June 30, 2022

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Nutrient-rich production medium of CHO cell line development platform-based

Nutrient-rich production medium of CHO cell line development platform-based

Nutrient-rich production medium of CHO cell line development platform-based.

Abstract:

With the help of nutrients for the expression of exogenous protein cells, a large amount of newly developed medium for mammalian cell culture is prepared, but this medium is generally limited to host cell culture, transfection, and cell cloning under low cell density conditions. And the growth of cells. In many cases, a suitable platform medium is the first choice for cell line development and then replaced with a rich medium during production. This study demonstrates a rich chemically defined medium for Chinese Hamster Ovary Cells (CHO), which can be used for both cell line development and the final development of culture processes.

Compared with the poorly prepared medium, the nutrient-prepared medium can be used for transfection, semi-solid medium optimization, mini-pool screening and single-cell cloning medium development, and can be supplemented in batches. The material culture mode obtains the final clone with high expression. The method developed in this research can abandon the need for cells to adapt to the production medium after the development of the cell line, and reduce the clonal problems associated with medium replacement. In addition, the established method has advantages over traditional methods, including saving resources and reducing the work and time of production process optimization.

 

Introduction of Nutrient-rich production medium of CHO cell line development platform-based

The cell culture medium is essential to provide nutrients for the growth of mammalian cells (such as Chinese hamster ovary cells (CHO)). The application of appropriate culture medium is essential for the development of high productivity and high quality cell lines and culture processes. Many culture media developed in the past few decades have caused changes in cell line development processes.

Generally, according to the composition of the medium, the medium is divided into serum medium (SM), serum-free medium (SFM), protein-free medium (PFM) and chemically defined medium (CDM) types. The development of traditional cell lines is carried out by adding serum to the classic medium, which brings problems to the development of the production process, such as low productivity (less than 100 mg/L) due to nutrient deficiency in the medium, and adherent cell culture technology Development issues and related issues arising from the use of serum.

The use of protein-free medium (PFM) allows cell lines to adapt to SFM to improve productivity, for suspension cell culture, and to solve the problem of serum use (Fig.1a). Current cell line development usually adopts SFM-adapted host cells and domesticates the cells under serum-free conditions to simplify process development and reduce the time for later cells to adapt to SFM (Fig.1b).

However, technically speaking, this requires optimizing the host cell medium, transfection medium, selection medium, and cloning medium used in each step of cell line development. Therefore, the use of SFM to establish a new cell line development platform will consume a lot of resources in each step to optimize the medium, and in a specific step, a medium different from the basic platform medium may be used.

Nutrient-rich production medium of CHO cell line development platform-based

Nutrient-rich production medium of CHO cell line development platform-based

Fig 1 Typical cell line development (CLD) workflow with media development

  • (A) Traditional cell line development: Cell line selection adopts classic medium (containing serum), and then domesticated to (serum-free) commercial medium;
  • (B) The latest changes in CLD: host cells adapt to commercial culture media, while the whole process of cell line development uses commercial media, but transfection and cloning require special media, and production also requires rich production media (the latest commercial Chemical medium) to achieve higher productivity
  • (C) The cell line development of this research plan: The rich production medium (the latest commercial medium) is used as the platform medium during the whole research process, thereby minimizing the use of different medium

Currently, newly developed cell culture processes tend to use the latest CHO medium rich in nutrients for development and final production in bioreactors, which can increase the possibility of high productivity. Generally, it is difficult to publicly obtain the formula components of commercial media, and even in the field of media development, it is very limited for individuals to obtain them. However, amino acids are generally a good indicator of the richness of the medium, and amino acids can be detected during cell culture.

This study evaluated the amino acid content in the culture medium described in the article or analyzed by the industry, and determined the growth trend of the medium amino acid content over time, which is usually related to the increase in productivity (Table 1). Therefore, in this study, a new classification of media was made in order to distinguish between traditional media (the media lacking nutritional supplements and insufficient for culturing cells), the earlier commercial media (relatively less nutritious) and the latest Commercial medium (relatively rich in nutrients).

Table 1 Medium characteristics and amino acid composition range
        

In most cases, cell line development is carried out on a relatively poor platform medium (earlier commercial medium), and then changed to a new, more nutritious medium (the latest commercial medium) during expression production. However, the FDA’s latest regulatory views emphasize the importance of cell line clonality. It is well known that the genome of CHO cells is unstable and heterogeneous, which can lead to frequent genotypic variation and phenotypic instability.

Therefore, regulatory agencies worry that this may lead to unstable process repeatability or consistency. Therefore, it is necessary to ensure that the production cell line is isolated from a single cell, and cloned during cell line development. In addition, the FDA recently pointed out that cells should be adapted to serum-free or suspension culture conditions for cloning, and the cloned cells need to undergo a new round of monoclonalization after adapting to the new conditions. In addition, the latest report also pays close attention to the genetic diversity of cell clones, and spontaneous mutations in CHO cells can occur quickly in a short period of time (even if cells divide, there is a risk of diversity).

In order to reduce this risk, this study tried to establish a new platform, using the latest commercial CHO medium from the beginning of cell line development, minimizing the replacement of the medium (Fig.1c). In the past, after establishing cell lines with poor media, they usually switched to richer media for production. However, in this study, after pre-adapting CHO host cells to each candidate production medium, each candidate production medium was used in the entire workflow for cell line development, and the selected clones were evaluated in the process.

 

Materials and Method

Cells and media

CHO-DG44 cells were obtained from Dr. Chasin (Columbia University, New York, USA), and subjected to serum-free adaptation in CDM4CHO (GE Healthcare, Bjokgatan, Uppsala, Sweden), and finally used as host cells for cell line development. CDM4CHO and two other chemically defined media HyCell CHO (GE Healthcare, Bjokgatan, Uppsala, Sweden) and ActiPro (GE Healthcare, Bjokgatan, Uppsala, Sweden) were tested as the basic medium for the development of IgG-producing cell lines. For convenience, CDM4CHO is named CDM1, HyCell CHO is named CDM2, and ActiPro is named CDM3.

Before the study, the host cells adapted to CDM1 were subcultured in CDM2 and CDM3 for about 20 times, and then used for comparative study of cell line development basic medium. Each medium was supplemented with 100x hypoxanthine and thymidine (HT) supplements (Thermo Fisher Scientific, Waltham, MA, US) at a concentration of 2x to culture host cells, and 6 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, US).

 

Transfection and cell culture

In the process of cell line development, the host cells are acclimated to the culture medium (CDM1, CDM2 or CDM3) at the seeding cell density (3~5×105cells/S, passage once every 2~3 days), and suspended in shake flasks (Merck KGaA, Darmstadt, Germany) subculture (140 rpm, 37°C, 5% CO2) until the cells grow normally (viability> 95%). When 1 mg/mL polyethyleneimine Mw 40,000 Da (Polyscience, Inc, Warrington, PA, USA) was used to transfect the IgG-expressing plasmid, the medium was temporarily changed to OptiPRO SFM (Thermo Fisher Scientific, Waltham, MA, US).

Then transfer the transfection pool to the initial basal medium (CDM1, CDM2 or CDM3), and culture in suspension for 48 hours in the presence of HT. After 48 hours, the cells were used for mini-pool evaluation or ClonePix2 (Molecular Devices, San Jose, CA, USA) experiment. Cells were further cultured without HT, and 20 nM methotrexate (MTX) was added until the viability was fully restored (>90%), and finally the productivity of the transfection pool was tested.

 

Clone screening and IgG quantification

The productivity of mini-pool or ClonePix2 screening clones was evaluated by Octet®QKe system (ForteBio, Fremont, CA, USA). Inoculate 1000 to 4000 transfected cells per well into a 96-well plate and culture in a 37°C, 5% CO2 incubator for 2-3 weeks to form a mini-pool. During the culture process, 20 nM MTX was added to the basal medium to promote the amplification of the target gene. Detect the IgG productivity of the mini-pool supernatant by using the Octet®QKe system.

In the case of ClonePix2 evaluation, the transfected cells were plated in a semi-solid medium and cultured in a 37°C, 5% CO2 incubator for 12-16 days. The semi-solid medium book is prepared by mixing concentrated basal medium (2x CDM1, 1.5x (CDM2 and CDM3)) and CloneMatrix reagent (Molecular Devices, San Jose, CA, USA). The final formula contains 20 nM MTX and recombinant CloneDetect reagent (Molecular Devices, San Jose, CA, USA; a fluorescein-labeled antibody with high affinity and specificity for human IgG), inoculated to 20,000 transfected cells/mL to a 6-well plate in.

While expressing the target IgG, the transfected cells will form clones with the same fluorescent spots in the semi-solid medium, and then select the strong fluorescent clones, transfer them to a 96-well plate, and use the Octet QKe system to detect and culture 3 ~7 days of supernatant.

The mini-pool or ClonePix2 screening clones cultured in 96-well plates are further expanded in 24-well and 6-well plates, and inoculated into 6-well plates at a fixed seeding density. After incubating at 5% CO2 and 37°C for 3 days, test Final productivity to obtain the clone with the highest IgG expression. The CEDEX HiRes analyzer (Roche, Mannheim, Germany) was used to measure the cell density during the process, and the specific cell productivity was calculated based on the Octet®QKe results.

Finally, single cells were isolated by limiting dilution cloning (LDC), and single clones were analyzed using Octet QQe in 96-well plates and 6-well plates to identify and screen candidate cell lines. The cell images were analyzed using a clone selection imager (Molecular Devices, San Jose, CA, USA) to observe wells with single isolated cells and growing into cell clusters.

 

Final clone evaluation

Use shake flasks in batch culture mode, or use Ambr® 15 micro bioreactor (Sartorius, Royston, UK) in fed-batch culture mode to test the final clones selected from each medium. During the batch culture, glucose was added to maintain the glucose concentration above about 2 g/L. For the fed-batch culture mode, follow the manufacturer’s recommendations and procedures, adding 4% Cell Boost 7a and 0.4% 7b (GE Healthcare, Bjokgatan, Uppsala, Sweden) respectively. The feeding procedure is: in the first feed Point (3rd day) after feeding, feeding every 2 days, feeding continuously four times. After the first feeding, the culture temperature dropped from 37°C to 32°C. Calculate cell specific productivity (Qp) based on growth rate (μ) and volumetric productivity.

The growth rate (μ) is calculated as follows:

N: living cell density; t: culture time

The specific cell productivity (Qp) is calculated as follows:

P: Volumetric productivity

 

Results

Host cell adaptation and transfection

This study tested the latest commercial CHO media developed by some large companies to evaluate whether the media cooperates well with cell line development platforms (such as host cells, vectors) (Table 2). CDM2, CDM3 and CD FortiCHO (Thermo Fisher Scientific, Waltham, MA, US) performed well in monoclonal antibody cell lines and recombinant protein cell lines, which were developed using CDM1 in the early stages.

This study tried to adapt CHO DG44 cells that had been adapted to CDM1 to these media, and the results showed that these cells could only adapt to CD FortiCHO (data not shown). After transfection of IgG-containing plasmids with polyethyleneimine, the productivity after 48 hours of culture was measured. The results showed that the results were different between experiments (Table 3). The reason for this variability is not clear, but it may be related to the host. The state of cell preparation is related.

Table 2 Evaluation of media performance based on platform media

However, when the transfection pool was continuously subcultured to restore cell growth, the adapted pool performance showed similar batch results, indicating that productivity results are not always directly related to the success or failure of transfection. In each experiment, the productivity of CDM3 was reduced 48 hours after transfection. The cell lines adapted to CDM2 and CDM3 respectively obtained higher productivity in the pool batch culture. The reason may be the increase in the supply of components in these richer media (Table 3
Nutrient-rich production medium of CHO cell line development platform-based

mini-pool cloning experiment medium comparison study

After the cell line is developed, it is resuscitated and cultured after transfection. Usually, the mini-pool is spread to a 96-well plate to select cells before non-producer cells grow beyond producer cells. Previous cell line development through CDM1 is effective for mini-pool growth. After 48 hours of transfection, a 96-well plate is generally inoculated with 1000-4000 cells/well according to the transfected gene. Research has found that 2000 cells/well is the most suitable for mini-pool clone growth.

For IgG expressing cells, after culturing for about 2 to 3 weeks (the general procedure for recovery of the process (Table 4)), about 10% of the seeded wells subsequently produced mini-pool clones. In the case of CDM2 culture, regardless of the inoculation conditions, such as inoculation of 1000, 2000 or 4000 cells/well will produce extremely high growth rates. On all test plates, 96% of the wells grew (Table 4). In contrast, even when seeded at high cell density, CDM3 does not grow clones (Table 4).

Table 4 Summary of min-pool screening results
Nutrient-rich production medium of CHO cell line development platform-based

In the case of CDM1, high seeding density cells may initially show a high growth rate, but they suffer damage due to depletion of certain clonal nutrients, and these cells will eventually show a shorter lifespan. CDM2 is more efficient, not only can generate mini-pool, but also can maintain clone growth for a longer time. However, further research is needed to determine whether these conditions can also grow non-productive cells, which is not the case due to the similar selection/growth ratio (~30%).

However, compared with CDM1, since CDM2 is a richer medium, higher volumetric productivity can be achieved at a lower specific cell productivity, so only selecting a standard larger than 10 mg/L may cause problems. Interestingly, the unfavorable growth of CDM3 may indicate that nutrient-rich media is not beneficial for cell growth inoculated with low cell density.

Comparative study on the culture medium of ClonePix2 cloning experiment

The latest ClonePix2 technology also tests the application of clone screening media. A semi-solid medium was prepared according to the ClonePix2 manual and used for high-yield clone growth and detection. In semi-solid media, it is important to determine the conditions for good colony growth. The supplier provides two products, and studies have shown that CloneMedia reagent (CloneMatrix and supplier media mixture) can work well in a variety of well plates and conditions. However, technically speaking, the high-yielding clones detected may be different when returning to the original medium. But it can be used in concentrated form by optimizing the mixing of CloneMatrix reagent and cell culture medium.

The reason for this is that it can be cultured with the initial cell culture medium, and potential problems caused by medium changes can be avoided. In this study, various media were tested, and it was found that some components in nutrient-rich media could not be dissolved when they were 2 times concentrated. Some of my colleagues could not produce clonal clusters when inoculated with 200-1000 cells/mL (data not shown). Under these circumstances, studies have found that formulating some nutrient-rich media at a concentration of 1.5x can solve this problem and cause sufficient colony growth.


Fig.2 ClonePix2 image comparison

  • (A) CDM1/CloneMatrix mixed culture
  • (B) CDM2/CloneMatrix mixed culture
  • (C) CDM3/CloneMatrix mixed culture

Adapted to CDM1, CDM2 and CDM3 cells respectively 48 hours after transfection, the transfected pool was inoculated into CloneMatrix medium (Fig. 2; Table 5). CDM1 produces the largest number of clones, and can obtain an expression level of about 10 mg/L or more in a 96-well plate. CDM2 also produced many clones, but most of the clones were too small or too close to nearby clones (neighboring clones), so they were not suitable for selection. The overall growth of CDM3 clones is poor.

At the same time, in 96-well plates, CDM3 clones also show poor growth recovery problems, and it is almost impossible to expand the culture in 6-well plates. The clones selected by CDM2 showed good growth, and many high-yielding clones (> 10mg/L) were successfully screened in 96-well plates, and they could be expanded to 6-well plates. However, since CDM2 is more abundant than CDM1, it is necessary to perform cell specific productivity analysis in a 6-well plate to determine whether the high-yielding clones grown in CDM2 are similar to those grown in CDM1.

Table 5 Summary image of ClonePix2 cloning results

Comparison of single cell separation and 6-well plate data

All clones screened by using mini-pool and Clone-Pix2 methods were individually expanded to 6-well plates, and the specific productivity of cloned cells was compared under uniform culture conditions (Fig. 3). Due to insufficient productivity of CDM3, no clone was selected. Only the first five clones from CDM1 and CDM2 were selected for single cell isolation.

Nutrient-rich production medium of CHO cell line development platform-based
Fig.3 Specific cell growth rate of 6-well plate

For CDM1 and CDM2 limiting dilution (LDC), a cloning medium capable of growing a single clone is required. Early research work has developed CDM1 cloning medium, which is composed of serum-free medium and its composition is similar to CDM1. In the case of CDM2, the reported commercial cloning medium was used to support the good growth of single clones. Studies have found that the commercial cloning medium can be used for monoclonal growth, but due to lack of nutrients, it cannot maintain cell viability for a long period of time (~2 weeks) in 96-well plates.

Therefore, this study tried to mix commercial cloning medium and CDM2. Facts have proved that 25% CDM2: 75% commercial cloning medium or mixing the two mediums at a ratio of 1:1 is sufficient to cultivate single clones (Fig. 4). In some cases, the addition of conditioned medium can enhance the secretion of potential growth factors, thereby promoting the growth of monoclonal cells. The addition of 10% conditioned medium can make the single clone grow slightly faster, but it does not seem to significantly increase the rate of clone formation.

Limiting dilution (LDC) of the best clones screened on the CDM1 and CDM2 platforms can produce similar clone formation rates (22.7% and 18.5%, respectively). The single clone obtained by limiting dilution (LDC) was tested in a 6-well plate to evaluate the specific cell productivity. The results of the monoclonal isolation successfully illustrated the presence of high- and low-yielding cells (Figure 3). When the overall specific productivity of CDM1 platform is higher than CDM2, both are better than CDM3.

Nutrient-rich production medium of CHO cell line development platform-based
Fig.4 Commercial cloning medium and CDM2 mixed culture test to successfully obtain a single clone (0.5 cells/well inoculated into 96-well plate and test for clone growth) (a) add 10% conditioned medium; (b) not add

 

 

Final clone evaluation

As a suspension culture, the host cells before transfection have been pre-adapted to each medium. Therefore, there is no need to re-suspension acclimatization in shake flasks and micro-bioreactors, and the final clone evaluation directly on the well plate.

Through batch culture, we tested the priority single-cell clones obtained by CDM1 and CDM2 (20 clones were selected for each medium). The batch culture of CDM1 could only last until the 7th day due to the subsequent decline in viability, while CDM2 The batch culture can continue until the 11th day before stopping. Both batch cultures were supplemented with glucose, so the decline in vitality was not due to glucose depletion, so this data shows that there is indeed a difference between poor and rich media. The average cell specific productivity between CDM1 single clones was nearly two times higher than that of CDM2 (CDM1 and CDM2 were 13.6±3.7 pg/cell/day and 6.9±2.2 pg/cell/day, respectively) (Fig. 3). On the contrary, the final expression level of CDM1 was 186.4±31.0mg/L, and CDM2 was 267.4±59.2mg/L, which indicated that the richer CDM2 had the advantage of production process development.

After confirming that the feed (Cell Boost 7a and 7b) can be beneficial to cell culture in both media, a batch feed experiment of the same feed mixture was performed in a micro-bioreactor. The results showed that the average final expression levels of the 3 batches of CDM1 and CDM2 were 2.3±0.1 g/L and 2.9±0.4 g/L, respectively.

 

Discuss of Nutrient-rich production medium of CHO cell line development platform-based

According to different process steps, the development of cell lines will have different requirements for the culture medium. The host cell culture medium needs to add other components according to the host cell line itself, such as dihydrofolate reductase (dhfr)-deficient cell lines (such as CHO-DG44, CHO-DUXB11) need to add HT, glutamine synthetase (GS) deficiency Type cell lines need to add glutamine. In addition, based on the experience of domestication of various host cells with various commercial media, some media will fail to domesticate. For example, CD FortiCHO is a powerful medium for the recombinant CHO-DG44 cell line (Table 2). The cell line has been tested on CDM1, CDM2 and CDM3, but the host cell growth was not possible due to cell aggregation, so this study was stopped (Data not shown).

Depending on the selection medium, the transfection conditions will vary greatly. It is more convenient to use the medium provided by the transfection kit, which can minimize the optimization settings of the transfection method. In this study, the polyethyleneimine transfection method requires OptiPRO SFM to successfully transfect, so this medium was temporarily used in the transfection step, and then changed to the platform medium originally planned. As a follow-up study, this study tested electroporation to set the transfection method without changing the medium. The results showed that this can be done, but the best electroporation conditions (voltage, pulse and CDM3) are among CDM1, CDM2 and CDM3. Shock numbers) are different (data not shown).

After the transfection, the selection medium is the main medium for the development of the entire cell line, and supplements required by the vector selection system are added. In this study, the IgG expression vector only needs to remove HT, so the cells inserted into the vector can survive the existence of the dhfr gene. The addition of MTX can expand the cells to increase specific cell productivity. According to the different designs of the vector, the resistance conferred by neo gene, Pac gene and Sh ble gene are used to select pressurized screening reagents, such as geneticin, puromycin and bleomycin. Using CDM1, CDM2 and CDM3 as the selection medium, mini-pool screening showed that the recovery rate of the 96-well plate of CDM1 was close to 10%, while CDM3 had no clone growth.

The excessive growth of each well of CDM2 may dilute non-productive cells. Mini-pool screening shows that almost all wells of CDM2 show excessive growth. This may mask the screening of truly high-yielding cells and miss these clones (unselected ). These phenomena can be explained by the principle of stoichiometry and the material balance of cell growth. After the cell is metabolized, the nutrient material of the medium is lacking, and the cell growth and production will be restricted by the limited nutrient material.

Therefore, not high-producing cells, but non-productive cells or low-producing cells usually show a high growth rate. In order to pass the CDM2 mini-pool screening and determine a reasonable growth ratio condition, it is necessary to reduce the cell seeding density of the 96-well plate or increase the sieving pressure of the medium. CDM3 may not grow at such a cell seeding density because the medium is too rich, so it is necessary to increase the seeding cell density.

Parallel to the mini-pool screening method, ClonePix2 screening showed that the CDM1/Clone-Matrix mixed medium can produce cell cloning clusters well and obtain a large number of selectable cloning clusters (selected 11.5%). Similar to mini-pool screening, the CDM2/CloneMatrix mixed medium produces too many cell clones, which are too small or too close to neighboring clones. Therefore, the proportion of selectable cloning groups is very low (40% selected).

Most of the colonies grown in CDM3/CloneMatrix mixed medium are selectable, but the total number of colonies is much less than other conditions. This study will continue to optimize the seeding cell density and the mixing ratio of CDM2 and CDM3/CloneMatrix. At the same time, more research is needed to obtain clear results. Studies have found that finding the appropriate cell density for inoculation based on the choice of basal medium or the mixing ratio is the most important factor in determining the selectable clones. From the comparison of the results of this study, CDM2/CloneMatrix mixed medium requires fewer seeded cells, while CDM3/CloneMatrix requires more seeded cells to get better results.

The cloning medium for single cell isolation is the most complicated medium to choose in the development of cell lines. Existing studies have shown that traditional methods only add serum to the culture medium, and most of them can obtain a clone recovery rate of about 50% to 70%. In the absence of serum, it is difficult to achieve good clone recovery.

Sealover et al. (2013) reported that CHO-DG44 is more difficult to clone and recover than CHO-S. Lim et al. (2013) reported that mixing the conditioned medium with the cloning medium is a good practice and research, and found that the cultured CHO cells can secrete growth factors, however, use the pre-tested medium in Table 2 to cultivate the recombinant CHO-DG44, no matter whether conditioned medium is added or not, clone recovery cannot be obtained. The best solution obtained in this study is to mix the platform medium (CDM1 or CDM2) with the commercial cloning medium, so now, when a new basic medium is applied to cell line development, a mixing ratio test is required. 

The overall evaluation of the single clones isolated from CDM1 and CDM2 were performed. Due to the comparison of different cell lines and media, the comparison between batch culture and fed-batch culture will be more complicated. The priority clones isolated from CDM1 and CDM2 need to be tested in batches for better comparison. The study found that the average specific productivity of CDM1 clones was higher (Fig. 3).

This may be due to the difference in screening results. ClonePix2 can select fewer clones or the high growth rate of mini-pool clones makes CDM2 less likely to find high-yielding clones. Since the fed-batch strategy shows that Cell Boost 7 performs well in CDM1 and CDM2, the number of clones tested in fed-batch test is less than that in batch culture (Fig.5a), but no matter what medium, comparison studies between clones can still be observed Different results.

The comparative study of the three clones in Fig.5b shows that the best working clone can be obtained by developing cell lines in batches. Even though the selected clones generally have lower specific cell productivity, the final expression level of CDM2 in fed batches is still higher than that of CDM1. If the CDM1 derived clones are fed-batch culture using a rich medium (the same feed mixture is used for cloning tests on CDM3), a final yield of 2-4 g/L is observed (data not shown). It can be concluded from the data that a richly formulated production medium (such as CDM2) can be successfully used as a basic medium for cell line development, which shows similar productivity to CDM1 cell lines.

Fig.5 Comparison of the culture data of CDM1 derived clones and CDM2 derived clones (a) Batch culture (no feed) data of CDM1 and CDM2 priority clones (the average of 20 clones each, the error distance is the standard deviation); (b) CDM1 Fed-batch culture data of CDM2 and CDM2 priority clones (average of 3 clones, standard deviation of error). CDM1 data uses CDM1 as the basal medium, and CDM2 data uses CDM2 as the basal medium. For fed-batch culture, both CDM1 and CDM2 culture are similar and use the same feed medium at the same time (Cell Boost 7a and 7b)

 

According to this study, one of the possible limitations or obstacles to the use of rich media for cell line development should be the growth restriction under low cell density conditions. It has been reported that the low cell density condition itself will hinder the cell growth rate due to the lower secretion of growth factors from the seeded cells. However, currently, there is no research or information about this more nutritious medium that can hinder low cell density culture. Zhu et al. (2012) showed that a 1:1 mixture of poor classic medium and SFM medium can achieve better cloning efficiency, which means that rich medium has this effect.

The results of this study also show some limitations of low cell density inoculation in rich media, such as 1.5× concentrated CDM2 and CloneMatrix mixed medium, or 1.5× concentrated CDM3 and CloneMatrix mixed culture, instead of 2× concentrated (compared to In contrast, the poorer CDM1 works well under 2× concentrated solution), the mini-pool and ClonePix2 screening of CDM3 require high cell seeding density, or the mixing of CDM2 with the poorer commercial cloning medium can achieve single Cell cloning. The medium needs to be optimized to better maintain cell growth at high cell seeding densities and ultimately make them more abundant. In the future, it is necessary to do more research and development for low cell density culture programs, such as cloning media.

This study shows that the latest commercial enriched CHO medium developed and formulated for production can be used as a basic medium for cell line development. This medium can be used for initial host cell growth, post-transfection screening and pressure screening, and final production Provide services. The method developed in this research can eliminate the requirement of cell lines to adapt to the production medium and reduce the clonal problems associated with medium replacement. This approach will also reduce time and effort, eliminating the need to adapt the cells in a new medium and optimize the production culture process. It will be very beneficial to develop cell lines in a short time and quickly.

 

Nutrient-rich production medium of CHO cell line development platform-based.

Nutrient-rich production medium of CHO cell line development platform-based.

Nutrient-rich production medium of CHO cell line development platform-based.

Nutrient-rich production medium of CHO cell line development platform-based.

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