density. [Figure 4A] Figure 1 shows the effect of high molecular weight PDADMAC aggregation for cell cultures that produce high lactate concentrations. Solid black diamonds indicate no lactate added. Dashed black squares represent lactate added at 3 g / L. Dashed black triangles represent lactate added at 6 g / L. Dashed black circles represent lactate added at 9 g / L. [Figure 4B] Figure 1 shows the effect of high molecular weight PDADMAC aggregation for cell cultures with high cell density as measured by packed cell volume (PCV). A dashed black diamond represents 44% PCV. A dashed black triangle represents 33% PCV. A dashed black square represents 22% PCV. A solid black circle represents 11% PCV. [Figure 5] Figure 1 shows the effect of various diluents on high molecular weight PDADMAC aggregation for cell culture methods with high lactate concentrations. Dotted and dashed filled triangles represent sucrose. Dashed filled squares represent cell culture medium without diluent. Dashed filled diamonds represent betaine. Dashed filled circles represent PEG 1,000. Solid filled triangles represent PEG 6,000. Dashed filled triangles represent dextran 70 and betaine. [Figure 6] Figure 1 shows the effect of PDADMAC / PEG aggregation on cell culture with a PCV of 43.2%. The dashed black diamond represents PDADMAC only. The dashed black square represents sucrose. The solid black triangle represents PDADMAC / PEG. [Figure 7]The effect of the order of adding PDADMAC and PEG is shown. Bolus addition of PDADMAC and PEG (one-step method) is indicated by a black triangle with a dashed line. PDADMAC addition followed by PEG addition (two-stem method) is indicated by a black square with a dashed line. PDADMAC alone is indicated by a black diamond with a solid line. [Figure 8] The effect of adding Triton X-100 is shown. PDADMAC / PEG addition is shown by a solid black square. PDADMAC / PEG / Triton X-100 addition is shown by a dashed black triangle. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides a simple harvest and flocculation technique designed to maximize the harvest operation of high cell mass cell culture processes. A mammalian cell culture harvest process is provided that utilizes a cationic polymer in combination with a nonionic polymer in the flocculation of cell culture broth. Also provided is the use of a cationic polymer in combination with both a nonionic polymer and a nonionic surfactant.

[0034] The present invention is based on the discovery that flocculating mammalian cell culture broth using a nonionic polymer, or a nonionic polymer and a nonionic surfactant in combination with a cationic polymer, reduced aggregate settling time from 24 hours or more to less than 1 hour, and in some cases, down to 15 minutes, regardless of the density (up to 44% packed cell volume) or lactate concentration (10 g / L) of the cell culture process. The use of a nonionic polymer also results in the removal of higher-order aggregates and host cell proteins that co-purify with the desired recombinant product. This simple recovery method maximizes the recovery operation of cell culture processes, especially high-cell-volume culture processes at commercial scale.

[0035] Flocculation is a method by which suspended particles form larger aggregates or clusters. In flocculation, particles are brought out of suspension in the form of flocs by the addition of a flocculation agent or flocculent. Flocculation agents can be anionic or cationic polymers. These include natural flocculating agents such as alginic acid or chitosan; inorganic flocculating agents such as colloidal clay and activated silica; and synthetic flocculating agents such as polyacrylamide and polydiallyldimethylammonium chloride. Synthetic flocculating agents can be manufactured to have specific molecular weights (based on chain length) and molecular weight distributions.

[0036] Cationic polymers interact with negatively charged particles, such as organic matter. In cell culture broth, cationic polymers interact with negatively charged particles, such as viable and nonviable cells, cell metabolites, and cellular debris, such as nucleic acids, proteins, and liposomes. Aggregation of negatively charged compounds found in cell culture broth with cationic polymers occurs through ionic interactions, either via cross-linking of negatively charged particles; patch binding of cationic polymers, leading to aggregation; or charge neutralization of large negatively charged particles, leading to the release of neutralized particles from solution. Aggregates formed by cationic polymer cross-linking of negatively charged particles have increased shear sensitivity, producing larger aggregates and leading to aggregate disruption, resulting in a higher concentration of smaller particles, which sediment more slowly. Through patching or charge neutralization, the high charge density cationic polymer interacts with anionic patches on particles in suspension, either neutralizing the charge on the particles or forming larger particles that release from solution. Aggregates formed in this manner have smaller aggregate particle volumes and are less susceptible to shear disruption. For example, the addition of polydiallyldimethylammonium chloride (PDADMAC) to cell culture broth aggregates negatively charged cells and cell debris into larger particles via an electrostatic patching mechanism (Ramsden et al. (1998), Biotechnology Techniques, 12(8):599-603). PDADMAC also aggregates negatively charged submicron particles, producing a feedstream with significantly higher recovery filter train throughput compared to typical centrifugation recovery feedstreams. Aggregation via ionic interactions can be disrupted by increasing salt concentration or altering pH.

[0037] The addition of cationic polymers such as polydiallyldimethylammonium chloride to mammalian cell culture media containing or formerly containing cells expressing recombinant proteins results in the aggregation of negatively charged particles, including cells (viable and nonviable), cell metabolites, and cell debris. These large aggregated particles can be removed by centrifugation or gravity settling. The settling rate or time required for the aggregated cells and cell debris to settle depends on the density of the cells, cell debris, and cell metabolites. At low cell densities, typical of batch cell culture methods, the aggregated material typically settles (without further settling) at some point over a period of 4 to 24 hours, typically about 20-24 hours. The settling rate is significantly reduced in cell culture processes that produce high cell densities (greater than 10% compacted cell volume), submicron cell debris, and / or high lactate concentrations (greater than 2-3 g / L) of biomass. Cell culture processes that produce high cell densities or increase lactate concentrations require large amounts of cell broth diluent for the aggregates to settle within 24 hours. Despite the ease of using cationic polymers such as PDADMAC as an alternative to traditional recovery methods, prolonged settling times can be even less desirable on a commercial scale.

[0038] The present invention provides that the aggregate particle size and particle size growth rate in cationic polymer flocculants are greatly enhanced in the presence of nonionic polymers and nonionic surfactants. When PDADMAC was used in combination with nonionic polymers such as polyethylene glycol and nonionic surfactants such as Triton X100, aggregate gravity settling times of less than 2 hours were routinely observed, despite high biomass / cell densities that significantly increased the aggregate settling time of PDADMAC alone. Shear forces that could disrupt aggregates and / or reduce the flocculation rate were tolerated by the addition of nonionic polymers and nonionic surfactants. Harvest recovery yields of 80-90% or greater were consistently obtained, with significantly reduced host DNA. Nonionic polymer addition also reduced host cell protein and some high molecular weight species.

[0039] As used herein, a "cationic polymer" is a positively charged polymer that binds to negatively charged suspended particles. Cationic polymers include, but are not limited to, polymers of diallyldimethylammonium chloride (DADMAC). In a preferred embodiment, polymerization of DADMAC forms PDADMAC (Figure 1), an N-substituted pyrrolidine structure. Cationic polymers also include polyethyleneimine (PEI), polyacrylamide (PAA), and chitosan.

[0040] Concentrations of PDADMAC ranging from 20 or about 20 pg to 90 or about 90 pg per total cell density resulted in low supernatant turbidity and good aggregate sedimentation. "Total cell density" refers to the sum of viable and nonviable cells as measured by trypan blue exclusion using a Cedex cell counter and analyzer. In one embodiment, for small cell lines, such as diploid cell lines, PDADMAC is added at 25 or about 25 pg per total cell density. In another embodiment, for larger cell lines, such as tetraploid cell lines, PDADMAC is added at 43 or about 43 to 57 or about 57 pg per total cell density.

[0041] PDADMAC with a molecular weight of 200,000 to 500,000 influences flocculation performance by increasing sedimentation rate and supernatant clarity compared to lower molecular weight forms. In one embodiment, the PDADMAC molecular weight is in the range of 400,000 to 500,000. In one embodiment, PDADMAC with a molecular weight in the range of 400,000 to 500,000 is used at a final concentration of 22 pg per total cell density. In another embodiment, PDADMAC with a molecular weight in the range of 400,000 to 500,000 is used at a final concentration of 25 pg per total cell density. In another embodiment, PDADMAC with a molecular weight in the range of 400,000 to 500,000 is used at a final concentration of 45 pg per total cell density.

[0042] As used herein, "sedimentation velocity," "gravitational settling velocity," and "flocculation, compaction settling velocity" are used interchangeably. Sedimentation velocity can be determined by methods known in the art and described herein. For example, gravitational settling is 1 g. Sedimentation velocity is determined by dividing the volume of the floc by the total volume, measured in a 0.5 L or 1 L glass graduated cylinder. The total volume is the volume of the cell broth, including all flocculation / sedimentation agents.

[0043] "Settling time" is the time it takes for the aggregates to settle. Settling time is achieved when the aggregate settling rate is 1% per hour or less. Settling times of only 15 minutes are described herein for PDADMAC flocculation combined with the administration of nonionic polymer or nonionic polymer in combination with nonionic surfactant. The fast settling rate occurred despite high biomass / cell density, which significantly increased the flocculation settling time of PDADMAC alone. Shear forces that disrupted the aggregates and / or reduced the flocculation rate were tolerated by the addition of nonionic polymer and nonionic surfactant.

[0044] The clarity of the supernatant is independent of the sedimentation rate but depends on the cationic polymer dosage concentration. Other factors, such as temperature, density, and viscosity of the cell culture medium, had little effect on the sedimentation rate or clarity of the supernatant. In particular, the PDADMAC dosage is a function of cell volume, total cell density (viable and nonviable), and the concentration of submicron cell debris particles.

[0045] As used herein, "nonionic polymer" refers to a hydrophilic polymer that enhances intermolecular interactions and promotes precipitation. Nonionic polymers include, but are not limited to, polyethylene glycol (PEG), maltodextran, starch, methylcellulose, and dextran.

[0046] Increased sedimentation rates were achieved when PEG or dextran was added to mammalian cell culture media simultaneously with or following the addition of the cationic polymer flocculant. Product recovery depended on the nonionic polymer concentration, PEG molecular weight, the order of addition of the nonionic polymer and PDADMAC (simultaneously or PDADMAC first, followed by the nonionic polymer), and the duration of cell culture or debris concentration in the cell culture broth.

[0047] PEG 3,000 is useful in the range of 3, or about 3 to 4.5 or about 4.5% (w / v). PEG 6,000 is useful in the range of 2.5, or about 2.5% to 3.5 or about 3.5% (w / v). In one embodiment, PEG 3,000 is used at a final concentration of 3% (w / v). In another embodiment, PEG 3,000 is used at a final concentration of 15% (w / v). In another embodiment, PEG 3,000 is used at a final concentration of 25% (w / v).

[0048] As used herein, "nonionic surfactant" refers to an organic compound that is amphiphilic, i.e., contains both hydrophobic and hydrophilic groups, and includes, but is not limited to, sapoin and Triton X 100. In one embodiment, Triton X-100 is used at a final concentration of 0.05% (w / v).

[0049] The nonionic polymer may be added alone or in combination with the nonionic surfactant. In either case, it may be added simultaneously with the cationic polymer or subsequent to the addition of the cationic polymer. Both the nonionic polymer and surfactant may be added quickly, with addition times of one minute or less.

[0050] Once the aggregates have settled (first sedimentation), a clarified supernatant can be collected. To increase recombinant product recovery, the aggregates can be washed or resuspended to remove any residual recombinant product. A suitable wash diluent includes sucrose, PEG, cell culture medium, and buffered saline. In one embodiment, the wash diluent is 9% sucrose. The aggregates and wash diluent are mixed for less than 1 minute to 60 minutes and allowed to settle for approximately 1 hour to 24 hours. Once the aggregates have settled (second sedimentation), a clarified second supernatant is collected. The supernatants from the first and second sediments may be combined or purified separately.

[0051] The clarified supernatant can be recovered by removing the supernatant by pumping or decanting, followed by filtration through a depth filter containing diatomaceous earth and then through a membrane filter cut to 0.2 μm or through a 0.2 μm filter alone.

[0052] Cationic polymer removal can be monitored by methods known in the art and described herein, such as assays for monitoring mammalian cell toxicity; assays for measuring the inhibition of DNA or RNA transcription by DNA polymerase or reverse transcription, and assays for measuring protein translation of mRNA. For example, PDADMAC removal from intermediates in recombinant protein purification processes can be monitored by the inhibition of DNA amplification using quantitative polymerase chain reaction (QPCR).

[0053] The cell culture broth may be used directly from the bioreactor or may be chilled prior to flocculation. In one preferred embodiment, the temperature range of the cell culture broth is from at or about 36° C. to 20° C., or about 20° C. In another embodiment, the cell culture broth is chilled to 20° C., or about 20° C.

[0054] The present invention provides a method for recovering recombinant proteins from mammalian cell culture. Typical methods used in commercial processes for the production of recombinant proteins by mammalian cell culture include batch culture, fed-batch culture, and perfusion culture. Batch culture is a discontinuous method in which cells are grown for a short period in a fixed volume of culture medium, followed by a total harvest. Harvesting generally occurs when the maximum cell density is achieved (usually 5-10 x 10). 6 Fed-batch cultures occur at a rate of 10 to 30 x 10 cells / mL. Fed-batch cultures provide a bolus or continuous medium supply to replenish these medium components as they are consumed. Because fed-batch cultures receive additional nutrients throughout their run, they can achieve much higher cell densities (>10 to 30 x 10 cells / mL) when compared to batch methods. 6 In perfusion culture, a typical large-scale commercial cell culture strategy involves high cell densities, 60-90 (or more) × 10, where the biomass is approximately 50 to more than half the reactor volume. 6 We strive to achieve a cell density of 1 x 10 cells / mL. 8 Very high cell densities of >100 cells / mL have been achieved, and even higher densities are expected.

[0055] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably throughout this specification and refer to molecules comprising two or more amino acid residues linked together by peptide bonds. Peptides, polypeptides, and proteins include modifications, including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. Polypeptides include protein-based drugs and can be of chemical or commercial interest. Polypeptides include, among others, antibodies, fusion proteins, and cytokines. Peptides, polypeptides, and proteins can be produced by recombinant animal cell lines using cell culture methods and can be referred to as "recombinant peptides," "recombinant polypeptides," and "recombinant proteins." Expressed proteins can be produced intracellularly or secreted into the culture medium, from which they can be recovered and / or collected.

[0056] Examples of polypeptides that can be recovered using the methods of the present invention include proteins that contain an amino acid sequence that is identical or substantially similar to all or a portion of one of the following proteins: tumor necrosis factor (TNF), flt3 ligand (WO 94 / 28391), erythropoietin, thrombopoietin, calcitonin, IL-2, angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322):55-60), receptor activator of NF-kappa B ligand (RANKL, WO 01 / 36637), tumor necrosis factor (TNF)-dependent apoptosis-inducing ligand (TRAIL, WO 97 / 01633), thymic stromal-derived lymphopoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF, Australian Patent No. 588819), mast cell growth factor, stem cell growth factor (U.S. Patent No. 6,204,363), epidermal growth factor, keratinocyte growth factor, megakaryocyte growth and development factor, RANTES, human fibrinogen-like 2 protein (FGL2; NCBI accession number NM_00682; Ruegg and Pytela (1995), Gene160:257-62) growth hormone, insulin, insulinotropin, insulin-like growth factor, parathyroid hormone, interferons including α-interferon, γ-interferon and consensus interferon (U.S. Pat. Nos. 4,695,623 and 4,897,471), nerve growth factor, brain-derived neurotrophic factor, synaptotagmin-like proteins (SLP1-5), neurotrophin-3, glucagon, interleukins, colony-stimulating factors, lymphotoxin-β, leukemia inhibitory factor, and oncostatin-M. Descriptions of proteins that can be produced according to the methods of the present invention are found, for example, in Human Cytokines: Handbook for Basic and Clinical Research, all volumes (Aggarwal and Gutterman, eds., Blackwell Science, Cambridge, Massachusetts, 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press, New York, 1993); and The Cytokine Handbook,Vols.1and2 (Thompson and Lotze, eds., Academic Press, San Diego, CA, 2003).

[0057] Additionally, the methods of the present invention will be useful for recovering receptors for any of the above-mentioned proteins, antagonists of such receptors, or proteins comprising all or part of the amino acid sequence of any of the above-mentioned proteins and / or proteins substantially similar to such receptors or antagonists. These receptors and antagonists include: both forms of tumor necrosis factor receptor (TNFR, referred to as p55 and p75, U.S. Patent Nos. 5,395,760 and 5,610,279), interleukin-1 (IL-1) receptor (type I and II; European Patent No. 0460846, U.S. Patent No. 4,968,607 and U.S. Patent No. 5,767,064), IL-1 receptor antagonists (U.S. Patent No. 6,337,072), IL-1 antagonists or inhibitors (U.S. Patent Nos. 5,981,713, 6,096,728 and 5,075,222), IL-2 receptor, IL-4 receptor (European ...0460846, U.S. Patent No. 0460846, U.S. Patent No. 0460846, U.S. Patent No. 0460846, U.S. Patent No. 0460846, U.S. Patent No. 0460846, U.S. Patent No. 0460846, U.S. Patent No. 0460846, U.S. Patent No. No. 0367566 and U.S. Pat. No. 5,856,296), IL-15 receptor, IL-17 receptor, IL-18 receptor, Fc receptor, granulocyte-macrophage colony-stimulating factor receptor, granulocyte colony-stimulating factor receptor, receptor for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO 01 / 36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No. 6,015,938), receptor for TRAIL (including TRAIL receptors 1, 2, 3, and 4), and death domain-containing receptors such as Fas or apoptosis-inducing receptor (AIR).

[0058] Other proteins that can be recovered using the present invention include proteins containing all or part of the amino acid sequence of differentiation antigens (referred to as CD proteins) or their ligands, or proteins substantially similar to either of these. Leukocyte Typing VI(Proceedings of the VIth International Workshop and Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996). Similar CD proteins have been disclosed at subsequent conferences. Examples of such antigens include CD22, CD27, CD30, CD39, CD40, and their ligands (CD27 ligand, CD30 ligand, etc.). Some CD antigens are members of the TNF receptor family, including 41BB and OX40. The ligands are often members of the TNF family, such as 41BB ligand and OX40 ligand.

[0059] Enzymatically active proteins or their ligands can also be recovered using the present invention. Examples include proteins containing all or a portion of, or substantially similar to, one of the following proteins or their ligands: members of the disintegrin and metalloprotease domain family, including TNF-alpha converting enzyme, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein AI, globin, IL-2 antagonist, alpha-1 antitrypsin, ligands for any of the above enzymes, and numerous other enzymes and their ligands.

[0060] The term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or to antigen-binding regions thereof, which compete with intact antibodies for specific binding, and includes, unless otherwise specified, human, humanized, chimeric, multispecific, monoclonal, polyclonal, and oligomeric antibodies or antigen-binding fragments thereof. Also included are proteins having antigen-binding fragments or regions, such as Fab, Fab', F(ab'), Fv, diabodies, Fd, dAb, maxibodies, single-chain antibody molecules, CDR fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an antibody globulin sufficient to provide a specific antigen binding target polypeptide. The term "antibody" includes, but is not limited to, antibodies prepared, expressed, generated, or isolated by recombinant methods, such as antibodies isolated from host cells transfected to express the antibody.

[0061] Examples of antibodies include, but are not limited to, those that recognize any one or combination of the above proteins and / or proteins including, but not limited to, the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-I alpha, IL-I beta, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-1 receptor ... -4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subunit, FGL2, PDGF-β and its analogs (see U.S. Patent Nos. 5,272,064 and 5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (U.S. Patent No. 6,235,883), VEGF receptor, hepatocyte growth factor, osteoprotegerin ligand, interferon gamma, B lymphocyte stimulatory factor (BlyS, also known as BAFF, THANK, TALL-1, and zTNF4; Do and Chen-Kiang (2002), Cytokine Growth Factor Rev, 13(1):19-25) C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (a gene product expressed in association with lung cancer), HER-2, HER-3, tumor-associated glycoprotein TAG-72, SK-1 antigen, tumor-associated epitopes present in high concentrations in the serum of patients with colon and / or pancreatic cancer, cancer-associated epitopes or proteins expressed on breast, colon, squamous cell, prostate, pancreatic, lung and / or kidney cancer cells and / or on melanoma, glioma or neuroblastoma cells, necrotic core of tumors, integrin alpha 4 beta 7, integrin VLA-4, B2 integrin, TRAIL receptors 1, 2, 3 and 4, RANK, RANK ligand, TNF-α, adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesion, platelet glycoprotein gp IIb / IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (factor VIIa - inhibitor of tissue factor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (cytotoxic T lymphocyte-associated antigen), Fc-gamma-1 receptor, HLA-DR10 beta, HLA-DR antigen, sclerostin, L-selectin, respiratory syncytial virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcus mutans, and Staphylococcus aureus.Specific examples of known antibodies that can be produced using the methods of the present invention include adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab, canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab, eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, labetuzumab, These include, but are not limited to, mapatumumab, matuzumab, mepolizumab, motavizumab, muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab, ranibizumab, rituximab, lovelizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, zalutumumab, and zanolimumab.

[0062] The present invention can also be used to recover recombinant fusion proteins, for example, comprising any of the proteins described above. For example, recombinant fusion proteins comprising one of the proteins described above and a multimerization domain such as a leucine zipper, coiled coil, or Fc portion of an immunoglobulin, or a substantially similar protein, can be produced using the methods of the present invention. See, e.g., WO 94 / 10308; Lovejoy et al. (1993); Science 259:1288-1293;Harbury et al. (1993), Science 262:1401-05;Harbury et al. (1994), Nature 371:80-83; Hakansson et al. (1999), Structure 7:255-64. Specifically, such recombinant fusion proteins include proteins in which portions of a receptor are fused to the Fc portion of an antibody, such as etanercept (p75 TNFR:Fc) and belatacept (CTLA4:Fc).

[0063] For purposes of the present invention, a cell culture medium is a medium suitable for the growth of animal cells, e.g., mammalian cells, in in vitro cell culture. Cell culture medium formulation is well known in the art. Generally, cell culture media consist of buffers, salts, carbohydrates, amino acids, vitamins, or trace elements. Cell culture media may or may not contain serum, peptones, and / or proteins. A variety of tissue culture media, including serum-free defined media, are commercially available. For example, any one or combination of the following cell culture media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as EX-CELL® Type 300 (JRH Biosciences, Lenexa, Kansas), among others. Depending on the requirements of the cells being cultured and / or the desired cell culture parameters, cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc.

[0064] Cell culture media may be serum-free, protein-free, and / or peptone-free. "Serum-free" refers to cell culture media that do not contain animal serum, such as fetal bovine serum. "Protein-free" refers to cell culture media that do not contain exogenously added proteins, such as transferrin, protein growth factor IGF-1, or insulin. Protein-free media may or may not contain peptone. "Peptone-free" refers to cell culture media that do not contain exogenous protein hydrolysates, such as animal and / or plant protein hydrolysates. Cell culture broth or similar terms refer to cell culture media that contain, inter alia, live and non-live mammalian cells, cellular metabolites, and cellular debris, such as nucleic acids, proteins, and liposomes.

[0065] Cell culture or "culturing" refers to the growth and proliferation of cells outside a multicellular organism or tissue. Suitable mammalian cell culture conditions are known in the art. See, for example, Animal Cell Culture: A Practical Approach, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or attached to a solid substrate. Fluidized-bed bioreactors, hollow-fiber bioreactors, roller bottles, shake flasks, or stirred-tank bioreactors, with or without microcarriers and operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes, can be used for mammalian cell culture.

[0066] Mammalian cells, such as CHO cells, may be grown in small scale cultures, e.g., in 100 ml, to large scale cell cultures, such as systems with culture sizes of thousands and tens of thousands of ml, for clinical and commercial manufacturing of protein therapeutics.

[0067] Cell lines (also called "host cells") are genetically engineered to express polypeptides of commercial or scientific interest. Cell lines are generally derived from lineages that arose from a primary culture that can be maintained in culture for an indefinite period of time. Genetic engineering of cell lines involves transfecting, transforming, or transducing the cells with a recombinant polynucleotide molecule and / or otherwise altering the host cells to express a desired recombinant polypeptide (e.g., by homologous recombination and gene activation or fusion of recombinant and non-recombinant cells). Methods or vectors for genetically engineering cells and / or cell lines to express a polypeptide of interest are well known to those skilled in the art; for example, various techniques are available. Current Protocols in Molecular Biology(Wiley & Sons, New York, 1988 and quarterly revision); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69.

[0068] A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, VA) and commercial distributors. Examples of mammalian cell lines commonly used in the industry include VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells are used for the production of complex recombinant proteins, such as cytokines, clotting factors, and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol Chem. 263:6352-6362;McKinnon et al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990), J. Immunol 145:3011-3016). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44 are desirable CHO host cell lines because efficient DHFR selectable and replicable gene expression systems allow high recombinant protein expression levels in these cells (Kaufman RJ (1990) Meth Enzymol 185:537-566). Furthermore, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells, and proteins recombinantly expressed in them, have been extensively characterized and approved by regulatory agencies for use in clinical and commercial manufacturing.

[0069] While the terminology used herein is standard within the art, definitions of certain terms are provided herein to ensure clarity and certainty in the meaning of the claims. Units, prefixes, and symbols may be denoted in SI-approved format. Numerical ranges recited herein are inclusive of the numbers defining the range and include and support each integer within the defined range. Unless otherwise indicated, the words "a" or "an" should be interpreted as meaning "at least one." Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The methods and techniques described herein generally follow conventional methods well known in the art and, unless otherwise indicated, are as described in the various general and more specific references cited and described throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1990). All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference.

[0070] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. [Example]

[0071] [Example 1] This experiment compares various molecular weight formulations of diallyldimethylammonium chloride (PDADMAC) for flocculating mammalian cell culture broth and their settling times.

[0072] CHO cells expressing recombinant monoclonal antibodies were grown in a 2,000 L bioreactor in fed-batch culture for 15 days. The cell culture broth was cooled to 10°C prior to testing. A series of spin flasks were set up, each containing 1 L of cell culture broth. PDADMAC was supplied as a 20% (w / v) liquid (Sigma-Aldrich, St. Louis, MO), and the processing stock solution used in all these experiments was prepared by diluting it to 10% (w / v) with purified water. PDADMAC with molecular weights of 100,000–200,000; 200,000–350,000; and 400,000–500,000 was added to each flask to achieve final PDADMAC concentrations between 29 and 86 pg per total cell density. The PDADMAC solution was added over a period of approximately 1 minute while stirring at 70-80 rpm at 10°C, and the mixture was incubated for 15 minutes. The aggregates were allowed to settle at ambient temperature. This material was used for sedimentation time determination.

[0073] The second fed-batch culture was grown for 15 days in a 1,000 L disposable reactor. The cell culture broth was maintained at approximately 36°C. A series of spin flasks were set up, each containing 1 L of cell culture broth. PDADMAC with molecular weights of less than 100,000, 100,000-200,000; 200,000-350,000; and 400,000-500,000 was added to each flask to achieve final PDADMAC concentrations between 25 and 76 pg per total cell density. The PDADMAC solution was added over a period of approximately 1 minute with stirring at 70-80 rpm at approximately 36°C, followed by a 15-minute incubation. Aggregates were allowed to settle at ambient temperature. This material was used for turbidity determination.

[0074] Total cell density was determined by adding the total number of viable cells to the total nonviable cells, as measured by trypan blue dye exclusion using a Cedex cell counter and analyzer (Roche Innovatis AG, Indianapolis, IN). The clumped solution was then transferred to a 1 L glass graduated cylinder, and clumping, compressible sedimentation velocity was determined. Measurements were taken at 15-minute intervals over a 90-minute period, and the relative clumped volume was calculated as the sedimented clump volume / total volume.

[0075] The supernatant was removed from the sedimented, aggregated cell mass by decanting followed by filtration through a 0.2 μm filter. Turbidity was measured using a 2100P turbidity meter (Hach, Loveland, CO).

[0076] 2A and 2B show that flocculation with PDADMAC having an average molecular weight greater than 200,000 but less than 500,000 leads to optimal settling time and clarity compared to PDADMAC with an average molecular weight less than 200,000.

[0077] [Example 2] This experiment compares the amount of PDADMAC required to flocculate mammalian cell culture broth expressing recombinant antibodies from small cell lines, such as diploid cells, and large cell lines, such as tetraploid cell lines.

[0078] Diploid and tetraploid cell lines were grown as described above. A series of spin flasks were set up, with each flask containing 1 L of cell culture broth from each diploid and tetraploid culture. In this and all subsequent experiments, unless otherwise specified, PDADMAC with an average molecular weight of 400,000-500,000 was used. PDADMAC was added to each flask to a final concentration as shown in Table 2. Total cell density was determined as described above.

[0079] [Table 1]

[0080] The PDADMAC solution was added continuously as above with stirring, and the aggregates were allowed to settle at ambient temperature.

[0081] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation and compressible settling velocity. Measurements were taken at 15-minute intervals from 90 to 120 minutes, and the relative flocculated volume was calculated as described above.

[0082] FIG. 3A shows that aggregation with PDADMAC at a concentration of 25 pg per whole cell had the fastest sedimentation time.

[0083] FIG. 3B shows that aggregation with PDADMAC at a concentration of 57 pg per whole cell had the fastest sedimentation time.

[0084] [Example 3] This experiment looks at the impact of PDADMAC aggregation for cell cultures that produce high lactate concentrations and / or have high cell densities.

[0085] CHO cells expressing recombinant monoclonal antibodies were grown for 14 days in a 1,000 L disposable bioreactor. The cell culture broth was at ambient temperature. Four spin flasks were set up, with each flask containing 1 L of cell culture broth. Prior to adding PDADMAC, each flask received either 3 g / L, 6 g / L, or 9 g / L of Na DL-lactate at 60% (w / w) (Sigma Aldrich, St. Louis, MO), or no lactate as a control. PDADMAC was then added to each flask to a final concentration of 25 pg per total cell density (PDADMAC stock solution, as described above). Total cell density was determined as described above.

[0086] PDADMAC was added and stirred continuously at ambient temperature as described above, and the aggregates were then allowed to settle at ambient temperature.

[0087] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation, compressible settling velocity. Measurements were taken at various intervals over 1500 minutes and the relative flocculated volume was calculated as described above.

[0088] A second batch of CHO cell culture was grown in a 1,000 L disposable reactor in perfusion culture for 20 days. The cell broth was allowed to cool to ambient temperature. Prior to the addition of PDADMAC, the cell broth was diluted to 25%, 50%, and 75% with cell culture medium. The cell broth, at 44% compressed cell volume, was not diluted prior to the addition of PDADMAC. The final PDADMAC concentration was 25–26 pg per total cell density. As described above, the PDADMAC addition rate was approximately 1 min with stirring and settling at ambient temperature.

[0089] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation, compressible settling velocity. Measurements were taken at various intervals over 1400 minutes and the relative flocculated volume was calculated as described above.

[0090] 4A and 4B show that sedimentation velocity is significantly reduced in cell culture methods that have high lactate concentrations (greater than 2-3 g / L) and / or produce high cell densities of biomass (greater than 10% compressed cell volume).

[0091] [Example 4] This experiment compares the settling times of various diluents used in combination with PDADMAC.

[0092] CHO cells expressing a recombinant monoclonal antibody were grown in perfusion culture in a 1,000 L disposable bioreactor for 18 days. Day 16 cell broth was fed for testing at 36°C and allowed to cool to ambient temperature for 2.5 hours before aggregation. Addition and settling were performed at ambient temperature. Before PDADMAC addition, the cell broth was diluted to 67% with various diluents. The lactate concentration was 5 g / L. The PDADMAC addition rate was approximately 1 minute with a 15-minute incubation period at a speed of 75-85 rpm. The PDADMAC stock solution was 10% (w / v) from the initial 20% (w / v) stock solution and purified water. The final PDADMAC concentration was 25 pg per total cell density. The aggregates were allowed to settle at ambient temperature.

[0093] A series of dilutions were prepared; the concentrations are shown in Table 3.

[0094] [Table 2]

[0095] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation, compressible settling velocity, and measurements were taken at various intervals over 1600 minutes, and the relative flocculated volume was calculated as described above.

[0096] As shown in Figure 5, the combination of PDADMAC and PEG 6,000 had the fastest sedimentation time. Combinations of PDADMAC with PEG 6,000, PEG 1,000, or dextran 70 / betaine had improved sedimentation rates compared to PDADMAC alone. The addition of a nonionic polymer significantly increased the aggregate sedimentation rate. Combinations of PDADMAC with either PEG or dextran also decreased the sedimentation time compared to PDADMAC alone, independent of the lactate concentration in the culture.

[0097] [Example 5] In this experiment, we look at the effect of a combination of PDADMAC and PEG 3,000 on sedimentation rate for cell cultures with high cell densities.

[0098] CHO cells expressing a recombinant monoclonal antibody were grown in perfusion culture in an 80 L bioreactor for 20 days. The cell culture broth was cooled to ambient temperature before testing. Before adding PDADMAC, the cell broth was diluted to 10% with 36% (w / v) sucrose or 25% (w / v) PEG 3,000 (both in purified water). The final cell broth sucrose concentration was 3.6% (w / v), and the final PEG 3,000 concentration was 2.5% (w / v). The PDADMAC addition rate was approximately 1 min with a 15-min incubation period at a speed of 75–85 rpm. The PDADMAC stock solution was 10% (w / v) from the initial 20% (w / v) stock solution and purified water. The final PDADMAC concentration was 22 pg / wt. cell density for PEG 3,000 and 25 pg / wt. cell density for sucrose and the control or undiluted cell broth. The control or undiluted cell broth PCV was 48%. The diluted cell broth PCV = (control PCV x dilution factor) = 43.2%.

[0099] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation, compaction settling velocity, and measurements were taken at various intervals over 240 minutes, and the relative flocculated volume was calculated as described above.

[0100] As shown in Table 6, the combination of PDADMAC and PEG 3,000 led to an even faster settling rate than PDADMAC alone. As seen in Example 3, the settling rate of PDADMAC alone decreased significantly with increasing cell density. The combination of PEG 3,000 and PDADMAC reduced aggregate settling time compared to that of PDADMAC alone, independent of the density of the cell culture process (43% packed cell volume in this case).

[0101] [Example 6] In this experiment, the effect of timing of PEG addition with PDADMAC on sedimentation rate is examined.

[0102] CHO cells expressing a recombinant monoclonal antibody were grown in an 80 L bioreactor for 19 days using the perfusion method. The cell culture broth was cooled to 21°C. Three spin flasks were set up, each containing 1 L of cell culture broth. PDADMAC (molecular weight 400,000-500,000) was added to one flask at a concentration of 45 pg per total cell density. PDADMAC at 45 pg per total cell density and PEG 3,000 at 15% (w / v) were added as a bolus to another flask (one-step method). PDADMAC was added to the third flask at a concentration of 45 pg per total cell density, followed by PEG 3,000 at a final concentration of 15% (w / v) (two-step method). The PDADMAC addition rate was approximately 1 min. The PDADMAC / PEG addition rate was approximately 5 min. All additions were made at ambient temperature. All flasks were incubated at 75-85 rpm for 15 minutes. Aggregates were allowed to settle at ambient temperature.

[0103] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation, compaction settling velocity, and measurements were taken at various intervals over 240 minutes, and the relative flocculated volume was calculated as described above.

[0104] As shown in Figure 7, the combination of PDADMAC and PEG 3,000, added either simultaneously or sequentially, decreased the aggregate settling time compared to that of PDADMAC alone.

[0105] Large-scale cultures were then prepared. CHO cells expressing recombinant monoclonal antibodies were grown in perfusion culture in an 80 L bioreactor for 19 days. The cell culture broth was cooled to 21°C. PDADMAC at a concentration of 45 pg per total cell density and PEG 3,000 at a final concentration of 15% (w / v) were added simultaneously at ambient temperature with an addition rate of 21 min, followed by a 5-minute incubation at 100 rpm. Aggregates were allowed to settle at ambient temperature.

[0106] Once the flocs had settled (first settling), the clarified supernatant was collected by pumping the liquid from the bioreactor followed by filtration through a depth filter containing diatomaceous earth and then a membrane filter cut to 0.2 μm.

[0107] The aggregates were washed in an equal volume of 9% sucrose solution to remove any residual recombinant protein and allowed to settle for 16 hours. Once the aggregates had settled (second precipitation), the clarified second supernatant was collected as described above.

[0108] Clarified and harvested cell culture supernatants from the above flocculation (both small-scale and large-scale) were purified using Protein A chromatography, followed by product quality determination. The Protein A eluate was not neutralized prior to product quality determination. Protein A eluate product quality attributes measured included molecular variants measured by SEC and host cell proteins measured by ELISA.

[0109] The Protein A purified material was then passed over a CEX column at pH 7.5.

[0110] [Table 3]

[0111] Product quality is similar between the control and first aggregate harvest at both scales. The PDADMAC / PEG first harvest tends to remove higher order aggregates, reflected by lower HMW concentrations in the Protein A pool. A decrease in host cell protein concentration was observed with the PDADMAC / PEG harvest. Resuspension with sucrose led to slightly higher CHOP and HMW concentrations compared to the first PDADMAC / PEG harvest, likely due to resolubilization of these impurities.

[0112] [Example 7] In this experiment, the effect of adding a surfactant along with PDADMAC and PEG on settling time is examined.

[0113] CHO cells expressing a recombinant monoclonal antibody were grown in perfusion culture in an 80 L bioreactor for 15 days. The cell broth on day 14 was cooled to 30°C for testing. Two spin flasks were set up, each containing 1 L of cell culture broth. PDADMAC and PEG 3,000 were added simultaneously to one flask. PDADMAC was added at a concentration of 25 pg per total cell density (molecular weight 400,000-500,000) and PEG 3,000 was added at a final concentration of 3% (w / v). The other flask contained the above concentrations of PDADMAC and PEG, plus Triton X-100 at a final concentration of 0.05% (v / v). (Triton X-100 stock solution was 10% (v / v) from an initial 20% (v / v) stock solution; Sigma Aldrich, St. Louis, MO.) The three components were added simultaneously. The mixture was incubated at 75-85 rpm for 15 minutes, with an addition rate of approximately 1 minute. All flasks were rotated as described in the previous example. Aggregates were allowed to settle at ambient temperature.

[0114] The flocculated solution was then transferred to a 1 L glass graduated cylinder to determine the flocculation, compaction settling velocity, and measurements were taken at various intervals over 240 minutes, and the relative flocculated volume was calculated as described above.

[0115] As shown in Figure 8, the addition of Triton X-100 together with PDADMAC and PEG 3,000 decreased the aggregate settling time compared to PDADMAC and PEG 3,000 alone.

Claims

1. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate aggregation; mixing the cell culture medium during aggregation; allowing the aggregates to settle; and Recovering the clarified supernatant A method comprising:

2. 2. The mammalian cell culture recovery method of claim 1, wherein the cationic polymer is polydiallyldimethylammonium chloride.

3. 2. The mammalian cell culture recovery method of claim 1, wherein the non-ionic polymer is selected from polyethylene glycol and dextran.

4. 2. The mammalian cell culture recovery method of claim 1, wherein the non-ionic polymer is selected from PEG 3,000 and PEG 6,000.

5. 10. The mammalian cell culture recovery method of claim 1, further comprising adding a non-ionic surfactant to the cell culture medium.

6. 6. The mammalian cell culture recovery method according to claim 5, wherein the nonionic surfactant is Triton X-100.

7. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding polydiallyldimethylammonium chloride and PEG 3,000 to the cell culture medium to initiate aggregation; mixing the cell culture medium during aggregation; allowing the aggregates to settle; and and recovering the clarified supernatant.

8. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding polydiallyldimethylammonium chloride, PEG 3,000, and Triton X-100 to the cell culture medium to initiate aggregation; agitating the cell culture medium during aggregation; allowing the aggregates to settle; and and recovering the clarified supernatant.

9. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate aggregation; mixing the cell culture medium during aggregation; allowing the agglomerate to settle for a first settling; collecting the first clarified supernatant; washing the first sedimented flocculate; allowing the washed flocculate to settle for a second settling; and recovering the second clarified supernatant.

10. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding a cationic polymer and a non-ionic polymer to the cell culture medium to initiate aggregation; mixing the cell culture medium during aggregation; allowing the agglomerate to settle for a first settling; collecting the first clarified supernatant; washing the first settled flocculate if the product recovery in the first clarified supernatant is less than 80%; allowing the washed flocculate to settle for a second settling; and recovering the second clarified supernatant.

11. 11. The mammalian cell culture recovery method according to claim 9 or 10, wherein the cationic polymer is polydiallyldimethylammonium chloride.

12. 11. The mammalian cell culture recovery method of claim 9 or 10, wherein the non-ionic polymer is selected from polyethylene glycol and dextran.

13. 11. The mammalian cell culture recovery method of claim 9 or 10, wherein the non-ionic polymer is selected from PEG 3,000 and PEG 6,000.

14. 11. The mammalian cell culture recovery method of claim 9 or 10, further comprising adding a non-ionic surfactant to the cell culture medium.

15. 15. The mammalian cell culture recovery method according to claim 14, wherein the nonionic surfactant is Triton X-100.

16. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding polydiallyldimethylammonium chloride and PEG 3,000 to the cell culture medium to initiate aggregation; mixing the cell culture medium during aggregation; allowing the agglomerate to settle for a first settling; collecting the first clarified supernatant; washing the first sedimented flocculate; allowing the washed flocculate to settle for a second settling; and recovering the second clarified supernatant.

17. 1. A mammalian cell culture recovery method comprising: culturing mammalian cells expressing the recombinant protein in a cell culture medium for a predetermined period of time or until a desired cell density and / or packed cell volume is achieved; adding polydiallyldimethylammonium chloride, PEG 3,000, and Triton X-100 to the cell culture medium to initiate aggregation; mixing the cell culture medium during aggregation; allowing the agglomerate to settle for a first settling; collecting the first clarified supernatant; washing the first sedimented flocculate; allowing the washed flocculate to settle for a second settling; and recovering the second clarified supernatant.

18. 18. The method of any of claims 1 to 17, wherein the cationic polymer and the non-ionic polymer are added simultaneously.

19. 19. The method of any of claims 1 to 18, wherein the cationic polymer is added first and mixed for at least 30 seconds before adding the non-ionic polymer.

20. 20. The method of any one of claims 1 to 19, wherein the cationic polymer, the nonionic polymer, and the nonionic surfactant are added simultaneously.

21. 21. The method of any of claims 1 to 20, wherein the cationic polymer is added first and mixed for at least 30 seconds before adding the non-ionic polymer and non-ionic surfactant.

22. 22. The method of any one of claims 1 to 21, wherein the cationic polymer is a polymer of diallyldimethylammonium chloride, polydiallyldimethylammonium chloride, polyethyleneimine, polyacrylamide or chitosan.

23. 23. The method of any one of claims 1 to 22, wherein the non-ionic polymer is polyethylene glycol or dextran.

24. 24. The method of any one of claims 1 to 23, wherein the non-ionic surfactant is sapoin or Triton X100.

25. 25. The method of any of claims 1 to 24, wherein polydiallyldimethylammonium chloride is added at a concentration of from at or about 20 to at or about 90 pg per total cell density.

26. 26. The method of any one of claims 1 to 25, wherein polydiallyldimethylammonium chloride is added at a concentration of 25 or about 25 pg per total cell density and the mammalian cells are derived from a diploid cell line.

27. 27. The method of any one of claims 1 to 26, wherein polydiallyldimethylammonium chloride is added at between 43 pg per total cell density and 57 pg per total cell density, and the mammalian cells are derived from a tetraploid cell line.

28. 28. The method of any one of claims 1 to 27, wherein the concentration of PEG 3,000 is from 3 or about 3% to 4.5 or about 4.5%.

29. 29. The method of any one of claims 1 to 28, wherein the concentration of PEG 6,000 is from 2.5 or about 2.5% to 3.5% or about 3.5%.

30. 30. The method of any one of claims 1 to 29, wherein the concentration of Triton X100 is 0.05% (w / v).

31. 31. The method of any one of claims 1 to 30, wherein the mammalian cell culture broth is between 36°C and 20°C.

32. 32. The method of any of claims 1 to 31, wherein the mammalian cell culture broth is at or above 20°C.

33. 33. The method of any one of claims 1 to 32, wherein the aggregates from the first precipitation are washed in a 9% sucrose solution.

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