Photo: GSK Marburg
Vaccines and animal cell technology
(last update: June 2013)
Vaccination is one of the most successful and cost-effective public health interventions of all. It has eradicated smallpox, lowered the global incidence of polio by 99% since 1988 and achieved dramatic reductions in diseases such as measles, diphtheria, whooping cough (pertussis), tetanus and hepatitis B. “Vaccination is the 20th century’s greatest public health triumph after improved access to clean, safe drinking water.” (1)
Prior to the advent of cell culture, viruses could be propagated only on whole organisms, animal or plants. Whole organisms could include the natural host and laboratory animals such as chicken embryonated eggs, rabbits, mice, rats and others. The development of cell culture techniques in the 1950’s opened the door to the manufacturing of a wide range of biological pharmaceutical products at industrial scale.
Tissue culture is the general term for the removal of cells, tissues or organs from an animal or plant and their subsequent placement into an artificial medium environment for maintaining cell viability.
The culture of whole organs or intact organ fragments with the intent to use cells as machinery to produce biological is called Organ Culture.
When the cells are removed from the organ fragments prior to or during cultivation thus disrupting their normal relationships with neighboring cell, the technology is called Cell Culture.
When cells are individually dissociated from an organism and placed into a suitable medium and support culture environment, they will attach, divide and grow. This cell culture is named Primary Culture. Cell culture may be initiated from normal, embryonic or malignant tissue. When cells in the primary culture vessel have grown and filled up all of the available culture substrate, they may be sub-cultured and give place for continued growth.
Following serial sub-culture of primary cells, cells that continue to grow are subjected to genetic reorganization and adapt to the new synthetic environment and proliferate serially, generating an immortalization event that results in a Continuous Cell Line over several passages or definitely immortal cells.
The English physicist Robert Hooke (1635-1703) viewed, through a microscope of 50x magnification, a thin cutting of cork and discovered empty spaces contained by walls and used for the first time the term “cell” to describe it. In 1670, Antonie van Leeuwenhoek (1632- 1723) described cells in a drop of pond water using a microscope, and was the first person to observe single-celled animals (protozoa) with a microscope.
In 1839, the German biologist Theodor Schwann reached, in agreement with Matthias Schleiden, the conclusion that animal tissue was composed of cells, ending speculations that plants and animals were fundamentally different in structure. The statement that cell is the basic unit of life was the second generalization concerning cells and is the most important in the development of biology. It became known as the “cell theory”.
After several empiric experiences in various countries, Edward Jenner demonstrated that infection with cowpox could protect a person from small pox infection. In order to ban the arm to arm transmission of inoculums, the vaccine material, produced on calves, came to be wide spreadly used in different parts of Europe and North America. Animals have been used for the production of human vaccines since vaccine farms were established to harvest the cowpox virus from calves in the late 1800s. From that point through the first half of the 20th century, most vaccines were developed with the use of animals, either by growing pathogens in live animals or by using embryonated chicken eggs.
By the late 1800’s, as bacteriology established itself, Pasteur developed the first live attenuated viral vaccine for rabies. Although the virus could not be cultured, Pasteur was able to take the infected spinal cord of a rabbit, inoculate another rabbit, take its spinal cord and inoculate another rabbit, and so on for multiple passages until when, after two weeks of desiccation, the virus was attenuated yet still immunogenic enough to serve as a vaccine. The first time it was successfully used was in 1885 on a 9-year old boy severely bitten by a rabid dog.
Animal cell culture was first successfully undertaken by Ross Harrison in 1907 with frog’s embryonic nerve tissue. He described his own work as an extension of Wilheim Roux (1885) experiments. Both scientists were interested in studying specific forms of cellular differentiation during embryonal development. It was not until late 1940’s to early 1950’s that several developments occurred that made cell culture widely available as a scientific tool.
In the history of cell culture and vaccine development, a large breakthrough was the use of embryonated chicken eggs for viral growth by Goodpasture’s in 1931. From this, came Theiler’s safe and effective minced chick tissue vaccine 17D virus strain against yellow fever that found enormous application in tropical countries.
The viral agent causing Foot and Mouth Disease (FMD) in cows and pigs was the first virus of vertebrates to be discovered in 1897, soon after the discovery of tobacco mosaic virus of plants. It was not until 1920 that a convenient animal model for the study of FMD virus was established by Waldmann and Pape, using guinea-pigs. Later the vigil bovine animals were used for the virus multiplication for veterinary medicine and for the development of an inactivated vaccine in 1938 that proved to be efficient and safe.
Development of in vitro techniques for the growth of the FMD virus achieved by Frenkel in 1947, has been critical for the large-scale production of vaccines demonstrating that large amounts of virus could be produced in surviving tongue epithelium and forming the basis for the vaccination program initiated in Europe in the 1950s.
Primary cell techniques began in 1949 with Enders, Weller and Robins who were honored with a Nobel price for their studies on the use of primary cells for virus propagation after years of using whole animal tissues and chick embryo tissues. First examples include Mumps virus and Poliovirus propagation on various cell production systems (1949).
In 1943, Earle established the L-cell mouse fibroblast cell line as the first continuous cell line. Further in 1951, the first human cell line was established by Gey which proved to be a successful in vitro model and perhaps the most famous human cell line, the HeLa, or Henrietta Lacks cell line from cervical cancer. The cell line was a scientific achievement with profound future benefit to medical research, and was found to be remarkably durable and prolific as illustrated by its contamination of many other cell lines used in research. At the same period, Dulbecco made use of trypsin for generating replicate subcultures.
Vaccine and cell culture history started in 1950’s with the M. Hilleman’s question about which cell substrate should be used to develop a live adenovirus vaccine. Essentially there were two choices: HeLa cells derived from a human carcinoma in which adenovirus grew to high titers, and primary monkey kidney cells in which the virus did not grow well. The government committee that reviewed these cell substrate options determined that HeLa cells were not acceptable and that normal cells should be used for vaccine production. Essentially by default, normality was linked with primary cells, which were then used widely for vaccine development.
A major development in cell substrates was the use of primary monkey kidney cells for the manufacture of the Salk inactivated polio vaccine (IPV). Trivalent killed Salk polio-vaccine was prepared using virus grown in primary Macacus monkey renal cell cultures and was licensed in 1955. This vaccine was faced with three immediate problems that related to: incompleteness of poliovirus inactivation, highly variable immunizing potency, and the discovery of a new endigenous contaminating monkey polyomavirus SV40. The African Cercopithecus monkey, free of endigenous SV40 virus contamination was then introduced to circumvent this problem.
Children exposed to SV40 were followed-up for medical surveillance, and after more than 30 years of surveillance the conclusion was that there was no evidence of an increased frequency of cancer. Nevertheless, concerns about endogenous viruses in non-human primates continued as more latent viruses were identified. The consequence was to develop closed and carefully monitored colonies of animals and in some cases specific pathogen free colonies from which primary cell culture could be derived.
Cell substrates have been a recurring focus of attention during the past 60 years, often with a great deal of anxiety attached. The two core issues that continue to be the focus of attention even today are the potential for transmitting infectious diseases and/or cancer. The first major decision regarding cell substrate was, in fact, to avoid the theoretical risk of transmitting cancer through live viral vaccines by restricting the acceptable types of production cells to those from primary tissues, and prohibiting the use of cells derived from human tumors, such as HeLa, for such purpose.
Cells used for vaccine production, and acceptability
Primary cell cultures (PCCs) can be established from safe animals or embryos, or from selected tissues from embryos, new born animals, or adult animals of almost any species. Primary cells do not expand much, but can be dissociated with protease and expanded in a new container for multiplication. The first requirements for cell substrates were published by the World Health Organization (WHO) in 1959 for the production of IPV vaccine derived from the kidney of clinically healthy monkeys. Those requirements were revised and re-published in 1966. The use of animals bred in a carefully controlled colony especially those that are specific pathogen free are strongly recommended.
Diploïd cell lines
The next major event in the cell substrate area was the development of human diploid cell (HDCs) in 1960s, proposed as an alternative to primary monkey kidney cells. Dr Leonard Hayflick developed a number of cell strains from normal human embryonic tissues. Acceptance of HDCs was based on a significant amount of characterization data showing a normal chromosomal constitution, ability to generate cryo-preserved banks, a finite lifespan and inability to form tumors in animal models. With such characterization and leadership, the diploid cell strain concept was accepted in Europe and several National Health Authorities approved Hayflick’s WI-38 cell line for vaccines use and several years later also the MRC-5 cell line.
Based on the experience gained with HDCs, the characterization of cells became a central feature in the evaluation of all new cell types. The United States initially maintained its opposition based mostly on a speculative fear that diploid cell strains might harbor a human leukemia virus, but only until 1972, when the US Regulatory Agency approved the use of WI-38 cell line for oral live polio vaccine production, and a license was granted for live rubella virus vaccine in 1977.
Continuous Cell Lines
Normal vertebrate cells cannot be passed indefinitely in culture. After a limited number of cell doublings depending on the age and species of the original tissue, cultured cells stop to divide and then degenerate and die in a phenomenon called “crisis” or senescence. At anytime during the culture cells may become transformed meaning that they are no longer subject to crisis and senescence but can be sub-cultured for an undefined period of time. The transformation is a complex phenomenon in which cells become immortalized and are named “cell line” or “continuous cell line” in which case cells can be indefinitely propagated by subculturing.
Several techniques can be used to obtain an immortalized cellular lineage from primary cells:
- Carry on passages of a normal cell culture and obtain continuous cell line, often described as spontaneous transformation (e.g., Vero, BHK-21, CHO, MDCK)
- Immortalization can be induced by treatment with chemical mutagens (e.g., QT35, LMH),
- Hybridization between host cell with an immortalized cell line (e.g., hybridoma),
- Transfection with ectopic expressed genes involved in the cell cycle, such as E1 adenovirus (e.g., HEK-293, PER-C6),
- Transfection with hTERT telomerase gene to enable indefinite replication of normal cells.
Under particular conditions, certain cells are able to spontaneously transform into immortal cell lines. The mechanisms by which cells gain immortalization are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Genetic modifications induced during the immortalization process can have consequences on the karyotype with cells becoming aneuploid and containing abnormalities in chromosome number and structure.
Continuous cell lines (CCLs) have been used for the production of safe and effective bio-therapeutics and vaccines since the 1970’s. To date a limited number of cell lines have been authorized by Health Authorities to produce vaccines, limited to CCLs obtained from normal tissues.
The Vero cell line, which was the first continuous mammalian cell line established from African green monkeys in 1962, is currently the most widely accepted by regulatory authorities for vaccine manufacturing. This is due to the fact that Vero-derived human vaccines have been used for nearly 40 years. The Vero cells can be grown and infected on micro-carrier beads in large-scale fermentors and in serum-free medium with no loss in productivity.
Then, in 1987, a report from theWHO Study Group on Biologicals rescinded the requirement for non-tumorigenicity for continuous cell lines. In its risk assessment, the Group concluded that “…continuous cell lines are acceptable as substrates for the production of biological, but the differences in the nature of the products and in the manufacturing processes must be taken into account in making a decision on the acceptability of a given product.”
A WHO Expert Committee and the International Alliance for Biological Standardization (IABS) concluded in 1996 that the presence of 10 ng of heterogeneous DNA of non tumorigenic CCL per dose of product poses negligible risk for safety and that “nuclease treatment of products during manufacture would probably add more concerns than it would remove.” The group concluded that every product must stand on its own merits for safety and efficacy and that the use of alternative manufacturing methods not involving the use of continuous cell lines would be irrelevant to the acceptance or rejection of a product derived from a continuous cell line.
The next step of cell acceptability, for therapeutic drug, was in 1970s when interferon (IFN), found to have an interesting therapeutic potential, had to be produced at large scale. At that time the only source of IFN was from primary human lymphocytes which had a very limited productivity. This issue could be solved by the use of human lymphoma cells (Namalva cell line). However, Namalva cells contain an integrated Epstein Barr virus and unrelated cancer-sequences. After considerable discussion there was agreement from the regulatory agencies in the US and Europe to allow the use of Namalva cells for the production of IFN. Nevertheless, all possible functional DNA would need to be eliminated from the biological product with DNAses treatment and purification steps during the manufacturing process.
Monoclonal antibody technology, through development of recombinant DNA, facilitated the development of continuous cell lines that could secrete antibody of defined specificity.
The first tumorigenic cell line to be considered for use in the production of a live viral vaccines was the PER.C6 cell line. It was used for the development and production of a replication-defective adenovirus vectored HIV-1 vaccine (VRBPAC 2001). While adventitious agents and residual cell-substrate DNA are potential concerns with all novel cell substrates, there may be a heightened concern when the cell substrate is tumorigenic or derived from a tumor. In particular, the potential risk of adventitious agent contamination (including TSE contamination, since PER.C6 cells are neural derived), of residual cell-substrate DNA, and of whole cells were considered.
The next tumorigenic cell line under consideration was the Madin-Darby canine kidney (MDCK) cell line, proposed for the production of inactivated influenza virus vaccines (VRBPAC 2005). Additionally, a non-tumorigenic MDCK cell line was considered for the manufacture of a live, attenuated influenza virus vaccine (VRBPAC 2008).
Cellular DNA emerged as a major safety issue. Thus, all possible functional DNA would need to be eliminated from the biological product with DNAses treatment and purification steps during the manufacturing process.
The current repertoire of cell substrates is inadequate for manufacture of certain types of new vaccines. To address this issue, the VRBPAC (2) recognized in 2012, that human tumor-derived cell lines could be an important addition to the repertoire of cell substrates for the manufacture of viral vaccines, and there was nothing that a priori precluded the use of such cells. This position was not confirmed by the European Medicines Agency (EMA).
In any case, the use of a specific tumorigenic cell substrate is generally discussed with the vaccines advisory committee before initiating a clinical trial. In particular, three major safety concerns need to be addressed: 1) the presence of residual live cells in the vaccine that might have the potential of being tumorigenic in humans; 2) the presence of residual DNA from the cell substrate; and 3) the potential presence of adventitious agents, including adventitious viruses that might have contributed to the tumorigenic phenotype.
Insect and avian cells
The first continuously growing insect cell cultures were established from lepidoptera insects around 1960. Since then, the Spodoptera frugiperda Sf9 and Trichoplusia ni Hi-5 cell lines are the most widely used. The latter two are susceptible to the baculovirus Autographa californica multiple capsid nucleopolyhedrovirus and are used for the expression of foreign genes, for example, for the production of subunit vaccines to produce proteins for gene delivery vectors for mammalian cells. The baculovirus-insect cell expression system, often referred to as BEVS, is a well known tool for producing complex proteins and virus-like particles (VLPs) antigens providing rapid access to biologically active proteins.
The first commercially available veterinary vaccine produced in insect cells was the swine fever virus (CSFV) vaccine, and for humans medicine the European Medicines Agency licensed in 2007 the bivalent human papilloma virus vaccine indicated for the prevention of cervical cancers.
Regarding avian cells, a stable duck cell line EB66 has been established from embryonic stems (ES) cells by spontaneous immortalization. After stabilization in a dedicated culture media, these cells were found to have an ultra structure similar to other ES cells, a large nucleus, expression of ES-specific markers on their cell surface, a stable karyotype, and to express the expected telomerase activity for a cell line derived from ES cells. A second category of duck cell line is the AGE1.CR1 which has been immortalized using E1 gene of adenovirus type 5, and which is grown in suspension cultures in serum-free media.
Stem Cells (SCs) differ from other cell types by sustaining a predominant stem-cell population that retains the capacity to produce cell progenitors of differentiated cell types of almost all human tissues.
Pluripotent SCs are well characterized and can be differentiated in different tissue specific cells showing some very interesting properties for virus productivity.
As for differentiated cell lines, the non transformed nature must be considered as a pre-requisite for vaccine production.
Future of cell culture technology for vaccine production
Vaccines are mainly prophylactic and sometimes therapeutic; they represent bio-pharmaceutical products making their development and commercialization complex. The WHO requires reports studies to ensure the safety of the population receiving vaccines.
Approximately 20 vaccines are currently in use, which represents a small number of products reported from the first human vaccine developed by Jenner in 1796 until today. This indicates the major difficulties encountered to develop new vaccines and the large number of trials and clinical assays needed to ensure maximum safety and efficacy to patients. The use of molecular biology, biotechnology, DNA recombination and genomics might play a large role in the development of novel vaccines for infectious agents.
Vaccines for polio, measles, mumps, rubella, chickenpox and more recently Rotavirus, HPV, are currently manufactured using cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is currently being funded by the United States government, including the use of mammalian, avian and insect cell- based processes as well as vegetal cell line and plants. As a consequence, currently all paths are open, including novel insights coming from the fields of recombinant DNA-based vaccines made with new vectors, and the use of novel adjuvants.
As a result, state-of-the-art technologies to simplify vaccine development and manufacturing are becoming evermore crucial. One important difference between the production of vaccines and other biopharmaceuticals is the risk-safety consideration related to working with pathogens and pathogenic antigens. As with all bio-molecules purified from crude biological material, the removal of contaminants (e.g., derivatives from host cell such as DNA, protein, or leachable), must be documented. However, the removal or inactivation of adventitious viruses remains a unique challenge. Risk assessment related to the use of cell culture techniques are essentially the same as those identified in 1950’s: transmissible agents (e.g. virus) and cellular components (e.g. DNA, HCP).
Nevertheless, scientific knowledge in developmental procedures and process characterization by sophisticated analysis are critical not only to improve yields, but also to determine the final product quality. From a regulatory perspective, Quality by Design (QbD) and Process Analytical Testing (PAT) are important initiatives that can be applied effectively to many types of vaccine processes.
Acceptance of cell substrates can be achieved by their evaluation and characterization on a case by case basis, and by considering the product acceptability on the basis of a risk and benefit analysis taking into account different factors such as risk mitigation through control of the production in the manufacturing process, the seriousness of the disease, and the availability of other therapies.
1. S. Plotkin in Vaccines fifth edition
2. Vaccines and Related Biological Products Advisory Committee
Updated by Dr. Jean-François Bouquet (June 2013).