Recombinant Protein Formation By CHO Cells Assignment Sample

Recombinant Protein Formation By CHO Cells Assignment

1.0 Introduction

“Chinese Hamster Ovary (CHO)” cells are the most safety standard-proven and popular cells used for industrial-level protein production. CHO cells are standardized and well-established for protein production of recombinant nature. These cells provide a basis which is optimal in the development of “continuous exchange cell-free (CECF)” systems. These cells also are used to screen DNA templates for an industrial approach. In pharmaceutical industries, these cells are specifically used to express recombinant proteins, through various methods of genetic engineering of the cells.

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2.0 Background

The “Chinese Hamster Ovary (CHO)” cells are the most commonly used cells that have been in the large scale production of therapeutic proteins in a commercial scale. These cells provide a basis which is optimal in the development of “continuous exchange cell-free (CECF)” systems. In the recent years, the invention of new technologies regarding the cell culture of rCHO cells have helped to increase the production of recombinant proteins with a titre value of more than 10g/L (https://onlinelibrary.wiley.com, 2020). This process now-a-days had been optimized according to the suitable situations by the commercial pharmaceutical industries. Around 70% production of recombinant therapeutic proteins around the world has been produced by these cells, and the estimated market value is around 20 billion dollars (https://books.google.co.in, 2022). “Chinese Hamster Overy (CHO)” cells are identified by their character of low efficiency of glucose catabolism, and lactate production. Therefore, it can be hypothesized that the low efficiency of glucose catabolism can be identified by measuring pyruvate transportation to mitochondria. The “mitochondrial pyruvate carrier (MPC)” is responsible for introducing pyruvate into mitochondria. MPC has two subunits, expressed by two different genes (Fouladiha et al. 2020). Moreover, these cells have internal functions similar to that of human cells. Like the glycosylation of preteins and the folding and assembling of proteins inside the human cells. For this reason, human proteins whose function is closely linked with “posttranslational modification” is best suited for commercial production by using CHO cells as a host. These cells were also extremely efficient in expressing large scale protein molecules, that were unable to be expressed by prokaryotic hosts.

The continuous maintenance of the cell culture is also not relatively hard. These cells are cultured using the technique of “batch fermentation” (Tran et al. 2019). But, with the increasing development of control mechanisms of fermentation, optimization of the culture medium and robust screening process of these CHO cells, a high concentration of cells (18 million/ml) can be sustained, which is economical for large scale protein production. [Refer to appendix 1]

Figure 1: CHO cells observed under confocal microscope

(Source: www.biopharma-reporter.com)

3.0 Research Aim and Objectives

The aim of the research is to researched about new framework and models regarding the production of recombinant proteins using the cell culture of “Chinese Hamster Ovary (CHO)” cells.

The objectives of the research is given below.

• To observe different currently used technologies used in the commercial production of recombinant proteins used by the CHO cells.

• To find in details about the currently used and under developing technologies for the production of recombinant proteins using CHO cells.

• To observe the genetic modification done on the CHO cells over the years to produce more recombinant proteins.

• To find out the conditions in the cell culture, to achieve the maximum protein yield.

• To find out the reasons behind the large-scale use of these cells for recombinant protein production.

4.0 Work Plan

4.1 Methodology

The commercial production of recombinant proteins were done using a “fed-batch fermentation” culture. This is the most efficient way currently available for the production of recombinant proteins. At first, cell tissue culture was prepared for these CHO cells in a sample specimen of mice. The low chromosome number had made these cells particularly useful for the studies. The preparation of CHO cell lines were developed. This was done through the use of DHFR selection system. In an industrial setting this process needs atleast six months (Prabhu et al. 2019). After the development of complete cell line of CHO cells, the transformation of those cells were completed using different methods like “calcium phosphate precipitation”, “retroviral transfection” or “electroporation” methods. The vector DNA after entering those CHO cells, the DNA gets integrated into the genome of the CHO cells and expressed accordingly through the synthesis of recombinant proteins (Nguyen et al. 2019). After the development of recombinant cell through the process of transformation, various methods were deployed to isolate the Cells and cloning of those cells. As the DHFR gene has a reduced enzymatic activity, a weak promoter sequence had been used to express those genes. After the cells got completely transformed, the cells were selected using low level of MTX and cultivated in a culture medium, where the molecules of glycine, thymidine and hypoxanthine is not present (Dahodwala, and Lee, 2019).. Due to the presence of marker genes, on the plasmid DNA, different antibiotics were used to express those cells in antibiotic medium. After that, the CHO cells were genetically transformed prior to the experiment to survive the conditions after foreign DNA integration, and this leads to increased copy number of DHFR cells and the recombin

Materials used	Cost
Petridish	$500
Centrifuge machine Fermentation tank	$400
$3000
Test tubes	$200
Antibiotic tablets	$100
Other essential equipment	$1000
Cell sample	$50
Refrigerator	$100
Materials used	Cost
Petridish	$500
Centrifuge machine Fermentation tank	$400
$3000
Test tubes	$200
Antibiotic tablets	$100
Other essential equipment	$1000
Cell sample	$50
Refrigerator	$100

ant protein. The cells produced after this process contains, genome where different integration sites are present, have a high copy number, and have varrying productivities. [Refer to appendix 2]

4.2 Expected outcome

The technology of recombinant protein production using the CHO cells is one of the most important process used by the pharmaceutical industry. In this experiment, the CHO cells were selected using the DHFR selection process. The DHFR acts as a selection marker for the proteins to be expressed. As the plasmid DNA segment which is inserted into the genome of the CHO cells which contains genes helps them to survive under the presence of antibiotics like ampicillin, hygromysin, the colonies which appears on the surface of the media are transformed (Chevallier et al. 2020). It was observed that, the productivity of these cells are as high as 10 to 100 fold under the transformed condition, rather than the ordinary cell culture. The recombinant protein which is produced are further analysed by the fact that it is produced in mass amount by the pharmaceutical industry.

5.0 Beneficiaries, contributions and impact

The main beneficiary of the project is the pharmaceutical industry leaders. The CHO cells were long been in the use for the production of recombinant proteins, like those used in different antibiotics, medicinal products among others. Prior to the invention and advancement of this technology, the recombinant protein production by the cell lysate are relatively hard. The protein produced by these cell lysate can be readily usable in the human body (https://www.voxco.com, 2021). As these cells have internal functions similar to that of human cells like the glycosylation of proteins and the folding and assembling of proteins inside the human cells.

6.0 Research Budget

The budget for the research is relatively moderate. Since the technology was standardised in the early 1990s. The budget required for the following project is given in the table below.

(Source: www.aiche.org)

Materials used	Cost
Petridish	$500
Centrifuge machine Fermentation tank	$400
$3000
Test tubes	$200
Antibiotic tablets	$100
Other essential equipment	$1000
Cell sample	$50
Refrigerator	$100
Materials used	Cost
Petridish	$500
Centrifuge machine Fermentation tank	$400
$3000
Test tubes	$200
Antibiotic tablets	$100
Other essential equipment	$1000
Cell sample	$50
Refrigerator	$100

Table 1: The cost of the experiment

(Self-created in MS Word)

7.0 Conclusion

In conclusion, it can be said that recombinant CHO cells are widely popular, and useful genetically designed methods for the expression of different recombinant proteins. The genes responsible for the expression of particular enzymes or other proteins is integrated into the genome of CHO cells through the process of transformation. These genes in the plasmid DNA are integrated into the host cell genome at specific binding sites. After, the gene integration the protein get expressed through normal cellular mechanisms. Researchers followed a secondary data collection method during their research work. They also followed the research design of descriptive nature.

References

Books

(https://books.google.co.in, 2022) Available at: Basic Laboratory methods for Biotechnology [Accessed on: 08.03.2023]

Journals

Chevallier, V., Andersen, M.R. and Malphettes, L., 2020. Oxidative stress?alleviating strategies to improve recombinant protein production in CHO cells. Biotechnology and bioengineering, 117(4), pp.1172-1186.

Coulet, M., Kepp, O., Kroemer, G. and Basmaciogullari, S., 2022. Metabolic Profiling of CHO Cells during the Production of Biotherapeutics. Cells, 11(12), p.1929.

Dahodwala, H. and Lee, K.H., 2019. The fickle CHO: a review of the causes, implications, and potential alleviation of the CHO cell line instability problem. Current opinion in biotechnology, 60, pp.128-137.

Fouladiha, H., Marashi, S.A., Torkashvand, F., Mahboudi, F., Lewis, N.E. and Vaziri, B., 2020. A metabolic network-based approach for developing feeding strategies for CHO cells to increase monoclonal antibody production. Bioprocess and Biosystems Engineering, 43, pp.1381-1389.

Nguyen, L.N., Baumann, M., Dhiman, H., Marx, N., Schmieder, V., Hussein, M., Eisenhut, P., Hernandez, I., Koehn, J. and Borth, N., 2019. Novel promoters derived from chinese hamster ovary cells via in silico and in vitro analysis. Biotechnology journal, 14(11), p.1900125.

Tran, K., Gurramkonda, C., Cooper, M.A., Pilli, M., Taris, J.E., Selock, N., Han, T.C., Tolosa, M., Zuber, A., Peñalber?Johnstone, C. and Dinkins, C., 2019. Cell?free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate. Biotechnology and bioengineering, 115(1), pp.92-102.

Prabhu, A., Gadre, R. and Gadgil, M., 2019. Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells. Applied microbiology and biotechnology, 102, pp.5989-5999.

Articles

(https://onlinelibrary.wiley.com, 2020) Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells. Available at: https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.27439. [Accessed on: 08.03.2023]

Website

(https://www.voxco.com, September 29, 2021) Descriptive Research Design. Available at: https://www.voxco.com/blog/descriptive-research-design/#:~:text=What%20is%20Descriptive%20Research%20Design,problem%20rather%20than%20the%20why. [Accessed on: 08.03.2023]