KR20160025622A - Improved process for production of monoclonal antibodies - Google Patents

Abstract

The present invention provides an improved method for obtaining a substantial amount of monoclonal antibody having a desired profile of charged variants. The method comprises initially culturing mammalian cells at a suitable temperature during production of the desired molecule, followed by reducing the temperature and optionally adding the appropriate amino acid (s) simultaneously. The present invention also provides antibodies with the desired profile of the glycans prepared above using the improved methods.

Description

[0001] IMPROVED PROCESS FOR PRODUCTION OF MONOCLONAL ANTIBODIES [0002]

The present invention relates to an improved process for obtaining a substantial amount of monoclonal antibodies having a desired profile of charged variants. In one embodiment, the process also provides an antibody having the desired profile of the glycan. The present process involves initially culturing mammalian cells at a suitable temperature during the production of the desired molecule, followed by reducing the temperature and optionally adding the appropriate amino acid (s) simultaneously.

Proteins are huge and complex molecules. They are required to exist in their natural confirmation to maintain their biological activity. In addition, at high concentrations, protein molecules in solution are subject to certain deformation or degradation or aggregation over time during storage. In one aspect, the present invention preferably provides an improved method for obtaining an increased amount of a desired quality product that is a monoclonal antibody. Monoclonal antibodies (mAbs) have gained considerable interest as therapeutic agents due to their high specificity for binding to their target antigen, their ability to initiate an immune response against the target antigen, and long-term serum persistence. There are a number of monoclonal antibodies directed against tumor-specific antigens. For example, some inherent characteristic features of each immunoglobulin, both charge and glycan structures, have been found to be important and specific for the mechanism of action. Like many other proteins, monoclonal antibodies have charge heterogeneity that optimizes electrostatic interactions and regulates their structure, stability, chemical and biological properties. During production, various forms of micro heterogeneity occur due to degradation, modification, or various enzymatic processes. Protein degradation occurs due to chemical instability or physical instability. Chemical instability can be primarily the result of deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Chemical instability causes the formation of a variety of charge mutants which alter the properties of biomolecules. Chemical modifications such as deamidation and sialylation each lead to an increase in the net negative charge on the mAbs and a decrease in the pI value. Other mechanisms of the generation of acidic variants are known in the art. The deamidated isoforms are susceptible to degradation by loss of activity and, therefore, they affect not only the significant activity but also the stability of the monoclonal antibody protein.

Similarly, N-glycosylation in the Fc region modulates the antibody-acting function of immunoglobulins and other Fc-containing molecules. Fc glycans may contain several different types of terminal sugars that affect the function of the antibody. The effect of terminal galactosylation is known to those skilled in the art. The galactosylation pattern of different immunoglobulins exhibits product-specific variability in, for example, immunoglobulins. It is important to note that mutations in terminal galactosylation affect antibodies that bind to the antigen and have a profound effect on the CDC activity of the molecule. On the other hand, although the degree of change in galactosylation has been known to have less effect on ADCC activity, afucosylation is extremely important for ADCC activity.

Several production processes of monoclonal antibodies have been known in the art. Such processes include osmotic pressure maintenance, addition of salt with reduction of temperature and the like.

US 5705364 adds alkanoic acid and salts thereof to the culture at a concentration of about 0.1 mM to about 20 mM and maintains the osmotic pressure of the culture at about 250 to about 600 mOsm. Discloses a cell culture process in which the amount of sialic acid present on the oligosaccharide side chain of the glycoprotein is controlled by maintaining the temperature of the culture at a temperature of about 30 캜 to 35 캜.

US 5976833 provides a method for improving productivity in the production of useful materials using animal cells. Which comprises the steps of (1) culturing animal cells at a temperature at which animal cells can grow; and (2) culturing the animal cells at a lower temperature .

WO 2014035475 discloses a method for controlling the oligosaccharide distribution of recombinant-expressed proteins, including supplementing cell culture media used in recombinant expression of said proteins using yeast hydrolytic supplements and plant hydrolyzate supplements. It also discloses a method of controlling the oligosaccharide distribution of an antibody by modulating the asparagine amino acid concentration of the cell culture medium; The present invention does not include the supplementation of these hydrolysates in the culture medium.

Although there is a difference in the availability of different processes for the production of monoclonal antibodies, there is still a need to identify cell culture processes for monoclonal antibody production, which can be used for any significant batch-to-batch variation of the glycans profile and the charged levels of the glycans. Such a process may also help to obtain a monoclonal antibody protein with the desired charge and / or glycan profile. The present invention provides such an improved process for monoclonal antibody production using a modified cell culture method. The process according to the present invention does not include either addition of a salt or maintenance of a suitable osmotic pressure. The present invention provides a novel method for the production of monoclonal antibodies having a desired profile of glycans and charged variants.

The present invention provides an improved process for obtaining a significant amount of glycans and monoclonal antibodies having a desired profile of charged variants using modified cell culture methods.

In one aspect, the cell culture method is characterized by maintaining cell culture production conditions at various temperatures, either at once or in a step-wise manner during the cell culture process.

In another aspect, the present invention provides a process for the production of a catalyst which performs a production process at an initial high temperature in a growing phase and thereafter, during mid-log to late-log phase or stationary phase Thereby providing an improved process for the production of monoclonal antibodies that reduce the temperature of the culture system to a second, lower temperature.

In another aspect, the present invention provides an improved process for the production of monoclonal antibodies by supplying an amino acid suitable for the culture system during mid-logarithm or rate-logarithmic or quiescent period.

In a further aspect, the amino acid (s) according to the invention are added to the cell culture medium at specific concentrations and at specific time intervals during the cell culture process.

In a preferred embodiment, the amino acids are selected from glutamine and asparagine or a combination thereof.

In a preferred embodiment, the present invention relates to a process for carrying out a process at an initial high temperature in a growing season, and thereafter, during simultaneous feeding of the amino acid (s) to the culture medium, during mid- Providing an improved upstream process for obtaining a significant amount of monoclonal antibody having a desired profile of glycan and charged variant by reducing the temperature of the culture system second below the initial temperature in either.

In a further aspect, the amino acid according to the invention is selected from basic amino acid-containing amide groups which are, for example, glutamine, asparagine, histidine, lysine, arginine and combinations thereof.

In a preferred embodiment, monoclonal antibodies are selected from anti-HER antibodies, anti-TNF antibodies, anti-VEGF antibodies and anti-CD20 antibodies.

In a more preferred embodiment, the monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab, ranibizumab, ) And rituximab.

Figure 1: shows the charged variant profile of purified adalimumat protein by HP-IEC.
Figure 2: shows the glycan profile of purified adalimumat protein by CE-LIF.

In one embodiment, the present invention performs the production process at an initial high temperature in the growing season and then, at a later time, in a stepwise fashion, either at once or during mid-log or rate- To provide a process for the production of monoclonal antibodies having a desired profile of charged variants while maintaining the desired glycan profile of the protein.

In a further embodiment, the cell culture method is characterized by maintaining cell culture production conditions at various temperatures, either once or in a step-wise manner during the cell culture process.

In another embodiment, the present invention preferably provides a substantial amount of a monoclonal antibody having the desired profile of glycan by feeding a suitable amino acid, such as an amide group, containing amino acid (s) and / or basic amino acid (s) To provide a process for the production of. Such amino acid (s) may be supplied at any stage during the mid-log period or the quiescent period.

In a further embodiment, the amino acid (s) according to the invention are added to the cell culture medium at specific concentrations and at specific time intervals during the cell culture process.

In a still further embodiment, the addition of the amino acid (s) according to the invention is carried out at two or more different intervals during the cell culture process.

In a preferred embodiment, the addition of the amino acid (s) to the cell culture medium according to the invention is carried out at a concentration lower than 20 mM, preferably lower than 10 mM each time.

In a preferred embodiment, the present invention relates to a process for carrying out a production process at an initial high temperature in a growing season and thereafter decreasing the temperature of the culture system to a second lower temperature during either mid-logarithmic or late- And provides a significant amount of glycans and monoclonal antibodies with the desired profile of charged variants by feeding the amino acid (s) to the culture system.

In a further aspect, the amino acid according to the invention is selected from basic amino acid-containing amide groups, for example glutamine, asparagine, histidine, lysine, arginine and combinations thereof.

In general, the initial high temperature of the culture system is maintained at 37 占 폚. The temperature of the culture system according to the present invention may be reduced to 30 占 폚 at any stage during the mid-log period or the stop period, at certain time intervals, either at once or in a staged manner. The process according to the present invention provides a significant amount of monoclonal antibodies with the desired profile of glycans and charged variants. In addition, the present process maintains the desired glycan profile of the monoclonal antibody.

In one of its embodiments, the invention provides a method for producing a desired glycan profile of a protein, preferably a monoclonal antibody, by supplying a suitable amino acid, such as glutamine and / or asparagine, to the culture system at any stage during the mid- to provide. The amount of amino acid (s) added is in the range of 1 to 4 mM, preferably 2 to 3 mM. It has been found that the supply of glutamine and / or asparagine according to the invention to the cell culture medium during production increases the formation of the desired glycane (s) moiety (s) in a product-specific manner in monoclonal antibody protein structures.

In a preferred embodiment, the invention relates to a process for carrying out a process at an initial high temperature in a growing season and thereafter decreasing the temperature of the culture system to a second lower temperature during either the mid-logarithmic or late- (S) (glutamine and / or asparagine) during the production of the desired protein to produce a significant amount of glycans and monoclonal antibodies with the desired profile of charged variants.

In one embodiment, the present invention provides a method for the production of an antibody having a desired profile of a glycan and a charged variant, wherein the glucose concentration is 0.5 g / L to 8 g / L, preferably 2 g / L to 4 g / L, more preferably about 2.5 g / L.

In another embodiment, the invention provides a process for the production of an antibody having a desired profile of a glycan and a charged variant, wherein the pH is adjusted to a pH of from about 6 to about 10, using a suitable buffer selected from sodium bicarbonate, sodium carbonate and HEPES buffer. pH < / RTI > 7.5.

In a further embodiment, the invention provides a process for the production of an antibody having a desired profile of glycans and charged variants, wherein the cell productivity is at least 0.5 g / L, preferably 1 - 4 g / L maintain.

In yet a further embodiment, the invention provides a process for the production of an antibody having a desired profile of glycan and charged variant, wherein the cell viability is at least 30%, preferably at least about 80% 95%.

In a more preferred embodiment, the monoclonal antibody is selected from Trastuzumab, Pertuzumab, Infliximab, Adalimuma, Bevacizumab, Ranibizumab, and Rituximab.

Justice:

Glycan - This term refers to polysaccharides or oligosaccharides. Glycans may be homo- or heteropolymers of monosaccharide residues and may be linear or branched. Glycans may also be used to refer to carbohydrate moieties of glycoconjugates, such as glycoproteins, glycolipids, or proteoglycans.

• The desired glycan profile - this can be defined as the distribution pattern of various glycan molecules attached to a protein essential for its biological activity.

Mid-logger - It is defined as the growth period of cells in the culture medium while the cell population exponentially increases. This group appears as a part of the growth curve, and when the logarithmic values of the cell population are plotted against time, it appears as a straight line, which is called the logarithmic phase and its midpoint is the mid- It is called.

• Rate-logger - it is defined as the growth period of cells in the rate-logger prior to conversion to stasis. This group appears as part of the growth curve, and when the logarithmic value of a population of cells is plotted against time, it appears as a straight line segment, which is called the logger and its end phase is called the rate-logger.

● Stasis - The plateau of the growth curve after log-phase growth of cells in the culture medium is called a stasis, and the population of cells is kept constant at that time. New cells are produced at the same rate as older cells die.

• Charge species variants - it is a specific property of proteins that optimize electrostatic interactions and control their structure, stability, and chemical and biological properties. It differs from protein to protein due to the specific distribution of the charged amino acids on the protein molecule.

Analytical method used in the present invention:

High Pressure Ion Exchange Chromatography (HP-IEC): For example, the separation of different charged variants of the azithromycin purified monoclonal antibody is carried out using analytical HP-weak cation exchange chromatography . The column was equilibrated in sodium phosphate buffer at pH 6.9 (mobile phase A). Elution of the charged species variants of the protein was performed using increasing salt concentration in mobile phase A (sodium chloride) at 0.5 mL / min.

Capillary Electrophoresis - Laser Induced Fluorescence (CE-LIF): Glycan analysis (glycosylation variants) of the preparation of a purified monoclonal antibody, for example, Carbohydrate moieties were separated and then performed by the CE-LIF method. After the enzymatic treatment, the carbohydrate moiety was labeled with APTS (8-aminopyrene 1,2,6-trisulfonate), and the derived glycans were then transferred to a capillary system (N- CHO coated; 50 cm x 50 [mu] m). Glycans were identified for the labeled glucose ladder standard detected by a LIF detector with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

The preferred manner of the monoclonal antibody production process according to the present invention is illustrated by the following examples, which should not be construed as limiting the scope of the invention in any way:

Example  One

Anti-TNFα Antibody Mammalian cells expressing adalimumab were generated by standard molecular biology techniques. The clones were marginally diluted and homogeneous populations from single cells were obtained. Cells were cryopreserved in the form of cell banks and used for further development. Cells were resuscitated and propagated into a series of inoculum development stages and inoculated into bioreactors containing appropriate growth media. Cell culture was performed in a controlled environment by maintaining pH 7.2 + -0.4 using CO ₂ gas and / or sodium bicarbonate as needed. Dissolved oxygen concentration was maintained at 40 ± 20% saturation by the injection of air and / or oxygen gas and by controlling the stirring speed in the bioreactor. The temperature was controlled at 37 占 폚. The growth medium comprises the following components:

ingredient density

CHO growth powder medium 19.8 g / L

Sodium hydrogencarbonate 2.2 g / L

Pluronic F-68 1.2 g / L

Cells were grown for 2 days under the conditions described above. From day 3, the supply was initiated and continued until the end of the batch. The following media components were fed to the cell culture medium as a common feed -

ingredient liter  Sugar concentration

CHO foundation powder medium 138.9 g

insulin 50 mg

Lipid supplement 1 × concentration

Iron citrate 1 × concentration

Polyethylene glycol 220 mg

Sodium hydrogencarbonate 10.8 g

The batches were harvested between 13 and 18 days of culture. After cell clarification, the supernatant containing adalimumab was readjusted to substantially match the next column column equilibrium condition. The desired protein was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analysis for each of the charged species variant and glycan profile, as shown in Tables 1 and 2. The process illustrated herein can be used for any desired antibody.

Example  2

Adal mawwat  Effect of reduced temperature from 37 ° C to 35 ° C

The experiment was carried out in a 30L bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions of the culture system except for the general feed medium and other process parameters. The temperature of the culture system was reduced from 37 占 폚 to 35 占 폚 in a rate logger. Adalimumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analysis for each charged species variant and glycan profile, as shown in Tables 1 and 2.

Example  3

Adal mawwat  Effect of glutamine supplementation

The experiment was carried out in a 30L bioreactor. Growth conditions were the same as in Example-1, except for the supply of glutamine amino acid in the culture system, except for the normal feed medium and other process parameters. Feeding of 2 mM glutamine started at the mid-log-phase of cell growth and continued to the end of production at specific intervals.

Adalimumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analysis for each charged species variant and glycan profile, as shown in Tables 1 and 2.

Example  4

Adal mawwat  Effect of Temperature Reduction and Glutamine-Feeding

The experiment was carried out in a 30L bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was reduced from 37 째 C to 35 째 C in the mid-logger and after that time the temperature of the culture system was further reduced to 33 째 C during the transition from the logger to the stator. Feeding of 3 mM glutamine started at the mid-logger and continued at specific intervals until the end of the production of the desired monoclonal antibody.

Adalimumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analysis for each charged species variant and glycan profile, as shown in Tables 1 and 2.

Example  5

Effect of cultivation at 37 ° C without glutamine supplementation on trastuzumab

The experiment was carried out in a bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was maintained at 37 캜 continuously for the batch duration. There was no additional glutamine feed except in the initial batch medium.

Trastuzumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  6

Effect of reduced temperature from 37 占 폚 to 33 占 폚 for Trastuzumab:

The experiment was carried out in a bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was reduced from 37 째 C to 33 째 C during the transition from log-group to stationary. Feeding of 2 mM glutamine started at the mid-logger and continued at specific intervals until the end of the production of the desired monoclonal antibody.

Trastuzumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  7

On bevacizumab  Effect of reduced temperature from 37 占 폚 to 35 占 폚 without feeding glutamine:

The experiment was carried out in a 30L culti-flask. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was reduced from 37 占 폚 to 35 占 폚 during the transition from log-group to stationary. There was no additional glutamine feed except in the initial batch medium.

Bevacizumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  8

On bevacizumab  ≪ tb > < SEP > 37 < SEP >

The experiment was carried out in a bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was maintained at 37 占 폚 during the entire batch. Feeding of 4 mM glutamine started at the mid-logger and continued at specific intervals until the end of the production of the desired monoclonal antibody.

Bevacizumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  9

To rituximab  ≪ tb > < SEP > 37 < SEP >

The experiment was carried out in a bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was maintained at 37 占 폚 during the entire batch. There was no additional glutamine supply other than that in the initial batch medium.

Rituximab was purified to satisfactory levels and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  10

To rituximab  About Glutamine Having a supply  Continued during batch Culture effect at 37 ° C:

The experiment was carried out in a bioreactor. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was maintained at 37 占 폚 during the entire batch. Feeding of 4 mM glutamine started at the mid-logger and continued at specific intervals until the end of the production of the desired monoclonal antibody.

Rituximab was purified to satisfactory levels and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  11

About Trastuzumab Glutamine Having a supply  Continued during batch Culture effect at 37 ° C:

The experiment was carried out in a 30L bioreactor (200L bioreactor). Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was maintained at 37 占 폚 during the entire batch. Feeding of 2 mM glutamine started at the mid-logger and continued at specific intervals until the end of the production of the desired monoclonal antibody.

Trastuzumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

Example  12

Effect of reduced temperature from 37 占 폚 to 35 占 폚 without feeding glutamine against trastuzumab:

The experiment was carried out in a 30L culti-flask. Growth conditions were the same as in Example-1, except for the temperature conditions in the culture system and the normal feed medium and other process parameters except for the supply of glutamine. The temperature of the culture system was reduced from 37 째 C to 35 째 C during the transition from log-group to stationary. There was no additional glutamine feed except in the initial batch medium.

Trastuzumab was purified to a satisfactory level and submitted to HP-IEC and CE-LIF analyzes for each of the charged species variants and glycan profiles, as shown in Tables 1 and 2.

result

variety Monoclonal antibody Charged  Bell In variants  Influence of temperature on Example
number Temperature Glutamine supply Charged species variant Main mutant (%) acid
Child small (%) Basic
Child small (%) Example-1 37 ℃ none 69.92 15.49 14.59 Example-2 37 ° C to 35 ° C none 75.37 5.22 19.42 Example-3 37 ℃ has exist 74.67 10.54 14.80 Example-4 37 ° C to 35 ° C to 33 ° C has exist 75.31 7.48 17.16 Example 5 37 ℃ none 75.96 14.33 9.71 Example-6 37 ° C to 33 ° C has exist 77.46 11.41 11.11 Example-7
37 ° C to 35 ° C none 68.53 24.76 6.70 Example-8 37 ℃ has exist 66.34 14.55 14.35 Example-9 37 ℃ none 69.98 10.53 19.48 Example-10 37 ℃ has exist 62.43 5.89 31.68 Example-11
37 ℃ has exist 74.64 17.68 7.67 Example-12
37 ° C to 35 ° C none 83.79
11.29
4.93

variety Monoclonal antibody Glycann  Effect of glutamine supply on profile Example
number Temperature Glutamine supply Glycane profile (%) Agalactose
(Agalactose)
Moiety
(G0 and G0F) The galactose moieties (G1F, G1'F and G2F) Example-1 37 ℃ none 69.14 30.86 Example-2 37 ° C to 35 ° C none 67.36 32.64 Example-3 37 ℃ has exist 85.46 14.54 Example-4 37 ° C to 35 ° C to 33 ° C has exist 75.49 24.51 Example 5 37 ℃ none 69.26 30.74 Example-6 37 ° C to 33 ° C has exist 73.76 26.24 Example-7
37 ° C to 35 ° C none 66.72 23.28 Example-8
37 ℃ has exist 81.40 18.42 Example-9 37 ℃ none 64.16 35.84 Example-10 37 ℃ has exist 79.00 21.00 Example-11
37 ℃ has exist 65.934 34.065 Example-12
37 ° C to 35 ° C none 51.40 34.103

The obtained product was subsequently purified and suitably formulated by known techniques.

Claims (15)

A process for producing an antibody having the required glycans and / or charge species variants profiles using a modified cell culture method, said method comprising:
a) maintaining cell culture conditions at suitable temperature conditions at specific intervals;
b) simultaneously or sequentially adding the specific amino acid (s) at specific intervals to the medium during the process. The method of claim 1, wherein the cell culture method maintains cell culture production conditions at a temperature that is reduced at a suitable temperature, a fixed temperature, or a step-wise manner during the cell culture process. . 3. The method of claim 2, wherein the suitable temperature conditions comprise maintaining the cell culture conditions at a second temperature and, optionally, at a third temperature condition up to the end of the process followed by cell growth at the first temperature condition to the mid-log phase. production process. The production process according to claim 3, wherein the first temperature condition is maintained at a temperature higher than the second and, optionally, the third temperature condition. The production process according to any one of claims 2 to 5, wherein the first temperature condition is about 37 ° C, and the second, optionally, the third temperature condition is in the range of 35 ° C to 30 ° C. The production process according to claim 1, wherein the amino acid (s) are added to the cell culture medium at a specific concentration and at specific time intervals during the cell culture process. The production process according to claim 7, wherein the addition of the amino acid (s) is carried out at two or more different intervals during the cell culture process. The production process according to claim 1, wherein the addition of the amino acid (s) to the cell culture medium is performed at a concentration lower than 10 mM each time. The production process according to claim 1, wherein the amino acid (s) is selected from basic amino acid containing an amide group which is, for example, glutamine, asparagine, histidine, lysine, arginine and combinations thereof. The production process according to any one of claims 1 to 9, wherein the glucose concentration is maintained in the range of 0.5 g / L to 8 g / L, preferably 2 g / L to 4 g / L. The production process according to any one of claims 1 to 10, wherein the pH is maintained within the range of pH 6 to pH 7.5 using a suitable buffer selected from sodium bicarbonate, sodium carbonate and HEPES buffer. The production process according to any one of claims 1 to 11, wherein the cell productivity is 0.5 g / L or more, preferably 1 to 4 g / L. The production process according to any one of claims 1 to 12, wherein the cell viability is maintained above 30%, preferably about 80%, more preferably 95%. The production process according to any one of claims 1 to 13, wherein the antibody is selected from an anti-HER antibody, an anti-TNF antibody, an anti-VEGF antibody and an anti-CD20 antibody. The method of any one of claims 1 to 14 wherein the antibody is selected from the group consisting of trastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab, Ranibizumab, and rituximab. ≪ RTI ID = 0.0 >

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