Intermittent perfusion combined fed-batch culture

Abstract

The invention provides a method for combining intermittent perfusion with fed-batch culture. The method comprises a fed-batch culture process and one or more periods of intermittent perfusion from a middle period to a later period so as to improve productivity and product quality.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a cell culture method for improving the yield and quality of recombinant protein products. Background Art

[0002] In the past decade, many cell culture modes have emerged in the field of recombinant protein production, such as monoclonal antibodies and fusion proteins. Among them, fed-batch culture has become the most widely used method for large-scale production due to its ease of operation and scalability. Perfusion culture is another commonly used culture strategy. This method requires continuous culture medium replacement and has a very large demand for culture medium. Usually, a special production plant design is required to meet the preparation and storage of a large amount of culture medium. It also requires a cell retention system and corresponding downstream processing capabilities to achieve continuous culture medium replacement and product harvesting. However, it has obvious advantages over fed-batch culture in improving product yield and quality.

[0003] Fed-batch culture usually lasts about 14 days, and the growth curve goes through four different stages: latent phase, exponential phase, stationary phase and cell death phase. The cell death phase is a direct reflection of the poor cell state, usually due to the accumulation of toxic metabolites during the cell culture process. The environmental conditions formed by the large-scale release of proteases, reductases, glycosylases and sialidases into the cell culture will further aggravate cell death, which may lead to protein degradation and changes in protein glycoform structure. Finally, cell death will also lead to reduced yield and premature termination of the culture process. In the fed-batch culture mode, the batch addition of nutrients can alleviate or delay cell death to a certain extent, but it cannot fundamentally solve all problems and difficulties.

[0004] At present, culture medium optimization is commonly used to improve cell viability and product yield. Cell viability and cell state can be better maintained by adjusting the feeding strategy or supplementing additives. However, culture medium optimization strategies may not be universally effective or suitable for all cell lines. Process parameter optimization, such as cooling strategies, is another method that can effectively improve cell performance. However, again, these adjustments are not universally feasible. Another way to maintain cell viability is to use genetic engineering methods to inhibit cell apoptosis, including, for example, overexpression of negative regulators of apoptosis (BCL-2, BCL-xL, and MCL-1), knockout of positive regulators of apoptosis (BAK and BAX), and overexpression of HSP27, HSP70, or both to attenuate apoptosis (Matthew N. Henry et al., Biotechnology and Bioengineering. 2020; 117: 1187–1203). However, genetic engineering of cell lines usually requires complex design, execution, and validation processes. In summary, none of the above methods can completely solve the problem of accumulation of harmful byproducts, which is a potential root cause of poor cell performance in the late stages of cell culture.

[0005] The popularity of perfusion technology is largely due to its ability to extend the cell culture cycle by continuously removing spent culture medium and replenishing fresh culture medium. However, current methods require expensive investment to build various necessary facilities, the preparation of culture medium is arduous, and the product production cost is high. The treatment of large amounts of spent culture medium is also a problem. Therefore, full perfusion culture may not be suitable for the production of all recombinant proteins.

[0006] Therefore, there is a need for better cell culture methods, especially mammalian cell culture methods, so that they can be more widely used in the production of more recombinant proteins to more effectively improve the yield and quality of products. Summary of the invention

[0007] In general, this article provides a new cell culture method based on the "Intermittent Perfusion Fed Batch (IPFB)" model of the present invention to improve the yield, quality and upstream economic benefits of recombinant protein production. Generally speaking, this new culture model is based on the traditional batch feeding model and introduces one or more perfusion operations in the later stage.

[0008] In one aspect, the present invention provides a cell culture method comprising batch feeding culture and at least a first perfusion phase therebetween, wherein the first perfusion phase is initiated 0-7 days after the temperature is lowered or 0-5 days after the viable cell density (VCD) reaches a peak value.

[0009] Optionally and preferably, in some cases, the method of the present invention further comprises one or more additional perfusion phases, wherein each additional perfusion is initiated 1 to 5 days after the end of the previous perfusion phase, for example, 2 days, 3 days or 4 days later.

[0010] Optionally and preferably, in some cases, the method of the present invention further comprises using perfusion culture and / or seed expansion culture of intensified fed-batch culture to provide an inoculum source for the fed-batch culture.

[0011] In some embodiments, the cell is an engineered host cell that recombinantly expresses a target product, and the method includes the step of harvesting the expressed product. The cell can be a mammalian cell, such as a CHO cell, and the target product can be a polypeptide, a monoclonal antibody. Therefore, the present invention provides a method for producing a target product, comprising culturing a cell expressing a target product using the method of the present invention and harvesting the expressed target product.

[0012] Compared with traditional typical perfusion culture, the IPFB culture method of the present invention can extend the culture period and better maintain the health of cells, thereby improving product yield and quality, while reducing culture medium consumption and achieving better cost control. In addition, the IPFB mode of the present invention can be adapted to various batch feed culture processes to improve product yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings are part of the specification and are used to further illustrate the relevant contents of the present invention. The following one or more drawings and detailed description of specific embodiments are helpful for better understanding of the present invention.

[0014] Figure 1 : Schematic diagram of an exemplary architecture of the IPFB method of the present invention.

[0015] Figure 2 : Timeline of the IPFB method of the present invention.

[0016] Figure 3 : One of the following examples is a cell growth curve of a shake tube culture with and without medium replacement.

[0017] Figure 4 : Cell viability curves of shake tube culture with and without medium replacement.

[0018] Figure 5 : Lactate curves of shake tube culture with and without medium replacement.

[0019] Figure 6 : Product yield curves of shake tube culture with and without medium replacement.

[0020] Figure 7 : One of the following examples, cell growth curves of IPFB culture and batch fed culture control in 3L culture tank.

[0021] Figure 8 : Cell viability curves of IPFB culture and fed-batch culture control in the 3L culture tank of this example.

[0022] Fig. 9 : Lactic acid curves of IPFB culture and fed-batch culture control in the 3L culture tank of this example.

[0023] Fig.10 : Product yield curve of IPFB culture and batch fed culture control in 3L culture tank of this example.

[0024] Fig.11 : The molecular weight variant-related product purity of the IPFB culture and batch fed culture control in the 3L culture tank of this example.

[0025] Fig.12 : The purity of the charge variant-related products of the IPFB culture and the fed-batch culture control in the 3L culture tank of this example.

[0026] Fig.13 : Cell growth curves of IPFB culture and batch fed culture control in 3L culture tank in intermittent perfusion rate study in Example 1 below.

[0027] Fig.14 : Cell viability curves of IPFB culture and fed-batch culture control in the 3L culture tank of this example.

[0028] Fig.15 : Lactic acid curves of IPFB culture and fed-batch culture control in the 3L culture tank of this example.

[0029] Fig.16 : Product yield curve of IPFB culture and batch fed culture control in 3L culture tank of this example.

[0030] Fig.17 : The cell growth curves of IPFB culture and batch fed culture control in 3L culture tanks in different intermittent perfusion mode studies in Example 1 below.

[0031] Fig.18 : Cell viability curves of IPFB culture and fed-batch culture control in the 3L culture tank of this example.

[0032] Fig.19 : Lactic acid curves of IPFB culture and fed-batch culture control in the 3L culture tank of this example.

[0033] Fig. 20 : Product yield curve of IPFB culture and batch fed culture control in 3L culture tank of this example. DETAILED DESCRIPTION

[0034] the term

[0035] Herein, unless otherwise specified, “a” or “1” before a name, quantifier or unit indicates the presence or quantity of at least one, thus encompassing a plural meaning.

[0036] Herein, unless otherwise specified, a numerical value or numerical range, whether or not preceded by the word "about", covers an approximate range equivalent to the numerical value or numerical range that can be understood by a technician in the relevant field, such as ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% of the numerical value or end value.

[0037] Herein, unless otherwise specified, the technical features in different implementations may be combined with each other, and the technical solutions or implementations thus formed still belong to the technical solutions or implementations of the present invention.

[0038] Herein, unless otherwise specified, all numerical ranges, including numerical ranges expressed with "between" the end values, include the end values. For example, the range expressed with "between 1 and 10" includes the end values ​​1 and 10.

[0039] In this article, when a duration, period or interval is expressed in days, and when a time point is expressed as a day or a certain day, it means that the duration or moment is measured or divided by days (days), and the numerical value does not represent an exact multiple of 24 hours.

[0040] Unless otherwise specified herein, the meanings of technical and scientific terms used are the same as those commonly understood by those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety to constitute the disclosure of this document. The citations are only used to illustrate the technical level of those skilled in the art and not the prior art prior to the present invention.

[0041] Specific implementation method description

[0042] The present invention introduces one or more perfusion operations in the middle and late stages on the basis of batch fed-batch culture. Figure 1 Schematic diagram of an exemplary process architecture of the present invention. As shown in the figure, a culture container (such as a culture tank) is connected to a retention system, and the retained cells (sometimes also including products) are returned to the culture container together with the culture medium, and the filtrate containing waste (sometimes also including products) is discharged or collected. The infusion device simultaneously replenishes fresh culture medium to the culture container to keep the culture volume constant.

[0043] Traditional fed-batch culture uses discontinuous batch replenishment of nutrients without discharging spent culture medium. Fed-batch culture is usually inoculated at a predetermined cell density, and then the culture is maintained by adding nutrients in batches. It is worth noting that since fed-batch culture usually does not replace spent culture medium, as cell culture proceeds, metabolite accumulation often has toxic side effects on cell proliferation or target protein production. In particular, at high cell density, cell apoptosis mechanisms can exacerbate poisoning.

[0044] In contrast, typical perfusion cultures perform continuous medium replacement, with fresh and spent medium pumped in and out at the same flow rate, while retaining a large number of viable cells in the culture. Cell retention techniques can be used to retain cells in the culture while removing spent medium. However, full perfusion cultures are expensive. The high costs of medium supply, equipment, maintenance, and spent medium disposal are the main issues limiting their applicability.

[0045] The intermittent perfusion combined with batch feeding (IPFB) mode of the present invention combines the advantages of the two modes, having both the ease of operation of batch feeding culture and the excellent cell persistence of perfusion culture. Specifically, a cell retention device is used to alternately introduce one or more culture medium replacements in the middle and late stages of batch feeding culture. This method can reduce toxic side effects, and since the cells are in a healthier state, the cell culture cycle can be further extended and product yield can be increased. Using a cell retention device for a limited number of culture medium replacements in the middle and late stages of batch feeding culture can better maintain cell viability and viable cell density (VCD) throughout the culture cycle, and can also extend the culture and production cycles, all of which help to obtain higher product yields and quality.

[0046] Herein, the term "intermittent perfusion (IP)" refers to one or more perfusion periods (i.e., a period of time) interspersed in a typical complete batch feed culture process, and such perfusion periods are therefore alternated with the batch feed culture process and do not constitute a full continuous perfusion culture. Since perfusion culture is mainly based on replacing the culture medium, while cells and products can also be retained in the culture tank, herein, "intermittent perfusion" and "medium replacement" with respect to IPFB culture are interchangeable synonymously.

[0047] Herein, for fed-batch culture and IPFB culture, “mid-late stage” refers to after the temperature drop or after the peak VCD.

[0048] Specifically, the present invention provides a method for cell culture, comprising batch feeding culture and at least a first phase of perfusion, wherein (a) the first phase of perfusion is started 0-7 days (e.g., 1, 2, 3, 4, 5, 6 days) after the temperature is lowered or (b) 0-5 days (e.g., 1, 2, 3, 4 days) after the viable cell density (VCD) reaches a peak value. Therefore, when there is a temperature lowering operation, the start of the first phase of perfusion is usually (not necessarily) determined according to item (a), and when there is no temperature lowering operation, it is determined according to item (b).

[0049] In some embodiments, the method of the present invention includes a cooling operation, depending on the specific cell line or clone. The cooling operation can be, for example, to below 37°C, for example, to below 33°C (including this number), for example, 31°C, 30°C or lower. In some embodiments, cooling is performed when VCD rises to at least about 50%, at least about 70%, at least about 80% or at least about 90% peak VCD, for example, cooling is performed when it rises to about 50%-95%, about 50%-75%, about 70%-90% or about 85%-95% peak. In some embodiments, cooling is performed on days 0 to 5 of the batch feed culture process, i.e., the IPFB culture process, such as the 1st, 2nd, 3rd, and 4th days, and the culture process is based on the inoculation day as the 0th day. Early cooling is preferred during high-density inoculation. The days and dates herein are counted by the batch feed culture process, i.e., the IPFB culture process, and the process is based on the inoculation day as the 0th day.

[0050] The "peak VCD" used to determine the timing of cooling or the start of the first phase of perfusion can be a pre-determined VCD peak, that is, a peak value determined in a pre-test without cooling. It is common practice to obtain parameters that are indicative of actual operation by matching pre-tests. The pre-determined VCD peak is the same as or very close to the actual peak.

[0051] In some embodiments, the first perfusion is initiated after day 2 (inclusive), such as day 3, 4, 5, 6, 7, 8, 9, 10 or later. For example, the first perfusion may be initiated on a day between day 2 and day 7 or a day between day 4 and day 8.

[0052] In some embodiments, the method of the present invention includes multi-phase perfusion. That is, the method of the present invention may also include one or more additional perfusions in addition to the aforementioned first phase perfusion, so as to further improve process performance and product quality. The additional perfusion (i.e., n-phase perfusion) may start 1 to 5 days after the end of the previous phase (i.e., n-1 phase) perfusion. For example, the method of the present invention may include at least one second phase perfusion, and may also include at least one third phase perfusion, wherein the second phase perfusion starts 1-5 days after the end of the first phase perfusion, and the third phase perfusion starts 1-5 days after the end of the second phase perfusion. In some embodiments, each additional perfusion starts 2 days, 3 days, or 4 days after the end of the previous phase. Figure 2 Shown is a timeline of the IPFB culture process of the present invention.

[0053] In some embodiments, additional perfusion, such as phase II perfusion, can start after the 3rd day, such as after the 5th day, after the 6th day, after the 7th day, after the 8th day, after the 9th day, after the 10th day, after the 11th day and / or after the 12th day, all of which are inclusive. In some embodiments, additional perfusion, such as phase II perfusion, can start on a day between the 5th and 12th day or between the 8th and 12th day. In some embodiments, the method of the present invention further comprises a third phase perfusion. The third phase perfusion can start after the 8th day, such as after the 9th day, after the 10th day, after the 11th day, after the 12th day, all of which are inclusive. In some embodiments, the third phase perfusion starts on a day between the 8th and 12th day.

[0054] Additional perfusion can further improve product yield and quality. It is worth noting that even if the IPFB method of the present invention includes multiple perfusion phases, the amount of culture medium used is still much lower than that of typical full perfusion culture.

[0055] The medium replacement rate during the IPFB culture process of the present invention can be as high as 100%. In some embodiments, the perfusion rate of each medium replacement, i.e., intermittent perfusion, is about 0.5-6VVT, and the duration does not exceed 72 hours. VVT is the volume of the culture tank / time (Vessel Volume each Time). This is equivalent to a replacement rate of 39%-100%, according to: Replacement rate = (1-1 / e m )×100%, "m"=perfusion rate in VVT. In some embodiments, the rate of each intermittent perfusion is about 1-3 VVT. In some embodiments, the duration of each intermittent perfusion is about 6 to 72 hours, such as about 12-48 hours or about 24-36 hours.

[0056] Intermittent perfusion replaces the waste-free cell-free waste culture medium with fresh culture medium, and the cells are trapped by a cell retention device connected to the culture tank through an external loop. Various cell retention techniques and devices used in perfusion culture systems can be used. In some embodiments, the cutoff value of cell retention is selected or set to be able to retain cells and target products such as polypeptides or monoclonal antibodies in the culture tank. For example, the cutoff value can be 50KD or less (including this number). In some embodiments, hollow fiber tangential flow filtration technology such as general tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) is used to retain cells. Hollow fiber tangential flow filtration is preferred for mammalian cells, especially ATF. In some embodiments, the pore size of the hollow fiber tangential flow filtration, i.e., the pore size (e.g., nominal pore size) of the filter material such as the filter element or filter column used, is 50KD or less (including this number). In some embodiments, intermittent perfusion uses ATF as a cell retention means and device.

[0057] The starting inoculation density of the fed-batch culture in the present invention can be about 0.3×106 Up to 10×10 6 cells / mL, about 0.3×10 6 Up to 50×10 6 The seeding density can be, for example, about 5±1.0×10 6 cells / mL, about 10±2.0×10 6 cells / mL, about 20±2.0×10 6 cells / mL, about 30±2.0×10 6 cells / mL, about 40±2.0×10 6 cells / mL or about 50±2.0×10 6 In some embodiments, about 5±1.0×10 6 -10±2.0×10 6 The high-density inoculation of 10 cells / mL can be carried out by high-density seed culture. The high-density seed culture can be achieved by enhanced seed expansion such as perfusion or enhanced batch feeding. Therefore, in some cases, the method of the present invention also includes seed expansion culture using perfusion culture and / or enhanced batch feeding culture to provide an inoculation source for the batch feeding process.

[0058] During the cultivation process, feed and carbon source (e.g., glucose) are added regularly, as in typical fed-batch culture. In some embodiments, feed is added once every two days, which can start from day 0. In some embodiments, the feed volume each time is 2%-4% of the cultivation volume. In some embodiments, the glucose level is maintained at about 3-10 g / L.

[0059] The method of the present invention can be used to harvest more and better biomass and / or target products. In some embodiments, the cultured cells are animal cells, such as mammalian cells, such as CHO cells.

[0060] In some embodiments, the engineered host cells expressing the target product are cultured, and the method includes the step of harvesting the expressed product. In some embodiments, the target product can be a polypeptide and a protein, such as an immunoglobulin and a monoclonal antibody or a fragment thereof. Therefore, the present invention also provides a method for producing the target product, comprising using the method of the present invention to culture cells expressing the target product and harvesting the expressed target product.

[0061] As used herein, "monoclonal" when modifying an antibody refers to the homogeneity of the antibody structure and activity, and has nothing to do with the method by which the antibody is prepared. Monoclonal antibodies can be monovalent or multivalent (e.g., bivalent), and can be monospecific or multispecific (e.g., bispecific). Antibody fragments include, for example, (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, Fc fragments, heavy chain variable region (HV) fragments, single-chain antibody fragments (e.g., single-chain variable region fragments scFv), single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments, and the like.

[0062] The present invention solves common problems and bottlenecks in batch fed-batch culture, such as low culture viability, decreased VCD, atypical lactic acid accumulation, and low product yield, and provides a more robust and efficient culture process, especially mammalian culture. As shown in the examples below, the IPFB culture of the present invention can reduce terminal lactic acid accumulation, improve VCD and cell viability sustainability, increase product yield, and obtain better product quality. As shown in the examples below, product yield can be increased by at least 30%, or even more than 150%. In terms of the scale and operability of large-scale biological preparation production, the IPFB culture of the present invention greatly reduces the complexity and equipment requirements associated with the preparation, storage, and waste disposal of a large amount of culture medium required for perfusion culture. In addition, the cell state in the IPFB culture harvest is healthier, which is also of great benefit to downstream purification, while also reducing potential safety risks.

[0063] Example

[0064] The following examples are only used to further illustrate the present invention and are not intended to limit the scope of the present invention.

[0065] Materials and methods

[0066] In the following examples, CHO-K1 cells expressing the recombinant target IgG1 monoclonal antibody were cultured. Unless otherwise specified, the basal medium was Actipro medium (Hyclone product number Cat No. SH31037) containing 1% HT supplement (HT Supplement, Gibco product number Cat No. 11067), and the feed medium was CB7a / CB7b (Hyclone product number Cat No. SH31026 and Cat No. SH31027).

[0067] Cell density and cell viability were monitored by trypan blue dye exclusion using a Vi Cell analyzer (software version Vi Cell XR 2.04), and lactate accumulation and monoclonal antibody (mAb) product titer were monitored using a CedexBio HT analyzer.

[0068] Example 1: Effect of medium replacement at different times

[0069] First, the effect of medium replacement at different times was studied, and the experiments are described in Table 1. Experiment No. 1 ("ST No. 1") without medium replacement was used as a control.

[0070] Table 1: Medium replacement in shake tube culture

[0071]

[0072] Cells were inoculated at the indicated inoculum density into 50 ml shake tubes containing 15 ml of basal medium. Feeds were made at the times and ratios indicated in Table 1. The medium was replaced by centrifugation at 250 g for 5 minutes, removing the supernatant and replacing it with an equal amount of fresh medium. This replacement was close to 100% medium replacement (excluding the volume of the cell pellet), which is equivalent to a perfusion rate of about 3 VVT used in subsequent culture tank experiments.

[0073] result

[0074] like Figure 3 and Figure 4 As shown in Figure 2, although the peak VCD of the medium replacement conditions at different times overlapped, it is worth noting that the culture with medium replacement showed higher VCD and cell viability and persistence in the later stage, especially after day 10. At the same time, medium replacement also reduced the accumulation of residual lactate ( Figure 5 ). The control culture (ST No. 1) was terminated early due to low cell viability. In contrast, the cultures that underwent one or more medium changes were able to continue for a longer period of time.

[0075] The final titer of the mAb product of the control was 2.451 g / L, and the cultures with one or more medium replacements showed significant improvements compared to this ( Figure 6 ). As shown in the figure, the titer increase of single medium replacement on days 4, 6, and 8 was similar, and the increase of single replacement on day 10 was slightly lower. Also, as shown in the figure, the product titer further increased with the increase in the number of replacements, among which the titer of test No. 8 ("D4, D8, D12-3VVT") was the highest, reaching 6.235 g / L. In general, the introduction of medium replacement can increase the product titer by 45% to 154%.

[0076] Example 2: IPFB culture in 3L culture tank

[0077] The IPFB culture system of this experiment is roughly as follows Figure 1 As shown, an alternating tangential flow filtration (ATF) system was included as a cell retention device. The experiment is described in Table 2.

[0078] Table 2: IPFB culture in 3L culture tanks

[0079]

[0080] Note:

[0081] 1: When VCD rises to this point, the temperature drops to 31.0℃

[0082] The inoculation density of the 3L culture tank is 11×10 6 The culture labeled “D 5–3VVT” started intermittent perfusion on day 5 at a rate of 3VVT for 24 hours; the culture labeled “D 5, D 9–3VVT” started intermittent perfusion on day 5 at a rate of 3VVT for 24 hours, and the culture labeled “D 5, D 9–3VVT” started intermittent perfusion on day 9 at a rate of 3VVT for 24 hours after the first perfusion on day 5 as described above.

[0083] result

[0084] like Figure 7 and Figure 8 As shown in Figure 2, although the peak VCDs of different culture processes were highly coincident, it is noteworthy that the perfused culture showed higher VCD and cell viability and persistence in the later stages. Fig. 9 As shown in Figure 3, control cultures began to show a late lactate recovery as early as day 7. In contrast, lactate recovery was delayed by as much as 4 days in the “D 5–3VVT” with one perfusion and by as much as 6 days in the “D 5,D 9–3VVT” with two perfusions.

[0085] Fig.10 The time curve of product titer. In the control culture, the titer rose slowly, entered the plateau phase on the 12th day, and the final titer was 4.866g / L. In contrast, in the two IPFB cultures, the titer rose rapidly and the rising period was longer, and continued to rise until the end of the culture. The final titers were 7.590g / L and 7.634g / L, respectively, that is, the yield increased by more than 55% and more than 67% compared with the control. In addition, as shown in the figure, the titer of the two intermittent perfusions was higher than that of the one.

[0086] Take a sample of the harvest from the 3L culture tank and purify it with protein A for product quality testing. Fig.11 and Fig.12 As shown in Table 3, intermittent perfusion improved the purity of products related to molecular weight variants and charge heterogeneity. In addition, the results of N-glycan analysis are shown in Table 3. The N-glycan status of the three experimental culture modes was similar. The above results show that IPFB not only improves product yield, but also helps to improve the quality of recombinant products.

[0087] Table 3: N-glycan type results of 3L culture tank experiment

[0088]

[0089] Example 3: Effect of intermittent perfusion rate

[0090] This example further studies the effect of perfusion rate. IPFB culture and control culture at two different perfusion rates in a 3L culture tank were compared. The experiment is described in Table 4.

[0091] Table 4: Perfusion rate study - 3L culture tank

[0092]

[0093] Note:

[0094] 1: When VCD rises to this point, the temperature drops to 31.0℃

[0095] result

[0096] like Fig.13 and Fig.14 As shown in Figure 2, despite the high overlap in peak VCD, the VCD and cell viability and persistence of IPFB cultures produced during the entire culture period were significantly enhanced in the 1VVT and 2VVT intermittent perfusion IPFB cultures compared to the control. The late lactate rise was delayed to a similar extent in both IPFB cultures using the lower perfusion rate ( Fig.15 ), which is consistent with what was seen in the previous examples. The increase in lactate in the late stage is a manifestation of mitochondrial function decline. According to the delay in the increase in lactate in the late stage, it can be seen that medium replacement can maintain healthier cell metabolism.

[0097] Thanks to the healthy cell state, the product titers of the two IPFBs continued to rise rapidly until the end of the culture, and the final protein yield was 30-40% higher than that of the control (7.220 g / L-7.904 g / L compared with 5.536 g / L, Fig.16 ). This study shows that even low-speed IPFB still has significant competitive advantages over traditional batch feeding. Moreover, the advantage of IPFB process is that it can be easily adjusted to meet the actual needs of product cost control and production facilities.

[0098] Example 4: Study of different intermittent perfusion modes

[0099] In this study, more different IP modes were evaluated, including different perfusion rates, durations, and / or intervals. The experiments are described in Table 5. Among them, the cultures marked as "D5 to D8-6VVT" underwent an intermittent perfusion (IP) starting on day 5, and 6VVT continued for 72 hours until day 8; "D4,D6,D9-0.5VVT" underwent 3 IPs on days 4, 6, and 9, each with 0.5VVT for 6 hours, and the second and third IPs started 2 and 3 days after the end of the previous IP; "D4,D5,D10-1.0VVT" underwent 3 IPs on days 4, 5, and 10, each with 1.0VVT for 6 hours, and the second and third IPs started 1 day and 5 days after the end of the previous IP; "Control" did not undergo IP.

[0100] Table 5: Different IP modes of culture in 3L culture vessels

[0101]

[0102] Note:

[0103] 1: When VCD rises to this point, the temperature drops to 31.0℃

[0104] result

[0105] like Fig.17 and Fig.18 As shown, the experimental groups showed continuous improvement in VCD and cell viability throughout the production culture compared to the control. Fig.19 As shown, the experimental group showed a delayed increase in lactate in the late stage, which is consistent with what was seen in the previous examples.

[0106] Thanks to the healthy cell state, the titer of the product cultured by IPFB continued to rise rapidly until the end of the culture, and the final titers were 6.214 g / L, 6.340 g / L and 6.636 g / L, respectively, that is, the protein yield was 36%-45% higher than that of the control (4.583 g / L). Fig. 20 ). This study shows that the IPFB culture of the present invention can be flexibly expanded and adjusted according to actual needs, and significantly improves cell growth performance and product yield.

Claims

1. A method for culturing cells, comprising a fed-batch culture process and comprising at least a first perfusion phase, wherein the first perfusion phase is initiated 0-7 days after the temperature is changed, or the first perfusion phase is initiated 0-5 days after the viable cell density (VCD) reaches a peak value.

2. The method of claim 1, wherein the temperature change is performed when the VCD rises to at least 50% of the peak value.

3. The method of claim 1, wherein the temperature change is performed on day 0 to day 5 of the fed-batch culture process.

4. The method of claim 1, wherein the first phase perfusion is initiated on or after day 2, or on day 2 to day 7, of the fed-batch culture process.

5. The method according to claim 1, further comprising one or more additional perfusion phases, wherein the additional perfusion phases are respectively started 1 to 5 days after the end of the previous perfusion phase.

6. The method of claim 5, wherein the one or more additional perfusion phases are initiated on or after the 5th day, or on the 5th to 12th day, of the fed-batch culture process.

7. The method of claim 1, wherein the perfusion comprises a retention device, such as an alternating tangential flow filtration (ATF) system or a tangential flow filtration (TFF) system.

8. The method of claim 1, wherein the perfusion rates of the first phase and the one or more additional phases of perfusion are each independently 0.5-6 VVT.

9. The method of claim 1, wherein the running time of the first phase and the one or more additional perfusion phases is independently 6 to 72 hours.

10. The method of claim 1, wherein the method further comprises a seed expansion stage to provide inoculation to the fed-batch culture process, the seed expansion stage comprising perfusion culture and / or intensified fed-batch culture.

11. The method of claim 1, wherein the fed-batch culture process is carried out with an inoculation density of 0.3×10 6 cells / ml to 50×10 6 Start with an inoculum of 10 cells / ml.

12. The method of claim 1, wherein the cell is a mammalian cell.

13. The method of claim 1, wherein the cell is a host cell transformed to recombinantly express a product of interest, and the method further comprises the step of harvesting the expression product. The method of claim 13 , wherein the target product is a polypeptide or a monoclonal antibody.

15. A method for producing a target product, comprising culturing cells expressing the target product according to the method of claim 1 and harvesting the expressed target product.

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