DESC:FIELD OF THE INVENTION

The present invention pertains to a cell culture medium comprising addition of lactate and methods of using thereof. The present invention further pertains to a method of producing a protein of interest in a large scale cell culture, comprising supplementing the cell culture with lactate.

BACKGROUND OF THE INVENTION

Over the last few decades, much research has focused on the production of recombinant proteins, e.g., monoclonal antibodies, and the work has taken a variety of angles. While much work in the literature has utilized media containing sera or hydrolysates, chemically defined media were also developed in order to eliminate the problematic lot-to-lot variation of complex components (Luo and Chen, Biotechnology and Bioengineering 97(6): 1654- 1659 (2007)). An improved understanding of the cell culture has permitted a shift to chemically defined medium without compromising on growth, viability, titer, etc. To date optimized chemically defined processes have been reported with titers as high as 7.5- 10 g/L (Huang et al, Biotechnology Progress 26(5): 1400-1410 (2010); Ma et al, Biotechnology Progress 25(5): 1353-1363 (2009); Yu et al, Biotechnology and Bioengineering 108(5): 1078- 1088 (2011). In general, the high titer chemically defined processes are fed batch processes with cultivation times of 11 -18 days. The process intensification has been achieved without compromising product quality while maintaining relatively high viabilities.
Development of such therapeutics of biological origin requires production at industrial scale thereby providing access to large amounts of recombinant proteins. Preferred expression systems are mammalian cell cultures which are superior to most other eukaryotic systems based on insect cells, yeast or the like, or even traditional prokaryotic expression systems.

However, mammalian cell culture includes tremendous challenges especially at the industrial scale. Production facilities for mammalian cell culture require thorough optimization of many process conditions.

One of the most important process parameters for controlling the overall production process is the medium in which cells are grown. Suitable cell culture media must provide cell cultures with all necessary nutrients, which is especially difficult if no components of animal origin like serum or proteins, e.g. growth factors, are added to the media.

Further, mammalian cell culture require particular supplement components at different stages of the polypeptide production process. Accordingly, cell culture media must provide the necessary substrates during a) initial growth and proliferation of the host cells at lower densities; b) subsequent cultivation of cells to high densities; c) the actual process of polypeptide formation in the cultured cells.

One of the critical quality attribute for biosimilar/ antibodies development is glycosylation patterns. It is well documented in literature that glycosylation modification of monoclonal antibody have profound impact on its therapeutic activity.

Accordingly, considerable efforts have been taken in the past to develop cell culture media with special emphasis on their use for large scale production of antibodies. Nevertheless, continuous improvement of cell culture media is still an important goal in order to further maximize polypeptide production in terms of product quality and quantitative yields.

Many components of cell culture media have been investigated in the past in terms of their role for biosimilar/ antibodies production. Possible targets are inorganic salts, amino acids, sources of carbon like glucose, or vitamins.

However, no prior precedent has been established for the selective use of lactate as a mammalian cell culture media supplement. Feeding lactate provided very interesting tools to influence the glycoprofile. This could be useful for the modulating the glycosylation pattern for biosimilar/ antibodies development. This work opens up the new avenues of alternative to glucose feeding strategies for modulating the glycoforms towards the elect target profile.

OBJECTS OF THE INVENTION

The principal object of the present invention is to provide method of producing a composition comprising antibody, said method comprising: culturing a host cell expressing antibody, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising antibody.

Another object of the present invention is to provide method for achieving a modulated glycosylation profile of antibody comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the antibody.

Another object of the present invention is to provide method of modulating the mannose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said antibody, thereby modulating the mannose content of said antibody.

Another object of the present invention is to provide method of modulating the galactose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said antibody, thereby modulating the galactose content of said antibody.

Another object of the present invention is to provide method of producing a composition comprising Trastuzumab, said method comprising: culturing a host cell expressing Trastuzumab, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising Trastuzumab.

Another object of the present invention is to provide method for achieving a modulated glycosylation profile of Trastuzumab comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the Trastuzumab.

Another object of the present invention is to provide method of modulating the mannose content of Trastuzumab, said method comprising: culturing a host cell expressing said Trastuzumab in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said Trastuzumab, thereby modulating the mannose content of said Trastuzumab.

Another object of the present invention is to provide method of modulating the galactose content of Trastuzumab, said method comprising: culturing a host cell expressing said Trastuzumab in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said Trastuzumab, thereby modulating the galactose content of said Trastuzumab.

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide method of producing a composition comprising antibody, said method comprising: culturing a host cell expressing antibody, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising antibody.

Another aspect of the present invention is to provide method for achieving a modulated glycosylation profile of antibody comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the antibody.

Another aspect of the present invention is to provide method of modulating the mannose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said antibody, thereby modulating the mannose content of said antibody.

Another aspect of the present invention is to provide method of modulating the galactose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said antibody, thereby modulating the galactose content of said antibody.

Another aspect of the present invention is to provide method of producing a composition comprising Trastuzumab, said method comprising: culturing a host cell expressing Trastuzumab, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising Trastuzumab.

Another aspect of the present invention is to provide method for achieving a modulated glycosylation profile of Trastuzumab comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the Trastuzumab.

Another aspect of the present invention is to provide method of modulating the mannose content of Trastuzumab, said method comprising: culturing a host cell expressing said Trastuzumab in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said Trastuzumab, thereby modulating the mannose content of said Trastuzumab.

Another aspect of the present invention is to provide method of modulating the galactose content of Trastuzumab, said method comprising: culturing a host cell expressing said Trastuzumab in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said Trastuzumab, thereby modulating the galactose content of said Trastuzumab.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the comparative process performance indicators of all 7 batches (Control & Experimental batches).
Figure 2 shows the comparative productivity profile of all 7 batches (Control & Experimental batches).
Figure 3 shows the comparative Glycan profile of all 7 batches (Control & Experimental batches).

DETAILED DESCRIPTION OF THE INVENTION

The principal embodiment of the present invention is to provide method of producing a composition comprising antibody, said method comprising: culturing a host cell expressing antibody, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising antibody.

Another embodiment of the present invention is to provide method for achieving a modulated glycosylation profile of antibody comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the antibody.

Another embodiment of the present invention is to provide method of modulating the mannose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said antibody, thereby modulating the mannose content of said antibody.

Another embodiment of the present invention is to provide method of modulating the galactose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said antibody, thereby modulating the galactose content of said antibody.

Another embodiment of the present invention is to provide method of producing a composition comprising Trastuzumab, said method comprising: culturing a host cell expressing Trastuzumab, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising Trastuzumab.

Another embodiment of the present invention is to provide method for achieving a modulated glycosylation profile of Trastuzumab comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the Trastuzumab.

Another embodiment of the present invention is to provide method of modulating the mannose content of Trastuzumab, said method comprising: culturing a host cell expressing said Trastuzumab in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said Trastuzumab, thereby modulating the mannose content of said Trastuzumab.

Another embodiment of the present invention is to provide method of modulating the galactose content of Trastuzumab, said method comprising: culturing a host cell expressing said Trastuzumab in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said Trastuzumab, thereby modulating the galactose content of said Trastuzumab.

The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, modified antibodies, subunits of antibodies, antibody derivatives, artificial antibodies, combinations of antibodies with proteins and antibody fragments sufficiently long to display the desired biological activity, The monoclonal antibodies as used herein may be human antibodies.

The term "cell density" as used herein refers to that number of cells present in a given volume of medium.

The terms "culture", "cell culture" and "eukaryotic cell culture" as used herein refer to a eukaryotic cell population that is suspended in a medium (see definition of "medium" below) under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms as used herein can refer to the combination comprising the mammalian cell population and the medium in which the population is suspended.

The term "seeding" as used herein refers to the process of providing a cell culture to a bioreactor or another vessel. In one embodiment, the cells have been propagated previously in another bioreactor or vessel. In another embodiment, the cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term refers to any number of cells, including a single cell.

The term "titer" as used herein refers to the total amount of recombinantly expressed polypeptide or protein produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of polypeptide or protein per milliliter of medium or in units of grams of polypeptide or protein per liter of medium.

The batch cell culture includes fed-batch culture or simple batch culture. The term “fed batch cell culture” refers to cell culture wherein cells and cell culture medium are supplied to the culturing vessel initially and additional culture nutrients are fed continuously or in discrete increments to the culture during the culturing process with or without periodic cell and/or product harvest before termination of the culture. The term “simple batch culture” relates to a procedure in which all components for cell culturing including the cells and the cell culture medium are supplied to the culturing vessel at the start of the culturing process.

As used in the present disclosure and claims, the singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise.

A medium comprises lactate in an amount sufficient to increase the lactate concentration in the culture by between about 0.01 g/L and about 5 g/L, about 0.01 g/L and about 4 g/L, about 0.01 g/L and about 3 g/L, about 0.01 g/L and about 2 g/L, about 0.01 g/L and about 1 g/L, about 0.01 g/L and about 0.5 g/L, about 0.01 g/L and about 0.25 g/L, about 0.05 g/L and about 5 g/L, about 0.05 g/L and about 4 g/L, about 0.05 g/L and about 3 g/L, about 0.05 g/L and about 2 g/L, about 0.05g/L and about 1 g/L, about 0.05 g/L and about 0.5 g/L, about 0.05 g/L and about 0.25 g/L, about 0.1 g/L and about 5 g/L, about 0.1 g/L and about 4 g/L, about 0.1 g/L and about 3 g/L, about 0.1 g/L and about 2 g/L, about 0.1 g/L and about 1 g/L, about 0.1 g/L and about 0.5 g/L, about 0.1 g/L and about 0.25 g/L, about 0.2 g/L and about 5 g/L, about 0.2 g/L and about 4 g/L, about 0.2 g/L and about 3 g/L, about 0.2 g/L and about 2 g/L, about 0.2g/L and about 1 g/L, about 0.2 g/L and about 0.5 g/L, about 0.2 g/L and about 0.25 g/L, about 0.25 g/L and about 5 g/L, about 0.25 g/L and about 4 g/L, about 0.25 g/L and about 3 g/L, about 0.25 g/L and about 2 g/L, about 0.25 g/L and about 1 g/L, or about 0.25 g/L and about 0.5 g/L.

A feed medium comprises lactate in an amount sufficient to increase the lactate concentration in the culture by about 0.01 g/L, about 0.02 g/L, about 0.03 g/L, about 0.04 g/L, about 0.05 g/L, about 0.06 g L, about 0.07 g/L, about 0.08 g/L, about 0.09 g/L, about 0.1 g/L, about 0.15 g/L, about 0.2 g/L, about 0.25 g/L, about 0.5 g/L, about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, or about 5 g/L. A skilled artisan readily understands that the absolute amount of lactate supplemented by a feed medium to a cell culture can be calculated from the volume of feed medium added to the culture and the lactate concentration of the feed medium.

A culture according to the invention can be contacted with a feed medium described herein at regular intervals. In one embodiment, the regular interval is about once a day, about once every two days, about once every three days, about once every 4 days, or about once every 5 days. In a specific embodiment, the culture is a fed batch culture.

In accordance with the present invention, the total volume of feed medium added to a cell culture should optimally be kept to a minimal amount. For example, the total volume of the feed medium added to the cell culture can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell culture prior to adding the feed medium.

The use of the cell culture medium according to the present invention for production of antibodies generally involves the cultivation of CHO cells for recombinant expression of antibodies. It is preferred that the cell culture medium is used for large-scale production of antibodies. Large-scale production of antibodies relates to the amounts typically required for the industrial production of antibodies used for preparation of therapeutically active biopharmaceuticals. Cell cultures of at least 500 L volume, at least 1000 L, least 5000 L or ever higher volumes typically represent large-scale production applications.

In order to understanding the impact of lactate supplementation on the glycosylation profile, CHO cells were fed with lactate in fed batch culture. In present work, experiments were executed at bioreactor system with lactate supplementation from early fed batch stage till the harvest. The impact of lactate feeding was evaluated on glycosylation profile of the monoclonal antibody and the cell culture parameters such as cell count, viability, metabolite profiles and titer. Lactate feeding reduce Man5 by 3 to 4% as compared to control cells (non-lactate feed cells). As compared to control cells, fucosylation increased by 1 to 2% in lactate feed cells, total Mannosylation reduced by 3 to 4 %, while total galactosylation improved by 3 to 5%. No adverse effect of lactate feeding observed on cell count and viability. This work represents feeding lactate during vital stage of the culture can serve as an alternative carbon source in the presence of glucose, modulating the glycosylation profile.

Major benefits for Lactate supplementation is as follows:-
• Reduces total mannosylation levels
• Increases total galactosylation
• Increase fucosylation
• High productivity

The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Example 1

PROCESS BACKGROUND

Experiment for the said invention carried out in Chinese hamster ovary cells (CHO) [clone ID: CHOSI Her5 D6dh25F3E12/11/KN#8728 procured form EUGENEX] which is an engineered cell line to produce trastuzumab by recombinant DNA technology.

Batches were executed at 5 L scale bioreactors with base process described in table 1. MAMPF7e as basal media with 30mg/L Putrescine, 70mg/L FAC and 4mM glutamine on Day 0 was used as production media and 1g/L galactose on day 6 and 11 added as supplement. Further, 3% U1 and 0.8% U2 feed addition was done on Day 2, 4, 6, 8, 10 and 12. The targeted seeding density to inoculate production batches was (0.5 ± 0.1) x 106 cells/mL. Seed for the experiments were generated from UCB vials and expansion was done according to the seed volume requirements for next stage. SF1000 were used as N-1 for the inoculation of 5L bioreactors. The details of the media, feeds and reagents used in the process are listed in the Error! Reference source not found..

Table 1: Description of base process
Sr. No Parameter Process/Setpoint
1 Scale 5L Bioreactor
2 Media MAMPF7e supplemented with 30mg/L Putrescine, 70mg/L FAC and 4mM glutamine
3 Seeding density (0.5 ± 0.1) x 106 cells/mL
4 Feed 3% U1 feed & 0.8% U2 feed
5 Feeding schedule Day 2,4,6,8,10,12,14
6 Galactose addition 1g/L on day 6 and 11
7 Glucose maintenance Upto 5g/L
8 pH setpoint 6.9 ± 0.2
Do setpoint 50%
9 Temperature shift Day 0-3: 37°C
Day 3-5: 32°C
Day 5-14 : 30°C
10 Agitation Day 0-6 : 275 RPM
Day 0-14 : 285 RPM
11 Gassing Day 0-6: Air + Oxygen mixture
Day 6-14: Only Oxygen

Process set parameters includes pH set point (6.9 ± 0.2), dissolved oxygen set point (50%), agitation (D0 to D5-275 RPM, D6 to D8-315 RPM and D9 till harvest day-335 RPM) and temperature shift from 37 °C on day 0 to 32°C from day 3 to day 5 and 30°C onwards from day 5 to till harvest day (Day 14). The gassing strategy was shifted to pure oxygen after day 6 from the initial combination of air and oxygen mixture.
Glucose was added to the culture to maintain the final glucose concentration in the range 4-5 g/L, from day 4 onwards. Harvesting was done by centrifugation at 4000 RPM (for 20 minutes) followed by filtration through 0.2 µm PES filter. Harvest criteria is day 14 or viability = 80%, whichever is achieved earlier.

LACTATE SUPPLEMENTATION

As the aim of experiment was to evaluate the effect of lactate supplementation on the glycan modulation, batches with and without lactate was evaluated. Control batches had the previously mentioned base process without any lactate addition whereas lactate supplementation or maintenance performed as the part the experimentation with the same base process. The brief description of the experiment is as mentioned in table 3.

Table 2: Experiment details
Sr .No Batch ID Batch description Lactate supplementation
1 P26U170 Control-1 No lactate addition
2 P26U171 Control-2 No lactate addition
3 P26U194 Experimental Lactate maintenance: 1.5 g/L
4 P26U197 Experimental Lactate maintenance: (0.5-1) g/L
5 P26U198 Experimental Lactate maintenance: 0.5 g/L
6 P26U206 Control-3 No lactate addition
7 P26U207 Experimental Lactate maintenance: 1g/L

As mentioned, in control process there was no lactate added and in experiment condition various levels of lactate maintenance from 0.5 to 2 g/L to evaluate the impact on the glycan modulation. In usual case lactate accumulates in cell culture during growth phase as metabolic by-product. Later during production phase it will gone into consumption mode and will be maintained almost negligible level till end of the process. Lactate supplementation will start from the production phase (i.e) from day 6 onwards till day 12.

RESULTS:

1. Cell culture, Metabolite and productivity profile:
Process performance were monitored for major cell culture attributes includes Growth, pH trend and metabolite profiles. Viable cell count and Viability measured using automated cell viability analyzer, pH online during the time of sampling was captured, and residual glucose & lactate were measured using Nova bioanalyzer.
Growth pattern was similar in all the lactate maintenance condition in comparison to control batches within variation of ±2 million cell/mL as shown in figure 1A. There was no significant difference in viability profile observed in all the batches as shown in figure 1B. Day 14 viability is above 95% in all the experiment and control conditions.
pH trend was also similar across the batches. Drop in pH observed on day 3 in all the condition which is due to the peak lactate accumulation from the culture. After temperature shift, pH start shoot up back to 7.1 and maintained throughout the batch as shown in figure 1C. There was no significant difference in lactate maintenance observed on the pH value of the culture.
Initial glucose concentration in media was about 10 g/L which will be consumed by cell within 4 to 5 day then onwards glucose will be maintained by feeding externally in order to maintained upto 5g/L. Residual glucose profile shown in figure 1D demonstrates the similar consumption pattern of the glucose which similar across the batches.
In control batches the usual lactate trend was observed to be accumulation of maximum peak on the day 3 and then onwards in consumption mode which goes almost as low as 0g/L at the later stages of the batch. Whereas in experimental condition, lactate was supplemented externally to maintain the required minimum residual concentration of lactate in the cell culture during the process from day 6 to day 12. In all the experiment case after day 12 there was resurge in lactate observed as shown in figure 1E.
Ammonia accumulation pattern was similar in both experiment and control condition expect lactate main. 1g/L which has lower accumulation of about 10mMol on day 14 as shown in figure 1F.
Productivity profile of the batches were estimated by Pro A HPLC. As shown in figure 2, average titre of control bathes were around ~1.4g/L. Increased in total productivity observed in 0.5g/L & (0.5-1) g/L lactate maintenance batches which could be due to the slightly higher growth observed in those two batches. Hence there is no considerable impact on the titre by addition of lactate.

2. Glycan profile
The primary aim of the experiment was to modulate the glycan species. The major glycoform were considered here to analyze the impact of lactate on the same. Total Gal in control batches was around ~30%, whereas on addition of lactate seems to increase the TGAL up to 35% which was one of the major outcome of this study as shown in figure 3A.

Secondly, High Mannose species significantly decreasing with the supplement of lactate. There was trend observed with the HM reduction and the residual concentration of lactate maintained during the process. Higher the lactate maintained in the process, lower the HM obtained as shown in figure 3B. This finding was not observed or not well reported so far in any of bio similar development cases. Since, HM species play major role in the serum clearance of the drug, it was very important to fine tune its level in antibodies. Lactate was evidently modulating the level of HM species at different concentration.
Afucosylation has its own important in the activity of the molecule as it play important role in ADCC activity of the antibodies such as Trastuzumab. Lactate shows ability to modulate the AF species significantly. In control process % AF was around ~ (7-8) %, whereas supplementing lactate reducing the AF species to ~ (5-6) % in the process as shown in figure 3C. Here, again the reduction of AF was directly depended on the concentration of lactate maintained during the process.

Another major observation was that the lactate supplementation had not significant impact on G0f species though it was changing the other glycoforms in the system as shown in figure 3D. Since G0f was the main peak for any antibody molecule, it was difficult to maintain G0f level during the modulation of other glycan species. But the lactate supplementing and maintaining strategy specifically modulating the TGAL, HM and AF without showing major change in the main peak (G0f). This was an added advantage of process with lactate where glycan modification was required without disturbing G0f distribution.

SUMMARY/CONCLUSION

Similarity of glycan profile was one of major challenge in development of any biosimilars or antibodies of therapeutic interest. These glycoform play important role in pharmacokinetics and activities of drug, hence it was very crucial to had a prominent tool to control. Lactate supplementation evaluated in this study evidently shown its capability to modulate the glycoform especially High mannose, Total Galactosylation and Afucosylation without affecting the G0f species. Based on the concentration of lactate maintained or supplemented, the process can lead to produce protein of interest with modulated or target glycan profile. ,CLAIMS:WE CLAIM,

1. A method of producing a composition comprising antibody, said method comprising: culturing a host cell expressing antibody, in cell culture media supplemented with a lactate supplement, thereby producing said composition comprising antibody.

2. A method for achieving a modulated glycosylation profile of antibody comprising providing lactate to a cell culture at a concentration that falls within a target lactate concentration range, wherein the cell culture comprises host cells producing the antibody.

3. A method of modulating the mannose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the mannose content of said antibody, thereby modulating the mannose content of said antibody.

4. A method of modulating the galactose content of antibody, said method comprising: culturing a host cell expressing said antibody in cell culture media supplemented with an amount of lactate supplement sufficient to modulate the galactose content of said antibody, thereby modulating the galactose content of said antibody.

5. The method according to any of preceding claims wherein the said antibody is selected from Trastuzumab, Ranibizumab or Bevacizumab.

6. The method according to any of preceding claims wherein the said cell culture media supplemented with an amount of lactate supplement in range of about 0.01 g/L to about 5 g/L.

7. The method according to any of preceding claims wherein the said feed media supplemented with an amount of lactate supplement in range of about 0.01 g/L to about 5 g/L.

8. The method according to any of preceding claims wherein the said feed media supplemented at the regular interval about once a day, about once every two days, about once every three days, about once every 4 days or about once every 5 days.