RU2672318C1 - Method for producing monoclonal antibodies of therapeutic purpose by continuous cultivation of cho cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a method for the continuous cultivation of the Chinese hamster ovary cell line CHO, which is the producer of antibodies, and the use of this method. Method includes the continuous cultivation of the Chinese hamster CHO cell line in the bioreactor, the acceleration of perfusion, which is in the range of 0.1 * day^-2 up to 0.12 * day^-2, and removal of the cell suspension from the bioreactor, carried out at a rate of 0.15 * day^-1. Further, this method is used for the production of therapeutic antibodies in the Chinese hamster ovary cell line CHO.

EFFECT: invention makes it possible to achieve high specific productivity and high viability (more than 95 %) of cells, and also to equalize the degree of glycosylation of the protein throughout the entire culture.

17 cl, 9 dwg, 3 ex

Description

The invention relates to pharmaceutical biotechnology. A method for perfusion culturing cells is proposed, which allows producing antibodies with a reduced level of variation of critical glycans in industrial volumes, in particular therapeutic antibodies affinity for the C5 complement component and anti-IgE antibodies.

Terms and definitions

Fed-Batch - the cultivation of microorganisms or cell cultures in a periodic mode with recharge.

Perfusion is a type of continuous cultivation with partial or complete separation of cells from the effluent of the culture fluid and returning them to the bioreactor.

The perfusion rate is the volume of the flow of culture fluid, can be expressed in liters per day (l / day, l * day ^-1 ).

Relative perfusion rate - the number of reactor volumes (N) that are changed per day. Dimension is defined in relative units as N / day or N * day ^-1

Acceleration of perfusion - a change in perfusion rate per unit time, achieved by increasing the flow rate of the medium at the initial stage of perfusion cultivation. It is defined as the first derivative of perfusion rate, i.e. perfusion rate divided by time, N / day ² , or N * day ^-2 .

Specific perfusion rate (USP) is the ratio of the flow rate of the nutrient medium to the cell concentration, measured in picoliters per cell per day, pl / cell / day. The stationary phase of perfusion cultivation is the phase of the perfusion process in which the growth of cells in the bioreactor is compensated by their selection, so that the concentration of cells for a long time is maintained at a certain level with slight deviations.

Blading - continuous or discrete selection of a cell suspension from a bioreactor in a stationary phase of perfusion cultivation to prevent overgrowth of culture and lack of nutrients, the dimension is determined in relative units - the number of reactor volumes taken per day, N * day ^-1 .

Continuous cell cultivation using perfusion processes is a promising method used in industrial biotechnology for the production of various proteins in cells, including for therapeutic purposes. At the same time, the short period of application of perfusion processes in the world and, accordingly, insufficient experimental verification of the features of the method, simultaneously with the appearance of new nutrient media, led to the appearance of a new technical problem discovered by the inventors. Nutrient media of earlier generations did not have a large impact on the cultivation process in the wide range of perfusion acceleration applied. However, the new generation environments that appeared relatively recently manifest themselves in a completely different way. Different perfusion accelerations cause a different reaction of cell cultures, quite often extremely negative. Therefore, the problem of optimizing perfusion acceleration requires careful handling and thorough research.

Currently, there are many different modifications of the process, and the process is still unknown, which with equal efficiency would allow to produce any necessary protein in any cell culture, that is, the choice of the final conditions of the method of protein production each time is a non-trivial task that requires the synthesis of various methods and experimental verification of new methods.

Description of the drawings.

FIG. 1. Graphs of changes in the flow velocity of the medium at various accelerations of perfusion

FIG. 2. Growth curves of CHO cell culture expressing an antibody having the amino acid sequence of eculizumab at various perfusion accelerations

FIG. 3. The dynamics of changes in the viability of the cell culture of Cho, expressing an antibody having the amino acid sequence of eculizumab, with various accelerations of perfusion

- 0,1* сутки^-1,

- 0,15* сутки^-1)FIG. 5. The influence of the speed of the blading on the content of Man5 glycan in the target protein (

- 0.1 * day ^-1 ,

- 0.15 * day ^-1 )

- 0,1* сутки^-1,

- 0,15 *сутки^-1)FIG. 6. The influence of the speed of the blading on the content of G0 glycan in the target protein (

- 0.1 * day ^-1 ,

- 0.15 * day ^-1 )

- 0,1* сутки^-1,

- 0,15* сутки^--1)FIG. 7. The influence of the speed of building on the productivity of the process of perfusion cultivation (

- 0.1 * day ^-1 ,

- 0.15 * day ^-1 )

- 0,1*сутки^-2,

- 0,15 *сутки^-2)FIG. 4. The dynamics of the productivity of a culture of CHO cells expressing an antibody having the amino acid sequence of eculizumab at various accelerations of perfusion (

- 0.1 * day ^-2 ,

- 0.15 * day ^-2 )

FIG. 8. Growth curves of a culture of CHO cells expressing an antibody having the amino acid sequence of omalizumab at various perfusion accelerations

FIG. 9. Dynamics of productivity of CHO cell culture expressing an antibody having the amino acid sequence of omalizumab at various accelerations of perfusion

The objective of the inventors was to develop a highly productive method for the production of several types of therapeutic antibodies in animal cell culture directed towards the C5 component of complement, as well as anti-IgE antibodies. In particular, eculizumab, omalizumab and their bioanalogs containing amino acid sequences, respectively, can be obtained in this way

The following solutions are known from the prior art that disclose perfusion culturing of cells for the production of therapeutic antibodies therein.

CN 104845880 A discloses a device and method for producing anti-IgE antibodies by continuous perfusion culture of CHO cells. To stabilize the physiological state of the culture, and, as a result, stabilize the entire cultivation process, a controlled selection of the cell suspension from the bioreactor is used. The disadvantage of this method is the relatively low productivity of the process, due to the high specific rate of cell growth (μ = 0.34 days ^-1 ), at which their specific productivity is far from maximum. Thus, with a high consumption of the nutrient medium, the productivity of the process is relatively low.

The publication “Optimization of perfusion acceleration is a key stage in the development of high-performance perfusion cultivation of CHO cells. (Morozov AN, et al. Biotechnology, 2016: 60-67) "discloses a method for optimizing the continuous cultivation of CHO cells expressing neutralizing monoclonal antibodies, which include, in particular, omalizumab, eculizumab and adalimumab. The experiment showed that a slow acceleration of perfusion (0.1 * day ^-2 ) leads to a significant suppression of cell proliferation, an increase in their volume (cell hypertrophy), an increase in specific productivity and, at the same time, a change in the glycosylation profile of the antibody in such a way that it can increase antibody-dependent cellular cytotoxicity (ADCC), and the pharmacokinetic properties of the target molecule may change towards a decrease in half-life. These changes are undesirable and may make the cultivation process unsuitable for industrial use.

US 20140255993 A1 discloses perfusion cultures of CHO cells in which at some point (before or during the production phase), the cell cycle is arrested and cell proliferation is suppressed, leading to an increase in specific cell productivity and an increase in the total yield of the target protein. The arrest of the cell cycle is achieved by limiting the content in the nutrient medium of the amino acid asparagine (not more than 5 mmol / l).

The disadvantages of the method include the need for individual selection of the optimal concentration of asparagine for each cell line producer; the potential insensitivity of some cell lines to this technical solution, a decrease in the viability of the cell culture obtained in this way during the cultivation process, which may negatively affect the technology and quality of the product. There may be problems with clarifying filtration due to the large number of cell debris; a high level of impurities of the host cell proteins and the need for additional chromatographic purification steps; enhanced proteolysis of the target protein.

Various perfusion cultivation options are also disclosed in documents WO 2004097006 A1, WO 2005095578 A1, WO 2009023562 A2, US 2016/0281044 A1, WO 2014059035 A1.

The closest analogues of the claimed invention can be considered the solution CN 104845880 A, which discloses the production of anti-IgE antibodies in a perfusion cultivation mode, a method for producing monoclonal antibodies by continuous cultivation in CHO cells (a line of immortalized Chinese hamster ovary cells) with increased specific productivity while significantly suppressing them proliferation (Morozov A.N. et al., Biotechnology, 2016: 60-67.), as well as the method disclosed in US 20140255993 A1, where CHO cells expressing recombination during perfusion nanth proteins cease to proliferate due to the low concentration of asparagine in the nutrient medium, which entails an increase in the specific productivity of the cells and the overall yield of the product.

The disadvantages of the method disclosed in document CN 104845880 A are associated with a lower concentration of the target protein in the culture fluid during the cultivation of CHO cells in perfusion mode compared to batch (fed-batch) processes. As a result, more resources are required for the preparation, use, storage and disposal of culture media, especially during a long perfusion process. The costs of media and recharge are significantly higher than for fed-batch processes. To solve this problem, it is necessary to increase the concentration of the target protein in the culture fluid. One way to do this is to increase the specific productivity of CHO cells, which is achieved, in particular, by suppressing cell proliferation and maintaining the culture in this state for a long time while maintaining high viability.

In the proposed solution, suppression of proliferation and an increase in the specific productivity of cells is achieved through the use of small perfusion acceleration values at the stage of biomass accumulation. Stability of the state of the cells is ensured by the continuous withdrawal of the cell suspension from the bioreactor at a rate of 0.1 * day ^-1 to 0.15 * day ^-1 . In this case, the use of special deficient media is not required; monitoring of individual components, such as amino acids, is not required. Low flow rates of the nutrient medium from the very beginning of the cultivation process provide a low specific growth rate and a significant suppression of cell proliferation in the production phase of the process (specific growth rate does not exceed 0.15 / day). At the same time, a new technical result is achieved - increasing cell viability to 95% and higher throughout the cultivation process, in contrast to the examples from Amgen patent (US 20140255993 A1).

A publication of Morozov et al. Discloses a similar method of cultivation, however, the use of this method leads to a significant deviation of the biological activity of the expressed target protein from the target due to changes in its glycosylation. This disadvantage is eliminated in the proposed technical solution due to the introduction of a number of changes to the cultivation process. In particular, the rate of selection of the cell suspension from the bioreactor was changed, which led to the stabilization of the glycosylation profile of the target protein. The selection of a cell suspension from a bioreactor - the so-called blading - helps prevent nutritional deficiencies and increase the stability of growth and production characteristics of cell culture.

The inventors have found optimal perfusion acceleration modes to achieve maximum cell productivity. The acceleration of perfusion is controlled by changing the feed rate of fresh nutrient medium into the bioreactor and the rate of extraction of the spent medium from the bioreactor from minimum to maximum during the initial stage of perfusion cultivation. Accelerated perfusion plays an important role in establishing an effective, highly productive perfusion cultivation process. It was found that the acceleration of perfusion of more than 0.1 * day ^-2 leads to intensive growth of the culture, a short doubling time and the rapid achievement of peak cell concentrations. The specific productivity of such cells was at an average level (about 10 pkg / cell / day). Viability when accelerated above 0.25 * day ^-2 after reaching a peak concentration decreased to 80-85%.

Acceleration of perfusion of less than 0.12 * day ^-2 did not cause a drop in cell viability, which remained high throughout the process (above 95%). In addition, under these conditions, the proliferation rate was significantly reduced and the specific cell productivity increased (up to 20 pkg / cell / day).

Varying the acceleration of perfusion to achieve an effective, highly productive cultivation process has not previously been disclosed in the prior art.

Materials and methods

As a model for the study, we used variants of the CHO cell line expressing monoclonal antibodies, which are bio-analogues of therapeutic antibodies - omalizumab, eculizumab and adalimumab.

Cells were grown in a Climo-Shaker ISF1-XC shaker incubator (Kuhner, Switzerland) in Erlenmeyer flasks on a commercial growth medium BalanCD CHO Growth A (Irvine Scientific, USA) containing 6 mM L-glutamine (Applichem, Germany) at 37 ° C 5% CO ₂ , 90 rpm. The resulting inoculum was introduced into a bioreactor containing a nutrient medium of the same composition. The composition of the perfusion medium: 90% BalanCD CHO Growth A, 10% BalanCD CHO Feed 3 (Irvine Scientific), 6 mM L-glutamine.

Perfusion cultivation of cells was carried out in disposable wave-type bioreactors Sartorius Flexsafe 10L perfusion (Sartorius, Germany) with an integrated perfusion membrane. The working volume of the suspension was 3.0 L. Sowing cell concentration - 0.5 ± 0.1 million / ml.

The concentration of dissolved oxygen in the bioreactors was maintained at a level of 50% of saturation. The flow rate of the gas mixture was 0.3 l / min. Also, pH-stating was carried out at a level of 6.9 during the entire cultivation process by supplying CO ₂ or a 5% sodium hydroxide solution. The swing frequency of the platform with the bioreactor was in the range of 22-28 kache / min. The inclination angle of the platform was 6 °. The cultivation temperature in the growth phase is 37 ° C. Upon reaching a perfusion rate of 0.5 * day ^-1 , a temperature was reduced to 34 ° C. The maximum perfusion rate was 1.0 * day ^-1 . Perfusion began on the third day of cultivation.

Two different perfusion acceleration modes were investigated: 0.1 * day ^-2 and 0.15 * day ^-2 . The blading speed was 0.1 * day ^-1 and 0.15 * day ^-1 .

The concentration of antibodies in the culture fluid was determined by isolating the target protein on a MabSelect affinity sorbent (GE Healthcare, Sweden) and measuring its amount on a BioPhotometer Plus spectrophotometer (Eppendorf, Germany) at a wavelength of 280 nm using a calculated extinction coefficient. The target protein glycosylation profile was analyzed using an Alliance E2695 HPLC system (Waters, USA) with a Multi λ Fluorecsence Detector 2475 (Waters) on a TSKgel Amide-80 column (Tosoh Corporation, Japan).

The inventors found that cell cultures expressing monoclonal antibodies or anti-IgE antibodies affinity for the C5 complement component while accelerating perfusion equal to 0.1 * day ^-2 by withdrawing a cell suspension from the bioreactor at a rate of from 0.1 * day ^-1 to 0.15 * day ^-1 , achieved 1.5 - 2 times higher specific productivity than in the control at 0.15 * day ^-2 , coupled with high vitality (more than 95%), and also showed the ability to stay for a long time in the production phase of perfusion cultivation. High specific productivity is achieved by suppressing the proliferative activity of cells. Due to the change in blading, the variation of critical glycans is significantly reduced and the target protein expressed by the cells has a stable glycosation profile throughout the entire cultivation process. This technical result allows to provide high specificity and biological activity of the resulting antibodies, necessary for inclusion in the composition of therapeutic drugs.

The above examples demonstrate the applicability of the method for the production of various types of antibodies in various modes. Using the data and patterns disclosed by the authors in the description, a qualified specialist can easily

can apply this method to produce various types of therapeutic antibodies in eukaryotic cell culture.

Example 1

As an example 1, two perfusion acceleration modes are given — 0.1 * day ^-2 and 0.15 * day ^-2 for a producer of an antibody containing the amino acid sequence of SEQ ID # 1 (eculizumab sequence) and the affinity complement component for the C5 component.

CHO cells expressing this monoclonal antibody were grown in a Climo-Shaker ISF1-XC shaker incubator (Kuhner, Switzerland) in Erlenmeyer flasks on a commercial BalanCD CHO Growth A nutrient medium (Irvine Scientific, USA) containing 6 mM L-glutamine (Applichem , Germany) at 37 ° С, 5% СО ₂ , 90 rpm. The resulting inoculum was introduced into a bioreactor containing a nutrient medium of the same composition. The composition of the perfusion medium: 90% BalanCD CHO Growth A, 10% BalanCD CHO Feed 3 (Irvine Scientific), 6 mM L-glutamine.

Cell perfusion was carried out in Sartorius Flexsafe RM 10L perfusion pro 1,2 my disposable wave-type bioreactors (Sartorius, Germany) with an integrated perfusion membrane. The working volume of the suspension was 3.0 L. Sowing cell concentration - 0.5 ± 0.1 million / ml.

The concentration of dissolved oxygen in the bioreactors was maintained at a level of 50% of saturation. The flow rate of the gas mixture was 0.3 l / min. Also, pH-stating was carried out at a level of 6.9 during the entire cultivation process by supplying CO ₂ or a 5% sodium hydroxide solution. The swing frequency of the platform with the bioreactor was in the range of 22-28 kache / min. The inclination angle of the platform was 6 °. The cultivation temperature in the growth phase is 37 ° C. Upon reaching a perfusion rate of 0.5 * day ^-1 , a temperature was reduced to 34 ° C. The maximum perfusion rate was 1.0 * day ^-1 . Perfusion began on the third day of cultivation.

Two perfusion acceleration modes were used: 0.1 * day ^-2 and 0.15 * day ^-2 . The blading speed was 0.1 * day ^-1 and 0.15 * day ^-1 .

From FIG. 2 and FIG. Figure 4 shows that a lower acceleration value slows down the proliferation of cells and increases their productivity. From FIG. Figure 3 shows that the slowing down of proliferation is accompanied by the preservation of high cell viability.

Example 2

In the next experiment, two cultivations were carried out. The experimental conditions are the same as in example 1, with the exception of the acceleration of perfusion and speed of building. In both cases, the acceleration of perfusion had the same value - 0.1 * day ^-2 ; the blading speed in the first embodiment was 0.1 * day ^-1 , in the second - 0.15 * day ^-1 .

In FIG. 5 and FIG. Figure 6 shows data on the dynamics of changes in the proportion of Man5 and G0 peaks in the glycosylation profile of the target protein, depending on the blading rate used. It can be noted that at a blading speed of 0.15 * day ^{-1, the} proportion of Man5 and G0 peaks does not change more than 2 times during the cultivation process. At the same time, at a blinding rate of 0.1 * day ^-1, there is a negative dynamics in the glycosylation profile of the antibody: the proportion of G0 glycan increased during cultivation by more than 6 times, Man5 - by more than 5 times.

In FIG. 7 shows data on the productivity of two cultivation processes, from which it can be seen that with an increase in the speed of building from 0.1 * day ^-1 to 0.15 * day ^-1 there is no significant drop in the yield of the target protein.

Example 3

In the next experiment, two cultivations were carried out. The experimental conditions are the same as in example 1, except for the used cell line. In this example, a CHO cell line was used expressing a monoclonal anti-IgE antibody containing the amino acid sequence of SEQ ID NO: 2 (omalizumab sequence).

In FIG. 8, it can be seen that when perfusion is accelerated 0.1 * day ^-2 on the 10-12th day, there is a significant suppression of cell proliferation, and the specific growth rate drops to values comparable to the blading speed (about 0.1 * day ^-1 -0.15 * day ^-1 ). With acceleration of perfusion 0.15 * day ^-2, cell proliferation is more intense, and their maximum concentration reaches 67 million / ml, which is 1.5 times higher than the maximum concentration in the process with acceleration of perfusion 0.1 * day ^-2 (43 million / ml). The specific productivity of cells in a process with accelerated perfusion of 0.1 * day ^-2 in the production phase is 2.2 times higher than the specific productivity observed in a process with accelerated perfusion of 0.15 * day ^-2 (Fig. 9).

Claims (17)

1. The method of continuous cultivation of the cell line of the ovary of the Chinese hamster CHO, which is an antibody producer, characterized in that the perfusion acceleration is in the range from 0.1 * day ^-2 to 0.12 * day ^-2 , and the suspension of cells from the bioreactor is carried out with the rate of 0.15 * day ^-1 . 2. The method according to p. 1, where the acceleration of perfusion occurs at a speed of 0.1 * day ^-2 . 3. The method according to p. 1, where the inoculum concentration of cells is 0.5 ± 0.1 million / ml. 4. The method according to any one of paragraphs. 1-3, where the cell line produces an antibody containing the amino acid sequence, which is the sequence of SEQ ID No. 1. 5. The method according to any one of paragraphs. 1-3, where the cell line produces an antibody containing the amino acid sequence, which is the sequence of SEQ ID No. 2. 6. The method according to any one of paragraphs. 1-3, where the cell line produces eculizumab. 7. The method according to any one of paragraphs. 1-3, where the cell line produces omalizumab. 8. The method according to any one of paragraphs. 1-3, where the cell line produces an antibody specific for the C5 complement component. 9. The method according to any one of paragraphs. 1-3, where the cell line produces an antibody specific for IgE. 10. The use of the method according to claim 1 for the production of therapeutic antibodies in the ovary cell line of the Chinese hamster CHO. 11. The use of claim 10, wherein the therapeutic antibody is eculizumab. 12. The use of claim 10, wherein the therapeutic antibody is omalizumab. 13. The use of claim 10, wherein the therapeutic antibody is specific for the C5 complement component. 14. The use of claim 10, wherein the therapeutic antibody is specific for IgE. 15. The use of claim 10, wherein the therapeutic antibody comprises an amino acid sequence that is SEQ ID NO: 1. 16. The use of claim 10, wherein the therapeutic antibody comprises an amino acid sequence that is SEQ ID NO: 2. 17. The use of claim 10, wherein a specific cell productivity of up to 20 pkg / cell / day is achieved.

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