DESC:DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention provides a cell culture process for culturing mammalian cells expressing an antibody, the process comprising culturing the cells at a pH range of 6.6 to 7.5 by lowering of temperature of the cell culture from a first temperature to a second temperature, and supplementation of galactose to obtain antibody composition comprising galactosylated variants.
Any mammalian cell or cell type which is suitable for expression of recombinant proteins in a cell culture medium may be used for the present invention. Non-limiting examples of mammalian cells that may be used with the present invention include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells and murine myeloma cells (NS0 and Sp2/0) human retinoblasts (PER.C6 cell line), human embryonic kidney cell line (HEK-293 cell line) (Dumont, J., et al., Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol, 2016. 36(6): p. 1110-1122). In a preferred embodiment, CHO cell lines expressing recombinant proteins may be used in accordance with the present invention.
Certain glycosylation profile of the pharmaceutical monoclonal antibody (mAb) are desirable based on its mechanism of action as the glycosylation profile effects the stability, safety and efficacy of the antibody. In an embodiment, the cell culture process of the present invention may be used to produce an antibody composition comprising galactosylated glycoform and/or G0F glycoform at a target/predetermined range.
Cell culture medium is understood by those skilled in the art to refer to a nutrient solution in which cells, such as animal or mammalian cells, are grown. A cell culture medium generally includes one or more of the following components: an energy source (e.g., a carbohydrate such as glucose); amino acids; vitamins; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements in the micromolar range. Cell culture medium can also contain additional components, such as hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, and the like); salts (e.g., calcium, magnesium and phosphate); sugars (e.g. mannose, galactose, fucose); amino acids (glutamine); buffers (e.g., HEPES); nucleosides and bases (e.g., adenosine, thymidine, hypoxanthine); antibiotics (e.g., gentamycin); and cell protective agents (e.g., a Pluronic polyol (Pluronic F68). Commercially available media can be utilized in accordance with the present invention, for example, Dulbecco's Modified Eagles Medium (DMEM, Sigma-Aldrich); RPMI-1640 Medium (Sigma-Aldrich); EX-CELL® Advanced CHO Fed-batch Medium (Sigma-Aldrich); Cell Boost™ 7a and 7b (GE Healthcare Bio-Sciences AB). One skilled in the art would appreciate that some cell culture media are suited to support cells through their initial growth phase (basal medium) while some sustain cells through the later growth phase and production phase of cell culture (feed medium), and would be able to choose appropriate culture medium.
The methods described in the present invention are in recognition of the fact that various parameters of the cell culture process may be used to obtain antibody composition of desired glycosylation profile. In an embodiment, the cell culture process envisages culturing the cells at a pH range of about 6.6 to about 7.5, performing a temperature shift and supplementation of galactose.
A person of ordinary skill in the art would be able to characterize and analyse the various antibody variants present in the antibody composition produced by the cell culture process described herein using the state of the art techniques.
In an embodiment, the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises
a) providing mammalian cells expressing the said antibody
b) culturing the cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature
d) supplementing culture media with galactose
e) recovering the said recombinant antibody composition
wherein the antibody composition produced comprises of galactosylated glycoform and/or G0F glycoform, and wherein the percentage of galactosylated glycoform in the antibody composition decreases with the decrease in the difference between the first culture temperature and the second culture temperature.
In an embodiment, the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises
a) providing mammalian cells expressing the said antibody
b) culturing cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature
d) supplementing culture media with galactose
e) recovering the said recombinant antibody composition
wherein the antibody composition produced comprises of galactosylated glycoform and/or G0F glycoform, and wherein the percentage of galactosylated glycoform in the antibody composition decreases with the decrease in the difference between the first temperature and the second temperature.
In another embodiment, the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises
a) providing/culturing the mammalian cell expressing the said antibody
b) culturing the cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature
d) supplementing culture media galactose
e) recovering the said recombinant antibody composition
wherein the obtained antibody composition is comprised of about 41% to about 47% galactosylated glycoform and/or about 47% to about 52% G0F glycoform.
In an embodiment, the present invention discloses a cell culture process for producing a monoclonal antibody composition having target/predetermined levels of the reference product of the antibody variants, wherein the cell culture process comprises
a) providing/culturing the mammalian cell expressing the said antibody
b) culturing the cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature
d) supplementing the cell culture with galactose
e) recovering the said recombinant antibody composition
wherein the antibody composition produced comprises of galactosylated glycoform, and wherein the percentage of galactosylated glycoform in the antibody composition decreases with the decrease in the difference between the first temperature and the second temperature.
In an embodiment, the present invention discloses a cell culture process for producing a monoclonal antibody composition having target/predetermined levels of the reference product of the antibody variants, wherein the cell culture process comprises
a) providing/culturing the mammalian cell expressing the said antibody
b) culturing the cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature
d) supplementing the cell culture with galactose
e) recovering the said recombinant antibody composition
wherein the antibody variants in the said composition comprised of about 41% to about 47% galactosylated glycoform and/or about 47% to about 52% G0F glycoform.
In any of the embodiments mentioned above, the antibody composition further comprises of afucosylated variants, high mannose variants and total afucosylated variants.
In any of the embodiments mentioned above, the cells are cultured at a pH range of 6.7 to 7.2 till day 4, performing a shift in pH range whereby the cells are cultured thereafter at a pH range of 6.7 to 7.4
In any of the embodiments mentioned above, the temperature shift is performed after the aforementioned shift in pH range.
In any of the embodiments mentioned above, the temperature shift is performed on day 5 of the cell culture, day 6 of the cell culture, or day 7 of the cell culture. In yet another embodiment, the temperature shift is performed on day 6 of the cell culture.
In any of the embodiments mentioned above, the difference between the first culture temperature and the second culture temperature of the cell culture ranges from about 6.5 to 2.5. In another embodiment, the culture temperature before the temperature shift is about 37°C and the culture temperature after the temperature shift is about 30°C-35°C. In yet another embodiment, the culture temperature before the temperature shift is about 37°C and the culture temperature after the temperature shift is selected from about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C.
In any of the embodiments mentioned above, the cumulative galactose supplementation in the present invention is 6 g/L, wherein galactose is supplemented as 3 g/L each on day 7 and 10.
In yet another embodiment, the antibody produced using the present invention is an anti a4ß7 integrin antibody. In a preferred embodiment, the antibody produced using the present invention is vedolizumab.
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
Example I
An anti-a4ß7 integrin antibody having a heavy chain and light chain as described in WO2007061679A1 was cloned and expressed in a recombinant CHO (rCHO) cell line using techniques described in detail in “Molecular Cloning: A Laboratory Manual (Fourth Edition)”. rCHO cells expressing the antibody were seeded at a density of about 0.5 million cells/mL in basal cell culture medium and cultured at an initial temperature of 36.5°C. The pH range of the cell culture is maintained between pH 6.6 to 7.5. The cell culture medium was supplemented with glucose. On day 6, a temperature shift was performed, thereby reducing the culture temperature to 30°C. The cell culture was supplemented with 3 g/L galactose each on day 7 and day 10. Feed medium was added on day 3, 5, 7, 9 and 11. The culture was harvested on day 13. The antibody composition and titer obtained is depicted in Table 1.
Example II:
The cell culture process described in Example I was carried with following modifications. The temperature shift applied on day 6 reduced the culture temperature to 32°C. The antibody composition and titer obtained is depicted in Table 1.
Example III:
The cell culture process described in Example I was carried with following modifications. The temperature shift applied on day 6 reduced the culture temperature to 34°C. The antibody composition and titer obtained is depicted in Table 1.
Example IV:
The cell culture process described in Example I was carried with following modifications. The cells were cultured at a pH 6.7 to 7.2 till day 4 and at pH 6.7 to 7.4 thereafter. The temperature shift applied on day 6 reduced the culture temperature to 32°C. The antibody composition and titer obtained is depicted in Table 1.
Table 1. The composition and titer of the anti-a4ß7 integrin antibody.
Examples GAL (%) G0F (%) Titer (g/L)
I 46.9 47.50 3.6
II 43.89 49.75 4.1
III 41.58 51.57 4.2
IV 43.5 48.9 3.7
,CLAIMS:We claim
1. A cell culture process for producing an antibody composition comprising galactosylated and G0F glycoforms, wherein the said process comprises
a) providing mammalian cells expressing the said antibody
b) culturing cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is about 2.5°C to 6.5°C lower than the first culture temperature
d) supplementing culture media with galactose
e) recovering the said recombinant antibody composition
wherein the percentage of galactosylated glycoform in the antibody composition decreases with the decrease in the difference between the first culture temperature and the second culture temperature.
2. A cell culture process of claim 1, wherein the antibody composition obtained comprises of about 41% to about 47% galactosylated glycoform and about 47% to about 52% G0F glycoform.
3. A cell culture process for producing a monoclonal antibody composition having target/predetermined levels of the reference product of the antibody variants, wherein the cell culture process comprises
a) providing the mammalian cell expressing the said antibody
b) culturing the cells at a pH range of about 6.6 to about 7.5
c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature
d) supplementing the cell culture with galactose
e) recovering the said recombinant antibody composition
wherein the antibody variants in the said composition comprises of about 41% to about 47% galactosylated glycoform and about 47% to about 52% G0F glycoform.
4. The cell culture process of claim 3, wherein the difference between the first culture temperature and the second culture temperature of the cell culture ranges from about 6.5°C to 2.5°C.
5. The cell culture process of claims 1 - 3, wherein the first culture temperature before the temperature shift is about 37°C and the second culture temperature after the temperature shift is selected from a range of about 30°C to about 35°C.
6. The cell culture process of claims 1 - 3, wherein the first culture temperature before the temperature shift is about 36.5°C and the second culture temperature after the temperature shift is selected from about 30°C, about 32°C, about 34°C.
7. A cell culture process of any of the preceding claims, wherein the cumulative galactose supplemented is about 6 g/L.
8. A cell culture process of any of the preceding claims, wherein the antibody composition obtained is an anti a4ß7 integrin antibody composition, vedolizumab.
9. A cell culture process of any of the preceding claims wherein the antibody composition obtained is an anti a4ß7 integrin antibody, vedolizumab.