Bioreactor process defined by ph and temperature for producing eptinezumab antibodies

Abstract

The invention provides a culture method for producing Eptinezumab in a mammalian cell culture, by comprising culturing mammalian cells, e.g., CHO cells in a bioreactor, e.g., a fed-batch bioreactor, wherein said mammalian cells comprise nucleic acid sequences which provide for the expression of Eptinezumab and wherein culturing is effected at a temperature between 33° C to 38º C and wherein the pH of about 7 (e.g., between 6.85 and 7.05), e.g., wherein the pH is maintained at about 7 for up to 14 days, or at 6.95. The inventive culturing method controls or enhances the cell growth rate, cell viability, viable cell density and / or antibody titer (Eptinezumab) of the mammalian cells.

Description

1295-US-PSP Bioreactor process for antibodies Related Applications The present invention relates to and claims benefit of priority to U.S. Provisional Application Number 63 / 667,187, filed on July 3, 2024, the contents of which are incorporated by reference in its entirety herein. Sequence Listing Disclosure

[0002] The contents of the electronic sequence listing (1295-WO-PCT.xml; Size: 11,166 bytes; and Date of Creation: June 26, 2025) is herein incorporated by reference in its entirety. Field of invention The present invention relates to the manufacture of recombinant proteins, particularly antibodies. More specifically, it relates to cell culture methods in a bioreactor which method increases the yield and quality during commercial scale manufacturing. Background Development of recombinant proteins as therapeutic proteins, such as therapeutic antibodies, requires production of the recombinant proteins at an industrial scale. In order to achieve this, different expression systems, both prokaryotic and eukaryotic systems, may be employed. Over the past two decades, however, the majority of the therapeutic proteins approved as therapeutic have been manufactured through mammalian cell cultures and such system remains the preferred expression system for producing large quantity of recombinant polypeptides for human use. Much effort is dedicated to establishing the optimal parameters of cell culture and recombinant expression to reach optimal cell growth through changes of the composition of the cell culture media and operating conditions and, development of large bioreactors. Bioreactor conditions, such as the composition of the medium and the growing conditions, including pH and temperature have been shown to impact the quality and yield of therapeutic proteins. However, each therapeutic protein requires dedicated work to find methods and conditions for the production of therapeutic proteins, and, in particular, therapeutic antibodies with minimal heterogeneity. The present disclosure is related to a method of improving the yield for a therapeutic antibody (Eptinezumab) used in the treatment of headache disorders such as migraine.1295-US-PSP Summary of the invention The present invention relates to a method for producing Eptinezumab comprising culturing mammalian cells in a bioreactor under conditions that comprises a temperature between 33oC to 38oC . The invention further relates to a pharmaceutical composition comprising Eptinezumab obtained by the method according to the invention. Brief description of the drawings Fig 1 shows the normalized titer (mg / L) in relation to the pH setting for Eptinezumab producing CHO cells in a bioreactor as described in Example 1. The pH optimum was determined to be 6.95. Normalized Harvest Titers by pH Setpoint. Harvest titers were normalized and plotted on y-axis. The pH settings used during the production run are shown on the X-axis. Data was plotted using JMP17 software (closed circles) and a quadratic line of fit added shown by solid blue line with 95% confidence interval shown by shaded area. Fig 2 shows Integral Viable Cell Density (IVCD) in relation to the pH setting for Eptinezumab producing CHO cells in a bioreactor as described in Example 1. The peak IVCD values were observed when the pH set point was between 6.85 to 6.95. Harvest IVCD values (x 1 million viable cell / mL hours) are plotted on y-axis by pH setting on X-axis. Data was plotted using JMP17 software (closed circles) and a quadratic line of fit added shown by solid blue line with 95% confidence interval shown by shaded area. Fig 3 shows the basic peak values as measured by Capillary Isoelectric Focusing (cIEF) assay. The basic peak values increased with pH above and below a pH setpoint of 6.95. Basic peaks analyzed by cIEF plotted by pH setting. One-step Protein A purified pre-harvest samples were analyzed by cIEF and Basic peak % is plotted on y-axis by pH setting on the x-axis. Data was plotted using JMP17 software (closed circles) and a quadratic line of fit added shown by solid blue line with 95% confidence interval shown by shaded area. Fig 4 shows the acidic peak values as measured by cIEF assay. The acidic species increase linearly, and main peak species decreased linearly with increasing pH. Acidic peaks analyzed by Capillary Isoelectric Focusing (cIEF) plotted by pH setting. One-step Protein A purified pre-harvest samples were analyzed by cIEF and Acidic peak % is plotted on y-axis by pH setting on the x-axis. Data was plotted using JMP17 software (closed circles) and a1295-US-PSP linear line of fit added shown by solid blue line with 95% confidence interval shown by shaded area. Fig 5 shows pH setting values and the corresponding titer which has been normalized. Fig 6 shows the DOE design for Example 2. Ten Factor Definitive Screening Design (DSD) Design of Experiment (DOE). The first column shows the bioreactor reference number. The remaining columns identify the ten parameters studied within the DOE. All other parameters were kept constant. Fig 7 shows the cIEF results by Temperature shift (oC) and shift day. Temperature Shift (oC) and Timing of Temperature Shift Effect on cIEF results. Post-shift temperature setting is shown on the bottom x-axis. Timing of Temperature shift is shown on upper X-axis (Day 5 shown in far-left panel, Day 7 shown in middle panel, and Day 9 shown in far right panel). Pre-Harvest samples sent for one-step Protein A purification and then submitted for cIEF analysis are shown on y-axis (Basic peak % top, Main peak % middle, Acidic peak % bottom). Detailed description of the invention The present disclosure provides method of improving the yield of a Eptinezumab by CHO cells, comprising culturing the CHO cells in a bioreactor under suitable conditions, wherein the suitable conditions include, but are not limited to, adjustment of a temperature set point, adjustment of a second temperature set point, adjustment of pH or adjustment of a pH shift or adjustment of any combination thereof. The CHO cells can be selected from e.g. CHO-S, CHO-K1 cells, CHO-DXB11 cells, or CHO- DG44 cells. Preferably the CHO cell is CHO-DG44 and is used to express Eptinezumab in a fed-batch bioreactor under the conditions described herein. As used herein, "temperature set point" refers to the temperature setting of a bioreactor or other upstream processing vessel used to grow cells and / or produce Eptinezumab. A temperature set point can be established at the outset of cell culture in the production bioreactor, where it can also be referred to as an "initial temperature set point". Subsequent changes in temperature during cell culture after the initial temperature set point can be referred to using ordinal numbering, i.e., a second temperature set point, or subsequently a third temperature set point. In some instances, a process can comprise an initial temperature set point and a second temperature set point (and final) temperature set point.1295-US-PSP As used herein, Capillary Isoelectric Focusing (cIEF) refers to a method used for the separation of proteins by their isoelectric points (pls). Imaged capillary isoelectric focusing (cIEF) separates and quantifies charge variants of eptinezumab under non-denaturing conditions. The method provides information about the composition and isoelectric points of acidic peaks, main peak and basic peaks. The assay can be used to monitor various post- translational and chemical changes including: deamidation, C-terminal and N-terminal clipping, N-terminal cyclization, fragmentation / clipping, disulfide bonding, glycosylation, and oxidation. These variants are frequently found migrating in the same peaks due to opposing effects on the isoelectric point value for the whole molecule. Peaks migrating before the main peak are acidic charge variants, while peaks eluting after the main peak are basic species. In certain embodiments the invention relates to the CGRP binding antibody Eptinezumab with the following sequences Heavy Chain CDRs for Eptinezumab CDR-H1: GYYMN SEQ ID No.: 1 CDR-H2: VIGINGATYYASWAKG SEQ ID No.: 2 CDR-H3: GDI SEQ ID No.: 3 The Variable Heavy Chain of Eptinezumab Comprises EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYA SWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS SEQ ID No.: 4 The Heavy Chain of Eptinezumab comprises EVQLVESGGG LVQPGGSLRL SCAVSGIDLS GYYMNWVRQA PGKGLEWVGV IGINGATYYA SWAKGRFTIS RDNSKTTVYL QMNSLRAEDT AVYFCARGDI WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDARVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY ASTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG SEQ ID No.: 51295-US-PSP A C-terminal lysine (K) may be present in case the Heavy Chain is not processed in a system that cleave this, e.g. in a yeast such as Pichia, but the c-terminal lysine will usually be cleaved in a CHO expression system: EVQLVESGGG LVQPGGSLRL SCAVSGIDLS GYYMNWVRQA PGKGLEWVGV IGINGATYYA SWAKGRFTIS RDNSKTTVYL QMNSLRAEDT AVYFCARGDI WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDARVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY ASTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK SEQ ID No.: 6 Light Chain CDRs for Eptinezumab CDR-L1: QASQSVYHNTYLA SEQ ID No.: 7 CDR-L2: DASTLAS SEQ ID No.: 8 CDR-L3: LGSYDCTNGDCFV SEQ ID No.: 9 The Variable Light Chain of Eptinezumab comprises QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR SEQ ID No.: 10 The Light Chain of Eptinezumab comprises QVLTQSPSSL SASVGDRVTI NCQASQSVYH NTYLAWYQQK PGKVPKQLIY DASTLASGVP SRFSGSGSGT DFTLTISSLQ PEDVATYYCL GSYDCTNGDC FVFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID No.: 11 Temperature The temperature of a production vessel such as a bioreactor can be an important aspect of bioproduction because the temperature of the bioreactor plays a role in cell growth, viable cell density, cell longevity and / or glycosylation activity of glycosylating enzymes inside a cell. Temperature changes can significantly affect the rate of enzymatic reactions within the cell, denature proteins, and / or cause other effects on a cell culture. Cells can be cultured at an initial temperature set point such as 37°C, for example, to encourage maximum viable cell1295-US-PSP density, and then the temperature can be modified to another temperature set point (i.e., a second temperature set point or a final temperature set point) to prolong cell longevity or to enhance desired production within the cell. One or more temperature set points can be used during the various phases of an upstream production process to improve the overall cell density, protein yield, or protein glycosylation profile of a protein of interest. In some aspects, the methods are directed to one or more temperature adjustments during protein production. Temperature adjustments can be a decrease of operating temperature during a manufacturing process. Temperature adjustments can also be an increase of operating temperature during a manufacturing process. In some aspects, the methods of the present disclosure use at least one or at least two temperature adjustments during a manufacturing process. The methods of the present disclosure are also related to controlling cell growth rate, cell viability, viable cell density and / or titer of cells for producing a protein. The initial temperature set point is important for creating reactor conditions conducive to cellular expansion and growth of the cells during the log growth phase. After the initial log phase, a second temperature set point that is lower than the initial set point is used to reduce the cellular expansion conditions to prevent overgrowth of the cell culture, which would lead to undesirable cell densities and a subsequent loss in total cell viability. In some aspects, the methods of the present disclosure involve culturing the cells in a bioreactor for an induction phase under an initial temperature set point of 36.5°C, subsequently culturing the cells in a second temperature set point of 34.5°C to finally culturing the cells. In some aspects, the methods of the present disclosure use at least two temperature set points, e.g., an initial temperature set point and a final set point. In some aspects, the initial temperature set point for the present method is between 35°C and 38 °C and a second temperature set point is lower than about 35°C. In some aspects, the initial temperature set point for the present method is between 35.5°C and 37.5 °C and a second temperature set point is lower than about 35°C, e.g., about 35°C or about 34°C. In some aspects, the initial temperature set point for the present method is 36.5 °C and a second temperature set point is 34.5°C.1295-US-PSP In some aspects, the present methods improve the Eptinezumab yield (e.g. as measured by the titer or IVCD) by at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, or at least about 10 fold higher than a method without the adjustment of the initial and the second temperature set points as described herein above. In some aspects the IVCD obtained is between 3000 and 4000 (1e6 viable cells / ml*hour) with the adjustment of the initial and the second temperature set points as described herein above. The timing of the temperature conditions may be such that the second temperature set point as described herein above occurs on or after day 5, day 6, day 7, day 8 or day 9 (day 5 to day 9) or after 156 to 180 hours. In some aspects the second temperature set point as described herein above occurs at day 7 or at 168 hours. pH one aspect, the methods of the present disclosure involve improving or controlling protein by controlling the pH of the process. The regulation of intracellular pH is a fundamental physiological process of great significance to the growth and metabolism of cells. Since intracellular pH has wide ranging consequences for the transport of nutrients and hormones, and for enzymatic reactions in the cells, cells devote a lot of energy to the regulation of cytoplasmic pH. Furthermore, pH plays a role in the glycosylation rates and profiles of protein produced by the cell. In some aspects, the pH set point is about pH 7.0. In some aspects, the pH is between pH 6.95 and pH 7.05 In some aspects, the pH set point is 6.95. Combination of Conditions In some aspects, the methods of the present disclosure comprise any combination of the above listed conditions.1295-US-PSP In some aspects, the methods comprise two or more conditions selected from the group consisting of: (i) an initial temperature set point between about 35°C and about 38°C, e.g., about 36.5°C, a second temperature set point about 34°C or 35°C, e.g., 34.5°C, (ii) a pH set point of about pH 7, e.g. between pH 6.95 and pH 7.05 or pH 6.95. In some aspects the IVCD obtained is between 3000 and 4000 (x1e6 viable cells / ml*hour) with the adjustment of the initial and the second temperature set points and the pH as described herein above. In some aspects, the methods comprise (i) initial temperature set point for the present method is between 35.5°C and 37.5 °C, e.g.36.5°C and a second temperature set point is lower than about 35°C, e.g.34.5°C and (ii) a pH set point of about pH 7, e.g. between pH 6.95 and pH 7.05 or pH 6.95. The timing of the temperature conditions may be such that the second temperature set point as described herein above occurs after day 5, day 6 day 7 or day 9 day (day 5 to day 9) or after 156 to 180 hours. In some aspect the second temperature set point as described herein above occurs at day 7 or at 168 hours. In some aspects, the methods comprise (i) an initial temperature set point of 36.5°C and a second temperature set point of 34.5 °C (ii) a pH set point of 6.95 and (ii) IVCD between 3000 and 4000 (1e6 viable cells / ml*hour). RESULTS The methods of the present disclosure are also useful for improving or controlling the growth of the cells during production as indicated by the mean harvest integral viable cell density (IVCD). The IVCD measure is an alternate way of measuring overall viable cell growth of an upstream production process, as opposed to the instantaneous measure of viable cell density. Since each cell has a variable protein production lifetime and various cell culture conditions affect viable cell rates over time, the IVCD is useful to estimate the total viable working cell hours during production phase of the process. In some aspects, the process exhibits a mean harvest integral viable cell density (IVCD) of between 3000 and 4000 (1e6 viable cells / ml*hour).1295-US-PSP The precent disclosure relates to a method for increasing the growth of cells, comprising culturing the cells in a bioreactor under suitable conditions, wherein the suitable conditions comprise an initial temperature set point of 36°C, a second temperature set point of 33°C, and a third temperature set point of 31°C. The initial pH set point is at 7.0 and may be adjusted (a second pH set point) to 6.9, when adjusting to the second temperature set point. The present disclosure is also related to a method of improving the yield of a protein by cells, comprising culturing the cells in a bioreactor under suitable conditions, wherein the suitable conditions comprise (i) an initial temperature set point of 36.5°C and a second (final) temperature set point 34.5°C, and a pH set point of about pH 7, e.g. between pH 6.95 and pH 7.05 or pH 6.95. In some aspects, the second (final) temperature set point occurs at about 156 to about 180 hours. In some aspects, the second (final) final temperature set point occurs at about 165 hours, about 166 hours, about 167 hours, about 168 hours, about 169 or about 170 hours after the initial temperature set point. In some aspects, the second temperature set point occurs at about 156 hours to about 180 hours. In some aspects, the second temperature set point occurs at about 150 hours, about 160 hours, about 170 hours, about 180 hours, about 190 hours, or about 200 hours, about 156 hours, about 162 hours, or about 168 hours. In some aspects, the second (final) temperature set point is 34.5°C and occurs after 168 hours.1295-US-PSP Examples Example 1. Identification of optimal pH setting for CHO Vyepti production. This experiment investigated the optimal pH setting for CHO Vyepti production yields, within the pH range of 6.75 to 7.10 over two run sets. Materials and Methods Cells and Medium Eptinezumab was produced in a fed-batch process using a suspension-adapted Chinese hamster ovary (CHO) cell line. The source was the working cell bank (WCB), which is derived from the master cell bank (MCB). For each production run set, a WCB vial was thawed, and contents transferred to a shake flask. The shake flask culture was expanded over several passages until sufficient viable cell mass was generated to inoculate the subsequent production bioreactors at the required initial viable cell density and working volume. Culture from the seed expansion stage was transferred to the production bioreactor where it was initially further expanded, followed by fed-batch production of eptinezumab (which is secreted into the culture fluid) where it was maintained until the end of the process, approximately at Day 14. Chemically defined media was used for seed expansion, production growth media, and starting on day 3 two nutrient feeds were bloused every other day for a total of 6 bolus additions each. Glucose and antifoam were supplemented as needed. All production reactor conditions in this pH study were equivalent except for starting pH setting. The pH deadband was set within a range of 0.02 to 0.05. If the online pH value was within the pH deadband of the pH setting no reactor intervention was required. If the online pH value was outside the pH setting + / - the pH deadband, then pH was adjusted by adding 1 M sodium hydroxide for base addition and CO2gas was sparged for acid addition. The pH settings tested were between 6.75 and 7.10. Daily samples were taken to monitor cell growth, viability, metabolites, and titer. A pre-harvest sample was taken and purified over a small-scale Protein A column and the neutralized eluate was submitted for product quality analyses including cIEF (Capillary Iso-Electric Focusing).1295-US-PSP Results The optimum pH setting for titer and product quality was determined to be 6.95. Maximum Integral Viable Cell Density (IVCD) values were observed when pH setpoint was 6.85 to 6.95 (Fig 2). Basic peak values measured by cIEF assay increased with pH setpoints below 6.95 (Fig 3). Acidic species increased linearly and main peak species from cIEF decreased linearly with increasing pH setpoint (Fig 4). Example 2. Identification of optimal temperature shift for CHO Vyepti production. A ten factor DSD (Definitive Screening Design) DOE (Fig 6) was conducted in ambr250 bioreactor vessels. a WCB vial was thawed, and contents transferred to a shake flask. The shake flask culture was expanded over several passages until sufficient viable cell mass was generated to inoculate the subsequent production bioreactors at the required initial viable cell density and working volume. Culture from the seed expansion stage was transferred to the production bioreactor where it was initially further expanded, followed by fed-batch production of Eptinezumab (which is secreted into the culture fluid) where it was maintained until the end of the process, approximately at Day 14. Chemically defined media was used for seed expansion, production growth media, and nutrient feeds. Nutrient feeds and Glucose were added according to DOE design and antifoam was supplemented as needed. All production reactor conditions in this DOE study were equivalent except as stated in Fig 3. The pH deadband was set within a range of 0.02. If the online pH value was within the pH deadband of the pH setting no reactor intervention was required. If the online pH value was outside the pH setting + / - the pH deadband, then pH was adjusted by adding 1 M sodium hydroxide for base addition and CO2gas was sparged for acid addition. The pH settings tested were between 6.75 and 7.15. Daily samples were taken to monitor cell growth, viability, metabolites, and titer. A pre-harvest sample was taken and purified over a small-scale Protein A column and the neutralized eluate was submitted for product quality analyses including cIEF (Capillary Iso-Electric Focusing). Performing a temperature shift on day 7 at 34.5oC has been shown to produce acceptable product quality by cIEF acidic species.

Claims

1295-US-PSP Claims 1. A method for producing Eptinezumab comprising culturing mammalian cells in a bioreactor under conditions that comprise a temperature between 33oC to 38oC .

2. The method according to claim 1, wherein the pH is about 7 (such as between 6.85 and 7.05).

3. The method according to any of the preceding claims, wherein the pH is kept at about 7 for up to 14 days.

4. The method according to any of the preceding claims, wherein the pH is 6.

95.

5. The method according to any of the preceding claims, wherein the bioreactor is a fed-batch bioreactor.

6. The method according to any of the preceding claims, wherein the temperature conditions comprise an initial temperature setpoint between 35oC to 38oC and a second temperature set point 34.5oC occurring between day 5 and 9.

7. The method according to any of the preceding claims, wherein the temperature conditions comprise an initial temperature setpoint between 35oC to 38oC and a second temperature set point 34.5oC at day 7.

8. The method according to any of the preceding claims, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

9. The method according to any of the preceding claims, wherein the cells are CHO-S, CHO-K1 cells, CHO-DXB11 cells, or CHO-DG44 cells.

10. The method according to any of the preceding claims, wherein the method controls the cell growth rate, cell viability, viable cell density and or / titer of mammalian cells.

11. The method according to any of the preceding claims wherein the integral viable cell density is between 3000 and 4000 (1e6 viable cells / ml*hour).

12. A pharmaceutical composition comprising Eptinezumab obtained by the method according to any one or all of claims 1-10.

13. The method or the pharmaceutical composition according to any one of the previous claims, wherein the six CDRs of Eptinezumab comprise or consist of the following CDR sequences: Light Chain CDR 1 SEQ ID NO.: 7, Light Chain CDR 2 SEQ ID NO.: 8, Light Chain CDR 3 SEQ ID NO.:9, Heavy Chain CDR 1 SEQ ID NO.:1, Heavy Chain CDR 2 SEQ ID NO.:2, and Heavy Chain CDR 3 SEQ ID NO.:

3.

14. The method or the pharmaceutical composition according to any one of the previous claims wherein Eptinezumab has a VH region having the amino acid sequence of SEQ ID NO.: 4 and a VL region having the amino acid sequence of SEQ ID NO.: 10.1295-US-PSP 15. The method or the pharmaceutical composition according to any one of the previous claims wherein Eptinezumab comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 5 or SEQ ID NO.: 6 and a light chain having the amino acid sequence of SEQ ID NO.:

11.

16. The method or the pharmaceutical composition according to any one of the previous claims wherein the promoter used to express Eptinezumab by the mammalian cells comprises any promoter suitable for use in CHO cells, e.g., a constitutive or inducible promoter, optionally a mammalian or viral constitutive or inducible promoter, further optionally a temperature inducible or non-temperature inducible promoter, further optionally a promoter selected from a CHEF promoter, e.g., the CHEF-1α promoter derived from the human elongation factor-1α gene, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, EF-1alpha (Elongation Factor 1-alpha) promoter, CHEF1α (Chinese hamster elongation factor-1α) promoter, PGK (Phosphoglycerate Kinase) promoter, UBC (Ubiquitin C) promoter, hEF1α (Human Elongation Factor 1-alpha) promoter, an Endogenous CHO-K1 promoter optionally one or more of Actb, Ctsz, Hmox1, Hspa5, Vim, RegCG, and Rps18 promoters.

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