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Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Cell substrates for the production of viral vaccines

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Franc¸oise Aubrit, Fabien Perugi, Arnaud Léon, Fabienne Guéhenneux, Patrick Champion-Arnaud, Mehdi Lahmar ∗ , Klaus Schwamborn

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Vaccines Research & Discovery Department, Valneva SE, 6 rue Alain Bombard, 44800 Saint-Herblain, France

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a r t i c l e

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a b s t r a c t

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Article history: Available online xxx

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Vaccines have been used for centuries to protect people and animals against infectious diseases. For vaccine production, it has become evident that cell culture technology can be considered as a key milestone and has been the result of decades of progress. The development and implementation of cell substrates have permitted massive and safe production of viral vaccines. The demand in new vaccines against emerging viral diseases, the increasing vaccine production volumes, and the stringent safety rules for manufacturing have made cell substrates mandatory viral vaccine producer factories. In this review, we focus on cell substrates for the production of vaccines against human viral diseases. Depending on the nature of the vaccine, choice of the cell substrate is critical. Each manufacturer intending to develop a new vaccine candidate should assess several cell substrates during the early development phase in order to select the most convenient for the application. First, as vaccine safety is quite naturally a central concern of Regulatory Agencies, the cell substrate has to answer the regulatory rules stringency. In addition, the cell substrate has to be competitive in terms of viral-specific production yields and manufacturing costs. No cell substrate, even the so-called “designer” cell lines, is able to fulfil all the requested criteria for all viral vaccines. Therefore, the availability of a variety of cell substrates for vaccine production is essential because it improves the chance to successfully respond to the current and future needs of vaccines linked to new emerging or re-emerging infectious diseases (e.g. pandemic flu, Ebola, and Chikungunya outbreaks). © 2015 Published by Elsevier Ltd.

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Keywords: Vaccine production Cell substrate Regulatory requirements Manufacturing process

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1. History of cell substrates in vaccine development

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The lifesaving potential of vaccines, for humans and animals, has been largely proven by the comparison of the infectious disease burden before and after the introduction of national vaccination

Abbreviations: BSE, bovine spongiform encephalopathy; CAP, CEVEC’samniocyte production; CEF, chicken embryo fibroblast; DNA, deoxyribonucleic acid; EP, European Pharmacopeia; FBS, fetal bovine serum; GCCP, good cell culture practice; GMP, good manufacturing practices; HCD, host cell DNA; HEK, human embryonic kidney; MCB, master cell bank; MDCK, Madin Darby canine kidney; MRC, Medical Research Council; NRA, national regulatory authority; PERT, product-enhanced reverse transcriptase; RA, regulatory agency; RMK, rhesus monkey kidney; SCID, severe combined immunodeficiency; SV40, simian virus 40; TEM, transmission electron microscopy; TPD50 , tumour-producing dose in 50 per cent of animals; TSE, transmissible spongiform encephalopathy; WCB, working cell bank; WHO, World Health Organisation; WI, Wistar Institute. ∗ Corresponding author. Tel.: +33 2 28073710; fax: +33 2 2807 3711. E-mail addresses: [email protected] (F. Aubrit), [email protected] (F. Perugi), [email protected] (A. Léon), [email protected] (F. Guéhenneux), [email protected] (P. Champion-Arnaud), [email protected] (M. Lahmar), [email protected] (K. Schwamborn).

programs [1,2]. Over time, there have been significant advances in vaccine manufacturing technologies, leading to achievement of greater productivity as well as production of safer and more immunogenic vaccines. This review will focus on cell substrates used for the manufacturing of viral vaccines based on replication−competent viruses (e.g. live attenuated, recombinant, chimeric, and inactivated vaccines) intended for human use. Cell substrates have been the result of active research for decades and have been implemented to improve the product safety, answer the increasing demand of vaccine production and decrease the associated manufacturing costs.

1.1. Evolution of vaccine production techniques First vaccines were derived from sick people and infected animals. Jenner’s first inoculation was made by injecting an 8-year old boy with the pus from a cowpox lesion on a milkmaid’s hand [3]. From that point, and through the early 20th century, vaccines were mostly produced by employing animal tissues, such as nervous tissues extracted from rabbit, sheep or goat, suckling animal brain (mouse, rat, or rabbit), or using blood serum of infected animals.

http://dx.doi.org/10.1016/j.vaccine.2015.06.110 0264-410X/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Aubrit F, et al. Cell substrates for the production of viral vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.110

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Since the 1930s, methods for growing viruses in the laboratory using embryonated hen eggs have been developed and used to produce and manufacture human and veterinary vaccines [4]. Eggs are still largely used, particularly in the seasonal flu vaccine manufacturing [5]. However, they pose a number of limitations including risk of insufficient supply, time-consuming processes with inconsistent yields, high costs of manufacture, and the potential for allergic responses to egg-components [6–8]. To overcome egg limitations, cell culture technology has been introduced, offering higher flexibility than the traditional manufacturing procedures. The first important step in the vaccine field was the production of polio vaccine using monkey kidney cells by Jonas Salk in 1954 [9,10]. Since then, the use of primary cell substrates, such as primary chicken embryo fibroblasts (CEFs), has greatly facilitated vaccine manufacturing (e.g. measles, and mumps) [11]. Primary cells were derived directly from an animal source and were not stored—or to a limited extent—as cell banks. However, their use raised concerns due to their limited self-renewal capacity and to the risk of contamination of primary cultures, as cells needed to be freshly prepared for each vaccine production lot. The increasing demands in vaccine production yields and safety have urged the development of safer, cheaper, and more efficient cell substrates. The first cell substrates developed to this aim included both diploid and continuous cell lines (Table 1) [12]. Diploid cell lines, such as human lung-derived MRC5 (Medical Research Council 5) and WI-38 (Wistar Institute) cells [13,14], were obtained from primary cultures. They have a normal or near normal karyotype but show a finite capacity for serial propagation, which ends in senescence and cease of replication. Conversely, continuous cell lines, such as MDCK (Madin Darby canine kidney) cells [15,16] and African green monkey kidney-derived Vero cells [17], display infinite self-renewal capability and can be readily available for production from cell bank systems, allowing extensive characterization and reproducibility of the cell populations for an indefinite period. Thanks to their indefinite lifespan, continuous cell lines can be adapted to modern culture technologies (e.g. culture vessels, large-scale fermenters, micro/macro-carriers, and media). However, depending on the passage number, genetic modifications may occur and lead to a tumorigenic phenotype of the cell substrate. For example, Vero cell line at high passage levels (passage 162) displays genetic instability and develops tumorigenic potential [18]. Thus, only low-passage non-tumorigenic Vero cells can be used for vaccine production, which might raise concerns as the seeds stocks are currently decreasing due to the extensive use of this cell substrate. Such limitations, combined with the appearance of new technologies for cell line development, have encouraged the establishment of new cell lines, including the so-called “designer” cell lines [19,20]. Several ways have been explored to obtain the currently available new cell substrates, including selection pressure (e.g. suspension MDCK cell line; duck embryonic stem cell-derived EB66® cells [21]) or genetic modification (e.g. human retinaderived PER.C6® cells [22,23], duck retina-derived AGE1.CR® cells [24], and human amniocyte-derived CAP® cells [25]). These cell substrates have been developed for specific applications, such as adenovirus production in gene therapy (PER.C6® cell line) or influenza vaccine production [26] and have been extensively characterized to fulfil regulatory and biosafety requirements. The long and cost-effective derivation process, as well as the high market pressure, hinders the development and the characterization of a new cell substrate for each vaccine indication. Therefore, currently available cell lines are considered as an option to produce new vaccine candidates and are tested for the replication of viruses not efficiently produced by old cell substrates. Table 1 summarizes the most common cell substrates that are currently used in the vaccine field. Their main biological characteristics and properties are displayed in Tables 2 and 3.

1.2. Criteria of selection of cell substrate for the manufacturing of viral vaccines The selection of a cell substrate is an important step for the development and manufacturing of a viral vaccine candidate that relies on several parameters (e.g. cell susceptibility and permissiveness to the viral pathogen, performance in terms of viral antigens quality and production yield, primary versus continuous cells, ethical point of view, tumorigenicity status, anchorage-dependent versus suspension culture, culture medium, manufacturing cost, free of adventitious agents, etc.). The format of the vaccines (e.g. inactivated versus live-attenuated viral vaccines; administration routes; preventive or therapeutic vaccines) has also to be taken into account for the cell substrate selection. Finally, safety and industrial considerations deeply impact the choice of the suitable/optimal cell substrate. They are further detailed in Parts 2 and 3 of this review.

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2. Regulatory considerations

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2.1. Evolution of regulatory requirements for vaccine safety

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Since the first-generation vaccines produced by employing animal tissues, the main concerns of regulatory agencies (RAs), manufacturers and public health authorities are the possible presence of adventitious agents or cell components, such as deoxyribonucleic acid (DNA) or transforming protein in vaccine products. Indeed, several significant cases of contamination have been evidenced during the last century [27], such as the discovery of simian virus 40 (SV40) in monkey kidney cells (rhesus monkey kidney [RMK] cells) used to produce polio vaccines in the 1960s [28,29], bacterial viruses identified in several live-attenuated viral vaccines manufactured with bovine sera containing bacteriophages, in the early 1970s [30,31] and more recently, the detection of porcine circovirus sequences in rotavirus vaccines (Rotarix® and RotaTeq® ) [32]. The advance in science and technology and the use of more powerful analytical methods able to evidence undetectable or previously unknown contaminants has led the RAs to implement new manufacturing and controlling practices edited as guidelines. The basic principle underlying the guidelines is that quality, safety, potency, purity, and efficacy of the vaccine rely on a comprehensive approach based on the risk assessment that impacts the selection and characterization of raw materials and starting materials, the control of intermediate and final product but also the design and validation of the manufacturing process. Specific guidelines are in place and periodically revised to provide manufacturers advices on the selection, characterization, and maintenance of cell substrates used for vaccine production [33–38]. In particular, guidelines have evolved to take into consideration issues related to new cell substrates, including “designer” cell lines, which often exhibit a tumorigenic phenotype [20]. An interesting example is provided by the duck EB66® cell line. As per current version of chapter 5.2.3 of the European Pharmacopeia (EP), the preparation of live vaccines in tumorigenic cell line is prohibited [38]. However, the use of EB66® has been now considered as suitable for the manufacturing of such vaccines according to an anticipated change of the EP that will be harmonized with the World Health Organisation (WHO) recommendations published in 2013 [Personal communication]. 2.2. Characterization of cell substrates used for the manufacturing of viral vaccines Regardless of the cell type (e.g. primary, diploid, stem, or continuous cells) and the type of vaccines to be manufactured, the cell

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Table 1

Q4 Most common cell substrates currently used for the production of human viral vaccines. Cell line

Primary cells CEF

Diploid cell lines MRC5

WI-38

Continuous cell lines MDCKa

Cell type and origin

Viral susceptibility (not exhaustive list)

Examples of vaccine candidate in development

Marketed vaccines for human use

Chicken embryo fibroblast

Yellow fever; rabies; TBE; measles; mumps

MVA-based vaccines; HIV; Q fever

Rabies (Rabipur® ); TBE (FSME-Immun® , Encepur® ); measles (Attenuvax® ); mumps (Mumpsvax® )

Human embryonic lung

Varicella zoster virus; polioviruses; rabies; hepatitis A

Rabies

Human embryonic lung

Rubella; adenoviruses



Varicella zoster virus (Varilrix® ; Biopox® ; ProQuad® ); polioviruses (Poliovax® ); rabies (Imovax® ); hepatitis A (VAQTA® ) Rubella (Meruvax® II); adenoviruses (Adenovirus Type 4 and Type 7 Vaccine, Live, Oral® )

Canine kidney

Influenza virus type A and B; Human coxsackievirus B3, B4 and B5; reovirus type 2; AAV 4 and 5; vaccinia virus; VSV; Human poliovirus 2 Influenza virus type A and B; Measles virus; vaccinia virus; rubella virus; mumps virus; NDV; polioviruses; arbovirus (including dengue); rabies; RSV; parainfluenza viruses; reoviruses

Seasonal & pandemic flu; enterovirus

Seasonal flu (Optaflu® , Flucelvax® )

Enterovirus; RSV; HFMD; influenza virus, rabies

Pandemic (Celvapan® ) & seasonal flu (Preflucel® ); smallpox (ACAM2000® ); JEV (Ixiaro® ,IMOJEV® ); polioviruses (OPV® , IMOVAX Polio® , PolioRIX® , Adacel® ); rabies (VERORAB® , Abhayrab® ), rotaviruses (RotaRIX® , RotaTeq® ) −

Vero

African green monkey kidney

PER.C6®

AGE1.CR®

Human embryonic retina Duck retina

EB66®

Duck embryos

Adenovirus; influenza virus; lentivirus; polioviruses; Ebola

Influenza virus; West Nile virus

Smallpox; fowlpox; influenza virus; alphaviruses Influenza virus; measles virus; mumps virus; HSV; poxviruses; NDV; Sindbis virus; Sendai virus; VEEV; yellow fever; VSV

MVA-based vaccines; influenza virus Pandemic & seasonal flu; NDV; MVA recombinant vaccines

− Pandemic flu (no commercial name yet)

Note: CEF, chicken embryo fibroblast; MRC, Medical Research Council; WI, Wistar Institute; MDCK, Madin Darby canine kidney; AAV, adeno-associated virus; VSV, vesicular stomatitis virus; NDV, Newcastle disease virus; RSV, respiratory syncytial virus; HSV, herpes simplex virus; MVA, modified vaccinia virus Ankara; JEV, Japanese encephalitis virus; HIV, human immunodeficiency virus; TBE, tick-borne encephalitis; HFMD, hand, foot and mouth disease; VEEV, Venezuelan equine encephalitis virus.

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substrate must be carefully documented, well-characterized, and qualified for its intended purpose. For all cell types, preparation of a master cell bank (MCB) and a working cell bank (WCB) according to the general principle of good manufacturing practice (GMP) and good cell culture practice (GCCP) [39] is considered as best practice to ensure a reliable and consistent supply of cells that can be fully characterized and safety tested before their use for production. Cell banks are stored under conditions suitable for long-term stability such as liquid nitrogen or ultra-low temperature freezer and are periodically tested to demonstrate sustainability of their phenotypic and genotypic characteristics. The testing program of the cell substrate relies on a riskassessment that takes into consideration: (1) source of the cell substrate: species and tissues origin; (2) history of the cell substrate: methods of isolation, results of testing or screening performed on donors, passage history, raw materials used, history of passage in animals; (3) characteristics of the cell substrate: cell growth characteristics, biochemical, genetic or cytogenetic patterns, as well as results of tests for infectious agents; and (4) assessment of tumorigenic and oncogenic activity. Finally, compatibility between cell substrate and vaccine virus has to be assessed. As selective pressure could alter the genotype or even more the phenotype of the virus during passage in cell culture, stability of vaccine virus must be demonstrated.

2.2.1. Source of the cell substrate Origin of the cell substrate is a key element to be considered as potential source of contamination. Nature of the contaminants

varies according to the species of origin of the cell substrate. As an example, avian species, and especially chickens, may be contaminated by retroviruses, such as avian leukosis viruses or reticuloendotheliosis virus, or by bacteria such as Mycoplasma gallisepticum or Mycoplasma synoviae, while contaminants specific to canine species may include canine distemper virus and canine adenoviruses. Tissue type and immortalization methods need to be considered as well. For instance, cells derived from a tumor can present oncogenic virus sequences, such as the human cervical cancer HeLa cells that contain human papilloma virus 18 DNA [40,41]. This is also the case for cells transformed by DNA from adenovirus type 5, such as the human embryonic kidney 293 (HEK-293) [42] and the PER.C6® cell lines [22,23].

2.2.2. History of the cell substrate History of the cell substrate begins with isolation or derivation and should include details of the medical history or pathogen status of the donor in the case of human- or animal-derived cell substrate, traceability of raw materials used during the derivation process, as well as any other agents (e.g. cells, and viruses) handled in the same environment before and during the time of cell culture. In addition, procedures have to be set up to mitigate risks of contamination with adventitious agents, adaptation, engineering, or cloning performed on the cell substrate since its isolation from donor(s). Use of raw materials of human or animal origin has to be thoroughly documented and analyzed in respect to the presence of potential contaminants. Since the emergence of bovine spongiform encephalopathy (BSE) in 1980s, special attention is given to bovine

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Table 2 Main characteristics of most common cell substrates currently used for the production of human viral vaccines–Biological characteristics. Cell line

Primary cells CEF [87]

Diploid cell lines MRC5 (ATCC CCL-171) [37] WI-38 (ATCC CCL-75) [37] Continuous cell lines MDCK [88–90] Vero [91] PER.C6® [92]

AGE1.CR® [93]

EB66®

Process of derivation

Karyotype

Genetic stability

Tumorigenicity/ Oncogenicity

RT activity from retroviral origin

Adventitious agents

Derived from embryonated eggs

2n = 76



NT/NT

Yes

Risk of endogenous retrovirus particles

Derived from a normal embryonic lung

46 (XY), polyploidy rate of 3.6%

Yes

No/NT

No

No

Derived from a normal embryonic lung

46 (XY)

Yes

No/NT

No

Micrococcus at P8 (cleared with neomycin)

Derived from canine kidney Derived from African green monkey kidney Embryonic retinal cells transformed by the insertion of Ad5 E1 genes Duck retinal cells immortalized with the Ad5 E1 genes Derived from Duck embryonic stem cell

Undisclosed (hyperdiploid) Hypodiploid (2n = 58)

Yes

Yesa /No

No

No

Yes

No/Nob

No

No

46 (XX)

-

Yesc /No

No

No



Yes

No/NT

No

No

Diploid (2n = 78)

Yes

Yesd /No

No

No

Note: RT, reverse transcriptase; NT, not tested; CEF, chicken embryo fibroblast; MRC, Medical Research Council; NT, not tested; WI, Wistar Institute; MDCK, Madin Darby canine kidney; ATCC, American Type Culture Collection; CCL, certified cell line; P, passage; Ad5, adenovirus 5. a Both high [88,89] and low [90] tumorigenicity have been reported depending on the tested subclone. b If passage level below p150. c Weakly tumorigenic: only after injection of 107 cells. d Weakly tumorigenic: only after injection of 107 cells (unpublished data).

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and porcine materials (e.g. serum from bovines, trypsin from pigs, etc. [43,44]). The European Commission published a guidance that applied to any material derived from “transmissible spongiform encephalopathy (TSE) relevant animal species”, which are used for the preparation of raw and starting materials required for production, including those used to prepare WCB or new MCB [45]. 2.2.3. Characteristics of the cell substrate and detection of adventitious agents An extensive characterization has to be performed either on the MCB or on the WCB. A representative biosafety testing scheme based on WHO guidance is given in Table 4. Manufacturers define the characterization program according to national regulatory authorities’ (NRA) requirements and develop a testing strategy based on the history and type of cells. Testing program comprises assays investigating identity, purity, stability, and sterility (e.g.

karyotyping, isoenzyme profile, DNA fingerprinting, growth rate, presence of specific chromosomic, or surface markers, tests for nonviral agents: bacteria, fungi, mycoplasma and mycobacteria, tests for viral agents, including in vitro cell-culture tests for cytopathic and haemadsorbing/hemagglutinating viruses using different cell lines and in vivo assays in several species, assays for retroviruses, such as transmission electron microscopy (TEM) and productenhanced reverse transcriptase (PERT) assay) [46–48]. Assays for species-specific adventitious viruses known to be present in the species of origin or in raw materials used during the derivation process are recommended; testing for the presence of latent viruses (DNA viruses and endogenous retroviruses) using chemical inducers as well [49]. Known and novel viruses may be detected using new methods such as massive sequencing and virus microarrays [50,51]. These new technologies have not been recommended for use until now due to technical challenges such as assays

Table 3 Main characteristics of most common cell substrates currently used for the production of human viral vaccines - Manufacturing characteristics. Cell line Primary cells CEF Diploid cell lines MRC5 WI-38 Continuous cell lines MDCK [94] Vero [95] PER.C6® [23] AGE1.CR® [96–98] EB66® [66]

Culture type

Doubling time (hours)

Maximum cell density (cells/mL)

Scalabilitya

Adherent

>24

-

-

Adherent Adherent

27 24

-

-

Suspension Microcarriers Suspension Suspension Suspension

20-30 25-44 15-18

2 × 106 (batch)20 × 106 (perfusion) 1,000 L

Note: CEF, chicken embryo fibroblast; MRC, Medical Research Council; WI, Wistar Institute; MDCK, Madin Darby canine kidney; P, passage; WCB, working cell bank. a Maximum scalability demonstrated for each specific cell type.

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Table 4 Representative biosafety testing scheme for cell substrates. Test

Master Cell Bank

Working Cell Bank

End-of-Production Cells

Identity & purity Genetic stability (karyotyping) Sterility Bacteria, fungi Mycoplasma, Spiroplasma Mycobacteria Adventitious agents In vitro cell cultures In vivo assays TEMa Antibody production assay Bovine viruses Porcine viruses Retrovirusesa Specific viruses Tumorigenicity Oncogenicity

+ (+)

(+) NA

(+) (+)

+ + (+)

+ + (+)

+ + (+)

+ + + (+) (+) (+) + (+) NA NA

+ NA NA NA (+) (+) NA (+) NA NA

+ + + NA (+) (+) + NA + (+)b

Note: +required stage for testing; (+) alternative stage for testing; NA, not applicable. a May be combined with virus induction assays as applicable. b Required if the tumorigenicity result is positive.

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standardization and validation, data interpretation, strategies for investigating positive results, and choice of manufacturing stage at which they should be applied.

lines derived from tumors. Therefore, extensive characterization for adventitious agents in the cell substrate and clearance strategies through the manufacturing process is required.

2.2.4. Assessment of tumorigenic and oncogenic potency The use of immortalized and tumorigenic cell lines to manufacture viral vaccines has been discussed since the 1990s [52,53]. The main concerns associated with the tumorigenicity of cell substrates are induction of tumor allografts, transfer of known or unknown viruses and transfer of oncogenic agents or cell components that might initiate tumors. Therefore, description of the tumorigenic activity has to be performed for all diploid (with the exception of the well-characterized MRC-5 and WI-38) and continuous cell lines, as cells could acquire tumorigenic activity with increasing passage levels [54]. Cell lines are classified as tumorigenic when they possess the capacity to form tumor after injection of intact cells into genetically immunocompromised animals (e.g. nude mice, severe combined immunodeficiency (SCID) mice, or animals immunocompromised by inactivating the T-cell functions with antithymocyte globulin, antithymocyte, or antilymphocytes serum) [34]. The tumorigenic phenotype of the cell substrate can be defined by evaluating the kinetics of tumor formation at dose of 107 , 105 , 103 and 10 cells/animal, over at least a 4-month observation. The determination of the tumor-producing dose in 50% of animals (TPD50 ), the time required for tumor development and the capacity to form metastases are parameters that must be evaluated for a given cell substrate. The current understanding is that a lower TPD50 (capacity to form tumors at 101 −104 cells/animal; examples include Hela cells) is correlating to a higher risk of inducing neoplastic development. There is no general rule for the cell substrate and/or the product acceptability in this respect but rather a discussion with the RAs on a case by case basis. Use of tumorigenic and tumor-derived cells for vaccine production requires an oncogenicity study that must be performed using cell DNA (≥100 ␮g) and cell lysate (obtained from 107 cells) injected in newborn (e.g.

Cell substrates for the production of viral vaccines.

Vaccines have been used for centuries to protect people and animals against infectious diseases. For vaccine production, it has become evident that ce...
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