Concentrated perfusion medium

Abstract

The present invention relates to a serum-free cell culture perfusion medium comprising medium components grouped into at least three separate aqueous concentrated feeds and a diluent, wherein the resulting serum-free cell culture perfusion medium is adjusted to neutral pH after mixing. Further provided is a method of preparing said serum-free cell culture perfusion medium. The present invention further relates to a method of using said serum-free cell culture perfusion medium for culturing mammalian cells or producing a protein of interest in perfusion culture, said method achieving high productivity at low specific perfusion rates. The present invention further relates to the use of the new and improved serum-free cell culture perfusion medium to control the osmolarity of perfusion cell cultures, wherein increasing the osmolarity results in an increase in overall productivity and / or specific productivity by inhibiting cell growth during cell culture, e.g. during the production phase of perfusion cell culture. Inhibiting cell growth in particular reduces or eliminates the need for wasteful cell bleed.

Description

Technical Field

[0001] The present invention relates to a serum-free cell culture perfusion medium comprising culture medium components grouped into at least three separate aqueous concentrate feeds and a diluent, wherein the serum-free cell culture perfusion medium adjusts the pH to a neutral pH after mixing. A method for preparing the serum-free cell culture perfusion medium is also provided. The present invention further relates to a method for using the serum-free cell culture perfusion medium to culture mammalian cells or produce a protein of interest in a perfusion culture, wherein the method achieves high productivity at low cell-specific perfusion rates. The present invention further relates to the use of a new and improved serum-free cell culture perfusion medium to control the osmotic pressure of a perfusion cell culture, wherein increasing the osmotic pressure results in an increase in total productivity and / or cell-specific productivity by inhibiting cell growth during the cell culture, for example during the production phase of the perfusion cell culture. Inhibiting cell growth particularly reduces or eliminates the need for wasteful cell discharge. Background Art

[0002] Three methods are commonly used in commercial production of recombinant proteins by mammalian cell culture: batch, fed-batch, and perfusion.

[0003] Perfusion-based methods provide potential improvements that are superior to batch and fed-batch methods, including improved product quality and stability, improved scalability, and increased cell-specific productivity. Unlike batch and fed-batch bioreactors, perfusion systems involve the continuous removal of spent culture medium. By continuously removing spent culture medium and replacing it with new culture medium, nutrient levels can be better maintained while optimizing growth conditions and removing cell waste. Reduced waste reduces toxicity to cells and expressed products. Therefore, perfusion bioreactors typically significantly reduce protein degradation and therefore obtain higher-quality products. Products can also be harvested and purified faster and more continuously, which is particularly effective when producing unstable products.

[0004] 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 thing, perfusion bioreactors are smaller and can produce the same productivity (i.e., product yield) with a smaller volume. It is generally believed that perfusion bioreactors can function at 5 to 20 times higher concentrations 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 a 1,000-liter perfusion bioreactor could replace a typical 10,000-liter traditional fed-batch bioreactor without negatively impacting overall productivity. This significant advantage translates into smaller space requirements when scaling up production. This can also translate into a range of advantages related to lower operating utilities, less infrastructure, less labor, reduced equipment complexity, continuous harvesting, and increased product yield.

[0005] The higher cell culture densities achieved partly explain the higher productivity of perfusion systems. In a typical large-scale fed-batch commercial cell culture process, 10-50x10 6 However, using perfusion-based bioreactors, cell densities >1x10 8 The results indicate that the perfusion bioreactor can achieve an extreme cell density of 10 cells / mL. In addition, in perfusion mode, high cell numbers can be maintained for longer periods of time by continuously replenishing spent medium. In perfusion bioreactors, higher cell densities are achieved over time, which in part explains the more efficient performance of perfusion bioreactors.

[0006] A typical perfusion culture begins with a batch culture start-up, lasting one day or longer to achieve rapid initial cell growth and biomass accumulation, followed by continuous, stepwise, and / or intermittent addition of fresh perfusion medium to the culture and simultaneous removal of spent medium, wherein the cells are retained throughout the culture growth and production phases. Various methods such as sedimentation, centrifugation, or filtration can be used to remove spent medium while maintaining the cells. Perfusion flow rates ranging from a small fraction of a working volume per day to many working volumes per day have been employed.

[0007] While continuous perfusion systems offer numerous advantages over traditional fed-batch and batch systems, many challenges remain before perfusion bioreactors gain wider acceptance and utilization in the biologics 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 traditional fed-batch systems. Specifically, the volume of culture medium required to maintain a perfusion rate of 1-3 vessel volumes per day (vvd) becomes logistically challenging, if not impossible, above pilot scale (~100 L bioreactors).

[0008] WO 92 / 22637 formulates concentrated culture medium subsets separated based on physicochemical properties. However, these concentrated culture medium subsets do not adjust pH after mixing and are therefore unsuitable for direct addition to cell cultures. Furthermore, the culture media disclosed in WO 92 / 22637, such as minimal media RPMI-1640, DMEM, and Ham's F-12, are less rich than modern cell culture media, with amino acid concentrations in the mM working concentration range rather than the μM working concentration range.

[0009] Another challenge facing continuous perfusion cell culture systems is maintaining a constant viable cell density and, therefore, a healthier, more productive cell culture. This is typically addressed by allowing "cell bleed." During cell bleed, cells are removed and discarded as waste at a rate sufficient to allow steady-state perfusion cell culture. This, in turn, maintains a constant viable cell density. Due to the cell bleed technique, a significant portion of the culture medium and product can be lost. This technique siphons off the proliferating cells and culture medium to maintain a constant, sustainable viable cell density within the bioreactor. As much as one-third of the harvestable material can be lost due to cell bleed. Consequently, using cell bleed reduces the product yield per run because the product within the fraction removed by cell bleed is not harvested. Therefore, any amount of cell bleed negatively impacts process efficiency, product recovery, and, most importantly, leads to product loss. The cell bleed rate is determined by the cell growth rate. Faster doubling times also require higher cell bleed to maintain a constant cell density, resulting in greater waste.

[0010] As an alternative to cell excretion, others have tried to use chemical additives to slow down cell growth rates. Reduced cell growth also typically 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. Similar disclosures are 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 teaches that CDK4 / 6 inhibitors specifically inhibit the cell cycle and do not affect other cell targets. However, it is necessary to avoid adding inhibitors and compounds that are not needed for cell growth and / or cell maintenance. Therefore, additional methods that effectively inhibit cell growth in a perfusion state and avoid the need for cell excretion will significantly help advance this area.

[0011] Osmotic pressure is a known lever for influencing cell growth. Existing techniques for influencing cell growth using osmotic pressure 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, the ability to control the cell culture process to a target osmotic pressure has never been established, particularly in perfusion culture. In addition, chemical additives affect the composition of the culture medium and / or need to be removed in subsequent purification steps, thereby increasing process complexity. Chemical additives, including salts, may also affect product quality.

[0012] Given the challenges facing perfusion cell culture, such as production media consumption and the desire to further increase productivity, media that improve logistical issues and methods that effectively inhibit cell growth under perfusion conditions and avoid the need for cell expulsion without further additives would significantly help advance the field. Summary of the Invention

[0013] The present invention relates in part to the following discovery: can be developed in a more concentrated form of feed culture medium through being separated, to reduce the volume of cell specific perfusion rate and the preparation culture medium consumed.These concentrated feeds are diluted in the bioreactor container.It is advantageous to use together with sterile deionized water as a diluent, and the diluent does not need to be prepared except filtering.In addition, the unique combination of 3 kinds of culture medium concentrates (acidic, alkaline and nearly neutral) designed in the present invention allows the use of higher multiple concentrates, thereby further reducing the total culture medium volume.By reducing the preparation volume of culture medium, a kind of perfusion process has been developed, and the perfusion process has eliminated the culture medium volume bottleneck, therefore can prove that the perfusion cell culture process is amplified to 1000L scale and possibly larger scale is reasonable.

[0014] Using a separate concentrated feed and diluent also allows cell growth to be controlled by culture osmotic pressure. Using the concept of mass balance, osmotic pressure balance is derived with the known osmotic pressure of each concentrated feed, feed rate, and calculated daily cell specific osmotic pressure consumption rate to predict the culture residual osmotic pressure as output. Using this method, the growth of cell culture can be controlled by raising the residual osmotic pressure to a physiological stress level (approximately 350-400mOsm or higher) and simultaneously maintaining a lower level of cytotoxicity (approximately 400mOsm or higher). Physiological stress levels and cytotoxicity levels can be cell line specific. However, this can easily be determined by measuring the viable cell concentration and viability under different osmotic pressure levels during the culture period, and can be determined in small scale (such as 3ml working volume). In the present invention, culture osmotic pressure is controlled via osmotic pressure balance, which includes the change of the culture concentrate feed rate relative to the daily benchmark under a fixed daily container volume (VVD) or the change of the VVD feed rate relative to the daily benchmark under a fixed culture concentrate feed rate. Osmotic balancing enables targeting higher or lower residual osmolality by adjusting the concentration and dilution rates, whereas chemical additive schemes used by others can only adjust osmolality in the increasing direction. Osmotic balancing as described herein has been found to be effective in inhibiting cell growth, and this growth inhibition results in increased cell specific productivity and helps maintain high viability in cell culture.

[0015] The cell growth inhibition induced by osmotic balance described herein not only results in increased cell specific productivity and sustained high cell viability, but in perfusion cell culture, such inhibition also reduces or eliminates the need to use cell expulsion techniques during the perfusion state to otherwise maintain cells in a stable growth state. This reduces or eliminates product losses due to wasteful and undesirable cell expulsion techniques.

[0016] The three-part highly concentrated feed medium provided herein can theoretically be used in conjunction 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 present invention is particularly suitable for continuous perfusion cell culture systems. Furthermore, the osmotic balance can theoretically be used in conjunction with any type of cell culture system. However, it is particularly advantageous when the cell culture system is a continuous perfusion cell culture system.

[0017] In one aspect, the present invention relates to a serum-free cell culture perfusion medium separated, comprising medium components grouped into 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 a neutral pH. In a preferred embodiment, 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. The diluent is preferably sterile water. In one embodiment, after mixing the at least three separate aqueous concentrate feeds and the diluent, the pH of the resulting 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. The compartmentalized serum-free cell culture perfusion medium according to the present 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 mixing the at least three separate aqueous concentrated feeds directly in the reaction vessel of the cell culture and / or bioreactor.

[0018] In certain embodiments, 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. The pH of the near-neutral concentrated feed is about 6.5 to about 8.5. Preferably, the pH of the alkaline concentrated feed is about 9 or higher, the pH of the acidic concentrated feed is about 5 or lower, and the pH of the near-neutral concentrated feed is about 7 to about 8.5. In addition, 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 resulting serum-free cell culture perfusion medium with a pH adjusted to a neutral pH; and the ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrated feeds in the resulting serum-free cell culture perfusion medium with a pH adjusted to a neutral pH determines the osmotic pressure of the serum-free cell culture perfusion medium.

[0019] The acidic concentrated feed may contain trace elements, trace metals, inorganic salts, chelating agents, polyamines and regulatory hormones. The acidic concentrated feed and / or the near-neutral concentrated feed may contain surfactants, antioxidants and carbon sources. Further, the alkaline concentrated 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 contains cysteine ​​and / or cystine and / or folic acid. The remaining amino acids are in the acidic and / or near-neutral concentrated feed, preferably in the acidic concentrated feed. Preferably, vitamins and metals are in separate feeds, preferably vitamins are in the near-neutral feed and metals are in the acidic feed. Vitamins that are poorly soluble in aqueous solution, such as choline chloride, are present in the neutral feed and the acidic feed.

[0020] The present invention also relates to an alkaline aqueous concentrated feed for use in combination with an acidic aqueous concentrated feed, a near-neutral aqueous concentrated 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 a neutral pH. In another embodiment, the present invention relates to an acidic aqueous concentrated feed for use in combination with an alkaline aqueous concentrated feed, a near-neutral aqueous concentrated 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 a neutral pH. In yet another aspect, the present invention relates to a near-neutral aqueous concentrated feed for use in combination with an alkaline aqueous concentrated feed, an acidic aqueous concentrated 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 a neutral pH.

[0021] In yet 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 of components based on solubility at alkaline, acidic, and neutral pH, (b) (i) dissolving the subgroup of components soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed; (ii) dissolving the subgroup of components soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; and (iii) dissolving the subgroup of components soluble at neutral pH in a neutral aqueous solution to form a near-neutral concentrated feed; (c) optionally storing the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed in separate containers; and (d) mixing the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed. The method of claim 1, wherein the at least three separate aqueous concentrated feeds and the near-neutral concentrated feeds and a diluent are added 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 just 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 concentrated feeds and the diluent, the pH of the resulting serum-free cell culture perfusion medium prepared by the method is between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2.

[0022] In certain embodiments, the at least three concentrated feeds are added dropwise to the reaction vessel of the cell culture and / or bioreactor through a separate port. Compared to the serum-free cell culture perfusion medium mixed and diluted before being added to the bioreactor, mixing and diluting the at least three separate aqueous concentrated feeds in the container allows the production of a culture medium consumption reduction of 50-90%, preferably 60-90%, within a 14-day culture period. Typically, the reaction vessel of the cell culture and / or bioreactor comprises mammalian cells. In addition, the method further comprises a step of sterilizing the concentrated feed before storage and / or addition to the reaction vessel of the cell culture and / or bioreactor.

[0023] In certain embodiments, the alkaline concentrated feed is a 2x to 80x concentrated feed, wherein the acidic concentrated feed is a 2x to 40x concentrated feed, and the near-neutral concentrated feed is a 2x to 50x concentrated feed. The pH of the near-neutral concentrated feed is 6.5-8.5. Preferably, the pH of the alkaline concentrated feed is 9 or higher, the pH of the acidic concentrated feed is 5 or lower, and the pH of the near-neutral concentrated feed is 7 to 8.5. In addition, 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 having a pH adjusted to a neutral pH 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 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 concentrated feeds are added to the reaction vessel of the cell culture and / or bioreactor to provide a serum-free cell culture perfusion medium having a pH adjusted to a near-neutral pH. In certain embodiments, the reaction vessel of the cell culture and / or bioreactor comprises 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.

[0024] Also provided is a serum-free cell culture perfusion medium obtainable by the method according to the present invention.

[0025] In another aspect, the present invention relates to a method of culturing mammalian cells expressing a heterologous protein in perfusion culture, the method comprising: (a) inoculating a bioreactor with mammalian cells expressing a heterologous protein in a serum-free cell culture medium; (b) culturing the mammalian cells in perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium feed and removing spent medium while maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a compartmentalized serum-free cell culture perfusion medium comprising medium components grouped into 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 (i) a serum-free cell culture perfusion medium obtainable by the method according to the present invention, wherein the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed of the serum-free cell culture perfusion medium feed 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 is premixed with one of the at least three separate aqueous concentrated feeds immediately before being added to the reaction vessel of the cell culture and / or bioreactor. The method generally further comprises harvesting the heterologous protein from the cell culture.

[0026] The mammalian cells may be initially cultured in a batch culture prior to the initiation of perfusion culture and / or the perfusion culture may be initiated from day 0 to day 3 of the batch culture (i.e., post-inoculation). Typically, the perfusion rate is increased after the initiation of perfusion until the target viable cell density is reached. In certain embodiments, the perfusion 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.

[0027] In certain embodiments, 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 a target viable cell density, preferably wherein the osmotic pressure level of the serum-free cell culture perfusion medium is increased gradually or stepwise starting from about half the target viable cell density. The target viable cell density is about 30×10 6 cells / ml or higher, approximately 60x10 6 cells / ml or higher, approximately 80x10 6cells / ml, preferably about 100x10 6 Cells / ml or higher.Osmotic pressure can be controlled using the following: (a) constant concentrated feed perfusion rate and different diluent perfusion rates, which result in different total perfusion rates; or (b) constant total perfusion rate and different concentrated feed perfusion rates; wherein the at least three concentrated feeds are added to each other with a fixed ratio (v / v / v) according to their multiple concentrations, to maintain the relative proportion of the medium components in the 1x serum-free cell culture perfusion medium. Therefore, osmotic pressure can be increased using the following: (a) constant concentrated feed perfusion rate and the diluent perfusion rate of reduction, which result in the total perfusion rate of reduction; or (b) constant total perfusion rate and the concentrated feed perfusion rate of increase and the diluent perfusion rate of reduction; wherein the at least three concentrated feeds are added to each other with a fixed ratio (v / v / v) according to their multiple concentrations, to maintain the relative proportion of the medium components in the 1x serum-free cell culture perfusion medium. Preferably, no further additive is added to the culture to increase osmotic pressure.

[0028] Those skilled in the art will know how to determine the optimal growth osmotic pressure level of the mammalian cells. In one embodiment, the optimal growth osmotic pressure level of the mammalian cells is approximately 280 to less than 350mOsm. Osmotic pressure is maintained at the level that is most suitable for growth, until reaching the target viable cell density of approximately half. Preferably, gradually or progressively increase osmotic pressure from the target viable cell density of approximately half, preferably increase to approximately 10-50% of the optimal growth osmotic pressure level. Osmotic pressure is increased to and maintained at the osmotic pressure level that suppresses the cell growth under the approximately target viable cell density, wherein in one embodiment, the osmotic pressure level that suppresses the cell growth of mammalian cells is approximately 350mOsm or higher, preferably approximately 380mOsm or higher. Increasing osmotic pressure reduces or eliminates the needs that cell is discharged during the production phase.

[0029] When osmotic pressure is increased, cell growth is inhibited to maintain a sustainable viable cell density without cell expulsion. The yield of the heterologous protein produced in the cell culture is increased by at least 5-50% relative to the yield of a control cell culture in which osmotic pressure is not increased.

[0030] Typically, using the methods of the present invention, the specific perfusion rate of cells (pl / cell / day) is reduced by at least 50% relative to the specific perfusion rate of cells in 1× serum-free cell culture medium.

[0031] In certain embodiments, the reaction vessel of the cell culture and / or bioreactor comprises 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.

[0032] The heterologous protein can be a therapeutic protein, an antibody, or a therapeutically effective fragment thereof. The mammalian cell can 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 cell can 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.

[0033] According to the method of the present invention, one or more supplements selected from the list consisting of an antifoaming agent, a base, glutamine and glucose may further be added separately (ie additionally) to the cell culture.

[0034] Also provided is a method of producing a therapeutic protein using the method according to the invention.

[0035] Also provided is the use of a serum-free cell culture perfusion medium according to the present invention or a serum-free cell culture perfusion medium obtainable by a method according to the present invention for culturing mammalian cells, in particular for culturing mammalian cells in perfusion culture. In certain embodiments, 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. Also provided is the use of a serum-free cell culture perfusion medium according to the present invention for adding the at least three separate aqueous concentrate feeds separately to a reaction vessel of a cell culture and / or bioreactor.

[0036] Furthermore, the present invention provides for controlling osmotic pressure in perfusion cell culture using a compartmented serum-free cell culture perfusion medium according to the present invention or a serum-free cell culture perfusion medium obtainable by a method according to the present invention. Increasing the osmotic pressure of the cell culture inhibits cell growth and increases heterologous protein production. The yield of the heterologous protein produced in the cell culture is increased by at least 5-50% relative to the yield of a control cell culture in which osmotic pressure was not increased. In one embodiment, the growth inhibition is sufficient to maintain a sustainable viable cell density without cell expulsion. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 . Bioreactor and feed setup showing separate inlet additions for: acidic, basic and neutral feeds and diluent.

[0038] Figure 2 . Reactor volume exchange or perfusion rate over time, expressed per liter of bioreactor (L br ) of culture medium (L 培养基 ) and days, e.g., typical operating perfusion rates for a perfusion culture with a feeding strategy using a combination of three medium concentrates (MC; lower dashed line), MC in combination with a diluent (solid line), and the potential maximum perfusion rate of the combined feeds.

[0039] Figure 3 Viable cell density (+ / - 3 standard deviations; solid lines) and viability (+ / - 3 SD; dashed lines) for three 100-L bioreactor runs using a concentrated medium feed + diluent feed regimen. The intrinsic peak VCD for this cell line (i.e., without hyperosmotic inhibition of growth) is > 200e6 c / mL. By increasing the pre-peak osmotic pressure, culture growth is inhibited and peak VCD is suppressed.

[0040] Figure 4 Osmolality (mOsm) for three 100-L bioreactor runs, showing a gradual increase in osmolality until approximately day 6, when the target viable cell density was reached. From peak VCD onward, osmolality was maintained at >380 mOsm to inhibit cell proliferation.

[0041] Figure 5 Figure 2. Reactor volume exchange (also known as perfusion rate) of three 100 L bioreactor runs performed with different diluent volumes using a concentrated medium feed fixed at 0.5 vessel volumes per day (VVD). Different diluent volumes controlled the residual osmotic pressure in the culture vessel.

[0042] Figure 6 Permeate productivity (g / L) from three 100-L bioreactor runs using different diluent volumes (total perfusion rate varied) and a concentrated medium feed fixed at 0.5 vessel volumes per day (VVD) 生物反应器 Permeate productivity was calculated by converting the daily instantaneous titer of permeate (g / L) as measured by Cedex bioanalyzer. 培养基 ) multiplied by the daily perfusion rate (L 培养基 / L 生物反应器 / day) to calculate.

[0043] Figure 7 Daily specific productivity (Qp, pg / cell / day) of a CHO cell culture expressing recombinant IgG from three 100-L bioreactor runs performed with different diluent volumes (total perfusion rate varied) using a concentrated medium feed fixed at 0.5 vessel volumes per day (VVD). Daily Qp was estimated by summing the total productivity of the bioreactor system (i.e., product recovered via permeate and product retained within the bioreactor) and dividing by the daily viable cell density (VCD).

[0044] Figure 8 Cell-specific perfusion rate (nL / cell / day) of CHO cells in three 100-L bioreactor runs performed with different diluent volumes (total perfusion rate varied) using a concentrated medium feed fixed at 0.5 vessel volumes per day (VVD).

[0045] Figure 9 Viable cell density (VCD, e5c / mL; solid line) and viability (%; dashed line) of three BI CHO cell lines A (◇), B (□), and C (Δ) expressing different recombinant IgG molecules. Data are from a 2-L bioreactor scale using three concentrated media feeds and different ratios of sterile water diluent to maintain the target residual osmotic pressure, with a constant perfusion rate of two vessel volumes per day (VVD).

[0046] Figure 10 Residual osmolality (mOsm) of three BI CHO cell lines, A (◇), B (□), and C (Δ), expressing different recombinant IgG molecules. Data are from a 2-L bioreactor scale using three concentrated media feeds and different ratios of sterile water diluent to maintain the target residual osmolality, with a constant perfusion rate of two vessel volumes per day (VVD).

[0047] Figure 11 Reactor volume exchange (also known as perfusion rate; L medium / L bioreactor / day) for three BI CHO cell lines A (◇), B (□), and C (Δ) expressing different recombinant IgG molecules. Data are from a 2-L bioreactor scale using three concentrated media feeds and different ratios of sterile water diluent to maintain the target residual osmotic pressure, with a constant perfusion rate of two vessel volumes per day (VVD).

[0048] Figure 12 Permeate productivity (g / L) of three BI CHO cell lines A (◇), B (□) and C (Δ) expressing different recombinant IgG molecules 生物反应器 Permeate productivity was calculated by converting the daily instantaneous titer of permeate (g / L) as measured by Cedex bioanalyzer. 培养基 ) multiplied by the daily perfusion rate (L培养基 / L 生物反应器 Data are from a 2-L bioreactor scale using three concentrated media feeds and different ratios of sterile water diluent to maintain target residual osmolality, with a constant perfusion rate of two vessel volumes per day (VVD).

[0049] Figure 13 Daily specific productivity (Qp, pg / cell / day) of three BI CHO cell lines A (◇), B (□), and C (Δ) expressing different recombinant IgG molecules. Daily Qp was estimated by adding the total productivity of the bioreactor system (i.e., product recovered via permeate and product retained within the bioreactor) and dividing by the daily viable cell density (VCD). Data are from a 2-L bioreactor scale using three concentrated media feeds and different ratios of sterile water diluent to maintain the target residual osmotic pressure, with a constant perfusion rate of two vessel volumes per day (VVD).

[0050] Figure 14 Cell-specific perfusion rate (CSPR; nL / cell / day) of three BI CHO cell lines A (◇), B (□) and C (Δ) expressing different recombinant IgG molecules. Data are from a 2-L bioreactor scale using three concentrated media feeds and different ratios of sterile water diluent to maintain the target residual osmotic pressure, with a constant perfusion rate of approximately two vessel volumes per day (VVD). The differences in CSPR between cell lines are due to differences in viable cell density (VCD) (for VCD and viability, see Figure 9 ). The ratio of the feeds relative to each other is kept constant, while the overall ratio of feed to diluent is adjusted based on a mass balance calculated based on osmotic pressure according to the following equation: Osmotic pressure input = Osmotic pressure output + Osmotic pressure consumption, where Osmotic pressure input is the osmotic pressure of the culture concentrate feed and diluent perfused into the bioreactor, Osmotic pressure output is the residual osmotic pressure of the bioreactor supernatant, and Osmotic pressure consumption is the osmotic pressure difference between input and output. Osmotic pressure consumption is used to calculate the osmotic pressure input required for a given desired osmotic pressure output. The respective concentrate feed and diluent perfusion rates are then calculated to achieve the required osmotic pressure input at a total perfusion rate of 2 vvd.

[0051] Figure 15. CHO DG44 cell line (cell line A, Δ) and two different CHO-K1 cell lines (cell line B □, ◇; cell line C x, x) run in duplicate were cultured in a 2-L bioreactor using three concentrated media feeds fixed at a total of 0.5 vessel volumes per day (VVD) using different diluent volumes. All cell lines expressed different recombinant IgG molecules. Shown are (A) viable cell density (VCD; e5c / mL); (B) viability (%); (C) permeate productivity (g / L / day) by converting the daily instantaneous titer of permeate (g / L) as measured by a Cedex bioanalyzer 培养基 ) multiplied by the daily perfusion rate (L 培养基 / L 生物反应器 / day); and (D) exchanged with the reactor volume (L 培养基 / L 生物反应器 / day) represents the perfusion rate.

[0052] Figure 16. CHO-K1 cell lines expressing recombinant IgG in a glutamine synthetase (GS) selection system were cultured in a 2L bioreactor. Operation was performed in "MC different, total VVD fixed" (◇) or "MC fixed, total VVD different" (□) perfusion control mode. "MC different, total VVD fixed" refers to a constant daily total container volume (VVD) perfusion rate, which is achieved by changing the perfusion rate of the combined culture medium concentrate (MC) and simultaneously changing the diluent rate to maintain 2VVD. "MC fixed, total VVD different" refers to a constant perfusion rate of MC at 0.5VVD and different diluent perfusion rates with respect to the overall fluctuating perfusion rate. Shown are (A) viable cell density (VCD, e5c / mL; primary axis) and viability (%; secondary axis), (B) osmotic pressure (mOsm), (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 DESCRIPTION

[0053] Definitions of certain terms are provided below. Generally, unless otherwise stated or defined, any term presented in this disclosure should be given its ordinary meaning in the art.

[0054] The general embodiment "comprising" or "comprised" encompasses the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting manner. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless expressly stated to refer to the singular only.

[0055] As used herein, the term "perfusion" refers to maintaining a cell culture bioreactor, wherein the culture medium of equivalent volume is added and removed from the reactor simultaneously, while the 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 the continuous removal of cell waste. Perfusion is commonly used to obtain a much higher cell density than conventional bioreactor batches or fed-batch conditions, and therefore obtains a higher volumetric productivity. Secretory protein products can be harvested continuously while cells are retained in the reactor, such as by filtration, alternating tangential flow (ATF), cell sedimentation, ultrasonic separation, hydrocyclone or any other method well 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 grow (homogeneous culture) or be attached to the surface or be embedded in different devices (heterogeneous culture) in suspension culture. To keep the working volume in the bioreactor constant, the harvest rate and cell drain (fluid removal) should be equal to the predetermined perfusion rate. Cultures are typically initiated via batch culture, and perfusion begins 2-3 days after inoculation, when the cells are still in the exponential growth phase and before nutrient limitation occurs. 6 Inoculation with 5 cells / ml or more) may require earlier or even immediate perfusion. Thus, perfusion may be started from day 0 to day 4 after inoculation, preferably from day 0 to day 3 after inoculation.

[0056] By adding fresh medium and removing spent medium simultaneously, perfusion-based methods offer potential improvements over batch and fed-batch methods. Large-scale commercial cell culture strategies can reach 60-90x10 6 High cell densities of >1x10 cells / mL have been achieved, where approximately one-third to more than half of the reactor volume may be biomass. 8Perfusion cultures are typically initiated with a batch culture start-up, which lasts for a day or more to achieve rapid initial cell growth and biomass accumulation, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture and simultaneous removal of spent medium, with cells retained throughout the growth and production phases of the culture. While maintaining the cells, various methods such as sedimentation, centrifugation, or filtration can be used to remove spent medium. Perfusion flow rates have been employed ranging from a fraction of a vessel volume per day (VVD) to many vessel volumes per day.

[0057] As used herein, the term "perfusion rate" is the volume added and removed, and is typically measured every day. Perfusion rate depends on cell density and culture medium. While ensuring sufficient nutrient addition rates and by-product removal rates, perfusion rate should be minimized to reduce the dilution of the product of interest, i.e., harvest titer. Perfusion typically begins on the 0-3 day after inoculation, when cells are still in the exponential growth phase, and therefore perfusion rate may increase during the culture period. The increase in perfusion rate can be incremental or continuous, i.e., based on cell density or nutrient consumption. It typically starts with 0.5 or 1 container volume per day (VVD) and can rise to approximately 5VVD. Preferably, the perfusion rate is between 0.5 and 2VVD. The increase can reach 0.5 to 1VVD per day. For the continuous increase of perfusion, a biomass probe can be connected to a harvest pump based on the desired cell specific perfusion rate (CSPR) so that the perfusion rate increases as a linear function of the cell density determined by the biomass probe. CSPR is equal to the perfusion rate per cell density, and the ideal CSPR depends on the cell line and cell culture medium. The 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 specific cell line. Using at least three separate aqueous concentrated feeds, the supply of nutrients is separated from the overall VVD and CSPR, allowing very low CSPR, such as 5 to 20 pL / cell / day, preferably even 5 to 10 pL / cell / day. 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, this significantly reduces culture medium consumption, especially preparation culture medium consumption, as in the 14-day culture period.

[0058] As used herein, the term "steady state" refers to a state in which the cell density and bioreactor environment remain relatively constant. This can be achieved by cell expulsion, nutrient limitation, 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 maintain the cells in steady state. Typical viable cell densities at steady state are 10 to 50 x 10 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 the bioreactor.

[0059] The terms "cell discharge (cell bleed)" and "cell discharge (cell bleeding)" are used interchangeably herein, and refer to removing cells and culture medium from a bioreactor to maintain constant, sustainable viable cell density in a bioreactor. Described constant, sustainable viable cell density can also be referred to as target cell density. This cell discharge can be carried out using a draw tube and a peristaltic pump with the flow velocity limited. The size of the pipeline should be suitable, because the pipe is too narrow to easily cause cell aggregation and obstruction, and if too large, the cell may settle. Cell discharge can be determined based on growth rate, and therefore viable cell density can be limited in a continuous manner to the desired volume. Alternatively, cells can be removed and replaced with culture medium with a specific frequency (for example, once a day), so that cell density is maintained in a predictable scope. Ideally, the cell discharge rate equals growth rate, to maintain stable cell density.

[0060] In some embodiments, the cell that is discharged from the cell is removed and the product of interest is discarded. In contrast to permeate, the cell discharge contains cells, which makes the product storage before purification more difficult and may have a negative impact on product quality. Therefore, before storage and product purification, cells must be removed continuously, which will be time-consuming and labor-intensive and cost-inefficient. For slow-growing cells, the cell discharge may be approximately 10% of the fluid removed, and for cells that grow rapidly, the cell discharge may be approximately 30% of the fluid removed. Therefore, the product loss caused by the cell discharge may be approximately 30% of the total product produced. As used herein, " permeate " refers to the harvested product of the cell that has been separated and will be retained in the culture vessel.

[0061] The terms "culture" or "cell culture" are used interchangeably and refer to a cell population maintained in a culture medium under conditions suitable for allowing the cell population to survive and / or grow. The present invention relates only to mammalian cell cultures, and in particular to mammalian perfusion cell cultures. Mammalian cells can be cultured in suspension or while attached to a solid support. As will be clear to those skilled in the art, a cell culture refers to a composition comprising a cell population and a culture medium in which the population is suspended. The specific type of cell culture is not particularly limited and can encompass all forms and techniques of cell culture, including but not limited to perfusion, continuous, finite, suspension, adherent or monolayer, anchorage-dependent, and 3D cultures. As used herein, the term cell culture refers to serum-free cell culture.

[0062] As used herein, the term "culturing" refers to the process of growing or maintaining mammalian cells under controlled conditions and under conditions that support cell growth and / or survival. As used herein, the terms "maintaining cells" and "culturing cells" are used interchangeably. Cultivating can also refer to the step of inoculating cells in a culture medium.

[0063] As used herein, the term "batch culture" is a discontinuous process in which cells are grown in a fixed volume of culture medium for a short period of time and then fully harvested. The cell density of cultures grown using batch methods increases until a maximum cell density is reached, after which the viable cell density decreases as the medium components are consumed and the levels of metabolic byproducts (such as lactate and ammonia) accumulate. Harvesting typically occurs after achieving a maximum cell density (typically 5-10 x 10 6 cells / mL, depending on media formulation, cell line, etc.) or shortly thereafter, typically about 3 to 7 days.

[0064] As used herein, the term "fed-batch culture" improves upon a batch process by providing a bolus or continuous feed of culture medium to replenish those culture medium components that have been consumed. Because fed-batch cultures receive additional nutrients throughout the culture, they have the potential to achieve higher cell densities (>10 to 30 x 10 cells) when compared to batch methods. 6In some embodiments, the feed-batch culture method is to use a feed-batch method to culture the cells of the cell culture medium in a manner that allows the production of a desired cell density (e.g., cells / ml, depending on the culture medium formulation, cell line, etc.) and an increased product titer. Unlike batch processes, biphasic cultures can be created and maintained by manipulating the feed-batch strategy and culture medium formulation 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, compared to batch culture, fed-batch culture is likely to achieve higher product titers. As with batch methods, over time, the accumulation of metabolic byproducts will lead to a decrease in cell viability because these byproducts will gradually accumulate in the cell culture medium, which will limit the duration of the production phase to about 1.5 to 3 weeks. Fed-batch culture is discontinuous and is typically harvested when the metabolic byproduct level or culture viability reaches a predetermined level.

[0065] The terms "polypeptide" or "protein" are used interchangeably herein with "amino acid residue sequence" and refer to polymers of amino acids. These terms also include proteins that are post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation, or protein processing. Modifications and changes can be made in the structure of a polypeptide, such as fusions with other proteins, amino acid sequence substitutions, deletions, or insertions, while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence, and a protein with the same properties can be obtained. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acid. The term "polypeptide" generally refers to a sequence having more than 10 amino acids, and the term "peptide" refers to a sequence of up to 10 amino acids in length.

[0066] As used herein, the term "heterologous protein" refers to a polypeptide derived from an organism or species different from the host cell. A heterologous protein is encoded by a heterologous polynucleotide that is experimentally placed into a host cell that does not naturally express the protein. A heterologous polynucleotide may also be referred to as a transgene. Therefore, it may be a gene or open reading frame (ORF) encoding a heterologous protein. When used with reference to a protein, the term "heterologous" may also indicate that the protein comprises amino acid sequences that are not identical in relationship to one another or that are not identical in length to one another in nature. Therefore, it also encompasses recombinant proteins. Heterologous may also refer to a polynucleotide sequence, such as a gene or transgene, or a portion thereof, that is inserted into the genome of a mammalian cell in a position in which it is not normally present. In the present invention, a heterologous protein is preferably a therapeutic protein.

[0067] 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, a cell culture medium provides the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal cell growth and / or survival, as well as buffers and salts. The culture medium may also contain supplemental 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 (inorganic compounds typically present at very low final concentrations) including trace metals, amino acids (including non-proteinogenic amino acids), lipids, antioxidants, glucose, and / or other energy sources, such as organic acids; as described herein. In addition, a surfactant may be included in the culture medium. In certain embodiments, the culture medium is advantageously formulated to a pH and salt concentration that are optimal for cell survival and proliferation. "Cell culture perfusion medium" or "perfusion medium" is a medium used for continuous perfusion. Those skilled in the art will appreciate that further components that are not part of the cell culture medium may be added to the cell culture during the culture period. For example, a defoaming agent may be added separately. Furthermore, glucose and / or glutamine may be added alone or in addition to the glucose provided with the cell culture medium. Finally, a base (e.g., sodium carbonate or sodium hydroxide) may be added to the cell culture to control the pH during the culture period.

[0068] Examples of amino acids in cell culture media include, but are not limited to, proteinogenic 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 salts or derivatives thereof, and non-proteinogenic amino acids such as hydroxyproline, ornithine, α-amino-n-butyric acid, and salts or derivatives thereof. Derivatives thereof 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 the like, and hydrates thereof. 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, and the like, 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, p-aminobenzoic acid, pyridoxal, pyridoxine, riboflavin, thiamine, tocopherol, vitamin B12, retinol (vitamin A), ascorbate, and the like, and their salts. Examples of polyamines include, but are not limited to, putrescine, spermidine, and spermine; organic acids include, but are not limited to, taurine or alternative carbon sources such as succinic acid, pyruvic acid, and citric acid; fatty acids include, but are not limited to, linoleic acid, linolenic acid, palmitic acid, and oleic acid; surfactants include, but are not limited to, pluronic F68; buffers include, for example, phosphate buffer (monobasic and dibasic phosphates); antioxidants include, for example, reduced glutathione or lipoic acid; and examples of chelating agents include, but are not limited to, citrate or ethylenediaminetetraacetic acid (EDTA). Energy sources include, for example, pyruvic acid or dextrose. Other compounds that may be present in the culture medium include, for example, ethanolamine, taurine, i-inositol, and proteins such as insulin or insulin-like growth factor. Compounds may also be added to prepare dry powder culture mediums, such as dextrose, which may be added only for grinding purposes rather than as a culture medium component.

[0069] Substratum according to the present invention is the serum-free perfusion medium (or serum-free cell culture perfusion medium) added after 0 to 4 days of inoculation, i.e. perfusion culture starts from the 0th day to the 4th day of cell culture. Therefore, it can also be referred to as the cell culture perfusion medium feeding, because it is usually added after inoculation. Perfusion cell culture can have different cultivation stages, including growth stage and production stage. The specific culture medium used during growth stage (growth medium) and production stage (production medium) can be specially designed to be implemented in the specific stage. Usually, before starting to perfuse with production medium, cells are seeded in growth medium. In addition, before replacing growth medium with production medium, perfusion may have begun. In certain embodiments, cell culture medium according to the present invention is production medium. However, two kinds of culture mediums (growth medium and production medium) are all complete mediums and allow the maintenance and / or growth (that is, not needing to mix with further culture medium) of cell culture. This is in contrast to the feed medium or fed-batch medium used in fed-batch culture, which is typically an incomplete medium that replenishes consumed nutrients, but is typically reduced in components such as salts and buffers to reduce the osmotic pressure of the medium and allow further concentration of the feed medium. Without mixing with the basal medium or inoculum medium, the medium is generally insufficient to support the maintenance of the cell culture.

[0070] 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. Since perfusion medium provides a stable source of fresh nutrients and is constantly removed from the bioreactor, it is a complete medium that allows the maintenance and / or growth of cell culture. The term "complete medium" refers to a nutrient solution containing all components of the culture medium intended to be present in the cell culture.

[0071] The serum-free cell culture perfusion medium according to the present invention is a complete medium and can exist in a separated form comprising at least three separate aqueous concentrated feeds and a diluent, wherein the first concentrated feed is an alkaline concentrated feed, the second concentrated feed is an acidic concentrated feed, and the third concentrated feed is a neutral concentrated feed, or exists as a resulting serum-free cell culture perfusion medium after mixing. The term "serum-free cell culture perfusion medium" that does not clearly characterize the culture medium being separated refers to the resulting serum-free cell culture perfusion medium formed after mixing. Since the separated culture medium is used to directly add to the reaction vessel of the cell culture and / or bioreactor, the resulting serum-free cell culture medium is not usually present in a pure or isolated form, but rather is a mixture with an already existing cell culture (i.e., culture medium and cells). Therefore, it is important that the separated culture medium regulates pH after mixing. However, since the pH of the culture may change during cell culture, it may still be necessary to maintain a constant pH using an alkali to regulate pH during culture.

[0072] 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 separated from serum derived from any animal or human. Various tissue culture media including defined culture media are commercially available, for example, any one or combination of the following cell culture media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal 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 medium such as EX-CELL TM 300 series (JRH Biosciences, Lenexa, Kansas), etc. Serum-free versions of these media are also available. Depending on the requirements of the cells to be 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.

[0073] As used herein, the term "protein-free" refers to a cell culture medium that does not contain any protein. Thus, it lacks proteins isolated from animals or humans, serum-derived proteins, or recombinantly produced proteins, such as those produced in mammalian, bacterial, insect, or yeast cells. Protein-free culture medium may contain a single recombinant protein, such as insulin or insulin-like growth factor, but only if such addition is explicitly stated.

[0074] As used herein, the term "chemically defined" refers to a culture medium that is serum-free and does not contain any hydrolysate (e.g., protein hydrolysate derived from yeast, plants, or animals). Preferably, the chemically defined medium is also protein-free or contains only selected recombinantly produced (non-animal derived) proteins, such as insulin or insulin-like growth factor. Chemically defined medium is composed of a mixture of characterized and purified substances. An example of a chemically defined medium is the CD-CHO culture medium from Invitrogen (Carlsbad, CA, US).

[0075] As used herein, the term "suspension cells" or "non-adherent cells" refers to cells cultured in suspension in liquid culture medium. Adherent cells (such as CHO cells) can be adapted to grow in suspension and thereby lose their ability to attach to the surface of a container or tissue culture dish.

[0076] As used herein, the term "bioreactor" means any container that can be used for cell culture growth. A bioreactor can have any size, as long as it can be used for culturing cells; Typically, a bioreactor is sized to the volume of the cell culture suitable for growing therein. Typically, a bioreactor will be at least 1 liter, and 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 liters. 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 relevant considerations, one of ordinary skill in the art will appreciate and will be able to select a suitable bioreactor for use in practicing the present invention. The cell culture used for the method of the present invention can be grown in any bioreactor suitable for perfusion culture. The specific type of bioreactor is not particularly limited and may encompass all types of bioreactors suitable for perfusion cell culture.

[0077] 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 (eg, trypan blue dye exclusion).

[0078] 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 fraction of cells that are alive at a particular time relative to the total number of living and dead cells in culture at that time.

[0079] 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. The titer can be expressed in milligrams or micrograms of polypeptide or protein per milliliter (or other volumetric measure) of culture medium.

[0080] As used herein, the term "yield" refers to the amount of heterologous protein produced in a perfusion culture over a specific period of time. "Total yield" refers to the amount of heterologous protein produced in a perfusion culture over the entire run.

[0081] As used herein, the terms "reduction," "reduced," or "decline" generally mean a decrease of at least 5% compared to a reference level, such as a decrease of 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 up to and including a 100% decrease, or any integer between 10-100%, compared to a control mammalian cell culture cultured under the same conditions using the same serum-free cell culture medium, such as wherein osmotic pressure is not increased during culture, particularly during perfusion culture.

[0082] As used herein, the terms "enhancement," "enhanced," "enhanced," "increase," or "increased" generally mean an increase of at least 5% compared to a control cell, 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 10-300%, compared to a control mammalian cell culture cultured under the same conditions using the same serum-free cell culture medium, such as wherein osmotic pressure is not increased during culture, particularly during perfusion culture.

[0083] As used herein, a "control cell culture" or "control mammalian cell culture" is a cell culture that is identical to the cell culture to which it is compared, using the same serum-free cell culture medium according to the present invention comprising medium components grouped into at least three aqueous concentrated feeds and a diluent, except that the osmotic pressure is not increased during the culture, in particular during perfusion culture.

[0084] As used herein, the term "mammalian cell" is a cell line suitable for producing heterologous proteins, preferably therapeutic proteins, more preferably secreted recombinant therapeutic proteins. Preferred mammalian cells according to the present 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 non-transformed cells or cells that are part of an organ structure. Preferred mammalian cells are BHK21, 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. Particularly preferred are CHO-DG44, CHO-K1 and BHK21, and even more preferred are CHO-DG44 and CHO-K1 cells. Most preferred are CHO-DG44 cells. Also contemplated are mammalian cells, particularly glutamine synthetase (GS)-deficient derivatives of CHO-DG44 and CHO-K1 cells. The mammalian cells may further comprise one or more expression cassettes encoding heterologous proteins, preferably recombinant secretory therapeutic proteins. The mammalian cells may also be murine cells, such as murine myeloma cells, such as NS0 and Sp2 / 0 cells or derivatives / progeny of any of these cell lines. However, derivatives / progeny of these cells, other mammalian cells (including but not limited to human, mouse, rat, monkey and rodent cell lines) may also be used in the present invention, particularly for the production of biopharmaceutical proteins.

[0085] As used herein, the term "growth phase" refers to the stage of cell culture, in which cells proliferate exponentially and the viable cell density in the bioreactor increases. The cells in culture typically follow a standard growth pattern for proliferation. After the culture is inoculated, it can be a lag phase, which is a slow growth period when cells adapt to the culture environment and prepare for rapid growth. The growth phase (also referred to as the logarithmic growth phase or logarithmic phase) is the period in which cells proliferate exponentially and consume the nutrients of the growth medium. It is then followed by the production phase.

[0086] The term "production phase" refers to the phase of cell culture that begins once harvesting begins, which can be when or before the target viable cell density is reached. 生物反应器 / day, harvesting is usually started. Typical target cell density is 10x10 6 cells / ml to about 120x10 6 The target cell density according to the present invention is therefore at least 30 x 106 cells / ml, at least 40x10 6 cells / ml, at least 50x10 6 cells / ml, at least 60x10 6 cells / ml, at least 80x10 6 cells / ml or at least 100x10 6 cells / ml. The target cell density can even be as high as 100x10 6 cells / ml to 200x10 6 cells / ml, preferably 120x10 6 cells / ml to 150x10 6 cells / ml.

[0087] In certain embodiments herein, when the production phase begins, the osmotic pressure of the cell culture is increased to the level that causes growth inhibition. Preferably, osmotic pressure increases gradually or progressively from the level that is most suitable for growth. Therefore, before reaching the target viable cell density, it is necessary to increase osmotic pressure. Preferably, starting from the target viable cell density of about half, osmotic pressure is increased gradually or progressively to the level that causes growth inhibition from the level that is most suitable for growth. This allows once reaching the target viable cell density, just reaching the osmotic pressure level that causes growth inhibition. Importantly, maintain high osmotic pressure (that is, the osmotic pressure level that causes growth inhibition) until cultivation ends. It will be understood by those skilled in the art that removing osmotic pressure will remove growth inhibition.

[0088] The terms "growth arrest," "growth inhibition," and "growth suppression" are used synonymously herein and refer to cells that stop increasing in number (i.e., stop dividing). The cell cycle comprises an interphase and a mitotic phase. The interphase consists of three phases: DNA replication is restricted to the S phase; G1 is the gap between the M and S phases, and G2 is the gap between the S and M phases. During the M phase, the nucleus divides, and then the cytoplasm divides. In the absence of mitotic signals to proliferate or in the presence of compounds that induce growth arrest, the cell cycle arrests. Cells can partially disassemble their cell cycle control system and exit the cycle to a specialized non-dividing state called G0. Growth suppression can be easily assessed by determining the viable cell density over time. Preferably, the cells are maintained at a viable cell density with a variation of ≤30%, more preferably ≤20%. More preferably, the cells are maintained at a target viable cell density with a variation of ≤30%, more preferably ≤20%.

[0089] Cell culture perfusion medium

[0090] In one aspect of the present disclosure, a serum-free cell culture perfusion medium is disclosed, comprising medium components grouped into 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 serum-free cell culture perfusion medium is pH-adjusted to a neutral pH. In a preferred embodiment, the at least three separate aqueous concentrate feeds are not premixed prior to addition to the reaction vessel of the cell culture and / or bioreactor. Premixing of two or more feeds is not ideal because precipitation may occur upon mixing. Thus, 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 mixing the at least three separate aqueous concentrated feeds directly in the reaction vessel of the cell culture and / or bioreactor. In a preferred embodiment, the serum-free cell culture perfusion medium comprises medium components grouped into at least three separate aqueous concentrated feeds as described and a diluent. This includes a serum-free cell culture perfusion medium consisting of the at least three separate aqueous concentrated feeds and a diluent. The 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 medium. Those skilled in the art will appreciate that perfusion culture is typically performed using mammalian cells, and thus the perfusion medium is a perfusion medium for mammalian cells.

[0091] Those skilled in the art will also appreciate that further components that are not part of the cell culture medium can be added to the cell culture during the culture period. For example, a defoaming agent can be added separately. Furthermore, glucose and / or glutamine feeds can be added separately, or in addition to the glucose and / or glutamine provided with the cell culture medium. Finally, a base (e.g., sodium carbonate or sodium hydroxide) can be added to the cell culture to control the pH during the culture period.

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

[0093] In order 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 concentrated feeds are mixed in a certain 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 multiple concentrations of the at least three separate aqueous concentrated feeds relative to each other, and the at least three separate aqueous concentrated feeds are diluted with the diluent. Although the diluent for the serum-free cell culture perfusion medium according to the present 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 does not require additional storage space for pre-made components. The ratio (v / v) of the cumulative volume of the diluent and the at least three independent aqueous concentrated feeds determines the multiple concentration of the serum-free cell culture perfusion medium described in the reaction vessel of cell culture and / or bioreactor, and the at least three independent aqueous concentrated feeds are added in the reaction vessel of cell culture and / or bioreactor to provide the gained serum-free cell culture perfusion medium with pH adjusted to nearly neutral pH. Therefore, the advantage of using the at least three independent aqueous concentrated feeds is that the multiple concentration of the substratum can adapt to viable cell density and nutritional needs (maintaining nutritional balance). Osmotic pressure can be used as an alternative indicator for estimating nutritional balance inside and outside the system. Therefore, osmotic pressure balance can be used for calculating the adjustment of concentrated feed cumulative volume (with their fixed ratio relative to each other) and diluent feed rate, to achieve desired residual osmotic pressure and nutritional level.

[0094] After mixing the at least three separate concentrated aqueous feeds and the diluent, the resulting serum-free cell culture perfusion medium is pH-adjusted to a neutral pH. This means that the pH is automatically adjusted after mixing without the need for adding titrants such as NaOH or HCl. After mixing the at least three separate concentrated aqueous feeds and the diluent, the pH of the medium should be a neutral pH, 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 concentrated feed can be 2x to 80x concentrated feed, preferably 20x to 40x concentrated feed, more preferably 20x to 30x concentrated feed, and most preferably 25x feed. Usually preferably higher concentrated feed. However, in order to obtain best results, such as the alkaline concentrated feed can be prepared as a concentrated feed that is not maximally concentrated, to better match the nearly neutral and / or acidic feed. This will also make the titrant safety in the concentrated feed (such as the alkaline concentrated feed). The nearly neutral concentrated feed can be 2x to 50x concentrated feed, preferably 10x to 40x concentrated feed, more preferably 20x to 30x concentrated feed, and most preferably 25x concentrated feed. The acidic concentrated feed can be 2x to 40x concentrated feed, 4x to 20x concentrated feed, 5x to 12x concentrated feed or 6x to 10x concentrated feed. Usually, preferably higher concentrated feed (alkaline, acidic and neutral, combined and single). However, to obtain the best results, for example, the alkaline concentrated feed can be prepared as a concentrated feed that is not maximally concentrated (e.g., less than 80x) to better match the near-neutral and / or acidic feed. This will also make the titrant in the concentrated feed (e.g., the alkaline concentrated feed) safe.

[0096] In one embodiment, 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, preferably the alkaline concentrated feed is a 20x to 40x concentrated feed, the acidic concentrated feed is a 4x to 20x concentrated feed, and the near-neutral concentrated feed is a 10x to 40x concentrated feed, more preferably the alkaline concentrated feed is a 20x to 30x concentrated feed, the acidic concentrated feed is a 5x to 12x concentrated feed, and the near-neutral concentrated feed is a 20x to 30x concentrated feed, and most preferably the alkaline concentrated feed is a 25x concentrated feed, the acidic concentrated feed is a 6x to 10x concentrated feed, and the near-neutral concentrated feed is a 25x concentrated feed. In a specific embodiment, the alkaline concentrated feed and the near-neutral concentrated feed are substantially similarly concentrated. Thus, for example, the alkaline concentrated feed is a 20x to 30x concentrated feed and the near neutral concentrated feed is a 20x to 30x concentrated feed, or the alkaline concentrated feed is a 25x concentrated feed and the near neutral concentrated feed is a 25x concentrated feed, and the acidic concentrated feed is maximally concentrated.

[0097] In the serum-free cell culture perfusion medium, 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 having a pH adjusted to a neutral pH. Thus, 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 medium components in the 1x serum-free cell culture perfusion medium (1x formulation). In other words, the ratios of the feeds to each other should be such that the original ratios of the 1x formulation are maintained. For example, if the alkaline concentrated feed is a 25x concentrated feed, the acidic concentrated feed is a 6x concentrated feed, and the near-neutral concentrated feed is a 25x concentrated feed, the concentrated feeds are added at a ratio of 1:4.2:1, or if the alkaline concentrated feed is a 30x concentrated feed, the acidic concentrated feed is a 10x concentrated feed, and the near-neutral concentrated feed is a 30x concentrated feed, the concentrated feeds are added at a ratio of 1:3:1. In addition, the ratio (v / v) of the diluent to the cumulative volume of the at least three separate aqueous concentrated feeds in the resulting serum-free cell culture perfusion medium adjusted to a neutral pH determines the osmotic pressure of the serum-free cell culture perfusion medium. The ratio (v / v) of the diluent to the cumulative volume of the at least three separate aqueous concentrated feeds in the serum-free cell culture perfusion medium adjusted to a neutral pH also determines the multiple concentration of the serum-free cell culture perfusion medium. The multiple concentration can be any value from 0.1x to the maximum multiple concentration, but is usually between 0.5x and 2x, preferably between 1x and 2x. The maximum multiple concentration (n 最大 X) can be calculated as follows:

[0098] 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)),

[0099] in

[0100] n 最大 X is the maximum multiple concentration after mixing the three separate aqueous concentrate feeds;

[0101] n 碱性 X is n times the concentration of the alkaline concentrated feed;

[0102] n 酸性X is n times the concentration of the acidic concentrated feed;

[0103] n 中性 X is n times the concentration of the near-neutral concentrated feed; and

[0104] * Represents the mathematical operation multiplication.

[0105] For example, if the alkaline concentrated feed is a 25x concentrated feed, the acidic concentrated feed is a 6x concentrated feed, and the near-neutral concentrated feed is a 25x concentrated feed, the maximum multiple concentration of the three separate aqueous concentrated feeds after mixing is 4.1x. Therefore, the production medium consumption is reduced by about 75%. If the alkaline concentrated feed is a 30x concentrated feed, the acidic concentrated feed is a 10x concentrated feed, and the near-neutral concentrated feed is a 30x concentrated feed, the maximum multiple concentration of the three separate aqueous concentrated feeds after mixing is 6x. Therefore, the production medium consumption is reduced by more than 80%. In addition, the use of concentrated feeds allows the multiple concentration of the serum-free cell culture medium in the cell culture and / or bioreactor to be adjusted, and therefore allows a higher viable cell density to be maintained at a similar or only moderately increased perfusion rate and thereby at a reduced cell-to-cell perfusion rate.

[0106] The serum-free cell culture perfusion medium comprises an alkaline concentrate, an acidic concentrate and a near-neutral concentrate. A near-neutral concentrate feed refers to a pH of 7.5±1.0. Therefore, the pH of the near-neutral concentrate feed is 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 pressure space in the resulting serum-free cell culture perfusion medium. Therefore, the pH of the near-neutral concentrate feed may be slightly alkaline at a pH of up to about 8.5. Preferably, the pH of the near-neutral pH is about 7 to about 8.5, more preferably about 7.5 to about 8.5.

[0107] The pH of the alkaline concentrate feed may be about 9 or higher, such as a pH of about 9 to about 11, preferably a pH of about 9.8 to about 10.8, and more preferably a pH of about 9.8 to about 10.5. The pH of the acidic concentrate feed may be about 5 or lower, such as a pH of about 2 to about 5, preferably a pH of about 3.6 to about 4.8, and more preferably a pH of 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.

[0108] In one embodiment, the pH of the alkaline concentrated feed is about 9 or higher, the pH of the acidic concentrated feed is about 5 or lower, and the pH of the near-neutral concentrated feed is about 7 to about 8.5. Preferably, the pH of the alkaline concentrated feed is about 9 to about 11, the pH of the acidic concentrated feed is about 2 to about 5, and the pH of the near-neutral concentrated feed is about 7 to about 8.5; more preferably, the pH of the alkaline concentrated feed is about 9.8 to about 10.8, the pH of the acidic concentrated feed is about 3.6 to about 4.8, and the pH of the near-neutral concentrated feed is about 7 to about 8.5; and most preferably, the pH of the alkaline concentrated feed is about 9.8 to about 10.5, the pH of the acidic concentrated feed is about 3.8 to about 4.5, and the pH of the near-neutral concentrated feed is about 7.5 to about 8.5.

[0109] The 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. In addition, medium components that are particularly insoluble in aqueous solution can be separated into separate feeds. For example, choline chloride can be provided with the near-neutral concentrated feed and the acidic concentrated feed to achieve the desired concentration in the final serum-free cell culture medium.

[0110] The near-neutral concentrated feed preferably contains all vitamins that are soluble at neutral pH. Further, the near-neutral concentrated feed preferably does not contain any metals. Since metals may interact with certain vitamins, the vitamins are preferably kept separate from the metals. Therefore, it is preferred that the vitamins are provided in the near-neutral concentrated feed and alternatively in the acidic concentrated feed. An exception is folic acid, which can also be provided with the alkaline feed. Therefore, in one embodiment, the vitamins and metals are provided in separate feeds, preferably the vitamins are in the near-neutral feed and the metals are in the acidic feed. However, vitamins that are poorly soluble in aqueous solutions at neutral pH can also be in the acidic feed. For example, vitamins such as pantothenate, thiamine, choline chloride and / or pyridoxine can also be provided in the acidic feed. In addition, vitamins that are generally poorly soluble in aqueous solutions, such as choline chloride, can be present in the neutral feed and the acidic feed. The neutral concentrated feed can also contain compounds such as L-α-amino-n-butyric acid, iso-inositol and / or the 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.

[0111] Salt and metal are preferably provided in the described acidic concentrated feed.For example, but not limited to, the described acidic concentrated feed can comprise trace element, trace metal, inorganic salt, iron source chelating agent, polyamine and / or regulation and control hormone, as insulin or insulin-like growth factor.The aminoacid that is selected from lower group is preferably in the described acidic concentrated feed, and the described group is made up of the following: alanine, arginine, asparagine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline(R), serine, threonine, tryptophan and valine.In addition, surfactant, antioxidant and carbon source and optional ethanolamine and / or lipid acid can be provided in the described acidic concentrated feed and / or the described nearly neutral concentrated feed.

[0112] The alkaline concentrated feed is mainly composed of amino acids with maximum solubility at an alkaline pH of about 9 or higher. Preferably, the alkaline concentrated feed contains at least aspartic acid, histidine, tyrosine and cysteine. Cysteine ​​is water-soluble, but is easily oxidized to cystine, which has poor water solubility, at neutral pH. Therefore, cysteine ​​and / or cystine are preferably in the alkaline concentrated feed. Another compound that is soluble at an alkaline pH of about 9 or higher is folic acid. Therefore, folic acid can also be provided together with the alkaline feed. The amino acids that are not provided together with the alkaline concentrated feed are preferably provided together with the acidic concentrated 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 that are not provided together with the alkaline feed) can be provided in the acidic and / or near-neutral concentrated feed, preferably in the acidic concentrated feed.

[0113] Those skilled in the art will appreciate that, compared to feed media, such as those used for fed-batch culture, complete media are more difficult to provide as concentrates because they contain more components, particularly salts and buffers that increase osmotic pressure and thus limit osmotic pressure space. Furthermore, compared to prior art media (such as RPMI 1640 and DMEM / F12, etc.), modern nutrient-rich media are more difficult to provide as concentrates. These more modern nutrient-rich media are particularly rich in amino acids, typically containing amino acids in the mM range rather than in the μM range. Therefore, the serum-free cell culture perfusion medium according to the present invention is a medium containing amino acids, the concentration of which in 1x serum-free cell culture perfusion medium exceeds 50mM, preferably exceeds 70mM, more preferably exceeds 100mM, and even more preferably exceeds 120mM. Since glutamine is sometimes added separately, the serum-free cell culture perfusion medium preferably contains natural amino acids other than glutamine, the concentration of which in the resulting serum-free cell culture perfusion medium exceeds 50mM, preferably exceeds 70mM, more preferably exceeds 100mM, and even more preferably exceeds 120mM. Natural amino acids except glutamine refer to 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, for example, alanine and glycine. Natural amino acids also include derivatives of natural amino acids, such as dipeptides or cystine.

[0114] Those skilled in the art optimize the various processes for cell culture medium composition and for other process characteristics and culture performance. For example, and particularly in the case of materials where cell density is not very high, they can be tested in shake flasks. Where a higher oxygenation rate is required, a rotary tube (as disclosed, for example, in Strnad et al., Biotechnol. Prog., 2010, Vol. 26, No. 3, pages 653-663) can be used, which stirs at a higher revolution per minute (rpm). Rotating tube bioreactors can be advantageously used as small-scale models for evaluating culture medium, various process parameters, and growth characteristics under high density (> 20e6c / 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 operations. The ability to centrifuge multiple rotary tubes for culture medium exchange enables perfusion cell culture on a small scale (working volume 15mL).

[0115] The at least three separate aqueous concentrate feeds are preferably sterile prior to storage and prior to mixing. In one embodiment, the at least three separate aqueous concentrate feeds are filter sterilized. In addition, with respect to mixing of the components, the at least three separate aqueous concentrate feeds are not premixed prior to addition 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 via separate entry points.

[0116] The entry 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 are therefore added simultaneously. They can be added from the bottom, from the top, or from the side of the bioreactor, and adjacent to each other or on different sides, as long as the culture is continuously mixed.

[0117] The diluent (e.g., sterile water) can be added separately to the reaction vessel of the cell culture and / or bioreactor. Therefore, the diluent is preferably added directly to the reaction vessel of the cell culture and / or bioreactor, preferably added through an entry point separated from the entry point of the at least three independent aqueous concentrated feeds. The entry point can be a valve or port in the bioreactor. Advantageously, the diluent is added continuously at a predetermined perfusion rate and is therefore added simultaneously with the at least three independent aqueous concentrated feeds. It can be added from the bottom, top, or side of the bioreactor and adjacent to or on different sides of one or all of the at least three independent aqueous concentrated feeds, as long as the culture is continuously mixed. Alternatively, the diluent can be pre-mixed with one of the at least three independent aqueous concentrated feeds before being added to the reaction vessel of the cell culture and / or bioreactor. Therefore, the diluent can be added to the reaction vessel of the cell culture and / or bioreactor together with one of the at least three independent aqueous concentrated feeds, preferably added through an entry point separated from the at least two other independent aqueous concentrated feeds. In one embodiment, the diluent is premixed with the alkaline concentrated feed just prior to addition to the reaction vessel of the cell culture and / or bioreactor.

[0118] In another aspect, the present invention also relates to the use of a compartmentalized serum-free cell culture perfusion medium according to the present invention for culturing mammalian cells, preferably in perfusion culture. In one embodiment, the cell culture medium according to the present invention is used to control the osmotic pressure of a cell culture, preferably a perfusion cell culture. In particular, the osmotic pressure is increased in perfusion cell culture. Increasing the osmotic pressure of the cell culture inhibits cell growth and increases heterologous protein production. 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.

[0119] 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 a control cell culture in which the osmotic pressure is not increased. Preferably, the yield is determined over a portion or the entire culture period.

[0120] By using the serum-free cell culture medium according to the present invention or the serum-free cell culture medium obtained by the method according to the present 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 1× serum-free cell culture medium. The cell specific perfusion rate (pl / cell / day) of the serum-free cell culture perfusion medium according to the present invention or the serum-free cell culture medium obtained by the method according to the present invention is preferably constant over part or all of the culture period.

[0121] The present invention also relates to an alkaline aqueous concentrated feed for use in combination with an acidic aqueous concentrated feed, a near-neutral aqueous concentrated 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 a neutral pH. In another embodiment, the present invention relates to an acidic aqueous concentrated feed for use in combination with an alkaline aqueous concentrated feed, a near-neutral aqueous concentrated 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 a neutral pH. In yet another aspect, the present invention relates to a near-neutral aqueous concentrated feed for use in combination with an alkaline aqueous concentrated feed, an acidic aqueous concentrated 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 a neutral pH. The basic aqueous concentrated feed, the acidic aqueous concentrated feed, the near-neutral aqueous concentrated feed, the diluent, and the serum-free cell culture perfusion medium can be further characterized as disclosed above.

[0122] Method for preparing serum-free cell culture perfusion medium.

[0123] In yet 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 subsets of components based on solubility at alkaline, acidic, and neutral pH, (b) (i) providing a subset of components soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed; (ii) providing a subset of components soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; and (iii) providing a subset of components soluble at neutral pH in a neutral aqueous solution to form a near-neutral concentrated feed; (c) optionally storing the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed in separate containers; and (d) mixing the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed. A concentrated feed and a near-neutral concentrated feed and a diluent are added 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 is premixed with one of the at least three separate aqueous concentrated feeds immediately before being added to the reaction vessel of the cell culture and / or bioreactor; wherein upon 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 a near-neutral pH. Thus, the serum-free cell culture perfusion medium prepared according to the method comprises the medium components and the diluent as disclosed for the compartmentalized serum-free cell culture perfusion medium according to the present invention, the medium components being grouped into at least three separate aqueous concentrated feeds. Typically, after adding the at least three separate concentrated aqueous feeds and the diluent, the reaction vessel of the cell culture and / or bioreactor comprises mammalian cells.

[0124] The method may comprise the step of sterilizing the concentrated feed, preferably by filtration sterilization, prior to storage and / or addition to a reaction vessel of a cell culture and / or bioreactor, wherein the reaction vessel of the cell culture and / or bioreactor comprises 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.

[0125] Preferably, the three concentrated feeds are added dropwise to the reaction vessel of the cell culture and / or bioreactor through separate ports. Compared to serum-free cell culture perfusion medium that is mixed and diluted before addition to the bioreactor, mixing and diluting the at least three separate aqueous concentrated feeds in the vessel allows for a 50-90%, preferably 60-90%, reduction in production medium consumption over a 14-day culture period. The maximum multiple concentration (n) of the at least three separate aqueous concentrated feeds after mixing can be calculated using the formula provided above. 最大 X) to calculate the reduction in production medium consumption and to calculate the percentage of the cumulative volume of the at least three separate aqueous concentrate feeds relative to the volume of 1× serum-free cell culture perfusion medium further comprising the diluent.

[0126] The separate addition of the at least three separate concentrated feeds and the diluent enables control of the osmotic pressure of the serum-free cell culture perfusion medium in the bioreactor. The osmotic pressure of the serum-free cell culture perfusion medium in the bioreactor can also be controlled if the diluent is premixed with one of the at least three separate concentrated aqueous feeds immediately prior to addition to the cell culture and / or bioreactor reaction vessel.

[0127] A constant concentrate feed perfusion rate and different diluent perfusion rates (which result in different total perfusion rates) can be used to control the osmotic pressure of the cell culture. The constant concentrate feed perfusion rate relates to the cumulative perfusion rate of the at least three separate aqueous concentrate feeds, which are 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 separate aqueous concentrate feeds and the diluent. Alternatively, a constant total perfusion rate and different concentrate feed perfusion rates can be used to control the osmotic pressure of the cell culture. This naturally results in different diluent perfusion rates. In another alternative, a constant diluent perfusion rate and different concentrate feed perfusion rates (which result in different total perfusion rates) can be used to control the osmotic pressure of the cell culture.

[0128] 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 medium components in the 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 that adjusts the pH to a neutral pH in the reaction vessel of the cell culture and / or bioreactor; and the ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrated feeds determines the osmotic pressure and / or multiple concentration of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor, and the at least three separate aqueous concentrated feeds are added to the reaction vessel of the cell culture and / or bioreactor to provide the serum-free cell culture perfusion medium that adjusts the pH to a near-neutral pH.

[0129] The osmotic pressure of the cell culture can be increased using: a constant concentrated 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 concentrated feed perfusion rate and a reduced diluent perfusion rate; or a constant diluent perfusion rate and an increased concentrated feed perfusion rate, resulting in an increased total perfusion rate; wherein the at least three concentrated feeds are added to each other at a fixed ratio (v / v / v) based on their multiple concentrations to maintain the relative proportions of the medium components in the 1× serum-free cell culture perfusion medium. In one embodiment, preferably, no further additives are added to the culture to increase the osmotic pressure.

[0130] In yet another aspect, the present invention relates to a serum-free cell culture perfusion medium obtainable by the method according to the present invention.

[0131] Cell culture methods

[0132] For the purpose of understanding, it will be appreciated by those skilled in the art that cell culture and culture runs for protein production can include at least three general types; that is, perfusion culture, batch culture, and fed-batch culture. In perfusion culture, for example, fresh culture medium supplements are provided to cells during the incubation period, while old culture medium is removed daily and, for example, products are harvested daily or continuously. In perfusion culture, perfusion culture medium can be added daily and can be added continuously, i.e., as a drip or infusion. For perfusion culture, as long as the cells remain alive and the environment and culture conditions are maintained, the cells can be kept in culture for a long time. Since the cells grow continuously, it is generally necessary to remove the cells during operation in order to maintain a constant viable cell density, which is referred to as cell discharge. The cells discharge the product in the culture medium removed together with the cells, which is usually discarded and therefore wasted. Therefore, it is advantageous to maintain a viable cell density during the production phase without or with only minimal cell discharge, and to increase the total yield of each run.

[0133] In batch culture, cells are initially cultured in a culture medium that is not removed, replaced, or supplemented, i.e., the cells are not "fed" with new culture 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 stages of fed-batch or perfusion culture. For perfusion culture, mammalian cells can, for example, be initially cultured in a batch culture before starting the perfusion culture.

[0134] For fed-batch culture, the culture run time is increased by replenishing the culture medium with fresh culture medium once or more daily (or continuously) during the run, i.e., the cells are "fed" with new culture medium ("feed medium") during the culture period. Fed-batch culture can include various feeding schemes and times as described above, for example, daily, every other day, every two days, etc., more than once a day, or less than once a day, etc. Further, fed-batch culture can be continuously fed with feed medium. The desired product is then harvested at the end of the culture / production run.

[0135] Mammalian cells can be cultured in perfusion culture. During heterologous protein production, it is desirable to have a controlled system in which cells are grown to a desired viable cell density and then switched to a high productivity state of growth arrest in which cells use energy and substrate to produce the heterologous protein of interest, rather than cell growth and cell division. Methods for accomplishing this goal, such as temperature fluctuations and amino acid starvation, are not always successful and may have undesirable effects on product quality. As described herein, by performing conventional cell discharge, the viable cell density during the production phase can be maintained at a desired level. However, this results in the discarding of the heterologous protein of interest. Cell growth arrest during the production phase results in a reduction in the need for cell discharge, and can even maintain cells under a more productive state.

[0136] In one aspect, a method of culturing mammalian cells expressing a heterologous protein in perfusion culture is provided, the method comprising: (a) inoculating a bioreactor with mammalian cells expressing a heterologous protein in a serum-free cell culture medium; (b) culturing the mammalian cells in perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium feed and removing spent medium while maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a compartmentalized serum-free cell culture perfusion medium comprising medium components grouped into 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. feed; and wherein after mixing the at least three separate aqueous concentrated feeds and the diluent in the resulting serum-free cell culture perfusion medium, the pH of the partitioned serum-free cell culture perfusion medium is adjusted to a neutral pH; and / or (ii) a serum-free cell culture perfusion medium obtained by the method according to the present invention, and wherein the alkaline concentrated feed, the acidic concentrated feed and the near-neutral concentrated feed of the partitioned serum-free cell culture perfusion medium feeds are added separately to the reaction vessel of a cell culture and / or bioreactor, and wherein the diluent is added separately to the reaction vessel of a cell culture and / or bioreactor or is premixed with one of the at least three separate aqueous concentrated feeds immediately before addition to the reaction vessel of a cell culture and / or bioreactor.

[0137] In one embodiment, before starting perfusion culture, mammalian cells are initially cultured in batch culture. Typically, in step (a), the serum-free cell culture medium is a growth medium. Step (a) may further include culturing mammalian cells in a growth medium and starting 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 a serum-free cell culture medium according to the present invention or a serum-free cell culture medium obtained by the method according to the present invention until a target cell density is reached; and further maintaining mammalian cells at a target cell density during the production phase by perfusion with a serum-free cell culture medium according to the present invention or a serum-free cell culture medium obtained by the method of the present invention. The serum-free cell culture perfusion medium for perfusion culture can be a production medium, and the perfusion culture is carried out by continuously feeding mammalian cells according to step (b) and removing spent culture medium while keeping the cells in culture. The method further comprises the step of harvesting the heterologous protein from the cell culture.

[0138] The production phase typically begins before the target cell density is reached. The target cell density depends on the cell line and the maximum viable cell density of the cell line and is typically about 15-45% of the maximum viable cell density. The production phase can start from 10x10 6 cells / ml to about 120x10 6 Preferably, the production phase starts with a cell density of at least 10 x 10 6 cells / ml, at least 20x10 6 cells / ml, at least 30x10 6 cells / ml, at least 40x10 6 cells / ml or at least 50x10 6 Cell density is usually 0.2 ± 0.1 g / L when cultured in permeate. 生物反应器 The production phase begins when the amount of heterologous protein is increased by 100 mg / day or more, and this is when purification of the heterologous protein begins.

[0139] According to the method of the present invention, in step (a), culturing mammalian cells can be limited to inoculating mammalian cells expressing heterologous proteins in serum-free medium, and therefore does not require but may include a culturing step before the start of perfusion, and further does not require but may include the start of perfusion culture. Typically, a growth medium is used in step (a), which is replaced by a culture medium according to the present invention or obtained in step (b) according to the method of the present invention, also known as a production medium. Further according to the method of the present invention, maintaining the mammalian cells by perfusion during the production phase includes culturing the mammalian cells by perfusion at a substantially constant viable cell density of about the target viable cell density during the production phase, wherein a substantially constant viable cell density means a variation within 30%, preferably 20%, and more preferably 10% of the viable cell density.

[0140] The present invention also relates to a method for producing a heterologous protein, said method comprising culturing mammalian cells expressing the heterologous protein in perfusion culture using the method according to the present invention. A person skilled in the art will understand that the method according to the present invention is an in vitro culture method.

[0141] In one embodiment, the serum-free cell culture perfusion medium can be chemically defined and / or free of hydrolysates. Preferably, the serum-free cell culture perfusion medium is protein-free or free of proteins other than recombinant insulin and / or insulin-like growth factor. Therefore, the serum-free cell culture perfusion medium can be a protein-free medium or a protein-free medium comprising recombinant insulin and / or recombinant insulin-like growth factor. More preferably, the serum-free perfusion medium is chemically defined and protein-free or free of proteins other than recombinant insulin and / or insulin-like growth factor. This also applies to the serum-free medium used in step (a) of the inventive method.

[0142] Prior to initiating perfusion culture, the mammalian cells may initially be cultured in a batch culture. Typically, perfusion culture is initiated 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.

[0143] As explained above, the method of the present invention may further comprise the step of maintaining the cell density by cell excretion at steady state. The cell density referred to in this context is the viable cell density, which can be determined by any method known in the art. For example, the calculation for controlling the cell excretion rate can be based on maintaining an INCYTE corresponding to the target VCD. TMLive cell density probe ( COMPANY) or FUTURA TM Biomass capacitance probe value ( instruments), or daily cell and viability counts can be performed offline using any cell counting device, such as a hemacytometer, VI-CELL XR TM (BECKMAN ), CEDEX HI-RES TM or VIACOUNT TM Determination (EMD GUAVA ). Using the methods of the present invention, cell expulsion can be eliminated or reduced by increasing the osmotic pressure compared to a 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 and without increasing the osmotic pressure of the cell culture according to the present invention. More specifically, cell expulsion can be reduced compared to a 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 and without increasing the osmotic pressure. A perfusion cell culture without cell expulsion can also be referred to as a "dynamic perfusion culture" or a "dynamic perfusion process". Preferably, the dynamic perfusion culture also comprises a high viable cell density, for example, greater than 80x10 6 cells / ml, higher than 100x10 6 cells / ml, higher than 120x10 6 cells / ml or even higher than 140x10 6 cells / ml and / or a relatively short culture time of less than 30 days, preferably 14-16 days.

[0144] In one embodiment, the osmotic pressure of the serum-free cell culture perfusion medium can be increased to a level above the optimal growth osmotic pressure, thereby resulting in growth inhibition of mammalian cells at a target viable cell density, preferably wherein the osmotic pressure level of the serum-free cell culture perfusion medium is increased gradually or stepwise starting from about half the target viable cell density. The target viable cell density can be about 30×10 6 cells / ml or higher, approximately 60x10 6 cells / ml or higher, approximately 80x10 6 cells / ml, preferably about 100x10 6 cells / ml or higher. The target viable cell density can even be as high as about 100x10 6 cells / ml to 200x10 6 cells / ml, preferably about 120x10 6 cells / ml to 150x106 cells / ml. For the intrinsic maximum viable cell density greater than 150x10 6 Cell lines with 200x10 cells / ml typically require cell growth inhibition to ensure adequate oxygen supply, avoid excessive cell aggregation (which could clog cell retention devices), minimize the effects of waste metabolite accumulation, etc., although 200x10 6 Target viable cell density of cells / ml.

[0145] Can use constant concentrated feed perfusion rate and different diluent perfusion rates (it causes different total perfusion rates) to control the osmotic pressure of cell culture.Constant concentrated feed perfusion rate relates to the accumulation or total perfusion rate of at least three independent aqueous concentrated feeds, and described at least three independent aqueous concentrated feeds are more specifically described alkaline concentrated feed, described acidic concentrated feed and described near neutral concentrated feed.Can for example with the constant total perfusion rate feeding described concentrated feed of 0.5VVD (for example, with 6x acidic feed of 0.33VVD, each with 25x alkaline and near neutral feed of 0.08VVD).Total perfusion rate is the cumulative perfusion rate of described at least three independent aqueous concentrated feeds and described diluent.Alternatively, can use constant total perfusion rate and different concentrated feed perfusion rates to control the osmotic pressure of cell culture.This naturally causes different diluent perfusion rates. In another alternative, constant diluent perfusion rate and different concentrated feed perfusion rates (which result in different total perfusion rates) can be used to control the osmotic pressure of cell culture. The at least three concentrated feeds are added to each other with a fixed ratio (v / v / v) according to their multiple concentrations, to maintain the relative proportions of the culture medium components in the 1x serum-free cell culture perfusion medium. In other words, the ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed and the nearly neutral concentrated feed is a fixed ratio (for each culture medium), to provide the serum-free cell culture perfusion medium with pH adjusted to neutral pH in the reaction vessel of cell culture and / or bioreactor. Preferably, constant concentrated feed perfusion rate and different diluent perfusion rates (which result in different total perfusion rates) are used to control the osmotic pressure of cell culture.

[0146] The osmotic pressure of the cell culture (and the multiple concentration of the serum-free cell culture perfusion medium) can be increased using the following: a constant concentrate feed perfusion rate and a reduced diluent perfusion rate, which results 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, which results in an increased total perfusion rate; wherein the at least three concentrate 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 the 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.

[0147] The ratio (v / v) of the cumulative volume of the diluent to the at least three separate aqueous concentrated feeds determines the osmotic pressure and the multiple concentration of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor, and the at least three separate aqueous concentrated feeds are added to the reaction vessel of the cell culture and / or bioreactor to provide the serum-free cell culture perfusion medium with the pH adjusted to a near-neutral pH. The multiple concentration of the serum-free cell culture medium can be any value from 0.1x to a maximum multiple concentration, which can be calculated as explained above. Using concentrated feeds allows the multiple concentration of the serum-free cell culture medium in the cell culture and / or bioreactor to be adjusted, and therefore, in addition to regulating growth inhibition by increasing osmotic pressure, it also allows the nutrient content in the culture medium to be increased by increasing the multiple concentration of the serum-free cell culture medium. This allows maintaining a higher viable cell density with a similar or only moderately increased perfusion rate and therefore a reduced cell-to-cell perfusion rate. The term "fold concentrated" refers to a concentrate (n>1) or dilution (n>1) of 1x serum-free cell culture perfusion medium, where 1x serum-free cell culture perfusion medium is the originally prepared or designed serum-free cell culture perfusion medium formulation.

[0148] The ratio (v / v) of the cumulative volume of the diluent and the at least three independent aqueous concentrated feeds also determines the multiple concentration (total nutrient content) of the serum-free cell culture perfusion medium described in the reaction vessel of cell culture and / or bioreactor, and the at least three independent aqueous concentrated feeds are added in the reaction vessel of cell culture and / or bioreactor to provide the gained serum-free cell culture perfusion medium with pH adjusted to nearly neutral pH. Therefore, the advantage of using concentrated feed is that the multiple concentration of the substratum can adapt to viable cell concentration and nutritional needs (maintaining nutritional balance). Osmotic pressure can be used as an alternative indicator for nutritional balance inside and outside the estimation system. Therefore, osmotic pressure balance can be used for calculating the adjustment of concentrated feed cumulative volume (with their fixed ratio relative to each other) and diluent feed rate, to achieve desired residual osmotic pressure and nutritional level.

[0149] Any feeding strategy must take into account the osmotic pressure added by any other feeds (such as glucose or alkaline titrants). The choice of osmotic pressure control scheme depends on the cell line and on 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, which is calculated on a daily basis. The osmotic pressure balance of daily osmotic pressure consumption can be calculated according to the following equation: osmotic pressure input-osmotic pressure output=osmotic pressure consumption, where osmotic pressure input is the osmotic pressure of the concentrated feed and diluent perfused into the bioreactor, osmotic pressure 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., 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 pressure consumption for the next day. This consumption rate and the expected osmotic pressure output can be used to calculate the osmotic pressure input required for the next day. The infusion rate of the diluent and / or the concentrated feed is then adjusted to match the osmotic pressure input target.

[0150] The optimal growth osmotic pressure level of cell culture depends on the cell line and can be between about 280mOsm and about 390mOsm, more preferably between 280 to less than about 350mOsm (mOsmol / kg water). Some cell lines can still grow optimally at an osmotic pressure higher than 390mOsm. The optimal growth osmotic pressure level of mammalian cells in cell culture depends on the mammalian cells used, and may also depend on the culture conditions. The optimal growth osmotic pressure level of mammalian cells can be easily determined by determining the viable cell density and viability under different osmotic pressures. The optimal osmotic pressure level does not depend on cell density, but is preferably determined at about the target viable cell density. The osmotic pressure should be maintained at a level that is most suitable for growth, at least until the target viable cell density of about half is reached.

[0151] In case the target viable cell density is reached, osmotic pressure can be increased to inhibit cell growth, such as increasing about 10-70%, about 10-60% or about 10-50% of the optimal growth osmotic pressure level of mammalian cells.Osmotic pressure should be increased gradually or stepwise, preferably starting from about half the target viable cell density (that is, about a colony, doubling from the target viable cell density), more preferably increased to about 10-70%, about 10-60% or about 10-50% of the optimal growth osmotic pressure level. In one embodiment, osmotic pressure is increased to about 350mOsm or higher, preferably to about 380mOsm or higher, to about 400mOsm or higher, to about 420mOsm or higher or to about 450mOsm or higher.Osmotic pressure is increased to the level that suppresses the cell growth of mammalian cells and can not produce cytotoxicity to mammalian cells. The osmotic pressure can be increased to and maintained at an osmotic pressure level that inhibits the cell growth of mammalian cells, preferably at about the target viable cell density, wherein the osmotic pressure level that inhibits the cell growth of mammalian cells is about 350mOsm or higher or about 380mOsm or higher. However, it is important that the cell viability of the mammalian cells is substantially unaffected. For most cell lines, the osmotic pressure level begins to become cytotoxic above about 400mOsm, but for individual cell lines, the osmotic pressure level can be increased to 450mOsm without affecting the cytotoxicity. Increasing the osmotic pressure to physiological pressure levels inhibits cell growth. The osmotic pressure that inhibits the cell growth of mammalian cells in cell culture depends on the mammalian cells used. By measuring the viable cell density and viability under different osmotic pressures, it is easy to determine the osmotic pressure that inhibits the cell growth of specific mammalian cells in cell culture without reaching cytotoxic levels. Preferably, the increased osmotic pressure results in maintaining the cells at about the target viable cell density during the production phase and does not affect viability. Therefore, increasing the osmotic pressure reduces or eliminates the need for cell discharge during the production phase. By increasing the osmotic pressure of the cell culture, cell growth can be inhibited to maintain sustainable viable cell densities without cell expulsion, particularly high viable cell densities without cell expulsion, such as <100x10 6 cells / ml, preferably <120x10 6 cells / ml, which can also be called dynamic perfusion culture.

[0152] 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 a control cell culture in which the osmotic pressure is not increased. Preferably, the yield is determined over a portion or the entire culture period.

[0153] By using the serum-free cell culture medium according to the present invention or the serum-free cell culture medium obtained by the method according to the present 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 1× serum-free cell culture medium.

[0154] In one embodiment of the method of the present invention, the reaction vessel of cell culture and / or bioreactor comprises at least about 100L serum-free cell culture perfusion medium, preferably at least about 1000L serum-free cell culture perfusion medium. Preferably, the volume of cell culture is at least about 100L and / or the volume of bioreactor is at least about 100L. More preferably, the volume of cell culture is at least about 1000L and / or the volume of bioreactor is at least about 1000L. Although the serum-free cell culture perfusion medium used in the inventive method or prepared by the inventive method is complete serum-free cell culture perfusion medium, culture can be further supplemented. Suitable fill-in that can be added separately in the cell culture is but not limited to defoamer, alkali, glucose and / or glutamine.

[0155] 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.

[0156] bioreactor

[0157] Serum-free cell culture perfusion medium can be used in any type of cell culture system, type, or format suitable for continuous perfusion.

[0158] Any cell perfusion bioreactor and cell retention device can be used for perfusion culture.The bioreactor for perfusion is not very different from the bioreactor for batch / fed-batch culture, except that the bioreactor size for perfusion is more compact and is connected to the cell retention device.Being mainly determined by cell whether to be attached to surface growth or to grow in single cell suspension or cell aggregate for the method for being retained in bioreactor inside. Although most mammalian cells are attached to surface or matrix growth (heterogeneous culture) in history, efforts have been made to adapt many industrial mammalian cell lines to suspension growth (homogeneous culture), mainly because suspension culture is more easily expanded in proportion. Therefore, the cell for the inventive method is preferably grown in suspension. In a non-restrictive manner, the exemplary retention system for the cell of suspension growth is a rotary filter, external filtration such as tangential flow filtration (TFF), alternating tangential flow (ATF) system, cell sedimentation (vertical sedimentation and inclined sedimentation), centrifugal, ultrasonic separation and hydrocyclone. Perfusion systems can be divided into two categories: filtration-based systems, such as spin filters, external filtration, and ATF; and open perfusion systems, such as gravity settlers, centrifuges, ultrasonic separation devices, and hydrocyclones. Filtration-based systems show a high degree of cell retention that does not vary with flow rate. However, filters can become clogged, and thus the length of a culture run is limited or requires filter replacement. An example of an ATF system is the Filtration-Based System from REPLIGEN. TM XCELL TM ATF system, and an example of a TFF system is a centrifugal pump from TFF system. Cross-flow filters (such as hollow fibers (HF) or flat plate filters) can be used together with ATF and TFF systems. In particular, hollow fibers made of modified polyethersulfone (mPES), polyethersulfone (PES) or polysulfone (PE) can be used together with ATF and TFF systems. The pore size range of HF can be from several hundred kDa to 15 μM. The open perfusion system will not clog, and therefore can operate indefinitely at least in theory. However, at higher perfusion rates, the degree of cell retention is reduced. There are currently three systems that can be used at an industrial scale: alternating tangential filters (ATF), gravity (particularly inclined settlers) and centrifuges. Cell retention devices suitable for heterologous or homologous culture 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 are incorporated herein by reference. Perfusion culture is not a true steady-state process; the total cell concentration and viable cell concentration only reach steady-state when the cell effluent is removed from the bioreactor.

[0159] The physical parameters (such as pH, dissolved oxygen and temperature) in the perfusion bioreactor should be monitored online and controlled in real time. Off-line or online sampling can be used to determine cell density, vigor, metabolites and product concentration. When the perfusion operation starts with continuous harvesting and feed supplement, the perfusion rate generally refers to the harvest flow rate, which can be manually set to the desired value. For example, the weight control for the bioreactor can activate the feed pump so that the constant volume in the bioreactor can be maintained. Alternatively, level control can be achieved by the culture volume being pumped out above a predetermined level. The perfusion rate in the bioreactor must be adjusted to deliver enough nutrients to the cells.

[0160] The perfusion rate can be controlled using, for example, cell density measurements, pH measurements, oxygen consumption or metabolite measurements. Cell density is the most important measurement for perfusion rate adjustment. 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 are known to those skilled in the art, such as capacitance probes, for example, the INCYTE TM Live cell density probe ( COMPANY) or FUTURA TM Biomass capacitance probe value ( Instruments). 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 bleed. Thus, cell bleed is determined by the specific growth rate of the mammalian cells in culture. Cell bleed is typically not harvested and is therefore considered waste.

[0161] The method of the present invention further comprises harvesting the heterologous protein from the perfusion cell culture. The present invention contemplates any suitable method for harvesting and purifying the protein of interest. Harvesting can also occur intermittently throughout the cell culture life cycle, 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 by a cell retention device. Since the product residence time of the product protein in the cell culture in the perfusion bioreactor is shorter than that in the fed batch, contact with proteases, sialidases and other degrading proteins is minimized, which may result in better product quality of the heterologous protein produced in the perfusion culture. Preferably, a method as described in U.S. Provisional Application 62827504, particularly the method described therein, is used. Figure 6 The harvested product is purified using the iSKID described in

[15] . The iSKID is an integrated skid that combines multiple unit operations in a highly automated manner and allows for complete continuous automated manufacturing.

[0162] Expression product

[0163] The heterologous protein produced by the methods and uses of the present invention can be any secreted protein, preferably a therapeutic protein. Since most therapeutic proteins are recombinant therapeutic proteins, they are most preferably recombinant therapeutic proteins. Examples of therapeutic proteins are, but are not limited to, antibodies, fusion proteins, cytokines, and growth factors.

[0164] Therapeutic proteins produced in mammalian cells according to the methods of the present invention include, but are not limited to, antibodies or fusion proteins, such as Fc fusion proteins. Other secreted recombinant therapeutic proteins can be, for example, enzymes, cytokines, lymphokines, adhesion molecules, receptors, and derivatives or fragments thereof, as well as any other polypeptides and scaffolds that can be used as agonists or antagonists and / or have therapeutic or diagnostic uses.

[0165] Other recombinant proteins of interest include, but are not limited to, insulin, insulin-like growth factor, hGH, tPA, cytokines such as interleukins (IL), such as interleukins 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 hormone growth factor and any other polypeptide that can be used as an agonist or antagonist and / or has therapeutic or diagnostic uses.

[0166] Preferred therapeutic proteins are antibodies or fragments or derivatives thereof, more preferably IgG1 antibodies.Thus, 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 the present 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.

[0167] Antibody fragments include, for example, "Fab fragments" (fragment antigen binding = Fab). Fab fragments consist of the variable regions of two chains held together by adjacent constant regions. These can be formed from conventional antibodies by protease digestion, for example with papain, but Fab fragments can similarly be produced by genetic engineering. Further antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

[0168] Using genetic engineering methods, it is possible to produce shortened antibody fragments, which are composed only of the variable regions of heavy chain (VH) and light chain (VL). These are referred to as Fv fragments (fragment variable=fragment of variable part). Since these Fv fragments lack the covalent bonding of two chains by the cysteine ​​of the constant chain, Fv fragments are often stable. It is advantageous to connect the variable regions of heavy chain and light chain by short peptide fragments, which for example have 10 to 30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of VH and VL, connected by a peptide linker, is obtained. This antibody protein is referred to as single-chain Fv (scFv). The example of scFv antibody protein is well known to those skilled in the art.

[0169] Preferred therapeutic antibodies according to the present invention are bispecific antibodies. 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 one molecule. Exemplary bispecific antibodies are, but are not limited to, diabodies, BiTE (bispecific T cell engagers) formats, and DART (dual affinity retargeting) formats. The diabody format separates the homologous variable domains of the heavy and light chains with two antigen binding specificities on two separate polypeptide chains, wherein the two polypeptide chains are non-covalently associated. The DART format is based on the diabody format, but it provides additional stabilization through a C-terminal disulfide bridge.

[0170] Another preferred therapeutic protein is a fusion protein, such as an Fc fusion protein. Therefore, the present invention can be advantageously used to produce a fusion protein, such as an Fc fusion protein. In addition, the method for increasing protein production according to the present invention can be advantageously used to produce a fusion protein, such as an Fc fusion protein.

[0171] The effector portion of the fusion protein can be the complete sequence of a natural or modified heterologous protein or any portion of the sequence, or the complete sequence of a natural or modified heterologous protein or a composition of any portion of the sequence. The immunoglobulin constant domain sequence can be obtained from any immunoglobulin subtype, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 subtype, or classification such as IgA, IgE, IgD or IgM. Preferentially, they are derived from human immunoglobulin, more preferably derived from human IgG, and even more preferably derived 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 the heavy chain immunoglobulin constant region comprising an N-linked glycosylation site. Fc fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site into another expression construct, the other expression construct comprising, for example, other immunoglobulin domains, enzymatically active protein portions, or effector domains. Thus, the Fc fusion protein according to the present invention further comprises a single-chain Fv fragment linked to the CH2 domain of a heavy chain immunoglobulin constant region containing, for example, an N-linked glycosylation site.

[0172] Recovery and preparation of expression products

[0173] In a further aspect, a method of producing a therapeutic protein is provided using the method of the present invention, and the method of producing a therapeutic protein optionally further comprises the steps of purifying the therapeutic protein and formulating it into a pharmaceutically acceptable formulation.

[0174] Therapeutic proteins, particularly antibodies, antibody fragments or Fc fusion proteins are preferably recovered / isolated from the culture medium as secreted polypeptides. It is necessary to purify the therapeutic protein from other recombinant proteins and host cell proteins to obtain a substantially homogenous preparation of the therapeutic protein. As a first step, cells and / or particulate cell debris are removed from the culture medium. Further, the therapeutic protein is purified from contaminant soluble proteins, polypeptides and nucleic acids, for example, 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.

[0175] expression vector

[0176] In one embodiment, the heterologous protein expressed using the method of the present invention is encoded by one or more expression cassettes, and the expression cassette comprises a heterologous polynucleotide encoding the heterologous protein. The heterologous protein can be placed under the control of an amplifiable genetic selection marker (such as dihydrofolate reductase (DHFR), glutamine synthetase (GS)). The amplifiable selection marker gene can be on an expression vector identical to the heterologous protein expression cassette. Alternatively, the amplifiable selection marker gene and the heterologous protein expression cassette can be on different expression vectors, but are very closely integrated into the genome of the host cell. For example, two or more vectors of co-transfection are often closely integrated into the genome of the host cell. Then, by adding an amplification reagent (for example, MTX for DHFR or MSX for GS) to the culture medium, the amplification of the genetic region containing the secretory therapeutic protein expression cassette is mediated.

[0177] A sufficiently high and stable level of heterologous protein expressed by mammalian cells can also be achieved by, for example, cloning multiple copies of a heterologous protein encoding polynucleotide into an expression vector. Cloning multiple copies of a heterologous protein encoding polynucleotide into an expression vector and amplifying the heterologous protein expression cassette as described above can be further combined.

[0178] Mammalian cell lines

[0179] 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 present 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 non-transformed 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 referred to 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. Most preferred are CHO-DG44 cells. Also contemplated are mammalian cells, particularly glutamine synthetase (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.

[0180] Mammalian cells may further comprise one or more expression cassettes encoding heterologous proteins, such as therapeutic proteins, preferably recombinant secretory therapeutic proteins. The host cell may also be a mouse-derived cell, such as a mouse-derived myeloma cell, such as a derivative / offspring of any of NS0 and Sp2 / 0 cells or such cell lines. Non-limiting examples of mammalian cells that may be used for the present invention are also summarized in Table 1. However, derivatives / offspring of these cells, other mammalian cells (including but not limited to humans, mice, rats, monkeys, and rodent cell lines) may also be used in the present invention, particularly for producing biopharmaceutical proteins.

[0181] Table 1: Mammalian production cell lines

[0182]

[0183]

[0184] 1CAP (CEVEC's Amniocyte Production) cells are an immortalized cell line based on primary human amniocytes. They are generated by transfecting these primary cells with a vector containing functional E1 and pIX of adenovirus 5. Due to authentic human post-translational modifications, CAP cells allow for the competitive and stable production of recombinant proteins with excellent biological activity and therapeutic efficacy.

[0185] Mammalian cells are most preferred when established, adapted, and fully cultured under serum-free conditions and, optionally, in a medium free of any proteins / peptides of animal origin. Commercially available culture 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 of appropriate nutrient solutions. Any culture medium may be supplemented with a variety of compounds as needed, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPES), nucleosides (e.g., 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. In order to grow and select genetically modified cells expressing a selectable gene, a suitable selection agent is added to the culture medium.

[0186] In view of the above, it will be understood that the present invention also encompasses the following provisions:

[0187] Item 1 provides a compartmentalized serum-free cell culture perfusion medium comprising medium components grouped into at least three separate aqueous concentrate feeds and a diluent, wherein the first concentrate feed is a basic concentrate feed, the second concentrate feed is an acidic concentrate feed, and the third concentrate feed is a near-neutral concentrate feed; wherein

[0188] After mixing the at least three separate aqueous concentrated feeds and the diluent in the resulting serum-free cell culture perfusion medium, the pH of the compartmentalized serum-free cell culture perfusion medium is adjusted to a neutral pH.

[0189] Item 2 specifically describes the compartmentalized serum-free cell culture perfusion medium as described in Item 1, 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 between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2.

[0190] Item 3 specifically describes the compartmentalized serum-free cell culture perfusion medium as described in Item 1 or 2, wherein the diluent is sterile water.

[0191] Item 4 specifically describes the serum-free cell culture perfusion medium separated as described in any one of items 1 to 3, wherein the serum-free cell culture perfusion medium separated is used for

[0192] (a) adding the alkaline concentrated feed, the acidic concentrated feed and the near-neutral concentrated feed separately to a reaction vessel of a cell culture and / or a bioreactor;

[0193] (b) 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

[0194] (c) mixing the at least three separate aqueous concentrated feeds directly in the reaction vessel of the cell culture and / or bioreactor.

[0195] Clause 5 specifically describes the compartmentalized 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.

[0196] Item 6 specifically describes the compartmentalized serum-free cell culture perfusion medium as described in Item 5, wherein

[0197] (a) the alkaline concentrated feed is a 20x to 40x concentrated feed, the acidic concentrated feed is a 4x to 20x concentrated feed, and the near-neutral concentrated feed is a 10x to 40x concentrated feed;

[0198] (b) the alkaline concentrated feed is a 20x to 30x concentrated feed, the acidic concentrated feed is a 5x to 12x concentrated feed, and the near-neutral concentrated feed is a 20x to 30x concentrated feed; and / or

[0199] (c) the alkaline concentrated feed is a 25x concentrated feed, the acidic concentrated feed is a 6x to 10x concentrated feed, and the near-neutral concentrated feed is a 25x concentrated feed.

[0200] Clause 7 specifies the compartmentalized serum-free cell culture perfusion medium of any of the preceding clauses, wherein the pH of the near-neutral concentrated feed is 6.5-8.5.

[0201] Item 8 specifically describes a compartmentalized serum-free cell culture perfusion medium as described in any of the preceding items, wherein the pH of the alkaline concentrated feed is 9 or higher, the pH of the acidic concentrated feed is 5 or lower, and the pH of the near-neutral concentrated feed is 7 to 8.5.

[0202] Item 9 specifically describes the compartmentalized serum-free cell culture perfusion medium as described in Item 8, wherein

[0203] (a) the pH of the alkaline concentrated feed is from 9 to 11, the pH of the acidic concentrated feed is from 2 to 5, and the pH of the near-neutral concentrated feed is from 7 to 8.5;

[0204] (b) the pH of the alkaline concentrated feed is from 9.8 to 10.8, the pH of the acidic concentrated feed is from 3.6 to 4.8, and the pH of the near-neutral concentrated feed is from 7 to 8.5; or

[0205] (c) the pH of the alkaline concentrated feed is from 9.8 to 10.5, the pH of the acidic concentrated feed is from 3.8 to 4.5, and the pH of the near-neutral concentrated feed is from 7.5 to 8.5.

[0206] Item 10 specifically describes a partitioned serum-free cell culture perfusion medium as described in any of the preceding items, wherein the obtained 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.

[0207] Item 11 specifies the compartmentalized serum-free cell culture perfusion medium as described in any one of the preceding items, wherein the obtained compartmentalized serum-free cell culture perfusion medium is a production medium.

[0208] Clause 12 specifically describes the compartmentalized serum-free cell culture perfusion medium as described in any one of the preceding clauses, wherein

[0209] (a) the ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed to the near-neutral concentrated feed is fixed to provide a resulting serum-free cell culture perfusion medium having a pH adjusted to a neutral pH; and

[0210] (b) 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 adjusted to a neutral pH determines the osmotic pressure of the serum-free cell culture perfusion medium.

[0211] Clause 13 specifies the compartmentalized serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein the acidic concentrated feed comprises trace elements, trace metals, inorganic salts, chelating agents, polyamines, and regulatory hormones.

[0212] Clause 14 specifies the compartmentalized serum-free cell culture perfusion medium of any of the preceding clauses, wherein the acidic concentrated feed and / or the near-neutral concentrated feed comprises a surfactant, an antioxidant, and a carbon source.

[0213] Item 15 specifically describes a compartmentalized serum-free cell culture perfusion medium as described in any of the preceding items, wherein the alkaline concentrated feed comprises amino acids having 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.

[0214] Item 16 specifies the compartmentalized serum-free cell culture perfusion medium as described in Item 15, wherein the remaining amino acids are in the acidic and / or near-neutral concentrated feed, preferably in the acidic concentrated feed.

[0215] Clause 17 specifies a compartmentalized serum-free cell culture perfusion medium as described in any of the preceding clauses, wherein vitamins and metals are in separate feeds, preferably vitamins are in the near-neutral feed and metals are in the acidic feed.

[0216] Item 18 specifically describes the compartmentalized serum-free cell culture perfusion medium as described in Item 17, wherein a vitamin that is poorly soluble in aqueous solution, such as choline chloride, is present in the neutral feed and the acidic feed.

[0217] Item 19 specifically describes an alkaline aqueous concentrate feed for combining 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 a neutral pH.

[0218] Item 20 specifies an acidic aqueous concentrate feed for combining with a basic 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 a neutral pH.

[0219] Item 21 specifies a near-neutral aqueous concentrate feed for combining with a basic 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 a neutral pH.

[0220] Item 22 specifically describes a method for preparing a serum-free cell culture perfusion medium, the method comprising:

[0221] (a) providing cell culture medium components in at least three component subgroups based on solubility at alkaline, acidic, and neutral pH,

[0222] (b)

[0223] (i) dissolving a subcomponent soluble at alkaline pH in an alkaline aqueous solution to form an alkaline concentrated feed;

[0224] (ii) dissolving a subcomponent soluble at acidic pH in an acidic aqueous solution to form an acidic concentrated feed; and

[0225] (iii) dissolving a subcomponent soluble at neutral pH in a neutral aqueous solution to form a near-neutral concentrated feed;

[0226] (c) optionally storing the prepared alkaline concentrated feed, acidic concentrated feed, and near-neutral concentrated feed in separate containers; and

[0227] (d) adding the prepared alkaline concentrated feed, acidic concentrated feed and near-neutral concentrated feed and diluent to a reaction vessel of a cell culture and / or bioreactor, wherein

[0228] (i) adding the alkaline concentrated feed, the acidic concentrated feed, and the near-neutral concentrated feed separately to a reaction vessel of a cell culture and / or bioreactor; and

[0229] (ii) adding the diluent separately to the reaction vessel of the cell culture and / or bioreactor, or premixing the diluent with one of the at least three separate concentrated aqueous feeds immediately before adding to the reaction vessel of the cell culture and / or bioreactor;

[0230] wherein upon 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 a neutral pH.

[0231] Item 23 specifically describes the method as described in Item 22, wherein after mixing the at least three separate aqueous concentrated feeds and the diluent, the pH of the serum-free cell culture perfusion medium after pH adjustment is between 6.7 and 7.5, between 6.9 and 7.4, and preferably between 6.9 and 7.2.

[0232] Item 24 specifies the method as described in Item 22 or 23, wherein the diluent is sterile water.

[0233] Clause 25 specifies the method of any one of clauses 22 to 24, wherein the at least three concentrated feeds are added dropwise to the reaction vessel of the cell culture and / or bioreactor through separate ports.

[0234] Clause 26 specifies a 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 reduction in manufactured medium consumption by 50-90%, preferably 60-90%, over a 14 day culture period, compared to serum-free cell culture perfusion medium that is mixed and diluted prior to addition to the bioreactor.

[0235] Item 27 specifies the method of any one of items 22 to 26, wherein the reaction vessel of the cell culture and / or bioreactor comprises mammalian cells.

[0236] Clause 28 specifies the method of any one of clauses 22 to 27, further comprising the step of sterilizing the concentrated feed prior to storage and / or addition to a reaction vessel of a cell culture and / or bioreactor.

[0237] Clause 29 specifies the process of any one of clauses 22 to 28, wherein the alkaline concentrated feed is a 2x to 80x concentrated feed, wherein the acidic concentrated feed is a 2x to 40x concentrated feed and the near-neutral concentrated feed is a 2x to 50x concentrated feed.

[0238] Clause 30 specifies the method as described in clause 29, wherein

[0239] (a) the alkaline concentrated feed is a 20x to 40x concentrated feed, the acidic concentrated feed is a 4x to 20x concentrated feed, and the near-neutral concentrated feed is a 10x to 40x concentrated feed;

[0240] (b) the alkaline concentrated feed is a 20x to 30x concentrated feed, the acidic concentrated feed is a 5x to 12x concentrated feed, and the near-neutral concentrated feed is a 20x to 30x concentrated feed; and / or

[0241] (c) the alkaline feed is a 25x concentrated feed, the acidic concentrated feed is a 6x to 10x concentrated feed, and the near-neutral concentrated feed is a 25x concentrated feed.

[0242] Clause 31 specifies the method of any one of clauses 22 to 30, wherein the pH of the near-neutral concentrated feed is 6.5-8.5.

[0243] Clause 32 specifies the method of 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.

[0244] Clause 33 specifies the method as described in clause 32, wherein

[0245] (a) the pH of the alkaline concentrated feed is from 9 to 11, the pH of the acidic concentrated feed is from 2 to 5, and the pH of the near-neutral concentrated feed is from 7 to 8.5;

[0246] (b) the pH of the alkaline concentrated feed is from 9.8 to 10.8, the pH of the acidic concentrated feed is from 3.6 to 4.8, and the pH of the near-neutral concentrated feed is from 7 to 8.5; or

[0247] (c) the pH of the alkaline concentrated feed is from 9.8 to 10.5, the pH of the acidic concentrated feed is from 3.8 to 4.5, and the pH of the near-neutral concentrated feed is from 7.5 to 8.5.

[0248] Item 34 specifically describes a method as described in Item 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.

[0249] Clause 35 specifies the method of any one of clauses 22 to 34, wherein separate addition of the at least three separate concentrated feeds and the diluent enables control of the osmotic pressure of the serum-free cell culture perfusion medium in the bioreactor.

[0250] Clause 36 specifies the method as described in any one of clauses 22 to 35, wherein

[0251] (a) the ratio (v / v / v) of the alkaline concentrated feed to the acidic concentrated feed to the near-neutral concentrated feed is fixed to provide the serum-free cell culture perfusion medium with the pH adjusted to neutral pH in a reaction vessel of a cell culture and / or bioreactor; and

[0252] (b) the ratio (v / v) of the diluent to the cumulative volume of the at least three separate aqueous concentrated feeds that are added to the reaction vessel of the cell culture and / or bioreactor to provide the serum-free cell culture perfusion medium with the pH adjusted to near neutral pH determines the osmotic pressure of the serum-free cell culture perfusion medium in the reaction vessel of the cell culture and / or bioreactor.

[0253] Clause 37 specifies the method of any one of clauses 22 to 36, wherein the acidic concentrated feed comprises trace elements, trace metals, inorganic salts, chelating agents, polyamines, and regulatory hormones.

[0254] Clause 38 specifies the method of any one of clauses 22 to 37, wherein the acidic concentrated feed and / or the near-neutral concentrated feed comprises a surfactant, an antioxidant, and a carbon source.

[0255] Clause 39 specifies a method as described in any of clauses 22 to 38, wherein the alkaline concentrated feed comprises amino acids having 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.

[0256] Item 40 specifies the method of Item 39, wherein the remaining amino acids are in the acidic and / or near-neutral concentrated feed, preferably in the acidic concentrated feed.

[0257] Clause 41 specifies the process of any one of clauses 22 to 40, wherein the vitamins and metals are in separate feeds, preferably the vitamins are in the near-neutral feed and the metals are in the acidic feed.

[0258] Item 42 specifies the method of Item 41, wherein a vitamin that is poorly soluble in aqueous solution, such as choline chloride, is present in the neutral feed and the acidic feed.

[0259] Item 43 specifies the method of any one of items 22 to 42, wherein the reaction vessel of the cell culture and / or bioreactor comprises 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.

[0260] Item 44 specifies a serum-free cell culture perfusion medium obtainable by the method according to items 22 to 43.

[0261] Item 45 specifies a method of culturing mammalian cells expressing a heterologous protein in perfusion culture, the method comprising:

[0262] (a) inoculating a bioreactor with mammalian cells expressing a heterologous protein in serum-free cell culture medium;

[0263] (b) culturing the mammalian cells in perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium feed and removing spent medium while maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a compartmentalized serum-free cell culture perfusion medium comprising culture medium components grouped into 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; and wherein upon mixing the at least three separate aqueous concentrate feeds and the diluent in the resulting serum-free cell culture perfusion medium, the compartmentalized serum-free cell culture perfusion medium has its pH adjusted to a neutral pH; and / or (ii) a serum-free cell culture perfusion medium according to clause 44, and

[0264] wherein the alkaline concentrated feed, the acidic concentrated feed and the near-neutral concentrated feed of the separated serum-free cell culture perfusion medium feed 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 is premixed with one of the at least three separate aqueous concentrated feeds immediately before addition to the reaction vessel of the cell culture and / or bioreactor.

[0265] Item 46 specifies the method of Item 45, wherein the mammalian cells are initially cultured in a batch culture prior to initiating the perfusion culture.

[0266] Item 47 specifies the method of Item 45 or 46, wherein the perfusion culture starts from day 0 to day 3 of the batch culture.

[0267] Clause 48 specifies the method of any one of clauses 45 to 47, wherein after perfusion is initiated, the perfusion rate is increased until a target viable cell density is reached.

[0268] Clause 49 specifies the method of clause 48, wherein the perfusion 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.

[0269] Item 50 specifically describes a method as described in any one of items 45 to 49, wherein the osmotic pressure of the serum-free cell culture perfusion medium is increased to above the optimal growth osmotic pressure level, thereby resulting in growth inhibition at a target viable cell density, preferably wherein 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.

[0270] Clause 51 specifies the method of any one of clauses 45 to 50, wherein the target viable cell density is about 30×10 6 cells / ml or higher, approximately 60x10 6 cells / ml or higher, approximately 80x10 6 cells / ml, preferably about 100x10 6 cells / ml or higher.

[0271] Clause 52 specifies the method of any one of clauses 45 to 51, wherein the osmotic pressure is controlled using:

[0272] (a) a constant concentrate feed infusion rate and varying diluent infusion rates, which result in varying total infusion rates; or

[0273] (b) constant total perfusion rate and different concentrate feed perfusion rates;

[0274] 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 medium components in the 1× serum-free cell culture perfusion medium.

[0275] Clause 53 specifies the method of any one of clauses 45 to 52, wherein the osmotic pressure is increased using:

[0276] (a) a constant concentrate feed infusion rate and a reduced diluent infusion rate, which results in a reduced overall infusion rate; or

[0277] (b) a constant total perfusion rate and an increased concentrate feed perfusion rate and a decreased diluent perfusion rate;

[0278] 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 medium components in the 1× serum-free cell culture perfusion medium.

[0279] Clause 54 specifies the method of any one of clauses 50 to 53, wherein no further additives are added to the culture to increase osmotic pressure.

[0280] Clause 55 specifies the method of any one of clauses 50 to 54, wherein the optimal growth osmotic pressure level is about 280 to less than 350 mOsm.

[0281] Clause 56 specifies the method of any one of clauses 50 to 55, wherein the osmotic pressure is maintained at a level optimal for growth until about half of the target viable cell density is reached.

[0282] Clause 57 specifies the method of any one of clauses 50 to 56, wherein the osmotic pressure is gradually or stepwise increased starting from about half the target viable cell density, preferably to about 10-50% of the optimal growth osmotic pressure level.

[0283] Clause 58 specifically describes a method as described in any of clauses 50 to 57, wherein the osmotic pressure is increased to and maintained at an osmotic pressure level that inhibits cell growth at about a 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.

[0284] Clause 59 specifies the method of any one of clauses 50 to 58, wherein increasing osmotic pressure reduces or eliminates the need for cell expulsion during the production phase.

[0285] Item 60 specifies the method of any one of items 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 a control cell culture in which osmotic pressure is not increased.

[0286] Clause 61 specifies the method of any one of clauses 50 to 60, wherein cell growth is inhibited to maintain a sustainable viable cell density without cell expulsion.

[0287] Item 62 specifies the method of any one of items 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 1× serum-free cell culture medium.

[0288] Item 63 specifies the method of any one of items 45 to 62, further comprising harvesting the heterologous protein from the cell culture.

[0289] Clause 64 specifies the method of any one of clauses 45 to 63, wherein the heterologous protein is a therapeutic protein, an antibody, or a therapeutically effective fragment thereof.

[0290] Clause 65 specifies the method as described in Clause 64, wherein the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody or a fragment thereof.

[0291] Clause 66 specifies the method as described in any one of clauses 45 to 65, wherein the mammalian cell comprises a Chinese hamster ovary (CHO) cell, a Jurkat cell, a 293 cell, a HeLa cell, a CV-1 cell or a 3T3 cell or a derivative of any of these cells, wherein the CHO cell can 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.

[0292] Clause 67 specifies the method of any one of clauses 45 to 66, wherein the reaction vessel of the cell culture and / or bioreactor comprises 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.

[0293] Clause 68 specifies the method of any one of clauses 45 to 67, wherein a further supplement selected from the list of antifoaming agents, base and glucose is added separately to the cell culture.

[0294] Item 69 specifies a method of producing a therapeutic protein using the method of any one of items 45 to 68.

[0295] Item 70 specifies use of the compartmentalized serum-free cell culture perfusion medium according to any one of Items 1 to 18 or the serum-free cell culture perfusion medium according to Item 44 for culturing mammalian cells.

[0296] Item 71 specifies use of the compartmentalized serum-free cell culture perfusion medium according to any one of items 1 to 18 or the serum-free cell culture perfusion medium according to item 44 for culturing mammalian cells in perfusion culture.

[0297] Item 72 specifies use of the compartmentalized serum-free cell culture perfusion medium of any one of items 1 to 18 or the serum-free cell culture perfusion medium of item 44 for controlling the osmotic pressure of a perfused cell culture.

[0298] Item 73 specifies the use according to Item 72, wherein increasing the osmotic pressure of the cell culture inhibits cell growth and increases heterologous protein production.

[0299] Item 74 specifically describes the use according to Item 73, wherein the yield of the heterologous protein produced in the cell culture is increased by at least 5-50% relative to the yield of a control cell culture in which the osmotic pressure is not increased.

[0300] Item 75 specifies the use as described in item 73 or 74, wherein the growth inhibition is sufficient to maintain a sustainable viable cell density without cell expulsion.

[0301] Item 76 specifically describes the use according to Item 70 or 75, wherein the cell specific perfusion rate (pl / cell / day) is reduced by at least 30% relative to the cell specific perfusion rate of 1× serum-free cell culture medium.

[0302] Clause 77 specifies the use of the compartmentalized serum-free cell culture perfusion medium according to any one of clauses 1 to 18 for separately adding the at least three separate aqueous concentrated feeds to the reaction vessel of a cell culture and / or bioreactor.

[0303] Example

[0304] method

[0305] Seed training and inoculum:

[0306] The Chinese hamster ovary (CHO) cell line expressing recombinant IgG was suspended in a Corning-Life Sciences shake flask (Oneonta, NY), which was expanded in a proprietary growth medium by 3e7 cell vials. The flask was inoculated with 0.5e6 cells / mL for 3 days, and with 0.8e6 cells / mL for 2 days, and grown in batch mode, stirred at 120 rpm until an N-3 3L shake flask was formed, which was stirred at 80 rpm with a 50 mm orbital radius. The culture incubator (Infors, Annapolis, MD) was maintained at 36.5 ° C, 5% CO2, without humidity control. The N-2 stage was inoculated with 1.0 ± 0.4e6 cells / mL, and grown in batch mode for 3 days with a 5L working volume in a GE wave (GE Healthcare). The N-1 stage was run in a perfusion mode in a GE Wave 25 system (GE Healthcare). The seeding density was 1.0 ± 0.4e6 cells / mL in a 25 L working volume. Perfusion was started at 0.5 vessel volumes per day (vvd) on day 1 of culture and increased by 0.5 vvd each day until reaching 2.0 vvd on day 4, which was maintained until day 5 or 6. Run duration was determined based on achieving the target viable cell density (VCD): 40e6c / mL.

[0307] Experimental bioreactor setup:

[0308] The perfusion N-1 culture was used to inoculate a 100 L disposable bioreactor (SUB) at a high density of 10 ± 2e6 cells / mL, as described in U.S. Provisional Application 62827504, particularly its Figure 6Custom ThermoFisher Hyclone (Logan, Utah) SUB bags were used with a DeltaV distributed control system (Emerson, St Louis, MO) to maintain the culture at 36.5°C with a target oxygen set point of 60% air saturation and a pH set point of 7.1, using a single marine-style impeller at 18 W / m2 per unit volume. 3 The cell culture was 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). The harvested cell culture fluid or permeate that passed through the TFF was directly transferred to the capture column of the iSkid purification unit operation. Growth medium, three concentrated medium feeds (acidic, alkaline, and neutral), 0.1 μm filtered sterile reverse osmosis deionized (RODI) water diluent, alkaline titrant (1 M sodium carbonate) to maintain pH during culture, glucose feed (500 g / L), and 1% medical antifoam C emulsion (Dow Corning, Midland, MI) were attached to the SUB via sterile welded tubing or sterile, corrosion-resistant quick connectors (Colder Products Company, St Paul, MN). All additions were separate to avoid precipitation, with the exception of the alkaline concentrate feed, which was manifolded with sterile water diluent and subsequently passed through an in-line mixer before reaching the bioreactor in a single tube.

[0309] The perfusion medium used (three concentrated medium feeds) was prepared as follows:

[0310] A 1x acid feed contains the following:

[0311] Proteinogenic amino acids not present in the alkaline feed and the nonproteinogenic amino acids hydroxyproline and ornithine, totaling 87.8 mM;

[0312] Inorganic salts including buffer salts (trace metal salts and iron sources are listed separately), totaling 21.4 mM;

[0313] organic acid taurine and alternative carbon sources, totaling 16.3 mM;

[0314] combined iron sources, totaling 0.25 mM;

[0315] 0.28 mM polyamine;

[0316] 0.28 mM ethanolamine;

[0317] Trace metals (excluding iron), totaling 0.1 mM;

[0318] 0.02m first antioxidant;

[0319] vitamins, 0.07 mM calcium pantothenate, 0.04 mM thiamine, and 0.3 mM pyridoxine;

[0320] Choline chloride added separately to the acidic and neutral feeds was 1.27 mM in the acidic feed;

[0321] 50 mM carbon source;

[0322] 2.4 μM of recombinant protein as a growth factor; and

[0323] 0.2 mM surfactant.

[0324] For the 6x concentrated acidic feed used in the examples, these concentrations were increased 6-fold. The final pH of the 6x concentrated acidic 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 basic powder before adding the carbon source, and 1 g / L glucose was added only for grinding purposes.

[0325] A 1x neutral feed contains the following:

[0326] 25 mM bicarbonate;

[0327] 4.1 mM inorganic buffer salt;

[0328] 1.69 mM inositol;

[0329] All other vitamins not already included in the acidic feed (but including remaining choline chloride), totaling 0.57 mM;

[0330] 0.01 mM of a second antioxidant;

[0331] 0.043 mM L-α-amino-n-butyric acid;

[0332] 0.2 mM surfactant; and

[0333] 5 μM linoleic acid.

[0334] For the 25x concentrated neutral feed used in the examples, these concentrations were increased by a factor of 25. Without the use of titrants, the final pH of the 25x concentrated neutral feed self-adjusted to 8.0 ± 0.1 and the osmolarity was 1500 ± 35 mOsm.

[0335] 1x Alkaline Feed contains:

[0336] Amino acids, aspartic acid, histidine, tyrosine, cysteine ​​(including cystine), total concentration 43 mM.

[0337] For the 25x concentrated alkaline feed used in the examples herein, the concentration was increased 25-fold.The final pH of the 25x concentrated alkaline feed was adjusted to 10.2 ± 0.1 using sodium hydroxide and the osmolarity was 1600 ± 50 mOsm.

[0338] The perfusion medium consisted of three separate aqueous concentrate feeds adjusted to pH 7.0 ± 0.1, where the acidic concentrate feed was a 6x concentrate feed with a pH of 4.2 ± 0.1 and an osmolarity of 1700 ± 50 mOsm, the neutral concentrate feed was a 25x concentrate feed with a pH of 8.0 ± 0.1 and an osmolarity of 1500 ± 35 mOsm, and the alkaline concentrate feed was a 25x concentrate feed with a pH of 10.2 ± 0.1 and an osmolarity of 1600 ± 50 mOsm.

[0339] Example 1

[0340] After inoculation at day 0, proprietary growth medium was used immediately to start perfusion at a rate of 1vvd. Perfusion rate increased by 0.5vvd every day until reaching 2.0vvd on the 2nd day. By adding and maintaining bioreactor working volume via bioreactor weight control culture medium. On the 2nd day, concentrated culture medium feeding and diluent replaced growth medium to begin " production phase ", that is, when being cultivated in permeate and reaching the product of 0.2 gram / Lbr / day, began to load capture column. During the production phase, with constant 0.5vvd total amount feed-bundling concentrated feed (acidic feed is 0.33vvd, and alkaline and neutral feed are each 0.08vvd). Feed rate was calculated using the following equation so that, compared with the complete 1x formulation of 2vvd, the nutrient ratio in every kind of feeding kept identical:

[0341] [1x]*2vvd=[6x]*Xvvd (Equation 1)

[0342] Where X is the perfusion rate in vvd of acidic feed necessary to maintain the same nutrient content as 2 vvd of the 1x concentration formulation.

[0343] Similarly,

[0344] [1x]*2vvd=[25x]*Xvvd (Equation 2)

[0345] Where X is the perfusion rate in vvd of alkaline or neutral feed necessary to maintain the same nutrient content as 2 vvd of the 1x concentration formulation.

[0346] The VCD maximum value of the cell line used in these experiments was approximately 140 ± 30e6 cells / mL, which is based on previous engineering runs, which showed that this range was the maximum sustainable VCD (results not shown). VCD counts were performed on Beckman Coulter Vi-cell (Indianapolis, IN). In order to achieve a target of approximately 15-45% lower than the peak growth capacity of the cell line (results not shown), the osmotic pressure of the culture was gradually increased to inhibit cell replication. Culture osmotic pressure was measured with a BioProfile FLEX analyzer (Nova Biomedical, Waltham, MA), and all other culture metabolites were measured with a Roche Cedex BioAnalyzer (Indianapolis, IN). The osmotic pressure increase was achieved by adjusting the diluent rate daily to achieve the target residual osmotic pressure of the culture while keeping the feed addition rate constant. Therefore, the total perfusion rate varied from day to day. The osmotic pressure balance of daily osmotic pressure consumption was calculated according to the following equation:

[0347] Osmotic pressure input - Osmotic pressure output = Osmotic pressure consumption (Equation 3)

[0348] Wherein osmotic pressure input is the osmotic pressure of the culture medium concentrated feed and diluent that are perfused into the bioreactor, osmotic pressure output is the residual osmotic pressure of the bioreactor supernatant, and osmotic pressure consumption is the osmotic pressure difference between input and output.Then this daily osmotic pressure consumption is normalized to the cell number in culture, i.e., every cell osmotic pressure consumption every day.Then this every cell daily consumption rate (or cell specific osmotic pressure consumption rate, CSOCR) is multiplied by the predicted VCD of the second day to predict the osmotic pressure consumption of the second day.Then, this consumption rate and expected osmotic pressure output are used in equation 3 to calculate the osmotic pressure input required for the second day.Therefore, while maintaining feeding, the perfusion rate of diluent is adjusted to match the osmotic pressure input target.According to Table 1, the expected osmotic pressure target and estimated perfusion rate every day are all different (the value of each run is different, resulting in the following range):

[0349] sky Estimated viable cell density (e6c / mL) Target osmotic pressure (mOsm) Estimated perfusion rate (VVD) 2 25 300-330 1.6-1.8 3 50 300-330 2 4 75 330-360 1.8-2 5 100 350-380 1.5-1.6 6 130-150 380-410 1.2-1.4 7 150-170 380-410 1.2-1.7 8 150-170 380-410 1.2-1.3 9 140-170 380-410 1.2-1.3 10 140-180 380-410 1.2-1.3 11 130-180 380-410 1.2-1.3 12 130-170 380-410 1.2-1.3 13 130-170 380-410 1.2-1.4 14 120-160 380-410 1.3-1.4

[0350] Daily glucose measurements were taken and separate glucose bolus feeds were added as needed to maintain residual glucose at or above 2 g / L. The culture was terminated at 14 days based on a business case that matched the run duration of a typical fed-batch culture. The results of 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 specific productivity) and Figure 8 (cell specific perfusion rate).

[0351] Example 2

[0352] Three CHO cell lines A (◇), B (□) and C (Δ) expressing different recombinant IgG molecules were cultured in 2 L bioreactors (see Figures 9 to 14 ). After inoculation at day 0, proprietary growth medium was used to start perfusion at a rate of 1 vvd. The perfusion rate was increased by 0.5 vvd every day until it reached 2.0 vvd on the 2nd day. The bioreactor working volume was maintained by controlling the addition of culture medium via the bioreactor weight. On the 2nd day, the concentrated culture medium feed and diluent replaced the growth medium to begin the "production phase," that is, when the culture reached a product of 0.2 grams / Lbr / day in the permeate, the capture column was loaded. The cells were fed with a constant volume of approximately 2 vvd using three types of concentrated culture medium feeds and sterile water diluent in different proportions. As explained in Example 1, the feed rate was calculated so that the nutrient ratio in each feed remained the same compared to the complete 1x formulation of 2 vvd.

[0353] The maximum VCD values ​​for cell lines A and B were approximately 180 ± 30e6 cells / mL and 140 ± 30e6 cells / mL, respectively, based on previous engineering runs that indicated this range as the maximum sustainable VCD for these cell lines (results not shown). The maximum peak VCD for cell line C was 100 ± 20e6 c / mL, so there was no need to inhibit the growth of this cell line, and osmotic pressure remained within the physiological optimal range of 330 ± 30 mOsm. VCD counts were performed on a Beckman Coulter Vi-cell (Indianapolis, IN). For cell lines A and B, the osmotic pressure of the culture was gradually increased to inhibit cell replication in order to achieve a target of approximately 15-45% below the peak growth capacity of these cell lines (results not shown). Culture osmotic pressure was measured using a BioProfileFLEX analyzer (Nova Biomedical, Waltham, MA), and all other culture metabolites were measured using a Roche Cedex BioAnalyzer (Indianapolis, IN). The osmotic pressure increase was achieved by adjusting the concentrate feed rate and diluent rate daily to achieve the target residual osmotic pressure of the culture while keeping the total VVD addition constant at two vvd. As explained in Example 1, the daily osmotic pressure consumption was calculated according to the following equation:

[0354] Osmolarity input - Osmolarity output + Osmolarity consumption.

[0355] As in 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, both the feed and diluent rates are adjusted (as opposed to only the diluent rate as in Example 1) to achieve the target osmotic pressure input at a total perfusion rate of 2 vvd.

[0356] Daily glucose measurements were taken and a separate glucose bolus feed was added as needed to maintain residual glucose at or above 2 g / L. Based on a business case matching the run duration of a typical fed-batch culture, the culture was terminated at 14 days without the need for cell removal. The results of 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 specific productivity) and Figure 14 (cell specific perfusion rate).

[0357] Example 3

[0358] CHO DG44 cell line (cell line A, Δ) and two different CHO-K1 cell lines (cell line B□, ◇; cell line C x, x) run in duplicate were cultured in a 2L bioreactor using three concentrated media feeds fixed at a total of 0.5 vessel volumes per day (VVD) with different diluent volumes. 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 synthetase (GS) selection system (see Figure 15). All cell lines expressed different recombinant IgG molecules. The bioreactor working volume was maintained by controlling the addition of media via the bioreactor weight. On day 2, the concentrated media feed and diluent replaced the growth media to start the "production phase", that is, when the culture reached 0.2 g / L in the permeate. 生物反应器 When the product of 1x / day is reached, the capture column is loaded. During the production phase, the feed is concentrated with a constant total feed volume of 0.5 vvd (0.33 vvd for the acidic feed and 0.08 vvd for each of the alkaline and neutral feeds). As explained in Example 1, the feed rate is calculated so that the nutrient ratios in each feed remain the same compared to the complete 1x formulation of 2 vvd.

[0359] Cell line A was cultured at the physiological optimal osmotic pressure (330 ± 30 mOsm) for the entire culture duration (12 days) to promote maximum cell culture growth (i.e., possible peak VCD). This was considered the "engineering" or development run for the cell line. Cell line B was targeted with a VCD maximum of 150 ± 30e6 cells / mL ± 20e6 c / mL, based on a previous engineering run that showed this range to be the maximum sustainable VCD for the cell line (results not shown). Cell line C had a maximum peak VCD < 100 ± 20e6 c / mL, so there was no need to inhibit the growth of the cell line, and the osmotic pressure was maintained within the physiological optimal range of 330 ± 30 mOsm. VCD counts were performed on a Beckman Coulter Vi-cell (Indianapolis, IN). For cell line B, in order to achieve a target of approximately 15-45% lower than the peak growth capacity of the cell line (results not shown), the osmotic pressure of the culture was gradually increased to inhibit cell replication. Culture osmolality 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). Osmolality was increased by adjusting the diluent rate daily to achieve the target residual osmolality of the culture while maintaining a constant feed addition rate. As explained in Example 1, daily osmolality expenditure was calculated according to the following equation:

[0360] Osmotic pressure input - Osmotic pressure output + Osmotic pressure consumption

[0361] As in 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 the next day.

[0362] Daily glucose measurements were taken and individual glucose bolus feeds were added as needed to maintain residual glucose at or above 2 g / L. As shown in Figure 15, the cultures were terminated at 11, 12, and 14 days. The results for the 2 L bioreactor runs are shown below: Figure 15A Viable cell density (VCD; e5c / mL) is shown; Figure 15B Vitality (%) is shown; Figure 15C The permeate productivity (g / L / day) is shown by dividing the permeate daily instantaneous titer (g / L) as measured by the Cedex bioanalyzer by the 培养基 ) multiplied by the daily perfusion rate (L 培养基 / L 生物反应器 / day); and Figure 15D The exchange rate is shown in terms of reactor volume (L 培养基 / L 生物反应器 / day) represents the perfusion rate.

[0363] Example 4

[0364] A CHO-K1 cell line expressing recombinant IgG in a glutamine synthetase (GS) selection system was cultured in a 2L bioreactor. As described in Examples 2 and 3, operations were performed in "MC different, total VVD fixed" (◇) or "MC fixed, total VVD different" (□) perfusion control mode. "MC different, total VVD fixed" refers to a constant daily total container volume (VVD) perfusion rate, which is achieved by varying the perfusion rate of the combined medium concentrate (MC) and simultaneously varying the diluent rate to maintain 2VVD. "MC fixed, total VVD different" refers to a constant perfusion rate of MC at 0.5VVD and different diluent perfusion rates with respect to the overall fluctuating perfusion rate. Both perfusion control modes are capable of manipulating the medium osmotic pressure to set a target ( Figure 16B ). Viability and viable cell density were comparable using both perfusion control modes for this cell line. Separation of the medium concentrate feed (i.e., nutrient delivery) from the diluent enables low perfusion rates (≤2VVD) with the ability to provide adequate nutrients at high cell densities by varying the ratio of medium concentrate to diluent. Consequently, residual culture osmolarity can be controlled at elevated levels above the physiological optimal range (which varies depending on the cell line; 300-330 mOsm for this cell line) without increasing the perfusion rate above 2VVD (the highest perfusion rate is believed to be scalable to >100 L bioreactors of the company). Peak viable cell density (VCD) can be suppressed when osmolarity is increased before reaching VCD (see Figure 3 and Figure 4 ).

[0365] Adjusted productivity ( Figure 16C ) was determined as the total productivity of the system, i.e., including the product in the permeate and retained in the bioreactor per day. For the cell line shown, the productivity of the two perfusion control modes was similar, so either perfusion mode could be selected as the process for this cell line. 培养基 / L 生物反应器 / day) or the perfusion rate is shown in Figure 16D Due to operator error on days 4 and 5 of the "MC varies, total VVD fixed" run, the target of 2 VVD was not achieved on these days. The target of 2 VVD was maintained for all remaining days of the production phase (> day 2). The "MC fixed, total VVD varies" run demonstrates the variable perfusion rate necessary to maintain target osmolality (Figure 16b).

Claims

1. A method of culturing mammalian cells expressing a heterologous protein in perfusion culture, the method comprising: (a) inoculating a bioreactor with mammalian cells expressing a heterologous protein in a serum-free cell culture medium; (b) culturing the mammalian cells in perfusion culture by continuously feeding the mammalian cells with a serum-free cell culture perfusion medium feed and removing spent medium while maintaining the cells in culture, wherein the serum-free cell culture perfusion medium feed is (i) a compartmentalized serum-free cell culture perfusion medium comprising medium components grouped into 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; and wherein upon mixing the at least three separate aqueous concentrate feeds and the diluent in the resulting serum-free cell culture perfusion medium, the The compartmentalized serum-free cell culture perfusion medium has a pH adjusted to a neutral pH; and wherein the alkaline concentrate feed, the acidic concentrate feed, and the near-neutral concentrate feed of the compartmentalized serum-free cell culture perfusion medium feeds are added separately to a reaction vessel of a cell culture and / or bioreactor, and wherein the diluent is added separately to the reaction vessel of the cell culture and / or bioreactor or is premixed with one of the at least three separate aqueous concentrate feeds just prior to addition to the reaction vessel of the cell culture and / or bioreactor, 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 a target viable cell density.

2. The process of claim 1, 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.

3. The method of claim 1, wherein the mammalian cells are initially cultured in a batch culture prior to initiating perfusion culture. The method of claim 3 , wherein the perfusion culture is started from day 0 to day 3 of the batch culture.

5. The method of claim 1, wherein after perfusion is initiated, the perfusion rate is increased until a target viable cell density is reached.

6. The method of claim 5, wherein the perfusion 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.

7. The method of claim 1, wherein the osmotic pressure level of the serum-free cell culture perfusion medium is increased gradually or stepwise starting from about half the target viable cell density.

8. The method of claim 1, wherein the target viable cell density is about 30 x 10 6 cells / ml or higher.

9. The method of claim 1, wherein the target viable cell density is about 60 x 10 6 cells / ml or higher.

10. The method of claim 1, wherein the target viable cell density is about 80 x 10 6 cells / ml.

11. The method of claim 1, wherein the target viable cell density is about 100 x 10 6 cells / ml or higher.

12. The method of claim 1, wherein osmotic pressure is controlled using: (a) a constant concentrated feed perfusion rate and different diluent perfusion rates, which result in different total perfusion rates; or (b) a constant total perfusion rate and different concentrated feed perfusion rates; wherein the at least three concentrated feeds are added to each other at a fixed ratio (v / v / v) based on their multiple concentrations to maintain the relative proportions of the medium components in the 1× serum-free cell culture perfusion medium.

13. The method of claim 1, wherein the osmotic pressure is increased using: (a) a constant concentrated feed perfusion rate and a reduced diluent perfusion rate, which results in a reduced total perfusion rate; or (b) a constant total perfusion rate and an increased concentrated feed perfusion rate and a reduced diluent perfusion rate; wherein the at least three concentrated feeds are added to each other in a fixed ratio (v / v / v) based on their multiple concentrations to maintain the relative proportions of the medium components in the 1× serum-free cell culture perfusion medium.

14. The method of claim 1, wherein no further additives are added to the culture to increase osmotic pressure.

15. The method of claim 1, wherein the optimal osmotic pressure level for growth is about 280 to less than 350 mOsm.

16. The method of claim 1, wherein the osmotic pressure is maintained at a level optimal for growth until about half of the target viable cell density is reached.

17. The method of claim 1, wherein the osmotic pressure is increased gradually or stepwise starting from about half the target viable cell density.

18. The method of claim 17, wherein the osmotic pressure is increased gradually or stepwise starting from about half the target viable cell density to about 10-50% of the optimal growth osmotic pressure level.

19. The method of claim 1, wherein osmotic pressure is increased to and maintained at an osmotic pressure level that inhibits cell growth at about a target viable cell density, wherein the osmotic pressure level that inhibits cell growth is about 350 mOsm or greater.

20. The method of claim 19, wherein the osmotic pressure level that inhibits cell growth is about 380 mOsm or greater.

21. The method of claim 1, wherein increasing osmotic pressure reduces or eliminates the need for cell excretion during the production phase.

22. The method of claim 1, wherein the yield of the heterologous protein produced in the cell culture is increased by at least 5-50% relative to the yield of a control cell culture in which osmotic pressure is not increased.

23. The method of claim 1, wherein cell growth is inhibited to maintain a sustainable viable cell density without cell expulsion.

24. The method of claim 1, wherein the cell specific perfusion rate (pl / cell / day) is reduced by at least 30% relative to the cell specific perfusion rate of 1× serum-free cell culture medium.

25. The method of claim 1, further comprising harvesting the heterologous protein from the cell culture.

26. The method of claim 1, wherein the heterologous protein is a therapeutic protein, antibody, or therapeutically effective fragment thereof.

27. The method of claim 26, wherein the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a fragment thereof.

28. The method of claim 1, wherein the mammalian cell comprises a Chinese hamster ovary (CHO) cell, a Jurkat cell, a 293 cell, a HeLa cell, a CV-1 cell, or a 3T3 cell, or a derivative of any of these cells.

29. The method of claim 28, wherein the CHO cell is selected from the group consisting of: CHO-DG44 cells, CHO-K1 cells, CHODXB11 cells, CHO-S cells, and CHO GS-deficient cells or mutants thereof.

30. The method of claim 1, wherein the reaction vessel of the cell culture and / or bioreactor comprises at least about 100 L of serum-free cell culture perfusion medium.

31. The method of claim 1, wherein the reaction vessel of the cell culture and / or bioreactor comprises at least about 1000 L of serum-free cell culture perfusion medium.

32. The method of claim 1, wherein a further supplement selected from the list of antifoaming agents, base and glucose is added separately to the cell culture.

33. A method of producing a therapeutic protein using the method of any one of claims 1-32.

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