One of the bestselling products on the medical market are biopharmaceuticals. This type of drugs and bioingredients are mainly produced in CHO cell lines. Thus, the pharmacists are focused on the improvement of the methods and processes for industrial production of biomolecules for pharmacological application.
Bacteria, yeast or mammalian cell lines – which is the best producer for therapeutic biomolecules?
These biomacromolecule substances represent nucleic acids and proteins which cannot be synthesized as final products after chemical reactions. These can be expressed as a result of the metabolic pathways in genetically modified bacteria, plant or mammalian cell cultures. First scientific reports for production of biopharmaceuticals described the use of bacterial strains that can be easily cultivated and can express high quantities of the desired biomolecule (recombinant protein) in short terms. However, scientists found out that the bacterial cell cannot express modified proteins in a process of glycosylation during the post-transcriptional phase. This specific function and the possibility for disulfide bonds formation are typical for eukaryotic cells like yeast cell lines. Their cultivation can be done easily and at low cost, but it is more difficult to control the expression in comparison with bacteria. Also, yeast-specific glycosylation patterns are different from those of mammalian cells. Thus, many of the most important modern biopharmaceuticals are produced in mammalian cell lines even if the cultivation is more difficult and more expensive than bacterial cells. Moreover, in contrast to microorganisms, mammalian cell lines only produce small quantities of recombinant proteins. Biopharmaceuticals such as Enbrel, Remicade, Rituxan, Avastin, Herceptin and Humira achieve annual revenues of more than 5 billion dollars. The Chinese hamster ovary (CHO) cell line is the most important mammalian cell line used for the production of therapeutic proteins.
Nowadays, all CHO cells that are stored in many hundreds of laboratories and production departments around the world originate from a single female Chinese hamster that lived and died in 1957 in Theodore T. Puck’s laboratory at the University of Denver, Colorado.
70% of all recombinant biopharmaceutical ingredients used for the production of human medicines are produced in cells that originate from the immortalized primary culture of the ovary cells of Puck’s hamster.
Advantages of CHO cell lines
- immortality;
- can be kept in suspension cultures;
- genetically stable (which is not valid for cancer cell lines);
- can be reproduced with expression vectors that contain the “gene of interest”;
- they can be transfected;
- they remain stable during the process of selection, amplification, single cell cloning and the characterization of the clone.
How can be increased the productivity of the CHO cell lines?
The quality of the glycoproteins and monoclonal antibodies produced commercially by CHO cell clones depend on the level of the clone’s productivity. Since this is an extremely competitive market, pharmaceutical companies focus their efforts on the optimization of the downstream processing. The steps of optimization include the kind/type of cell culture, its characteristics of the expression system used for the gene of interest or for transfection, the composition of the culture medium, the cultivation (batch, feed-batch) and the duration of the culture passages, pH value, temperature and cell concentration. All these aspects affect the viability and productivity levels of the cell lines. Over the last 20 years, the optimization has led to an increase of the number of cells that can be kept in culture 20-fold. Now it is possible to cultivate around 10 million protein-expressing cells per ml culture medium for 3 times.
The characterization of a stable clone is a time-consuming process, but it can be further reduced through the involvement of modern technologies. For example, quantitative PCR provides rapid selection of cells and the image-based automated analysis of microtitre plates enables the identification and cloning of the most suitable cells from the amplified cell population.
The protein level that can be achieved with bioreactors is a key cost factor. At present, it is possible to obtain therapeutic antibody titres of 3 – 5 g/l. Most researches are focused on increasing this value even further. The general trend is moving towards the use of smaller bioreactors and single-use plastic systems rather than stainless steel systems.
Which are the advantages of the single-use plastic systems/bioreactors?
- flexibility;
- lower investments;
- quick construction and operation;
- inexpensive sterilization and cleaning
In comparison with standardized large-scale automated stainless-steel systems, personnel costs are higher because the semi-automated processes associated with the use of single-use systems require highly trained users and a larger number of manual steps. Further analyses will be performed in order to achieve higher levels of biopharmaceuticals from mammalian cell lines.