CN115287251A

CN115287251A - Intermittent perfusion combined batch feeding culture

Info

Publication number: CN115287251A
Application number: CN202211027235.0A
Authority: CN
Inventors: 向绍勋; 于乐; 汤自然; 田军; W·王; 周伟昌
Original assignee: Wuxi Yaoming Biotechnology Co ltd
Current assignee: Wuxi Yaoming Biotechnology Co ltd
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2022-11-04

Abstract

A cell culture method by combining intermittent perfusion with batch fed-batch culture mode comprises batch fed-batch culture and alternately performing one-phase or multi-phase perfusion in the middle and later stages to improve the yield and quality of products.

Description

Intermittent perfusion combined batch feeding culture

Technical Field

The invention relates to the field of cell culture, in particular to a cell culture method for improving the yield and quality of a recombinant protein product.

Background

Over the past decade, numerous cell culture models have emerged in the field of recombinant protein production, such as monoclonal antibodies and fusion proteins. Among them, batch fed-batch culture is the most widely adopted method for large-scale production due to its simple operation and scalability. Perfusion culture is another common culture strategy, which requires continuous medium replacement, requires a very large amount of medium, usually requires specialized production plant design to accommodate the formulation and storage of large amounts of medium, and also requires a cell retention system and corresponding downstream processing capabilities to achieve continuous medium replacement and product harvest, but has significant advantages over batch fed-batch culture in terms of improved product yield and quality.

Batch fed-batch cultures usually last around 14 days, with the growth curve going through 4 different phases: latency, exponential, stationary and decline phases. The cell death phase is a direct reflection of poor cell status, usually due to the accumulation of toxic metabolites during cell culture. Environmental conditions resulting from the release of large amounts of proteases, reductases, glycosylases, sialidases, etc. into cell cultures can further exacerbate cell death, possibly leading to protein degradation and changes in protein glycoform structure. Finally, cell death also leads to reduced yields and premature termination of the culture process. In the batch fed-batch culture mode, the batch addition of nutrients can relieve or delay cell death to a certain extent, but cannot fundamentally solve all problems and difficulties.

Currently, medium optimization is commonly used to improve cell viability and product yield. Cell viability and cell status can be better maintained by adjusting the feeding strategy or supplementing additives. However, media optimization strategies are not necessarily universally effective or appropriate for all cell lines. Process parameter optimization, such as cooling strategies, is another method that can be effective in improving cell performance. Again, however, these adjustments are not universally feasible. Another approach to maintaining cell viability is to use genetic engineering methods to inhibit apoptosis, including, for example, overexpression of negative regulators of apoptosis (BCL-2, BCL-xL, and MCL-1), knock-out 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 often requires complex design, implementation and validation procedures. In summary, none of the above methods completely solves the problem of accumulation of harmful by-products, which is a potential root cause of poor cell performance in the middle and late stages of cell culture.

The popularity of perfusion processes has greatly benefited from their ability to extend the cell culture cycle by continuously removing spent media and replenishing fresh media. However, the current methods require expensive investment to construct various necessary facilities, the culture medium preparation work is heavy, and the production cost of the product is high. The treatment of large quantities of waste culture media is also a puzzlement and problem. Therefore, the full perfusion culture is not necessarily suitable for the production of all recombinant proteins.

Thus, there is a need for better cell culture methods, particularly mammalian cell culture methods, that can be more widely used for the production of more recombinant proteins to more effectively improve product yield and quality.

Disclosure of Invention

In general, a novel cell culture method based on the "Intermittent Perfusion combined Batch feeding" (also referred to as "IPFB") mode of the present invention is provided herein to improve the yield, quality and upstream economics of recombinant protein production. In general, the new culture mode is to introduce one or more stages of perfusion operation in the middle and later stages of the culture mode on the basis of the traditional fed-batch mode.

In one aspect, the invention provides a cell culture method comprising fed-batch culture and at least a first perfusion phase therebetween, the first perfusion phase being initiated 0-7 days after cooling down or 0-5 days after the Viable Cell Density (VCD) has risen to a peak.

Alternatively, it is preferred that in some cases the method of the invention further comprises one or more additional transfusions, each of which is initiated 1 to 5 days, e.g. 2, 3 or 4 days, after the end of the previous perfusion.

Alternatively, and preferably, in some instances, the methods of the invention further comprise providing a source of inoculation to the fed-batch culture using a perfusion culture and/or a seed expansion culture that enhances the fed-batch culture.

In some embodiments, the cell is an engineered host cell recombinantly expressing a product of interest, and the method comprises the step of harvesting the expression product. The cell may be a mammalian cell, such as a CHO cell, and the product of interest may be a polypeptide, a monoclonal antibody. Thus, provided herein are methods of producing a product of interest comprising culturing a cell expressing the product of interest using the methods of the invention and harvesting the expressed product of interest.

Compared with the traditional typical perfusion culture, the IPFB culture method can prolong the culture period and better maintain the healthy state of cells, thereby improving the yield and quality of products, reducing the consumption of culture medium and realizing better cost control. In addition, the IPFB mode of the present invention can adapt to various fed-batch culture processes to improve product yield and quality.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, further illustrate the invention and, together with the description, serve to explain the principles of the invention. The invention may be better understood with reference to the following detailed description of one or more of the accompanying drawings.

FIG. 1: schematic diagram of an exemplary architecture of the IPFB method of the present invention.

FIG. 2: timeline for the IPFB method of the present invention.

FIG. 3: in one of the examples below, cell growth curves were run in shake-tube culture with and without medium replacement controls.

FIG. 4: cell viability curves for controls with and without medium replacement in shake tube culture.

FIG. 5: lactate profiles with and without medium replacement control in shake tube culture.

FIG. 6: product yield curves with and without medium replacement control in shake tube culture.

FIG. 7 is a schematic view of: cell growth curves for IPFB and fed-batch culture controls in 3L culture tanks, one of the examples below.

FIG. 8: this example 3 cell viability curves for IPFB culture and fed-batch culture controls in L culture tanks.

FIG. 9: this example 3 lactic acid profile of IPFB culture and fed-batch culture control in L culture tank.

FIG. 10: this example 3 product yield curve for IPFB culture and fed-batch culture controls in L culture tanks.

FIG. 11: this example 3 molecular weight variant related product purity for IPFB culture and fed-batch culture control in L-pot.

FIG. 12: this example 3 charge heteroplasmon-related product purity for IPFB culture and batch fed culture control in L culture tanks.

FIG. 13: cell growth curves for IPFB culture in 3L culture tanks and fed-batch culture controls in one of the following examples, intermittent perfusion rate studies.

FIG. 14 is a schematic view of: this example 3 cell viability curves for IPFB culture and fed-batch culture controls in L culture tanks.

FIG. 15 is a schematic view of: this example 3 lactic acid profile of IPFB culture and fed-batch culture control in L culture tank.

FIG. 16: this example 3 product yield curve for IPFB culture and fed-batch culture controls in L culture tanks.

FIG. 17: cell growth curves for IPFB culture and fed-batch culture controls in 3L culture tanks in a different intermittent perfusion mode study in one of the examples below.

FIG. 18: this example 3 cell viability curves for IPFB culture and fed-batch culture controls in L culture tanks.

FIG. 19: this example 3 lactic acid profile of IPFB culture and fed-batch culture control in L culture tank.

FIG. 20: this example 3 product yield curve for IPFB culture and fed-batch culture controls in L culture tanks.

Detailed Description

Term(s)

As used herein, unless otherwise indicated, the word "a" or "1" preceding a name, quantity, or unit indicates the presence, i.e., a quantity, of at least one, and thus is intended to encompass the plural.

Unless otherwise indicated herein, a value or range of values, whether or not accompanied by the antecedent "about," encompasses approximate ranges equivalent to the value or range of values as would be understood by a person skilled in the relevant art, e.g., ranges of values or endpoints of ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%.

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

As used herein, all numerical ranges include values expressed as "between" the two endpoints, unless otherwise specified. For example, a range expressed as "between 1 and 10" includes the

endpoints

1 and 10.

Herein, when a time period, period or interval is expressed in days, when a time point is expressed in a day or a day, it means that the time period or time is counted or distinguished by day (day), wherein the numerical values do not represent multiples of exactly 24 hours.

Unless defined otherwise herein, technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety into the disclosure herein. The citation is for illustration only of the state of the art to which the present invention pertains and not prior art to the present invention.

Description of the preferred embodiments

The invention introduces one or more perfusion operations in the middle and later stages on the basis of fed-batch culture. FIG. 1 is a schematic diagram of an exemplary flow architecture of the present invention. As shown, the culture vessel (e.g., culture tank) is connected to a retention system, the retained cells (and sometimes the product) are returned to the culture vessel along with the culture medium, and the filtrate containing waste (and sometimes the product) is drained or collected. The culture vessel is simultaneously replenished with fresh medium by means of an infusion device, keeping the culture volume constant.

The traditional batch fed-batch culture adopts discontinuous batch supplement of nutrient components, and does not discharge waste culture medium. Batch fed-batch cultures are usually seeded at a predetermined cell density and then maintained by batch supplementation with nutrients. It is noteworthy that, since batch fed-batch feeding is usually not performed with waste medium replacement, metabolite accumulation tends to generate toxic side effects on cell proliferation or target protein production as cell culture proceeds. In particular, at high cell densities, the mechanisms of apoptosis exacerbate the toxic phenomenon.

In contrast, typical perfusion cultures undergo continuous medium replacement, with fresh and spent media being pumped in and out at the same flow rate, while retaining large amounts of viable cells in the culture. Cell retention means may be employed to retain the cells in culture while the spent media is removed. However, complete perfusion culture is costly. The high cost of media supply, equipment, maintenance, disposal of spent media, etc. is a major problem limiting its applicability.

The intermittent perfusion combined fed batch (IPFB) mode combines the advantages of the two modes, and has the advantages of simple operation of fed batch culture and excellent cell continuity of perfusion culture. Specifically, the medium replacement is alternately introduced once or more times by using a cell retention device in the middle and later stages of the fed-batch culture. The method can reduce toxic and side effects, and prolong cell culture period and increase product yield due to healthier cell state. Medium replacement with a cell retention device for a limited number of times during the middle and late stages of fed-batch culture better maintains cell viability rate and Viable Cell Density (VCD) throughout the culture cycle and also extends the culture and production cycle, all contributing to higher product yields and quality.

Herein, the term "Intermittent Perfusion (IP)" is one or more perfusion segment periods (i.e. a period of time) interspersed in what would otherwise typically be a complete fed-batch culture process, such perfusion segment periods thus alternating with a fed-batch culture process and not constituting a full-time continuous perfusion culture. Since perfusion culture is dominated by replacement of the medium while the cells, and possibly also the product, are retained in the culture tank, herein "intermittent perfusion" and "medium replacement" in the context of IPFB culture may be exchanged synonymously.

Herein, "middle-late" with respect to batch fed cultures and IPFB cultures refers to after cooling or after peak VCD.

In particular, the invention provides a method of cell culture comprising fed-batch culture and at least a first perfusion, wherein the first perfusion is initiated (a) 0-7 days (e.g. 1, 2, 3, 4, 5, 6 days) after cooling down or (b) 0-5 days (e.g. 1, 2, 3, 4 days) after the Viable Cell Density (VCD) has risen to a peak. Therefore, the start of the first-phase perfusion is usually (but not necessarily) determined in accordance with the item (a) in the case of the cooling operation, and is determined in accordance with the item (b) in the case of the absence of the cooling operation.

In some embodiments, the methods of the invention include a cooling operation, depending on the particular cell line or clone. The temperature reduction may be, for example, to below 37 ℃, for example to below 33 ℃ (inclusive), for example 31 ℃, 30 ℃ or less. In some embodiments, the decrease in temperature is performed when the VCD is increased to at least about 50%, at least about 70%, at least about 80%, or at least about 90% of the peak VCD, e.g., increased to about 50% -95%, about 50% -75%, about 70% -90%, or about 85% -95% of the peak VCD. In some embodiments, the temperature reduction is performed on days 0 to 5, e.g.,

days

1, 2, 3, and 4, of the batch fed batch culture process, i.e., the IPFB culture process, with the seeding day being day 0. Early cooling is preferred for high density inoculation. The days and dates herein were counted as the batch fed culture process, i.e., the IPFB culture process, which was based on the day of inoculation as day 0.

The "peak VCD" used to determine the cooling opportunity or first-phase perfusion start may be a previously determined VCD peak, i.e. a peak determined without cooling matching a pre-experiment. It is customary practice to obtain parameters of indicative significance for the actual operation by means of matching preliminary experiments. The pre-measured VCD peak is the same as or very close to the actual peak.

In some embodiments, the first phase of perfusion is initiated after day 2 (inclusive), e.g., 3, 4, 5, 6, 7, 8, 9, 10, or later. For example, the start of first phase perfusion may be on day 2 to day 7 or on day 4 to day 8.

In some embodiments, the methods of the invention comprise multi-phase perfusion. That is, the method of the present invention may further include additional perfusion in one or more stages other than the first perfusion stage, thereby further improving process performance and product quality. The additional perfusion (i.e., the n-phase perfusion) may be started 1 to 5 days after the end of the previous-phase (i.e., the n-1 phase) perfusion, respectively. For example, the method of the present invention may comprise at least one second-stage perfusion starting 1 to 5 days after the first-stage perfusion is ended, and may further comprise at least one third-stage perfusion starting 1 to 5 days after the second-stage perfusion is ended. In some embodiments, the additional perfusion begins 2 days, 3 days, or 4 days after the end of the previous phase. FIG. 2 shows a timeline of the IPFB culture process of the present invention.

In some embodiments, the additional perfusion, e.g., the second perfusion, can be started after day 3, e.g., after day 5, after day 6, after day 7, after day 8, after day 9, after day 10, after day 11, and/or after day 12, all inclusive. In some embodiments, the additional perfusion, e.g., the second-phase perfusion, may begin on days 5 to 12 or days 8 to 12. In some embodiments, the methods of the invention further comprise a third phase of perfusion. The third perfusion period may be started after day 8, for example, after

day

9, 10, 11, and 12, inclusive. In some embodiments, the third perfusion phase begins on day 8 to day 12.

The product yield and quality can be further improved by adding perfusion. It is noteworthy that even though the IPFB method of the present invention involves multi-phase perfusion, the medium usage is still much lower than for typical full perfusion cultures.

The replacement rate of the culture medium in each time in the IPFB culture process can reach 100 percent. In some embodiments, the perfusion rates for each medium replacement, i.e., intermittent perfusion, are each about 0.5 to 6VVT for no more than 72 hours. VVT is the culture tank Volume/Time (Vessel Volume reach Time). This corresponds to a degree of substitution of 39% to 100%, according to: substitution rate = (1-1/e) ^m ) X 100%, "m" = perfusion rate in VVT units. In some embodiments, the rate of each intermittent perfusion is about 1 to about 3VVT each. In some embodiments, the duration of each intermittent perfusion is about 6 to 72 hours each, for example about 12-48 hours or about 24-36 hours.

Intermittent perfusion replaces waste cell-free medium with fresh medium, and cells are retained by a cell retention device connected to the culture tank via an external circuit. Various cell retention techniques and devices used in perfusion culture systems can be employed. In some embodiments, the cut-off value for the cell cut-off is selected or set to be capable of cutting off the cells and the desired product, such as a polypeptide or a monoclonal antibody, in the culture tank. For example, the cut-off value may be below 50kD (inclusive). In some embodiments, cell retention is performed using a hollow fiber tangential flow filtration technique, such as general Tangential Flow Filtration (TFF) or alternating tangential flow filtration (ATF). Preferred for mammalian cells is hollow fiber tangential flow filtration, especially ATF. In some embodiments, the pore size of the hollow fiber tangential flow filtration, that is, the pore size (e.g., nominal pore size) of the filter medium such as a filter element or a filter column used therein, is 50KD or less (inclusive). In some embodiments, intermittent perfusion employs ATF as a cell retention means and device.

The priming density of the batch fed-batch culture of the present invention may be about 0.3X 10 ⁶ To 10X 10 ⁶ About 0.3X 10 cells/mL ⁶ To 50X 10 ⁶ Individual cells/mL, or higher. The seeding density may be, for example, about 5. + -. 1.0X 10 ⁶ Individual cell/mL, about 10. + -. 2.0X 10 ⁶ About 20. + -. 2.0X 10 cells/mL ⁶ About 30. + -. 2.0X 10 cells/mL ⁶ Individual cell/mL, about 40. + -. 2.0X 10 ⁶ Individual cell/mL or about 50. + -. 2.0X 10 ⁶ Individual cells/mL. In some embodiments, about 5. + -. 1.0X 10 is used ⁶ -10±2.0×10 ⁶ High density seeding of individual cells/mL. High density inoculation can be from high density seed culture. High density seed culture can be achieved by enhanced seed amplification such as perfusion or enhanced batch flow. Thus, in some instances, the methods of the invention further comprise providing a source of inoculation to the fed-batch process using perfusion culture and/or seed expansion culture that enhances fed-batch culture.

During the culture, the feeding and carbon source (e.g., glucose) are periodically added as in the typical fed-batch culture. In some embodiments, feeding is performed every third day, which may begin on day 0. In some embodiments, the feed volume per feed is 2% to 4% of the incubation volume, respectively. In some embodiments, the glucose level is maintained at about 3-10g/L.

The method of the invention can be used to harvest more, better biomass and/or target products. In some embodiments, the cultured cells are animal cells, e.g., mammalian cells, e.g., CHO cells.

In some embodiments, cultured is an engineered host cell that recombinantly expresses a product of interest, and the method comprises the step of harvesting the expression product. In some embodiments, the product of interest may be polypeptides and proteins, such as immunoglobulins and monoclonal antibodies or fragments thereof. Thus, also provided herein are methods of producing a product of interest comprising culturing a cell expressing the product of interest using the methods of the invention and harvesting the expressed product of interest.

As used herein, "monoclonal" when modifying an antibody means the uniformity of antibody structure and activity, regardless of the method of antibody preparation. 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.

The present invention solves the common problems and bottlenecks of batch fed-batch culture such as low culture viability, reduced VCD, atypical lactate accumulation and low product yield, providing a more robust and efficient culture process, particularly for mammalian culture. As shown in the examples below, the IPFB culture of the present invention can reduce end-stage lactate accumulation, improve VCD and cell viability persistence, increase product yield, and achieve better product quality. As shown in the examples below, product yield can be improved by at least 30%, and even more than 150%. In terms of scale-up and operability of large-scale biological agent production, the IPFB culture of the present invention greatly reduces the complexity and equipment requirements associated with the large-scale media preparation, storage and waste disposal required for perfusion culture. Furthermore, the cell status in IPFB culture harvest is healthier, which also benefits downstream purification, while also reducing potential safety risks.

Examples

The following examples are intended to further illustrate the invention and are not to be construed as limiting the scope of the invention.

Materials and methods

In the following examples, CHO-K1 cells recombinantly expressing IgG1 monoclonal antibodies of interest were cultured. Unless otherwise stated, the basal medium was Actipro medium (Hyclone product No. Cat No. SH31037) containing 1% HT additive (HT Supplement, gibco product No. Cat No. 11067) and the feed medium was CB7a/CB7b (Hyclone product Nos. Cat No. SH31026 and Cat No. SH31027).

Cell density and Cell viability were monitored by trypan blue dye exclusion with a Vi Cell analyzer (software version Vi Cell XR 2.04). Lactate accumulation and monoclonal antibody (mAb) product titers were monitored with a CedexBio HT analyzer.

Example 1: effect of Medium replacement at different times

The effect of medium replacement at different times was first investigated and the experiments are described in table 1. Experiment No.1 ("ST No. 1") without medium replacement was used as a control.

Table 1: medium replacement in shake tube culture

Cells were seeded at the indicated seeding density into 50ml shake tubes containing 15ml basal medium. The feeds were carried out for the times and ratios indicated in Table 1. Medium replacement was performed by removing the supernatant after centrifugation at 250g for 5 minutes and replacing it with an equal amount of fresh medium. This displacement was close to 100% medium displacement (not counting cell pellet volume), which corresponds to a perfusion rate of about 3VVT used in subsequent culture tank experiments.

Results

As shown in fig. 3 and 4, although the peak VCDs for the different time plating conditions are highly coincident, it is noted that the media-plated cultures exhibited higher VCDs and cell viability and persistence at the later stages, especially after day 10. At the same time, medium replacement also reduced residual lactic acid accumulation (fig. 5). The control culture (ST No. 1) was terminated early due to low cell viability. In contrast, culturing with one or more medium replacements can last longer.

The mAb product titer of the control was finally 2.451g/L, compared to which all cultures with one or more medium replacements were significantly improved (fig. 6). As shown, the titer increases were similar for the single medium replacement on

days

4, 6 and 8, and slightly lower for the single replacement on day 10. And, as shown, the product titer further improved with increasing number of substitutions, with the titer in test No. 8 ("D4, D8, D12-3 VVT") being the highest, up to 6.235g/L. Overall, the introduction of medium replacement can increase product titer by 45% to 154%.

Example 2: IPFB culture in 3L culture pots

The IPFB culture system of this experiment, shown generally in FIG. 1, included an alternating tangential flow filtration (ATF) system as the cell retention device. The experiments are described in table 2.

Table 2: IPFB culture in 3L culture pots

Note:

1: when the VCD is raised to the point, the temperature is reduced to 31.0 DEG C

The inoculation density of the 3L culture tank was 11X 10 ⁶ Individual cells/mL, which benefits from the seed stage using perfusion culture. The culture identified as "D5-3 VVT" started intermittent perfusion at day 5, with a perfusion rate of 3VVT, for 24 hours; "D5, D9-3 VVT" after the first perfusion on day 5 as described above, a second perfusion was performed on day 9 at a perfusion rate of 3VVT for 24 hours.

Results

As shown in fig. 7 and 8, although the peak VCDs for the different culture processes are highly coincident, it is noteworthy that the perfused culture shows higher VCD and cell viability and persistence in the later stages. As shown in fig. 9, the control culture started to develop late lactate level back-rise as early as day 7. In contrast, lactate return was delayed by as much as 4 days for "D5-3 VVT" with one perfusion and by as much as 6 days for "D5, D9-3 VVT" with two perfusions.

FIG. 10 is a time curve of product titer. In the control culture, the titer rose slowly, entering the plateau phase on day 12, and the final titer was 4.866g/L. In contrast, in both IPFB cultures, titers increased rapidly and for a longer period of time, continuing to increase until the end of the culture, with final titers of 7.590g/L and 7.634g/L, respectively, i.e., yields increased by more than 55% and more than 67% over the control, respectively. In addition, as shown, the titer was higher for two intermittent perfusions than for one.

And (3) taking a 3L culture tank harvest sample, purifying by using protein A, and then carrying out product quality detection. As shown in fig. 11 and 12, intermittent perfusion improved the molecular weight variant-related product purity and the charge heteroplasmon-related product purity. 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 increases product yield, but also contributes to improving the quality of the recombinant product.

Table 3: n-glycan results of 3L jar experiments

Example 3: effect of intermittent perfusion Rate

This example further investigates the effect of perfusion rate. IPFB cultures and control cultures at two different perfusion rates in 3L culture tanks were compared. The experiments are described in table 4.

Table 4: perfusion speed research-3L culture tank

Note:

1: when the VCD is raised to this point, the temperature is lowered to 31.0 DEG C

Results

As shown in fig. 13 and 14, despite the high coincidence of peak VCDs, VCDs and cell viability and persistence throughout the production culture of 1VVT and 2VVT intermittently perfused IPFB cultures were significantly enhanced compared to the control. In both IPFB cultures with lower perfusion rates, late lactate elevation was delayed to a similar extent (fig. 15), consistent with what was seen in the previous examples. Later lactic acid elevation is a manifestation of mitochondrial function decline. Medium replacement is able to maintain healthier cellular metabolism as evidenced by delayed late lactate elevation.

Thanks to the healthy cell state, the product titers of both IPFBs continued to rise rapidly until the end of the culture, with a final protein yield 30-40% higher than the control (7.220-7.904 g/L versus 5.536g/L, fig. 16). This study shows that even low-speed IPFB still has a significant competitive advantage over traditional fed-batch culture. Moreover, the IPFB process has the advantage that the IPFB process can be conveniently adjusted to meet the actual requirements of product cost control, production facilities and the like.

Example 4: study of different intermittent perfusion modes

The study evaluated more different IP patterns, including different perfusion rates, durations and/or intervals. The experiments are described in table 5. Wherein, the culture marked as "D5 to D8-6VVT" is performed with one Intermittent Perfusion (IP), starting from day 5, and the 6VVT is continuously operated for 72 hours till day 8; "D4, D6, D9-0.5VVT" was IP 3 times at 4, 6 and 9, respectively, each time 0.5VVT for 6 hours, 2 nd and 3 rd times starting at 2 days and 3 days after the end of the previous time, respectively; "D4, D5, D10-1.0VVT" was IP 3 times at 4, 5 and 10, respectively, each time 1.0VVT for 6 hours, 2 nd and 3 rd times beginning at 1 day and 5 days after the end of the previous time, respectively; "control" was not IP.

Table 5: different IP mode culture in 3L culture tank

Note:

Results

As shown in fig. 17 and fig. 18, both experimental groups showed continuous improvement in VCD and cell viability throughout the production culture compared to the control. As shown in fig. 19, the experimental group showed a delay in late lactate elevation, consistent with that seen in the previous examples.

Thanks to the healthy cell state, the product titer of IPFB cultures continued to rise rapidly until the end of the culture, with final titers of 6.214g/L, 6.340g/L and 6.636g/L, respectively, i.e. protein yields 36% -45% higher than the control (4.583 g/L) (fig. 20). The research shows that the IPFB culture can be flexibly expanded and regulated according to actual requirements, and the cell growth performance and the product yield are obviously improved.

Claims

1. A method of cell culture comprising fed-batch culture and at least a first perfusion, wherein the first perfusion is initiated 0-7 days after a decrease in temperature or 0-5 days after a rise in Viable Cell Density (VCD) to a peak.

2. The method of claim 1, wherein said decreasing is performed when VCD rises to at least 50% of peak value, and/or said decreasing is performed on days 0 to 5 of said fed-batch culture.

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

4. The method according to claim 1, further comprising one or more additional perfusions, each of which is initiated 1 to 5 days after the end of the previous perfusion.

5. The method of claim 4, wherein the one or more phase superfusion is initiated after day 5 or on days 5 to 12 of the fed-batch culture.

6. The method of claim 1, wherein the perfusion rates of the first phase and the chase perfusion are each 0.5-6VVT; and/or the duration of the first phase and the superfusion are each 6 to 72 hours.

7. The method of claim 1, further comprising one or more of the following features:

the perfusion comprises a cell retention device such as an alternating tangential flow filtration system or a tangential flow filtration system;

adopting perfusion culture and/or seed amplification culture of enhanced batch fed-batch culture to provide an inoculation source for the batch fed-batch culture;

the inoculation density of the batch fed-batch culture is 0.3 multiplied by 10 ⁶ Cell/ml to 50X 10 ⁶ Individual cells/ml.

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

9. The method of claim 1, wherein the cell is an engineered host cell recombinantly expressing a product of interest, the method comprising the steps of harvesting the expression product; such as a polypeptide or a monoclonal antibody.

10. A method of producing a product of interest comprising culturing a cell expressing a product of interest using the method of claim 1 and harvesting the expressed product of interest.