Photo: GSK Marburg
Animal Cell Technology: Becoming more and more important
(last update: March 2016, Matthieu Stettler and Chantra Eskes)
By definition animal cell technology is that part of biotechnology which is based on the use of animal (including human) cells, propagated in vitro, for the manufacture of biological molecules, e.g. biopharmaceuticals, and as toolbox in the discovery and testing of medicines. The main field of application of animal cell technology is the medical and pharmaceutical arena. The members of ACTIP, the Animal Cell Technology Industrial Platform, want to support and reinforce further development of animal cell technology which will lead to increased utilization of animal and human cells to be propagated in vitro.
As early as the 19th century, animal cell technology already formed the basis for providing vaccines against small pox and rabies, although these vaccines were actually produced in living animals. In the 20th century, knowledge in many areas of the life sciences increased tremendously. In the early 1950s, this led to the Salk and Sabin processes of in vitro cultivation of cells for the production of highly effective vaccines against polio virus. Other vaccines against a wide variety of human and animal viral diseases like rubella, measles, foot and mouth disease etc. followed soon after. In addition, novel animal cell technologies made it possible to develop high throughput assays for the screening of new candidate pharmaceuticals and as diagnostic tools.
Breakthroughs in the 1970s and 1980s
A major breakthrough in the field of animal cell technology came in the late 1970s and early 1980s with the development of hybridoma and recombinant DNA technology. The hybridoma technology makes it possible to elicit, select and produce, in vitro, so called monoclonal antibodies of consistent quality, high specificity and unlimited quantities, whereas previously antibodies could only be produced in living animals with a varying degree of quality. Besides the more “classical” use of antibodies in passive immunization of patients against infectious diseases, especially monoclonal antibodies are nowadays widely used as a therapeutic mean to treat cancer, chronic and autoimmune diseases as well as for diagnostic purposes like blood group typing and disease testing. The development of recombinant DNA technology or heterogeneous gene expression allows for example the production of human proteins in animal cells at a relatively efficient production scale. Previously, many of these human proteins could not be produced or isolated at all.
Need for animal cells
The recombinant DNA technology – also called genetic engineering – makes it possible to transfer a gene from one species into cells of another, and to let these transformed cells produce the encoded gene product. For example, the encoding gene for human insulin has been successfully transfected into the easy to grow bacterium Escherichia coli. However, with more complex and larger gene products than insulin, it is no longer possible to use bacteria like E. coli as host cells for the production. The reason is that these bacteria are unable to perform the complicated posttranslational processing steps such as glycosylation, folding and trimming, which is often a prerequisite to exert biological functions. Alternatively, such proteins can be expressed successfully and correctly in cultured animal cells. For example, the bioactivity of erythropoietin, tissue plasminogen activator or factor VIII is strongly dependent on a correct posttranslational processing. Development of novel cell lines has resulted in an increased frequency in which animal cells are being used. In the early eighties the majority of biopharmaceuticals was produced in E. coli (86%), but nowadays the majority of biopharmaceuticals are produced in animal cells, where Chinese Hamster Ovary (CHO) has become the gold standard. This development has been mainly driven by the production of therapeutic monoclonal antibodies. Using animal cells as production factories, however, had the disadvantage that the production costs are relatively high in comparison with production in cultivated bacteria or yeast cells, and related to this, the relatively small production scale. Addressing this situation was and still is in the focus of development activities on animal cells. Aims herein are development of robust high yielding cell lines, improvement of process conditions and media, etc
Therapeutics: demand for large scale manufacturing facilities
At present, the majority of therapeutic biopharmaceuticals has been produced using animal cell technology and include proteins used for the treatment of cardiovascular diseases (tissue plasminogen activator: tPA, reteplase), cystic fibrosis (DNases), anaemia (erythropoietin: EPO), haemophilia (coagulation factors VIII and IX), cancer and viral infections (interferons and interleukins), multiple sclerosis (interferon-beta2) and dwarfism (human growth hormone: hGH). Monoclonal antibodies are used for the treatment of a rapidly expanding list of diseases, e.g. to reduce the rejection of transplanted organs by the recipient (e.g. Simulect and Zenapax), to treat metastasized breast cancer (Herceptin), to treat non-Hodgkin lymphoma’s (Rituxan), to inhibit allergic asthma (Xolair), and many others (see monograph on monoclonal antibodies). These different antibody indications have in common that effective patient dosing requires relatively large amounts of antibody-protein, which translates into a huge demand for large scale manufacturing capacity.
New opportunities with biosimilars
As the patent protection of some of the currently commercialized biologics expired or will expire in the near future, the development and commercialization of so called biosimilars has been recently initiated. According to the EMA, a biosimilar is a therapeutic biopharmaceutical that is “similar to another biological medicine that has already been authorized for use”. It can be a relatively small molecule such as human insulin or erythropoietin, or a complex molecule such as a monoclonal antibody. During the development of a biosimilar, studies are performed in order to demonstrate that the proposed molecule does not have any meaningful differences from the reference medicine in terms of quality, safety or efficacy. One of the main challenge when developing a biosimilar process, is the fact that the manufacturer does not have access to the originator’s cell bank and manufacturing process. Despite this and other uncertainties, an increasing number of biosimilars of successful biologics are likely to be approved for commercial use in the near future.
Cell and Gene therapy
Culturing of animal cells forms also an important basis for cell and gene therapy. This may drive the development of more specific and personalized patient interventions. Obviously this has an influence on manufacturing conditions.
Cell therapy is the use of human cells to treat patients, such as autologous applications where cells from a patient are removed, enriched and/or modified and finally re-injected in the patient. Applications are tissue regeneration after injury, rebuilding the immune system after chemotherapy, and others. In addition, with the increased ability to isolate and culture embryonic stem cells as well as the creation of induced pluripotent stem cells, new types of treatments for neurodegenerative diseases, diabetes, heart disease and other conditions may be envisioned. The field itself is currently developing and manufacturing and regulatory questions need to be addressed to shape and design this new area.
In contrast to direct treatment of patients with recombinant biopharmaceuticals, gene therapy is based on the delivery of the corresponding gene to a patient, in such a way that the patient may produce the functional gene product (i.e. the protein) intracellularly. In principle, such an approach is considered to result in long-lasting treatment or cure, e.g. modification of patients’ genome. For the delivery of relevant genes into specific tissues in patients recombinant viruses are being used. These viruses are genetically modified and produced in vitro in animal cells. Gene therapy is very promising since it may yield a number of advantages. When we consider for example patients suffering from a bleeding disorder such as Haemophilia A, these patients have a deficient gene encoding for clotting Factor VIII. Current treatment is based on the lifelong treatment with the recombinant protein Factor VIII. Gene therapy might make it possible to administer the gene encoding Factor VIII into the liver cells of these patients. These cells are the natural producer cells of Factor VIII and, thus, may behave naturally after transfection with the functional gene encoding Factor VIII.
The evolution of the drug discovery and development process has been revolutionized by the implementation of high-throughput technologies, combinatorial chemistry and genomics-based target identification and validation. Animal and human cells have become an essential part of the cell-based assays and models, which form the basis of target identification and validation experiments, HT-screening of gene (sequence) libraries for lead finding and optimization purposes and HT-testing for ADME/tox of candidate drugs. Given the fact that these areas are still in rapid development, it will be obvious that improvement of cell culture procedures remains a prerequisite. In this respect, multidisciplinary R&D programs remain crucial to feed further progress in the tool box needed for efficient drug discovery.
How are animal cells propagated?
Animal cells are cultivated in vitro by mimicking the same conditions as they have in their natural environment, the body. They need complex and balanced culture media which contain substrates such as glucose, amino acids, vitamins, salts, trace elements and others. For biopharmaceuticals manufacturing this is usually achieved through the use of media free of serum additives or protein growth factors in order to ensure high reproducibility and safety standards.
Small scale cultures are performed in tissue culture flasks or shake flasks in incubators with controlled atmosphere. Large scale cultivation for industrial purposes is mainly performed in bioreactors which are designed to monitor and control optimal physicochemical conditions (e. g. mixing, temperature, aeration). This is normally done in stainless steel tanks which can be sanitized and sterilized. Alternatively, cells can also be grown in bioreactors which are fitted with single use cultivation bags. In these systems, cells grow, multiply and, in the case of recombinant cells, release human proteins into the culture medium from which the protein product can be purified, formulated and packaged.
The design of safe, reliable and consistent manufacturing processes is one of the key challenges for biopharmaceutical companies and a regulatory expectation. This is best outlined in the pharmaceutical quality by design (QbD) concept. The focus of this concept is that quality should be built into a product with an understanding of the product and process by which it is developed and manufactured. The biopharmaceutical industry is also actively looking at opportunities to improve the cost of goods when implementing efficient platform processes or improving the production technologies without compromising quality and patient safety.