US 20080274507A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0274507 A1 Gomes et al. (43) Pub. Date: Nov. 6, 2008
(54) METHODS OF PROTEIN PRODUCTION Related U.S. Application Data USING ANTI-SENESCENCE COMPOUNDS (60) Provisional application No. 60/913,382, filed on Apr. (75) Inventors: Jose Manuel Gomes, Chelmsford, 23, 2007. MA (US); Yen-Tung Luan, Chelmsford, MA (US); Gregory Publication Classification Walter Hiller, Wakefield, MA (51) Int. C. (US); Wenge Wang, North CI2P 2L/04 (2006.01) Chelmsford, MA (US) (52) U.S. Cl...... 435/69.7 Correspondence Address: CHOATE, HALL & STEWART LLP/WYETH (57) ABSTRACT PATENT GROUP, TWO INTERNATIONAL PLACE Methods of producing a protein in cell culture comprising an anti-senescence compound. Such as the antioxidant carnos BOSTON, MA 02110 (US) ine, are provided. According to teachings of the present inven (73) Assignee: WYETH, Madison, NJ (US) tion, cells grown in a cell culture medium comprising an anti-senescence compound exhibit increased viability and (21) Appl. No.: 12/107,533 productivity. Furthermore, cell cultures grown in the presence of an anti-senescence compound exhibit decreased levels of (22) Filed: Apr. 22, 2008 high molecular weight aggregates in the cell culture medium.
Effect of Carnosine On Acidic Peaks for MYO-29.
Effect of Carnosine on Acidic peaks for Myo-029
16.00 14. OO . 12.00 1O.OO . 8.OO . 6.00 - 4.OO . 2.00 O.OO . s s Patent Application Publication Nov. 6, 2008 Sheet 1 of 11 US 2008/0274507 A1
Figure 1a. Effect of Carnosine on Acidic Peaks for MYO-29.
Effect of Carnosine on Acidic peaks for Myo-029
16.OO . 14.OO . 12.OO . 1O.OO . 8.OO . 6.OO . 4.OO . 2.OO . O.OO . s
Figure 1b. Effect of Carnosine on High Molecular Weight Aggregates.
HMW 6 of Myo-029 in bioreactors
16.OO .
12.OO .
8.OO .
4.OO .
O.OO Day 7 control Day 7 control Carnosine Carnosine Patent Application Publication Nov. 6, 2008 Sheet 2 of 11 US 2008/0274507 A1
Figure 1c. Effect of Carnosine Additions on Different Days on Acidic Peaks.
Effect of Carnosine on Acidic peaks for Myo-029
O S. O E. s E. 9. s 9. s (s I
Figure 1d. Effect of Carnosine Additions on Different Days on High Molecular Weight Aggregates.
HMW 9% of Myo-029 in bioreactors
8 g is aa as a Patent Application Publication Nov. 6, 2008 Sheet 3 of 11 US 2008/0274507 A1
Figure 2a. Effect of Carnosine on Daily Viable Cell Density.
Daily Viable Cell Density (Cedex)
14.OO -- Control AD 10 Feed 12.00 a. 1 O.OO - - 20mM Carnosine D1 O Feed 8.OO - Control B D10 Feed 6.OO a 20mM Carnosine 4.OO ... 2.OO subo. Control C 0.00 O 2 4 6 8 10 12 14 16 Culture Time (Days)
Figure 2b. Effect of Carnosine on Daily Cell Viability.
Daily Cell Viability (Cedex)
1 OO --Control AD 10 Feed
90 is user2OnM CarnOSine D1 O Feed 8O Control B D10 Feed
70 OmM Carnosine
60 an Control C 50 4 6 8 10 12 14 16 Culture Time (Days) Patent Application Publication Nov. 6, 2008 Sheet 4 of 11 US 2008/0274507 A1
Figure 2c. Effect of Carnosine on Daily Titer.
Daily Titer Pro A HPLC
are Control D10 Feed P1 ocese Carnosine D10 Feed P2 Contro D10 Feed P3 · Carnosine P4 --Control P5
O 2 4 6 8 10 12 14 16 18 Culture Time (Days)
Figure 2d. Effect of Carnosine on Cumulative Specific Cellular Productivity.
Cumulative Specific Cellular Productivity
35.0 3O.O on 25.0 Control AD10 Feed g 2O.O OmM Carnosine D10 Feed Control B D10 Feed s 15.0 - OnM Carnosine 10.0 Control C 5.0 - O.O. Day 12 Day 14 Culture Time (Days) Patent Application Publication Nov. 6, 2008 Sheet 5 of 11 US 2008/0274507 A1
Figure 2e. Effect of Carnosine on High Molecular Weight Aggregates.
% HMW Aggregate by SEC (ADS)
Control AD 10 Feed
20mM Carnosine D10 Feed
Control B D10 Feed
OmM Carnosine
Control C
Day 12 Day 14 Culture Time (Days)
Figure 3a. Effect of Different Concentrations of Carnosine on Viable Cell Density.
Viable Cell Density (Cedex)
12
10 - MMMMMMMM 8 - - - Contro P2 5 6 - 5%. Feed Contro T5 co -- 20mM CarnOSine c 4 MMM 2 Add Carnosine
O O 2 4. 8 1O 12 14 Culture Time (Days) Patent Application Publication Nov. 6, 2008 Sheet 6 of 11 US 2008/0274507 A1
Figure 3b. Effect of Different Concentrations of Carnosine on Daily Cell Viability.
Daily Cell Viability (Cedex)
1OO .
90 MWWWMMM MMM CD so to Control P2 80 Control T5 70 mo 20mM CarnOSine SS * 40mM Carnosine 60 50 O 2 4. 6 8 1O 12 14 Culture Time (Days)
Figure 3c. Effect of Different Concentrations of Carnosine on Daily Titer.
Daily Titer Pro A HPLC
-e-control P2 Contro T5 ona 20mM CarnOSine
O 2 4. 6 8 10 12 14 Culture Time (Days) Patent Application Publication Nov. 6, 2008 Sheet 7 of 11 US 2008/0274507 A1
Figure 3d. Effect of Different Concentrations of Carnosine on Cumulative Specific Cellular Productivity.
Cumulative Specific Cellular Productivtity
30.0 -
a Control P2 Control T5 20mM Carnosine 40mM Carnosine
12 Culture Time (Days)
Figure 3e. Effect of Different Concentrations of Carnosine on High Molecular Weight Aggregates.
HMW Aggregate by SEC (ADS)
COntrol P2 Control T5
220mM Carnosine 40mM Carnosine
12 Culture Time (Days) Patent Application Publication Nov. 6, 2008 Sheet 8 of 11 US 2008/0274507 A1
Figure 4. Viable Cell Density Profiles of a Chinese Hamster Ovary (CHO) Cell Line Producing Recombinant TNFR Fusion Protein Grown in Media Containing or Lacking Carnosine.
Cell Growth 16E--O7
14E--Of MMWWMMMWM -1.-1s-1
12E--O7 s
1.OE+O7 t
8.OE-06 s
4.OE-06 -o- Control (N=4)
as a 20 nM Carrosine
O.OE--OO - O 2 4 6 8 1O 12 Culture Timed
Figure 5. Cell Viability Profiles of a Chinese Hamster Ovary (CHO) Cell Line Producing Recombinant TNFR Fusion Protein in Media Containing or Lacking Carnosine. OO% Cell Viability
95% win.
a 90%
85% WM
-H Control (N=4) 80% of our - C - 20 raw Carlosine
75% —r.TrrormerM O 2 4. 6 8 1 O 12 Culture Timed Patent Application Publication Nov. 6, 2008 Sheet 9 of 11 US 2008/0274507 A1
Figure 6. Effect of Carnosine on the Percentage of Aggregated/Misfolded TNFR Fusion Protein. Misfolded and/or Aggregated Product 3 O 22 2 1 , 8 5 1 2
-- Control (Ne4)
a C 20 m Carnosine
2 4 6 8 10 12 Culture Timed
Figure 7. Effect of Carnosine on the Percentage of High Molecular Weight (HMW) Aggregates Produced by a Chinese Hamster Ovary (CHO) Cell Line Producing Recombinant TNFR Fusion Protein. High Molecular Weight Aggregated Product
14 WWWMMMMMMMMMMMWWMMMMMMMMMM MMMMMMMMMMWWMMMMMMMMMWWMMMMMMMMMMMMM
2 4. 6 8 10 12 Culture Time Id Patent Application Publication Nov. 6, 2008 Sheet 10 of 11 US 2008/0274507 A1
Figure8. Product Titer Profiles of a Chinese Hamster Ovary (CHO) Cell Line Producing Recombinant TNFR Fusion Protein Grown in Media Containing or Lacking Carnosine.
OOO Product Titer
900 -- 8 O O WWWW 5 O O i 34 OO OO 2OO -o- Control (Nr. 4)
1OO - G - 20 mM Carnosine -
2 4. 6 8 10 12 Culture Timed Figure 9. Specific Cellular Productivity Profiles of a Chinese Hamster Ovary (CHO) Cell Line Producing Recombinant TNFR Fusion Protein Grown in Media Containing or Lacking Carnosine.
8 Cumulative Average Specific Productivity
7
6
5
4 -o- Control (N=4)
a 20 W. Carrosie 3 2 4 6 8 10 12 Culture Time d Patent Application Publication Nov. 6, 2008 Sheet 11 of 11 US 2008/0274507 A1
Figure 10. Total Sialylation Profiles, Expressed as a Percentage of the Reference Material, of Recombinant TNFR Fusion Protein Produced by a Chinese Hamster Ovary (CHO) Cell Line Grown in Media Containing or Lacking Carnosine.
120
60
-o- Control (N=4) -ms-O- 20 M Carrhosie 20 5 6 7 s s io f 12 13 Days Figure 11. Distribution of Sialylated N-linked Oligiosaccharides, Expressed as a Percentage of Total N-Linked Oligosaccharides, of Recombinant TNFR Fusion Protein Produced by a Chinese Hamster Ovary (CHO) Cell Line Grown in Media Containing or Lacking Carnosine.
60 5 O - - , , A. O
3 O
2 O w
O m -- Control (N=4) —O– 20 mM Carrosine
5 6 7 8 9 10 11 12 13 Days US 2008/0274507 A1 Nov. 6, 2008
METHODS OF PROTEIN PRODUCTION interest is enhanced. In certain embodiments, such an anti USING ANTI-SENESCENCE COMPOUNDS senescence compound comprises carnosine. 0007. In certain embodiments, the present invention pro CROSS REFERENCE TO RELATED vides compositions that enhance production of a protein of APPLICATIONS interest. Any of a variety of proteins may be produced in 0001. This application is copending with, shares at least accordance with the methods and compositions of the present invention. For example, in certain embodiments, methods and one common inventor with, and claims priority to U.S. pro compositions of the present invention are used to produce an visional patent application No. 60/913,382, filed Apr. 23. antibody. In certain embodiments, methods and compositions 2007, the contents of which are hereby incorporated by ref of the present invention are used to produce a receptor, erence in their entirety. optionally linked to one or more additional protein moieties. BACKGROUND OF INVENTION For example, methods and compositions of the present inven tion may be used to produce a TNFR fusion protein. 0002 This disclosure relates generally to the production 0008. In certain embodiments, the present invention pro of proteins in mammalian cell cultures. Particularly this dis vides a cell culture medium comprising an anti-senescence closure relates to culturing mammalian cells in the presence compound that enhances production of a protein of interest of an anti-senescence compound, e.g. carnosine, to maintain expressed in a host cell. In certain embodiments, such an viability and increase productivity with superior quality. Cul anti-senescence compound comprises carnosine. In certain turing cells has produced many protein products. These prod embodiments, genetically manipulated host cells are com ucts, such as hybridoma-produced monoclonal antibodies, bined with an inoculum medium to form a cell culture can be used for therapeutic, research or other applications. medium, which is grown in a bioreactor. During a production Animal cells, notably mammalian cells, are often used to run of the desired protein product, conditions of the bioreac produce proteins. Unfortunately, using animal cells causes tor may be altered and/or supplements may be added in order the production process to be time consuming and costly. to increase the productivity and/or maintain viability. Supple 0003. Adding a chemical agent to a cell culture medium ments may include a feed medium and/or one or more addi can increase cell productivity by inducing cells to produce a tives Such as in the instant disclosure, carnosine and/or other product, thereby increasing overall yield. The optimal agent anti-senescence compounds. to use varies depending on a number of factors, including the 0009 Mammalian host cells, for example, Chinese ham desired protein product and cell type. Similar factors also sterovary (CHO) cells, can experience a reduction in viability affect the amount of the chosen agent added and when the nearing the end of a production run in a bioreactor. It has been agent is added to the cell culture medium. Examples of agents discovered that the addition of an anti-senescence agent, Such are alkanoic acids or salts, urea derivatives, or dimethylsul as carnosine and analogs thereof, to a cell culture medium phoxide (DMSO). Chemical agents, such as sodium butyrate, helps maintain a higher viable cell number and cell viability can have diverse effects on protein production. The addition until the protein is harvested. of an agent can increase the specific productivity of the cells, 0010. In addition, methods for increasing productivity, but also have cytotoxic effects and can inhibit cell growth and Such as altering the temperature after the growth phase and/or viability. during the production phase of the production run may be 0004 As cells produce a protein, typically, the protein is used in accordance with the present invention. To give but one secreted into the cell culture medium. The specific protein, example, during the production of a protein product, specifi however, is not the only matter in the medium; high molecular cally the antibody for growth differentiation factor-8 (GDF weight aggregates, acidic species, and other materials are also 8), the temperature was shifted downwards to help initiate and often in the medium, which can make the process of purifi increase the productivity. In certain embodiments, addition of cation more laborious and costly. Techniques and methods an anti-senescence compound Such as carnosine helps are available to improve product quality, enabling more effi increase the productivity of the cell culture. In certain cient protein purification; including, among others, altering embodiments, an anti-senescence compound may be added the conditions of the bioreactor or using a different cell line. before, during and/or after Such a temperature shift. However, there nevertheless remains a need in the field for protein production techniques and methods that lead to 0011. It has also been discovered that the addition of an improved purification processes. anti-senescence compound Such as carnosine to a cell culture medium increases the overall quality of protein product. Dur 0005. Therefore, what is needed is a chemical agent that is ing the production of the protein, high molecular weight added to the cell culture medium that can enhance the expres aggregates, along with other unwanted species, are in the cell sion of a protein of interest while maintaining high cell viabil culture medium. The addition of carnosine decreases the ity. What is further needed is an agent that increases the amount of high molecular aggregates and increases product product quality of the protein by decreasing the amount of quality. In certain embodiments, addition of an anti-senes high molecular weight aggregates and acidic species in the cence compound other than carnosine decreases accumula cell culture medium. tion of such high molecular weight aggregates and improves SUMMARY OF THE INVENTION product quality. In certain embodiments, carnosine is added in combination with one or more additional anti-senescence 0006. In certain embodiments, the present disclosure compounds. relates to a process for enhanced production of a protein 0012. The concentration of anti-senescence compound product. For example, in certain embodiments, the present (e.g., carnosine) added to the cell culture medium can vary invention provides methods of culturing host cells expressing depending on many factors of the process, including, for a protein of interest in a medium comprising an anti-senes example, the cell type, the desired product, and the conditions cence compound Such that overall production of the protein of of the bioreactor, among others. Also, carnosine can be Sub US 2008/0274507 A1 Nov. 6, 2008 stituted with its analogues; acetyl-carnosine, homo-carnos Fusion Protein Grown in Media Containing or Lacking Car ine, anserine, and beta-alanine. In certain embodiments, car nosine. The Control Condition is the Average of 4 Control nosine is provided in combination with one or more other Bioreactor Runs. anti-senescence compounds. In certain embodiments, the 0031 FIG. 6. Effect of Carnosine on the Percentage of concentration of anti-senescence agent (e.g., carnosine) in a Aggregated/Misfolded TNFR Fusion Protein. The Control cell culture medium is about 5 mM to about 100 mM. In Condition is the Average of 4 Control Bioreactor Runs. certain embodiments, the concentration is about 10 mM to 0032 FIG. 7. Effect of Carnosine on the Percentage of about 40 mM. In certain embodiments, the concentration is High Molecular Weight (HMW) Aggregates Produced by a about 20 mM. Chinese Hamster Ovary (CHO) Cell Line Producing Recom 0013 Any suitable culture procedures and inoculum binant TNFR Fusion Protein. The Control Condition is the medium may be used to culture the cells in the process of Average of 4 Control Bioreactor Runs. protein production. Both serum and serum free media may be 0033 FIG.8. Product Titer Profiles of a Chinese Hamster used. In addition, culturing methods may be used to culture Ovary (CHO) Cell Line Producing Recombinant TNFR the cells as appropriate for the specific cell type and protein Fusion Protein Grown in Media Containing or Lacking Car product. Such procedures are known and understood by those nosine. The Control Condition is the Average of 4 Control of ordinary skill in the cell culture art. Bioreactor Runs. 0014. Other features and advantages of the disclosure will 0034 FIG. 9. Specific Cellular Productivity Profiles of a be apparent from the following description, and from the Chinese Hamster Ovary (CHO) Cell Line Producing Recom claims. binant TNFR Fusion Protein Grown in Media Containing or Lacking Carnosine. The Control Condition is the Average of BRIEF DESCRIPTION OF THE DRAWINGS 4 Control Bioreactor Runs. 0015 FIG. 1a. Effect of Carnosine on Acidic Peaks for 0035 FIG. 10. Total Sialylation Profiles, Expressed as a MYO-29. Percentage of the Reference Material, of Recombinant TNFR 0016 FIG. 1b. Effect of Carnosine on High Molecular Fusion Protein Produced by a Chinese Hamster Ovary (CHO) Weight Aggregates. Cell Line Grown in Media Containing or Lacking Carnosine. 0017 FIG. 1c. Effect of Carnosine Additions on Different The Control Condition is the Average of 4 Control Bioreactor Days on Acidic Peaks. Runs. 0018 FIG. 1d. Effect of Carnosine Additions on Different 0036 FIG. 11. Distribution of Sialylated N-linked Oli Days on High Molecular Weight Aggregates. gosaccharides, Expressed as a Percentage of Total N-Linked 0019 FIG.2a. Effect of Carnosine on Daily Viable Cell Oligosaccharides, of Recombinant TNFR Fusion Protein Density. Produced by a Chinese Hamster Ovary (CHO) Cell Line 0020 FIG.2b. Effect of Carnosine on Daily Cell Viability. Grown in Media Containing or Lacking Carnosine. The Con 0021 FIG.2c. Effect of Carnosine on Daily Titer. trol Condition is the Average of 4 Control Bioreactor Runs. 0022 FIG. 2d. Effect of Carnosine on Cumulative Specific Cellular Productivity. The bars on days 12 and 14 represent, DEFINITIONS from left to right: Control A D10 Feed, 20 mM.Carnosine D10 0037. Following long-standing convention, the terms “a Feed, Control B D10 Feed, 20 mM Carnosine (No D10 Feed), and “an’ mean “one or more' when used in this application, and Control C (No D10 Feed). including the claims. Even though the invention has been 0023 FIG. 2e. Effect of Carnosine on High Molecular described with a certain degree of particularity, it is evident Weight Aggregates. The bars on days 12 and 14 represent, that many alternatives, modifications, and variations will be from left to right: Control A D10 Feed, 20 mM.Carnosine D10 apparent to those skilled in the art in light of the disclosure. Feed, Control B D10 Feed, 20 mM Carnosine (No D10 Feed), Accordingly, it is intended that all Such alternatives, modifi and Control C (No D10 Feed). cations, and variations, which fall within the spirit and scope 0024 FIG.3a. Effect of Different Concentrations of Car of the invention, be embraced by the defined claims. nosine on Viable Cell Density. 0038. The term “anti-senescence compound” as used 0025 FIG. 3b. Effect of Different Concentrations of Car herein refers to any agent or compound that, when added to a nosine on Daily Cell Viability. cell culture, promotes viability, growth, and/or lifespan of a 0026 FIG. 3c. Effect of Different Concentrations of Car cell grown therein. In certain embodiments, use of Such an nosine on Daily Titer. anti-senescence compound in cell culture results in increased 0027 FIG. 3d. Effect of Different Concentrations of Car titer, increased cell specific productivity, increased cell nosine on Cumulative Specific Cellular Productivity. The viability, increased integrated viable cell density, decreased bars represent, from left to right: Control P2. Control P5, 20 accumulation of high molecular weight aggregates, and/or mM Carnosine, and 40 mM Carnosine. decreased accumulation of acidic species than would be 0028 FIG. 3e. Effect of Different Concentrations of Car observed under otherwise identical culture conditions that nosine on High Molecular Weight Aggregates. The bars rep lack the anti-senescence compound. Non-limiting examples resent, from left to right: Control P2, Control P5, 20 mM of anti-senescence compounds that may be used in accor Carnosine, and 40 mM Carnosine. dance with methods and compositions of the present inven 0029 FIG. 4. Viable Cell Density Profiles of a Chinese tion include carnosine, acetyl-carnosine, homo-carnosine, Hamster Ovary (CHO) Cell Line Producing Recombinant anserine, and beta-alanine. In certain embodiments, two or TNFR Fusion Protein Grown in Media Containing or Lack more anti-senescence compounds may be used in accordance ing Carnosine. The Control Condition is the Average of 4 with compositions and methods of the present invention. Control Bioreactor Runs. 0039. The phrase “host cell” refers to cells which are 0030 FIG. 5. Cell Viability Profiles of a Chinese Hamster capable of being genetically manipulated and/or are capable Ovary (CHO) Cell Line Producing Recombinant TNFR of growth and survival in a cell culture medium. Typically, the US 2008/0274507 A1 Nov. 6, 2008 cells can express a large quantity of an endogenous or heter ments, a cell line comprises primary host cells that have not ologous protein of interest and can either retain the protein or been transfected with exogenous DNA and express an endog secrete it into the cell culture medium enous protein of interest. 0040 Host cells are typically “mammalian cells,” which 0046. The “growth phase' of a cell culture medium refers comprise the nonlimiting examples of vertebrate cells, to the period when the cells are undergoing rapid division and including baby hamster kidney (BHK), Chinese hamster growing exponentially, or close to exponentially. Typically, ovary (CHO), human kidney (293), normal fetal rhesus dip cells are cultured in conditions optimized for cell growth for loid (FRhL-2), and murine myeloma (e.g., SP2/0 and NSO) generally 1-4 days. Growth phase conditions may include a cells. One of ordinary skill in the art will be aware of other temperature at about 35°C. to 42°C., generally about 37°C. host cells that may be used in accordance with methods and The length of the growth phase and the culture conditions in compositions of the present invention. the growth phase can vary but are generally known to a person 0041. The term “cell culture medium” refers to a solution of ordinary skill in the cell culture art. In certain embodi containing nutrients to Support cell Survival under conditions ments, a cell culture medium in a growth phase is Supple in which cells can grow and produce a desired protein. The mented with a feed medium. phrases "inoculation medium' or “inoculum medium” refer 0047. The “transition phase' occurs during the period to a solution or Substance containing nutrients in which a when the cell culture medium is being shifted from conditions culture of cells is initiated. In certain embodiments a “feed consistent with the growth phase to conditions consistent medium' contains similar nutrients as the inoculation with the production phase. During the transition phase, fac medium, but is a solution or substance with which the cells are tors like temperature, among others, are often changed. In fed after initiation of the culture. In certain embodiments, a certain embodiments, a cell culture medium in a transition feed medium contains one or more components not present in phase is Supplemented with a feed medium. an inoculation medium. In certain embodiments, a feed 0048. The “production phase' occurs after both the medium lacks one or more components present in an inocu growth phase and the transition phase. The exponential lation medium. A person of ordinary skill in the cell culture growth of the cells has ended and protein production is the art will know without undue experimentation what compo principal objective. The cell culture medium can be supple nents make-up the inoculation and feed mediums. Typically, mented to initiate production. In certain embodiments, a cell these solutions provide essential and non-essential amino culture medium in a production phase is Supplemented with a acids, vitamins, energy sources, lipids, and trace elements feed medium. In addition, the temperature of the cell culture required by a cell for growth and survival. In certain embodi medium during the production phase may be lower, generally, ments, an inoculation medium, a feed medium or both com than during the growth phase, which typically encourages prise an anti-senescence compound. production. The production phase continues until a desired 0042. The term “cell culture characteristic” as used herein endpoint is achieved. refers to an observable and/or measurable characteristic of a 0049. The phrase “viable cell density” refers to the total cell culture. Methods and compositions of the present inven number of cells that are surviving in the cell culture medium tion are advantageously used to improve one or more cell in a particular Volume, generally per ml. The phrase “cell culture characteristics. In certain embodiments, improvement viability” refers to number of cells, which are alive compared of a cell culture characteristic comprises increasing the mag to the total number of cells, both dead and alive, expressed as nitude of a cell culture characteristic. In certain embodiments, a percentage. improvement of a cell culture characteristic comprises 0050 “Integrated Viable Cell Density”, “IVCD’: The decreasing the magnitude of a cell culture characteristic. As terms “integrated viable cell density' or “IVCD as used non-limiting examples, a cell culture characteristic may be herein refer to the average density of viable cells over the titer, cell specific productivity, cell viability, integrated viable course of the culture multiplied by the amount of time the cell density, accumulation of high molecular weight aggre culture has run. When the amount of protein produced is gates, and/or accumulation of acidic species. One of ordinary proportional to the number of viable cells present over the skill in the art will be aware of other cell culture characteris course of the culture, integrated viable cell density is a useful tics that may be improved using methods and compositions of tool for estimating the amount of protein produced over the the present invention. course of the culture. 0043. The term “defined medium' as used herein refers to 0051. The term “high molecular weight aggregates’ refers a medium in which the composition of the medium is both to generally mis-folded proteins oran improperassociation of known and controlled. Defined media do not contain complex at least two polypeptides. The association may arise by any additives Such as serum or hydrolysates that contain unknown method including, but not limited to, covalent, non-covalent, and/or uncontrolled components. disulfide, or nonreducible cross linking. In certain embodi 0044) The term “complex medium' as used herein refers ments, methods and compositions of the present invention are to a medium that contains at least one component whose advantageously utilized to reduce the accumulation of high identity or quantity is either unknown or uncontrolled. molecular weight aggregates. 0045. The phrase “cell line' refers to, generally, primary 0.052 The phrase “antioxidant refers to a compound that host cells that express a protein of interest. In some embodi can prevent oxidative damage to lipids, proteins, DNA and ments, the cells have been transfected with exogenous DNA other essential macromolecules by blocking free radicals. coding for a desired protein and/or containing control 0053 “Therapeutic protein': A “therapeutic protein' is a sequences that activate expression of linked sequences, protein or peptide that has a biological effect on a region in the whether endogenous or heterologous. In certain embodi body on which it acts or on a region of the body on which it ments, cells derived from such genetically modified cells remotely acts via intermediates. A therapeutic protein can be, form a cell line and are placed in a cell culture medium to for example, a secreted protein, such as, an antibody, an grow and produce the protein product. In certain embodi antigen-binding fragment of an antibody, a soluble receptor, a US 2008/0274507 A1 Nov. 6, 2008
receptor fusion, a cytokine, a growth factor, an enzyme, or a 0.136049, 2005/0175614, 2005/0180970, 2005/0186216, clotting factor, as described in more detail herein below. The 2005/0202012, 2005/0202023, 2005/0202028, 2005/ above list of proteins is merely exemplary in nature, and is not 0202534, and 2005/0238646, and related patent family mem intended to be a limiting recitation. One of ordinary skill in bers thereof, all of which are hereby incorporated by refer the art will understand that any protein may be used in accor ence herein in their entireties. dance with the present invention and will be able to select the 0059 Single domain antibodies can include antibodies particular protein to be produced based as needed. whose complementary determining regions are part of a 0054 As used in the specification, the terms polypeptide, single domain polypeptide. Examples include, but are not protein and peptide are synonymous and are used inter limited to, heavy chain antibodies, antibodies naturally changeably. Accordingly, as used herein, the size of a protein, devoid of light chains, single domain antibodies derived from peptide or polypeptide generally comprises more than 2 conventional 4-chain antibodies, engineered antibodies and amino acids. For example, a protein, peptide or polypeptide single domain scaffolds other than those derived from anti can comprise from about 2 to about 20 amino acids, from bodies. Single domain antibodies may be any of the art, or any about 20 to about 40 amino acids, from about 40 to about 100 future single domain antibodies. Single domain antibodies amino acids, from about 100 amino acids to about 200 amino may be derived from any species including, but not limited to acids, from about 200 amino acids to about 300 amino acids, mouse, human, camel, llama, goat, rabbit, bovine. According and so on. to one aspect of the invention, a single domain antibody as 0055 As used herein, an amino acid refers to any naturally used herein is a naturally occurring single domain antibody occurring amino acid, any amino acid derivative oranyamino known as heavy chain antibody devoid of light chains. Such acid mimic known in the art. In certain embodiments, the single domain antibodies are disclosed in WO 9404678 for residues of the protein or peptide are sequential, without any example. For clarity reasons, this variable domain derived non-amino acid interrupting the sequence of amino acid resi from a heavy chain antibody naturally devoid of light chain is dues. In other embodiments, the sequence may comprise one known hereinas a VHH or nanobody to distinguish it from the or more non-amino acid moieties. In particular embodiments, conventional VH of four chain immunoglobulins. Such a the sequence of residues of the protein or peptide may be VHH molecule can be derived from antibodies raised in Cam interrupted by one or more non-amino acid moieties. elidae species, for example in camel, llama, dromedary, 0056 “Antibody”: The term “antibody” is used to refer to alpaca and guanaco. Other species besides Camelidae may any antibody-like molecule that has an antigen binding produce heavy chain antibodies naturally devoid of light region, and includes antibody fragments such as Fab', Fab, chain; such VHHs are within the scope of the invention. F(ab'). Sub.2, single domain antibodies (DABs). Fv, sclv 0060 Examples of binding fragments encompassed (single chain FV), and the like. Techniques for preparing and within the term “antigen-binding fragment of an antibody using various antibody-based constructs and fragments are include (i) a Fab fragment, a monovalent fragment consisting well known in the art. Means for preparing and characterizing of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, antibodies are also well known in the art (See, e.g., Harlow a bivalent fragment comprising two Fab fragments linked by and Lane, Antibodies: A Laboratory Manual, Cold Spring a disulfide bridge at the hinge region; (iii) a Fd fragment Harbor Laboratory, 1988; incorporated herein by reference). consisting of the VH and CH1 domains; (iv) a Fv fragment For example, an antibody can include at least one, and pref consisting of the VL and VH domains of a single arm of an erably two full-length heavy chains, and at least one, and antibody, (v) a dAb fragment, which consists of a VH domain; preferably two light chains. The term “antibody” as used (vi) a camelid or camelized variable domain, e.g., a VHH herein includes an antibody fragment or a variant molecule domain; (vii) a single chain Fv (sclv); (viii) a bispecific Such as an antigen-binding fragment (e.g., an Fab., F(ab')2, Fv, antibody; and (ix) one or more fragments of an immunoglo a single chain Fv fragment, a heavy chain fragment (e.g., a bulin molecule fused to an Fc region. Furthermore, although camelid VHH) and a binding domain-immunoglobulin fusion the two domains of the Fv fragment, VL and VH, are coded (e.g., SMIPTM). for by separate genes, they can be joined, using recombinant 0057 The antibody can be a monoclonal or single-speci methods, by a synthetic linker that enables them to be made as ficity antibody. The antibody can also be a human, human a single protein chain in which the VL and VH regions pair to ized, chimeric, CDR-grafted, or in vitrogenerated antibody. form monovalent molecules (known as single chain FV In yet other embodiments, the antibody has a heavy chain (scFV); see, e.g., Bird et al. (1988) Science 242:423-26: Hus constant region chosen from, e.g., IgG1, IgG2, IgG3, or IgG4. ton et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83). In another embodiment, the antibody has a light chain chosen Such single chain antibodies are also intended to be encom from, e.g., kappa or lambda. In one embodiment, the constant passed within the term “antigen-binding fragment of an region is altered, e.g., mutated, to modify the properties of the antibody. These antibody fragments are obtained using con antibody (e.g., to increase or decrease one or more of Fc ventional techniques known to those skilled in the art, and the receptor binding, antibody glycosylation, the number of cys fragments are evaluated for function in the same manner as teine residues, effector cell function, or complement func are intact antibodies. tion). Typically, the antibody specifically binds to a predeter 0061. Other than “bispecific' or “bifunctional antibod mined antigen, e.g., an antigen associated with a disorder, ies, an antibody is understood to have each of its binding sites e.g., a neurodegenerative, metabolic, inflammatory, autoim identical. A “bispecific' or “bifunctional antibody' is an arti mune and/or a malignant disorder. ficial hybrid antibody having two different heavy/light chain 0058 Small Modular ImmunoPharmaceuticals (SMIPTM) pairs and two different binding sites. Bispecific antibodies provide an example of a variant molecule comprising a bind can be produced by a variety of methods including fusion of ing domain polypeptide. SMIPs and their uses and applica hybridomas or linking of Fab' fragments. See, e.g., Songsiv tions are disclosed in, e.g., U.S. Published Patent Application. ilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Nos. 2003/0118592, 2003/0133939, 2004/0058445, 2005/ Kostelny et al., J. Immunol. 148, 1547-1553 (1992). US 2008/0274507 A1 Nov. 6, 2008
0062. The phrase “bioreactor refers to a vessel in which a media. The present invention encompasses the Surprising cell culture medium can be contained and internal conditions finding that it is advantageous to use an anti-senescence com of which can be controlled during the culturing period, e.g., pound (including, but not limited to, carnosine) in cell culture pH and temperature. to produce a protein of interest. In certain embodiments, 0063 A “fed batch culture” refers to a method of culturing using such an anti-senescence compound in cell culture to cells in which cells are first inoculated in a bioreactor with an produce a protein of interest results in one or more improved inoculum medium. The cell culture medium is then Supple cell culture characteristics including, but not limited to, mented at one or more points throughout the production run increased titer, increased cell specific productivity, increased with a feed medium containing nutritional components and/ cell viability, increased integrated viable cell density, or other Supplements. decreased accumulation of high molecular weight aggre 0064. A “batch culture” refers to a method of culturing gates, and/or decreased accumulation of acidic species. cells in which cells are inoculated in a bioreactor with all the 0071. It has been demonstrated that carnosine is cytotoxic necessary nutrients and Supplements for the entirety of the to human or rodent transformed and neoplastic cells in Mini production run. No nutrient additions are made to the cell mal Essential Medium (MEM, Sigma), which has lower glu culture medium throughout the duration of the production. cose levels, but not in Dulbecco's Modified Eagle's Medium 0065. A “perfusion culture” refers to a method of culturing (DMEM, Sigma), which contains 1 mM pyruvate. (Holliday cells that is different from a batch or fed-batch culture etal, Biochemistry (Moscow), 65:843-848,846). In addition, method, in which the culture is not terminated, or is not necessarily terminated, prior to isolating and/or purifying an dialyzed fetal calf serum with low molecular weight com expressed protein of interest, and in which new nutrients and pounds removed increased the cytotoxic effects of carnosine. other components are periodically or continuously added to Id. It was also determined that 1 mM oxaloacetate and 1 mM the culture, during which the expressed protein is periodically of O-ketoglutarate had comparable effects as pyruvate, nei or continuously harvested. The composition of the added ther of which are components of the inoculum or feed medi nutrients may be changed during the course of the cell culture, ums used with the carnosine examples. Id. Sodium pyruvate, depending on the needs of the cells, the requirements for however, is an original component in the inoculum medium at optimal protein production, and/or any of a variety of other a concentration of 0.5 mM, not for carnosine additions but factors known to those of ordinary skill in the art. rather to better mimic in vivo conditions in a bioreactor sys 0066. The phrase “expression” refers to the transcription tem and as a potential alternate energy source. The inoculum and the translation that occurs within a host cell. The level of medium is also serum free, which would imply that carnosine expression relates, generally, to the amount of protein being would have cytotoxic effects. According to the reference, the produced by the host cell. addition of carnosine to a cell culture medium would have 0067 “Cell specific productivity”, and the like, refer to the similar cytotoxic effects as seen in the MEM medium in specific, as in per cell, product expression rate. The cell spe Holliday. By utilizing methods and/or compositions of the cific productivity is generally measured in micrograms of present invention, such cytotoxicity is reduced or eliminated protein produced per 10° cells per day or in picograms of and cell viability and protein production are improved. protein produced per 10° cells per day. 0072. In certain embodiments, carnosine is provided in a 0068. The term “titer as used herein refers to the total cell culture medium at a concentration of between about 5 amount of recombinantly expressed protein produced by a mMandabout 100 mM. In certain embodiments, carnosine is cell culture in a given amount of medium Volume. Titer is provided in a cell culture medium at a concentration of about typically expressed in units of milligrams or micrograms of 10 mM to about 40 mM, for example at a concentration of protein per milliliter of medium. about 20 mM. In certain embodiments, carnosine is provided 0069. One of skill in the art will recognize that the methods in a cell culture medium at a concentration of about 5, 6, 7, 8, disclosed herein may be used to culture many of the well 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, known mammalian cells routinely used and cultured in the 26, 27, 28, 29, 30, 31, 32,33, 34, 35,36, 37,38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48,49, 50,55, 60, 65,70, 75,80, 85,90, 95, art, i.e., the methods disclosed herein are not limited to use 100 mM, or higher. In certain embodiments, such concentra with only the instant disclosure. tions of carnosine are achieved in cell culture by adding DETAILED DESCRIPTION OF CERTAIN carnosine at multiple times during the cell culture process, for EMBODIMENTS OF THE INVENTION example, in one or more feed media. The concentration of carnosine utilized depends on the cell culture medium and the 0070. It has been discovered that using an anti-senescence cell line being used, among other factors, including the compound Such as carnosine modifies the viability and pro desired effects being sought on the cell line or product. Ana ductivity of a cell culture. For example, addition of carnosine logs of carnosine, e.g. acetyl-carnosine, homo-carnosine, maintains cell viability, and improves productivity of cells, anserine, and beta-alanine, may also be provided in a cell and improves product quality of the desired protein product. culture medium for a similar effect. One or more of these Carnosine is an antioxidant and an anti-senescence com analogs may be provided in a cell culture medium. In certain pound that is also a naturally occurring dipeptide present at embodiments, such analogs are provided in a cell culture high levels (up to 20 mM) in muscle and nerve tissues in medium that lacks carnosine. In certain embodiments, such animals. Being an antioxidant, carnosine also is a free radical analogs are provided in a cell culture medium in combination Scavenger and glycation inhibitor. Generally, carnosine trans with carnosine. In certain embodiments, such analogs are forms reactive species into non-reactive species thereby pro provided at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, tecting proteins, DNA, and other essential macromolecules. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, As an anti-senescence compound, carnosine can extend the 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41,42, 43,44, 45,46, lifespan of human diploid fibroblasts and human fetal lung 47, 48,49, 50, 55, 60, 65,70, 75, 80, 85,90, 95, 100 mM, or (primary cell lines) at a concentration of 20 mM in culture higher. US 2008/0274507 A1 Nov. 6, 2008
0073. In certain embodiments, in order to produce a pro cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 tein of interest, initially, host cells are transfected or trans (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line formed with exogenous DNA coding for a protein to Supply (Hep G2). transformed cells, which constitutively produce the desired protein product. In certain embodiments, a nucleic acid mol Polypeptides ecule introduced into the cell encodes the protein desired to be 0077. Any polypeptide that is expressible in a host cell expressed according to the present invention. In certain may be produced in accordance with the present invention. embodiments, a nucleic acid molecule contains a regulatory The polypeptide may be expressed from a gene that is endog sequence or encodes a gene product that induces or enhances enous to the host cell, or from a heterologous gene that is the expression of the desired protein by the cell. As a non introduced into the host cell. The polypeptide may be one that limiting example, such a gene product may be a transcription occurs in nature, or may alternatively have a sequence that factor that increases expression of the protein of interest. was engineered or selected by the hand ofman. A polypeptide 0.074. In certain embodiments, a nucleic acid that directs to be produced according to the present invention may be expression of a protein is stably introduced into the host cell. assembled from polypeptide fragments that individually In certain embodiments, a nucleic acid that directs expression occur in nature. Additionally or alternatively, the engineered of a protein is transiently introduced into the host cell. One of polypeptide may include one or more fragments that are not ordinary skill in the art will be able to choose whether to naturally occurring. Proteins or peptides may be made by any technique known to those of skill in the art, including the stably or transiently introduce the nucleic acid into the cell expression of proteins, polypeptides or peptides through stan based on experimental, commercial or other needs. dard molecular biological techniques, the isolation of pro 0075. A gene encoding a protein of interest may optionally teins or peptides from natural sources, or the chemical Syn be linked to one or more regulatory genetic control elements. thesis of proteins or peptides. The coding regions for known In some embodiments, a genetic control element directs con genes may be amplified and/or expressed using the tech stitutive expression of the protein. In some embodiments, a niques disclosed herein or as would be known to those of genetic control element that provides inducible expression of ordinary skill in the art. Alternatively, various commercial a gene encoding the protein of interest can be used. Use of an preparations of proteins, polypeptides and peptides are inducible genetic control element (e.g., an inducible pro known to those of skill in the art. moter) allows for modulation of the production of the protein 0078. Therapeutic proteins that may desirably be in the cell. Non-limiting examples of potentially useful induc expressed in accordance with the present invention will often ible genetic control elements for use in eukaryotic cells be selected on the basis of an interesting or useful biological include hormone-regulated elements (see e.g., Mader, S, and or chemical activity. For example, the present invention may White, J. H., Proc. Natl. Acad. Sci. USA 90:5603-5607, be employed to express any pharmaceutically or commer 1993), synthetic ligand-regulated elements (see, e.g. Spencer, cially relevant enzyme, clotting factor, receptor, antibody, D. M. et al., Science 262:1019-1024, 1993) and ionizing hormone, regulatory factor, antigen, binding agent, etc. The radiation-regulated elements (see e.g., Manome, Y. et al., following list of therapeutic proteins that can be produced Biochemistry 32:10607-10613, 1993: Datta, R. et al., Proc. according to the present invention is merely exemplary in Natl. Acad. Sci. USA 89:10149-10153, 1992). Additional nature, and is not intended to be a limiting recitation. One of cell-specific or other regulatory systems known in the art may ordinary skill in the art will understand that any polypeptide may be expressed in accordance with the present invention be used in accordance with methods and compositions and will be able to select the particular polypeptide to be described herein. produced based on his or her particular needs. 0076 Any host cell susceptible to cell culture, and to expression of proteins, may be utilized in accordance with the Fusion Proteins present invention. The host cells are generally mammalian cells, more particularly animal cells, such as Chinese hamster 007.9 Fusion proteins generally have all or a substantial ovary (CHO) cells. Other non-limiting examples of mamma portion of a targeting peptide, linked at the N- or C-terminus, lian cells that may be used in accordance with the present to all or a portion of a second polypeptide or protein. For invention include BALB/c mouse myeloma line (NSO/I, example, fusions may employ leader sequences from other ECACC No: 851 10503); human retinoblasts (PER.C6 (Cru species to permit the recombinant expression of a protein in a Cell, Leiden, The Netherlands)); monkey kidney CV1 line heterologous host. Another useful fusion includes the addi transformed by SV40 (COS-7, ATCC CRL 1651); human tion of an immunologically active domain, Such as an anti embryonic kidney line (293 or 293 cells subcloned for growth body epitope, to facilitate purification of the fusion protein. A in suspension culture, Graham et al., J. Gen Virol. 36:59 fusion protein can include a targeting moiety, e.g., a soluble (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); receptor fragment or a ligand, and an immunoglobulin chain, Chinese hamster ovary cells--/-DHFR (CHO, Urlaub and an Fc fragment, a heavy chain constant regions of the various Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse isotypes, including for example: IgG1, IgG2, IgG3, IgG4. sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 IgM, IgA1, IgA2, Ig), and IgE. For example, the fusion (1980)); monkey kidney cells (CV1 ATCCCCL 70); African protein can include the extracellular domain of a receptor, green monkey kidney cells (VERO-76, ATCC CRL-1587); and, e.g., fused to, a human immunoglobulin Fc chain (e.g., human cervical carcinoma cells (HelLa, ATCC CCL 2): human IgG1 or human IgG4, or a mutated form thereof). In canine kidney cells (MDCK, ATCCCCL34); buffalo ratliver one embodiment, the human Fc sequence has been mutated at cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, one or more amino acids, e.g., mutated at residues 254 and ATCC CCL 75); human liver cells (Hep G2, HB 8065); 257 from the wild type sequence to reduce Fc receptor bind mouse mammary tumor (MMT 060562, ATCCCCL51); TRI ing. The fusion proteins may additionally include a linker US 2008/0274507 A1 Nov. 6, 2008
sequence joining the first moiety to the second moiety, e.g., BRI Natalizumab, VECTIBIX Panitumumab, VER the immunoglobulin fragment. For example, the fusion pro LUMA Nofetumomab, XOLAIR Omalizumab, ZANA tein can include a peptide linker, e.g., a peptide linker of about PAX Daclizumab, ZEVALIN Ibritumomab Tiuxetan, etc. 4 to 20, more preferably, 5 to 10, amino acids in length; in 0084. In certain embodiments, the monoclonal, chimeric, certain embodiments, the peptide linker is 8 amino acids in or humanized antibodies described above contain amino acid length. For example, the fusion protein can include a peptide residues that do not naturally occur in any antibody in any linker having the formula (Ser-Gly-Gly-Gly-Gly)y whereiny species in nature. These foreign residues can be utilized, for is 1, 2, 3, 4, 5, 6, 7, or 8. In other embodiments, additional example, to confer novel or modified specificity, affinity or amino acid sequences can be added to the N- or C-terminus of effector function on the monoclonal, chimeric or humanized the fusion protein to facilitate expression, steric flexibility, antibody. detection and/or isolation or purification. 0080 Inclusion of a cleavage site at or near the fusion Clotting Factors junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of I0085 Clotting factors have been shown to be effective as functional domains, such as active sites from enzymes, gly pharmaceutical and/or commercial agents. Hemophilia B is a cosylation domains, cellular targeting signals or transmem disorder in which the blood of the sufferer is unable to clot. brane regions. Examples of proteins or peptides that may be Thus, any Small wound that results in bleeding is potentially incorporated into a fusion protein include cytostatic proteins, a life-threatening event. Given the importance of recombinant cytocidal proteins, pro-apoptosis agents, anti-angiogenic clotting factors in the treatment of diseases Such as hemo agents, hormones, cytokines, growth factors, peptide drugs, philia, production of clotting factors in accordance with the antibodies, Fab fragments antibodies, antigens, receptor pro present invention is of particular interest. In certain embodi teins, enzymes, lectins, MHC proteins, cell adhesion proteins ments, the present invention may be used to produce clotting and binding proteins. Methods of generating fusion proteins factors in a cell culture wherein the misfolding and/or aggre are well known to those of skill in the art. Such proteins can be gation of the produced clotting factors are reduced. produced, for example, by chemical attachment using bifunc I0086 For example, Coagulation Factor IX (Factor IX or tional cross-linking reagents, by de novo synthesis of the “FIX) is a single-chain glycoprotein whose deficiency complete fusion protein, or by attachment of a DNA sequence results in Hemophilia B. FIX is synthesized as a single chain encoding the targeting peptide to a DNA sequence encoding Zymogen that can be activated to a two-chain serine protease the second peptide or protein, followed by expression of the (Factor IXa) by release of an activation peptide. The catalytic intact fusion protein. domain of Factor IXa is located in the heavy chain (see Chang et al., J. Clin. Invest., 100:4, 1997, incorporated herein by Antibodies reference in its entirety). Other clotting factors that can be produced in accordance with the present invention include 0081 Antibodies are proteins that have the ability to spe tissue factor, and von Willebrands factor and/or commercially cifically bind a particular antigen. Given the large number of available blood-clotting factors. Representative commer antibodies currently in use or under investigation as pharma cially available blood-clotting factors that can be produced in ceutical or other commercial agents, production of antibodies accordance with the present invention include, for example, in accordance with the present invention is of particular inter ALTEPLASE Tissue Plasminogen Activator; t-PA), BEN est. For example, the present invention may be used to pro EFIX Factor IX, HEMOFIL Antihemophilic Factor: Fac duce antibodies in a cell culture wherein the misfolding and/ tor XIII), RECOMBINATE (Recombinant Antihemophiliac or aggregation of the produced antibodies are reduced. Factor), etc. 0082 In certain embodiments, methods and/or composi tions of the present invention are employed to produce an Enzymes antibody against growth differentiation factor-8 (GDF-8). Nonlimiting examples of GDF-8 antibodies include Myo29, I0087 Another class of polypeptides that have been shown Myo28, and Myo22. In certain embodiments, GDF-8 anti to be effective as pharmaceutical and/or commercial agents bodies produced in accordance with the present teachings are and that can desirably be produced according to the teachings produced in the form of human IgG isotopes. In certain of the present invention includes enzymes. Given the impor embodiments, methods and/or compositions of the present tance of recombinant enzymes in the treatment of diseases invention are employed to produce a MYO29 antibody such and other commercial and pharmaceutical uses, production of as described in international patent application, publication enzymes in accordance with the present invention is of par number WO 2004/037861, entitled Neutralizing Antibodies ticular interest. For example, the present invention may be Against GDF-8 and Uses Thereof, incorporated herein by used to produce enzymes in a cell culture wherein the mis reference in its entirety. folding and/or aggregation of the produced enzymes are 0083. Other representative commercially available thera reduced. Representative commercially available enzymes peutic proteins that can be produced in accordance with the that can be produced in accordance with the present invention present invention include, for example, AVASTIN Bevaci include, for example, ACTIVASE Recombinant alteplase, Zumab, CAMPATH Alemtuzumab, ERBITUX Cetux CEREDASE Alglucerase, CEREZYME Imiglucerase, imab, HERCEPTIN TRASTUZUMAB), HUMIRA Ad PULMOZYME DNasel, etc. alimumab), LUCENTIS Ranibizumab, MYLOTARG gemtuzumab ozogamicin), MYCSCINT Imicromab Pen Growth Factors and Other Signaling Molecules etate), PROSTASCINT Capromab Pendetide, RAPTIVA I0088 Another class of polypeptides that have been shown Efalizumab, REMICADE Infliximab, REOPRO Abcix to be effective as pharmaceutical and/or commercial agents imab, RITUXANRituximab, SIMULECSTBasilximab, and that can desirably be produced according to the teachings SOLIRIS Eculizumab, SYNAGIS Palivizumab. TYSA of the present invention includes growth factors and other US 2008/0274507 A1 Nov. 6, 2008 signaling molecules. Growth factors are often glycoproteins tors in a cell culture wherein the misfolding and/or aggrega that are secreted by cells and bind to and activate receptors on tion of the produced receptors are reduced. other cells, initiating a metabolic or developmental change in 0091 Receptors are typically trans-membrane glycopro the receptor cell. Given the biological importance of growth teins that function by recognizing an extra-cellular signaling factors and other signaling molecules and their importance as ligand. Receptors often have a protein kinase domain in addi potential therapeutic agents, production of these molecules in tion to the ligand recognizing domain. This protein kinase accordance with the present invention is of particular interest. domain initiates a signaling pathway by phosphorylating tar For example, the present invention may be used to produce get intracellular molecules upon binding the ligand, leading growth factors or other signaling molecules in a cell culture to developmental or metabolic changes within the cell. In wherein the misfolding and/or aggregation of the produced certain embodiments, an extracellular domain of a transmem growth factors or other signaling molecules are reduced. brane receptor is produced in accordance with methods and 0089. Non-limiting examples of mammalian growth fac systems disclosed herein. In certain embodiments, an intrac tors and other signaling molecules include cytokines; epider ellular domain of a transmembrane receptor is produced in mal growth factor (EGF); platelet-derived growth factor accordance with methods and systems disclosed herein. (PDGF): fibroblast growth factors (FGFs) such as aFGF and 0092. In certain embodiments, tumor necrosis factor bFGF: transforming growth factors (TGFs) such as TGF inhibitors, in the form of tumor necrosis factor alpha and beta alpha and TGF-beta, including TGF-beta 1, TGF-beta 2. receptors (TNFR-1; EP417,563 published Mar. 20, 1991; and TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth TNFR-2, EP 417,014 published Mar. 20, 1991, each of which factor-I and -II (IGF-I and IGF-II): des(1-3)-IGF-I (brain is incorporated herein by reference in its entirety) are IGF-I), insulin-like growth factor binding proteins; CD pro expressed in accordance with Systems and methods of the teins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin present invention (for review, see Naismith and Sprang, J. (representative commercially available erythropoietins Inflamm.47(1-2):1-7, 1995-96, incorporated herein by refer include, for example, ARANESEP Idarbepoetin: CEA ence in its entirety). According to some embodiments, a SCAN Arcitumomab, EPOGEN epoetin alfa: PROCRIT tumor necrosis factor inhibitor comprises a soluble TNF epoetin alfa), etc.); osteoinductive factors; immunotoxins; a receptor. In certain embodiments, a tumor necrosis factor bone morphogenetic protein (BMP); an interferon such as inhibitor comprises a soluble TNFR fused to any portion of an interferon-alpha, -beta, and -gamma (representative commer immunoglobulin protein, including the Fc region of an immu cially available interferons include, for example, noglobulin. In certain embodiments, TNF inhibitors of the ACTIMUNNE Interferon gamma-1b), AVONEX Inter present invention are soluble forms of TNFR I and TNFR II. feron beta-1a, REBIF Interferon beta-1a, BETASERON In certain embodiments, TNF inhibitors of the present inven Interferon beta-1b), etc.); colony stimulating factors tion are soluble TNF binding proteins. In certain embodi (CSFs), e.g., M-CSF, GM-CSF, and G-CSF (other represen ments, the TNF inhibitors of the present invention are TNFR tative commercially available colony stimulating factors Fc, for example, etanercept. As used herein, “etanercept.” include, for example, GRANUCYTE, Lenograstim, LEUK refers to a TNFR-Fc, which is a dimer of two molecules of the INE Sargramostim), etc.); interleukins (TLS), e.g., IL-1 to extracellular portion of the p75 TNF-C. receptor, each mol IL-10; tumor necrosis factor (TNF) alpha and beta; insulin ecule consisting of a 235 amino acid Fc portion of human A-chain; insulin B-chain; proinsulin; follicle stimulatinghor IgG1. In accordance with the invention, an anti-senescence mone, calcitonin; luteinizing hormone; glucagon; clotting compound, Such as carnosine, is used to decrease the amount factors such as factor VIIIC, factor IX, tissue factor, and von of misfolded and/or aggregated protein during the production Willebrands factor; anti-clotting factors such as Protein C: of TNFR-FC. atrial natriuretic factor, lung Surfactant; a plasminogen acti vator, such as urokinase or human urine or tissue-type plas 0093. In some embodiments, receptors to be produced in minogen activator (t-PA); bombesin; thrombin, hemopoietic accordance with the present invention are receptor tyrosine growth factor; enkephalinase; RANTES (regulated on activa kinases (RTKs). The RTK family includes receptors that are tion normally T-cell expressed and secreted); human mac crucial for a variety of functions numerous cell types (see, rophage inflammatory protein (MIP-1-alpha); mullerian-in e.g., Yarden and Ullrich, Ann. Rev. Biochem. 57:433-478, hibiting Substance; relaxin A-chain; relaxin B-chain; 1988: Ullrich and Schlessinger, Cell 61:243-254, 1990, each prorelaxin; mouse gonadotropin-associated peptide; neu of which is incorporated herein by reference). Non-limiting examples of RTKs include tumor necrosis factor alpha and rotrophic factors such as bone-derived neurotrophic factor beta receptors, members of the fibroblast growth factor (FGF) (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or receptor family, members of the epidermal growth factor NT-6), or a nerve growth factor such as NGF-beta. One of receptor (EGF) family, platelet derived growth factor (PDGF) ordinary skill in the art will be aware of other growth factors receptor, tyrosine kinase with immunoglobulin and EGF or signaling molecules that can be expressed in accordance homology domains-1 (TIE-1) and TIE-2 receptors (Sato et with methods and compositions of the present invention. al., Nature 376(6535):70-74, 1995, incorporated herein by reference in its entirety) and c-Met receptor, some of which Receptors have been suggested to promote angiogenesis, directly or 0090 Another class of polypeptides that have been shown indirectly (Mustonen and Alitalo, J. Cell Biol. 129:895-898, to be effective as pharmaceutical and/or commercial agents 1995). Other non-limiting examples of RTK's include fetal and that can desirably be produced according to the teachings liver kinase 1 (FLK-1) (sometimes referred to as kinase insert of the present invention includes receptors. Given the biologi domain-containing receptor (KDR) (Terman et al., Oncogene cal importance of receptors and their importance as potential 6:1677-83, 1991) or vascular endothelial cell growth factor therapeutic agents, production of these molecules in accor receptor 2, VEGFR-2), fms-like tyrosine kinase-1 (Flt-1) dance with the present invention is of particular interest. For (DeVries et al. Science 255; 989-991, 1992: Shibuya et al., example, the present invention may be used to produce recep Oncogene 5:519–524, 1990), sometimes referred to as vascu US 2008/0274507 A1 Nov. 6, 2008
lar endothelial cell growth factor receptor 1 (VEGFR-1), medium may be supplemented with pyruvate, oxaloacetate neuropilin-1, endoglin, endosialin, and AXl. Those of ordi and/or C-ketoglutarate. One of ordinary skill in the art will be nary skill in the art will be aware of other receptors that can be able to alter a base medium for use with methods and com expressed in accordance with the present invention. positions of the present invention without undue experimen 0094. In certain embodiments, the receptor to be produced tation. in accordance with the present invention is a G-protein 0099. In certain embodiments, cells are cultured in any of coupled receptor (GPCR). GPCRs are a major target for drug a variety of chemically defined media containing an anti action and development. In fact, receptors have led to more senescence compound, wherein the components of the media than half of the currently known drugs (Drews, Nature Bio are both known and controlled. For example, defined media technology, 14:1516, 1996) and GPCRs represent the most typically do not contain complex additives such as serum or important target for therapeutic intervention with 30% of hydrolysates. In certain embodiments, cells are cultured in clinically prescribed drugs eitherantagonizing or agonizing a any of a variety of complex media containing an anti-senes GPCR (Milligan, G. and Rees, S., TIPS, 20:118-124, 1999). cence compound, in which not all components of the medium Since these receptors have an established, proven history as are known and/or controlled. In certain embodiments, such an therapeutic targets, production of GPCRs in accordance with anti-senescence compound comprises carnosine. the present invention is also of particular interest. 0100. The conditions of the bioreactor are controlled typi 0095. In general, practitioners of the present invention will cally, with the pH set between about 6.5 to about 7.5. The pH select their protein or polypeptide of interest, and will know is adjusted using an acid, generally CO, or a base, such as its precise amino acid sequence. Any given polypeptide that is sodium bicarbonate. The dissolved oxygen is controlled to be expressed in accordance with the present invention will between about 5 and 90% of air saturation, and the tempera have its own particular characteristics and may influence the ture is held between 30°C. to 42°C., during the growth phase. cell density or viability of the cultured cells, and may be A person of ordinary skill in the cell culture art can modify the expressed at lower levels than another polypeptide or protein conditions of the bioreactor based upon the cell line and grown under identical culture conditions. One of ordinary methods being employed to achieve the desired results with skill in the art will be able to appropriately modify inventive out undue experimentation. media and methods described herein in order to optimize cell 0101 Compositions and methods of the present invention growth, titer, folding or any other property of a given may be used with any cell culture method or system that is expressed polypeptide or protein. amenable to the expression of proteins. For example, cells 0096. One of ordinary skill in the art will be aware of other expressing a protein of interest may be grown in batch or useful and/or desirable proteins that may be expressed in fed-batch cultures, wherein the culture is terminated after accordance with methods and compositions of the present sufficient expression of the protein, after which the expressed invention. protein is harvested and optionally purified. Alternatively, 0097 Cell lines may be cultured using a variety of tech cells expressing a protein of interest may be grown in perfu niques to produce the desired protein product. The cell culture sion cultures, wherein the culture is not terminated and new can be done on a small or large scale, depending on the nutrients and other components are periodically or continu purpose of the cell culture medium or use of the product. For ously added to the culture, during which the expressed protein example, cells may be grown in a bioreactor. In certain is periodically or continuously harvested. embodiments, the volume of the bioreactor is at least 1 liter 0102. After the cells are seeded they go through a growth and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, phase during which the number of cells generally increases 10,000, 12,000 liters or more, or any volume in between. In exponentially. During the growth phase, the temperature or addition, bioreactors that may be used include, but are not temperature range of the cell culture will be selected based limited to, a stirred tank bioreactor, fluidized bed reactor, primarily on the temperatures or range of temperatures at hollow fiber bioreactor, or roller bottle. The systems can also which the cell culture remains viable, at which a high level of operate either in a batch, fed-batch, or continuous/perfusion protein is produced, at which production or accumulation of mode. The bioreactor and mode in which to control and metabolic waste products is minimized, and/or any combina monitor the cell culture medium will be known to one of tion of these or other factors deemed important by the prac ordinary skill in the cell culture art. In the instant example, the titioner. As one non-limiting example, CHO cells grow well system utilized is a stirred tank bioreactor and operated in and produce high levels or protein at approximately 37°C. In fed-batch mode. general, most mammaliancells grow well and/or can produce 0098. The bioreactor is generally seeded with an inoculum high levels or protein within a range of about 25°C. to 42°C., medium and a chosen cell line, for example a TNFR fusion although methods taught by the present disclosure are not protein cell line, which may be transfected to stably express limited to these temperatures. Certain mammalian cells grow and produce the desired protein product. Commercially avail well and/or can produce high levels of protein within the able medium such as Minimal Essential Medium (MEM, range of about 35° C. to 40°C. In certain embodiments, the Sigma), Ham's F10 (Sigma), or Dulbecco's Modified Eagle's cell culture is grown at a temperature of 20, 21, 22, 23, 24, 25, Medium (DMEM, Sigma) may be used as the base medium. 26, 27, 28, 29, 30, 31, 32,33, 34, 35,36, 37,38, 39, 40, 41, 42, These base mediums may then be supplemented with amino 43, 44, or 45° C. at one or more times during the growth phase. acids, vitamins, trace elements, and/or other components to Those of ordinary skill in the art will be able to select appro produce the inoculum or feed mediums used during the pro priate temperature or temperature range in which to grow duction run. In certain embodiments, a base medium is cells, depending on the needs of the cells and the production altered to permit robust growth of cells, to increase cell viabil requirements of the practitioner. ity, to increase cell productivity, to increase integrated viable 0103) Following the growth phase is a transition phase cell density, and/or to improve the quality of the produced during which the cells adapt to any changes occurring in the protein in the presence of carnosine. For example, a base Surroundings, such as a temperature change. The changes US 2008/0274507 A1 Nov. 6, 2008
occurring are typically parameters for the production phase. inoculation medium or a feed medium, increases cell viability In the instant examples, on day 4, the temperature decreased and/or specific protein production, thus improving the overall from about 37° C. to about 31° C. The temperature shift, yield of produced protein. however, can occur more than once and does not need to 0107 Aspects of a protein production process are deter necessarily go in the downward direction. Moreover, the tran mined by one of ordinary skill in the cell culture art. The sition phase and the temperature shift can occur on any day parameters such as seed density, duration of the production during the production run. Although most methods of pro culture, operating conditions during harvest, among others, duction include multi-phase processes, carnosine also may be including those mentioned above are functions of the cell line utilized in a single-phase process. and the cell culture medium. Therefore, the parameters can be determined without undue experimentation by a person of 0104. When shifting the temperature of the culture, the ordinary skill in the cell culture art. temperature shift may be relatively gradual. For example, it 0108. As with the temperature or temperature range dur may take several hours or days to complete the temperature ing the growth phase, the temperature or temperature range of change. Alternatively, the temperature shift may be relatively the cell culture during the production phase will be selected abrupt. The temperature may be steadily increased or based primarily on the temperature or temperature range at decreased during the culture process. Alternatively, the tem which the cell culture remains viable, at which a high level of perature may be increased or decreased by discrete amounts protein is produced, at which production or accumulation of at various times during the culture process. The Subsequent metabolic waste products is minimized, and/or any combina temperature(s) or temperature range(s) may be lower than or tion of these or other factors deemed important by the prac higher than the initial or previous temperature(s) or tempera titioner. In general, most mammalian cells remain viable and ture range(s). One of ordinary skill in the art will understand produce high levels or protein within a range of about 25°C. that multiple discrete temperature shifts are encompassed in to 42°C., although methods taught by the present disclosure these embodiments. For example, the temperature may be are not limited to these temperatures. In certain embodiments, shifted once (either to a higher or lower temperature or tem mammalian cells remain viable and produce high levels or perature range), the cells maintained at this temperature or protein within a range of about 25° C. to 35° C. In certain temperature range for a certain period of time, after which the embodiments, the cell culture is grown at a temperature of 20, temperature may be shifted again to a new temperature or 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, temperature range, which may be either higher or lower than 38, 39, 40, 41,42, 43,44, or 45° C. at one or more times during the temperature or temperature range of the previous tem the production phase. Those of ordinary skill in the art will be able to select appropriate temperature(s) or temperature range perature or temperature range. The temperature of the culture (s) in which to grow cells during the production phase, after each discrete shift may be constant or may be maintained depending on the particular needs of the cells and the particu within a certain range of temperatures. lar production requirements of the practitioner. The cells may 0105 Finally, there is the production phase where the cell be grown for any amount of time, depending on the needs of number does not substantially increase, but rather the cells the practitioner and the requirement of the cells themselves. produce the desired protein product. One of ordinary skill in 0109. In certain embodiments, batch or fed-batch cultures the art will understand, however, that in certain embodiments, are terminated once the culture achieves one or more relevant cells may continue to grow and increase in number during the culture conditions, as determined by the needs of the practi production phase. During this phase the environment of the tioner. In certain embodiments, batch or fed-batch cultures bioreactor is controlled at conditions in which the cells are are terminated once the expressed protein reaches a suffi more likely to be productive. For example, the temperature is ciently high titer, once the cell density reaches a sufficiently generally held at a temperature different than that of the high level, once the expressed protein reaches a Sufficiently growth phase, which is conducive to the production of a high cell density, and/or to prevent undesirable production or protein product, e.g. 31° C. Throughout the production run, accumulation of metabolic waste products (e.g., lactate and/ the cells may be fed a feed medium containing nutrients and or ammonium). One of ordinary skill in the art will be aware Supplements the cells may need. For example, in certain of other relevant culture conditions that may be used to deter cases, it may be beneficial or necessary to Supplement the cell mine when a batch or fed-batch culture should be terminated, culture during the Subsequent production phase with nutrients based on experimental, commercial, and/or other consider or other medium components that have been depleted or ations. metabolized by the cells. As non-limiting examples, it may be 0110. In certain embodiments, following a production run, beneficial or necessary to supplement the cell culture with the protein product is recovered from the cell culture medium hormones and/or other growth factors, particular ions (such and further isolated using traditional separation techniques. as sodium, chloride, calcium, magnesium, and phosphate), For example, the protein may initially be separated by cen buffers, vitamins, nucleosides or nucleotides, trace elements trifugation, retaining the Supernatant containing the protein. (inorganic compounds usually present at very low final con Additionally or alternatively, the protein product may be centrations), amino acids, lipids, or glucose or other energy bound to the surface of the host cell. In such embodiments, the Source. These Supplementary components may all be added to media is removed and the host cells expressing the protein are the cell culture at one time, or they may be provided to the cell lysed as a first step in the purification process. Lysis of mam culture in a series of additions. In certain embodiments, an malian host cells can be achieved by any number of means anti-senescence compound is provided in a feed medium at known to those of ordinary skill in the art, including physical one or more times during the production phase. disruption by glass beads and exposure to high pH conditions. 0106. According to certain embodiments, use of an anti 0111. Using conventional protein purification methods, senescence compound, e.g. carnosine, in the cell culture the protein may be additionally isolated. Methods by which to medium during the production phase, whether provided in an isolate and purify the desired protein product are known US 2008/0274507 A1 Nov. 6, 2008
within the cell culture art. Specific methods depend on the cell efficiently decreases the cost to produce the protein product. line used and the product sought. In certain embodiments, an anti-senescence compound. Such 0112 The anti-senescence compound, e.g. carnosine, may as carnosine, is used to decrease the amount of misfolded be added to the culture medium at a time optimal for the and/or aggregated protein. In certain embodiments, an anti specific cell culture process. For the instant examples, the senescence compound other than carnosine is used to addition of carnosine occurs after the growth phase is Sub decrease the amount of high molecular weight aggregates stantially complete and is in the transition phase. During the and/or acidic species. In certain embodiments, two or more addition, the cell culture medium is adapting to the new anti-senescence compounds are used to decrease the amount temperature resulting from the temperature shift. The transi of high molecular weight aggregates and/or acidic species. tion phase is generally when agents are added to help initiate 0116. In certain embodiments, cells are grown in accor the production phase. Carnosine, however, may be added at dance with any of the cell culture methods described in U.S. any point during the production run that generates optimal patent application Ser. Nos. 11/213,308, 11/213,317 and results, including the growth phase and the production phase. 1 1/213,633 each of which was filed Aug. 25, 2005, and each Carnosine may also be added in combination with other com of which is herein incorporated by reference in its entirety. ponents, such as a feed medium. In certain embodiments, an For example, in certain embodiments, the cells may be grown anti-senescence compound is provided in an inoculation in a culture medium in which the cumulative amino acid medium and is present in the cell culture during the entire cell concentration is greater than about 70 mM. In certain embodi culture process. In certain embodiments, two or more anti ments, the cells may be grown in a culture medium in which senescence compounds are provided in the cell culture the molar cumulative glutamine to cumulative asparagine medium. In certain embodiments, two or more anti-senes ratio is less than about 2. In certain embodiments, the cells cence compounds are provided in an inoculation medium and may be grown in a culture medium in which the molar cumu are present in the cell culture during the entire cell culture lative glutamine to cumulative total amino acid ratio is less process. In certain embodiments, two or more anti-senes than about 0.2. In certain embodiments, the cells may be cence compounds are provided, wherein one anti-senescence grown in a culture medium in which the molar cumulative compound is provided in an inoculation medium and is inorganic ion to cumulative total amino acid ratio is between present in the cell culture during the entire cell culture pro about 0.4 to 1. In certain embodiments, the cells may be cess, while another anti-senescence compound is provided grownina culture medium in which the combined cumulative after the cell culture has begun. glutamine and cumulative asparagine concentration is 0113. In certain embodiments, the concentration of an between about 16 and 36 mM. In certain embodiments, the anti-senescence compound present in the cell culture is dif cells may be grown in a culture medium that contains two, ferent for varying cell types and products. In certain embodi three, four or all five of the preceding medium conditions. Use ments, the concentration of carnosine present is different for of Such media allows high levels of protein production and varying cell types and products. Generally, the concentration lessens accumulation of certain undesirable factors such as is enough to enhance the productivity and quality without ammonium and/or lactate. toxic effects. For the instant examples, the range includes, but 0117. In some embodiments, the cells are grown under one is not limited to, 5 mM to 100 mM. It will be appreciated that or more of the conditions described in U.S. Provisional Patent the concentration of carnosine used may vary depending on Application Ser. No. 60/830,658, filed Jul. 13, 2006 and the cell culture medium. The appropriate concentration of incorporated herein by reference in its entirety. For example, carnosine for a particular cell line may need to be determined in Some embodiments, cells are grown in a culture medium with routine Small-scale experiments, such as, for example, a that contains manganese at a concentration between approxi 2L bioreactor, using conventional methods. One of ordinary mately 10 and 600 nM. In some embodiments, cells are grown skill in the art will be able to determine an advantageous or in a culture medium that contains manganese at a concentra optimal concentration of carnosine or other anti-senescence tion between approximately 20 and 100 nM. In some embodi compound without undue experimentation using cell culture ments, cells are grown in a culture medium that contains techniques and diagnostic methods that are known in the art. manganese at a concentration of approximately 40 nM. Use 0114. One advantage to adding carnosine or another anti of such media in growing glycoproteins results in production senescence compound, rather than chemical agents, is the ofaglycoprotein with an improved glycosylation pattern (e.g. effect on viability. Typically, cell growth ceases and the viable a greater number of covalently linked Sugar residues in one or cell number decreases with the addition of a chemical agent, more oligosaccharide chains). like sodium butyrate. (Kim et al. Biotechnol Bioeng, 71: 0118. In certain embodiments of the invention, proteins 184-193, 184). Examples below, however, demonstrate that produced according to one or more methods of the present addition of carnosine to a cell culture medium results in invention will have pharmacologic activity and will be useful higher viability at the time of harvest than is observed in a cell in the preparation of pharmaceuticals. Proteins produced culture grown in the absence of an anti-senescence compound according to one or more methods of the present invention Such as carnosine. Furthermore, Such carnosine-containing may be administered to a subject or may first be formulated cell cultures exhibit an increase in specific productivity. With for delivery by any available route including, but not limited the positive effects on the cell viability and specific produc to parenteral (e.g., intravenous), intradermal, Subcutaneous, tivity, the overall yield is higher. oral, nasal, bronchial, opthalmic, transdermal (topical), trans 0115. Another advantage is that an anti-senescence com mucosal, rectal, and vaginal routes. Inventive pharmaceutical pound Such as carnosine decreases the amount of high compositions typically include a purified protein expressed molecular weight aggregates and/or the number of acidic from a mammalian cell line, a delivery agent (i.e., a cationic species. Decreasing the amount of high molecular weight polymer, peptide molecular transporter, Surfactant, etc., as aggregates and acidic species simplifies purification of the described above) in combination with a pharmaceutically protein product. Enabling the protein to be isolated more acceptable carrier. As used herein the language “pharmaceu US 2008/0274507 A1 Nov. 6, 2008
tically acceptable carrier includes solvents, dispersion serum free inoculation medium. All the bioreactors were fed media, coatings, antibacterial and antifungal agents, isotonic 5% (v/v) of a feed medium on days 3, 5, 7, and 12 of a 14-day and absorption delaying agents, and the like, compatible with run. A day 10 feed of 5% (v/v) of the feed medium was added pharmaceutical administration. Supplementary active com to two of the control bioreactors and one containing carnos pounds can also be incorporated into the compositions. ine. The conditions of the bioreactors were held at a tempera 0119) A pharmaceutical composition is formulated to be ture of 37°C., a pH of 7.00, and a dissolved oxygen level at compatible with its intended route of administration. Such 30% air saturation. The agitation rate was 200 rpm and the formulations will be known by those of skill in the art. In certain embodiments, a protein produced according to the sparge gas had a combination of air and 7% carbon dioxide. present invention is formulated in oral and/or parenteral form. 0.124 All cells were cultured for 4 days at which point 20 In certain embodiments, for ease of administration and uni mM of carnosine was added to two of the bioreactors, the formity of dosage. Such oral and/or parenteral forms are for controls did not have carnosine added, and the temperature mulated as unit dosage form, wherein each unit contains a was shifted to 31° C. in all of the bioreactors on day 4 also. predetermined quantity of active protein calculated to pro The bioreactors were harvested on day 14 of the production duce the desired therapeutic effect in association with the run. Samples were taken throughout the run to monitor the required pharmaceutical carrier. One of ordinary skill in the progress of the cell culture medium. The controls performed art will be aware of unit dosage formulations appropriate for as expected. proteins produced according to the present invention. (0.125 FIG.2a shows the daily viable cell density. FIG.2b 0120 Certain embodiments and aspects are discussed in shows that the daily cell viability of the bioreactors with detail above. The present disclosure is further illustrated by carnosine was higher upon being harvested compared to two the following, non-limiting examples. Those of ordinary skill bioreactors without it. FIG. 2c shows the daily titer of the in the art will understand, however, that various modifications bioreactors and that the two bioreactors with carnosine to these embodiments are within the scope of the appended claims. It is noted that the addition of carnosine and/or other present had higher titer at the time of harvest. The cultures anti-senescence compounds is equally applicable to other with carnosine had better cumulative specific cellular produc mammaliancell cultures and protein products. It is the claims tivity shown in FIG. 2d. FIG. 2e shows the amount of high and equivalents thereof that define the scope of the present molecular weight aggregates and shows a decrease of high invention, which is not and should not be limited to or by this molecular weight aggregates in the bioreactors with carnos description of certain embodiments. ine present. EXAMPLES Example 3 Example 1 Dish Scale Carnosine Experiments Effect of Different Concentration of Carnosine Addi 0121 A MYO-29 cell line was cultured in a serum free tions production medium in a bioreactor with a 1 L working Vol ume and was temperature shifted from 37°C. to 31° C. on day (0.126 Four bioreactors were inoculated with 0.4x10 4. The pH of the bioreactor was held at 7.00 and the dissolved cells/ml with a 1 L working volume of a MYO-29 cell line in oxygen was at 30% air saturation. Cell culture medium was a serum free inoculation medium. All the bioreactors were all then taken from the bioreactor on day 4, day 7, and day 10 and fed 5% (v/v) of a feed medium on day 7 of the 14-day run. The put into culture dishes with an 8 ml working Volume and conditions of the bioreactor were held at a temperature of 37 placed into a 31°C. incubator, where the dish cultures were C., a pH of 7.00, and a dissolved oxygen level at 30% air cultured until day 12. The cells were supplemented with a saturation. The agitation rate was 200 rpm and the sparge gas feed medium on days 5 and 7. On day 5, 10% (v/v) of feed had a combination of air and 7% carbon dioxide. medium was added to the cell cultures and on day 7, 5% (v/v) of feed medium was added to the cell cultures. 10 mM of I0127. All cells were cultured for four days at which point carnosine was added on day 4, day 7, and day 10, respectively, the temperature was shifted in all of the bioreactors to 31°C. to the dishes and the cell culture medium was harvested on Also on day 4, 20 mM of carnosine was added to one biore day 12. actor, a second had 40 mM of carnosine added, and the 0122 FIG. 1a shows the effect of the carnosine additions controls did not have any carnosine added. All bioreactors on the amount of acidic peaks in the culture on day 7. FIG.1b were harvested on day 12 of the production run. Samples shows the effect of the carnosine on the high molecular were taken throughout the run to monitor the progress of the weight aggregates on the day 7 culture. FIG. 1c illustrates the cell culture medium. The controls performed generally as effect of carnosine additions on day 4, Versus day 7, Versus expected, except one control had slightly lower daily viabili day 10 on the acidic peaks. FIG. 1d shows the same experi ties than previously seen. ment's results but for high molecular weight aggregates. I0128 FIG. 3a shows the daily viable cell density, the Overall, carnosine had a positive effect by decreasing both the different bioreactors are fairly similar with the exception of acidic peaks and the high molecular weight aggregates in the bioreactor with 4 mM of carnosine. FIG. 3b shows that the cultured dishes. daily cell viability of the bioreactors with carnosine had a Example 2 higher viability upon being harvested compared to two con trol bioreactors without it. FIG. 3c shows the daily titer; the Effects of Carnosine Addition to Cell Culture bioreactors with the carnosine additions and one of the con Medium trols were similar. The bioreactor with 40 mM of carnosine (0123 Five bioreactors were inoculated with 0.9x10 cells/ had a higher cumulative specific cellular productivity. (FIG. ml with a 1 L working volume of a MYO-29 cell line in a 3d). FIG. 3e shows the amount of high molecular weight US 2008/0274507 A1 Nov. 6, 2008
aggregates; overall there is a decrease in high molecular cell specific productivity, increased cell viability, weight aggregates with the carnosine additions compared to increased integrated viable cell density, decreased accu the controls. mulation of high molecular weight aggregates, decreased accumulation of acidic species, and combina Example 4 tions thereof. Use of Carnosine in Mammalian Cell Culture to 2. The method of claim 1, wherein the anti-senescence Improve Product Characteristics of a Recombinant compound is selected from the group consisting of carnosine, TNFR Fusion Protein acetyl-carnosine, homo-carnosine, anserine, and beta-ala nine and combinations thereof. 0129. A TNFR fusion protein cell line was cultured in a 3. The method of claim 1, wherein the anti-senescence serum free production medium in a bioreactor with a 1 L compound comprises carnosine. working volume. On day 1, the temperature was shifted from 4. The method of claim 1, wherein the anti-senescence 37° C. to 29.5° C., sodium butyrate was added to a final compound is present in the cell culture medium at a concen concentration of 1 mM, and HMBA was added to a final tration of between about 5 mM and about 100 mM. concentration of 3 mM. The pH of the bioreactor was held at 5. The method of claim 1, wherein the cell culture is further 6.95 and the dissolved oxygen was at 60% air saturation. On provided with Supplementary components. day 2, carnosine was added to the culture to a final concen 6. The method of claim 5, wherein the supplementary tration of 20 mM. The cells were supplemented with a 5% components are provided in a feed medium. (v/v) feed medium on days 3, 6, 8, and 10. The cell culture 7. The method of claim 5, wherein the supplementary medium was harvested on day 12. Four separate control components are selected from the group consisting of hor bioreactors were run in which CHO cells expressing a recom mones and/or other growth factors, particular ions (such as binant TNFR fusion protein were grown under conditions Sodium, chloride, calcium, magnesium, and phosphate), buff identical to those described above, except that carnosine was ers, vitamins, nucleosides or nucleotides, trace elements (in not added on day 2. The control data in FIGS. 4-11 is the organic compounds usually present at very low final concen average of the four control bioreactor runs. trations), amino acids, lipids, glucose or other energy source, 0130. As can be seen in FIGS. 4 and 5, cell growth and cell and combinations thereof. viability were not significantly affected by carnosine addi 8. The method of claim 5, wherein the supplementary tion. FIG. 6 shows that the amount of misfolded and/or aggre components include an anti-senescence compound. gated TNFR fusion protein, as measured by hydrophobic 9. A method for producing a TNFR fusion protein com interaction chromatography (HIC), was significantly reduced prising steps of: when the cells were grown in media containing carnosine. culturing mammalian cells that contain a gene encoding FIG. 7 shows that the amount of high molecular weight the TNFR fusion protein in a cell culture medium, which (HMW) aggregates, as measured by size exclusion chroma gene is expressed under conditions of cell culture, at a tography (SEC), was also significantly reduced when cells first temperature or temperature range conducive for cell expressing TNFR fusion protein were grown in media con growth during a growth phase; taining carnosine. FIGS. 8 and 9 show that product titer and shifting the temperature or temperature range of the cell specific cellular productivity, respectively, were increased culture medium to a second temperature or temperature when cells expressing TNFR fusion protein were grown in range conducive for protein production; media containing carnosine. Finally, FIGS. 10 and 11 show culturing the host cells in the cell culture medium at the that glycosylation of the produced TNFR fusion protein was second temperature or temperature range through a tran not significantly different when the cells were grown in media sition phase and into a production phase; containing carnosine. wherein an anti-senescence compound is added to the cell 0131 Although the some embodiments of the disclosure culture and such that the cell culture exhibits an have been described herein, the above description is merely improved cell culture characteristic that differs from a illustrative. Further modification of the embodiments herein corresponding cell culture characteristic that would be disclosed will occur to those skilled in the cell culture art and observed under otherwise identical conditions in anoth all such modifications are deemed to be within the scope of erwise identical medium that lacks the anti-senescence the embodiments as defined by the appended claims. compound; What is claimed is: wherein the improved cell culture characteristic is selected 1. A method of producing a TNFR fusion protein in cell from the group consisting of increased titer, increased culture comprising steps of: cell specific productivity, increased cell viability, culturing mammalian cells that contain a gene encoding increased integrated viable cell density, decreased accu the TNFR fusion protein, which gene is expressed under mulation of high molecular weight aggregates, conditions of cell culture, in a cell culture medium com decreased accumulation of acidic species, and combina prising an anti-senescence compound; and tions thereof. maintaining the culture under conditions and for a time 10. The method of claim 9, wherein the anti-senescence Sufficient to permit expression of the protein; compound is added to the cell culture medium at the begin wherein the cell culture exhibits an improved cell culture ning of the cell culture process. characteristic that differs from a corresponding cell cul 11. The method of claim 9, wherein the anti-senescence ture characteristic that would be observed under other compound is added to the cell culture medium during the wise identical conditions in an otherwise identical growth phase. medium that lacks the anti-senescence compound; 12. The method of claim 9, wherein the anti-senescence wherein the improved culture characteristic is selected compound is added to the cell culture medium during the from the group consisting of increased titer, increased transition phase. US 2008/0274507 A1 Nov. 6, 2008
13. The method of claim 9, wherein the anti-senescence 20. The method of claim 17, wherein the supplementary compound is added to the cell culture medium during the components include an anti-senescence compound. production phase. 21. The method of claim 1 or 9, wherein the produced 14. The method of claim 9, wherein the anti-senescence TNFR fusion protein is heterologous to the mammalian cells. 22. The method of claim 1 or 9, wherein the mammalian compound is selected from the group consisting of carnosine, cells are CHO cells. acetyl-carnosine, homo-carnosine, anserine, and beta-ala 23. The method of claim 1 or 9, wherein the TNFR fusion nine and combinations thereof. protein comprises TNFR-Fc. 15. The method of claim 9, wherein the anti-senescence 24. The method of claim 23, wherein the TNFR-Fc is compound comprises carnosine. etanercept. 16. The method of claim 9, wherein the anti-senescence 25. A TNFR fusion protein produced according to the compound is present in the cell culture medium at a concen method of claim 1 or 9. tration of between about 5 mM and about 100 mM. 26. The protein of claim 25, wherein the TNFR fusion protein comprises TNFR-Fc. 17. The method of claim 9, wherein the cell culture is 27. A method for preparing a protein according to claim 1 further provided with Supplementary components. or 9, further comprising the step of isolating the protein from 18. The method of claim 17, wherein the supplementary the cell culture medium. components are provided in a feed medium. 28. A method according to claim 27, wherein the protein is 19. The method of claim 17, wherein the supplementary further purified or processed for formulation. components are selected from the group consisting of hor 29. A method according to claim 28, wherein the protein is mones and/or other growth factors, particular ions (such as formulated into a pharmaceutical composition. Sodium, chloride, calcium, magnesium, and phosphate), buff 30. A method for preparing a protein according to claim 1 ers, vitamins, nucleosides or nucleotides, trace elements (in or 9, wherein the high molecular weight aggregates comprise organic compounds usually present at very low final concen misfolded protein. trations), amino acids, lipids, glucose or other energy source, and combinations thereof.