Use of rapamycin for improved antibody production

Abstract

Disclosed herein are fed-batch methods of producing an antibody in rapamycin- containing cell culture medium.

Description

USE OF RAPAMYCIN FOR IMPROVED ANTIBODY PRODUCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 637,763, filed on April 23, 2024, the disclosure of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] Disclosed herein are fed-batch methods of producing an antibody in rapamycin- containing cell culture medium.BACKGROUND

[0003] Lactate is produced by the cells as a metabolic waste during antibody production in a fed-batch mode. In mammalian cell culture, lactate usually reaches a plateau which may be followed by lactate consumption by the cells due to metabolic shifts in cell metabolism. However, during cell culture development and often with process scale up, lactate production may continue and even accelerate, which can negatively impact antibody production. Neutralizing bases can be added to the culture medium to control pH due to increased lactate production. The continued addition of neutralizing bases, however, increases osmolarity, thereby stressing the cells and ultimately leads to the premature termination of mammalian cell culture.

[0004] The increased acidity caused by the continued production of lactate can also have deleterious effects on the antibody quality, such as the production of charged variants. The acidic environment, for example, can increase the number of acidic species of the antibody (e.g., glycates, etc.).

[0005] The alteration in redox potential can also cause direct damage to the antibody. The redox potential under high lactate concentration, for example, stimulates the production of hostcell reductases and can impact product quality due to cell lysis during cell culture or harvest process (see, e.g., Dionne et al., “A low redox potential affects monoclonal antibody assembly and glycosylation in cell culture,” J. Biotechnology, Feb 2017: 246). These reductases can reduce the disulfide-bonds and can significantly decrease antibody yield while creating burden on downstream purification process to remove the reduced species.SUMMARY

[0006] Disclosed herein are fed-batch methods of producing an antibody, the methods comprising: culturing cells comprising a nucleic acid molecule that encodes the antibody in an initial cell culture medium; prior to stationary phase of the cell growth, adding rapamycin to the cell culture medium to form a rapamycin-containing cell culture medium; and continuing to culture the cells in the rapamycin-containing cell culture medium to thereby produce the antibody.

[0007] Also disclosed herein are methods of reducing an amount of lactate produced by cells, increasing an amount of lactate consumed by cells, reducing an amount of harvested cell culture fluid (HCCF) impurity levels from cells, increasing stability of an antibody produced by cells, increasing antibody titer, increasing specific productivity of the cell culture, increasing specific productivity of the cell culture, reducing lactate concentration at end of culture, reducing a glucose consumption ratio of cells, or any combination thereof, the methods comprising: culturing cells in an initial cell culture medium; and prior to stationary phase of the cell growth, adding rapamycin to the initial cell culture medium to form a rapamycin-containing cell culture medium, wherein the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin, the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin, the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium is increased relative to the stability of an antibody produced by cells cultured in medium without rapamycin, the antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin, the specific productivity of the rapamycin-containing cell culture is increased relative to the specific productivity of cell culture without rapamycin, the lactate concentration at end of culture from the cells in the rapamycin- containing cell culture medium is reduced compared to a lactate concentration at end of culture from cells cultured in medium without rapamycin, the glucose consumption ratio of the cells inthe rapamycin-containing cell culture medium is reduced relative to a glucose consumption ratio of cells cultured in medium without rapamycin, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. The drawings show exemplary embodiments of the methods, but the methods are not limited to the specific embodiments. In the drawings:

[0009] FIG. 1 illustrates an exemplary process flow diagram using 250mL microscale, 3L, and 200 L production bioreactors.

[0010] FIG. 2A, FIG. 2B, and FIG.2C illustrate the results from experiments on cells producing mAb A Clone 1. Cells were cultured in medium without rapamycin (“control”), cultured in medium to which rapamycin was added to the initial cell culture medium, or cultured in medium to which rapamycin was added on day 3, day 7, or day 10 as a single bolus dose from a 20pM working solution, to a final rapamycin concentration of lOOnM. These experiments were conducted in 500mL shake flasks. FIG. 2A shows lactate concentration (g / L), FIG. 2B shows the viable cell density (VCD) over time, and FIG. 2C shows the titer of antibody in harvested cell culture fluid (HCCF) after 14-day culture.

[0011] FIG. 3A, FIG. 3B, and FIG. 3C illustrates the results from experiments performed on cells producing mAb B that were cultured in medium without rapamycin (“control”), or cultured in medium to which rapamycin was first added as 200nM dose on day 3 (Table 2, Method 5) or day 6 (Table 2, Method 6). These experiments were conducted in a 3L bench scale bioreactor. An additional 200nM dose of rapamycin was added to the cell culture on day 15 for a total of 400nM of rapamycin. FIG 3A illustrates the lactate concentration over time in cells grown in fed-batch bench scale bioreactors, FIG 3B illustrates the viable cell density (VCD), and FIG 3C illustrates the titer of antibody mAb B in harvested cell culture (HCCF) at day 20.

[0012] FIG. 4 illustrates the lactate concentration in a fed-batch culture of CHO cells producing mAb A Clone 1 in a 3L bench scale bioreactor. Cells were grown in medium without rapamycin (“control”) or in medium to which rapamycin was fed in 200nM doses on day 3 and day 8 each (400nM in total, Table 2, Method 1).

[0013] FIG. 5 illustrates the lactate concentration in a fed-batch culture of CHO cells producing mAb B in a 3L bench scale bioreactor in medium without rapamycin (“control”) or in medium to which rapamycin was supplemented daily from day 3 to day 15 at 2.7% of postinoculation reactor weight each day using nutrient feed containing 800nM of rapamycin for a total addition of 210nM rapamycin in a 16 day process (Table 2, Method 10).

[0014] FIG. 6 illustrates the lactate concentration in a fed-batch culture of CHO cells producing mAb C in a 3L bench scale bioreactor in medium without rapamycin (“control”) or in medium to which rapamycin was supplemented in 200nM doses on each of day 3 and day 8 (400nM in total, Table 2, Method 1).

[0015] FIG. 7 illustrates the lactate concentration in a fed-batch culture of CHO cells producing mAb A in a 3L bioreactor in medium without rapamycin (“control”); in medium to which 80nM of total rapamycin was fed (25nM dose on day 3 followed by 5nM doses each day from day 8 to 18, Table 2, Method 2) (condition 1); and in medium to which 240nM of total rapamycin was fed (80nM dose on day 3 followed by 15nM doses each day from day 8 to 18, Table 2, Method 3) (condition 2).

[0016] FIG. 8 illustrates the lactate concentration in a fed-batch culture of CHO cells producing mAb A in a 200L pilot scale bioreactor in medium without rapamycin (“control”) or in medium to which 1 lOnM of rapamycin was fed as 50nM dose on day 3 followed by lOnM doses each day from day 8 to 13 (Table 2, Method 4).

[0017] FIG. 9 illustrates the viable cell density (VCD) of a fed-batch culture of cells producing mAb A Clone 2 in a 3L bench scale bioreactor in medium without rapamycin (“control”) or in medium to which rapamycin was fed as 200nM doses on each of day 3 and day 8 (400nM in total, Table 2, Method 1).

[0018] FIG. 10 illustrates the VCD of a fed-batch culture of cells producing mAb B in a 3L bench scale bioreactor in medium without rapamycin (“control”) or in medium to which nutrient feed containing 800nM of rapamycin was added daily from day 3 to day 15 at 2.7% of post-inoculation reactor weight (a total addition of 210nM rapamycin in a 16 day process, Table 2, Method 10).

[0019] FIG. 11 illustrates the VCD of a fed-batch culture of cells producing mAb C in a 3L bench scale bioreactor in medium without rapamycin (“control”) or in medium to whichrapamycin was fed as 200nM doses on day 3 and day 8 each (400nM in total, Table 2, Method 1).

[0020] FIG. 12 illustrates the VCD of a fed-batch culture of cells producing mAb A Clone 1 in a 200L pilot scale in medium without rapamycin (“control”), in medium to which 80nM of rapamycin was fed (25nM dose on day 3 followed by 5nM doses from day 8 to day 18, Table 2, Method 2), or in medium to which 240nM of rapamycin was fed (80nM dose on day 3 followed by 15nM doses from day 8 to day 18, Table 2, Method 3).

[0021] FIG. 13 illustrates the VCD of a fed-batch culture of cells producing mAb A Clone 1 in a 200L pilot scale in medium without rapamycin (“control”) or in medium to which 1 lOnM of rapamycin was fed (50nM dose on day 3 followed by lOnM doses from day 8 to day 13, Table 2, Method 4).

[0022] FIG. 14 illustrates the VCD of a fed-batch culture of cells producing mAb B in a 200L pilot scale bioreactor in medium without rapamycin (“control”) or in medium to which nutrient feed containing lOOOnM of rapamycin was added daily from day 3 to day 14 at 2.7% of post-inoculation reactor weight (a total addition of 215nM rapamycin in a 14 day process, Table 2, Method 7).DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0023] The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure.

[0024] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the herein disclosure. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower endby a non-zero value. Tt is not intended that the scope of the methods be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.

[0025] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term “about” is used to encompass variations of ± 10% or less from the specified value.

[0026] As used herein, the singular forms “a,” “an,” and “the” include the plural.

[0027] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0028] The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of’ and “consisting of;” similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0029] The term “antibody” is meant in a broad sense and includes full length immunoglobulin molecules, antigen-binding fragments thereof, and antibodies conjugated to another molecule, such as antibody-protein fusions and antibody-drug conjugates. Antibody, as used herein, also includes within its scope multispecific (e.g., bispecific, trispecific, etc.) antibodies and multivalent antibodies.

[0030] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda ( ), based on the amino acid sequences of their constant domains.

[0031] “Antigen-binding fragment” refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full-length antibody (i.e., “antigenbinding fragment thereof’). Exemplary antigen binding fragments can have: heavy chain complementarity determining regions (CDR) 1, 2, and / or 3; light chain CDR 1, 2, and / or 3; aheavy chain variable region (VH); a light chain variable region (VL); and combinations thereof. Antigen binding fragments include: a Fab fragment (a monovalent fragment consisting of the VL, VH, constant light (CL), and constant heavy 1 (CHI) domains); a F(ab)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); a Fd fragment (consisting of the VH and CHI domains); a Fv fragment (consisting of the VL and VH domains of a single arm of an antibody); and a domain antibody (dAb) fragment (Ward et al., Nature 341 :544-546, 1989), (which consists of a VH domain or a VL domain). VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody, described in, e.g., Int’l Pub. Nos. W01998 / 44001, WO1988 / 01649, WO1994 / 13804, and W01992 / 01047. These antibody fragments are obtained using techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.

[0032] In the present application, the disclosed methods are performed using a range of exemplary recombinant antibodies as shown below.

[0033] Disclosed herein are fed-batch methods of producing an antibody, the methods comprising: culturing cells comprising a nucleic acid molecule that encodes the antibody in an initial cell culture medium; prior to stationary phase of the cell growth, adding rapamycin to the cell culture medium to form a rapamycin-containing cell culture medium; and continuing to culture the cells in the rapamycin-containing cell culture medium to thereby produce the antibody.

[0034] “Fed-batch” refers to the culturing of cells in medium to which glucose, nutrients, and / or other supplements required for cell growth are added, and in which the pH is adjusted.The supplementing and pH adjustment can be continuous or can occur periodically (daily, or every two to four days) beginning prior to the stationary phase of the culture. Preferably, the supplementation occurs early in log phase.

[0035] The term “lag phase,” “log phase,” “stationary phase,” and “death phase” are well- known in the art of cell culture and can be derived from the plot of the log of viable cell numbers in culture over time. At initial inoculation of cells into a volume of fresh medium, there may be a “lag phase,” a period in which there is little to no cell growth while the cells adjust to the presence of lower cell numbers, more nutrients, and / or less toxic metabolites. Next, cells enter a period of growth and division, and cell numbers double at regular intervals, leading to the term “log phase” (also referred to as exponential phase). Eventually the growth in cell numbers slows, and the numbers plateau in “stationary phase.” Stationary phase may be triggered by a variety of factors, including cell numbers, shortage of nutrients or oxygen, change in culture pH, redox potential, and / or accumulation of metabolites such as lactate. After an extended period in stationary phase, cell death may occur in “death phase.” Cell death and lysis releases host cell proteins that can potentially degrade recombinant proteins secreted into the media. Hence, recombinant proteins are typically harvested from bioprocess reactors before extensive cell death. Harvest typically involves solid-liquid separation to separate cells from the cell culture broth. One such method is centrifugation. Solid-liquid separation may also be followed by depth filtration and microfiltration to remove residual cell fragments and debris. The resulting fluid is “harvested cell culture fluid” (HCCF).

[0036] “End of cell culture” is defined as the initiation of cell culture cooling before the solid-liquid separation process. The culture is typically chilled to 10-20°C, and oxygen or air continues to be provided.

[0037] In some embodiments, the rapamycin is added at about day three of culturing the cells in the initial cell culture medium.

[0038] In some embodiments, the rapamycin is added during log phase of the cells.

[0039] Rapamycin can be added to the cell culture medium in the form of a working solution. Suitable concentrations of rapamycin working solution that can be added to the cell culture medium include, for example, about lOOnM to about 20pM. Upon addition of the rapamycin working solution to the cell culture medium, the cell culture medium can comprise, for example, about 3nM to about 460nM of rapamycin. The rapamycin working solution can beadded directly to the cell culture, to the feed media, or both. Upon addition of the rapamycin working solution to the initial cell culture medium and / or to the rapamycin-containing cell culture medium, the rapamycin-containing cell culture medium can comprise about 3nM to about 460nM of rapamycin.

[0040] The rapamycin can be added in the form of a bolus addition from a working solution or by supplementing nutrient feeds for addition to the medium on the required culture days. The rapamycin can be added to the cell culture medium continuously or in multiple doses on different days. In some embodiments, the rapamycin is added in multiple doses on different days of the culture duration. The rapamycin can be added, for example, on one or more of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or from day 3 until the end of culture via formulation in nutrient feeds. In some embodiments, the rapamycin is added on day 3 and day 15. In some embodiments, the rapamycin is added on day 3 and daily on each of days from day 8 onwards until the end of culture. In some embodiments, the rapamycin is added daily from day 3 until the end of culture. For example, the rapamycin can be added daily on each of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or until the end of culture. In some embodiments, the concentration of rapamycin in the cell culture after a first addition is between about 3nM to about 200nM.

[0041] Suitable cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293), mouse myeloma (NS0), and baby hamster kidney (BHK) cells. In some embodiments, the cells are CHO cells.

[0042] Addition of rapamycin to the cell culture medium can reduce the amount of lactate produced by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced by cells cultured in medium without rapamycin. Addition of rapamycin to the cell culture medium can increase the amount of lactate consumed by the cells in the rapamycin- containing cell culture medium relative to an amount of lactate consumed by cells cultured in medium without rapamycin. Addition of rapamycin to the cell culture medium can reduce the amount of lactate produced by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced by cells cultured in medium without rapamycin and can increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate consumed by cells cultured in medium without rapamycin. The rapamycin can induce the cells to reverse from lactate production to lactate consumption.

[0043] Addition of rapamycin to the cell culture medium can reduce the amount of harvested cell culture fluid (HCCF) impurity levels from the cells in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin. Addition of rapamycin to the cell culture medium can increase the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium during HCCF storage relative to the stability of an antibody produced by cells cultured in medium without rapamycin during HCCF storage. Addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels from the cells in the rapamycin- containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin and increase the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin.

[0044] Addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin. Addition of rapamycin to the cell culture medium can increase the specific productivity of the rapamycin-containing cell culture relative to the specific productivity of cell culture without rapamycin. Addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin. Addition of rapamycin to the cell culture medium can reduce the lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin. Addition of rapamycin to the cell culture medium can reduce the glucose consumption ratio of the cells in the rapamycin- containing cell culture medium relative to a glucose consumption ratio of cells cultured in medium without rapamycin.

[0045] Addition of rapamycin to the cell culture medium can lead to any combination of the following: reduce the amount of lactate produced by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced by cells cultured in medium without rapamycin;increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate consumed by cells cultured in medium without rapamycin; increase the antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin; reduce the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin; increase the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin; increase the specific productivity of the rapamycin-containing cell culture relative to the specific productivity of cell culture without rapamycin; reduce the peak VCD from the cells in the rapamycin-containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin; reduce the lactate concentration at end of culture from the cells in the rapamycin- containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin; and reduce the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium relative to a glucose consumption ratio of cells cultured in medium without rapamycin.

[0046] In some embodiments, the reduction in the amount of HCCF impurity levels comprises a reduction in the amount of lactate dehydrogenase (LDH). In some embodiments, the reduction in the amount of HCCF impurity levels comprises a reduction in the amount of residual DNA (rDNA). In some embodiments, the reduction in the amount of HCCF impurity levels comprises a reduction in the amount of LDH and a reduction in the amount of rDNA.

[0047] Addition of rapamycin can reduce the amount of HCCF impurity levels. In some embodiments, a reduction in the amount of HCCF impurity levels can be achieved by preventing or decreasing antibody fragmentation. In some embodiments, a reduction in the amount of HCCF impurity levels can be achieved by increasing sheer stress resistance during centrifugation during cell culture harvest. In some embodiments, a reduction in the amount of HCCF impurity levelscan be achieved by preventing or decreasing antibody fragmentations during antibody storage in HCCF.

[0048] Addition of rapamycin to the cell culture medium can reduce the peak concentration of lactate (i.e., the highest concentration of lactate produced during the stationary phase) produced by the cells in the rapamycin-containing cell culture medium relative to a peak amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak amount of lactate produced by the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a peak amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak amount of lactate produced by the cells in the rapamycin-containing cell culture medium by about 20% to about 70%, about 25% to about 65%, or about 30% to about 60% relative to a peak amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak amount of lactate produced by the cells in the rapamycin-containing cell culture medium by about 30% to about 60% relative to a peak amount of lactate produced by cells cultured in medium without rapamycin.

[0049] Addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium by about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100% relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin. In someembodiments, addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium by about 80% to about 95% relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin.

[0050] Addition of rapamycin to the cell culture medium can increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of lactate consumed by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium by about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% relative to an amount of lactate consumed by cells cultured in medium without rapamycin.

[0051] Addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium by about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, or about 40% to about 50% relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium by about 15% to about 35%.

[0052] Addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels (c. ., residual DNA) from the cells in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium withoutrapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels by about 10% to about 85%. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of LDH from the cells in the rapamycin-containing cell culture medium by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of LDH from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of LDH from the cells in the rapamycin-containing cell culture medium by about 10% to about 55% relative to an amount of LDH from cells cultured in medium without rapamycin. In some embodiments, the amount of LDH is reduced by about 10%. In some embodiments, the amount of LDH is reduced by about 55%. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of rDNA from the cells in the rapamycin-containing cell culture medium by about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or by about 100% relative to an amount of rDNA from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of rDNA from the cells in the rapamycin-containing cell culture medium by about 40% to about 90% relative to an amount of rDNA from cells cultured in medium without rapamycin. In some embodiments, the amount of rDNA is reduced by about 75%. In some embodiments, the amount of rDNA is reduced by about 80%.

[0053] Addition of rapamycin to the cell culture medium can increase the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium results in a less than an about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% decrease in stability of the antibody produced by the cells in the rapamycin- containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin.

[0054] Addition of rapamycin to the cell culture medium can decrease the fragmentation of the antibody produced in the cells in the rapamycin-containing cell culture medium by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when the antibody is stored in HCCF relative to an amount of fragmentation of antibody produced by cells cultured in medium without rapamycin.

[0055] Addition of rapamycin to the cell culture medium can increase the specific productivity of the rapamycin-containing cell culture by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to the specific productivity of cell culture without rapamycin.

[0056] Addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a peak VCD from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium by about 10% to about 50%.

[0057] Addition of rapamycin to the cell culture medium can maintain the peak VCD from the cells in the rapamycin-containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin.

[0058] Addition of rapamycin to the cell culture medium can reduce the lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin.

[0059] Addition of rapamycin to the cell culture medium can reduce the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium relative to aglucose consumption ratio of cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a glucose consumption ratio of cells cultured in medium without rapamycin. Glucose consumption ratio can be calculated by dividing the total amount of glucose solution added from day 3 to end of culture by the post inoculation culture weight and multiplying by 100, as shown in the herein disclosed examples.

[0060] Disclosed herein are methods of reducing an amount of lactate produced by cells, increasing an amount of lactate consumed by cells, reducing an amount of HCCF impurity levels from cells, increasing stability of an antibody produced by cells, increasing antibody titer, increasing specific productivity of the cell culture, reducing peak viable cell density (VCD), reducing lactate concentration at end of culture, reducing a glucose consumption ratio of cells, or any combination thereof, the methods comprising: culturing cells in an initial cell culture medium; and prior to stationary phase of the cells, adding rapamycin to the initial cell culture medium to form a rapamycin-containing cell culture medium, wherein the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin, the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin, the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium is increased relative to the stability of an antibody produced by cells cultured in medium without rapamycin, the antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin, the specific productivity of the rapamycin-containing cell culture is increased relative to the specific productivity of cell culture without rapamycin, the peak VCD from the cells in the rapamycin-containing cell culture medium is reduced relative to a peak VCD from cells cultured in medium without rapamycin, thelactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium is reduced compared to a lactate concentration at end of culture from cells cultured in medium without rapamycin, the glucose consumption ratio of the cells in the rapamycin- containing cell culture medium is reduced relative to a glucose consumption ratio of cells cultured in medium without rapamycin, or any combination thereof.

[0061] The disclosed methods can induce the cells to reverse from lactate production to lactate consumption.

[0062] The cells can comprise a nucleic acid molecule that encodes a protein of interest. In some embodiments, the protein of interest is an antibody.

[0063] In some embodiments, the rapamycin is added at about day three of culturing the cells in the initial cell culture medium.

[0064] In some embodiments, the rapamycin is added during log phase of the cells.

[0065] Rapamycin can be added to the cell culture medium in the form of a bolus addition from a working solution or by supplementing nutrient feeds for addition to the medium on the required days. Suitable concentrations of rapamycin working solution that can be added to the cell culture medium include, for example, about 200nM to about 20pM. Upon addition of the rapamycin working solution or the rapamycin-supplemented nutrient feed to the cell culture medium, the cell culture medium can comprise, for example, about 3nM to about 460nM of rapamycin. The rapamycin working solution or the rapamycin-supplemented nutrient feed can be added to the initial cell culture medium, to the rapamycin-containing cell culture medium, or both. Upon addition of the rapamycin working solution or the rapamycin-supplemented nutrient feed to the initial cell culture medium and / or to the rapamycin-containing cell culture medium, the rapamycin-containing cell culture medium can comprise about 3nM to about 460nM of rapamycin.

[0066] The rapamycin can be added to the cell culture medium continuously or in multiple doses on different days. In some embodiments, the rapamycin is added in multiple doses on different days of the culture duration. The rapamycin can be added, for example, on one or more of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or from day 3 until the end of culture. In some embodiments, the rapamycin is added on day 3 and day 15. In some embodiments, the rapamycin is added on day 3 and daily on each of days from day 8 until the end of culture duration. In some embodiments, the rapamycin is added daily from day 3 until the end of culture.For example, the rapamycin can be added daily on each of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, the concentration of rapamycin in the cell culture medium after a first addition is between about 3nM to about 200nM.

[0067] Suitable cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293), mouse myeloma (NSO), and baby hamster kidney (BHK) cells. In some embodiments, the cells are CHO cells.

[0068] In some embodiments, the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin. In some embodiments, the antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin and the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin. In some embodiments, the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin and the antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin and the antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin, andthe antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin.[00691 In some embodiments, the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin. In some embodiments, the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium is increased relative to the stability of an antibody produced by cells cultured in medium without rapamycin. In some embodiments, the amount of HCCF impurity levels from the cells in the rapamycin- containing cell culture medium is reduced relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin and the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium is increased relative to the stability of an antibody produced by cells cultured in medium without rapamycin.

[0070] In some embodiments, the antibody titer from the cells in the rapamycin- containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, the specific productivity of the rapamycin- containing cell culture is increased relative to the specific productivity of cell culture without rapamycin. In some embodiments, the peak VCD from the cells in the rapamycin-containing cell culture medium is reduced relative to a peak VCD from cells cultured in medium without rapamycin. In some embodiments, the lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium is reduced relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin. In some embodiments, the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium is reduced relative to a glucose consumption ratio of cells cultured in medium without rapamycin.

[0071] Addition of rapamycin to the cell culture medium can lead to any combination of the following: reduce the amount of lactate produced by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced by cells cultured in medium without rapamycin; increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate consumed by cells cultured in medium without rapamycin;increase the antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin; reduce the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin; increase the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin; increase the specific productivity of the rapamycin-containing cell culture relative to the specific productivity of cell culture without rapamycin; reduce the peak VCD from the cells in the rapamycin-containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin; reduce the lactate concentration at end of culture from the cells in the rapamycin- containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin; and reduce the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium relative to a glucose consumption ratio of cells cultured in medium without rapamycin.

[0072] In some embodiments, the reduction in the amount of HCCF impurity levels comprises a reduction in the amount of LDH. In some embodiments, the reduction in the amount of HCCF impurity levels comprises a reduction in the amount of rDNA. In some embodiments, the reduction in the amount of HCCF impurity levels comprises a reduction in the amount of LDH and a reduction in the amount of rDNA.

[0073] Addition of rapamycin can reduce the amount of HCCF impurity levels. In some embodiments, a reduction in the amount of HCCF impurity levels can be achieved by preventing or decreasing antibody fragmentation. In some embodiments, a reduction in the amount of HCCF impurity levels can be achieved by increasing sheer stress resistance during centrifugation during cell culture harvest.

[0074] Addition of rapamycin to the cell culture medium can reduce the peak amount of lactate (z.e., the highest amount of lactate produced during the culturing) produced by the cells in the rapamycin-containing cell culture medium relative to a peak amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to thecell culture medium can reduce the peak amount of lactate produced by the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a peak amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak amount of lactate produced by the cells in the rapamycin-containing cell culture medium by about 20% to about 70%, about 25% to about 65%, or about 30% to about 60% relative to a peak amount of lactate produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak amount of lactate produced by the cells in the rapamycin-containing cell culture medium by about 30% to about 60% relative to a peak amount of lactate produced by cells cultured in medium without rapamycin.

[0075] Addition of rapamycin to the cell culture medium can reduce the concentration of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium by about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100% relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of lactate produced at end of culture by the cells in the rapamycin-containing cell culture medium by about 80% to about 95% relative to an amount of lactate produced at end of culture by cells cultured in medium without rapamycin.

[0076] Addition of rapamycin to the cell culture medium can increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 100% relative to an amount of lactate consumed by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium by about 5% to than 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% relative to an amount of lactate consumed by cells cultured in medium without rapamycin.

[0077] Addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium by about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, or about 40% to about 50% relative to an antibody titer from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can increase the antibody titer from the cells in the rapamycin-containing cell culture medium by about 15% to about 35%.

[0078] Addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of HCCF impurity levels by about 10% to about 85%. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of LDH from the cells in the rapamycin-containing cellculture medium by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of LDH from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of LDH from the cells in the rapamycin-containing cell culture medium by about 10% to about 55% relative to an amount of LDH from cells cultured in medium without rapamycin. In some embodiments, the amount of LDH is reduced by about 10%. In some embodiments, the amount of LDH is reduced by about 55%. In some embodiments, addition of rapamycin to the cell culture medium can reduce the amount of rDNA from the cells in the rapamycin-containing cell culture medium by about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to an amount of rDNA from cells cultured in medium without rapamycin. The addition of rapamycin to the cell culture medium can reduce the amount of rDNA from the cells in the rapamycin-containing cell culture medium by about 40% to about 90% relative to an amount of rDNA from cells cultured in medium without rapamycin. In some embodiments, the amount of rDNA is reduced by about 75%. In some embodiments, the amount of rDNA is reduced by about 80%.

[0079] Addition of rapamycin to the cell culture medium can increase the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium results in a less than an about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% decrease in stability of the antibody produced by the cells in the rapamycin- containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin.

[0080] Addition of rapamycin to the cell culture medium can decrease the fragmentation of the antibody produced in the cells in the rapamycin-containing cell culture medium by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when the antibody is stored in HCCF relative to an amount of fragmentation of antibody produced by cells cultured in medium without rapamycin.

[0081] Addition of rapamycin to the cell culture medium can increase the specific productivity of the rapamycin-containing cell culture by about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to the specific productivity of cell culture without rapamycin.

[0082] Addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a peak VCD from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the peak VCD from the cells in the rapamycin-containing cell culture medium by about 10% to about 50%.

[0083] Addition of rapamycin to the cell culture medium can reduce the lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin.

[0084] Addition of rapamycin to the cell culture medium can reduce the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium relative to a glucose consumption ratio of cells cultured in medium without rapamycin. In some embodiments, addition of rapamycin to the cell culture medium can reduce the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% relative to a glucose consumption ratio of cells cultured in medium without rapamycin.EXAMPLES

[0085] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Example 1 - Effects of Rapamycin on lactate metabolism, cell growth, glucose consumption, antibody yield, and specific productivity

[0086] The mammalian / mechanistic target of rapamycin (mTOR) associates with other proteins to form the mTOR complex 1 (mTORCl), which acts as a regulator of protein production in response to environmental signals. In response to cellular signals including nutrients, energy levels, and redox levels, mTORCl activates mRNA translation, and thereby increases protein production. Rapamycin, a GMP-compliant, non-beta lactam antibiotic, binds to and inhibits mTORCl and thereby can affect various protein production machinery, and arrest cells in G1 / G0 phase. Rapamycin can also trigger autophagy, a class of processes that allow the cell to recycle older cellular material for energy production or the generation of new biomaterials. The timing, dosage(s), and target cells can influence the response to rapamycin.

[0087] The disclosed experiments show that appropriate timing and dosage of rapamycin to fed-batch CHO cell culture reduces cell growth, reduces cell death, increases antibody yield, increased specific productivity, improves lactate metabolism, and reduces glucose consumption. The disclosed experiments also show that rapamycin switches cells from lactate production to lactate consumption.Methods

[0088] FIG. 1 illustrates an exemplary process flow diagram using ambr250 microscale, 3L bench scale, and 200 L pilot scale for production bioreactors.Media and Solutions

[0089] Rapamycin (sirolimus) powder was dissolved in 200 proof ethanol to a concentration of 1 ImM to create a stock solution. Stock solution was then diluted in water for injection (WFI) to create working solution. Working solution concentration was made from 5- 20pM depending on desired final concentration in the culture medium. Alternatively, the 1 ImMstock solution was added in Feed A or FeedM to a concentration of 200-1600nM for daily addition to the culture medium. See Table 1.Fed-batch Culture in Bioreactors

[0090] A clonal CHO cell line expressing either mAh A, mAh B, mAh C, or mAh D was cultured in a fed-batch mode in serum-free, chemically defined Gibco Dynamis™ medium (THERMO FISHER SCIENTIFIC®) at an initial target of 0.5 xlO6cells / ml, with over 90% viability. For all experiments, the same seed cells were used to inoculate the production bioreactors for the control and rapamycin condition. Cells were grown with continuous agitation, temperature control, dissolved oxygen (DO) control, and pH control. Air and oxygen sparging was used to control DO. pH control was obtained through the addition of CO2 to the air / oxygen feed to lower the pH, and a solution of Na2COs was used to raise the pH. On day 3 and onwards, bioreactors were fed with nutrient feeds, glucose, and rapamycin, as applicable. Molecule specific process parameters and feeding schemes are provided in Table 1.Table 1. Reactor OperationTable 2. Rapamycin Delivery MethodsaBolus addition refers to rapamycin addition to cell culture media via an aqueous working solution (5-20pM) to the described final concentration. Reactor weight on described day is used to calculate concentration (e.g., 200nM bolus addition for 2kg culture weight would require 20mL of 20 pM working solution).bTable 1 describes Feed A and Feed M feeding schedules. All Rapamycin containing Feed A or Feed M was fed at 2.7% post inoculation reactor weight from day 3 to end of culture.ResultsEffects of Rapamycin on Fed-Batch Cell Culture are Time Dependent Effects of Rapamycin are Maximized when added in Early Log Phase

[0091] The cells used in this experiment contained a nucleic acid molecule encoding mAb A Clone 1 and the process was carried out in 500mL shake flasks in a fed-batch mode. A 20pM Rapamycin working solution was either added to initial batch medium or added as a bolus addition on day 3,7, or 10 to achieve lOOnM rapamycin final concentration in the culture. These days were selected for rapamycin addition as initial media, early log phase (day 3), stationary phase (day 7), and death phase (day 10). The presence of rapamycin in the initial medium, or theaddition of rapamycin on day 3 or 10 led to decreased lactate production in the culture (Figure 2C). Because this experiment was conducted in shake flasks, the limited DO control and onesided pH control with CO2 prevented lactate consumption. The presence of rapamycin in the initial medium or the addition of rapamycin on day 3 reduced growth rate. The control culture yielded a harvest titer of 4.87g / L. The presence of rapamycin in initial medium did not improve harvest titer compared to the control (4.94g / L). The addition of rapamycin on day 3, 7, or 10 improved harvest titers from 4.87 g / L(control) to 5.70g / L (lOOnM bolus addition on day 3), 5.33g / L (lOOnM bolus addition on day 7), and 5.09g / L (lOOnM bolus addition on day 10) respectively. Addition of rapamycin to the culture was narrowed to log phase for futured experiments. See FIG 2A-FIG. 2C.

[0092] The cells used in this experiment contained a nucleic acid molecule encoding mAb B and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was added to achieve 200nM doses at each addition (400nM total) from a 20pM working solution. The first dose was administered on day 3 (Method 5 as described in Table 2) or 6 (Method 6 as described in Table 2) followed by a secondary dose on day 15. Day 3 and 6 were selected to introduce rapamycin to the cell culture during early and late log phase growth. Adding the primary dose of rapamycin on day 3 reduced peak lactate from 5.94g / L to 2.41g / L, while the addition of rapamycin on day 6 had no effect. Addition of rapamycin lowered end of culture lactate from 14.01g / L to 1.91g / L (1stRapamycin addition on day 3) and 7.11 g / L (1stRapamycin addition on day 6). End of culture titer was improved from 9.38g / L to 12.86g / L (1stRapamycin addition on day 3) and 11.61 g / L (1stRapamycin addition on day 6). Day 3 was identified as a preferred additional point of primary rapamycin dose and used for future experiments. See FIG 3A-FIG. 3C.Rapamycin Effects on Lactate MetabolismEffects of Rapamycin on Lactate Metabolism are not Molecule Dependent

[0093] The cells used in this experiment contained a nucleic acid molecule encoding mAb A Clone 1 and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was added to achieve 200nM doses (each on day 3 and 8, 400nM total) from a 20pM working solution (Method 1, Table 2). With the addition of rapamycin, peak lactate concentration was reduced from 4.62g / L (observed on day 8) to 2.66g / L (observed on day 5). Lactate concentrationremained low through the duration of the culture. Rapamycin reduced end of culture lactate concentration from 3.3 Ig / L to 0.19 g / L on day 14. See FIG. 4 and Table 3.Table 3. mAb A Clone 1 Lactate Metabolism Summary

[0094] The cells used in this experiment contained a nucleic acid molecule encoding mAb B antibody and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at 800nM concentration and fed daily from day 3 to day 15 at 2.7% of the post-inoculation reactor weight (210nM total final concentration, Method 10, Table 2). With the addition of rapamycin, peak lactate concentration was reduced from 5.44g / L (observed on day 7) for control to 2.25g / L (observed on day 5). In addition, lactate concentration remained low throughout the process. End of culture lactate concentration with rapamycin was 0.79g / L as compared with 10.41g / L in the control. See FIG. 5 and Table 4.Table 4. mAb B Lactate Metabolism Summary

[0095] The cells used in this experiment contained a nucleic acid molecule encoding mAb C and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was added as 200nM doses (each on day 3 and 8, 400nM total) from a 20pM working solution (Method 1, Table 2). See Table 1. With the addition of rapamycin, peak lactate concentration was reduced from 3.25g / L (observed on day 8) to 1.74g / L (observed on day 4). Lactate concentrationremained low through the duration of the culture. Rapamycin reduced end of culture lactate concentration from 8.25 g / L to 0.47 g / L on day 15. See FIG. 6 and Table 5.Table 5. mAb C Lactate Metabolism SummaryEffects of Rapamycin on Lactate Metabolism are Dose Dependent

[0096] The cells used in this experiment contained a nucleic acid molecule encoding mAb A Clone 1 and the process was carried out in 3L bioreactors in fed-batch mode. All conditions followed seed expansion and reactor operations as described above. 80nM total rapamycin was added as a 25nM dose on day 3 followed by 5nM doses from day 8-18 (Method 2, Table 2). 240nM total rapamycin dose was added as 75nM dose on day 3 followed by 15nM doses from day 8-18 (Method 3, Table 2). Increasing rapamycin concentrations decreased peak lactate concentration from 4.09g / L to 2.74g / L (total 80nM rapamycin) to 2.20g / L (240nM rapamycin). Additionally, lactate peaked on day 7 with 240nM rapamycin vs day 8 for 80nM and control. Lactate concentration remained low throughout culture irrespective of concentration. Day 19 lactate concentration was 1.03g / L for 80nM and 0.84g / L for 240nM vs 5.24g / L for the control. See FIG. 7 and Table 6.Table 6. mAb A Clone 1 Dose Response Lactate Metabolism SummaryEffects of Rapamycin on Lactate Metabolism During Process Scale-up

[0097] The cells used in this experiment contained a nucleic acid molecule encoding mAh A Clone 1 and the process was carried out in 200L bioreactors in fed-batch mode. All conditions followed seed expansion and reactor operations as described above. 1 lOnM total rapamycin dose was added as a 50nM dose on day 3 followed by lOnM doses from day 8-13 (Method 4, Table 2). 1 lOnM rapamycin concentration decreased peak lactate concentration from 4.94g / L to 2.28g / L. Additionally, lactate peaked on day 6 with 1 lOnM rapamycin vs day 8 for control. Lactate concentration remained low throughout culture. Day 14 lactate concentration was 0.21g / L vs 4.04g / L for the control. See FIG. 8 and Table 7.Table 7. mAb A Clone 1 Pilot Scale Lactate Metabolism SummaryRapamycin Effects on Cell Growth

[0098] Described below are two methods used to evaluate cell growth. The first method was peak VCD as measured directly by a Beckman Coulter Vi-Cell XR cell counter. The second method was average specific growth over the mid log phase. Mid log phase is defined as day 4 to one day before peak VCD (linear portion of the growth curve). In the event control and rapamycin treated cultures reached peak VCD on different days, the shorter duration was used to analyze all cultures. The following formula was used to calculate daily specific growth rate (p).In (fVCD2■ Cell Culture W eighty / ((V CD1■ Cell Culture Weightf ) - - ■ - ■ - time2— time1Daily specific growth rate was averaged over the described duration to calculate average specific growth rate. A lower average specific growth rate indicated slower cell growth.Effects of Rapamycin on Cell Growth are not Molecule Dependent

[0099] The cells used in this experiment contained a nucleic acid molecule encoding mAb A clone 2 and the process was carried out in 3L bench scale bioreactors in fed-batch mode. Rapamycin was added as 200nM doses (each on day 3 and 8, 400nM total) from a 20 pM working solution (Method 1, Table 2). Rapamycin delivery resulted in reduced peak viable cell density (VCD) and growth rate. Peak VCD was reduced from 45.04 x 106cells / mL (observed on day 9) to 24.2 x 106cells / mL (observed on day 9) with the addition of rapamycin. In addition, average specific growth rate was reduced to 0.34 hr1with rapamycin from 0.45 hr1for the control. See FIG. 9 and Table 8.Table 8. mAb A Clone 2 Cell Growth Summary

[0100] The cells used in this experiment contained a nucleic acid molecule encoding mAb B antibody and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at 800nM concentration and fed daily from day 3 to day 15 at 2.7% of the post-inoculation reactor weight (210nM total final concentration, Method 10, Table 2). Rapamycin addition resulted in reduced peak VCD and growth rate. Peak VCD was reduced from 35.01 x 106cells / mL (observed on day 8) to 24.82 x 106cells / mL (observed on day 9) with rapamycin. In addition, average specific growth rate was reduced to 0.34 hr'1with rapamycin from 0.49 hr'1for the control. See FIG. 10 and Table 9.Table 9. mAb B Cell Growth Summary

[0101] The cells used in this experiment contained a nucleic acid molecule encoding mAb C and the process was carried out in bench scale bioreactors in feed batch mode. Rapamycin was added as 200nM doses (each on day 3 and 8, 400nM total) from a 20pM working solution (Method 1, Table 2). With the addition of rapamycin, peak VCD was reduced from 24.36 x 106cells / mL (observed on day 9) to 18.66 x 106cells / mL (observed on day 9). Although rapamycin and the control culture both reached peak VCD on day 9, the average specific growth rate during mid log phase was reduced from 0.31 hr1to 0.26 hr1with the addition of rapamycin. See FIG. 11 and Table 10.Table 10. mAb C Cell Growth SummaryEffects of Rapamycin on Cell Growth are Dose Dependent

[0102] The cells used in this experiment contained a nucleic acid molecule encoding mAb A Clone 1 and the process was carried out in bench scale bioreactors in fed-batch mode. Control, 80nM rapamycin, and 240nM rapamycin conditions were fed as described in Table 1. All rapamycin was added using a 5pM working solution. 80nM total rapamycin dose was added as 25nM dose on day 3 followed by 5nM doses from day 8 to 18 (Method 2, Table 2). 240nM total rapamycin dose was added as 80nM dose on day 3 followed by 15nM doses from day 8 to 18 (Method 3, Table 2). Rapamycin decreased the average specific growth rate from 0.57hr-1(control) to 0.50hr ' at both rapamycin concentrations. With both concentrations of rapamycin, peak VCD was observed on day 9 vs day 7 for the control. The data show that peak VCD can be manipulated with rapamycin. Both the control and 80nM rapamycin condition reached ~18 x 106cells / mL peak VCD while 240nM rapamycin reached only 15.89 x 106cells / mL. Thissuggests that peak VCD can be tuned with altered rapamycin concentrations and reduced peak VCD is not required. See FIG. 12 and Table 11.Table 11. mAb A Clone 1 Dose Response Cell Growth SummaryEffects of Rapamycin on Cell Growth During Process Scale-up

[0103] The cells used in this experiment contained a nucleic acid molecule encoding mAb A Clone 1 and the process was carried out in pilot scale bioreactors in fed-batch mode. Control and 1 lOnM condition were fed as described in Table 1. Rapamycin was added to the reactor from a 5pM working solution. 1 lOnM total rapamycin was added as a 50nM dose on day 3 followed by lOnM doses from day 8 to 13 (Method 4, Table 2). 1 lOnM rapamycin concentration was selected based on the dose response experiment described above. This was done to provide insight into how effect of rapamycin concentration would vary at production scale. This rapamycin strategy did not lead to a decreased peak VCD, but rapamycin led to a delayed peak VCD and slowed cell growth. Peak VCD was observed on day 7 for the control vs day 10 with rapamycin. Average specific growth rate was reduced from 0.54hr_1for the control to O.SOhr’1with rapamycin. This activity was similar to the bench scale 80nM condition described in FIG12. See FIG. 13 and Table 12.Table 12. mAb A Clone 1 200L Pilot Scale Cell Growth Summary

[0104] The cells used in this experiment contained a nucleic acid molecule encoding mAh B and the process was carried out in 200L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration lOOOnM and fed daily from day 3 to 13 (215nM total final concentration, Method 7, Table 2). Rapamycin addition resulted in reduced peak VCD and growth rate. Peak VCD was reduced from 35.01 x 106cells / mL (observed on day 8) to 24.82 x 106cells / mL (observed on day 9) with rapamycin. In addition, average specific growth rate was reduced to 0.34hr-1with rapamycin from 0.49hr-1for the control. See FIG. 14 Table 13.Table 13. mAb B Pilot Scale Cell Growth SummaryEffects of Rapamycin on Glucose Consumption

[0105] Glucose was fed to each bioreactor up to the glucose target amount on each day as per the feeding scheme outlined in Table 1. A 45% glucose solution was used to feed bioreactors for all experiments. Glucose feed amount each day was calculated as below:Glucose Feed Amount (g):A = ((Culture Broth Weight on the day of addition at sampling (kg) + Feed A amount (kg) + Feed B amount (kg)) x Target Glucose Concentration (g / kg)B = ((Culture Broth Weight on the day of addition at sampling (kg) x BGA Glucose (g / L) + (Feed A to be added x 75 (g / kg))45% Glucose Solution to be added (g) = [(A-B) / 0.45] x 1.163

[0106] Glucose consumption ratio was calculated as below:Glucose Consumption Ratio = (Total 45% Glucose Solution Added from day 3 to End of Culture (g) / Post Inoculation Culture Weight (g)) x 100

[0107] Post inoculation bioreactor weight is used to avoid any influence by variable inputs (z.e., glucose and base). A lower glucose consumption ratio indicates less glucose consumption by the culture.

[0108] Glucose utilization ratio was calculated as below:Total Batch Size (g) = End of culture batch weight (kg) x End of Culture titer (g / L)Glucose Utilization Ratio = Total 45 % Glucose solution added from day 3 to End of Culture (g) / Total Batch Size (g))

[0109] A lower glucose utilization ratio indicates more efficient conversion of glucose to mAb.Effects of Rapamycin on Glucose Consumption are not Molecule Dependent

[0110] The cells used in this experiment contained a nucleic acid molecule encoding mAb A clone 2 and the process was carried out in 3L bench scale bioreactors in fed-batch mode. Rapamycin was added as 200nM doses (each on day 3 and 8, 400nM total) from a 20 pM working solution (Method 1, Table 2). The control culture had a glucose consumption ratio of 7.49. With rapamycin, glucose consumption ratio was decreased to 5.09 (-40% decrease). The control culture had a glucose utilization ratio 8.49. With rapamycin, glucose utilization ratio was decreased to 4.36 (-50% decrease). See Table 14.Table 14. mAb A Clone 2 Bench Scale Glucose Consumption Summary[OHl] The cells used in this experiment contained a nucleic acid molecule encoding mAb B antibody and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at 800nM concentration and fed daily from day 3 to day 15 at 2.7% of the post-inoculation reactor weight (210nM total final concentration, Method 10, Table 2). The control culture had a glucose consumption ratio of 10.78. With rapamycin, glucoseconsumption ratio was decreased to 5.32 (-50% decrease). The control culture had a glucose utilization ratio 8.49. With rapamycin, glucose utilization ratio was decreased to 3.40 (-60% decrease). See Table 15.Table 15. mAb B Bench Scale Glucose Consumption SummaryEffects of Rapamycin on Glucose Consumption are Dose Dependent

[0112] The cells used in this experiment contained a nucleic acid molecule encoding mAb D and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a concentration of lOOnM (25nM final Concentration, Method 13, Table 2), 200nM (50nM final concentration, Method 8, Table 2), or 800nM (200nM total final concentration, Method 10, Table 2) and fed daily from day 3 to 13. The control culture had a glucose consumption ratio of 4.90. This ratio decreased in presence of rapamycin to 2.89 (25nM, -40% decrease from control), 1.35 (50nM, -70% decrease from control), and 0.80 (200nM, -85% decrease from control). This data suggests that glucose consumed by the cells decrease with increasing rapamycin concentration in the cell culture.

[0113] The control culture had a glucose utilization ratio of 4.13. This was decreased to 2.30 (25nM, -45% decrease from control), 1.10 (50nM, -75% decrease from control), and 0.67 (200nM, -85% decrease from control). 200nM Rapamycin resulted in more mAb product than glucose inputs as denoted by a utilization ratio less than 1. See Table 16.Table 16. mAb D Bench Scale Dose Response Glucose Consumption SummaryEffects of Rapamycin on Cell Growth During Process Scale-up

[0114] The cells used in this experiment contained a nucleic acid molecule encoding mAb B and the process was carried out in 200L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration lOOOnM and fed daily from day 3 to 13 (215nM total final concentration, Method 7, Table 2). The control culture had a glucose consumption ratio of 7.52. With rapamycin, glucose consumption ratio was decreased to 4.79 (-35% decrease). The control culture had a glucose utilization ratio of 6.73. With rapamycin, glucose utilization ratio was decreased to 3.63 (-45% decrease). See Table 17.Table 17. mAb B Pilot Scale Glucose Consumption SummaryRapamycin Effects on Specific Productivity / End of culture Titer

[0115] Specific productivity was measured using two separate methods. The first method measured end of culture titer with ROCHE BioHT or HPLC. A single analytical method was used to compare conditions of the same experiment, and the two methods were not compared with each other.

[0116] The second method averaged specific productivity from day 8 through the end of culture. The following formula was used to calculate daily specific productivity.

[0117] IVCD (integral viable cell density) was calculated using the trapezoidal method. Daily titer measurements for specific productivity calculations were conducted with Roche BioHT. Daily specific productivity was averaged over the described duration to calculateaverage specific productivity. A higher average specific productivity indicates increased antibody production for individual cells in the culture.Effects of rapamycin on productivity are not molecule specific.

[0118] The cells used in this experiment contained a nucleic acid molecule encoding mAb A Clone 2 and the process was carried out in bench scale bioreactors in fed-batch mode. Rapamycin was added as 200nM doses (each on day 3 and 8, 400nM total) from a 20 pM working solution (Method 1, Table 2). Titer was measured on day 14 and at end of culture. Rapamycin reactor remained viable until day 20 whereas control culture reached limit of being sustainable on day 16. Day 14 titer saw no significant difference between the control and rapamycin condition, but average specific productivity was improved from 20.48pg / cell / day to 36.74pg / cell / day with rapamycin. End of culture titer was 9.33g / L (observed on day 20, ~3g / L improvement from day 14) with rapamycin vs. 6.91g / L (observed on day 16, ~0.3g / L improvement from day 14) for the control. See Table 18.Table 18. mAb A Clone 2 Bench Scale Productivity Summary

[0119] The cells used in this experiment contained a nucleic acid molecule encoding mAb B and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at 800nM concentration and fed daily from day 3 to day 15 at 2.7% of the post-inoculation reactor weight (210nM total final concentration, Method 10, Table 2). Rapamycin addition resulted in increased end of culture titer and specific productivity. Titer was measured on day 14 and 16. 14 days was the platform culture duration, but prolonged culture with rapamycin was investigated. Day 14 titer was increased from 8.61g / L (control) to 10.83g / L (rapamycin). Rapamycin supplemented cultures also maintained high productivity for 2 additional days. End of culture titer on day 16 was 12.27g / L (rapamycin, ~1.5g / L improvement from day 14), compared to 9.10g / L (control, ~0.5g / L improvement from day 14). See Table 19.Table 19. mAb B Bench Scale Productivity SummaryEffects of rapamycin on productivity are dose dependent.

[0120] The cells used in this experiment contained a nucleic acid molecule encoding mAb B and the process was carried out in ambr250 automated microscale reactors in fed-batch mode. See Table 1 for operations. Rapamycin was formulated in nutrient feeds at 200nM (Method 8, Table 2), 400nM (Method 9 , Table 2), 800nM (Method 10, Table 2), 1200nM (Method 11 , Table 2), or 1600nM ((Method 12, Table 2), concentration and fed daily from day 3 to day 15 at 2.7% of the post-inoculation reactor weight (57nM, 115nM, 230nM, 345nM, and 460nM total final concentration respectively). Titer was measured at end of culture. Titer increased significantly with increasing rapamycin concentration. Titers are summarized in the table below. One way ANOVA analysis on rapamycin concentration effect on titer yielded a p value of <0.0001. See Table 20.Table 20. mAb B microscale Dose Response Productivity SummaryaOne way ANOVA analysis yielded a p-value of <0.0001 indicating that rapamycin concentration had a statistically significant impact on titer.Effects of Rapamycin on Productivity During Process Scale-Up

[0121] The cells used in this experiment contained a nucleic acid molecule encoding mAh B and the process was carried out in 200L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration lOOOnM and fed daily from day 3 to 13 (215nM total final concentration, Method 7, Table 2). Titer was measured at end of culture on day 14. Titer increased from 7.87g / L to 9.56g / L. Average specific productivity (from day 8 to 14) increased from 29.40pg / cell / day to 50.10 pg / cell / day. See Table 21.Table 21. mAb B Pilot Scale Productivity SummaryImpact of rapamycin on soluble impurities, product stability, and product quality

[0122] To determine the impact of rapamycin on soluble impurities produced during antibody production process and the impact of rapamycin on antibody quality, antibodies produced with and without rapamycin are compared as mentioned below.

[0123] At the end of culture, the cell culture is chilled and centrifuged to remove cells. In the resulting HCCF (harvested cell culture fluid), the proteins are examined to determine the relative amount of antibody, other proteins which are assumed to be host-cell proteins, and residual DNA amount.

[0124] Aliquots of HCCF are incubated at room temperature for 72 hours. At the conclusion of the incubation period, the product mAb is purified with Protein A Chromatography. The purified sample is assayed using reduced and non-reduced gel electrophoresis. The presence of host cell reductases can be deduced from the loss of main species antibody (z.e., HHLL) and increase in species lacking one or more of the H or L chains (e.g., HHL, HL, L and H species) over time.

[0125] After purification of antibody from the HCCF, the antibody is examined with size exclusion chromatography, gel electrophoresis, and imaged capillary isoelectric focusing to identify and quantify variant forms of the antibody, including reduced, oxidized, glycated, truncated, and different glycosylation isoforms.Summary

[0126] The data presented herein show that rapamycin has a dose-dependent effect on antibody titer, cell growth, lactate production, and glucose consumption. The effect of rapamycin was observed for each antibody format tested (IgGl -cytokine fusion, IgG4-YTE modified, and IgGl-YTE modified) and was observed when cells were grown in shake flasks, 3L bench scale bioreactors, Ambr250 microscale bioreactors, and 200L pilot scale bioreactors in a fed-batch process.

[0127] As observed herein, rapamycin forces the cell to reverse from lactate production to lactate consumption. This not only decreases culture fluid lactate, but it also improves antibody yield.Example 2 - Effect of rapamycin on harvested cell culture fluid (HCCF) impurity levels and mAh stability

[0128] Often harvested cell culture fluid (HCCF) is required to be stored before processing of the subsequent steps. Fragmentation of mAbs and decreased purity, decreasing overall yields, are observed in HCCF.

[0129] Rapamycin was tested as a mitigation strategy for two potential failure modes during mAb Production:HCCF impurity level- mAb fragmentation during HCCF hold. Fragmentation is used to broadly describe both reduction and proteolytic degradation of the protein of interest (or product).MethodsBench Scale Harvest

[0130] After the production process in the 3L bioreactor, the cell culture was cooled to room temperature. IL of cell culture was removed from the bioreactor and placed in 2x 500mL conical centrifuge bottles. Cells were sedimented in a bench top centrifuge. Supernatant was decanted. 900mL of supernatant was filtered with a lab scale depth filter (3M, BC0025S120ZB05A) and membrane filter (Sartorius, 5445307HV — LX) to generate HCCF (Harvested Cell Culture Fluid) at bench scale.Pilot Scale Harvest

[0131] After the production process in the 200L bioreactor, the cell culture was cooled below 22°C in the bioreactor. Cells were sedimented with a disc stack centrifuge (Alfa Laval, MBPX-404). The centrifuge was operated at three different bowl speeds (low, medium, and high) with constant flow rate. At each flow rate, material at two discharge intervals were collected and pooled from the centrifuge outlet. IL of the centrate pool was removed and filtered with a lab scale depth filter (3M, BC0025S120ZB05A) and membrane filter (Sartorius, 5445307HV — LX) to generate HCCF. Table 22 provides the pilot scale centrifuge conditions.Table 22. Pilot Scale Centrifuge ConditionsHCCF Hold Studies

[0132] Approximately 50mL of HCCF was aliquoted into a 50mL conical centrifuge tube. Tubes were filled to approximately 100% capacity to reduce headspace and prevent oxygenation of the HCCF. For each harvest condition, two tubes were set up. One tube was immediately frozen at -80°C. The remaining tube was stored at room temperature on the bench top without light protection. After a 72hr hold period, HCCF was frozen at -80°C. This required each hold condition to undergo one freeze thaw cycle before purification with protein A chromatography and analysis by ceSDS, and iciEF.ResultsEffects of Rapamycin on HCCF Impurity Levels

[0133] The cells used in this experiment contained a nucleic acid molecule encoding mAb D and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration of 800nM and fed daily from day 3 to 13 at 2.7% post inoculation reactor wight (200nM total final concentration, Method 10, Table 2). After bench scale harvest (see methods section above), fresh HCCF was assayed for LDH (lactate dehydrogenase), and rDNA (residual DNA). Supplementing cell culture with rapamycindecreased LDH by 55% (4740u / L to 2095u / L). Supplementing cell culture with rapamycin reduced rDNA by about 80% (524ppm to 94ppm). See Table 23.Table 23. mAb D HCCF Impurities Summary

[0134] The cells used in this experiment contained a nucleic acid molecule encoding mAb B and the process was carried out in 200L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration of lOOOnM and fed daily from day 3 to 13 at 2.7% post inculcation reactor weight (215nM total final concentration, Method 7, Table 2). After pilot scale harvest (see methods section above), fresh HCCF was assayed for LDH (lactate dehydrogenase), and rDNA (residual DNA). Increasing the bowl speed from low to high caused about a 10% increase in LDH in the control condition (5845 to 6292u / L) but only about a 4% increase in LDH in the rapamycin condition (5096 to 5322u / L). LDH was about 15% lower in the rapamycin condition compared to control for low, medium, and high settings (5096u / L vs 5845u / L, 5079u / L vs 6092u / L, and 5322u / L vs 6392u / L respectively). rDNA was relatively not affected by centrifuge bowl speed for both the control and rapamycin condition. Supplementing cell culture with rapamycin decreased rDNA by about 75% regardless of the centrifuge setting. See Table 24.Table 24. mAb B HCCF Impurities SummaryEffects of Rapamycin on mAb purity in HCCF

[0135] The cells used in this experiment contained a nucleic acid molecule encoding mAb D and the process was carried out in 3L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration 800nM and fed daily from day 3 to 13 (200nM total final concentration, Method 10, Table 2). After bench scale harvest (see methods section), fresh HCCF was aliquoted and stored as described above (see methods section). This resulted in a Ohr and 72hr sample. After a 72hr hold, control main peak decreased 6-fold from 93.4% to 15.3% as measured by NR-ceSDS. Main peak charge variant was decreased over 2-fold from 76.11% to 32.09%. After 72 hours, rapamycin condition main peak remained effectively unchanged (91.8% vs 91.3%) as measured by NR-ceSDS. Main peak charge variant remained effectively equal (74.78% vs 72.96%) as measured by iciEF. See Table 25.Table 25. mAb D Stability Summary

[0136] The cells used in this experiment contained a nucleic acid molecule encoding mAb B and the process was carried out in 200L bioreactors in fed-batch mode. Rapamycin was formulated in nutrient feeds at a final concentration lOOOnM and fed daily from day 3 to 13 (215nM total final concentration, Method 7, Table 2). After pilot scale harvest (see methods section above), fresh HCCF was aliquoted and stored as described above (see methods sectionabove). This resulted in a Ohr and 72hr sample. At low, center, and high bowl speed, main peak was decreased at 72hr compared to Ohr as measured by NR-ceSDS for the control condition. Low bowl speed resulted in about a 25% decrease in main peak (52.8% vs 73.0%) as measured by NR-ceSDS. Center bowl speed resulted in about a 40% decrease in main peak (64.2% to 36.5%) as measured by NR-ceSDS. High bowl speed resulted in about a 75% decrease in main peak (63.5% to 16.9%) as measured by NR-ceSDS. At low, center, and high bowl speed, main peak was effectively equal at 72hr compared to Ohr as measured by NR-ceSDS for the rapamycin condition (64.2% to 65.3%, 63.5% to 63.5%, and 61.7% to 61.8% respectively for low, center, and high bowl speed). Bowl speed and supplementing cell culture with rapamycin had no effect on main charge variant as measured by iciEF. All samples measured about 70% main charge variant. See Table 26.Table 26. mAb B Stability SummarySummary

[0137] The data presented herein show that rapamycin has a positive effect on both HCCF impurity levels (LDH and rDNA) and product stability. The effect was observed for both antibodies tested, and was observed for cells grown in 3L bench scale bioreactors and 200L pilot scale bioreactors.

[0138] The data presented herein also show that rapamycin contributes to reduction in cell lysis during centrifugation during cell culture harvest. This reduces the number of impurities in the HCCF and improves stability of the product during HCCF storage.

[0139] The data presented herein further show that rapamycin can prevent product fragmentation during HCCF storage at room temperature for up to 72hrs.

[0140] The below table summarizes the results of the individual experiments performed herein.Table 27. Results Summary

[0141] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments disclosed herein and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.EMBODIMENTS

[0142] The following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.Embodiment 1. A fed-batch method of producing an antibody, the method comprising:culturing cells comprising a nucleic acid molecule that encodes the antibody in an initial cell culture medium; prior to stationary phase of the cells, adding rapamycin to the initial cell culture medium to form a rapamycin-containing cell culture medium; and continuing to culture the cells in the rapamycin-containing cell culture medium to thereby produce the antibody.Embodiment 2. The method of embodiment 1, wherein the rapamycin is added at about day 3 of cell culture process.Embodiment 3. The method of embodiment 1 or 2, wherein the rapamycin is added during log phase of the cells.Embodiment 4. The method of any one of the previous embodiments, wherein the rapamycin concentration in cell culture fluid ranges from about 3nM to about 460nM.Embodiment 5. The method of any one of the previous embodiments, wherein the rapamycin is added in multiple doses on different days of the culture duration.Embodiment 6. The method of embodiment 5, wherein the rapamycin is added on day 3 and day 15.Embodiment 7. The method of embodiment 5, wherein the rapamycin is added on day 3 and daily from day 8 until the end of culture.Embodiment 8. The method of embodiment 5, wherein the rapamycin is added daily from day 3 until the end of culture.Embodiment 9. The method of any one of the previous embodiments, wherein the concentration of rapamycin after a first addition is between 3nM to 200nM.Embodiment 10. The method of any one of the previous embodiments, wherein the cells are Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293), mouse myeloma (NSO), or baby hamster kidney (BHK) cells.Embodiment 11. The method of any one of the previous embodiments, wherein the addition of the rapamycin reduces the amount of lactate produced by the cells in the rapamycin- containing cell culture medium relative to an amount of lactate produced by cells cultured in medium without rapamycin, increases the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate consumed by cells cultured in medium without rapamycin, reduces the amount of impurities inharvested cell culture fluid (HCCF) in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin, increases the stability of the antibody produced by the cells in the rapamycin- containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin, increases antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin, increases the specific productivity of the rapamycin- containing cell culture relative to the specific productivity of cell culture without rapamycin, reduces peak viable cell density (VCD) from the cells in the rapamycin- containing cell culture medium relative to a peak VCD from cells cultured in medium without rapamycin, reduces lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin, reduces the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium relative to a glucose consumption ratio of cells cultured in medium without rapamycin, or any combination thereof.Embodiment 12. A method of reducing an amount of lactate produced by cells, increasing an amount of lactate consumed by cells, reducing an amount of harvested cell culture fluid (HCCF) impurity levels from cells, increasing stability of an antibody produced by cells, increasing antibody titer, increasing specific productivity of the cell culture, reducing peak viable cell density (VCD), reducing lactate concentration at end of culture, reducing a glucose consumption ratio of cells, or any combination thereof, the method comprising: culturing cells in an initial cell culture medium; and prior to stationary phase of the cells, adding rapamycin to the initial cell culture medium to form a rapamycin-containing cell culture medium, wherein the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin, the amount of HCCF impuritylevels from the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin, the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium is increased relative to the stability of an antibody produced by cells cultured in medium without rapamycin, the antibody titer from the cells in the rapamycin- containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin, the specific productivity of the rapamycin- containing cell culture is increased relative to the specific productivity of cell culture without rapamycin, the peak VCD from the cells in the rapamycin-containing cell culture medium is reduced relative to a peak VCD from cells cultured in medium without rapamycin, the lactate concentration at end of culture from the cells in the rapamycin- containing cell culture medium is reduced compared to a lactate concentration at end of culture from cells cultured in medium without rapamycin, the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium is reduced relative to a glucose consumption ratio of cells cultured in medium without rapamycin, or any combination thereof.Embodiment 13. The method of embodiment 12, wherein the cells comprise a nucleic acid molecule that encodes a protein of interest.Embodiment 14. The method of embodiment 13, wherein the protein of interest is an antibody.Embodiment 15. The method of any one of embodiments 12-14, wherein the rapamycin is added at about day 3 of the culture.Embodiment 16. The method of any one of embodiments 12-15, wherein the rapamycin is added during log phase of the cells.Embodiment 17. The method of any one of embodiments 12-16, wherein the rapamycin- containing cell culture medium comprises from about 5nM to about 460nM of total rapamycin.Embodiment 18. The method of any one of embodiments 12-17, wherein the rapamycin is added in multiple doses on different days of the culture duration.Embodiment 19. The method of embodiment 18, wherein the rapamycin is added on day 3 and day 15.Embodiment 20. The method of embodiment 18, wherein the rapamycin is added on day 3 and daily from day 8 until the end of culture.Embodiment 21. The method of embodiment 18, wherein the rapamycin is added daily from day 3 until the end of culture.Embodiment 22. The method of any one of embodiments 12-21, wherein the concentration of rapamycin in the culture after a first addition is between 3nM to 200nM.Embodiment 23. The method of any one of embodiments 12-22, wherein the cells are Chinese hamster ovary (CHO) cells.

Claims

What is claimed:

1. A fed-batch method of producing an antibody, the method comprising: culturing cells comprising a nucleic acid molecule that encodes the antibody in an initial cell culture medium; prior to stationary phase of the cells, adding rapamycin to the initial cell culture medium to form a rapamycin-containing cell culture medium; and continuing to culture the cells in the rapamycin-containing cell culture medium to thereby produce the antibody.

2. The method of claim 1, wherein the rapamycin is added at about day 3 of cell culture process.

3. The method of claim 1 or 2, wherein the rapamycin is added during log phase of the cells.

4. The method of any one of the previous claims, wherein the rapamycin concentration in cell culture fluid ranges from about 3nM to about 460nM.

5. The method of any one of the previous claims, wherein the rapamycin is added in multiple doses on different days of the culture duration.

6. The method of claim 5, wherein the rapamycin is added on day 3 and day 15.

7. The method of claim 5, wherein the rapamycin is added on day 3 and daily from day 8 until the end of culture.

8. The method of claim 5, wherein the rapamycin is added daily from day 3 until the end of culture.

9. The method of any one of the previous claims, wherein the concentration of rapamycin after a first addition is between 3nM to 200nM.

10. The method of any one of the previous claims, wherein the cells are Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293), mouse myeloma (NSO), or baby hamster kidney (BHK) cells.

11. The method of any one of the previous claims, wherein the addition of the rapamycin reduces the amount of lactate produced by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate produced by cells cultured in medium without rapamycin, increases the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium relative to an amount of lactate consumed by cells cultured in medium without rapamycin, reduces the amount of impurities in harvested cell culture fluid (HCCF) in the rapamycin-containing cell culture medium relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin, increases the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium relative to the stability of an antibody produced by cells cultured in medium without rapamycin, increases antibody titer from the cells in the rapamycin-containing cell culture medium relative to an antibody titer from cells cultured in medium without rapamycin, increases the specific productivity of the rapamycin-containing cell culture relative to the specific productivity of cell culture without rapamycin, reduces lactate concentration at end of culture from the cells in the rapamycin-containing cell culture medium relative to a lactate concentration at end of culture from cells cultured in medium without rapamycin, reduces the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium relative to a glucose consumption ratio of cells cultured in medium without rapamycin, or any combination thereof.

12. A method of reducing an amount of lactate produced by cells, increasing an amount of lactate consumed by cells, reducing an amount of harvested cell culture fluid (HCCF) impurity levels from cells, increasing stability of an antibody produced by cells, increasing antibody titer, increasing specific productivity of the cell culture, reducing lactate concentration at end of culture, reducing a glucose consumption ratio of cells, or any combination thereof, the method comprising: culturing cells in an initial cell culture medium; andprior to stationary phase of the cells, adding rapamycin to the initial cell culture medium to form a rapamycin-containing cell culture medium, wherein the amount of lactate produced by the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of lactate produced by cells cultured in medium without rapamycin, the amount of lactate consumed by the cells in the rapamycin-containing cell culture medium is increased relative to an amount of lactate consumed by cells cultured in medium without rapamycin, the amount of HCCF impurity levels from the cells in the rapamycin-containing cell culture medium is reduced relative to an amount of HCCF impurity levels from cells cultured in medium without rapamycin, the stability of the antibody produced by the cells in the rapamycin-containing cell culture medium is increased relative to the stability of an antibody produced by cells cultured in medium without rapamycin, the antibody titer from the cells in the rapamycin-containing cell culture medium is increased relative to an antibody titer from cells cultured in medium without rapamycin, the specific productivity of the rapamycin-containing cell culture is increased relative to the specific productivity of cell culture without rapamycin, the lactate concentration at end of culture from the cells in the rapamycin- containing cell culture medium is reduced compared to a lactate concentration at end of culture from cells cultured in medium without rapamycin, the glucose consumption ratio of the cells in the rapamycin-containing cell culture medium is reduced relative to a glucose consumption ratio of cells cultured in medium without rapamycin, or any combination thereof.

13. The method of claim 12, wherein the cells comprise a nucleic acid molecule that encodes a protein of interest.

14. The method of claim 13, wherein the protein of interest is an antibody.

15. The method of any one of claims 12-14, wherein the rapamycin is added at about day 3 of the culture.

16. The method of any one of claims 12-15, wherein the rapamycin is added during log phase of the cells.

17. The method of any one of claims 12-16, wherein the rapamycin-containing cell culture medium comprises from about 5nM to about 460nM of total rapamycin.

18. The method of any one of claims 12-17, wherein the rapamycin is added in multiple doses on different days of the culture duration.

19. The method of claim 18, wherein the rapamycin is added on day 3 and day 15.

20. The method of claim 18, wherein the rapamycin is added on day 3 and daily from day 8 until the end of culture.

21. The method of claim 18, wherein the rapamycin is added daily from day 3 until the end of culture.

22. The method of any one of claims 12-21, wherein the concentration of rapamycin in the culture after a first addition is between 3nM to 200nM.

23. The method of any one of claims 12-22, wherein the cells are Chinese hamster ovary (CHO) cells.

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