Introduction

As a nutritional supplement for fedbatch, feeds are generally composed of components with high consumption, such as amino acids, vitamins and so on, with high concentration and high osmotic pressure, which are crucial in batch supplementation. Therefore, the development is very challenging, requiring appropriate components and suitable concentration. At present, there are not many types of commercial supplements on the market, and the market is basically monopolized by the industry's three giants (Gibco, Hyclone, Merck). Forced by the high price of imported products, as well as for the future of the continuous supply of concerns, the rise of localized feeds have been the trend.

01 Significance of Feeds

As for how important the feeds are, I will first post a set of data, choose a top commercial basal medium for batch culture, and at the same time use commercial feeds for fedbatch as a control group. The test cells are CHO-K1 monoclonal cell lines expressing a certain double antibody.

There is nothing more convincing than the data. Batch cultured cells also have logarithmic, plateau and apoptosis phases, but the apoptosis phase viability declined rapidly, with only 82.4% viability left on the 12th day, and the expression amount was 1.24 g/L. However, in the control group, the expression amount reached 4.52 g/L on the 11th day, and the viability on the 14th day was still 88%. This shows the importance of supplementation in cell culture and protein expression. Next, I changed a basal medium (HealSun medium) and carried out the same experiment.

Obviously, after changing the basal medium, feeds were still indispensable. But this time, after using HealSun basal medium, the state of cell growth was slightly improved. The expression on the 11th day was 2.91 g/L, which can reach 61% of the control group, but from the 11th day onwards the expression was not moving at all, and the cells were only surviving, not expressing. By comparing the two small experiments, it can be found that even with the same basal medium for batch culture, the growth and expression differed largely. This may be led by the different components in the basal medium and their concentration. But we can not be specific to determine that because the components of commercial basal media are unclear.

02 Combinations of Feeds

The general feeds are a main body feeding with a basic feeding, and the basic feeding has a clear composition, with several insoluble amino acids, such as tyrosine. I used HealSun feed only containing amino acids, vitamins and a few salts, based on HealSun medium, and compared with the above commercial feelings.

Using HealSun basal medium with only commercial feeds and no basic feeds, the final expression was 2.65 g/L, which was close to or even lower than the results in the batch assay. The cell growth and viability maintenance in the early stage was similar to the other two groups, and the maintenance in the late stage was very poor, and the viability at the end was even lower than the level of batch. The expression was stopped after the 11th day, and it was guessed that the commercial feeds did not contain one or several amino acids in the basic feeds at all, resulting in that the cells were able to utilize the content in the basic for growth and proper expression in the early stage, and the nutrients that were constantly replenished in the later stage became burdensome due to the inability to utilize them instead. Looking at the HealSun feeds, the expression could reach 5.36 g/L without the basic feeds, while the expression was 6.04 g/L after the basic feeds, and the ratio reached 88.7%. It may be due to the fact that the developmental feeds contains a certain concentration of insoluble amino acids, but the concentration was not enough. It has been reported in the papers that reducing the concentration of cysteine in normal feeds by 15% results in a decrease in antioxidants such as glutathione and taurine, leading to a shaprly reduced ability of cells to maintain redox at a later stage, a decrease in viability, and ultimately, a decrease in protein expression, which is consistent with the findings of the present experiments.

03 Effects of Feeds on Titer

On the 14th day of HealSun feeds, the expression amount was 6.04 g/L, which could reach the level of more than 90% of the commercial feeds. This time, I still used HealSun basal medium and set two concentrations of 20 amino acids and 11 vitamins in the above feeds, most of the amino acid concentration difference was small, between 2-5 times, individual special amino acids, such as alanine, glycine and tyrosine, concentration design difference was larger, up to more than 6-7 times. Vitamins’ concentration difference was wider, ranging from 3 times to 100 times. A total of 32 groups of PB (Plackett-Burman) tests were designed, plus a control group (No. 33, HealSun basal medium + commercial supplement). Medium preparation was complex, the experimental results were not satisfying, all the experimental groups were lower than the control group, the highest was only 5.15 g/L, which only contained the control group was 80.7%, while the lowest was 2.32 g/L, which did not reach the expression level of batch.

Although the experiment did not have the expected results, I did not dig up any valuable information, only found that the addition of different concentrations of amino acids and vitamins did not have that great an effect on cell growth, but the effect on protein expression was very great. I tried again, keeping the amino acid concentration unchanged, explored the vitamins individually, but the concentration range was adjusted to between 2-200 times, and designed 12 groups of PBs with one group of control (No. 13, HealSun basal + commercial feeds).

Let's analyze the control group first. This combination of HealSun basal medium with commercial feeds, from 6.54 g/L in the second experiment, to 6.38 g/L in the fourth experiment, to 6.34 g/L in this one. It can be said to be very stable. The viability of the experimental group was basically the same up to day 11, the last day showed a big difference, the lowest viability on day 14 was 19%, the highest viability had 91%, the situation of viable cell density was similar, the last day went from 22.7×106 cells/ml to 4.23×106 cells/ml. Different vitamin concentration had a big effect on the maintenance of the cell's state in the later stage, which was analyzed and concluded that the single most important factor for the maintenance of viable density and protein expression was choline chloride, which is consistent with the papers.

04 Significance of Custom Development of Feeds to Host Cells

Although it has been reported in the papers for a long time, I was still surprised that there were at least two feeds that could be comparable to the commercial feeds, and fedbatch was able to reach 6.4 g/L on the last 14th day. So I continued, and this time, I used CHOK1 monoclonal cells expressing another protein, and chose the above mentioned No. 11 (HealSun feeding 1) and No. 12 (HealSun feeding 2) to carry out the test.

HealSun basal medium with commercial feeds still performed well with the highest expression of 4.86 g/L, but for only HealSun feeding 1 or HealSun feeding 2, the experiment ended up with a final expression of only 3.5 g/L. So it can be seen that even though it was the same host cell, the disparity between different projects reveals the importance of the tailor-made customization of the development of different projects.

Sharing Experiences and Insights

Until now, cell metabolism is complex, so our knowledge of it is limited. As developers, we are trying to overcome the difficulties on the way of development, and hope to solve all the doubts.

References：

1.Shinobu Kuwae , Ichiko Miyakawa , Tomohiro Doi , Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content. Cytotechnology .2018 Jun;70(3):939-948.

2.Amr S. Ali , Rachel Chen , Ravali Raju , Rashmi Kshirsagar. Multi-Omics Reveals Impact of Cysteine Feed Concentration and Resulting Redox Imbalance on Cellular Energy Metabolism and Specific Productivity in CHO Cell Bioprocessing. Biotechnol J.2020 Aug;15(8):e1900565.