CHIRONVACCINES

 

 

 

 

 

 

Influenza Cell Culture Subunit Vaccine

Vaccines and Related Biological Products

Advisory Committee (VRBPAC)

 

Bethesda, MD

November 16, 2005

 

 

 

 

 

 

 

Property of Chiron Corporation. For media inquiries, contact Corporate Communications at (510) 923-6500 or at communications@chiron.com.


TABLE OF CONTENTS

1.0 EXECUTIVE SUMMARY.. 3

2.0 BACKGROUND.. 3

2.1 Egg-based Influenza Virus Vaccines. 3

2.2 Cell Culture-Derived Influenza Vaccines. 5

2.3 Cell Culture-Derived Pharmaceuticals and Vaccines. 6

Table 2.3-1 Continuous cell line and corresponding approved  pharmaceutical products. 7

3.0 CHIRON’S CELL CULTURE-BASED VACCINE.. 8

3.1 Selection of MDCK Cells as the Production Substrate. 8

3.2 Risk Assessment and Management of the MDCK-based Production System.. 9

3.2.1 Studies of MDCK cells, Lysates, and DNA.. 10

Table 3.2-1 Program of tumorigenicity and oncogenicity studies. 10

3.2.2 Defined Risks Assessment (DRA) 11

3.3 Status of Clinical Development 13

4.0 CONCLUSIONS. 13


1.0 EXECUTIVE SUMMARY

An increased demand for influenza vaccines and manufacturers’ inability to supply an adequate number of doses for the currently-licensed egg-derived influenza vaccines, along with the threat of an avian-strain influenza pandemic, have prompted the U.S. Department of Health and Human Services (DHHS), and other global public health agencies, to foster the development of alternative substrates for influenza vaccine production. Chiron Vaccines has developed a cell culture influenza vaccine that utilizes MDCK (Madin Darby Canine Kidney) cells for manufacturing. These cells offer significant advantages for routine influenza vaccine manufacturing, as well as being able to meet the unique challenges of pandemic vaccine manufacturing posed by virulent avian strain candidates. Because FDA defines these cells as ‘neoplastic’ (immortalized cell lines that are either tumorigenic or nontumorigenic), an extensive risk assessment for the product was carried out based upon the approach outlined in the 1999 FDA document “A Defined-Risks Approach to the Regulatory Assessment of the Use of Neoplastic Cells for Viral Vaccine Manufacture” in conjunction with discussions with the Office of Vaccines Research and Review (OVRR) review staff. The extensive program of studies demonstrates that the manufacturing process eliminates intact MDCK cells from the vaccine and reduces any potential risk associated with either residual MDCK-cell DNA or from occult adventitious agents to limits that are acceptable for the production of an influenza vaccine. This vaccine has been tested in more than 3000 human subjects in clinical trials outside the United States and has compared favorably in both its safety profile and in its ability to induce appropriate antibody responses when compared to conventional egg-derived influenza vaccines. On the basis of these findings, Chiron plans to vigorously pursue approval of this vaccine in the United States, Europe, and other markets.

2.0 BACKGROUND

2.1 Egg-based Influenza Virus Vaccines

Influenza vaccine manufacturing has traditionally relied on embryonated hens’ eggs to grow the viruses. The basic manufacturing process for these vaccines has changed very little since they were first introduced in the 1940s, aside from adding steps for virus disruption (”splitting”) or partial purification, or both. Egg-derived vaccines have had an extraordinarily long and successful track record of safety and efficacy in the United States and elsewhere, where annual vaccination has been shown consistently to reduce the incidence of illness and its complications and mortality when there is a reasonably close match between circulating influenza viruses and the strains contained in the vaccine.

Despite the remarkable success of egg-derived influenza virus vaccines during the past 50+ years, current circumstances affecting both supply and demand considerations underscore a critical and urgent need to pursue alternative approaches for vaccine production. Fulfilling this need has become a high priority for the U.S. government. These circumstances include:

·        Increased demand for vaccination. In recent years, there has been an emphasis on the annual vaccination of increasing numbers of people, with recommendations having targeted approximately 183 million people in the U.S. during the 2003-2004 influenza season. The recommendations include persons age 65 years old or greater (35.9 million), persons with underlying chronic illnesses age 2-64 years (42.4 million), pregnant women (4 million), household contacts of high risk persons (70.3 million), children age 6-23 months of age (6 million), health care workers < 65 years of age (7 million), and healthy 50-64 year old persons not covered in other categories (17.7 million). As a consequence of these recommendations and abundant, evidence-based literature supporting the value of vaccination, the demand for influenza vaccine has increased from ~20 million doses in the mid-1980’s to more than 80 million doses by 2001. Vaccination levels among healthy adults wishing to reduce their risk of acquiring influenza have also increased in recent years, to ~20% in 2003. Because of highly successful educational efforts and delivery programs and the proven value of annual immunization, the Advisory Committee on Immunization Practices (ACIP) is now strongly considering an even more dramatic shift in policy towards universal vaccination. Thus, in the not-too-distant-future, the actual demand for influenza vaccine in the United States could easily exceed 200 million doses annually, a figure well beyond current production capacity.

·        Reduced and/or variable vaccine supply. Relative to this large and increasing demand for influenza vaccine, U.S. manufacturers have exhibited increasing difficulty in providing sufficient numbers of doses. Significant vaccine shortages or delays in distribution have occurred in 3 of the past 5 years. Additionally, within the past 8 years, two manufacturers – King Pharmaceuticals and Wyeth – have ceased production, based largely on difficulties in ramping up production in the face of increasing standards required for current Good Manufacturing Practices (cGMP). A third manufacturer, Chiron, was unable to market its doses during the 2004-2005 influenza season because of concerns about product quality. The sudden withdrawal of this vaccine from the market forced the medical and public health communities to adopt a complex vaccine prioritization scheme in which certain groups were denied immunization. Although vaccine supplies during the current (2005-2006) influenza season may be increased relative to last year by the recent approval of GlaxoSmithKline’s (GSK) Fluarix®, the many factors contributing to the fragility of the supply chain remain and underscore the need for new and more reliable manufacturing paradigms to consistently meet current future demand.

·        Legitimate threat of pandemic influenza. Increased and more widespread circulation of avian influenza A/H5N1 viruses in Asia has been documented in recent years, with sporadic transmission of severe infection to over 100 persons, roughly 50% of whom have died. These viruses have been increasingly detected in migratory ducks and other birds, and also isolated from pigs. Most experts believe that it will only be a matter of time before the virus undergoes sufficient mutations to become more easily transmissible from human-to-human, or to undergo re-assortment with another human or animal virus to become immediately adapted to the human host. Because this virus and many other avian viruses are highly lethal in chickens (and eggs), and because a pandemic strain may emerge at a time when egg supplies are out-of-sync with the usual manufacturing cycle, alternative cell substrates suitable for production of a variety of influenza virus strains are urgently needed.

2.2 Cell Culture-Derived Influenza Vaccines

In response to the growing need for alternative means for influenza vaccine production, a number of companies, including Chiron, have carefully considered new approaches to satisfy seasonal and pandemic vaccine demand. In Chiron’s view, the ideal characteristics of such an approach include:

·        Continuing to use the same basic and highly effective manufacturing scheme used in egg-based production systems – i.e., production of multiple disrupted (“split”) or more highly purified influenza virus antigens – but growing the virus in an alternative cell substrate.

·        Utilizing a mammalian tissue culture cell line with a seed lot system to allow for (1) a more robust, consistent and reproducible means of vaccine production, utilizing a scalable bioreactor process; (2) uniformity and more detailed characterization of the cell substrate used for production; (3) further reductions in the risk of introduction of exogenous or endogenous adventitious agents during the production process; and (4) expression of influenza antigens, especially the hemagglutinin, in a more native (natural) conformation, potentially improving the specificity and potential avidity of antibody formation.

·        Increasing production capacity via (1) equivalent or higher influenza virus yields in comparison to eggs; (2) faster initiation of manufacturing, which can begin at any time; (3) easily scalable and large-scaled processes; and (4) continuous, year-round manufacturing as needed.

·        Converting the current and in parts largely open manufacturing process to a more modern, consistent, more easily controlled, and largely closed process in which (1) the risk of adventitious agents can be further reduced; and (2) the capability to upgrade the process to class III bio-safety standards can be added, which may be required for the management of a pandemic strain.

·        Developing the capability of producing influenza vaccines of avian origin, which generally cannot grow in eggs without genetic modification. Also, in the face of an avian influenza pandemic, the breeding flocks and eggs necessary for vaccine production might be destroyed, resulting in no vaccine at all for that season.

With respect to the information compiled below, Chiron believes that the MDCK cell line adapted to suspension growth fulfills these ideals.

2.3 Cell Culture-Derived Pharmaceuticals and Vaccines

To help meet the needs for increased influenza vaccine for seasonal and pandemic use, Chiron is in the advanced stages of developing an influenza vaccine using mammalian cell culture methodology to grow the virus. Although historically influenza vaccines have been produced in embryonated eggs, manufacturing of a number of licensed pharmaceutical products using mammalian cells has been a reality for many years. Therefore it is a logical progression that mammalian cells be considered for influenza vaccine manufacturing.

Several vaccines are produced in mammalian diploid cell strains. These are cells that have a finite capacity to replicate, they are anchorage dependent and they have the karyology of the tissue of origin. The list of products produced in such cells (MRC cells) includes hepatitis A, varicella, rabies, and inactivated polio vaccines.

Another group of mammalian cells, characterized as continuous cell lines, are increasingly used for pharmaceuticals and at least one vaccine (inactivated poliovirus vaccine). These cells have the potential to be sub-cultured ad infinitum (i.e., are immortal) and have the capacity to be grown in suspension. These cell lines are considered neoplastic and nearly all have been shown to be tumorigenic in immunosuppressed animals at some point in their in vitro life. Approved pharmaceutical products made from such cells include monoclonal antibodies, therapeutic proteins, hormones, and one vaccine, listed in Table 2.3-1.


 

Table 2.3-1 Continuous cell line and corresponding approved pharmaceutical products

Cell line

Number of products approved

Product Examples (Chemical Name)

African green monkey kidney epithelial (VERO)

1

 

-          Inactivated poliovirus vaccine

 

Baby hamster kidney (BHK)

2

-          Antihemophilic factor (recombinant)

-          Coagulation Factor VIIa (recombinant)

Chinese hamster ovary (CHO)

20

-          Alteplase

-          Antihemophilic factor (recombinant)

-          Erythropoeisis stimulating protein

-          Bevacizumab

-          Interferon beta-1a

-          Coagulation factor IX

-          Alemtuzumab

-          Imiglucerase

-          Etanercept

-          Erythropoietin alpha

-          Trastuzumab

-          Dornase alfa

-          Efalizumab

-          Rituximab

-          Tenecteplase

-          Omalizumab

-          Follicle-stimulating hormone (FSH)

Human embryonic kidney 293

1

 

-          Drotrecogin alfa (activated)

 

Myeloma

2

-          Cetuximab

-          Basiliximab

NS0

1

-          Palivisumab

SP2/0

2

-          Infliximab

-          Abciximab

 

Although all products have some side reactions, none of the adverse events associated with these products have ever been attributed to the use of the particular neoplastic cell substrate used for production.

The advantages of continuous cells lines over primary and diploid cell culture are numerous. Of considerable importance is the ability of continuous cell lines to become conditioned to grow in single cell suspension, free of added serum or proteins. From a manufacturing perspective, growth in suspension simplifies scale-up via the use of large bioreactors and contributes to a largely closed and consistent process. Continuous cell lines can be adapted to serum-free, protein-free growth media that eliminates animal derived reagents and their associated risks, such as the presence of occult adventitious agents like the agents of the transmissible spongiform encephalopathy.

3.0 CHIRON’S CELL CULTURE-BASED VACCINE

3.1 Selection of MDCK Cells as the Production Substrate

The selection of the specific cell substrate for influenza vaccine production is influenced by the need for a robust and commercially viable manufacturing process and numerous specific production requirements unique to influenza. Continual updating of influenza strains into the vaccine formulation requires that any cell line considered must be permissive to a wide range of influenza strains and produce high yields of virus. After careful consideration of a variety of possibilities, based on both reported experience in the scientific literature and in-house experiments, Chiron selected Madin Darby Canine Kidney (MDCK) cells as the production cell substrate:

·        Influenza Virus Permissiveness. The MDCK cell line is the preferred cell substrate for isolation and propagation of human influenza strains in WHO and U.S. surveillance centers and research laboratories. These cells are also permissive for a wide range of avian, swine, and equine influenza strains, including all of the recent H5N1 isolates from Asia.

·        Influenza Virus Yield and Consistency of Yield. Of the various available continuous mammalian cell lines (PER.C6, NIH-3T3, BHK, Vero and MDCK), Chiron’s investigations demonstrated that only Vero and MDCK have yielded influenza virus titers that are sufficiently high to be considered commercially viable. Of the two cell lines, virus titers were consistently higher in MDCK cells (3-10 times higher than Vero) and generally comparable to those achieved in eggs. Vero cells also required a higher virus inoculum (multiplicity of infection [MOI]), repeated addition of animal-derived trypsin to maintain cycles of infection, and longer periods of infection to achieve peak titers, thereby increasing the cost, time and complexity of vaccine production. In-house experiments by Chiron have demonstrated consistently high yields with MDCK cells for more than 20 influenza vaccine candidate strains covering the period 1996-2005, indicative of a high degree of permissiveness of these cells for both Type A and B influenza viruses.

·        Ability to Propagate Single Cells in Suspension. In order to develop a process suitable for large-scale industrial production, cells should ideally be adapted to grow in suspension. This will permit a fermentor-based system capable of rapid scale-up, and eliminates the many manual labor-intensive steps required to efficiently handle, inoculate, incubate and harvest allantoic fluid from millions of embryonated eggs. The cell suspension system developed by Chiron also enables the entire MDCK cell membrane to form microvilli, which are required for influenza virus budding and release. In contrast, microvilli do not form on those portions of the MDCK cell membranes that are adherent to a glass surface (e.g., microcarrier beads), and so those surfaces do not actively shed virus.

·        Reduction in the Risk of Cell Contamination by Adventitious Agents. In contrast to non-sterile eggs, which are a source of bioburden and other potential adventitious agents, Chiron’s continuous cell line is able to grow in a largely closed system, utilizing chemically-defined, serum-free media. The Master Cell Bank, Working Cell Bank and End-of Production Cells have been extensively characterized and tested for the presence of adventitious agents according to FDA and EMEA guidance documents. Moreover, in contrast to Vero cells, MDCK cells will not support the growth of other common respiratory pathogens that may enter the production train via co-infected seed virus preparations, including respiratory syncytial virus (RSV), coronaviruses, rhinoviruses, Mycoplasma sp. or Chlamydia sp. Similarly, MDCKs are not permissive for a number of avian viruses that may be present in embryonated eggs, including avian birnavirus, avian retrovirus, avian picornavirus, and avian polyomavirus.

·        Attainment of Proof of Principle. The manufacturing process has been validated at critical process stages and the resulting data indicate that it is reliable, robust, and well controlled. A total of 18 monovalent bulks were produced using 6 different seed viruses. All bulk and final product lots met release specifications; 5 trivalent lots were filled and have been or will be used in clinical studies. Thus far, over 3000 subjects have been immunized with MDCK-derived vaccine, including young adults and elderly adults. The safety and immunogenicity profiles of the MDCK-derived vaccine have compared favorably to a conventional egg-derived vaccine licensed in Europe (Agrippal, Chiron Vaccines – Siena, Italy; see additional details in Section 3.3 below). Thus, the new tissue culture-based process appears to be commercially viable, and has thus far met all major goals of the program.

 

3.2 Risk Assessment and Management of the MDCK-based Production System

Because MDCK cells were shown to grow and form tumors (consisting of MDCK cells) in immunodeficient mice, and because the molecular basis for MDCK cell-associated tumor formation is unknown, it was necessary to assess the potential risk of inducing a tumor as a consequence of administering a vaccine made in these cells. To ensure that such a risk with the MDCK-cell derived influenza vaccine is vanishingly small, Chiron designed a series of studies that were carried out following consultation with and approval by FDA reviewers. These studies, which are described in greater detail in Section 3.2.1, include (1) tumorigenicity studies, in which intact MDCK cells that were passaged beyond the end of production level, were inoculated into immunodeficient (athymic nude) mice at dosage levels ranging from 101-107 cells per injection; and (2) oncogenicity studies, in which MDCK cell lysates and purified DNA derived from MDCK cells were inoculated into three different neonatal rodent species, which were then monitored for tumor formation for 5 months. Following completion of these studies, Chiron then carried out a “Defined Risks Assessment” (DRA), using the approach recommended by the FDA for neoplastic mammalian cell lines proposed for vaccine production (see 1999 Guidance Document). As explained further in Section 3.2.2 below, the DRA addresses, in detail, the safety of Chiron’s MDCK cell-derived influenza vaccine, including the residual risk, if any, associated with the presence of intact cells, residual cell DNA, and/or residual oncogenic adventitious agents. Chiron has concluded from this assessment that manufacturing process provides robust and reliable methods to eliminate theoretical risk events by many orders of magnitude, and that the vaccine is appropriate for use in humans.

3.2.1 Studies of MDCK cells, Lysates, and DNA

The safety evaluation of neoplastic cell lines for the manufacturing of vaccines has been evolving over the past 20 years. The initial tumorigenicity testing carried out by Chiron consisted of establishing whether MDCK cells were tumorigenic. Testing of intact MDCK cells was performed in adult nude mice and in the anti-thymocyte rat model with 107 viable cells per injection (standard at the time). In order to meet current expectations, Chiron has conducted seven new animal studies to assess the tumorigenic potential of viable MDCK cells and the oncogenic potential of MDCK cell lysates and high molecular weight DNA. These new studies were conducted with end-of-production whole cells or lysates or DNA prepared from end-of-production cells. The testing program is summarized in Table 3.2-1 below.

Table 3.2-1 Program of tumorigenicity and oncogenicity studies

Age/Species

Test Article(s) a

Control Article(s)

Adult nude mice

1´101 – 1´107 MDCK cells

1´107 MRC-5 or HeLa cells

Neonatal nude mice

MDCK cell lysates b

Tris/BPL buffer

Neonatal rats

MDCK cell lysates b

Tris/BPL buffer

Neonatal hamsters

MDCK cell lysates b

Tris/BPL buffer

Neonatal nude mice

MDCK DNA c

Murine DNA

Neonatal rats

MDCK DNA c

Murine DNA

Neonatal hamsters

MDCK DNA c

Murine DNA

a        End of production cells were used to prepare all test articles in this program of studies

b     Test articles were clarified MDCK cell lysate and BPL-treated MDCK cell lysate

c     Test articles were MDCK cell DNA, influenza-infected MDCK cell DNA and BPL-treated influenza-infected MDCK cell DNA

As anticipated, intact viable MDCK cells grew and formed tumors consisting of MDCK cells when inoculated into immunodeficient (athymic nude) adult mice. Tumors were observed in some animals in all titration groups (1x101 to 1x107cells). Tumor tissue specimens were unequivocally identified as canine (MDCK cell) in origin by PCR. The microscopic morphology (epithelial) was consistent with that of MDCK cells.

Again, as anticipated, disruption of MDCK cells eliminated their tumorigenic potential in neonatal immunocompromised mice (the same model as above) as well as in neonatal rats and hamsters. Neither cell lysates nor purified full-length DNA caused tumors (oncogenicity) in three species of neonatal rodents, as described in the following experiments:

Neonatal nude mice, rats, and hamsters (£ 4 days old) were inoculated with untreated lysates of MDCK cells or lysates from BPL-treated cells and observed for 5 months. The amount of lysate inoculated into each animal represented the content of 5x106 to 1x107 cells (0.5 to 1 times the average number of cells required to make 1 human vaccine dose). These animals were all negative for any sign of oncogenicity both clinically as well as by macroscopic and microscopic postmortem examinations.

DNA prepared from untreated MDCK cells, influenza-infected MDCK cells, and influenza-infected BPL-treated cells were all negative for oncogenicity when inoculated into neonatal nude mice, rats, and hamsters that were observed for 5 months. The mice received from 28 to 35 mg of DNA and the rats and hamsters received 55 to 70 mg per animal. Therefore, mice received from 2,800 to 3,500 times, and rats and hamsters received 5,500 to 7,000 times more DNA than that contained in a dose of vaccine (<10 ng). These animals were all negative for any sign of oncogenicity both clinically as well as by macroscopic and microscopic postmortem examination.

Based on the results of the in vivo tumorigenicity/oncogenicity testing program, safety or risk reduction factors have been calculated to address the major risks identified by FDA in their guidance document that are associated with the use of neoplastic cell lines for the production of vaccines. The risk reduction factors calculated based upon experimental data are far in excess of those deemed acceptable by the FDA guidance.

3.2.2 Defined Risks Assessment (DRA)

In order to examine the risk of using MDCK cells as a cell substrate, Chiron followed the assessment model proposed in the FDA document “A Defined-Risks Approach to the Regulatory Assessment of the Use of Neoplastic Cells as Substrates for Viral Vaccine Manufacture” as presented at the symposium “Evolving Scientific and Regulatory Perspectives on Cell Substrates Used for Vaccine Development” (Rockville, MD, 1999). In this assessment, the impact of intact cells, residual MDCK DNA, and potential adventitious agents has been considered in depth.

·        The manufacturing process has been validated and demonstrated to remove intact, viable MDCK cells such that the risk of exposure to an intact cell is less than one per 1034 doses. Effectively, there is no risk from exposure to an intact cell.

·        Cell substrate DNA is reduced during manufacture of the vaccine to less than 10 ng/dose. This residual DNA has been further characterized and found be highly fragmented. Thus, all DNA detectable by capillary electrophoresis is < 200 base pairs in length and, based on a statistical model reviewed by the FDA, more than 99.99% of DNA is less than 561 base pairs in length (i.e., considerably smaller than any known oncogene). In addition, these small DNA fragments have a reduced capacity to function as a transcription template or to integrate into vaccine recipient cell DNA. The inactivation of the DNA by size reduction and its decreased ability to function as a template can be attributed to the effects of b-propiolactone (BPL) treatment, which is used in the manufacturing process for virus inactivation. Additional PCR testing of the production cells also reveals no evidence of any infectious or active oncogenic sequences. Thus, the residual DNA, at a specification of less than 10 ng per dose (average of 0.34 ng / dose over 18 commercial-scale lots) can be considered biologically inert and at such low levels as to render any oncogenic risk as extraordinarily remote.

·        The risk of exposure to potential adventitious agents (including oncogenic agents) from the cells is effectively eliminated by use of:

1.      Well characterized Master and Working Cell Banks that have been extensively tested for known and unknown adventitious agents. Exclusion tests and PCR results have revealed no evidence that the cells contain any infectious agent or viruses that are known to have oncogenic potential, namely retroviruses, polyomaviruses, herpesviruses and adenoviruses.

2.      Processes for defined and controlled raw materials, use of a well-defined, animal-protein-free media, and PCR testing of all virus seeds.

3.      Limited ability for the most likely viral contaminants that may enter the process to propagate in MDCK cells.

4.      Validated virus inactivation step with the use of BPL during downstream processing.

5.      Detergent treatment and virus subunit purification steps that provide for additional levels of viral clearance.

On the basis of this assessment, Chiron concludes that the manufacturing process and testing scheme developed for MDCK cell-derived vaccine provides robust and reliable means of eliminating known and theoretical risks and offers a safe alternative to current egg-derived influenza vaccines.

3.3 Status of Clinical Development

The clinical development plan for the MDCK cell-derived vaccine includes a sequence of clinical trials aimed at providing comprehensive information on its safety, tolerability, and immunogenicity. Completion of these investigational studies will address important clinical questions regarding the vaccine and will provide data to support licensure of the vaccine for the initial (proposed) indication in adults age 18 years and older, including those with chronic underlying disease. Thus far, the clinical program has been focused primarily on approval of the product in Europe. Chiron has worked with EMEA guidance via the scientific advice process and has gained acceptance of the use of the cell line for vaccine development. This is consistent with formal EMEA guidelines on cell culture use in vaccines. Chiron therefore plans to seek product approval by the EMEA in Europe. Initial studies in the United States (under IND) are expected to be underway in fall, 2005.

The currently available clinical trial experience for the vaccine includes more than 3000 subjects immunized in controlled clinical trials performed in Europe and New Zealand and includes young adults and elderly adults. The safety and immunogenicity profile of the investigational, MDCK cell-derived vaccine have compared favorably to a licensed egg-derived control vaccine.

4.0 CONCLUSIONS

In summary, data gathered to date conclusively demonstrate the essential elimination of potential risks associated with the use of the Chiron MDCK cells for influenza vaccine production. The principal risk was identified as the theoretical presence of intact MDCK cells in the vaccine; several independent and redundant manufacturing steps that remove and/or lyse cells have effectively eliminated this risk. In terms of a second theoretical risk, oncogenicity arising from exposure to cellular macromolecules (although lysates and DNA were not oncogenic in neonatal animals even at high doses), the extensive removal and inactivation steps during the manufacturing process result in risk reduction factors that are far in excess of those deemed acceptable by FDA guidance. Any residual DNA has been shown to be essentially biologically inert due to BPL treatment. The third theoretical risk, exposure to adventitious agents, has been countered by the use of defined, serum-free media, extensive ongoing testing of process intermediates, and a process that incorporates two robust viral inactivation steps. These factors all contribute to control the risk of adventitious agent contamination. Chiron, therefore, believes that the influenza vaccine produced in MDCK cells has been demonstrated to be appropriate to proceed with clinical development for eventual licensure by FDA. Furthermore, the influenza cell-culture derived vaccine offers significant, tangible advantages over the currently available egg-derived vaccines. The benefits of cell-derived vaccine include an increased capacity to supply the demand for vaccine during inter-pandemic years along with the ability to respond to a pandemic strain should one emerge in humans.