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Special Report
Future Microbiology
influenza vaccines
Jens-Peter Gregersen1, Heinz-Josef Schmitt1, Heidi Trusheim1 & Michael Bröker†1
1
Novartis Vaccines & Diagnostics GmbH, Emil-von-Behring-Strasse 76, D-35041 Marburg, Germany
†
Author for correspondence: Tel.: +49 642 139 2912 n Fax: +49 642 139 4667 n michael.broeker@novartis.com
After more than 60 years, the conventional production of influenza vaccines
employing fertilized chicken eggs has reached its limits – both in terms of temporal
flexibility and vaccine production volume. This problem is compounded by the
fact that the pandemic-driven situation in 2009 has roughly doubled the overall
vaccine demand. Modern cell culture technology has significant advantages
over the conventional method of manufacturing influenza vaccines employing
embryonated chicken eggs, and enables manufacturers to respond rapidly to
the increasing worldwide seasonal and pandemic-driven need for influenza
vaccines. Recent articles in the popular press claiming that cell culture-based
influenza vaccines can cause tumors have fomented uncertainty among the
general population and physicians, and also discredit officially accepted test
results and product licensing. This article provides an overview of the safety profile
of the cell culture technology, of the cells and of the final vaccine product.
10.2217/FMB.10.161 © 2011 Future Medicine Future Microbiol. (2011) 6(2), 143–152 ISSN 1746-0913 143
Special Report Gregersen, Schmitt, Trusheim & Bröker
In the 1980s, it was recognized that the isola- Clinical influenza virus
tion of influenza viruses in chicken eggs leads
to viral selection of mutations at the antibody
binding sites of the hemagglutinin gene, and in
many cases can result in altered viral antigenic
properties [6–8] . In MDCK cells no such selec- Egg
tion takes place, so that fidelity with the origi- MDCK cells
nal clinical isolate is assured (Figure 1) . This may
even lead to improved vaccine efficacy [9] . At
present, the licensing authorities are evaluating
how the currently applied structures and tech-
nical conditions to generate the annual vaccine
candidate strains need to be adapted to make
cell culture-based influenza isolates available Virus variant selection Unchanging virus (HA)
for vaccine manufacture [5,10,11,104,105] . While
in principle different cell lines are considered
to be suitable for isolating vaccine candidate
strains, MDCK cells can be considered the
most favored option for several reasons: there
is an excellent qualitative knowledge about
the characteristics of MDCK cells and they
possess optimal influenza replicating proper-
ties, making these cells the preferred cell sub-
strate for isolating influenza viruses within
the WHO inf luenza surveillance network
and in academic and diagnostic laboratories.
Furthermore, these cells can be made avail- Selective immune answer Broader answer
able from cell banks that have been produced Figure 1. Virus selection in eggs versus authentic copy in MDCK cells.
in accordance with good manufacturing prac- Representation of the difference between virus replication in chicken eggs and in
tice guidelines and which have been tested for MDCK cells: while the starting material (human isolate) is only selectively
purity, identity and for the absence of contami- reproduced in eggs, reproduction in MDCK cells is unchanged. This authentic copy
may be expected to have the potential for a higher immunogenicity of the
nating viruses as required by FDA, EMA and cell-based vaccine.
WHO guidelines [12,13,101,106] . HA: Hemagglutinin; MDCK: Madin–Darby canine kidney.
For the above-mentioned reasons, various English translations reprinted with permission of Wissenschaftliche
organizations involved in the generation, formal Verlagsgesellschaft, Stuttgart, Germany [26] .
recommendation and distribution of influenza
vaccine candidate strains, but also organiza- cell cultures, the process capacity is limited only
tions involved in setting quality standards and by the size and number of the bioreactors and
in licensure of influenza vaccines or in national the production volume can readily be scaled. By
health programs support the cell-based manu- contrast, for a single egg-based vaccination dose,
facture of influenza vaccines. Because MDCK 1–2 embryonated chicken eggs are required,
cells are permissive for all currently known influ- thus large-scale influenza vaccine manufacture
enza vaccine viruses, including avian, porcine needs many millions of fertilized, embryo-
and equine strains, and achieve high virus yields, nated eggs, which have to be ordered months
the majority of manufacturers developing cell in advance. Since they have to be infected with
culture-based influenza vaccines have decided the virus at an exactly determined time point in
in favor of MDCK cells on which to cultivate the embryonation process, exact timing of the
their respective vaccine virus (Table 3) . chicken flocks to match vaccine production is
In contrast to the conventional egg-based critical. Starting materials for cell culture pro-
process, the production method employing cesses are stored in advance and are available
MDCK cell lines utilizes a closed fermenter for immediate use, enabling vaccine production
system, which is associated with a reduced risk to be initiated at any time and throughout the
of contamination, and obviates the need for the year. Last, but not least, the fully characterized
addition of antibiotics. The production process cells from a uniform cell bank are all identical
is both readily controllable and standardized. and do not have the individual differences of
Particularly applicable to the MDCK suspension embryonated eggs.
For pandemic influenza viruses a higher bio- the suspension into a larger volume of fresh
safety level is required, unless genetically modi- medium. This means that the production
fied strains with a proven, lower pathogenicity capacity can be increased readily and rapidly in
can be provided very rapidly. Owing to the larger bioreactors.
partly open infection and harvesting steps for In adherent MDCK cells the virus buds only
the egg-based vaccine manufacture, but also due from the polar surfaces that bear microvilli, thus
to the high investment needed, an upgrade of microvilli are necessary for the budding and
conventional vaccine manufacturing plants to liberation of the influenza viruses. Free-floating
the required biosafety level has not been consid- MDCK cells form microvilli over the entire sur-
ered. Closed cell culture fermentation processes face of the cells (see cell depicted in Figure 2 ). A
are much better suited to establish high contain- higher number of microvilli and the enlarged
ment conditions. The Novartis cell culture plant budding surface permits high virus yields.
in Germany (Marburg) can operate at Biosafety Recent studies have shown that small scale
Level III, and is thus suitable for the produc- MDCK suspension cultures can also be used
tion of pandemic influenza viruses. Indeed, the very efficiently for the isolation of influenza
facilities were used in 2009 to produce a H1N1 viruses. After adaptation of some equipment,
pandemic vaccine. maintenance and handling of such cultures can
be accomplished faster and easier than adher-
Special features of the MDCK suspension ent cultures. Comparable or higher influenza
cell line isolation rates and good growth of influenza
As shown in Table 3, only a single MDCK suspen- strains was also observed for A/H3N2-strains,
sion cell line is currently being employed on an of which only a few replicate readily in eggs.
industrial scale – namely the MDCK 33016-PF During those studies it was also shown that
cell line developed by Novartis. These cells are passages in MDCK 33016-PF cells preserved
used to manufacture a seasonal influenza vac- the isolates` antigen specificity. Egg passages
cine (Optaflu®, licensed throughout the EU), led to rapid changes of amino acids of the
and also a pandemic H1N1 vaccine (Celtura®, hemagglutinin molecule, while MDCK pas-
licensed in various countries, including Japan). sage viruses were replicated accurately and
Compared with adherently growing cell lines, authentically [14] .
such suspension cell lines have various advantages. MDCK 33016-PF cells are grown in serum-
MDCK 33016-PF-cells grow freely suspended free medium and only require supplementation
in the culture medium. They need no surface with very few defined proteins, such as insu-
or carriers to adhere to, which also means that lin. This greatly reduces the potential risk of
adherence factors, which are normally contained contamination with animal viruses or TSEs.
in serum-derived medium supplements, are not Moreover, the cells were intensively studied for
required. Trypsin or other protease digestion their permissiveness for various relevant viruses.
steps are not needed to detach the adherent These studies showed that MDCK 33016-PF
cells from their supporting surface when the cells, like embryonated chicken eggs, are refrac-
cultures are to be passaged. Suspension cell tory to many other viruses, (i.e., they do not
cultures can be multiplied simply by diluting support their growth). This virus filter function
Figure 2. Authorization requirements for new cell lines. For permanent cell lines suitability for
use as cell substrate in accordance with the guidelines of International Committee of Harmonization,
FDA and EMA must be shown.
Reprinted with permission of the National Academy of Engineering, Washington, DC, USA [27] .
excludes the replication of various respiratory that might be associated with residual liv-
pathogens that might possibly be contained in ing cells, DNA or contaminating oncogenic
the influenza virus isolate. Furthermore, and in viruses (Figure 2) :
contrast to eggs, MDCK cells do not permit the n Determination of the capacity of living cells
chemical virus inactivation and virus-splitting n Absence of any detectable virus in the cell banks
by detergent treatment. The risk of a MDCK n Testing to exclude foreign viruses in the seed
cell being present in a dose of vaccine is cal-
virus
culated to be less than 1 per 1034 doses for
the Optaflu vaccine, which means that it is n The growth properties of potential adventi-
virtually impossible that the end product con- tious viruses in the cell substrate
tains living cells capable of inducing a tumor. n Risks of contamination with extraneous
A statistician has translated the safety margin
viruses during the manufacturing process
of 1:1034 into more practical terms: if every
person who has ever lived, and all those who n Inactivation and removal of potential con-
will live in the future until the sun burns out, taminants during the manufacturing process
were to receive a yearly vaccination for a period
of 100 years, the risk of introducing a tumor For the production of an influenza vaccine,
cell is 1:1012 or 1 in a trillion [102] . so-called seed viruses or working seeds are
As opposed to tumorogenity, oncogenicity is made. These are derived from WHO reference
defined as the ability to induce host cell tumors. strains, which were isolated from human nasal
The results of comprehensive series of labora- or throat swabs and were grown in embryo-
tory tests investigating the oncogenicity risk, nated eggs. The clinical material might thus be
carried out by both manufacturers were iden- contaminated with human pathogens, which
tical, and in agreement with experience with may be transferred to the seed virus. During
other cell lines, demonstrated that neither high egg passages avian viruses may also be intro-
doses of MDCK-cell lysates, nor cellular DNA duced into the reference strains. Further risks
were oncogenic. For studies with the MDCK of contamination are associated with the open
33016-PF cell line, the test animals were injected egg-based manufacturing process. For the most
with up to 7000 times more MDCK DNA than part, the virus safety concept applicable to the
the 10 ng maximum level recommended by the conventional egg-based influenza vaccines relies
WHO and permitted by the EMA. For Optaflu on the limited growth of human viruses in eggs
the amount of residual DNA is below that com- (virus filter effect) and on the inactivation of the
monly accepted level. Furthermore, the cellular viruses during the production process.
DNA is degraded by treatment with b-propiolac- In contrast to manufacturing in embryo-
tone. Residual DNA fragments were on average nated eggs, permanent cell lines can be applied
less than 300 base pairs. Typical oncogenes have in a closed manufacturing process. Master and
greater than 1000 base pairs and the average size working cell banks of the MDCK 33016-PF
of human oncogenes is 1925 base pairs [18] . cell line have been extensively characterized. A
These investigations, which are also described wide range of in vitro and in vivo test methods,
and discussed in more detail in a specific paper [16], including PCR tests were conducted in accor-
show that influenza vaccines manufactured using dance with international guidelines. In all tests
MDCK cell-culture technology carry no oncoge- the MDCK 33016-PF cells and its ancestors,
nicity risk for the vaccinee. In light of these results, were found to be negative for human and ani-
approval was granted by the authorities, and the mal viral contaminants. Likewise, the seed
EMA noted: “In conclusion, the data provided viruses that have been prepared for vaccine
are regarded to sufficiently address the issue of production (egg isolates with subsequent pas-
oncogenicity/tumorigenicity of the MDCK cell sages in MDCK 33016-PF cells) were exten-
substrate and to resolve any corresponding safety sively investigated for the presence of unde-
concern regarding the vaccine” [107] . sired viruses and mycoplasma. The results of
these tests – including numerous PCR tests
Viral safety conducted on seeds made for all recommended
In order to demonstrate the suitability of a per- vaccine seed virus strains since 1999 – were
manent cell line for use as a substrate for virus always negative.
cultivation, not only the risk from residual cells In growth studies with a wide range of differ-
or DNA, but also that of a theoretical contami- ent viruses it was shown that MDCK33016-PF
nation with adventitious viruses and agents must cells only support the growth of a few viruses
be evaluated as laid down in ICH, FDA and and thus represent an effective barrier to con-
EMA guidelines (Figure 2) . To this end, the fol- taminating agents. Risk assessments dem-
lowing aspects were investigated and results have onstrated that the introduction of MDCK
been published [15,16,19,20] . cells as a further cell substrate to passage egg
isolates did not increase the risks of contami- age groups [21,22] . In several of these studies,
nating viruses but in fact reduced these. The immunogenicity and safety of Optaflu have also
risk may even be reduced further if influenza been demonstrated by direct comparison with
viruses were isolated in MDCK cells and the an egg-based vaccine [21–23,107] .
use embryonated eggs was entirely omitted A recent observer-blinded and placebo-
[19] . In quantitative risk assessments, calcula- controlled efficacy study, involving more than
tions were made to enumerate the amount of 11,000 test subjects, showed a clinical efficacy
residual virus in the final vaccine in more than (endpoint: laboratory-confirmed influenza of
20 different virus families or species should a matching type) of 83.8%, as compared with
there ever be a contamination. For the Optaflu placebo [24] .
process and under worst-case conditions, the
theoretical contaminating virus was reduced Future perspective
to 10 -6 –10 -16 infectious units per dose. This A wealth of experience has been accumulated
result does not mean that one in a million in the production and application of human
doses of vaccine might contain an infectious medicines made by cell culture technolo-
amount of virus, which would be unaccept- gies. Considering the fact that for almost six
able. Rather, it means that, in the worst case decades influenza vaccine could only be made
of an unrecognized contamination, a vaccinee from embryonated eggs, it is a revolutionary
would have to receive a million doses of vac- step forward that cell culture technology can
cine for an infectious amount of virus to be now also be applied for the production of safe
transmitted [15] . In light of the recent detection and effective influenza vaccines on an indus-
of porcine circovirus in attenuated rotavirus trial scale. For the influenza vaccine manufac-
vaccines [108] , it may be of interest that porcine tured with the MDCK 33016-PF suspension
circovirus has also been studied. It was demon- cell line and authorized throughout the EU,
strated that this virus does not grow in MDCK data confirming its acceptable tolerability and
33016-PF cells and is effectively inactivated by high immunogenicity were collected from
b-propiolactone [15] . more than 12,000 clinical trial subjects. More
A recent study has investigated the theoretical than 30 million doses of the related pandemic
risk that the MDCK 33016-PF cell line might H1N1 vaccine have now been produced, were
be infected by TSE-triggering prions and, if distributed and have been administered to
this were indeed so, whether these can then large numbers of subjects.
replicate. In contrast to cats and mice, dogs The overall capacity for seasonal influenza
do not appear to be infectable by TSE agents. vaccines worldwide is currently estimated to be
This apparent resistance to prions has also been approximately 800 million doses [25] . Assuming
demonstrated for MDCK cells. No evidence of a world population of some 6.75 billion, the
transmission has been found in MDCK cells calculated coverage would amount to approxi-
that were inoculated with the high doses of mately 10–12% of the overall requirement [25] .
scrapie prions or with Creutzfeldt-Jakob disease Although not every potential vaccinee would
material. The cells neither supported prion rep- wish to be immunized or have the opportunity
lication, nor were they capable of transmitting to do so, it is clear that with the aid of cell-cul-
TSE [20] . ture technology the vaccine production capac-
ity can be increased. In the event of an acutely
Clinical safety, immunogenicity increased requirement, cell culture technology
& efficacy can accelerate vaccine availability.
The safety, tolerability and efficacy of cell- Comprehensive studies performed over
derived inf luenza vaccines have also been decades have repeatedly shown that, compared
investigated in clinical trials in humans. For with all other available cell substrates, MDCK
the EU approval process for the Optaflu subunit cells are an ideal cell substrate for the isolation
vaccine and also thereafter, clinical data were and replication of influenza vaccines. It is only
collected from more than 12,000 test subjects. a logical consequence that, once the MDCK-
Data obtained from vaccine approval studies based vaccine technology has been established
revealed that both, cell- and egg-based influenza and licensed, those cells are also used to isolate
vaccines, are equally well tolerated [107] . The the influenza vaccine candidate strains. The use
consistent, good tolerability of the vaccine was of MDCK cells for isolation of vaccine candi-
further confirmed by the results of recent clini- date strains offers several advantages that are
cal studies involving trial subjects in different not yet exploited, as various regulatory aspects
still need to be clarified. Furthermore, there are In the longer term, however, the main advan-
also many technical aspects to be considered, for tage of MDCK-cell-derived vaccine strains
example the availability of approved cell lines at without prior egg passage is their unchanged
the WHO Collaborating Centers who do the antigenicity and better match with circulating
strain isolation and selection, or the provision field strains. As described above, studies dem-
of matching reagents, should there be different onstrated not only better serological responses
cell lines used for manufacture [5] . to such authentic cell culture strains but also
For the annually changing vaccine strains, improved protection in animal challenge stud-
and particularly in the case of newly arising ies [7,9] . However, owing to the complexity of
pandemic influenza viruses, it is of upmost influenza vaccine strain selection and vaccine
importance to rapidly provide adequate vac- manufacture with ever changing virus strains,
cine strains to manufacturers. It would greatly the required changes are not readily imple-
reduce the time required to find, adapt and mented and approved by the licensing authori-
characterize adequate virus strains if the strains ties. Furthermore, it might take several years
isolated in MDCK cells could be directly rec- and very complex and expensive efficacy stud-
ommended for vaccine manufacture, rather ies to convincingly substantiate the protective
than trying to find a matching strain that also advantages of cell-based vaccine strains.
grows in embryonated eggs. The latter has
become increasingly difficult, and the egg iso- Acknowledgements
late frequently needs to be reassorted with high This article is a modified version of a contribution
yielding donor strains to qualify for vaccine which has originally been published in German in the
virus production. journal Medizinische Monatsschrift für Pharmazeuten.
Executive summary
Vaccine cell substrates
n For influenza vaccines manufactured from embryonated eggs, virus strains are isolated in eggs. Only a small percentage of influenza
isolates can be grown in embryonated eggs.
n Permanent cell lines are now considered to be suitable substrates for the production of many biological medicinal substances. A causal
association between a tumor and the use of a drug manufactured from a permanent cell line has never been established.
n Intact cells from most permanent cell lines are tumorigenic, (i.e., they continue to grow if inoculated into immunocompromised
laboratory model animals). Thus, intact cell must be reliably removed from any drug product derived from permanent cells.
Experience with Madin–Darby canine kidney cells
n Madin–Darby canine kidney (MDCK) cells are the preferred cell substrate for influenza virus isolation and characterization and are
routinely used by the Global Influenza Surveillance Network.
n Isolation of influenza viruses in chicken eggs leads to viral selection of mutations at the antibody binding sites of the hemagglutinin
gene. In MDCK cells no such selection takes place, so that fidelity with the original clinical isolate is assured.
n In laboratory studies and in human serological studies it has been demonstrated that MDCK-isolated influenza viruses, when compared
with egg-passaged virus, generate higher specific immune responses and may even result in better vaccine protection.
Safety aspects of MDCK cells
n Intact MDCK cells were removed, inactivated and destroyed by numerous consecutive steps during purification of an influenza vaccine.
n In agreement with experience with other cell lines, neither high doses of MDCK-cell lysates, nor cellular DNA preparations
were oncogenic.
n For Optaflu® cellular DNA is reduced to below 10 ng per dose and is further degraded by treatment with b-propiolactone.
Viral safety
n MDCK33016-PF cells represent an effective barrier to contaminating agents. The introduction of MDCK cells as a cell substrate to
passage egg isolates reduces the risk of virus contamination.
n Quantitative risk assessments for the Optaflu process demonstrated that the theoretical amount of contaminating viruses would be
(endpoint: laboratory-confirmed influenza) of the cell-based vaccine of 83.8%, compared with placebo.
Financial & competing interests disclosure authors have no other relevant affiliations or financial
The authors are full-time employees of Novartis Vaccines involvement with any organization or entity with a
and Diagnostics GmbH. Part of the work at Novartis financial interest in or financial conflict with the subject
described here has been funded with Federal funds from matter or materials discussed in the manuscript apart
the Office of Public Health Emergency Preparedness, from those disclosed.
Office of Research and Development Coordination, No writing assistance was utilized in the production
under Contract No. HHS0100200600012C. The of this manuscript.
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