Concentrated perfusion medium

Abstract

The present invention relates to a serum-free cell culture perfusion medium comprising medium components that make up at least three separate aqueous concentrated feeds and a diluent wherein the resulting serum-free cell culture perfusion medium, upon mixing, adjusts the pH to a neutral pH. The invention also provides a method for preparing the serum-free cell culture perfusion medium. The invention further relates to methods of culturing mammalian cells or producing proteins of interest in perfusion cultures using the serum-free cell culture perfusion media, which achieve high productivity at low cell specific perfusion rates. The invention further relates to the use of novel and improved serum-free cell culture perfusion media to control the osmotic pressure of a perfusion cell culture, where an increase in total productivity and / or cell specific productivity is caused by inhibiting cell growth, increasing osmotic pressure during cell culture, for example during the production phase of perfusion cell culture. Inhibiting cell growth particularly reduces or eliminates the need for wasted cell expulsion.

Description

[0001] This application is a divisional application of the invention patent application filed on September 25, 2020, with application number 202080081171.5 and invention title "Concentrated Perfusion Culture Medium". Technical Field

[0002] This invention relates to a serum-free cell culture perfusion medium comprising at least three separate aqueous concentrated feed media components and a diluent, wherein the pH of the serum-free cell culture perfusion medium is adjusted to neutral after mixing. A method for preparing the serum-free cell culture perfusion medium is also provided. The invention further relates to a method for culturing mammalian cells or producing proteins of interest in perfusion culture using the serum-free cell culture perfusion medium, the method achieving high productivity at low cell-specific perfusion rates. The invention further relates to the use of novel and improved serum-free cell culture perfusion media for controlling the osmotic pressure of perfused cell cultures, wherein by inhibiting cell growth during cell culture, such as during the production phase of perfused cell culture, increasing osmotic pressure results in increased overall productivity and / or cell-specific productivity. Inhibiting cell growth particularly reduces or eliminates the need for waste cell removal. Background Technology

[0003] The following three methods are typically used in the commercialization of recombinant proteins produced through mammalian cell culture: batch culture, fed-batch culture, and perfusion culture.

[0004] Perfusion-based methods offer potential improvements over batch and fed-batch methods, including improved product quality and stability, improved scalability, and increased cell-to-cell productivity. Unlike batch and fed-batch bioreactors, perfusion systems involve the continuous removal of used culture medium. By continuously removing used medium and replacing it with fresh medium, nutrient levels can be better maintained, growth conditions optimized, and cellular waste removed. Reduced waste lowers toxicity to cells and expressed products. Therefore, perfusion bioreactors typically result in significantly reduced protein degradation and thus higher quality products. Products can also be harvested and purified more quickly and continuously, which is particularly effective when unstable products are produced.

[0005] Perfusion bioreactors are also easier to scale. Compared to traditional batch or fed-batch systems, perfusion bioreactors offer several advantages in terms of scalability and / or growing demand. For one, perfusion bioreactors are smaller and can produce the same productivity (i.e., product yield) in a smaller volume. It is generally believed that perfusion bioreactors can operate at concentrations 5 to 20 times higher than fed-batch bioreactors. For example, a 100-liter perfusion bioreactor can produce the same product yield as a 1,000-liter fed-batch bioreactor. Therefore, it is conceivable that the use of a 1,000-liter perfusion bioreactor can replace a typical 10,000-liter conventional fed-batch bioreactor without negatively impacting overall productivity. This significant advantage translates into smaller space requirements when scaling up production. This also translates into a range of advantages associated with lower operating utilities, less infrastructure, less labor, reduced equipment complexity, continuous harvesting, and increased product yield.

[0006] The ability to achieve high cell culture densities partially explains the higher productivity of perfusion systems. In typical large-scale fed-batch commercial cell culture processes, densities of 10-50 x 10⁻⁶ cells can be achieved. 6 Cell densities of 1 x 10^6 cells / mL have been achieved. However, using perfusion-based bioreactors, >1 x 10^6 cells / mL has been achieved. 8 The extreme cell density is [number of cells / mL]. Furthermore, in perfusion mode, high cell numbers can be maintained for a longer period by continuously replenishing the used culture medium. The increasing cell density over time in perfusion bioreactors partially explains why perfusion bioreactors are more efficient.

[0007] Typical perfusion culture begins with a batch culture start-up, lasting one day or longer to achieve rapid initial cell growth and biomass accumulation. Fresh perfusion medium is then added to the culture continuously, gradually, and / or intermittently, while used medium is removed simultaneously, with cells retained throughout the growth and production phases. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used medium while maintaining cell numbers. Perfusion flow rates have ranged from a small portion of the working volume per day to many working volumes per day.

[0008] While continuous perfusion systems offer numerous advantages over conventional fed-batch and batch systems, several challenges remain before perfusion bioreactors gain wider acceptance and utilization in the biopharmaceutical manufacturing industry. For example, due to the continuous cycle of culture medium removal and replenishment, perfusion bioreactors consume significantly larger volumes of culture medium than conventional fed-batch systems. Specifically, maintaining a perfusion rate of 1–3 vessel volumes (vvd) per day above pilot-scale (~100 L bioreactor) becomes logically challenging, if feasible.

[0009] WO 92 / 22637 formulated concentrated medium subgroups based on their physicochemical properties. However, these concentrated medium subgroups do not adjust the pH upon mixing and are therefore unsuitable for direct addition to cell cultures. Furthermore, the media disclosed in WO 92 / 22637, such as the basal media RPMI-1640, DMEM, and Ham's F-12, are less enriched than modern cell media, with amino acid concentrations in the mM working range rather than the μM working range.

[0010] Another challenge facing continuous perfusion cell culture systems is maintaining a constant viable cell density and, consequently, healthier, more productive cell cultures. This is typically addressed by allowing “cell ejection.” During cell ejection, cells are removed at a rate sufficient to allow for steady-state perfusion cell culture and discarded as waste. This, in turn, keeps the viable cell density constant. Due to cell ejection techniques, a significant portion of the culture medium and products may be lost; this technique siphons proliferating cells and culture medium to maintain a constant, sustainable viable cell density within the bioreactor. Up to one-third of the harvestable material may be lost due to cell ejection. Therefore, using cell ejection reduces product yield per run because the product within the portion removed by cell ejection is not harvested. Thus, any amount of cell ejection negatively impacts process efficiency, product recovery, and, most importantly, leads to product loss. The cell ejection rate is determined by the cell growth rate. Faster doubling times also require higher cell ejection rates to maintain a constant cell density, thus resulting in more waste.

[0011] As an alternative to cell ejection, others have attempted to use chemical additives to slow cell growth rates. Reduced cell growth often also increases cell specific productivity. For example, Du et al. (Biotechnology and Bioengineering, Vol. 112, No. 1, January 2015) reported the use of small-molecule cell cycle inhibitors to control growth and improve cell culture productivity. A similar disclosure is found in WO 2014 / 109858, which discloses the use of CDK4 inhibitors in cell cultures such as batch, fed-batch, and perfusion cultures. Du et al. further taught that CDK4 / 6 inhibitors specifically inhibit the cell cycle without affecting other cellular targets. However, it is important to avoid adding inhibitors and compounds that are not needed for cell growth and / or cell maintenance. Therefore, methods to effectively inhibit cell growth in perfusion states and avoid the need for additional cell ejection would significantly help advance the field.

[0012] Osmotic pressure is a known lever influencing cell growth. Existing techniques for using osmotic pressure to influence cell growth are known in the literature (Zhu, et al. (2005) Biotechnology Progress 21, 70-77; Han, Koo and Lee (2009) Biotechnology Progress 25, 1440-1447; Hu and Aunins (1997) Current Opinion in Biotechnology, 148-153). However, particularly in perfusion culture, the ability to control the cell culture process to a target osmotic pressure has never been established. Furthermore, chemical additives affect the culture medium composition and / or need to be removed in subsequent purification steps, increasing process complexity. Chemical additives, including salts, can also affect product quality.

[0013] Given the challenges of perfused cell culture, such as the consumption of culture medium and the desire to further improve productivity, improved logic-based culture media and methods to effectively inhibit cell growth in the perfused state and avoid the need for cell effluent without the need for further additives will significantly help advance the field. Summary of the Invention

[0014] This invention relates in part to the discovery that feed media can be developed in a more concentrated form through separation, thereby reducing the specific perfusion rate of cells and the volume of preparative media consumed. These concentrated feeds are diluted in the bioreactor vessel. They are advantageously used with sterile deionized water as a diluent, which requires no preparation other than filtration. Furthermore, the unique combination of the three media concentrates (acidic, alkaline, and near-neutral) designed in this invention allows for the use of higher concentrations of concentrate, thereby further reducing the total media volume. By reducing the volume of preparative media, a perfusion process has been developed that eliminates the media volume bottleneck, thus demonstrating the rationale for scaling up perfused cell culture processes to 1000 L and potentially larger scales.

[0015] Using separate concentrate feeds and diluents also allows for control of cell growth via culture osmolarity. Using the concept of mass balance, osmolarity balance is derived from the known osmolarity of each concentrate feed, the feed rate, and a calculated daily cell specific osmolarity consumption rate to predict the residual osmolarity of the culture as output. Using this method, cell culture growth can be controlled by raising the residual osmolarity to physiological stress levels (approximately 350–400 mOsm or higher) while simultaneously maintaining it below cytotoxic levels (approximately 400 mOsm or higher). Both physiological stress and cytotoxicity levels can be cell line-specific. However, this can be readily determined by measuring viable cell concentration and viability at different osmolarity levels during culture, and can be determined on a small scale (e.g., 3 ml working volume). In the present invention, culture osmolarity is controlled via osmolarity balance, which includes the variation of the culture medium concentrate feed rate relative to a daily baseline at a fixed daily container volume (VVD) or the variation of the VVD feed rate relative to a daily baseline at a fixed culture medium concentrate feed rate. Osmotic equilibrium can target higher or lower residual osmotic pressure by adjusting the concentrate and dilution rate, whereas other chemical additive regimens can only modulate osmotic pressure in the direction of increase. Osmotic equilibrium, as described herein, has been found to be effective in inhibiting cell growth, and this growth inhibition leads to increased cell specific productivity and helps maintain high viability in cell culture.

[0016] The cell growth inhibition induced by osmotic balance as described herein not only results in increased specific cell productivity and sustained high cell viability, but also, in perfused cell culture, reduces or eliminates the need for cell ejection techniques during perfusion to otherwise maintain cells in a stable growth state. This reduces or eliminates product loss due to wasteful and undesirable cell ejection techniques.

[0017] The three-part highly concentrated feed medium provided herein can theoretically be used with any type of cell culture system, but is particularly advantageous in continuous perfusion cell culture systems. Therefore, the serum-free cell culture perfusion medium according to the invention is particularly suitable for continuous perfusion cell culture systems. Furthermore, osmotic equilibrium can theoretically be used with any type of cell culture system. However, it is particularly advantageous when the cell culture system is a continuous perfusion cell culture system.

[0018] In one aspect, the present invention relates to a separated serum-free cell culture perfusion medium comprising a culture medium component consisting of at least three separate aqueous concentrates and a diluent, wherein a first concentrate is an alkaline concentrate, a second concentrate is an acidic concentrate, and a third concentrate is a near-neutral concentrate; wherein the separated serum-free cell culture perfusion medium is pH-adjusted to neutral after the at least three separate aqueous concentrates and the diluent are mixed in the resulting serum-free cell culture perfusion medium. In a preferred embodiment, the at least three separate aqueous concentrates are not premixed before being added to the reaction vessel of the cell culture and / or bioreactor. The diluent is preferably sterile water. In one embodiment, after the at least three separate aqueous concentrates and the diluent are mixed, the resulting serum-free cell culture perfusion medium has a pH between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2. The segregated serum-free cell culture perfusion medium according to the invention is suitable for adding the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed separately to the reaction vessel of the cell culture and / or bioreactor; adding the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed directly to the reaction vessel of the cell culture and / or bioreactor without prior premixing; and / or directly mixing the at least three separate aqueous concentrated feeds in the reaction vessel of the cell culture and / or bioreactor.

[0019] In some embodiments, the alkaline concentrate feed is a 2x to 80x concentrate feed, the acidic concentrate feed is a 2x to 40x concentrate feed, and the near-neutral concentrate feed is a 2x to 50x concentrate feed. The pH of the near-neutral concentrate feed is about 6.5 to about 8.5. Preferably, the pH of the alkaline concentrate feed is about 9 or higher, the pH of the acidic concentrate feed is about 5 or lower, and the pH of the near-neutral concentrate feed is about 7 to about 8.5. Furthermore, the ratio (v / v / v) of the alkaline concentrate feed to the acidic concentrate feed to the near-neutral concentrate feed is a fixed ratio to provide a serum-free cell culture perfusion medium with pH adjusted to neutral; and the ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrate feeds in the serum-free cell culture perfusion medium with pH adjusted to neutral determines the osmotic pressure of the serum-free cell culture perfusion medium.

[0020] The acidic concentrate feed may contain trace elements, trace metals, inorganic salts, chelating agents, polyamines, and regulatory hormones. The acidic concentrate feed and / or the near-neutral concentrate feed may contain surfactants, antioxidants, and carbon sources. Further, the alkaline concentrate feed contains amino acids that have maximum solubility at an alkaline pH of 9 or higher, preferably at least aspartic acid, histidine, and tyrosine, and optionally cysteine ​​and / or cystine and / or folic acid. The remaining amino acids are in the acidic and / or near-neutral concentrate feeds, preferably in the acidic concentrate feed. Preferably, vitamins and metals are in separate feeds, preferably vitamins in the near-neutral feed and metals in the acidic feed. Vitamins that are poorly soluble in aqueous solutions, such as choline chloride, are present in both the neutral and acidic feeds.

[0021] The present invention also relates to an alkaline aqueous concentrate feed, which is used in combination with an acidic aqueous concentrate feed, a near-neutral aqueous concentrate feed, and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the serum-free cell culture perfusion medium is automatically adjusted to neutral pH. In another embodiment, the present invention relates to an acidic aqueous concentrate feed, which is used in combination with an alkaline aqueous concentrate feed, a near-neutral aqueous concentrate feed, and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH. In yet another aspect, the present invention relates to a near-neutral aqueous concentrate feed, which is used in combination with an alkaline aqueous concentrate feed, an acidic aqueous concentrate feed, and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

[0022] In another aspect, the present invention relates to a method for preparing a serum-free cell culture perfusion medium, the method comprising: (a) providing cell culture medium components in at least three subgroups based on their solubility at alkaline, acidic, and neutral pH levels; (b) (i) dissolving the subgroup components soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed; (ii) dissolving the subgroup components soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; and (iii) (c) Dissolve the subcomponents soluble at neutral pH in a neutral aqueous solution to form a near-neutral concentrated feed; (d) optionally store the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed in separate containers; and (e) add the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed, along with a diluent, to the reaction vessel of the cell culture and / or bioreactor, wherein (i) the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed are added separately to the reaction vessel of the cell culture and / or bioreactor; and (ii) the diluent is added separately to the reaction vessel of the cell culture and / or bioreactor, or the diluent is premixed with one of the at least three separate aqueous concentrated feeds before being added to the reaction vessel of the cell culture and / or bioreactor; wherein after mixing the at least three separate aqueous concentrated feeds and the diluent, the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to approximately neutral pH. The diluent is preferably sterile water. In one embodiment, after mixing the at least three separate aqueous concentrate feeds and the diluent, the resulting serum-free cell culture perfusion medium prepared by the method has a pH between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2.

[0023] In some embodiments, the at least three concentrated feeds are added dropwise to the reaction vessel of the cell culture and / or bioreactor through separate ports. In-vessel mixing and dilution of the at least three separate aqueous concentrated feeds allows for a 50-90% reduction in preparation medium consumption, preferably 60-90%, over a 14-day culture period compared to mixing and diluting serum-free cell culture perfusion medium before addition to the bioreactor. Typically, the reaction vessel of the cell culture and / or bioreactor contains mammalian cells. Furthermore, the method further includes a step of sterilizing the concentrated feeds before storing and / or adding them to the reaction vessel of the cell culture and / or bioreactor.

[0024] In some embodiments, the alkaline concentrate feed is a 2x to 80x concentrate feed, the acidic concentrate feed is a 2x to 40x concentrate feed, and the near-neutral concentrate feed is a 2x to 50x concentrate feed. The near-neutral concentrate feed has a pH of 6.5-8.5. Preferably, the alkaline concentrate feed has a pH of 9 or higher, the acidic concentrate feed has a pH of 5 or lower, and the near-neutral concentrate feed has a pH of 7 to 8.5. Furthermore, the ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed to the near-neutral concentrated feed is a fixed ratio to provide a serum-free cell culture perfusion medium with pH adjusted to neutral in the reaction vessel of the cell culture and / or bioreactor; and the ratio (v / v) of the diluent to the cumulative volume of the at least three separate aqueous concentrated feeds added to the reaction vessel of the cell culture and / or bioreactor to provide a serum-free cell culture perfusion medium with pH adjusted to near neutral. In some embodiments, the reaction vessel of the cell culture and / or bioreactor contains at least about 100 L of serum-free cell culture perfusion medium, preferably at least about 1000 L. Preferably, the volume of the cell culture is at least about 100 L and / or the volume of the bioreactor is at least about 100 L. More preferably, the volume of the cell culture is at least about 1000 L and / or the volume of the bioreactor is at least about 1000 L.

[0025] A serum-free cell culture perfusion medium that can be obtained by the method according to the invention is also provided.

[0026] On another front, the present invention relates to a method for culturing mammalian cells expressing a heterologous protein in a perfusion culture, the method comprising: (a) inoculating a bioreactor with mammalian cells expressing a heterologous protein in a serum-free cell culture medium; and (b) culturing the mammalian cells in a perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium and removing the used medium while simultaneously maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a separated serum-free cell culture perfusion medium comprising a culture medium component comprising at least three separate aqueous concentrate feeds and a diluent, wherein the first concentrate feed is an alkaline concentrate feed, the second concentrate feed is an acidic concentrate feed, and the third concentrate feed is a near-aqueous concentrate feed. A neutral concentrate; and wherein, after mixing the at least three separate aqueous concentrates and the diluent in the resulting serum-free cell culture perfusion medium, the separated serum-free cell culture perfusion medium is pH-adjusted to neutral; and / or (ii) a serum-free cell culture perfusion medium obtainable by the method according to the invention, wherein the alkaline concentrate, the acidic concentrate, and the near-neutral concentrate of the serum-free cell culture perfusion medium are added separately to the reaction vessel of the cell culture and / or bioreactor, and wherein the diluent is added separately to the reaction vessel of the cell culture and / or bioreactor, or premixed with one of the at least three separate aqueous concentrates before being added to the reaction vessel of the cell culture and / or bioreactor. The method typically further comprises harvesting the heterologous protein from the cell culture.

[0027] Prior to the initiation of perfusion culture, the mammalian cells may initially be cultured in batches and / or perfusion culture may begin from day 0 to day 3 of the batch culture (i.e., post-inoculation). Typically, the perfusion rate is increased after the start of perfusion until the target viable cell density is reached. In some embodiments, the perfusion rate is increased from less than or equal to 0.5 container volumes per day to approximately 5 container volumes per day, or from less than or equal to 0.5 container volumes per day to approximately 2 container volumes per day.

[0028] In some embodiments, the osmotic pressure of the serum-free cell culture perfusion medium is increased above the optimal growth osmotic pressure level, resulting in growth inhibition at the target viable cell density. Preferably, the osmotic pressure level of the serum-free cell culture perfusion medium is gradually or stepwise increased starting from about half of the target viable cell density. The target viable cell density is about 30 x 10⁻⁶ cells / mL. 6 10 cells / ml or higher, approximately 60 x 10 6 10 cells / ml or higher, approximately 80 x 10 6cells / ml, preferably about 100 x 10⁻⁶ 6 Cells / ml or higher. Osmolarity can be controlled using: (a) a constant concentrate perfusion rate and different diluent perfusion rates, resulting in different total perfusion rates; or (b) a constant total perfusion rate and different concentrate perfusion rates; wherein the at least three concentrates are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the culture medium components in 1x serum-free cell culture perfusion medium. Therefore, osmolarity can be increased using: (a) a constant concentrate perfusion rate and a decreased diluent perfusion rate, resulting in a decreased total perfusion rate; or (b) a constant total perfusion rate and an increased concentrate perfusion rate and a decreased diluent perfusion rate; wherein the at least three concentrates are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the culture medium components in 1x serum-free cell culture perfusion medium. Preferably, no further additives are added to the culture to increase osmolarity.

[0029] Those skilled in the art will know how to determine the optimal osmotic pressure level for the growth of the mammalian cells. In one embodiment, the optimal osmotic pressure level for mammalian cell growth is about 280 to less than 350 mOsm. The osmotic pressure is maintained at the level most suitable for growth until about half of the target viable cell density is reached. Preferably, the osmotic pressure is gradually or stepwise increased from about half of the target viable cell density, preferably to about 10-50% of the optimal growth osmotic pressure level. The osmotic pressure is increased to and maintained at an osmotic pressure level that inhibits cell growth at about the target viable cell density, wherein in one embodiment, the osmotic pressure level that inhibits cell growth of mammalian cells is about 350 mOsm or higher, preferably about 380 mOsm or higher. Increasing the osmotic pressure reduces or eliminates the need for cell removal during the production phase.

[0030] When osmotic pressure is increased, cell growth is inhibited to maintain a sustainable viable cell density without cell expulsion. The yield of heterologous proteins produced in the cell cultures is increased by at least 5-50% compared to the yield of the control cell cultures, without an increase in osmotic pressure.

[0031] Typically, using the method of the present invention, the cell specific perfusion rate (pl / cell / day) is reduced by at least 50% relative to the cell specific perfusion rate of 1x serum-free cell culture medium.

[0032] In some embodiments, the reaction vessel for the cell culture and / or bioreactor contains at least about 100 L of serum-free cell culture perfusion medium, preferably at least about 1000 L. Preferably, the volume of the cell culture is at least about 100 L and / or the volume of the bioreactor is at least about 100 L. More preferably, the volume of the cell culture is at least about 1000 L and / or the volume of the bioreactor is at least about 1000 L.

[0033] The heterologous protein may be a therapeutic protein, antibody, or a therapeutically effective fragment thereof. The mammalian cell may be any cell line selected from the group consisting of: Chinese hamster ovary (CHO) cells, Jurkat cells, 293 cells, HeLa cells, CV-1 cells, or 3T3 cells, or derivatives of any of these cells. The CHO cells may be further selected from the group consisting of: CHO-DG44 cells, CHO-K1 cells, CHO DXB11 cells, CHO-S cells, and CHO GS-deficient cells, or mutants thereof.

[0034] According to the method of the invention, one or more supplements selected from the list of defoamers, alkalis, glutamines and glucose can be added separately (i.e. additionally) to the cell culture.

[0035] A method for producing therapeutic proteins using the method according to the invention is also provided.

[0036] Also provided is the use of the separated serum-free cell culture perfusion medium according to the invention, or a serum-free cell culture perfusion medium obtainable by the method according to the invention, for culturing mammalian cells, particularly for culturing mammalian cells in perfusion culture. In some embodiments, the cell specific perfusion rate (pl / cell / day) is reduced by at least 30% relative to the cell specific perfusion rate of 1x serum-free cell culture medium. Also provided is the use of the separated serum-free cell culture perfusion medium according to the invention for individually adding the at least three separate aqueous concentrated feeds to the reaction vessel of the cell culture and / or bioreactor.

[0037] Furthermore, the present invention provides for controlling osmotic pressure in perfused cell cultures using a segregated serum-free cell culture perfusion medium according to the invention or a serum-free cell culture perfusion medium obtainable by the method according to the invention. Increasing the osmotic pressure of the cell culture inhibits cell growth and increases the production of heterologous proteins. The yield of heterologous proteins produced in the cell culture is increased by at least 5-50% relative to the yield of the control cell culture, wherein the osmotic pressure is not increased. In one embodiment, growth inhibition is sufficient to maintain a sustainable viable cell density without cell extrusion. Attached Figure Description

[0038] Figure 1 The bioreactor and feed setup are illustrated with separate inlet additions for the following: acidic, alkaline, and neutral feeds, as well as diluents.

[0039] Figure 2 The reactor volume exchange or injection rate over time, expressed as per liter of bioreactor (L). br ) of culture medium volume ( L培养基 The values ​​are given in days, for example, the typical operating perfusion rate (upper dashed line; VVD means “container volume per day”) for perfusion cultures with a fed-batch strategy that uses a combination of three culture medium concentrates: a combination of the three culture medium concentrates (MC; lower dashed line), an MC combined with a diluent (solid line), and the potential maximum perfusion rate of the combined feed.

[0040] Figure 3 Viable cell density (+ / -3 standard deviations; solid line) and viability (+ / -3 SD; dashed line) of three 100L bioreactor runs using a concentrated culture medium feed + diluent feed protocol. The cell line's intrinsic peak VCD (i.e., without inhibition of growth by high osmotic pressure) is >200e6 c / mL. Increasing the pre-peak osmotic pressure inhibits growth and suppresses peak VCD.

[0041] Figure 4 The osmotic pressure (mOsm) of three 100L bioreactor runs shows a gradual increase in osmotic pressure until approximately day 6, at which point the target viable cell density is reached. From the peak VCD, the osmotic pressure was maintained >380 mOsm to inhibit cell proliferation.

[0042] Figure 5 Reactor volume exchange (also known as perfusion rate) was performed in three 100L bioreactor runs using different diluent volumes and a fixed feed of concentrated culture medium at 0.5 container volumes (VVD) per day. The different diluent volumes controlled the residual osmotic pressure in the culture vessels.

[0043] Figure 6 Permeate productivity (g / L) of three 100L bioreactor runs using different diluent volumes (different total perfusion rates) and concentrated culture medium fed at a fixed rate of 0.5 container volumes (VVD) per day. 生物反应器 / day). Permeate productivity is measured by the daily instantaneous titer of permeate (g / L) as measured by a Cedex bioanalyzer. 培养基 Multiply by the daily perfusion rate (L) 培养基 / L 生物反应器 Calculated by ( / day).

[0044] Figure 7 The daily specific productivity (Qp, pg / cell / day) of CHO cell cultures expressing recombinant IgG was determined by three 100L bioreactor runs using concentrated medium fed at a fixed 0.5 container volumes (VVD) per day with different diluent volumes (different total perfusion rates). Daily Qp was estimated by summing the total productivity of the bioreactor system (i.e., products recovered via permeate and products retained in the bioreactor) and dividing by the daily viable cell density (VCD).

[0045] Figure 8 The cell-to-cell perfusion rate (nL / cell / day) of CHO cells was measured in three 100L bioreactor runs using different diluent volumes (different total perfusion rates) and concentrated culture medium fed at a fixed rate of 0.5 container volumes (VVD) per day.

[0046] Figure 9 Three BI CHO cell lines A (expressing different recombinant IgG molecules) Viable cell density (VCD, e5 c / mL; solid line) and viability (%); dashed line) for B(□) and C(Δ). Data are from a 2L bioreactor scale using three concentrated culture media feeds and varying proportions of sterile water diluent to maintain target residual osmotic pressure, employing a constant perfusion rate of two container volumes (VVD) per day.

[0047] Figure 10 Three BI CHO cell lines A (expressing different recombinant IgG molecules) The residual osmotic pressure (mOsm) of B(□) and C(Δ) were obtained. Data were obtained from a 2L bioreactor scale using three concentrated culture media feeds and different proportions of sterile water diluent to maintain the target residual osmotic pressure, with a constant perfusion rate of two container volumes (VVD) per day.

[0048] Figure 11 Three BI CHO cell lines A (expressing different recombinant IgG molecules) The reactor volume exchange (also known as perfusion rate; L culture medium / L bioreactor / day) for B(□) and C(Δ) is given. Data are from a 2L bioreactor scale using three concentrated culture media feeds and different proportions of sterile water diluent to maintain the target residual osmotic pressure, employing a constant perfusion rate of two container volumes (VVD) per day.

[0049] Figure 12 Three BI CHO cell lines A (expressing different recombinant IgG molecules) Permeate productivity (g / L) of B(□) and C(Δ) 生物反应器 / day). Permeate productivity is measured by the daily instantaneous titer of permeate (g / L) as measured by a Cedex bioanalyzer. 培养基 Multiply by the daily perfusion rate (L) 培养基 / L 生物反应器 The calculation is based on a per-day (VVD) ratio. The data comes from a 2L bioreactor scale that uses three concentrated culture media feeds and different proportions of sterile water diluent to maintain the target residual osmotic pressure, employing a constant perfusion rate of two container volumes (VVD) per day.

[0050] Figure 13 Three BI CHO cell lines A (expressing different recombinant IgG molecules) The daily specific productivity (Qp, pg / cell / day) of B(□) and C(Δ) was calculated. Daily Qp was estimated by summing the total productivity of the bioreactor system (i.e., products recovered via permeate and products retained in the bioreactor) and dividing by the daily viable cell density (VCD). Data were obtained from a 2L bioreactor scale using three concentrated culture media feeds and varying proportions of sterile water diluent to maintain the target residual osmotic pressure, employing a constant perfusion rate of two vessel volumes (VVD) per day.

[0051] Figure 14 Three BI CHO cell lines A (expressing different recombinant IgG molecules) Cell-specific perfusion rates (CSPR; nL / cell / day) for B(□) and C(Δ) cell lines were used. Data were obtained from a 2L bioreactor scale using three concentrated culture media feeds and varying proportions of sterile water diluent to maintain the target residual osmotic pressure, employing a constant perfusion rate of approximately two vessel volumes (VVD) per day. Differences in CSPR between cell lines are due to variations in viable cell density (VCD) (see [link to relevant documentation] for VCD and viability). Figure 9 The feed ratios relative to each other are kept constant, while the total feed-to-diluent ratio is adjusted for osmotic pressure-based mass balance according to the following equation: Osmotic Input = Osmotic Output + Osmotic Consumption, where osmotic input is the osmotic pressure of the concentrated feed and diluent infused into the bioreactor, osmotic output is the residual osmotic pressure of the bioreactor supernatant, and osmotic consumption is the osmotic pressure difference between input and output. Osmotic consumption is used to calculate the osmotic input required for a given desired osmotic output. The respective concentrated feed and diluent infusion rates are then calculated to achieve the required osmotic input at a total infusion rate of 2 vvd.

[0052] Figure 15. CHO DG44 cell line (cell line A, Δ) and two different CHO-K1 cell lines (cell line B, Δ) cultured in a 2L bioreactor using three concentrated culture media fed at a fixed total of 0.5 container volumes (VVD) per day with different diluent volumes. Cell lines C(x, x) were used, wherein the CHO DG44 cell line was expressed in a dihydrofolate reductase (dhfr) selection system, and the two different CHO-K1 cell lines were expressed in a glutamine synthase (GS) selection system. All cell lines expressed different recombinant IgG molecules. The figures shown are (A) viable cell density (VCD; e5 c / mL); (B) viability (%); and (C) permeate productivity (g / L / day), which is expressed as the daily instantaneous titer of permeate (g / L) as measured by a Cedex bioanalyzer. 培养基 Multiply by the daily perfusion rate (L) 培养基 / L 生物反应器 Calculated in terms of / day; and (D) in terms of reactor volume exchange (L 培养基 / L 生物反应器 The infusion rate is expressed as / day.

[0053] Figure 16. CHO-K1 cell lines expressing recombinant IgG in a glutamine synthase (GS) selection system cultured in a 2 L bioreactor. (The text then abruptly shifts to a discussion of total VVD fixation based on MC levels.) The perfusion control mode is operated under either "Constant MC, Different Total VVD" (□). "Different MC, Constant Total VVD" refers to a constant daily total container volume (VVD) perfusion rate, achieved by varying the perfusion rate of the combined culture medium concentrate (MC) while simultaneously varying the diluent rate to maintain 2 VVD. "Constant MC, Different Total VVD" refers to a constant perfusion rate of MC at 0.5 VVD and different diluent perfusion rates, with respect to overall fluctuating perfusion rates. The figures show (A) viable cell density (VCD, e5 c / mL; principal axis) and viability (%); secondary axis, (B) osmolarity (mOsm), and (C) adjusted productivity (g / L). 生物反应器 / day) and (D) reactor volume exchange (L 培养基 / L 生物反应器 / sky). Detailed Implementation

[0054] Definitions of certain terms are provided below. Generally, unless otherwise stated or defined, any term presented in this disclosure shall be given its ordinary meaning in the art.

[0055] The general implementation method “comprising” or “comprised” covers the more specific implementation method “consisting of”. Furthermore, the singular and plural forms are used without limitation. As used herein, unless explicitly stated otherwise, the singular forms “a,” “an,” and “the” refer to both the singular and plural forms.

[0056] As used herein, the term "perfusion" refers to maintaining a cell culture bioreactor in which an equivalent volume of culture medium is added to and removed from the reactor while cells are retained in the reactor. Perfusion culture can also be referred to as continuous culture. This provides a stable source of fresh nutrients and continuous removal of cellular waste. Perfusion is often used to obtain much higher cell densities than conventional batch or fed-batch conditions in bioreactors, and thus higher volumetric productivity. Secretory protein products can be continuously harvested while cells are retained in the reactor, for example by filtration, alternating tangential flow (ATF), cell sedimentation, sonication, hydrocyclone, or any other method known to those skilled in the art or as described by Kompala and Ozturk (Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, (2006), Taylor & Francis Group, LLC, pages 387-416). Mammalian cells can be grown in suspension culture (homogeneous culture) or attached to a surface or embedded in different devices (heterogeneous culture). To keep the working volume in the bioreactor constant, the harvest rate and cell discharge (fluid removal) should be equal to the predetermined perfusion rate. Cultures are typically initiated in batches, with perfusion beginning 2-3 days post-inoculation, when cells are still in the exponential growth phase and before nutrient limitation occurs. High seeding densities (5 x 10⁻⁶) are used. 6 Inoculation with cells / ml or higher may require earlier or even immediate perfusion. Therefore, perfusion can begin from day 0 to day 4 post-inoculation, preferably from day 0 to day 3 post-inoculation.

[0057] By adding fresh culture medium while simultaneously removing used medium, perfusion-based methods offer potential improvements over batch and fed-batch methods. Large-scale commercial cell culture strategies can achieve 60-90 x 10⁶ cells / years. 6 A high cell density of [number] cells / mL means that approximately one-third to more than half of the reactor volume can be biomass. Using perfusion-based culture, >1x10 [cells / mL] has been achieved. 8Extreme cell densities of cells / mL. Typical perfusion cultures begin with batch culture initiation, lasting one day or longer to achieve rapid initial cell growth and biomass accumulation, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture while simultaneously removing used medium, with cells retained throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used medium while maintaining cell numbers. Perfusion flow rates have ranged from a small volume per day (VVD) to many volumes per day.

[0058] As used herein, the term "perfusion rate" refers to the volume added and removed, and is typically measured daily. The perfusion rate depends on cell density and culture medium. While ensuring adequate nutrient addition and byproduct removal rates, the perfusion rate should be minimized to reduce dilution of the product of interest, i.e., the harvest titer. Perfusion typically begins on days 0–3 post-inoculation, when cells are still in an exponential growth phase, and therefore the perfusion rate may increase during culture. Increases in the perfusion rate can be incremental or continuous, based on cell density or nutrient consumption. It typically starts at 0.5 or 1 container volume (VVD) per day and can rise to approximately 5 VVD. Preferably, the perfusion rate is between 0.5 and 2 VVD. Increases can reach 0.5 to 1 VVD per day. For continuous increases in perfusion, a biomass probe can be connected to a harvest pump based on a desired specific cell perfusion rate (CSPR), such that the perfusion rate increases as a linear function of the cell density determined by the biomass probe. CSPR equals the perfusion rate per cell density, and the ideal CSPR depends on the cell line and cell culture medium. An ideal CSPR should result in optimal growth rate and productivity. A CSPR of 50 to 100 pL / cell per day may be a reasonable starting range, which can be adjusted to find the optimal rate for a particular cell line. Using at least three separate aqueous concentrate feeds, the nutrient supply is separated from the overall VVD and CSPR, thus allowing very low CSPRs, such as 5 to 20 pL / cell / day, preferably even 5 to 10 pL / cell / day. This significantly reduces medium consumption, particularly preparative medium consumption, during a 14-day culture period compared to serum-free cell culture perfusion medium mixed and diluted before being added to the bioreactor, or compared to conventional 1x serum-free cell culture perfusion medium.

[0059] As used herein, the term "steady state" refers to a state in which cell density and bioreactor environment remain relatively constant. This can be achieved through cell expulsion, nutrient restriction, and / or temperature reduction. In most perfusion cultures, nutrient supply and waste removal will allow for constant cell growth and productivity, and cell expulsion is required to maintain a constant viable cell density or to keep cells in a steady state. Typical viable cell densities at steady state are 10 to 50 x 10⁻⁶. 6 Cells / ml. Viable cell density can vary depending on the perfusion rate. Higher cell densities can be achieved by increasing the perfusion rate or by optimizing the culture medium used for perfusion. At very high viable cell densities, perfusion culture becomes difficult to control within a bioreactor.

[0060] The terms “cell bleed” and “cell bleeding” are used interchangeably herein and refer to the removal of cells and culture medium from a bioreactor to maintain a constant, sustainable viable cell density within the bioreactor. This constant, sustainable viable cell density may also be referred to as the target cell density. Such cell bleed can be performed using a suction tube and a peristaltic pump at a defined flow rate. The tubing size should be appropriate, as tubes that are too narrow can lead to cell aggregation and blockage, while tubes that are too large may cause cells to settle. Cell bleed can be determined based on the growth rate, thus allowing the viable cell density to be continuously controlled within a desired volume. Alternatively, cells can be removed and replaced with culture medium at a specific frequency (e.g., once daily) to maintain the cell density within a predictable range. Ideally, the cell bleed rate equals the growth rate to maintain a stable cell density.

[0061] Typically, the product of interest removed with cell extrusion is discarded and thus lost at harvest. In contrast to permeate, cell extrusion contains cells, which makes product storage prior to purification more difficult and can adversely affect product quality. Therefore, continuous cell removal prior to storage and product purification is time-consuming, labor-intensive, and cost-inefficient. For slow-growing cells, cell extrusion may account for approximately 10% of the fluid removed, while for rapidly growing cells, it may account for approximately 30%. Therefore, product loss due to cell extrusion may be approximately 30% of the total product produced. As used herein, “permeate” refers to the harvest from which cells have been separated to be retained in the culture vessel.

[0062] The terms "culture" or "cell culture" are used interchangeably and refer to a population of cells maintained in a culture medium under conditions suitable for allowing the cell population to survive and / or grow. This invention relates only to mammalian cell cultures, and particularly to mammalian perfused cell cultures. Mammalian cells may be cultured in suspension or simultaneously attached to a solid support. As will be apparent to those skilled in the art, a cell culture refers to a composition comprising a population of cells and a culture medium in which said population is suspended. There are no particular limitations on the specific type of cell culture and it may encompass all forms and techniques of cell cultures, including but not limited to perfusion, continuous, limited, suspension, adherent or monolayer, adherence-dependent, and 3D cultures. As used herein, the term cell culture refers to a serum-free cell culture.

[0063] As used herein, the term "culture" refers to the process by which mammalian cells are grown or maintained under controlled conditions and under conditions that support cell growth and / or survival. As used herein, the terms "maintenance cells" and "cultured cells" are used interchangeably. Culture can also refer to the step of seeding cells in a culture medium.

[0064] As used herein, the term "batch culture" is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time, followed by complete harvesting. Cultures grown using the batch method increase in cell density until reaching maximum cell density, after which the viable cell density decreases as culture medium components are consumed and metabolic byproducts such as lactic acid and ammonia levels accumulate. Harvesting typically occurs when maximum cell density (typically 5–10 x 10⁻⁶) is achieved. 6 The number of cells / mL (depending on the culture medium preparation, cell line, etc.) usually occurs at or shortly thereafter, typically around 3 to 7 days.

[0065] As used herein, the term "feed-batch culture" improves upon the batch process by providing a push or continuous feed of medium to replenish those medium components that have already been consumed. Because fed-batch culture receives additional nutrients throughout the culture process, it has the potential to achieve higher cell densities (>10 to 30 x 10⁻⁶) compared to batch methods. 6Cells / ml (depending on culture medium formulation, cell line, etc.) and increased product titers. Unlike batch culture, fed-batch culture can be created and maintained by manipulating feeding strategies and culture medium formulations to distinguish between the cell proliferation phase (growth phase) to achieve the desired cell density and the suspension or slow cell growth phase (production phase). Therefore, fed-batch culture has the potential to achieve higher product titers compared to batch culture. As with batch methods, the accumulation of metabolic byproducts over time will lead to a decline in cell viability, as these byproducts gradually accumulate in the cell culture medium, limiting the duration of the production phase to approximately 1.5 to 3 weeks. Fed-batch culture is discontinuous and is typically harvested when metabolic byproduct levels or culture viability reach predetermined levels.

[0066] The terms “polypeptide” or “protein” are used interchangeably with “amino acid residue sequence” herein and refer to polymers of amino acids. These terms also include proteins that have undergone post-translational modifications, including but not limited to glycosylation, acetylation, phosphorylation, or protein processing. Modifications and alterations can be made to the structure of a polypeptide while maintaining its biological function, such as fusion with other proteins, amino acid sequence substitution, deletion, or insertion. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence, and proteins with the same properties can be obtained. These terms also apply to amino acid polymers, where one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acids. The term “polypeptide” generally refers to a sequence having more than 10 amino acids, and the term “peptide” refers to a sequence of at most 10 amino acids.

[0067] As used herein, the term "heterologous protein" refers to a polypeptide derived from an organism or species different from the host cell. Heterologous proteins are encoded by heterologous polynucleotides that are experimentally inserted into host cells that do not naturally express the protein. Heterologous polynucleotides can also be referred to as transgenes. Therefore, it may be a gene encoding the heterologous protein or an open reading frame (ORF). When used as a reference protein, the term "heterologous" can also indicate that the protein contains amino acid sequences that are not naturally related to each other or are of different lengths. Therefore, it also covers recombinant proteins. Heterologous can also refer to a polynucleotide sequence, such as a gene or transgene, or a portion thereof, inserted into the genome of a mammalian cell in a location where it would not normally be present. In this invention, the heterologous protein is preferably a therapeutic protein.

[0068] The terms “medium,” “cell culture medium,” and “culture medium” are used interchangeably herein and refer to a nutrient solution that nourishes cells (particularly mammalian cells). Cell culture medium formulations are well known in the art. Typically, cell culture media provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements, as well as buffers and salts, required for minimum cell growth and / or survival. Culture media may also contain supplementary components that enhance growth and / or survival above the minimum rate, including, but not limited to, hormones and / or other growth factors (such as insulin or insulin-like growth factor), specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements including trace metals (inorganic compounds typically present in very low final concentrations), amino acids (including non-proteinogenic amino acids), lipids, antioxidants, glucose, and / or other energy sources, such as organic acids; as described herein. Furthermore, surfactants may be included in the culture medium. In some embodiments, the culture medium is advantageously formulated to a pH and salt concentration optimal for cell survival and proliferation. "Cell culture perfusion medium" or "perfusion medium" is a culture medium used for continuous perfusion. Those skilled in the art will understand that further components not part of the cell culture medium may be added to the cell culture during culture. For example, an antifoaming agent may be added alone. Furthermore, glucose and / or glutamine may be added alone, or glucose and / or glutamine may be added alone in addition to the glucose provided with the cell culture medium. Finally, an alkali (e.g., sodium carbonate or sodium hydroxide) may be added to the cell culture to control the pH during culture.

[0069] Examples of amino acids in cell culture media include, but are not limited to, proteogenic amino acids such as glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, and their salts or derivatives, as well as non-proteogenic amino acids such as hydroxyproline, ornithine, α-aminobutyric acid, and their salts or derivatives. Derivatives include, for example, oxidized dimers or dipeptides of cystine and cysteine, preferably alanyl or glycyl dipeptides of amino acids such as glutamine, tyrosine, or cysteine. Examples of inorganic salts include, but are not limited to, calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate, sodium metasilicate, trace metal salts, and their hydrates. Examples of trace metals include, but are not limited to, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, and manganese and their salts, such as ammonium molybdate, copper sulfate, sodium selenite, manganese chloride, manganese sulfate, zinc chloride, zinc sulfate, etc., and their hydrates. Examples of iron sources include, but are not limited to, ferric citrate, ferric nitrate, ferrous sulfate, ferrous chloride, ferric chloride, and ferrous phosphate. Examples of vitamins include, but are not limited to, biotin, choline chloride, choline, pantothenate, D-calcium, folic acid, nicotinamide, para-aminobenzoic acid, pyridoxal, pyridoxine, riboflavin, thiamine, tocopherol, vitamin B12, retinol (vitamin A), ascorbate, etc., and their salts. Examples of polyamines include, but are not limited to, putrescine, spermidine, and spermine; organic acids can be taurine or alternative carbon sources such as succinic acid, pyruvate, and citric acid; fatty acids can be linoleic acid, linolenic acid, palmitic acid, and oleic acid; surfactants can be pluronic F68; buffers can be, for example, phosphate buffers (dihydrogen phosphate and dihydrogen phosphate); antioxidants can be, for example, reduced glutathione or lipoic acid; and examples of chelating agents are not limited to citrate or ethylenediaminetetraacetic acid (EDTA). Energy sources can be pyruvate or dextrose, etc. Other compounds that can be present in the culture medium are ethanolamine, taurine, isinositol, and proteins such as insulin or insulin-like growth factor. Compounds can also be added for the formulation of dry powder culture media; for example, dextrose can be added solely for grinding purposes rather than as a component of the culture medium.

[0070] The culture medium according to the invention is a serum-free perfusion medium (or serum-free cell culture perfusion medium) added from day 0 to day 4 after inoculation, i.e., perfusion culture begins from day 0 to day 4 of cell culture. Therefore, it can also be referred to as cell culture perfusion medium feed, as it is typically added after inoculation. Perfusion cell culture can have different culture phases, including a growth phase and a production phase. Specific media used during the growth phase (growth medium) and the production phase (production medium) can be specifically designed for implementation in said specific phase. Typically, cells are inoculated in growth medium before perfusion begins with production medium. Furthermore, perfusion may have already begun before replacing the growth medium with production medium. In some embodiments, the cell culture medium according to the invention is a production medium. However, both media (growth medium and production medium) are complete media and allow for the maintenance and / or growth of the cell culture (i.e., no further mixing with other media is required). This contrasts with feed media or fed-batch media used for fed-batch cultures, which are typically incomplete media that replenish consumed nutrients but usually reduce components such as salts and buffers to lower osmotic pressure and allow for further concentration. Without mixing with basal or inoculation media, the medium is generally insufficient to sustain cell culture.

[0071] The term "perfusion medium" refers to a nutrient solution that nourishes cells (particularly mammalian cells) and is used for perfusion culture. It can be a growth medium and / or a production medium. It is typically designed to support perfusion culture during the production phase. Because perfusion medium provides a stable source of fresh nutrients and is continuously removed from the bioreactor, it is a complete medium that allows for the maintenance and / or growth of cell cultures. The term "complete medium" refers to a nutrient solution containing all the components of a medium intended to be present in cell cultures.

[0072] The serum-free cell culture perfusion medium according to the invention is a complete culture medium and can exist in a separated form comprising at least three separate aqueous concentrates and a diluent, wherein the first concentrate is an alkaline concentrate, the second concentrate is an acidic concentrate, and the third concentrate is a neutral concentrate, or exists as the resulting serum-free cell culture perfusion medium after mixing. The term "serum-free cell culture perfusion medium," which does not explicitly characterize the separation of the culture media, refers to the resulting serum-free cell culture perfusion medium formed after mixing. Since the separated culture media are used for direct addition to cell cultures and / or the reaction vessel of a bioreactor, the resulting serum-free cell culture medium is generally not present in a pure or isolated form, but rather as a mixture with the existing cell culture (i.e., culture medium and cells). Therefore, it is important to adjust the pH of the separated culture medium after mixing. However, since the pH of the culture may change during cell culture, pH adjustment using an alkali during culture may still be necessary to maintain a constant pH.

[0073] As used herein, the term "serum-free" refers to a cell culture medium that does not contain animal or human serum (such as fetal bovine serum). Preferably, the serum-free medium does not contain proteins isolated from any animal or human-derived serum. Various tissue media, including defined media, are commercially available; for example, any one or a combination of the following cell media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's modified Eagle's medium (DMEM), Eagle Minimum Essential Medium, 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. TM The 300 series (JRH Biosciences, Lenexa, Kansas, etc.) are also available. Serum-free versions of these media are also available. Depending on the requirements of the cells being cultured and / or the desired cell culture parameters, cell culture media can 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.

[0074] As used herein, the term "protein-free" refers to a cell culture medium that contains no proteins whatsoever. Therefore, it lacks proteins isolated from animals or humans, proteins derived from serum, or recombinant proteins, such as those produced in mammalian, bacterial, insect, or yeast cells. Protein-free media may contain a single recombinant protein, such as insulin or insulin-like growth factor, but only if such addition is explicitly stated.

[0075] As used herein, the term "chemically defined" refers to a serum-free culture medium that contains no hydrolysates (such as protein hydrolysates derived from yeast, plants, or animals). Preferably, a chemically defined medium is also protein-free or contains only selected recombinant (non-animal-derived) proteins, such as insulin or insulin-like growth factor. A chemically defined medium consists of a mixture of characterized and purified substances. An example of a chemically defined medium is, for instance, CD-CHO medium from Invitrogen (Carlsbad, CA, US).

[0076] As used herein, the terms “suspension cells” or “non-adherent cells” refer to cells cultured in suspension in a liquid culture medium. Adherent cells (such as CHO cells) can be adapted to grow in suspension and thus lose their ability to adhere to the surface of a container or tissue culture dish.

[0077] As used herein, the term "bioreactor" means any container that can be used for the growth of cell cultures. Bioreactors can be of any size, as long as they can be used to culture cells; typically, bioreactors are sized to accommodate the volume of cell cultures grown within them. Typically, a bioreactor will be at least 1 liter, but can be 2 or more, 5 or more, 10 or more, 50 or more, 100 or more, 200 or more, 250 or more, 500 or more, 1,000 or more, 1,500 or more, 2,000 or more, 2,500 or more, 5,000 or more, 8,000 or more, 10,000 or more, 12,000 or more. Preferably, the bioreactor will be at least 100 liters, more preferably at least 1,000 liters. The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during the culture period. Based on the relevant considerations, those skilled in the art will recognize and be able to select a suitable bioreactor for use in practicing this invention. Cell cultures used in the methods of this invention can be grown in any bioreactor suitable for perfusion culture. There are no particular restrictions on the specific type of bioreactor, and it can cover all types of bioreactors suitable for perfusion cell culture.

[0078] As used herein, “cell density” refers to the number of cells in a given volume of culture medium. “Viable cell density” 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 exclusion assay).

[0079] As used herein, the term "cell viability" means the ability of cells in culture to survive under a given set of culture conditions or experimental variations. As used herein, the term also refers to the portion of cells that are alive at a given time, relative to the total number of living and dead cells in culture at that time.

[0080] As used herein, the term "titer" refers to the total amount of a polypeptide or protein of interest (which may be naturally occurring or recombinant) produced by a cell culture in a given volume of culture medium. Titers can be expressed in milligrams or micrograms of polypeptide or protein per milliliter (or other volumetric measure) of culture medium.

[0081] As used herein, the term "yield" refers to the amount of heterologous protein produced in perfusion culture within a specific time period. "Total yield" refers to the amount of heterologous protein produced in perfusion culture throughout the entire run.

[0082] As used herein, the terms “reduction,” “reduced,” or “decline” generally mean a reduction of at least 5% compared to a reference level, such as at least 10% compared to a reference level, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 90%, or at most and including 100%, or any integer reduction between 10% and 100% compared to a control mammalian cell culture cultured under the same conditions using the same serum-free cell culture medium, such that the osmotic pressure does not increase during culture, particularly during perfusion culture.

[0083] As used herein, the terms “enhancement,” “enhanced,” “enhanced,” “increase,” or “increased” generally mean an increase of at least 5% compared to control cells, such as a decrease of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least 300%, or any integer decrease between 10% and 300% compared to control mammalian cell cultures cultured under the same conditions using the same serum-free cell culture medium, such that the osmotic pressure does not increase during culture, particularly during perfusion culture.

[0084] As used herein, “control cell culture” or “control mammalian cell culture” is the same cell culture as the cell culture to which it is compared, which uses the same serum-free cell culture medium according to the invention, except that the osmotic pressure does not increase during culture, particularly during perfusion culture, said serum-free cell culture medium comprising culture medium components consisting of at least three aqueous concentrated feeds and a diluent.

[0085] As used herein, the term "mammalian cell" refers to a cell line suitable for producing heterologous proteins, preferably therapeutic proteins, and more preferably secreted recombinant therapeutic proteins. Preferred mammalian cells according to the invention are rodent cells, such as hamster cells. Mammalian cells are isolated cells or cell lines. Mammalian cells are preferably transformed and / or immortalized cell lines. They are suitable for continuous passage in cell culture and do not include primary untransformed cells or cells that are part of an organ structure. Preferred mammalian cells are BHK21 and BHK TK. - CHO, CHO-K1, CHO-S cells, CHO-DXB11 (also known as CHO-DUKX or DuxB11), and CHO-DG44 cells, or derivatives / progeny of any of these cell lines, are included. CHO-DG44, CHO-K1, and BHK21 are particularly preferred, and even more preferred are CHO-DG44 and CHO-K1 cells. CHO-DG44 cells are the most preferred. Mammalian cells, particularly glutamine synthetase (GS) deficient derivatives of CHO-DG44 and CHO-K1 cells, are also included. Mammalian cells may further comprise one or more expression cassettes encoding heterologous proteins, preferably recombinant secreted therapeutic proteins. Mammalian cells may also be murine cells, such as murine myeloma cells, such as NSO and Sp2 / O cells, or derivatives / progeny of any of these cell lines. However, derivatives / progeny of these cells, and other mammalian cells (including but not limited to human, mouse, rat, monkey, and rodent cell lines), can also be used in this invention, particularly for the production of biopharmaceutical proteins.

[0086] As used herein, the term "growth phase" refers to a stage in cell culture in which cells proliferate exponentially and the density of live cells in the bioreactor increases. Cells in culture typically proliferate following a standard growth pattern. The post-inoculation phase can be a lag phase, a slow growth period as cells adapt to the culture environment and prepare for rapid growth. The growth phase (also known as the logarithmic growth phase or logarithmic growth period) is the period in which cells proliferate exponentially and consume the nutrients in the growth medium. This is followed by the production phase.

[0087] The term "production stage" refers to the cell culture phase that begins once harvesting commences, which can be at or before the target viable cell density is reached. This occurs when the heterologous protein concentration in the permeate reaches approximately 0.2 g / L. 生物反应器 Harvesting typically begins around 10 x 10⁶ cells per day. A typical target cell density is 10 x 10⁶. 6 Cells / ml to approximately 120x10 6 The target cell density is in the range of cells / ml, but can be even higher. Therefore, the target cell density according to the invention is at least 30 x 10⁻⁶ cells / ml.6 10 cells / ml, at least 40 x 10 6 Cells / ml, at least 50 x 10 6 Cells / ml, at least 60 x 10 6 10 cells / ml, at least 80 x 10 6 100 cells / ml or at least 100 x 10 6 Cells / ml. Target cell density can even reach 100x10⁶ cells / ml. 6 Cells / ml to 200x10 6 Cells / ml, preferably 120x10 6 Cells / ml to 150x10 6 Cells / ml

[0088] In some embodiments described herein, at the start of the production phase, the osmotic pressure of the cell culture is increased to a level that causes growth inhibition. Preferably, the osmotic pressure is increased gradually or stepwise from the level most suitable for growth. Therefore, the osmotic pressure needs to be increased before the target viable cell density is reached. Preferably, starting from about half of the target viable cell density, the osmotic pressure is gradually or stepwise increased from the level most suitable for growth to a level that causes growth inhibition. This allows the osmotic pressure level causing growth inhibition to be reached once the target viable cell density is achieved. Importantly, the high osmotic pressure (i.e., the osmotic pressure level causing growth inhibition) is maintained until the end of the culture. Those skilled in the art will understand that removing the osmotic pressure will remove the growth inhibition.

[0089] The terms “growth arrest,” “growth inhibition,” and “growth suppression” are used synonymously herein and refer to cells that have stopped increasing in number (i.e., stopped dividing). The cell cycle consists of interphase and mitotic phase. Interphase consists of three phases: DNA replication is restricted to S phase; G1 is the gap between M and S phases; and G2 is the gap between S and M phases. In M phase, nuclear division occurs, followed by cytoplasmic division. Cell cycle arrest occurs in the absence of mitotic signals for proliferation or in the presence of compounds that induce growth arrest. Cells can partially dismantle their cell cycle control systems and exit the cycle into a specialized non-dividing state known as G0. Growth inhibition can be readily assessed by determining the live cell density over time. Preferably, cells are maintained at a live cell density with a variation of ≤30%, more preferably ≤20%. More preferably, cells are maintained at a target live cell density with a variation of ≤30%, more preferably ≤20%.

[0090] Cell culture perfusion medium In one aspect of this disclosure, a separated serum-free cell culture perfusion medium is disclosed, the separated serum-free cell culture perfusion medium comprising a culture medium component consisting of at least three separate aqueous concentrates and a diluent, wherein the first concentrate is an alkaline concentrate, the second concentrate is an acidic concentrate, and the third concentrate is a near-neutral concentrate; wherein after mixing the at least three separate aqueous concentrates and the diluent in the resulting serum-free cell culture perfusion medium, the pH of the separated serum-free cell culture perfusion medium is adjusted to neutral. In a preferred embodiment, the at least three separate aqueous concentrates are not premixed before being added to the reaction vessel of the cell culture and / or bioreactor. Premixing two or more feeds is not ideal because precipitation may occur after mixing. Therefore, the separated serum-free cell culture perfusion medium is suitable for adding the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed separately to the reaction vessel of the cell culture and / or bioreactor; adding the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed directly to the reaction vessel of the cell culture and / or bioreactor without prior premixing; and / or directly mixing the at least three separate aqueous concentrated feeds in the reaction vessel of the cell culture and / or bioreactor. In a preferred embodiment, the serum-free cell culture perfusion medium comprises a culture medium component consisting of at least three separate aqueous concentrated feeds as described, and a diluent. This includes a serum-free cell culture perfusion medium composed of the at least three separate aqueous concentrated feeds and a diluent. The culture medium components are distributed primarily according to their inherent properties, such as solubility at neutral pH and / or improved solubility at alkaline or acidic pH. In a preferred embodiment, the serum-free cell culture perfusion medium is a production culture medium. Those skilled in the art will understand that perfusion culture is typically performed using mammalian cells, and therefore the perfusion medium is a perfusion medium for mammalian cells.

[0091] Those skilled in the art will also understand that further components not part of the cell culture medium may be added to the cell culture during culture. For example, an antifoaming agent may be added alone. Furthermore, glucose and / or glutamine may be added alone, or in addition to the glucose and / or glutamine provided with the cell culture medium. Finally, an alkali (e.g., sodium carbonate or sodium hydroxide) may be added to the cell culture to control the pH during culture.

[0092] In one embodiment, the serum-free cell culture perfusion medium may be chemically defined and / or free of hydrolysates. Free of hydrolysates means that the medium does not contain protein hydrolysates from animals, plants (soybeans, potatoes, rice), yeast, or other sources. Typically, chemically defined mediums are free of hydrolysates. In any case, the serum-free perfusion medium should be free of compounds derived from animal sources, particularly proteins or peptides derived from and isolated from animals (this does not include recombinant proteins produced from cell cultures). Preferably, the serum-free cell culture perfusion medium is protein-free or protein-free except for recombinant insulin and / or insulin-like growth factor. Therefore, the serum-free cell culture medium may be a protein-free medium or a protein-free medium containing recombinant insulin and / or recombinant insulin-like growth factor. Those skilled in the art will understand that protein-free mediums are generally chemically defined and / or free of hydrolysates. More preferably, the serum-free cell culture perfusion medium is chemically defined and protein-free or protein-free except for recombinant insulin and / or insulin-like growth factor. This also applies to serum-free culture perfusion media used in the method or prepared according to the method of the invention. This applies to both initial growth media and production media if used.

[0093] To adjust the separated serum-free cell culture perfusion medium to the desired "working" concentration, appropriate volumes of each of the at least three separate aqueous concentrates are mixed at a ratio to provide the serum-free cell culture perfusion medium, i.e., the serum-free cell culture perfusion medium at the working concentration, the ratio being determined by the multiples of the concentrations of the at least three separate aqueous concentrates relative to each other, and the at least three separate aqueous concentrates are diluted with the diluent. Although the diluent used for the serum-free cell culture perfusion medium according to the invention can theoretically also be an aqueous salt solution and / or an aqueous buffer, it is preferably sterile water. Sterile water is advantageous because it does not require preparation or mixing, and therefore eliminates the need for additional storage space for pre-prepared components. The ratio (v / v) of the diluent to the cumulative volume of the at least three separate aqueous concentrates in the cell culture and / or bioreactor reaction vessel determines the fold concentration of the serum-free cell culture perfusion medium added to the cell culture and / or bioreactor reaction vessel to provide a serum-free cell culture perfusion medium with pH adjusted to near neutral. Therefore, the advantage of using the at least three separate aqueous concentrates is that the fold concentration of the medium can be adapted to live cell density and nutrient requirements (maintaining nutrient balance). Osmolarity can be used as a proxy for estimating the nutrient balance inside and outside the system. Therefore, osmolarity balance can be used to calculate adjustments to the cumulative volume of the concentrates (at a fixed ratio relative to each other) and the diluent feed rate to achieve desired residual osmolarity and nutrient levels.

[0094] After mixing the at least three separate aqueous concentrates and the diluent, the resulting serum-free cell culture perfusion medium is adjusted to neutral pH. This means that the pH is automatically adjusted after mixing without the need for the addition of titrants such as NaOH or HCl. After mixing the at least three separate aqueous concentrates and the diluent, the pH of the medium should be neutral, between about 6.7 and about 7.5, preferably between about 6.9 and about 7.4, and more preferably between about 6.9 and about 7.2.

[0095] The alkaline concentrate feed can be a 2x to 80x concentrate feed, preferably a 20x to 40x concentrate feed, more preferably a 20x to 30x concentrate feed, and most preferably a 25x concentrate feed. Higher concentration feeds are generally preferred. However, for optimal results, the alkaline concentrate feed can be prepared as a partially concentrated feed to better match the near-neutral and / or acidic feeds. This also ensures the safety of the titrant in the concentrate feed (such as the alkaline concentrate feed). The near-neutral concentrate feed can be a 2x to 50x concentrate feed, preferably a 10x to 40x concentrate feed, more preferably a 20x to 30x concentrate feed, and most preferably a 25x concentrate feed. The acidic concentrate feed can be a 2x to 40x concentrate feed, a 4x to 20x concentrate feed, a 5x to 12x concentrate feed, or a 6x to 10x concentrate feed. Generally, higher concentration feeds (alkaline, acidic, and neutral, combined and single) are preferred. However, for optimal results, the alkaline concentrate feed can be prepared as a less concentrated concentrate (e.g., less than 80x) to better match the near-neutral and / or acidic feeds. This also ensures the safety of the titrant in the concentrate feed (such as the alkaline concentrate feed).

[0096] In one embodiment, the alkaline concentrate feed is a 2x to 80x concentrate feed, the acidic concentrate feed is a 2x to 40x concentrate feed, and the near-neutral concentrate feed is a 2x to 50x concentrate feed. Preferably, the alkaline concentrate feed is a 20x to 40x concentrate feed, the acidic concentrate feed is a 4x to 20x concentrate feed, and the near-neutral concentrate feed is a 10x to 40x concentrate feed. More preferably, the alkaline concentrate feed is a 20x to 30x concentrate feed, the acidic concentrate feed is a 5x to 12x concentrate feed, and the near-neutral concentrate feed is a 20x to 30x concentrate feed. Most preferably, the alkaline concentrate feed is a 25x concentrate feed, the acidic concentrate feed is a 6x to 10x concentrate feed, and the near-neutral concentrate feed is a 25x concentrate feed. In a particular embodiment, the alkaline concentrate feed and the near-neutral concentrate feed are concentrated in a substantially similar manner. Therefore, for example, the alkaline concentrate feed is a 20x to 30x concentrate feed, and the near-neutral concentrate feed is a 20x to 30x concentrate feed, or the alkaline concentrate feed is a 25x concentrate feed, and the near-neutral concentrate feed is a 25x concentrate feed, and the acidic concentrate feed is concentrated to the maximum extent.

[0097] In the serum-free cell culture perfusion medium, the ratio (v / v / v) of the alkaline concentrate to the acidic concentrate to the near-neutral concentrate is a fixed ratio to provide a serum-free cell culture perfusion medium with pH adjusted to neutral. Therefore, the at least three concentrates are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the medium components in the 1x serum-free cell culture perfusion medium (1x formulation). In other words, the ratios between the concentrates should maintain the original proportions of the 1x formulation. For example, if the alkaline concentrate is a 25x concentrate, the acidic concentrate is a 6x concentrate, and the near-neutral concentrate is a 25x concentrate, the concentrates are added at a ratio of 1:4.2:1; or if the alkaline concentrate is a 30x concentrate, the acidic concentrate is a 10x concentrate, and the near-neutral concentrate is a 30x concentrate, the concentrates are added at a ratio of 1:3:1. Furthermore, the osmotic pressure of the serum-free cell culture perfusion medium is determined by the ratio (v / v) of the cumulative volume of the diluent to the at least three individual aqueous concentrates in the serum-free cell culture perfusion medium adjusted to neutral pH. The ratio (v / v) of the cumulative volume of the diluent to the at least three individual aqueous concentrates in the serum-free cell culture perfusion medium adjusted to neutral pH also determines the fold concentration of the serum-free cell culture perfusion medium. The concentration factor can be any value from 0.1x to the maximum concentration factor, but is typically between 0.5x and 2x, preferably between 1x and 2x. The maximum concentration factor (n) after mixing the three separate aqueous concentrate feeds is... 最大 X) can be calculated as follows: n 最大 X=(n 碱性 X*n 酸性 X*n 中性 X) / ((n 碱性 X*n 酸性 X)+(n 碱性 X*n 中性 X)+(n 酸性 X*n 中性 X)), in n 最大 X is the maximum concentration multiple after mixing the three separate aqueous concentrate feeds; n 碱性 X is n times the concentration of the alkaline concentrate feed; n 酸性 X is n times the concentration of the acidic concentrate feed; n 中性 X is n times the concentration of the near-neutral concentrate feed; and * It represents the mathematical operation of multiplication.

[0098] For example, if the alkaline concentrate is 25x, the acidic concentrate is 6x, and the near-neutral concentrate is 25x, the maximum concentration of the mixture of the three individual aqueous concentrates is 4.1x. Therefore, the culture medium consumption is reduced by approximately 75%. If the alkaline concentrate is 30x, the acidic concentrate is 10x, and the near-neutral concentrate is 30x, the maximum concentration of the mixture of the three individual aqueous concentrates is 6x. Therefore, the culture medium consumption is reduced by more than 80%. Furthermore, using concentrates allows for adjustment of the concentration of the serum-free cell culture medium in the cell culture and / or bioreactor, and thus allows for maintaining a high viable cell density with similar or only moderately increased perfusion rates and thus with reduced specific perfusion rates.

[0099] The serum-free cell culture perfusion medium comprises an alkaline concentrate, an acidic concentrate, and a near-neutral concentrate. A near-neutral concentrate feed is defined as having a pH of 7.5 ± 1.0. Therefore, the pH of the near-neutral concentrate feed is from about 6.5 to about 8.5. The near-neutral concentrate feed preferably does not contain any additional titrant. Avoiding the use of titrants saves osmotic space in the resulting serum-free cell culture perfusion medium. Therefore, the pH of the near-neutral concentrate feed may be slightly alkaline at pH levels up to about 8.5. Preferably, the near-neutral pH is from about 7 to about 8.5, more preferably from about 7.5 to about 8.5.

[0100] The pH of the alkaline concentrate feed can be about 9 or higher, such as pH from about 9 to about 11, preferably pH from about 9.8 to about 10.8, and more preferably pH from about 9.8 to about 10.5. The pH of the acidic concentrate feed can be about 5 or lower, such as pH from about 2 to about 5, preferably pH from about 3.6 to about 4.8, and more preferably pH from about 3.8 to about 4.5. Although the pH can be adjusted quite precisely, a typical pH change is a change of 0.5.

[0101] In one embodiment, the pH of the alkaline concentrate feed is about 9 or higher, the pH of the acidic concentrate feed is about 5 or lower, and the pH of the near-neutral concentrate feed is about 7 to about 8.5. Preferably, the pH of the alkaline concentrate feed is about 9 to about 11, the pH of the acidic concentrate feed is about 2 to about 5, and the pH of the near-neutral concentrate feed is about 7 to about 8.5; more preferably, the pH of the alkaline concentrate feed is about 9.8 to about 10.8, the pH of the acidic concentrate feed is about 3.6 to about 4.8, and the pH of the near-neutral concentrate feed is about 7 to about 8.5; and most preferably, the pH of the alkaline concentrate feed is about 9.8 to about 10.5, the pH of the acidic concentrate feed is about 3.8 to about 4.5, and the pH of the near-neutral concentrate feed is about 7.5 to about 8.5.

[0102] The culture medium components are distributed primarily based on their inherent properties, such as solubility at neutral pH and / or improved solubility at alkaline or acidic pH. Furthermore, culture medium components that are particularly insoluble in aqueous solutions can be separated into separate feeds. For example, choline chloride can be provided together with the near-neutral concentrated feed and the acidic concentrated feed to achieve the desired concentration in the final serum-free cell culture medium.

[0103] The near-neutral concentrate feed preferably contains all vitamins soluble at neutral pH. Further, the near-neutral concentrate feed preferably does not contain any metals. Since metals can interact with some vitamins, vitamins are preferably kept separate from metals. Therefore, vitamins are preferably provided in the near-neutral concentrate feed and alternatively in the acidic concentrate feed. An exception is folic acid, which may also be provided with the alkaline feed. Thus, in one embodiment, vitamins and metals are provided in separate feeds, preferably vitamins in the near-neutral feed and metals in the acidic feed. However, vitamins that are poorly soluble in aqueous solutions at neutral pH may also be provided in the acidic feed. For example, vitamins such as pantothenate, thiamine, choline chloride, and / or pyridoxine may also be provided in the acidic feed. Furthermore, vitamins that are generally poorly soluble in aqueous solutions, such as choline chloride, may be present in both the neutral and acidic feeds. The neutral concentrate feed may also contain compounds such as L-α-amino-n-butyric acid, isoisinolate, and / or fatty acid linoleic acid. Furthermore, bicarbonate is preferably provided together with the neutral concentrate feed. In one embodiment, the neutral concentrate feed does not contain any additional titrant for pH adjustment.

[0104] Salts and metals are preferably provided in the acidic concentrate feed. For example, but not limited to, the acidic concentrate feed may contain trace elements, trace metals, inorganic salts, iron chelating agents, polyamines, and / or regulatory hormones, such as insulin or insulin-like growth factor. Amino acids selected from the group consisting of alanine, arginine, asparagine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine are preferably provided in the acidic concentrate feed and / or the near-neutral concentrate feed. Furthermore, surfactants, antioxidants, and carbon sources, as well as optional ethanolamines and / or fatty acids, may be provided in the acidic concentrate feed and / or the near-neutral concentrate feed.

[0105] The alkaline concentrate feed primarily contains amino acids that have maximum solubility at an alkaline pH of about 9 or higher. Preferably, the alkaline concentrate feed contains at least aspartic acid, histidine, tyrosine, and cysteine. Cysteine ​​is water-soluble but readily oxidized to poorly water-soluble cystine at neutral pH. Therefore, cysteine ​​and / or cystine are preferably included in the alkaline concentrate feed. Another compound soluble at an alkaline pH of about 9 or higher is folic acid. Therefore, folic acid may also be provided with the alkaline feed. Amino acids not provided with the alkaline concentrate feed are preferably provided with the acidic concentrate feed. Therefore, in one embodiment, the remaining amino acids (i.e., amino acids that do not have maximum solubility at an alkaline pH of about 9 or higher and / or amino acids not provided with the alkaline feed) may be provided in the acidic and / or near-neutral concentrate feeds, preferably in the acidic concentrate feed.

[0106] Those skilled in the art will understand that complete culture media are more difficult to provide as concentrates compared to feed media, such as those used for fed-batch culture, because they contain more components, particularly salts and buffers that increase osmotic pressure and thus limit osmotic space. Furthermore, modern nutrient-rich media are more difficult to provide as concentrates compared to prior art media such as RPMI 1640 and DMEM / F12. These more modern nutrient-rich media are particularly rich in amino acids, typically containing amino acids in the mM range rather than the µM range. Therefore, the serum-free cell culture perfusion medium according to the invention is a medium containing amino acids at a concentration of more than 50 mM, preferably more than 70 mM, more preferably more than 100 mM, and even more preferably more than 120 mM in 1x serum-free cell culture perfusion medium. Since glutamine is sometimes added alone, the serum-free cell culture perfusion medium preferably contains natural amino acids other than glutamine, at a concentration of more than 50 mM, preferably more than 70 mM, more preferably more than 100 mM, and even more preferably more than 120 mM in the resulting serum-free cell culture perfusion medium. Natural amino acids other than glutamine include alanine, glycine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, histidine, serine, threonine, tryptophan, tyrosine, and valine. However, not all natural amino acids need to be present in the serum-free cell culture perfusion medium, such as alanine and glycine. Natural amino acids also include derivatives of natural amino acids, such as dipeptides or cysteine.

[0107] Those skilled in the art can optimize various processes regarding cell culture medium composition and for other process characteristics and culture performance. For example, and especially in the case of materials with relatively low cell density, they can be tested in shake flasks. Where higher oxygenation rates are required, spin tubes (such as those disclosed, for example, in Srenard et al., Biotechnol. Prog., 2010, Vol. 26, No. 3, pages 653-663) can be used, which agitate at higher revolutions per minute (rpm). Spin tube bioreactors can be advantageously used as small-scale models for evaluating culture media, various process parameters, and growth characteristics at high densities (>20e6 c / mL). They can also reduce the time and effort required for process development by alleviating the need for large-scale culture medium preparation and laboratory-scale bioreactor operation. The ability to centrifuge multiple spin tubes for culture medium exchange enables perfusion cell culture at small scales (working volume 15 mL).

[0108] The at least three separate aqueous concentrate feeds are preferably sterile before storage and before mixing. In one embodiment, the at least three separate aqueous concentrate feeds are filtered and sterilized. Furthermore, regarding the mixing of components, the at least three separate aqueous concentrate feeds are not premixed before being added to the reaction vessel of the cell culture and / or bioreactor. Therefore, the aqueous concentrate feeds are preferably added directly to the reaction vessel of the cell culture and / or bioreactor, preferably through a separate inlet point.

[0109] The inlet point can be a valve or port in the bioreactor. Preferably, the at least three separate aqueous concentrate feeds are added dropwise. Advantageously, the at least three separate aqueous concentrate feeds are added continuously at a predetermined perfusion rate, and therefore simultaneously. They can be added from the bottom, from the top, or from the side of the bioreactor, and may be adjacent to each other or on different sides, as long as the culture is continuously mixed.

[0110] The diluent (e.g., sterile water) can be added separately to the reaction vessel of the cell culture and / or bioreactor. Therefore, it is preferable to add the diluent directly to the reaction vessel of the cell culture and / or bioreactor, preferably through an inlet point separate from the inlet points of the at least three separate aqueous concentrate feeds. The inlet point can be a valve or port in the bioreactor. Advantageously, the diluent is added continuously at a predetermined perfusion rate, and thus simultaneously with the at least three separate aqueous concentrate feeds. It can be added from the bottom, top, or side of the bioreactor, and adjacent to or on a different side from one or all of the at least three separate aqueous concentrate feeds, as long as the culture is continuously mixed. Alternatively, the diluent can be premixed with one of the at least three separate aqueous concentrate feeds before being added to the reaction vessel of the cell culture and / or bioreactor. Therefore, the diluent can be added together with one of the at least three separate aqueous concentrate feeds to the reaction vessel of the cell culture and / or bioreactor, preferably through an inlet point separate from the at least two other separate aqueous concentrate feeds. In one embodiment, the diluent is premixed with the alkaline concentrated feed before being added to the reaction vessel of the cell culture and / or bioreactor.

[0111] In another aspect, the invention also relates to the use of the separated serum-free cell culture perfusion medium according to the invention for culturing mammalian cells, preferably in a perfusion culture. In one embodiment, the cell culture medium according to the invention is used to control the osmotic pressure of the cell culture, preferably the perfused cell culture. In particular, the osmotic pressure is increased in the perfused cell culture. Increasing the osmotic pressure of the cell culture inhibits cell growth and increases the production of heterologous proteins. By increasing the osmotic pressure of the cell culture, cell growth can be inhibited to maintain a sustainable viable cell density without cell expulsion, which can also be referred to as dynamic perfusion culture.

[0112] By increasing the osmotic pressure of the cell culture, the yield of the heterologous protein produced in the cell culture can be increased by at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, or about 5-50%, preferably about 10-100%, relative to the yield of the control cell culture, without increasing the osmotic pressure. Preferably, the yield is determined for a portion or the entire culture period.

[0113] By using the serum-free cell culture medium according to the invention or the serum-free cell culture medium obtained by the method according to the invention, and optionally further increasing the osmotic pressure of the cell culture, the cell specific perfusion rate (pl / cell / day) is reduced by at least about 25%, at least 30%, or at least about 50% relative to the cell specific perfusion rate of 1x serum-free cell culture medium. The cell specific perfusion rate (pl / cell / day) of the serum-free cell culture perfusion medium according to the invention or the serum-free cell culture medium obtained by the method according to the invention is preferably constant throughout part or the entire culture period.

[0114] The present invention also relates to an alkaline aqueous concentrate feed, which is used in combination with an acidic aqueous concentrate feed, a near-neutral aqueous concentrate feed, and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the serum-free cell culture perfusion medium is automatically adjusted to neutral pH. In another embodiment, the present invention relates to an acidic aqueous concentrate feed, which is used in combination with an alkaline aqueous concentrate feed, a near-neutral aqueous concentrate feed, and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH. In yet another aspect, the present invention relates to a near-neutral aqueous concentrate feed, which is used in combination with an alkaline aqueous concentrate feed, an acidic aqueous concentrate feed, and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH. The alkaline aqueous concentrate feed, the acidic aqueous concentrate feed, the near-neutral aqueous concentrate feed, the diluent, and the serum-free cell culture perfusion medium can be further characterized as disclosed above.

[0115] A method for preparing serum-free cell culture perfusion medium.

[0116] In another aspect, the present invention relates to a method for preparing a serum-free cell culture perfusion medium, the method comprising: (a) providing cell culture medium components in at least three subgroups based on their solubility at alkaline, acidic, and neutral pH levels; (b)(i) providing the subgroup components soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed; (ii) providing the subgroup components soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; and (iii) (c) soluble subcomponents in neutral aqueous solution at neutral pH to form a near-neutral concentrated feed; (d) optionally storing the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed in separate containers; and (e) adding the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed, along with a diluent, to a reaction vessel of a cell culture and / or bioreactor, wherein (i) the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed are added separately to the reaction vessel of the cell culture and / or bioreactor; and (ii) the diluent is added separately to the reaction vessel of the cell culture and / or bioreactor, or the diluent is premixed with one of the at least three separate aqueous concentrated feeds before being added to the reaction vessel of the cell culture and / or bioreactor; wherein after mixing the at least three separate aqueous concentrated feeds and the diluent, the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to near-neutral pH. Therefore, the serum-free cell culture perfusion medium prepared according to the method comprises culture medium components and diluents as disclosed for the separated serum-free cell culture perfusion medium according to the invention, the culture medium components being composed of at least three separate aqueous concentrate feeds. Typically, after adding the at least three separate aqueous concentrate feeds and the diluent, the cell culture and / or bioreactor reaction vessel contains mammalian cells.

[0117] The method may include a step of sterilizing the concentrated feed, preferably by filtration sterilization, before storing and / or adding it to the reaction vessel of the cell culture and / or bioreactor. The reaction vessel of the cell culture and / or bioreactor contains at least about 100 L of serum-free cell culture perfusion medium, preferably at least about 1000 L of serum-free cell culture perfusion medium.

[0118] Preferably, the three concentrated feeds are added dropwise to the reaction vessel of the cell culture and / or bioreactor through separate ports. In-vessel mixing and dilution of the at least three separate aqueous concentrated feeds allows for a 50-90% reduction in preparation medium consumption, preferably 60-90%, over a 14-day culture period compared to mixing and diluting serum-free cell culture perfusion medium before addition to the bioreactor. The maximum fold concentration (n) of the mixture of the at least three separate aqueous concentrated feeds can be calculated using the methods provided above. 最大 The formula for X) is used to calculate the reduction in the consumption of the prepared culture medium, and to calculate the cumulative volume of the at least three separate aqueous concentrate feeds relative to the volume percentage of 1x serum-free cell culture perfusion medium further containing the diluent.

[0119] Adding the at least three separate concentrated feeds and the diluent individually allows for control of the osmotic pressure of the serum-free cell culture perfusion medium in the bioreactor. Control of the osmotic pressure of the serum-free cell culture perfusion medium in the bioreactor can also be achieved by premixing the diluent with one of the at least three separate aqueous concentrated feeds before adding it to the cell cultures and / or the reaction vessel of the bioreactor.

[0120] The osmotic pressure of cell cultures can be controlled using a constant concentrate feed perfusion rate and different diluent perfusion rates (which result in different total perfusion rates). The constant concentrate feed perfusion rate involves the cumulative perfusion rate of the at least three individual aqueous concentrate feeds, more specifically the alkaline concentrate feed, the acidic concentrate feed, and the near-neutral concentrate feed. The total perfusion rate is the cumulative perfusion rate of the at least three individual aqueous concentrate feeds and the diluent. Alternatively, the osmotic pressure of cell cultures can be controlled using a constant total perfusion rate and different concentrate feed perfusion rates. This naturally results in different diluent perfusion rates. In another alternative, the osmotic pressure of cell cultures can be controlled using a constant diluent perfusion rate and different concentrate feed perfusion rates (which result in different total perfusion rates).

[0121] The at least three concentrated feeds are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the culture medium components in 1x serum-free cell culture perfusion medium. In one embodiment, the ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed to the near-neutral concentrated feed is a fixed ratio to provide the serum-free cell culture perfusion medium with pH adjusted to neutral in the reaction vessel of the cell culture and / or bioreactor; and the ratio (v / v) of the diluent to the cumulative volume of the at least three individual aqueous concentrated feeds determines the osmotic pressure and / or fold concentration of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor, the at least three individual aqueous concentrated feeds being added to the reaction vessel of the cell culture and / or bioreactor to provide the serum-free cell culture perfusion medium with pH adjusted to near-neutral.

[0122] The osmotic pressure of the cell culture can be increased using: a constant concentrate feed perfusion rate and a reduced diluent perfusion rate, resulting in a reduced total perfusion rate; or a constant total perfusion rate and an increased concentrate feed perfusion rate and a reduced diluent perfusion rate; or a constant diluent perfusion rate and an increased concentrate feed perfusion rate, resulting in an increased total perfusion rate; wherein the at least three concentrates are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the culture medium components in a 1x serum-free cell culture perfusion medium. In one embodiment, preferably, no further additives are added to the culture to increase the osmotic pressure.

[0123] In another aspect, the present invention relates to a serum-free cell culture perfusion medium that can be obtained by the method according to the invention.

[0124] Cell culture methods For purposes of understanding, skilled practitioners will understand that cell culture and culture runs used for protein production can include at least three general types: perfusion culture, batch culture, and fed-batch culture. In perfusion culture, for example, fresh culture medium is provided to the cells during culture while old medium is removed daily and products are harvested, for example, daily or continuously. In perfusion culture, the perfusion medium can be added daily and can be added continuously, i.e., by dripping or infusion. For perfusion culture, cells can be maintained in culture for the desired long period as long as the cells remain viable and the environment and culture conditions are maintained. Because cells grow continuously, it is often necessary to remove cells during runs to maintain a constant viable cell density; this is called cell shedding. Cell shedding contains products from the culture medium removed along with the cells, which are usually discarded and thus wasted. Therefore, maintaining a viable cell density during production phases with little or no cell shedding increases the overall yield per run.

[0125] In batch culture, cells are initially cultured in a medium that is not removed, replaced, or replenished; that is, the cells are not "fed" with fresh medium during or before the end of the culture run. The desired product is harvested at the end of the culture run. Batch culture can also refer to the initial stage of fed-batch or perfusion culture. For perfusion culture, mammalian cells may be initially cultured, for example, in batch mode before perfusion culture begins.

[0126] For fed-batch culture, the culture run time is increased by replenishing the culture medium with fresh medium once or more daily (or continuously) during the run; that is, the cells are "fed" with fresh medium ("feed medium") during the culture period. Fed-batch culture can include various feeding protocols and times as described above, such as daily, every other day, every two days, more than once a day, or less than once a day, etc. Furthermore, fed-batch culture can be continuously fed with feed medium. The desired product is then harvested at the end of the culture / production run.

[0127] Mammalian cells can be cultured in perfusion culture. During heterologous protein production, a controlled system is desired in which cells are grown to a desired viable cell density and then converted to a high-productivity state of growth arrest, where cells use energy and substrate to produce the heterologous protein of interest, rather than through cell growth and division. Methods used to achieve this, such as temperature variations and amino acid starvation, are not always successful and can have undesirable effects on product quality. As described herein, viable cell density can be maintained at a desired level during the production phase by performing routine cell evoked loss. However, this results in the discarding of the heterologous protein of interest. Cell growth arrest during the production phase reduces the need for cell evoked loss and may even allow cells to be maintained in a more productive state.

[0128] In one aspect, a method is provided for culturing mammalian cells expressing a heterologous protein in a perfusion culture, the method comprising: (a) seeding a bioreactor with mammalian cells expressing a heterologous protein in a serum-free cell culture medium; and (b) culturing the mammalian cells in a perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium feed and removing the used medium while simultaneously maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a separated serum-free cell culture perfusion medium comprising a culture medium component comprising at least three separate aqueous concentrate feeds and a diluent, wherein a first concentrate feed is an alkaline concentrate feed, a second concentrate feed is an acidic concentrate feed, and a third concentrate feed is a near-neutral concentrate feed. The method of the invention provides for: (i) a serum-free cell culture perfusion medium, wherein the at least three separate aqueous concentrates and the diluent are mixed in the resulting serum-free cell culture perfusion medium, and the pH of the separated serum-free cell culture perfusion medium is adjusted to neutral; and / or (ii) a serum-free cell culture perfusion medium obtained by the method of the invention, wherein the alkaline concentrate, the acidic concentrate, and the near-neutral concentrate of the separated serum-free cell culture perfusion medium are added separately to the reaction vessel of the cell culture and / or bioreactor, and wherein the diluent is added separately to the reaction vessel of the cell culture and / or bioreactor, or the diluent is premixed with one of the at least three separate aqueous concentrates before being added to the reaction vessel of the cell culture and / or bioreactor.

[0129] In one embodiment, mammalian cells are initially cultured in batch culture before perfusion culture begins. Typically, in step (a), the serum-free cell culture medium is a growth medium. Step (a) may further include culturing mammalian cells in the growth medium and initiating perfusion culture using the growth medium. In step (b), culturing mammalian cells in perfusion culture comprises: culturing mammalian cells during the production phase by perfusion with the serum-free cell culture medium according to the invention or a serum-free cell culture medium obtained by the method according to the invention until a target cell density is reached; and further maintaining the mammalian cells at the target cell density during the production phase by perfusion with the serum-free cell culture medium according to the invention or a serum-free cell culture medium obtained by the method according to the invention. The serum-free cell culture perfusion medium used for perfusion culture may be a production medium, and the perfusion culture is performed by continuously feeding mammalian cells according to step (b) and removing the used medium while simultaneously maintaining the cells in culture. The method further includes the step of harvesting the heterologous protein from the cell culture.

[0130] The production phase typically begins before the target cell density is reached. The target cell density depends on the cell line and its maximum viable cell density, and is typically about 15-45% of the maximum viable cell density. The production phase can start from 10x10... 6 Cells / ml to approximately 120x10 6 Start with a cell density of 10 cells / ml or even higher. Preferably, the production phase begins at a cell density of at least 10 x 10⁻⁶ cells / ml. 6 cells / ml, at least 20 x 10 6 Cells / ml, at least 30 x 10 6 10 cells / ml, at least 40 x 10 6 10 cells / ml or at least 50 x 10 6 Cell density of cells / ml. Typically, this is achieved when cultured in osmotic medium to reach 0.2 ± 0.1 g / L. 生物反应器 The production phase begins when the amount of heterologous protein reaches a certain level (e.g., 1 day or more), at which point the purification of the heterologous protein begins.

[0131] According to the method of the invention, in step (a), culturing mammalian cells may be limited to inoculating mammalian cells expressing heterologous proteins in a serum-free medium, and therefore does not require but may include a culture step prior to the start of perfusion, and further does not require but may include the start of perfusion culture. Growth medium, also referred to as production medium, is typically used in step (a), which is replaced by the medium according to the invention or obtained in step (b) according to the method of the invention. Further according to the method of the invention, maintaining mammalian cells by perfusion during the production phase includes culturing mammalian cells during the production phase by perfusion at a substantially constant viable cell density of approximately the target viable cell density, wherein a substantially constant viable cell density means a variation within 30%, preferably 20%, more preferably 10% of the viable cell density.

[0132] The present invention also relates to a method for producing heterologous proteins, the method comprising culturing mammalian cells expressing heterologous proteins in perfusion culture according to the present invention. Those skilled in the art will understand that the method according to the present invention is an in vitro culture method.

[0133] In one embodiment, the serum-free cell culture perfusion medium may be chemically defined and / or free of hydrolysates. Preferably, the serum-free cell culture perfusion medium is protein-free or protein-free except for recombinant insulin and / or insulin-like growth factor. Therefore, the serum-free cell culture perfusion medium may be a protein-free medium or a protein-free medium containing recombinant insulin and / or recombinant insulin-like growth factor. More preferably, the serum-free perfusion medium is chemically defined and protein-free or protein-free except for recombinant insulin and / or insulin-like growth factor. This also applies to the serum-free medium used in step (a) of the method according to the invention.

[0134] Before starting perfusion culture, mammalian cells can initially be cultured in batches. Typically, perfusion culture begins from day 0 to day 5 of the batch culture, preferably from day 0 to day 4, and more preferably from day 0 to day 3. After perfusion begins, the perfusion rate is increased until the target viable cell density is reached. The perfusion rate can be increased, for example, from less than or equal to 0.5 container volumes per day to about 5 container volumes per day, preferably from less than or equal to 0.5 container volumes per day to about 2 container volumes per day.

[0135] As explained above, the method of the present invention may further include a step of maintaining cell density through steady-state cell shedding. The cell density mentioned in this context refers to viable cell density, which can be determined by any method known in the art. For example, the calculation of controlling the cell shedding rate can be based on maintaining the INCYTE™ viable cell density probe (HAMILTON® COMPANY) or FUTURA™ biomass capacitance probe value (ABER) corresponding to the target VCD. ® Instruments), or daily cell and viability counts can be performed offline via any cell counting device, such as a hemocytometer, VI-CELL XR™ (BECKMAN COULTER®), CEDEX HI-RES™ (ROCHE®), or VIACOUNT™ assay (EMDMILLIPORE® GUAVA EASYCYTE®). Compared to control perfusion cell culture, the method of the present invention can eliminate or reduce cell extrusion by increasing osmotic pressure, wherein the control perfusion cell culture is a perfusion cell culture under the same conditions using the same serum-free perfusion medium without increasing the osmotic pressure of the cell culture according to the present invention. More specifically, cell extrusion can be reduced compared to control perfusion cell culture, wherein the control perfusion cell culture is a perfusion cell culture under the same conditions using the same serum-free perfusion medium without increasing the osmotic pressure. Perfusion cell cultures without cell extrusion can also be referred to as “dynamic perfusion culture” or “dynamic perfusion process”. Preferably, dynamic perfusion culture also comprises a high viable cell density, for example, above 80 x 10⁻⁶ cells / cm². 6 Cells / ml, higher than 100x10 6 Cells / ml, higher than 120x10 6 140 x 10 cells / ml or even higher 6 Cells / ml and / or less than 30 days, preferably a relatively short culture time of 14-16 days.

[0136] In one embodiment, the osmotic pressure of the serum-free cell culture perfusion medium can be increased above the optimal growth osmotic pressure level, resulting in inhibition of mammalian cell growth at a target viable cell density. Preferably, the osmotic pressure level of the serum-free cell culture perfusion medium is gradually or stepwise increased starting from about half of the target viable cell density. The target viable cell density can be about 30 x 10⁻⁶ cells / day. 6 10 cells / ml or higher, approximately 60 x 10 6 10 cells / ml or higher, approximately 80 x 10 6 cells / ml, preferably about 100 x 10⁻⁶ 6 Cells / ml or higher. Target viable cell density can even reach approximately 100 x 10⁻⁶ cells / ml. 6Cells / ml to 200x10 6 cells / ml, preferably about 120 x 10⁻⁶ 6 Cells / ml to 150x10 6 Cells / ml. For intrinsic maximum viable cell density greater than 150 x 10⁻⁶ cells / ml. 6 Cell lines with a density of 100 cells / ml typically require inhibition of cell growth to ensure adequate oxygen supply, avoidance of excessive cell condensation (which can clog cell retention devices), and minimization of the impact of waste metabolite accumulation, although 200 x 10⁻⁶ cells / ml has been achieved. 6 The target live cell density is cells / ml.

[0137] The osmotic pressure of cell cultures can be controlled using a constant concentrate feed perfusion rate and different diluent perfusion rates (which result in different total perfusion rates). A constant concentrate feed perfusion rate involves the cumulative or total perfusion rate of at least three separate aqueous concentrates, more specifically the alkaline concentrate, the acidic concentrate, and the near-neutral concentrate. The concentrates can be fed, for example, at a constant total perfusion rate of 0.5 VVD (e.g., 0.33 VVD of 6x acidic feed, and 0.08 VVD each of 25x alkaline and near-neutral feeds). The total perfusion rate is the cumulative perfusion rate of the at least three separate aqueous concentrates and the diluent. Alternatively, a constant total perfusion rate and different concentrate feed perfusion rates can be used to control the osmotic pressure of cell cultures. This naturally results in different diluent perfusion rates. In another alternative, the osmotic pressure of the cell culture can be controlled using a constant diluent perfusion rate and different concentrate perfusion rates (which result in different total perfusion rates). The at least three concentrates are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the culture medium components in a 1x serum-free cell culture perfusion medium. In other words, the ratio (v / v / v) of the alkaline concentrate to the acidic concentrate to the near-neutral concentrate is a fixed ratio (for each medium) to provide the serum-free cell culture perfusion medium with pH adjusted to neutral in the reaction vessel of the cell culture and / or bioreactor. Preferably, the osmotic pressure of the cell culture is controlled using a constant concentrate perfusion rate and different diluent perfusion rates (which result in different total perfusion rates).

[0138] The osmotic pressure of the cell culture (and the fold concentration of the serum-free cell culture perfusion medium) can be increased using: a constant concentrate feed perfusion rate and a reduced diluent perfusion rate, resulting in a reduced total perfusion rate; or a constant total perfusion rate and an increased concentrate feed perfusion rate and a reduced diluent perfusion rate; or a constant diluent perfusion rate and an increased concentrate feed perfusion rate, resulting in an increased total perfusion rate; wherein the at least three concentrates are added to each other at a fixed ratio (v / v / v) according to their fold concentrations to maintain the relative proportions of the medium components in 1x serum-free cell culture perfusion medium. Preferably, no further additives (such as NaCl) for increasing osmotic pressure are added to the culture. Preferably, a constant concentrate feed perfusion rate and a reduced diluent perfusion rate (which results in a reduced total perfusion rate) are used to increase the osmotic pressure of the cell culture. In a preferred embodiment, no further additives for increasing osmotic pressure are added to the culture.

[0139] The osmotic pressure and fold concentration of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor are determined by increasing the cumulative volume ratio (v / v) of the diluent to the at least three separate aqueous concentrated feeds added to the reaction vessel of the cell culture and / or bioreactor to provide the serum-free cell culture perfusion medium with pH adjusted to near neutral. The fold concentration of the serum-free cell medium can be any value from 0.1x to a maximum fold concentration, which can be calculated as explained above. Using concentrated feeds allows adjustment of the fold concentration of the serum-free cell medium in the cell culture and / or bioreactor, and therefore, in addition to regulating growth inhibition by increasing osmotic pressure, it also allows for an increase in the nutrient content in the medium by increasing the fold concentration of the serum-free cell medium. This allows for maintaining a high viable cell density with similar or only moderately increased perfusion rates and therefore with reduced specific perfusion rates. The term “multiple concentration” refers to a concentrate (n>1) or dilution (n>1) of 1x serum-free cell culture perfusion medium, wherein the 1x serum-free cell culture perfusion medium is an original prepared or designed serum-free cell culture perfusion medium formulation.

[0140] The ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrate feeds also determines the fold concentration (total nutrient content) of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor, which is added to the reaction vessel of the cell culture and / or bioreactor to provide a resulting serum-free cell culture perfusion medium with pH adjusted to near neutral. Therefore, the advantage of using concentrate feeds is that the fold concentration of the medium can be adapted to live cell concentrations and nutrient requirements (maintaining nutrient balance). Osmolarity can be used as a proxy for estimating nutrient balance within and outside the system. Therefore, osmolarity balance can be used to calculate adjustments to the cumulative volume of the concentrate feeds (at a fixed ratio relative to each other) and the diluent feed rate to achieve desired residual osmolarity and nutrient levels.

[0141] Any feeding strategy must take into account the osmotic pressure added by any other feed (such as glucose or an alkaline titrant). The choice of osmotic control scheme depends on the cell line and the sensitivity of each cell line to osmotic pressure and waste accumulation. The lowest possible perfusion rate is preferred. The feed rate can be determined based on the known concentrated feed osmotic pressure and the assumed cell specific osmotic pressure consumption rate, calculated on a daily basis. The osmotic balance of daily osmotic pressure consumption can be calculated according to the following equation: Osmotic Input - Osmotic Output = Osmotic Pressure Consumption, where osmotic input is the osmotic pressure of the concentrated feed and diluent of the culture medium perfused into the bioreactor, osmotic output is the residual osmotic pressure of the bioreactor supernatant, and osmotic pressure consumption is the osmotic pressure difference between input and output. This daily osmotic pressure consumption is then normalized to the number of cells in the culture, i.e., daily osmotic pressure consumption per cell. This daily consumption rate per cell (or cell specific osmotic pressure consumption rate, CSOCR) is then multiplied by the predicted VCD for the next day to predict the osmotic pressure consumption for the next day. This consumption rate, along with the desired osmotic pressure output, can be used to calculate the required osmotic pressure input for the next day. Then adjust the infusion rate of the diluent and / or the concentrate feed to match the osmotic pressure input target.

[0142] The optimal osmotic pressure level for cell culture growth depends on the cell line and can range from about 280 mOsm to about 390 mOsm, more preferably from 280 to less than about 350 mOsm (mOsmol / kg water). Some cell lines can still grow optimally at osmotic pressures above 390 mOsm. The optimal osmotic pressure level for mammalian cells in cell cultures depends on the mammalian cells used and may also depend on the culture conditions. The optimal osmotic pressure level for mammalian cells can be readily determined by determining the viable cell density and viability at different osmotic pressures. The optimal osmotic pressure level does not depend on cell density, but is preferably determined at approximately the target viable cell density. The osmotic pressure should be maintained at the level most suitable for growth, at least until about half of the target viable cell density is reached.

[0143] Once the target viable cell density is reached, the osmotic pressure can be increased to inhibit cell growth, such as by increasing the osmotic pressure to about 10-70%, about 10-60%, or about 10-50% of the optimal growth osmotic pressure level for mammalian cells. The osmotic pressure should be increased gradually or incrementally, preferably starting from about half of the target viable cell density (i.e., approximately one population, doubled from the target viable cell density), more preferably increasing to about 10-70%, about 10-60%, or about 10-50% of the optimal growth osmotic pressure level. In one embodiment, the osmotic pressure is increased to about 350 mOsm or higher, preferably to about 380 mOsm or higher, about 400 mOsm or higher, about 420 mOsm or higher, or about 450 mOsm or higher. The osmotic pressure is increased to a level that inhibits mammalian cell growth without causing cytotoxicity to the mammalian cells. The osmotic pressure can be increased to and maintained at an osmotic pressure level that inhibits cell growth of mammalian cells, preferably at a target viable cell density, wherein the osmotic pressure level inhibiting cell growth of mammalian cells is about 350 mOsm or higher, or about 380 mOsm or higher. However, it is important that the cell viability of the mammalian cells is substantially unaffected. For most cell lines, osmotic pressure levels above about 400 mOsm begin to become cytotoxic, but for individual cell lines, the osmotic pressure level can be increased to 450 mOsm without affecting cytotoxicity. Increasing the osmotic pressure to physiological stress levels inhibits cell growth. The osmotic pressure that inhibits cell growth of mammalian cells in a cell culture depends on the mammalian cells used. By measuring viable cell density and viability at different osmotic pressures, the osmotic pressure that inhibits cell growth of specific mammalian cells in a cell culture without reaching a cytotoxic level can be easily determined. Preferably, the increased osmotic pressure results in maintaining cells at approximately the target viable cell density during the production phase without affecting viability. Therefore, increasing the osmotic pressure reduces or eliminates the need for cell removal during the production phase. By increasing the osmotic pressure of cell cultures, cell growth can be inhibited to maintain a sustainable viable cell density without cell leakage, especially at high viable cell densities, such as <100 x 10⁻⁶ cells / mL. 6 Cells / ml, preferably <120x10 6 Cells / ml, also known as dynamic perfusion culture.

[0144] By increasing the osmotic pressure of the cell culture, the yield of the heterologous protein produced in the cell culture can be increased by at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, or about 5-50%, preferably about 10-100%, relative to the yield of the control cell culture, without increasing the osmotic pressure. Preferably, the yield is determined for a portion or the entire culture period.

[0145] By using the serum-free cell culture medium according to the invention or the serum-free cell culture medium obtained by the method according to the invention and optionally further increasing the osmotic pressure of the cell culture, the cell specific perfusion rate (pl / cell / day) is reduced by at least about 25%, at least about 30%, or at least about 50% relative to the cell specific perfusion rate of 1x serum-free cell culture medium.

[0146] In one embodiment of the method of the present invention, the reaction vessel for the cell culture and / or bioreactor contains at least about 100 L of serum-free cell culture perfusion medium, preferably at least about 1000 L of serum-free cell culture perfusion medium. Preferably, the volume of the cell culture is at least about 100 L and / or the volume of the bioreactor is at least about 100 L. More preferably, the volume of the cell culture is at least about 1000 L and / or the volume of the bioreactor is at least about 1000 L. Although the serum-free cell culture perfusion medium used in or prepared by the method of the present invention is a completely serum-free cell culture perfusion medium, the culture can be further supplemented. Suitable supplements that can be added alone to the cell culture are, but are not limited to, antifoaming agents, alkalis, glucose, and / or glutamine.

[0147] The heterologous protein can be any protein, preferably a therapeutic protein, such as an antibody or a therapeutically effective fragment thereof, a fusion protein or a cytokine, or any heterologous protein described herein. The antibody can be a monoclonal antibody, a bispecific antibody, a multimeric antibody, or a fragment thereof.

[0148] bioreactor Serum-free cell culture perfusion media can be used for any type, type, or format of cell culture system suitable for continuous perfusion.

[0149] Any cell perfusion bioreactor and cell retention device can be used for perfusion culture. Bioreactors used for perfusion are not significantly different from those used for batch / feed-to-batch culture, except that bioreactors used for perfusion are more compact and connected to a cell retention device. The method for retaining cells inside the bioreactor is primarily determined by whether the cells are grown attached to a surface or in a single-cell suspension or cell aggregates. While historically most mammalian cells have grown attached to surfaces or substrates (heterogeneous culture), efforts have been made to adapt many industrial mammalian cell lines to suspension growth (homogeneous culture), mainly because suspension culture is easier to scale up. Therefore, cells used in the methods of the present invention are preferably grown in suspension. Exemplary retention systems for cells grown in suspension include rotary filters, external filtration such as tangential flow filtration (TFF), alternating tangential flow (ATF) systems, cell sedimentation (vertical sedimentation and inclined sedimentation), centrifugation, ultrasonic separation, and hydrocyclones. Perfusion systems can be divided into two categories: filtration-based systems, such as rotary filters, external filters, and ATF; and open perfusion systems, such as gravity settlers, centrifuges, ultrasonic separators, and hydrocyclones. Filtration-based systems exhibit high cell retention, which does not change with flow rate. However, filters can become clogged, thus limiting the length of culture runs or requiring filter replacement. An example of an ATF system is the XCELL™ ATF system from REPLIGEN™, and an example of a TFF system is the TFF system from LEVITRONIX® using a centrifugal pump. Cross-flow filters (such as hollow fiber (HF) or flat plate filters) can be used with both ATF and TFF systems. In particular, hollow fibers made of modified polyethersulfone (mPES), polyethersulfone (PES), or polysulfone (PE) can be used with both ATF and TFF systems. HF pore sizes range from several hundred kDa to 15 μM. Open perfusion systems do not clog and therefore can operate indefinitely, at least theoretically. However, at higher perfusion rates, cell retention decreases. Currently, three systems exist that can be used on an industrial scale: alternating tangential filters (ATF), gravity (especially tilting settlers), and centrifuges. Cell retention devices suitable for heterologous or homologous cultures are described in more detail by Kompala and Ozturk (Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, (2006), Taylor & Francis Group, LLC, pages 387-416), which is incorporated herein by reference. Perfusion culture is not a truly steady-state process; steady-state total cell concentration and viable cell concentration are only achieved when the cell outflow is removed from the bioreactor.

[0150] Physical parameters (such as pH, dissolved oxygen, and temperature) in a perfusion bioreactor should be monitored online and controlled in real time. Cell density, viability, metabolite, and product concentrations can be determined using offline or online sampling. When a perfusion operation begins with continuous harvesting and feeding, the perfusion rate typically refers to the harvest flow rate, which can be manually set to a desired value. For example, weight control for a bioreactor can activate the feed pump to maintain a constant volume within the bioreactor. Alternatively, level control can be achieved by pumping the culture volume above a predetermined level. The perfusion rate in the bioreactor must be adjusted to deliver sufficient nutrients to the cells.

[0151] Perfusion rates can be controlled, for example, using cell density measurements, pH measurements, oxygen consumption, or metabolite measurements. Cell density is the most important measurement for adjusting the perfusion rate. Depending on how the cell density measurement is performed, the perfusion rate can be adjusted daily or in real-time. Several online probes have been developed for estimating cell density, and these online probes are known to those skilled in the art, such as capacitance probes, such as the INCYTE™ live cell density probe (HAMILTON). ® (COMANY) or FUTURA™ Biocapacitance Probe Value (ABER) ® These cell density probes can also be used to control cell density at a desired set point by removing excess cells from the bioreactor, i.e., cell shedding. Therefore, cell shedding is determined by the specific growth rate of mammalian cells in the culture. Cell shedding is typically not harvested and is therefore considered waste.

[0152] The method of the present invention further comprises harvesting the heterologous protein from the perfused cell culture. The present invention considers any suitable method for harvesting and purifying the protein of interest. Harvesting may also occur intermittently throughout the cell culture lifecycle or at the end of the cell culture. Preferably, harvesting is performed continuously from the permeate, which is the supernatant produced after the cells are recovered via a cell retention device. Because the product retention time of the product protein in the cell culture within the perfused bioreactor is shorter compared to fed-batch processing, contact with proteases, sialidases, and other degrading proteins is minimized, which may result in a better quality product of the heterologous protein produced in the perfused culture. Preferably, methods such as U.S. Provisional Application 62827504, particularly its… Figure 6 The iSKID described herein is used to purify the harvested product. The iSKID is an integrated skid that combines multiple unit operations in a highly automated manner and enables fully continuous automated manufacturing.

[0153] Expression products The heterologous protein produced by the method and use of this invention can be any secreted protein, preferably a therapeutic protein. Since most therapeutic proteins are recombinant therapeutic proteins, it is most preferably a recombinant therapeutic protein. Examples of therapeutic proteins are, but are not limited to, antibodies, fusion proteins, cytokines, and growth factors.

[0154] Therapeutic proteins produced in mammalian cells by the method according to the invention include, but are not limited to, antibodies or fusion proteins, such as Fc fusion proteins. Other secreted recombinant therapeutic proteins may be, for example, enzymes, cytokines, lymphokines, adhesion molecules, receptors and their derivatives or fragments, as well as any other peptides and scaffolds that can be used as agonists or antagonists and / or have therapeutic or diagnostic uses.

[0155] Other recombinant proteins of interest include, but are not limited to: insulin, insulin-like growth factor, hGH, tPA, cytokines such as interleukins (ILs), such as interleukin IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) α, IFNβ, IFNγ, IFNω, or IFNτ, tumor necrosis factor (TNF), such as TNFα and TNFβ, TNFγ, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1, and VEGF. Also included is the production of erythropoietin or any other hormonal growth factor and any other polypeptides that can be used as agonists or antagonists and / or have therapeutic or diagnostic uses.

[0156] The preferred therapeutic protein is an antibody or a fragment or derivative thereof, more preferably an IgG1 antibody. Therefore, the present invention can be advantageously used to generate antibodies, such as monoclonal antibodies, multispecific antibodies or fragments thereof, preferably monoclonal antibodies, bispecific antibodies or fragments thereof. Exemplary antibodies within the scope of this invention include, but are not limited to, anti-CD2, anti-CD3, anti-CD20, anti-CD22, anti-CD30, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD44v6, anti-CD49d, anti-CD52, anti-EGFR1 (HER1), anti-EGFR2 (HER2), anti-GD3, anti-IGF, anti-VEGF, anti-TNFα, anti-IL2, anti-IL-5R, or anti-IgE antibodies, and are preferably selected from the group consisting of: anti-CD20, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2 / neu (erbB2), anti-EGFR, anti-IGF, anti-VEGF, anti-TNFα, anti-IL2, and anti-IgE antibodies.

[0157] Antibody fragments include, for example, "Fab fragments" (fragment antigen binding = Fab). Fab fragments consist of variable regions of two strands held together by adjacent constant regions. These can be formed from conventional antibodies by proteolytic digestion, for example, with papain, but similarly, Fab fragments can also be produced through genetic engineering. Further antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

[0158] Using genetic engineering methods, it is possible to produce shortened antibody fragments consisting only of variable regions of the heavy chain (VH) and light chain (VL). These are called Fv fragments (fragment variable = fragment with variable portion). Because these Fv fragments lack the covalent bonding of the two chains through cysteine ​​residues of the constant chain, they are often stable. It is advantageous to link the variable regions of the heavy and light chains using short peptide fragments, for example, having 10 to 30 amino acids, preferably 15. In this way, a single peptide chain consisting of VH and VL linked by a peptide linker is obtained. This antibody protein is called a single-chain Fv (scFv). Examples of scFv antibody proteins are known to those skilled in the art.

[0159] The preferred therapeutic antibody according to the invention is a bispecific antibody. Bispecific antibodies typically combine the antigen-binding specificity of target cells (e.g., malignant B cells) and effector cells (e.g., T cells, NK cells, or macrophages) in a single molecule. Exemplary bispecific antibodies are, but are not limited to, biantibodies, BiTE (bispecific T cell adaptor) formats, and DART (dual affinity retargeting) formats. The biantibody format separates homologous variable domains of a heavy chain and a light chain with two different antigen-binding specificities on two separate polypeptide chains, wherein the two polypeptide chains are non-covalently linked. The DART format is based on the biantibody format but provides additional stabilizing effects through a C-terminal disulfide bridge.

[0160] Another preferred therapeutic protein is a fusion protein, such as an Fc fusion protein. Therefore, the present invention can be advantageously used to produce fusion proteins, such as Fc fusion proteins. Furthermore, the method for increasing protein production according to the present invention can be advantageously used to produce fusion proteins, such as Fc fusion proteins.

[0161] The effector portion of a fusion protein can be the complete sequence or any portion of a sequence of a native or modified heterologous protein, or a combination of the complete sequence or any portion of a sequence of a native or modified heterologous protein. Immunoglobulin constant domain sequences can be obtained from any immunoglobulin subtype, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, or classes such as IgA, IgE, IgD, or IgM. Preferably, they are derived from human immunoglobulins, more preferably from human IgG, and even more preferably from human IgG1 and IgG2. Non-limiting examples of Fc fusion proteins are MCP1-Fc, ICAM-Fc, EPO-Fc, and scFv fragments, which are coupled to the CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site. Fc fusion proteins can be constructed using a genetic engineering approach that involves introducing a CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site into another expression construct, which includes, for example, other immunoglobulin domains, enzymatically active protein portions, or effector domains. Therefore, the Fc fusion protein according to the invention further comprises a single-chain Fv fragment linked to a CH2 domain of a heavy chain immunoglobulin constant region containing, for example, an NN-linked glycosylation site.

[0162] Recovery and formulation of expression products In a further aspect, the method of the present invention provides a method for generating therapeutic proteins, and the method for generating therapeutic proteins optionally further includes the steps of purifying the therapeutic protein and formulating it into a pharmaceutically acceptable formulation.

[0163] Therapeutic proteins, particularly antibodies, antibody fragments, or Fc fusion proteins, are preferably recovered / isolated from the culture medium as secreted peptides. It is necessary to purify the therapeutic proteins from other recombinant proteins and host cell proteins to obtain substantially homogeneous formulations of the therapeutic proteins. As a first step, cellular and / or microparticle cell debris is removed from the culture medium. Further, for example, the therapeutic proteins are purified from contaminant-soluble proteins, peptides, and nucleic acids by fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, Sephadex chromatography, and chromatography on silica or cation exchange resins (such as DEAE). Methods for purifying heterologous proteins expressed by mammalian cells are known in the art.

[0164] expression carrier In one embodiment, the heterologous protein expressed using the method of the present invention is encoded by one or more expression cassettes containing heterologous polynucleotides encoding the heterologous protein. The heterologous protein may be placed under the control of an amplifiable genetic selection marker (such as dihydrofolate reductase (DHFR) or glutamine synthase (GS)). The amplifiable selection marker gene may be on the same expression vector as the heterologous protein expression cassette. Alternatively, the amplifiable selection marker gene and the heterologous protein expression cassette may be on different expression vectors but integrated very closely into the genome of the host cell. For example, two or more vectors co-transfected simultaneously often integrate very closely into the genome of the host cell. The amplification of the genetic region containing the secretory therapeutic protein expression cassette is then mediated by adding amplification reagents (e.g., MTX for DHFR or MSX for GS) to the culture medium.

[0165] Sufficiently high and stable levels of heterologous proteins expressed by mammalian cells can also be achieved, for example, by cloning multiple copies of the heterologous protein-encoding polynucleotide into an expression vector. The cloning of multiple copies of the heterologous protein-encoding polynucleotide into an expression vector and the amplification of the heterologous protein expression cassette, as described above, can be further combined.

[0166] mammalian cell lines As used herein, mammalian cells are mammalian cell lines suitable for producing secretory recombinant therapeutic proteins, and therefore may also be referred to as “host cells.” Preferred mammalian cells according to the invention are rodent cells, such as hamster cells. Mammalian cells are isolated cells or cell lines. Mammalian cells are preferably transformed and / or immortalized cell lines. They are suitable for continuous passage in cell culture and do not include primary untransformed cells or cells that are part of an organ structure. Preferred mammalian cells are BHK21, BHK TK-, Jurkat cells, 293 cells, HeLa cells, CV-1 cells, 3T3 cells, CHO, CHO-K1, CHO-DXB11 (also known as CHO-DUKX or DuxB11), CHO-S cells, and CHO-DG44 cells, or derivatives / progeny of any of these cell lines. Particularly preferred are CHO cells, such as CHO-DG44, CHO-K1, and BHK21, and even more preferred are CHO-DG44 and CHO-K1 cells. The most preferred type is CHO-DG44 cells. Also included are mammalian cells, particularly glutamine synthase (GS) deficient derivatives of CHO-DG44 and CHO-K1 cells. In one embodiment of the invention, the mammalian cells are Chinese hamster ovary (CHO) cells, preferably CHO-DG44 cells, CHO-K1 cells, CHO DXB11 cells, CHO-S cells, CHOGS deficient cells, or derivatives thereof.

[0167] Mammalian cells may further comprise one or more expression cassettes encoding heterologous proteins, such as therapeutic proteins, preferably recombinant secreted therapeutic proteins. The host cell may also be a murine cell, such as murine myeloma cells, such as NSO and Sp2 / O cells, or derivatives / progeny of any such cell line. Non-limiting examples of mammalian cells that can be used in the sense of this invention are also summarized in Table 1. However, derivatives / progeny of these cells, and other mammalian cells (including but not limited to human, mouse, rat, monkey, and rodent cell lines), may also be used in this invention, particularly for the production of biopharmaceutical proteins.

[0168] Table 1: Mammalian Production Cell Lines

[0169] 1 CAP (CEVEC's Amniocyte Production) cells are immortalized cell lines based on primary human amniotic fluid cells. They are generated by transfecting these primary cells with a vector containing functional E1 and pIX of adenovirus 5. Due to real human post-translational modifications, CAP cells allow for the competitive and stable production of recombinant proteins with excellent biological activity and therapeutic efficacy.

[0170] Mammalian cells are preferred when established, adapted, and fully cultured under serum-free conditions, and optionally in a medium free of any animal-derived proteins / peptides. Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's modified Eagle's medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's modified Dulbecco's medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-Invitrogen, serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are exemplary suitable nutrient solutions. Any culture medium can be supplemented with a variety of compounds as needed. Non-limiting examples of these compounds include recombinant hormones and / or other recombinant growth factors (such as insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics, and trace elements. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. For the growth and selection of genetically modified cells expressing selectable genes, suitable selection reagents are added to the culture medium.

[0171] In view of the foregoing, it will be understood that the present invention also encompasses the following provisions: Clause 1 provides a separated serum-free cell culture perfusion medium comprising culture medium components consisting of at least three separate aqueous concentrate feeds and a diluent, wherein the first concentrate feed is an alkaline concentrate feed, the second concentrate feed is an acidic concentrate feed, and the third concentrate feed is a near-neutral concentrate feed; wherein After mixing the at least three separate aqueous concentrate feeds and the diluent in the resulting serum-free cell culture perfusion medium, the pH of the separated serum-free cell culture perfusion medium is adjusted to neutral pH.

[0172] Clause 2 specifically describes a separated serum-free cell culture perfusion medium as described in Clause 1, wherein the pH of the resulting serum-free cell culture perfusion medium, after mixing the at least three separate aqueous concentrate feeds and the diluent, is between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2.

[0173] Clause 3 specifically describes a segregated serum-free cell culture perfusion medium as described in Clause 1 or 2, wherein the diluent is sterile water.

[0174] Clause 4 specifically describes the segregated serum-free cell culture perfusion medium as described in any one of Clauses 1 to 3, wherein the segregated serum-free cell culture perfusion medium is used for (a) The alkaline concentrate feed, the acid concentrate feed and the near-neutral concentrate feed are added separately to the reaction vessel of the cell culture and / or bioreactor; (b) The alkaline concentrate, the acidic concentrate, and the near-neutral concentrate are added directly to the reaction vessel of the cell culture and / or bioreactor without prior premixing; and / or (c) The at least three separate aqueous concentrate feeds are directly mixed in the reaction vessel of the cell culture and / or bioreactor.

[0175] Clause 5 specifically describes the separated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the alkaline concentrated feed is a 2x to 80x concentrated feed, the acidic concentrated feed is a 2x to 40x concentrated feed, and the near-neutral concentrated feed is a 2x to 50x concentrated feed.

[0176] Clause 6 specifically describes the segregated serum-free cell culture perfusion medium as described in Clause 5, wherein (a) The alkaline concentrate feed is a 20x to 40x concentrate feed, the acidic concentrate feed is a 4x to 20x concentrate feed, and the near-neutral concentrate feed is a 10x to 40x concentrate feed; (b) The alkaline concentrate feed is a 20x to 30x concentrate feed, the acidic concentrate feed is a 5x to 12x concentrate feed, and the near-neutral concentrate feed is a 20x to 30x concentrate feed; and / or (c) The alkaline concentrate feed is a 25x concentrate feed, the acidic concentrate feed is a 6x to 10x concentrate feed, and the near-neutral concentrate feed is a 25x concentrate feed.

[0177] Clause 7 specifically describes a segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the near-neutral concentrated feed has a pH of 6.5-8.5.

[0178] Clause 8 specifically describes a segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the alkaline concentrate feed has a pH of 9 or higher, the acidic concentrate feed has a pH of 5 or lower, and the near-neutral concentrate feed has a pH of 7 to 8.5.

[0179] Clause 9 specifically describes the segregated serum-free cell culture perfusion medium as described in Clause 8, wherein (a) The pH of the alkaline concentrate feed is 9 to 11, the pH of the acidic concentrate feed is 2 to 5, and the pH of the near-neutral concentrate feed is 7 to 8.5. (b) The pH of the alkaline concentrate feed is 9.8 to 10.8, the pH of the acidic concentrate feed is 3.6 to 4.8, and the pH of the near-neutral concentrate feed is 7 to 8.5; or (c) The pH of the alkaline concentrate feed is 9.8 to 10.5, the pH of the acidic concentrate feed is 3.8 to 4.5, and the pH of the near-neutral concentrate feed is 7.5 to 8.5.

[0180] Clause 10 specifically describes the separated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the resulting serum-free cell culture perfusion medium is (a) a chemically defined medium, (b) a hydrolysate-free medium, and / or (c) a protein-free medium or a protein-free medium containing recombinant insulin and / or recombinant insulin-like growth factor.

[0181] Clause 11 specifically describes the separated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the resulting separated serum-free cell culture perfusion medium is a production medium.

[0182] Clause 12 specifically describes the segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein (a) The ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed to the near-neutral concentrated feed is a fixed ratio to provide a serum-free cell culture perfusion medium with pH adjusted to neutral; and (b) The osmotic pressure of the serum-free cell culture perfusion medium is determined by the ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrate feeds in the resulting serum-free cell culture perfusion medium with pH adjusted to neutral pH.

[0183] Clause 13 specifically describes a segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the acidic concentrated feed contains trace elements, trace metals, inorganic salts, chelating agents, polyamines, and regulatory hormones.

[0184] Clause 14 specifically describes a segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the acidic concentrate feed and / or the near-neutral concentrate feed comprises a surfactant, an antioxidant, and a carbon source.

[0185] Clause 15 specifically describes a segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the alkaline concentrated feed contains amino acids that have maximum solubility at an alkaline pH of 9 or higher, preferably including at least aspartic acid, histidine, and tyrosine, and optionally including cysteine ​​and / or cystine and / or folic acid.

[0186] Clause 16 specifically describes a segregated serum-free cell culture perfusion medium as described in Clause 15, wherein the remaining amino acids are in the acidic and / or near-neutral concentrated feed, preferably in the acidic concentrated feed.

[0187] Clause 17 specifically describes a segregated serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein vitamins and metals are in separate feeds, preferably vitamins in the near-neutral feed and metals in the acidic feed.

[0188] Clause 18 specifically describes the separated serum-free cell culture perfusion medium as described in Clause 17, wherein vitamins that are poorly soluble in aqueous solutions, such as choline chloride, are present in the neutral feed and the acid feed.

[0189] Clause 19 specifically describes an alkaline aqueous concentrate feed for use in combination with an acidic aqueous concentrate feed, a near-neutral aqueous concentrate feed and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

[0190] Clause 20 specifically describes an acidic aqueous concentrate feed for use in combination with an alkaline aqueous concentrate feed, a near-neutral aqueous concentrate feed and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

[0191] Clause 21 specifically describes a near-neutral aqueous concentrate feed, which is used in combination with an alkaline aqueous concentrate feed, an acidic aqueous concentrate feed and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

[0192] Clause 22 specifically describes a method for preparing serum-free cell culture perfusion medium, the method comprising: (a) Provide cell culture medium components in at least three component subgroups based on their solubility at alkaline, acidic, and neutral pH conditions. (b) (i) Dissolve the subcomponents soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed; (ii) Dissolving a subcomponent soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; and (iii) Dissolve the subcomponents soluble at neutral pH in a neutral aqueous solution to form a near-neutral concentrated feed; (c) Optionally, the prepared alkaline concentrate feed, acidic concentrate feed, and near-neutral concentrate feed may be stored in separate containers; and (d) The prepared alkaline concentrate, acidic concentrate, and near-neutral concentrate, along with the diluent, are added to the reaction vessel of the cell culture and / or bioreactor, wherein... (i) The alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed are added separately to the reaction vessel of the cell culture and / or bioreactor; and (ii) Add the diluent separately to the reaction vessel of the cell culture and / or bioreactor, or premix the diluent with one of the at least three separate aqueous concentrate feeds before adding it to the reaction vessel of the cell culture and / or bioreactor; In this process, after mixing the at least three separate aqueous concentrate feeds with the diluent, the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

[0193] Clause 23 specifically describes the method as described in Clause 22, wherein after mixing the at least three separate aqueous concentrate feeds and the diluent, the pH of the pH-adjusted serum-free cell culture perfusion medium is between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2.

[0194] Clause 24 provides for methods as described in Clauses 22 or 23, wherein the diluent is sterile water.

[0195] Clause 25 specifically describes a method as described in any one of Clauses 22 to 24, wherein the at least three concentrated feeds are added dropwise through separate ports to the reaction vessel of the cell culture and / or bioreactor.

[0196] Clause 26 specifically describes the method as described in any one of Clauses 22 to 25, wherein in-vessel mixing and dilution of the at least three separate aqueous concentrate feeds allows for a 50-90%, preferably 60-90%, reduction in the consumption of the prepared culture medium over a 14-day culture period compared to mixing and diluting serum-free cell culture perfusion medium before it is added to the bioreactor.

[0197] Clause 27 specifically describes a method as described in any one of Clauses 22 to 26, wherein the reaction vessel of the cell culture and / or bioreactor contains mammalian cells.

[0198] Clause 28 specifically describes a method as described in any one of Clauses 22 to 27, the method further comprising the step of sterilizing the concentrated feed prior to storage and / or addition to the reaction vessel of the cell culture and / or bioreactor.

[0199] Clause 29 specifically describes a method as described in any one of Clauses 22 to 28, wherein the alkaline concentrate feed is a 2x to 80x concentrate feed, the acid concentrate feed is a 2x to 40x concentrate feed, and the near-neutral concentrate feed is a 2x to 50x concentrate feed.

[0200] Clause 30 provides a specific description of the methods described in Clause 29, wherein (a) The alkaline concentrate feed is a 20x to 40x concentrate feed, the acidic concentrate feed is a 4x to 20x concentrate feed, and the near-neutral concentrate feed is a 10x to 40x concentrate feed; (b) The alkaline concentrate feed is a 20x to 30x concentrate feed, the acidic concentrate feed is a 5x to 12x concentrate feed, and the near-neutral concentrate feed is a 20x to 30x concentrate feed; and / or (c) The alkaline feed is a 25x concentrate feed, the acidic concentrate feed is a 6x to 10x concentrate feed, and the near-neutral concentrate feed is a 25x concentrate feed.

[0201] Clause 31 specifically describes the method as described in any one of Clauses 22 to 30, wherein the pH of the near-neutral concentrate feed is 6.5-8.5.

[0202] Clause 32 specifically describes a method as described in any one of Clauses 22 to 32, wherein the pH of the alkaline concentrate feed is 9 or higher, the pH of the acidic concentrate feed is 5 or lower, and the pH of the near-neutral concentrate feed is 7 to 8.5.

[0203] Clause 33 provides a specific description of the methods described in Clause 32, wherein (a) The pH of the alkaline concentrate feed is 9 to 11, the pH of the acidic concentrate feed is 2 to 5, and the pH of the near-neutral concentrate feed is 7 to 8.5. (b) The pH of the alkaline concentrate feed is 9.8 to 10.8, the pH of the acidic concentrate feed is 3.6 to 4.8, and the pH of the near-neutral concentrate feed is 7 to 8.5; or (c) The pH of the alkaline concentrate feed is 9.8 to 10.5, the pH of the acidic concentrate feed is 3.8 to 4.5, and the pH of the near-neutral concentrate feed is 7.5 to 8.5.

[0204] Clause 34 specifically describes the method as described in Clause 33, wherein the serum-free cell culture perfusion medium is (a) a chemically defined medium, (b) a hydrolysate-free medium, and / or (c) a protein-free medium or a protein-free medium containing recombinant insulin and / or recombinant insulin-like growth factor.

[0205] Clause 35 specifically describes a method as described in any one of Clauses 22 to 34, wherein the at least three separate concentrated feeds and the diluent are added separately to enable control of the osmotic pressure of the serum-free cell culture perfusion medium in the bioreactor.

[0206] Clause 36 specifically describes the method as described in any one of Clauses 22 to 35, wherein (a) The ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed to the near-neutral concentrated feed is a fixed ratio to provide the serum-free cell culture perfusion medium with pH adjusted to neutral in the reaction vessel of the cell culture and / or bioreactor; and (b) The ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrate feeds determines the osmotic pressure of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor, wherein the at least three separate aqueous concentrate feeds are added to the reaction vessel of the cell culture and / or bioreactor to provide the serum-free cell culture perfusion medium with pH adjusted to near neutral pH.

[0207] Clause 37 specifically describes a method as described in any one of Clauses 22 to 36, wherein the acidic concentrate feed comprises trace elements, trace metals, inorganic salts, chelating agents, polyamines, and regulatory hormones.

[0208] Clause 38 specifically describes a method as described in any one of Clauses 22 to 37, wherein the acidic concentrate feed and / or the near-neutral concentrate feed comprises a surfactant, an antioxidant, and a carbon source.

[0209] Clause 39 specifically describes a method as described in any one of Clauses 22 to 38, wherein the alkaline concentrate feed contains amino acids that have maximum solubility at an alkaline pH of 9 or higher, preferably containing at least aspartic acid, histidine, and tyrosine, and optionally containing cysteine ​​and / or cystine and / or folic acid.

[0210] Clause 40 specifically describes the method as described in Clause 39, wherein the remaining amino acids are in the acidic and / or near-neutral concentrate feed, preferably in the acidic concentrate feed.

[0211] Clause 41 specifically describes a method as described in any one of Clauses 22 to 40, wherein vitamins and metals are in separate feeds, preferably vitamins in the near-neutral feed and metals in the acidic feed.

[0212] Clause 42 specifically describes the method as described in Clause 41, wherein vitamins that are poorly soluble in aqueous solutions, such as choline chloride, are present in the neutral feed and the acidic feed.

[0213] Clause 43 specifically describes a method as described in any one of Clauses 22 to 42, wherein the reaction vessel of the cell culture and / or bioreactor contains at least about 100 L of serum-free cell culture perfusion medium, preferably at least about 1000 L of serum-free cell culture perfusion medium.

[0214] Clause 44 specifically describes a serum-free cell culture perfusion medium that can be obtained by means of the methods described in Clauses 22 to 43.

[0215] Clause 45 specifically describes a method for culturing mammalian cells expressing heterologous proteins in perfusion culture, the method comprising: (a) Inoculating a bioreactor with mammalian cells expressing heterologous proteins in serum-free cell culture medium; (b) The mammalian cells are cultured in a perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium and removing the used medium while simultaneously maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a separated serum-free cell culture perfusion medium comprising a medium component consisting of at least three separate aqueous concentrate feeds and a diluent, wherein the first concentrate feed is an alkaline concentrate feed, the second concentrate feed is an acidic concentrate feed, and the third concentrate feed is a near-neutral concentrate feed; and wherein the separated serum-free cell culture perfusion medium is pH-adjusted to neutral pH after mixing the at least three separate aqueous concentrate feeds and the diluent in the resulting serum-free cell culture perfusion medium; and / or (ii) a serum-free cell culture perfusion medium according to Clause 44, and The alkaline concentrate, acidic concentrate, and near-neutral concentrate of the separated serum-free cell culture perfusion medium are individually added to the reaction vessel of the cell culture and / or bioreactor, and the diluent is individually added to the reaction vessel of the cell culture and / or bioreactor, or the diluent is premixed with one of the at least three separate aqueous concentrates before being added to the reaction vessel of the cell culture and / or bioreactor.

[0216] Clause 46 specifically describes the method as described in Clause 45, wherein the mammalian cells are initially cultured in batches before perfusion culture begins.

[0217] Clause 47 provides a specific description of the method described in Clause 45 or 46, wherein perfusion culture begins from day 0 to day 3 of batch culture.

[0218] Clause 48 specifically describes the method as described in any one of Clauses 45 to 47, wherein the perfusion rate is increased after the initiation of perfusion until the target viable cell density is reached.

[0219] Clause 49 specifically describes the method as described in Clause 48, wherein the infusion rate is increased from less than or equal to 0.5 container volumes per day to about 5 container volumes per day, or from less than or equal to 0.5 container volumes per day to about 2 container volumes per day.

[0220] Clause 50 specifically describes a method as described in any one of Clauses 45 to 49, wherein the osmotic pressure of the serum-free cell culture perfusion medium is increased to a level above the optimal growth osmotic pressure, thereby resulting in growth inhibition at the target viable cell density, preferably wherein the osmotic pressure level of the serum-free cell culture perfusion medium is gradually or progressively increased starting from about half of the target viable cell density.

[0221] Clause 51 specifically describes the method as described in any one of Clauses 45 to 50, wherein the target live cell density is approximately 30 x 10⁻⁶. 6 10 cells / ml or higher, approximately 60 x 10 6 10 cells / ml or higher, approximately 80 x 10 6 cells / ml, preferably about 100 x 10⁻⁶ 6 Cells / ml or higher.

[0222] Clause 52 specifies the method as described in any one of Clauses 45 to 51, wherein osmotic pressure is controlled using the following: (a) A constant concentrate feed injection rate and different diluent injection rates result in different total injection rates; or (b) Constant total infusion rate and different concentrated feed infusion rates; The at least three concentrated feeds are added to each other at a fixed ratio (v / v / v) according to their multiple concentrations to maintain the relative proportions of the culture medium components in 1x serum-free cell culture perfusion medium.

[0223] Clause 53 specifically describes the method as described in any one of Clauses 45 to 52, wherein the osmotic pressure is increased using the following: (a) A constant concentrate feed infusion rate and a reduced diluent infusion rate, resulting in a reduced overall infusion rate; or (b) Constant total infusion rate and increased concentrate feed infusion rate and decreased diluent infusion rate; The at least three concentrated feeds are added to each other at a fixed ratio (v / v / v) according to their multiple concentrations to maintain the relative proportions of the culture medium components in 1x serum-free cell culture perfusion medium.

[0224] Clause 54 specifies the method as described in any one of Clauses 50 to 53, wherein no further additives are added to the culture to increase osmotic pressure.

[0225] Clause 55 specifies the method as described in any one of Clauses 50 to 54, wherein the optimal growth osmotic pressure level is about 280 to less than 350 mOsm.

[0226] Clause 56 specifically describes the method as described in any one of Clauses 50 to 55, wherein the osmotic pressure is maintained at a level most suitable for growth until approximately half of the target live cell density is reached.

[0227] Clause 57 specifically describes the method as described in any one of Clauses 50 to 56, wherein the osmotic pressure is gradually or stepwise increased from about half of the target viable cell density, preferably to about 10-50% of the optimal growth osmotic pressure level.

[0228] Clause 58 specifically describes a method as described in any one of Clauses 50 to 57, wherein the osmotic pressure is increased to and maintained at an osmotic pressure level that inhibits cell growth at approximately the target viable cell density, wherein the osmotic pressure level that inhibits cell growth is preferably about 350 mOsm or higher, more preferably about 380 mOsm or higher.

[0229] Clause 59 specifically describes the method as described in any one of Clauses 50 to 58, wherein increasing osmotic pressure reduces or eliminates the need for cell removal during the production phase.

[0230] Clause 60 specifically describes the method as described in any one of Clauses 50 to 59, wherein the yield of the heterologous protein produced in the cell culture is increased by at least 5-50% relative to the yield of the control cell culture, wherein the osmotic pressure is not increased.

[0231] Clause 61 specifically describes a method as described in any one of Clauses 50 to 60, wherein cell growth is inhibited to maintain a sustainable density of living cells without cell expulsion.

[0232] Clause 62 specifically describes the method as described in any one of Clauses 45 to 61, wherein the cell specific perfusion rate (pl / cell / day) is reduced by at least 30% relative to the cell specific perfusion rate of 1x serum-free cell culture medium.

[0233] Clause 63 specifically describes a method as described in any one of Clauses 45 to 62, the method further comprising harvesting heterologous proteins from cell cultures.

[0234] Clause 64 provides a method as described in any one of Clauses 45 to 63, wherein the heterologous protein is a therapeutic protein, an antibody, or a therapeutically effective fragment thereof.

[0235] Clause 65 provides a specific description of the method described in Clause 64, wherein the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a fragment thereof.

[0236] Clause 66 specifically describes a method as described in any one of Clauses 45 to 65, wherein the mammalian cells comprise Chinese hamster ovary (CHO) cells, Jurkat cells, 293 cells, HeLa cells, CV-1 cells, or 3T3 cells, or derivatives of any of these cells, wherein the CHO cells may be further selected from the group consisting of: CHO-DG44 cells, CHO-K1 cells, CHO DXB11 cells, CHO-S cells, and CHO GS-deficient cells or mutants thereof.

[0237] Clause 67 specifically describes a method as described in any one of Clauses 45 to 66, wherein the reaction vessel of the cell culture and / or bioreactor contains at least about 100 L of serum-free cell culture perfusion medium, preferably at least about 1000 L of serum-free cell culture perfusion medium.

[0238] Clause 68 specifically describes the method as described in any one of Clauses 45 to 67, wherein a further supplement selected from the list of defoamers, alkalis, and glucose is added separately to the cell culture.

[0239] Clause 69 specifically describes a method for producing therapeutic proteins using any one of Clauses 45 to 68.

[0240] Clause 70 specifically describes the use of a segregated serum-free cell culture perfusion medium as described in any one of Clauses 1 to 18 or a serum-free cell culture perfusion medium as described in Clause 44 for culturing mammalian cells.

[0241] Clause 71 specifically describes the use of a segregated serum-free cell culture perfusion medium as described in any one of Clauses 1 to 18 or a serum-free cell culture perfusion medium as described in Clause 44 for culturing mammalian cells in perfusion culture.

[0242] Clause 72 specifically describes the use of a segregated serum-free cell culture perfusion medium as described in any one of Clauses 1 to 18 or a serum-free cell culture perfusion medium as described in Clause 44 for controlling the osmotic pressure of perfused cell cultures.

[0243] Clause 73 specifies the uses described in Clause 72, wherein increasing the osmotic pressure of cell cultures inhibits cell growth and increases the production of heterologous proteins.

[0244] Clause 74 specifies the use as described in Clause 73, wherein the yield of heterologous proteins produced in the cell culture is increased by at least 5-50% relative to the yield of the control cell culture, without an increase in osmotic pressure.

[0245] Clause 75 specifies the use as described in Clauses 73 or 74, wherein growth inhibition is sufficient to maintain a sustainable live cell density without cell expulsion.

[0246] Clause 76 specifies the use as described in Clause 70 or 75, wherein the specific perfusion rate (pl / cell / day) is reduced by at least 30% relative to the specific perfusion rate of 1x serum-free cell culture medium.

[0247] Clause 77 specifically describes the use of a segregated serum-free cell culture perfusion medium as described in any one of Clauses 1 to 18 for individually adding the at least three separate aqueous concentrated feeds to the reaction vessel of the cell culture and / or bioreactor.

[0248] Example method Seed training and inoculum: Chinese hamster ovary (CHO) cell lines expressing recombinant IgG were cultured in suspension in Corning-Life Sciences shake flasks (Oneonta, NY), which were expanded from 3e7 cell vials in proprietary growth medium. Flasks were seeded at 0.5e6 cells / mL for 3 days and then at 0.8e6 cells / mL for 2 days, and grown in batches, agitated at 120 rpm until N-3 3L shake flasks were formed, agitated at 80 rpm with a 50 mm orbital radius. Culture incubators (Infors, Annapolis, MD) were maintained at 36.5°C and 5% CO2, without humidity control. The N-2 phase was seeded at 1.0 ± 0.4e6 cells / mL and grown in batches for 3 days in a GE Wave 25 system (GE Healthcare) at a working volume of 5 L. The N-1 phase was run in perfusion mode in a GE Wave 25 system (GE Healthcare). In a 25 L working volume, the seeding density was 1.0 ± 0.4 e6 cells / mL. Perfusion began on day 1 of culture at 0.5 container volumes (vvd) per day, increasing by 0.5 vvd each day until reaching 2.0 vvd on day 4, which was maintained until day 5 or 6. The run duration was determined based on achieving the target viable cell density (VCD): 40 e6 c / mL.

[0249] Experimental bioreactor setup: N-1 cultures were perfused and seeded at a high density of 10 ± 2e6 cells / mL in a 100 L single-use bioreactor (SUB), in accordance with U.S. Provisional Application No. 62827504, particularly its Figure 6 The Boehringer Ingelheims proprietary iSKID (an integrated, continuous biological treatment system) is described in the paper. Custom-designed ThermoFisher Hyclone (Logan, Utah) SUB bags are used with a DeltaV distributed control system (Emerson, St Louis, MO) to maintain the culture at 36.5°C, with a target oxygen setpoint of 60% air saturation and a pH setpoint of 7.1, where a single marine impeller operates at 18 W / m³ per unit volume. 3Operating at power. Cell cultures were recirculated at 13 liters per minute (LPM) using a low-shear centrifugal pump (Levitronix, Zurich, Switzerland) through a 0.2 μm pore size polyethylene sulfone (PES) tangential flow filtration (TFF) cell retention device (Repligen, Waltham, MA). Harvested cell cultures or permeate from the TFF were directly transferred to the capture column operated by iSkid's purification unit. Growth medium, three concentrated culture media feeds (acidic, alkaline, and neutral), 0.1 µm filtered sterile reverse osmosis deionized (RODI) water diluent, alkaline titrant (1M sodium carbonate) to maintain pH during culture, glucose feed (500 g / L), and 1% medical antifoaming agent C emulsion (Dow Corning, Midland, MI) were attached to the SUB via sterile welded tubing or sterile antiseptic quick connectors (Colder Products Company, St Paul, MN). All additives are added separately to avoid precipitation, except for the alkaline concentrate feed, which is diverted in multiple branches with sterile water diluent, then passes through an online mixer in the pipeline, and finally reaches the bioreactor in a single tube.

[0250] The perfusion culture medium (three concentrated culture media feeds) used was prepared as follows: A 1x acid feed includes the following: • Protein amino acids not present in alkaline feed, as well as non-proteinogenic amino acids hydroxyproline and ornithine, totaling 87.8 mM; • Inorganic salts, including buffer salts (trace metal salts and iron sources are listed separately), totaling 21.4 mM; • Organic acid taurine and alternative carbon sources, totaling 16.3 mM; • Combined iron source, totaling 0.25 mM; • 0.28 mM polyamine; • 0.28 mM ethanolamine; • Trace metals (excluding iron), totaling 0.1 mM; • 0.02 μm of the primary antioxidant; • Vitamins, 0.07 mM calcium pantothenate, 0.04 mM thiamine and 0.3 mM pyridoxine; Choline chloride added alone to acidic and neutral feeds is 1.27 mM in acidic feeds; • 50 mM carbon source; • 2.4 µM of recombinant protein as a growth factor; and • 0.2 mM surfactant.

[0251] For the 6x concentrated acid feed used in the examples, these concentrations were increased 6-fold. The final pH of the 6x concentrated acid feed was adjusted to 4.2 ± 0.1 with sodium hydroxide, and the osmotic pressure was 1700 ± 50 mOsm. Although not required, the culture medium was prepared as a basal powder before the addition of the carbon source, and 1 g / L glucose was added only for grinding purposes.

[0252] A 1x neutral feed includes the following: • 25 mM bicarbonate; • 4.1 mM inorganic buffer salt; • 1.69 mM inositol; • All other vitamins not yet included in the acidic feed (but including the remaining choline chloride), totaling 0.57 mM; • 0.01 mM of secondary antioxidant; • 0.043 mM L-α-amino-n-butyric acid; • 0.2 mM surfactant; and • 5 µM linoleic acid.

[0253] For the 25x concentrated neutral feed used in the examples, these concentrations were increased 25-fold. Without the use of titrants, the final pH of the 25x concentrated neutral feed self-adjusted to 8.0 ± 0.1, and the osmotic pressure was 1500 ± 35 mOsm.

[0254] 1x of alkaline feed includes: • Amino acids, aspartic acid, histidine, tyrosine, cysteine ​​(including cystine), total concentration 43 mM.

[0255] For the 25x concentrated alkaline feed used in the examples herein, this concentration is increased by 25 times. The final pH of the 25x concentrated alkaline feed was adjusted to 10.2 ± 0.1 using sodium hydroxide, and the osmotic pressure was 1600 ± 50 mOsm.

[0256] The perfusion medium, consisting of three separate aqueous concentrates, was adjusted to a pH of 7.0 ± 0.1. The acidic concentrate was a 6x concentrate with a pH of 4.2 ± 0.1 and an osmotic pressure of 1700 ± 50 mOsm; the neutral concentrate was a 25x concentrate with a pH of 8.0 ± 0.1 and an osmotic pressure of 1500 ± 35 mOsm; and the alkaline concentrate was a 25x concentrate with a pH of 10.2 ± 0.1 and an osmotic pressure of 1600 ± 50 mOsm.

[0257] Example 1 Immediately after inoculation on day 0, perfusion was initiated using proprietary growth medium at a rate of 1 vvd. The perfusion rate was increased by 0.5 vvd daily until reaching 2.0 vvd on day 2. The bioreactor working volume was maintained by controlling medium addition via bioreactor weight control. On day 2, concentrated medium feed and diluent were used to replace the growth medium to begin the “production phase,” that is, when the product reached 0.2 g / Lbr / day in the permeate, the capture column was loaded. During the production phase, concentrated feed was fed at a constant total amount of 0.5 vvd (0.33 vvd for acidic feed, and 0.08 vvd each for alkaline and neutral feeds). Feed rates were calculated using the following equation to maintain the same nutrient proportions in each feed compared to a full 1x formulation of 2 vvd: [1x]*2 vvd=[6x]*X vvd (Equation 1) Where X is the infusion rate in units of vvd of acidic feed necessary to maintain the same nutrient content as a 1x concentration formulation with 2vvd.

[0258] Similarly, [1x]*2 vvd=[25x]*X vvd (Equation 2) Where X is the infusion rate in units of vvd of alkaline or neutral feed necessary to maintain the same nutrient content as a 1x concentration formulation with 2vvd.

[0259] The cell lines used in these experiments had a maximum VCD of approximately 140 ± 30 e6 cells / mL, based on previous engineering runs that showed this range as the maximum sustainable VCD (results not shown). VCD counting was performed on a Beckman Coulter Vi-cell (Indianapolis, IN). To achieve a target of approximately 15–45% lower than the peak growth capacity of the cell lines (results not shown), the osmolarity of the cultures was gradually increased to inhibit cell replication. Culture osmolarity was measured using a BioProfile FLEX analyzer (Nova Biomedical, Waltham, MA), and all other culture metabolites were measured using a Roche Cedex BioAnalyzer (Indianapolis, IN). The osmolarity increase was achieved by adjusting the diluent rate daily to achieve the target residual osmolarity of the cultures while maintaining a constant feed addition rate. Therefore, the total perfusion rate varied daily. The osmolar balance of daily osmolar consumption was calculated according to the following equation: Osmotic pressure input - osmotic pressure output = osmotic pressure consumption (Equation 3) The osmotic input is the osmotic pressure of the concentrated feed and diluent infused into the bioreactor, the osmotic output is the residual osmotic pressure of the bioreactor supernatant, and the osmotic consumption is the osmotic pressure difference between the input and output. This daily osmotic consumption is then normalized to the number of cells in the culture, i.e., the daily osmotic pressure consumption per cell. This daily consumption rate per cell (or cell-specific osmotic pressure consumption rate, CSOCR) is then multiplied by the predicted VCD for the next day to predict the osmotic consumption for the following day. This consumption rate, along with the expected osmotic output, is then used in Equation 3 to calculate the required osmotic input for the next day. Therefore, the diluent infusion rate is adjusted to match the osmotic input target while maintaining the feed. According to Table 1, the expected osmotic pressure target and estimated infusion rate vary each day (the values ​​differ for each run, resulting in the following ranges):

[0260] Daily glucose measurements were performed, and individual glucose boluses were added as needed to maintain residual glucose at or above 2 g / L. Based on a commercial case matching the run duration of typical fed-batch cultures, the culture was terminated at 14 days. Results from three 100 L bioreactor runs are shown in... Figure 3 (VCD) Figure 4 (osmotic pressure) Figure 5 (reactor volume exchange) Figure 6 (Permeate productivity) Figure 7 (Daily productivity) and Figure 8 (Cellular perfusion rate)

[0261] Example 2 Three CHO cell lines A(A, B, C) expressing different recombinant IgG molecules were cultured in a 2 L bioreactor. B(□) and C(Δ) (see Figures 9 to 14 Immediately after inoculation on Day 0, perfusion was initiated using proprietary growth medium at a rate of 1 vvd. The perfusion rate was increased by 0.5 vvd daily until reaching 2.0 vvd on Day 2. The working volume of the bioreactor was maintained by controlling the addition of medium via bioreactor weight control. On Day 2, concentrated medium feed and diluent replaced the growth medium to begin the “production phase,” that is, when the culture reached a product concentration of 0.2 g / Lbr / day in the permeate, the capture column was loaded. Cells were fed at a constant volume of approximately 2 vvd using three different proportions of concentrated medium feed and sterile water diluent. As explained in Example 1, feed rates were calculated such that the nutrient proportions in each feed remained the same compared to 2 vvd of a complete 1x formulation.

[0262] The maximum VCD for cell lines A and B were approximately 180 ± 30 e6 cells / mL and 140 ± 30 e6 cells / mL, respectively, based on previous engineering runs that showed this range to be the maximum sustainable VCD for these cell lines (results not shown). Cell line C had a maximum peak VCD of 100 ± 20 e6 c / mL, therefore growth inhibition of this cell line was unnecessary, and the osmolarity was maintained within the physiologically optimal range of 330 ± 30 mOsm. VCD counting was performed on a Beckman Coulter Vi-cell (Indianapolis, IN). For cell lines A and B, culture osmolarity was gradually increased to inhibit cell replication in order to achieve a target of approximately 15–45% lower than the peak growth capacity of these cell lines (results not shown). Culture osmolarity was measured using a BioProfile FLEX analyzer (Nova Biomedical, Waltham, MA), and all other culture metabolites were measured using a Roche Cedex BioAnalyzer (Indianapolis, IN). The increase in osmotic pressure is achieved by adjusting the concentrate feed rate and diluent rate daily to reach the target residual osmotic pressure of the culture while maintaining a constant total VVD addition of two VVDs. As explained in Example 1, the daily osmotic pressure consumption is calculated according to the following equation: Osmotic pressure input - osmotic pressure output + osmotic pressure consumption.

[0263] Similar to Example 1, the daily osmotic pressure consumption rate is determined and then used to calculate the osmotic pressure input necessary to achieve the new desired osmotic pressure output for the next day. However, in the case of the osmotic pressure control strategy of Example 2, the rates of both the feed and diluent are adjusted (as opposed to the case in Example 1 where only the diluent rate is adjusted) to achieve the target osmotic pressure input at a total perfusion rate of 2 vvd.

[0264] Daily glucose measurements were performed, and individual glucose boluses were added as needed to maintain residual glucose at or above 2 g / L. Based on a commercial case matching the run duration of typical fed-batch cultures, the culture was terminated at 14 days without cell efflux. Results from three 2 L bioreactor runs are shown in... Figure 9 (VCD) Figure 10 (osmotic pressure) Figure 11 (reactor volume exchange) Figure 12 (Permeate productivity) Figure 13 (Daily productivity) and Figure 14 (Cellular perfusion rate)

[0265] Example 3 Using different diluent volumes and three concentrated culture media fed at a fixed total of 0.5 container volumes (VVD) per day, CHO DG44 cell lines (cell line A, Δ) and two different CHO-K1 cell lines (cell line B, Δ) were cultured in a 2L bioreactor. Cell lines C(x, x) were used, with the CHO DG44 cell line expressing folate reductase (dhfr) in a dihydrofolate reductase (DHFR) selection system and the two different CHO-K1 cell lines expressing glutamine synthase (GS) in a glutamine synthase (GS) selection system (see Figure 15). All cell lines expressed different recombinant IgG molecules. The working volume of the bioreactor was maintained by adding medium via bioreactor weight control. On day 2, concentrated medium was fed and the growth medium was replaced with diluent to begin the “production phase,” that is, when the culture reached 0.2 g / L in osmotic solution. 生物反应器 When the product is produced per day, the capture column is loaded. During the production phase, the feed is concentrated by a constant total feed rate of 0.5 vvd (0.33 vvd for acidic feed, and 0.08 vvd each for alkaline and neutral feeds). As explained in Example 1, the feed rate is calculated such that the nutrient content in each feed remains the same compared to a complete 1x formulation of 2 vvd.

[0266] Cell line A was cultured at physiologically optimal osmolarity (330 ± 30 mOsm) for the entire culture duration (12 days) to promote maximum cell culture growth (i.e., the possible peak VCD). This was considered the “engineering” or development run for this cell line. Cell line B was targeted for a maximum VCD of 150 ± 30 e6 cells / mL ± 20 e6 c / mL, based on a previous engineered run that showed this range as the maximum sustainable VCD for this cell line (results not shown). Cell line C had a maximum peak VCD < 100 ± 20 e6 c / mL, therefore no growth inhibition was necessary, and the osmolarity was maintained within the physiologically optimal range of 330 ± 30 mOsm. VCD counting was performed on a Beckman Coulter Vi-cell (Indianapolis, IN). For cell line B, to achieve a target of approximately 15–45% lower than the cell line’s peak growth capacity (results not shown), the osmolarity of the culture was gradually increased to inhibit cell replication. Culture osmolarity was measured using a BioProfile FLEX analyzer (Nova Biomedical, Waltham, MA), and all other culture metabolites were measured using a Roche Cedex BioAnalyzer (Indianapolis, IN). Osmolarity increases were achieved by adjusting the diluent rate daily to reach the target residual osmolarity of the culture while maintaining a constant feed addition rate. Daily osmolarity consumption was calculated according to the following equation, as explained in Example 1: Osmotic pressure input - Osmotic pressure output + Osmotic pressure consumption As in Example 1, the daily osmotic pressure consumption rate is determined and then used to calculate the osmotic pressure input required to achieve the new desired osmotic pressure output for the next day.

[0267] Daily glucose measurements were performed, and separate glucose boluses were added as needed to maintain residual glucose at or above 2 g / L. As shown in Figure 15, the culture was terminated at days 11, 12, and 14. Results from the 2 L bioreactor operation are shown below: Figure 15A The live cell density (VCD; e5 c / mL) is shown. Figure 15B Show vitality (%); Figure 15C The permeate productivity (g / L / day) is shown, which is expressed as the daily instantaneous titer of permeate (g / L) as measured by a Cedex bioanalyzer. 培养基 Multiply by the daily perfusion rate (L) 培养基 / L 生物反应器 Calculated by / day; and Figure 15D This shows the exchange of reactor volume (L) 培养基 / L 生物反应器 The infusion rate is expressed as / day.

[0268] Example 4 CHO-K1 cell lines expressing recombinant IgG in a glutamine synthase (GS) selection system were cultured in a 2 L bioreactor. As described in Examples 2 and 3, the total VVD was fixed at different MC values. The system operates under either "Constant MC, Different Total VVD" (□) perfusion control mode. "Different MC, Constant Total VVD" refers to a constant daily total container volume (VVD) perfusion rate, achieved by varying the perfusion rate of the combined culture medium concentrate (MC) while simultaneously varying the diluent rate to maintain 2 VVD. "Constant MC, Different Total VVD" refers to a constant perfusion rate of MC at 0.5 VVD and varying diluent perfusion rates, with respect to overall fluctuating perfusion rates. Both perfusion control modes allow manipulation of culture medium osmotic pressure to set a target ( Figure 16B Using two perfusion control modes with this cell line, viability and live cell density were comparable. Separation of the concentrated culture feed (i.e., nutrient delivery) from the diluent enabled a low perfusion rate (≤2 VVD), which has the ability to provide sufficient nutrients at high cell densities by varying the ratio of concentrated to diluent. Therefore, residual culture osmolality can be controlled at elevated levels above the physiological optimum (which varies depending on the cell line; for this cell line, it is 300–330 mOsm) without increasing the perfusion rate above 2 VVD (the highest perfusion rate is considered scalable to >100L bioreactors of this company). Peak VCD (see [link to relevant documentation]) can be suppressed when osmolality is increased before reaching peak live cell density (VCD). Figure 3 and Figure 4 ).

[0269] Adjusted productivity ( Figure 16C The total productivity of the system was defined as including both the permeate and the products retained in the bioreactor each day. For the cell line shown, the productivity of both perfusion control modes was similar, therefore either perfusion mode could be selected as the treatment process for that cell line. Reactor volume exchange (L) for cell culture 培养基 / L 生物反应器 ( / day) or infusion rate is shown in Figure 16D The target 2 VVD was not achieved on days 4 and 5 of the "MC Different, Total VVD Fixed" run due to operator errors. All remaining days of the production phase (>day 2) were maintained at the target 2 VVD. The "MC Fixed, Total VVD Different" run demonstrates the variable infusion rate necessary to maintain the target osmotic pressure (Figure 16b).

Claims

1. A separated serum-free cell culture perfusion medium, said separated serum-free cell culture perfusion medium comprising three separate aqueous concentrate feeds and a diluent, wherein the first concentrate feed is an alkaline concentrate feed, the second concentrate feed is an acidic concentrate feed, and the third concentrate feed is a near-neutral concentrate feed; wherein After mixing the three separate aqueous concentrate feeds and the diluent in the obtained serum-free cell culture perfusion medium, the pH of the separated serum-free cell culture perfusion medium is adjusted to neutral pH.

2. The separated serum-free cell culture perfusion medium as claimed in claim 1, wherein the pH of the resulting serum-free cell culture perfusion medium after mixing the three separate aqueous concentrate feeds and the diluent is between 6.7 and 7.5, between 6.9 and 7.4, preferably between 6.9 and 7.

2.

3. The separated serum-free cell culture perfusion medium as described in claim 1 or 2, wherein the diluent is sterile water.

4. The separated serum-free cell culture perfusion medium according to any one of claims 1 to 3, wherein the separated serum-free cell culture perfusion medium is used for (a) The alkaline concentrate feed, the acid concentrate feed and the near-neutral concentrate feed are added separately to the reaction vessel of the cell culture and / or bioreactor; (b) The alkaline concentrate feed, the acidic concentrate feed, and the near-neutral concentrate feed are added directly to the reaction vessel of the cell culture and / or bioreactor without prior premixing; and / or (c) The at least three separate aqueous concentrate feeds are directly mixed in the reaction vessel of the cell culture and / or bioreactor.

5. The separated serum-free cell culture perfusion medium according to any one of the preceding claims, wherein the alkaline concentrate feed is a 2x to 80x concentrate feed, the acidic concentrate feed is a 2x to 40x concentrate feed, and the near-neutral concentrate feed is a 2x to 50x concentrate feed.

6. The separated serum-free cell culture perfusion medium according to any one of the preceding claims, wherein the pH of the alkaline concentrate feed is 9 or higher, the pH of the acidic concentrate feed is 5 or lower, and the pH of the near-neutral concentrate feed is 7 to 8.

5.

7. An alkaline aqueous concentrate feed, said alkaline aqueous concentrate feed being used in combination with an acidic aqueous concentrate feed, a near-neutral aqueous concentrate feed and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

8. An acidic aqueous concentrate feed, said acidic aqueous concentrate feed being used in combination with an alkaline aqueous concentrate feed, a near-neutral aqueous concentrate feed and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

9. A near-neutral aqueous concentrate feed, said near-neutral aqueous concentrate feed being used in combination with an alkaline aqueous concentrate feed, an acidic aqueous concentrate feed and a diluent to form a serum-free cell culture perfusion medium, wherein the pH of the resulting serum-free cell culture perfusion medium is automatically adjusted to neutral pH.

10. A method for preparing serum-free cell culture perfusion medium, the method comprising: (a) Cell culture medium components are provided in three subgroups based on their solubility at alkaline, acidic, and neutral pH levels. (b) (i) Dissolve the subcomponents soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed; (ii) Dissolve the subcomponents soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; as well as (iii) Dissolve the subcomponents soluble at neutral pH in a neutral aqueous solution to form a near-neutral concentrated feed; (c) Optionally, the prepared alkaline concentrate feed, acidic concentrate feed and near-neutral concentrate feed may be stored in separate containers; as well as (d) The prepared alkaline concentrate, acidic concentrate, and near-neutral concentrate, along with the diluent, are added to the reaction vessel of the cell culture and / or bioreactor, wherein... (i) The alkaline concentrate feed, the acid concentrate feed and the near-neutral concentrate feed are added separately to the reaction vessel of the cell culture and / or bioreactor; and (ii) Add the diluent separately to the reaction vessel of the cell culture and / or bioreactor, or premix the diluent with one of the three separate aqueous concentrate feeds before adding it to the reaction vessel of the cell culture and / or bioreactor; The pH of the serum-free cell culture perfusion medium is automatically adjusted to neutral after the three separate aqueous concentrate feeds are mixed with the diluent.

11. A serum-free cell culture perfusion medium that can be obtained by the method according to claim 10.

12. A method for culturing mammalian cells expressing heterologous proteins in perfusion culture, the method comprising: (a) Inoculating a bioreactor with mammalian cells expressing heterologous proteins in serum-free cell culture medium; (b) The mammalian cells are cultured in perfusion culture by continuously feeding the mammalian cells with serum-free cell culture perfusion medium and removing the used medium while maintaining the cells in culture. The serum-free cell culture perfusion medium feed is (i) a separated serum-free cell culture perfusion medium composed of three separate aqueous concentrate feeds and a diluent, wherein the first concentrate feed is an alkaline concentrate feed, the second concentrate feed is an acidic concentrate feed, and the third concentrate feed is a near-neutral concentrate feed; and wherein after mixing the three separate aqueous concentrate feeds and the diluent in the resulting serum-free cell culture perfusion medium, the pH of the separated serum-free cell culture perfusion medium is adjusted to neutral; and / or (ii) the serum-free cell culture perfusion medium according to claim 11, and The alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed of the separated serum-free cell culture perfusion medium are added separately to the reaction vessel of the cell culture and / or bioreactor, and the diluent is added separately to the reaction vessel of the cell culture and / or bioreactor, or the diluent is premixed with one of the three separate aqueous concentrated feeds before being added to the reaction vessel of the cell culture and / or bioreactor.

13. A method for producing a therapeutic protein using the method of claim 12.

14. Use of the separated serum-free cell culture perfusion medium as described in any one of claims 1 to 6 or the serum-free cell culture perfusion medium as described in claim 11 for culturing mammalian cells.

15. Use of the separated serum-free cell culture perfusion medium as described in any one of claims 1 to 6 or the serum-free cell culture perfusion medium as described in claim 11 for culturing mammalian cells in perfusion culture.

16. Use of the separated serum-free cell culture perfusion medium as described in any one of claims 1 to 6 or the serum-free cell culture perfusion medium as described in claim 11 for controlling the osmotic pressure of the perfused cell culture.

17. The use of the separated serum-free cell culture perfusion medium as described in any one of claims 1 to 6 for individually adding the three separate aqueous concentrated feeds to the reaction vessel of the cell culture and / or bioreactor.

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