DESC:A CELL CULTURE PROCESS TO PRODUCE AN ANTIBODY COMPOSITION
FIELD OF INVENTION
The present invention relates to a cell culture process for producing recombinant biotherapeutics in mammalian cell culture.
BACKGROUND OF THE INVENTION
Recombinant therapeutic antibodies are among the fastest growing biotherapeutics used for the treatment of various malignancies and diseases. They are usually produced using mammalian cells, and Chinese Hamster Ovary (CHO) cell line is usually preferred for the production of most recombinant therapeutic antibodies as these cells are not only well adapted as suspension culture but also have post-translational modification (PTM) machinery, thereby producing antibodies that are biocompatible to humans.
Among the various PTMs, glycosylation is one of the major quality attributes of the therapeutic antibodies and it plays a vital role in their biological activity via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). (Zimmermann et. al. Antibodies 2019, 8, 9; Radhakrishnan et. al. Antibodies 2018, 7, 1; Zeng et. al. mAbs 2011 3:6, 568-576). Therefore, presence or absence of certain oligosaccharides can impact the effector functions of the antibodies. For example, presence of terminal galactose enhances the effect of CDC. Also, fucose bound to the antibody reduces the ADCC since it weakens the binding of the antibodies to the Fc?III receptors on the natural killer cells. Afucosylated therapeutic antibodies are preferred for improved therapeutic antibodies which have ADCC as its mechanism of action. Therefore, various strategies have been tried in order to modulate the glycosylation profile of therapeutic antibodies. These strategies include varying cell culture parameters such as dissolved oxygen concentration, partial pressure of carbon dioxide, temperature, pH, osmolality, manganese concentration, carbon sources etc. (Ivarson et. al. Journal of Biotechnology 188, 2014, 88–96; Mori et. al. Cytotechnology 2007, 55:109–114; Zhang et. al. Metabolic Engineering 65, 2021, 135–145). In studies with a chimeric antibody undertaken by Kim et al., it was found that lowering the culture pH and temperature enhanced the antibody galactosylation and sialylation (Biotechnology and Bioprocess Engineering 2018, 23: 456-464). Sou et. al. reported that lowering of temperature of IgG-producing CHO cell culture led to an increase in antibody titer and G0, G0F glycoforms, while the G1F and G2F glycoforms decreased (Biotechnology and Bioengineering, 2014, 112:6 1165-1176).
Rituximab, an anti-CD20 monoclonal antibody, is marketed for the treatment of various diseases including Non-Hodgkin’s Lymphoma (NHL) and Rheumatoid Arthritis (RA). Studies have shown that afucosylated rituximab exhibits enhanced ADCC compared to fucosylated rituximab. (Li et. al. 2017, PNAS, 114 (13) 3485-3490; Pereira et. al., 2018, MABS, 10:5, 693–711). WO2013114164A1 discloses that lowering of cell culture temperature combined with an increase in pH increases the afucosylated variants in a rituximab composition. A cell culture process using a temperature shift to lower values so as to obtain rituximab composition having target levels of high mannose, afucosylated and G0F glycans has been disclosed in WO2013114240A2.
‘Biosimilars’, sometimes called ‘similar biological medicinal product’ or ‘follow-on biologic’ or ‘subsequent entry biologic’, are therapeutic antibody products which are similar to already licensed therapeutic antibody product (the ‘reference product’). For a therapeutic antibody to get approved as a biosimilar, regulatory agencies mandate that it should have no clinically meaningful differences from the reference product (Sulivan et. al. Am J Health-Syst Pharm 2017, 74:568-79). Hence, in order to be designated as a biosimilar, the therapeutic antibody is required to have target/predetermined glycosylation profile as that of the reference product. Thus, the modulation of cell culture conditions, process parameters and media supplement is an important tool employed to achieve the target/predetermined glycosylation profile for the therapeutic antibody.
The present invention relates to a cell culture process for production of therapeutic monoclonal antibodies, the process comprising reverse temperature shift and to obtain an antibody composition having a target/predetermined glycosylation profile. In particular, the present invention discloses a cell culture process comprising a reverse temperature shift to obtain an antibody composition comprising a low % total afucosylated (TAF) glycans and high % galactosylated (Gal) glycans as compared to the antibody compositions obtained from cell cultures with normal temperature shift.
SUMMARY OF THE INVENTION
The present invention relates to cell culture process for culturing mammalian cell producing a recombinant therapeutic antibody. The invention provides mammalian cell culture process, comprising reverse temperature shift, for producing antibody compositions having a target/predetermined glycosylation profile. In particular, the invention provides a cell culture process comprising reverse temperature shift to obtain an antibody composition from mammalian cell culture, wherein the composition comprises low % total afucosylated glycans and high % galactosylated glycans as compared to antibody composition obtained by the same process but with normal temperature shift.
DESCRIPTION OF THE INVENTION
Definitions
The term “about” refers to a range of values that are similar to the stated reference value to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value.
The term “afucosylated glycans” refers to antibodies wherein fucose is not linked to the non reducing end of N-acetlyglucosamine and includes M3NAG, G0, G1A, G1B and G2. Further, the term “total afucosylated glycans” refers to glycans wherein fucose is not linked to the non-reducing end of N-acetlyglucosamine and includes mannose glycans. Without limitation, examples of total afucosylated glycans include G0, G1A, G1B, G2, M3-M9NAG, M3-M9 (see Table 1).
The term “antibody” or “monoclonal antibody” refers to an intact antibody or an antigen binding fragment thereof.
The term “antibody composition” refers to a population of antibody molecules or fragments thereof that is produced by mammalian cell culture. The population of antibody molecules may have one or several post translational modifications (PTM), imparting the antibody molecules a different molecular weight, charge, solubility or combinations thereof.
The term "biosimilar" refers to a recombinant protein, commonly with identical amino acid sequence to a reference product that contains, similar, very similar to or same post-translational modifications as the reference product yielding no clinically meaningful difference in terms of safety, purity and potency.
The terms “cell culture medium”, “culture medium”, "media", "medium", as used herein refer to a solution containing nutrients which are required to support the growth of the cells in cell culture.
The term “cell culture process” as used herein refers to a process of culturing a population of cells that are capable of producing recombinant protein of interest or antibody.
The term “G0F glycans” refers to antibodies with fucose linked to the non-reducing end of N-acetlyglucosamine, and does not contain any terminal galactose residues (see Table 1).
The term “galactosylated glycans” refer to antibodies containing terminal galactose residues such as G1A, G1B, G1AF, G1BF, G2, G2F and G2SF (see Table 1).
The term “high mannose glycans” refers to antibodies containing unsubstituted terminal mannose sugars (see Table 1). High mannose glycans contain more than 4 mannose residues attached to the GlcNAc2 core.
The term “IVCD” or “Integral viable cell density” refers to cell growth over time or integral of viable cells with respect to culture time that is used for calibration of specific protein production. The integral of viable cell density can be increased either by increasing the viable cell concentration or by lengthening the process time.
The term “normal temperature shift” refers to a temperature shift wherein the cells are cultured at a first temperature for a certain period of time and are shifted to a second temperature, wherein the second temperature is lower than the first temperature.
The term "reference product" refers to a currently or previously marketed recombinant protein, also described as the "originator product" or "branded product" serving as a comparator in the studies.
The term “reverse temperature shift” refers to a temperature shift wherein the cells are cultured at a first temperature for a certain period of time and are shifted to a second temperature, wherein the second temperature is higher than the first temperature.
The term “target/predetermined glycosylation profile” refers to the glycosylation profile of the ‘reference product’.
The term “temperature shift” refers to the change in temperature applied during the cell culture process in order to produce the therapeutic antibody.
Table 1: Representative table of various antibody glycans.


DESCRIPTION OF THE EMBODIMENTS
The present invention provides a mammalian cell culture process for producing antibody compositions that comprise various glycan variants of the antibody expressed in the cells. In particular, the present invention provides a mammalian cell culture process for producing antibody compositions comprised of target/predetermined glycosylation profiles defined in terms of levels of mannose glycans, afucosylated glycans and G0F glycans. The present invention provides mammalian cell culture process comprising a temperature shift for producing antibody composition having target/predetermined glycosylation profile. In an embodiment, the cell culture process of the present invention would comprise more than one temperature shift, wherein the individual temperature shift might result in subsequent lower temperature or higher temperature. In particular, the invention provides a cell culture process comprising a temperature shift to obtain an antibody composition from mammalian cell culture, wherein the composition comprises high mannose glycans, afucosylated glycans and G0F glycans. In a preferred embodiment, the invention discloses a cell culture process comprising a single temperature shift wherein, preferably, the temperature shift of the present invention is a reverse temperature shift and allows production of antibody composition having the target/predetermined glycosylation profile.
In an embodiment the present invention provides a mammalian cell culture process for producing antibody composition having target/predetermined glycosylation profile, the process comprising a temperature shift, wherein the temperature shift is carried out on attainment of a particular IVCD by the cell culture.
In an embodiment, the present invention discloses a cell culture process for the production of a biosimilar recombinant monoclonal antibody composition, wherein the cell culture process comprises a temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a second temperature, when the cell culture attains a particular IVCD,
c) wherein the temperature shift is a reverse temperature shift,
d) recovering the recombinant antibody
thereby obtaining an antibody composition that has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody.
In an embodiment, the present invention discloses a cell culture process for the production of a biosimilar recombinant monoclonal antibody composition, wherein the cell culture process comprises a temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a second temperature, when the cell culture attains IVCD of 6.6 million cell/mL,
c) wherein the temperature shift is a reverse temperature shift,
d) recovering the recombinant antibody
wherein the antibody composition so obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody.
In an embodiment, the present invention discloses a cell culture process for producing a biosimilar monoclonal antibody, the cell culture process comprising a reverse temperature shift, wherein
a) the cell culture is performed at a temperature of about 35oC for a first period of time,
b) subjecting the cell culture to a second temperature of about 37oC, when the cell culture attains IVCD of 6.6 million cell/mL,
d) recovering the recombinant antibody
thereby obtaining a monoclonal antibody composition with a target/predetermined glycosylation profile defined in terms of levels of high mannose glycans, afucosylated glycans and G0F glycans.
In an embodiment, the present invention discloses a cell culture process for the production of a recombinant antibody composition, wherein the cell culture process comprises a temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a temperature shift when the cell culture attains a particular IVCD,
c) wherein the temperature shift is a reverse temperature shift,
d) recovering the recombinant antibody,
so as to obtain an antibody composition comprising low % total afucosylated glycans and high % galactosylated glycans as compared to an antibody composition obtained by the same process but with normal temperature shift.
In an embodiment, the present invention discloses a cell culture process for the production of a recombinant antibody composition, wherein the cell culture process comprises a temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a second temperature when the cell culture attains IVCD of 6.6 million cell/mL,
c) wherein the temperature shift is a reverse temperature shift,
d) recovering the recombinant antibody
wherein the antibody composition so obtained is comprised of low % total afucosylated glycans and high % galactosylated glycans as compared to antibody composition obtained by the same process but with normal temperature shift.
In an embodiment, the present invention discloses a cell culture process for the production of a recombinant antibody composition, wherein the cell culture process comprises a reverse temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a temperature shift, when the cell culture attains IVCD of 6.6 million cell/mL,
c) wherein the increase in temperature is about 1oC to about 2oC
d) recovering the recombinant antibody
thereby obtaining an antibody composition comprising low % total afucosylated glycans and high % galactosylated glycans as compared to an antibody composition obtained by the same process but with normal temperature shift.
In a further embodiment, the present invention discloses a cell culture process comprising a reverse temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a second temperature, when the cell culture attains IVCD of 6.6 million cell/mL,
c) wherein the second temperature is higher than the first temperature and the increase in temperature is about 1oC to about 2oC
d) recovering the recombinant antibody
thereby obtaining an antibody composition comprising about 3.0 % total afucosylated glycans and about 53 % galactosylated glycans.
In an embodiment, the present invention discloses a cell culture process comprising a reverse temperature shift, wherein
a) the cell culture is performed at a first temperature of about 35oC for a first period of time,
b) subjecting the cell culture to a second temperature of about 37oC, when the cell culture attains a particular IVCD ,
c) recovering the recombinant antibody
thereby obtaining an antibody composition comprising low % total afucosylated glycans and high % galactosylated glycans as compared to antibody composition obtained by the same process but with normal temperature shift.
In an embodiment, the present invention discloses a cell culture process comprising a reverse temperature shift, wherein
a) the cell culture is performed at a first temperature of about 35oC for a first period of time,
b) subjecting the cell culture to a second temperature of about 37oC, when the cell culture attains IVCD of 6.6 million cell/mL,
c) recovering the recombinant antibody
thereby obtaining an antibody composition comprising low % total afucosylated glycans and high % galactosylated glycans as compared to antibody composition obtained by the same process but with normal temperature shift.
In an embodiment, the present invention discloses a cell culture process comprising a reverse temperature shift, wherein
a) the cell culture is performed at a first temperature of about 35oC for a first period of time,
b) subjecting the cell culture to a second temperature of about 37oC, when the cell culture attains a particular IVCD,
c) recovering the recombinant antibody
thereby obtaining an antibody composition comprising about 3.0 % total afucosylated glycans and about 53 % galactosylated glycans.
In an embodiment, the present invention discloses a cell culture process comprising a reverse temperature shift, wherein
a) the cell culture is performed at a first temperature of about 35oC for a first period of time,
b) subjecting the cell culture to a second temperature of about 37oC, when the cell culture attains IVCD of 6.6 million cell/mL,
c) recovering the recombinant antibody
thereby obtaining an antibody composition comprising about 3.0 % total afucosylated glycans and about 53 % galactosylated glycans.
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: Impact of normal temperature shift on % TAF glycans and % galactosylated glycans.
An anti-CD20 antibody 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 0.65 million cells/mL in basal cell culture medium. The medium is further supplemented with 6 g/L galactose. The cell culture is initiated at 37o C and pH 7.05 ± 0.02. When the cell culture attains an IVCD of about 4.6 million cells/mL, the cell culture temperature was lowered to 35o C and feed was added simultaneously. The cells were harvested at day 10. The % total afucosylated glycans, % galactosylated glycans and the titre of the antibody compositions so produced are given in table 2.
Example II: Impact of reverse temperature shift on % TAF glycans and % galactosylated glycans.
The cell culture process was carried out as described in Example I with following modifications. The cell culture was initiated at 35.8oC and pH 7.04 ± 0.02. The cell culture temperature was increased to 37oC when cell culture attains an IVCD of about 6.6 million cell/mL instead of lowering the cell culture temperature. The % total afucosylated glycans, % galactosylated glycans and the titre of the antibody compositions so produced are given in table 2.
Example III: Impact of reverse temperature shift on % TAF glycans and % galactosylated glycans.
The cell culture process was carried out as described in Example I with following modifications. The cell culture was initiated at 35.2oC and pH 7.04 ± 0.02. The cell culture temperature was increased to 37oC when the cell culture attains an IVCD of about 6.6 million cell/mL. The % total afucosylated glycans, % galactosylated glycans and the titre of the antibody compositions so produced are given in table 2.
Table 2. The impact of temperature shift during the cell culture on the % total afucosylated glycans and % galactosylated glycans
Examples Temperature shift TAF (%) GAL (%) Titre (g/L)
Example I
n=29 37o C to 35 o C 4.0±0.4 49±1 1.2± 0.1
Example II
n=18 35.8o C to 37o C 3.0±0.4 * 53 ±1* 1.0± 0.1*
Example III
n=06 35.2oC to 37o C 3.6±0.2** 53 ±1** 1.0± 0.1**
* p<0.001, between example I and example II
** p<0.001, between example I and example III

Date: Signature: ______________
V.R. Srinivas, Ph.D., LL.B
Head – IPM, Biologics
For, Dr. Reddy’s Laboratories Limited
,CLAIMS:We claim:
1. A cell culture process for the production of a biosimilar recombinant monoclonal antibody composition in mammalian cells, wherein the cell culture process comprises a temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a second temperature, when the cell culture attains IVCD of 6.6 million cell/mL,
c) wherein the temperature shift is a reverse temperature shift,
d) recovering the recombinant antibody
wherein the antibody composition so obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody.
2. A cell culture process for the production of a recombinant antibody composition in mammalian cells, wherein the cell culture process comprises a temperature shift, wherein
a) the cell culture is performed at a first temperature for a first period of time,
b) subjecting the cell culture to a temperature shift when the cell culture attains IVCD of 6.6 million cell/mL,
c) wherein the temperature shift is a reverse temperature shift,
d) recovering the recombinant antibody,
wherein the antibody composition so obtained comprises low % total afucosylated glycans and high % galactosylated glycans as compared to an antibody composition obtained by the same process but with normal temperature shift.
3. The cell culture process as claimed in the preceding claims, wherein the reverse temperature shift is the shift in the culture temperature from a first temperature to a second temperature, wherein the second temperature is higher than the first temperature and the increase in temperature is about 1oC to about 2oC.
4. The cell culture process as claimed in the preceding claims, the temperature shift is from about 35°C to 37°C.
5. The cell culture process as claimed in claims 1 and 2, wherein the obtained antibody composition comprises about 3.0 % total afucosylated glycans and about 53 % galactosylated glycans.
6. The antibody as claimed in the preceding claims is anti-CD20 antibody.
7. The mammalian cells used in the claimed cell culture is recombinant Chinese hamster ovary (CHO) cells.