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Safety of MDCK cell culture-based influenza vaccines

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Safety of MDCK cell culture-based

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.

Vaccine cell substrates karyology (nuclear characteristics, in particu-


Vaccines are administered prophylactically to lar with regard to DNA replication) is that of
healthy individuals and have to meet quality the donor tissue. Experience with primary cell
and safety requirements, which are consid- culture technology, for example, has been accu-
erably more stringent than those that apply mulating over many years, namely in the pro-
to classical medicinal products. Before the duction of vaccines against rabies, tick-borne
product is released to the market, each indi- encephalitis, measles, mumps, German measles
vidual batch of vaccine is quality-tested by an (rubella) and polio (Table 1) . However, primary
independent public supervisory institute in cells and diploid cell lines are deemed unsuit-
accordance with the guidelines of the relevant able for influenza vaccine manufacture since
licensing authority. While this applies to all these do not support high growth of influenza
vaccines, quality and safety considerations may viruses and are not amenable to high volume
emphasize different aspects, depending on the and rapid throughput virus production, which is
source/type of production. Below we discuss absolutely essential for influenza vaccines.
cell culture-based production.
For several decades, influenza vaccines have Permanent cell lines
exclusively been made using embryonated eggs Many permanent (or continuous) cell lines also
as cell substrate for virus propagation. For most originate from a natural tissue, but have under-
other viral vaccines and for drug manufactur- gone changes enabling them to grow and divide
ing the use of cell cultures has been established unendingly, that is, they are ‘immortalized’
as a routine technology and already has a long and now have the great advantage of unlimited
history. A distinction is made between pri- availability. There are also cell lines derived from
mary cells, diploid cell lines and permanent tumor tissues, such as mouse myeloma cells,
cell lines. which are used for the production of mono­­clonal
antibody drug products. Numerous pharma-
Primary cells & diploid cell lines ceutical drugs that have been used in human
Primary cells and diploid cell lines are derived medicine for years are manufactured with the
from natural tissue and thus have a limited aid of permanent cell lines. They include coagu-
capacity to divide, and a relatively short life­ lation factors, IFN‑b, monoclonal antibodies,
span. Primary cells must, from time to time erythropoetins and an inactivated polio vaccine Keywords
or for each batch, be collected afresh from the (Table 2) . Modified accordingly, permanent cell n cell culture n influenza
respective tissues. Diploid cell lines have a lon- lines have the important advantage of being able n MDCK cells n vaccines
n vaccine safety
ger, but finite, lifespan and are amenable to cell to grow in serum- and protein-free medium and
banking. Primary and diploid cell cultures are in suspension. As a result, a closed, highly flex- part of
characterized by adherent growth, which means ible production process employing bioreactors
they need a surface on which to grow, and their can be achieved.

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

nude mice). For the manufacture of medical


Table 1. Approved vaccines produced with primary cells and diploid
products from permanent cell lines the tech-
cell lines.
nical production process must reliably remove
Primary cell lines Vaccine against intact cells. To date, a causal association between
Primary monkey kidney cells Pox virus a tumor and the use of a drug manufactured
Polio with the aid of a permanent cell line has never
Chick embryo cells Tick-borne encephalitis been established, and permanent cell lines are
Measles now considered to be suitable substrates for the
Mumps production of many biological medicinal sub-
Rabies
stances and possess distinct advantages over
Human diploid cells primary and diploid cells substrates [101] .
MRC-5 Hepatitis A
WI-38 Polio Experience with Madin–Darby canine
(embryonic lung fibroblast cells) German measles
kidney cells
Rabies
Varicella
The original, adherent Madin–Darby canine
An English translation reprinted with permission of Wissenschaftliche Verlagsgesellschaft, Stuttgart, kidney (MDCK) cell line stems from the kidney
Germany [26]. of a healthy cocker spaniel and was established
in 1958 by SH Madin and NB Darby, who in
In  vitro investigations have shown that 1964 deposited it at the American Type Culture
almost all permanent cell lines are tumorigenic, Collection. Following numerous studies on the
(i.e., they continue to grow if inoculated into virological aspects and the physiological proper-
immunocompromised model animals, such as ties of the cells conducted mainly in the 1980s,
the MDCK cell line can be considered to be
one of the most exhaustively investigated and
Table 2. Approved medicinal products for human use produced with
best characterized epithelial cell lines which,
permanent cell lines.
after 30 years of in vitro culture, still maintains
Permanent cell line Sample products (substance or brand names) functional characteristics of renal epithelium [1] .
293 (human embryonic Drotrecogin a (activated) In 1991, in accordance with the transmissible
kidney) spongiform encephalopathy (TSE)-guidelines,
Baby hamster kidney Antihemophilic factor (recombinant) American Type Culture Collection (ATCC)
Coagulation factor VIIa (recombinant) sub-cultivated the working cell bank MDCK
Chinese hamster ovary Alteplase CCL-34, which for many investigators, authori-
Alemtuzumab ties and manufacturers represented the starting
Bevacizumab
material or reference (parent line) for compara-
Coagulation factor IX
Dornase a tive studies of further newly developed MDCK
Efalizumab cell lines [2,3,102,103] .
Erythropoesis-stimulating protein For decades, scientific institutes and research
Erythropoetin a, b, z laboratories worldwide have been working with
Etanercept adherent MDCK cells as a means of isolating and
Follicle-stimulating hormone replicating human influenza viruses for epide-
Imiglucerase
miological studies and diagnostic purposes. The
IFN‑b-1a
Omalizumab majority of the 122 national influenza centers
Rituximab of the Global Influenza Surveillance Network
Tenecteplase and the four WHO Collaboration Centers
Trastuzumab and reference laboratories in Atlanta, London,
MDCK Inactivated subunit influenza vaccine (Optaflu®, Celtura®) Melbourne and Tokyo favor the use of MDCK
Myeloma Basiliximab cells as the cell substrate for the monitoring of
NS0 Palivizumab viral variability and for isolating influenza virus
SF9 Bivalent human papillomavirus vaccine strains from clinical specimens [4,5] . However,
SP2/0 Abciximab the annual reference strains for vaccine produc-
Cetuximab tion are in accordance with the guidelines of the
Infliximab licensing authorities – still isolated from chicken
Vero Inactivated polio vaccine eggs and, in the case of those viruses exhibiting
MDCK: Madin Darby Canine Kidney; NS0: Mouse myeloma; SF9: Moth ovary; SP2/0: Mouse poor growth (e.g., the A/H1N1 and A/H3N2
spleen myeloma.
An English translation reprinted with permission of Wissenschaftliche Verlagsgesellschaft, Stuttgart, strains) these are converted into reassortants, a
Germany [26]. combination of two influenza viruses.

144 Future Microbiol. (2011) 6(2) future science group


Safety of MDCK cell culture-based influenza vaccines Special Report

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.

future science group www.futuremedicine.com 145


Special Report Gregersen, Schmitt, Trusheim & Bröker

Table 3. Manufacturers and developers of influenza vaccines employing


permanent cell lines for manufacture and development.
Manufacturer Country Cell substrate Origin
Baxter Austria Vero (adherent) Monkey kidney
GlaxoSmithKline Belgium MDCK (adherent) Canine kidney
Kaketsuken Japan EBx (suspension) Avian embryonic stem cells
Medimmune USA MDCK (adherent) Canine kidney
Nobilon-Schering- Netherlands MDCK (adherent) Canine kidney
Plough
Novartis Germany MDCK (suspension) Canine kidney
Sanofi Pasteur France PER.C6 (suspension) Human embryonic retina
Solvay Netherlands MDCK (adherent) Canine kidney
MDCK: Madin–Darby canine kidney.
An English translation reprinted with permission of Wissenschaftliche Verlagsgesellschaft, Stuttgart, Germany [26].

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

146 Future Microbiol. (2011) 6(2) future science group


Safety of MDCK cell culture-based influenza vaccines Special Report

Cells DNA Foreign viral


contamination

Elimination of intact cells No oncogenicity Absence of inherent


No transformability Acceptable DNA elimination foreign viruses
(no oncogenicity of cell lysate) and/or inactivation – Infectious
– Oncogenic
Elimination of potential
foreign viruses

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

replication of many avian viruses [15,16] . to develop tumor nodules in immunodeficient


experimental animals, for example in nude
Safety aspects of MDCK cells mice lacking a thymus, and characterization
Information about the results of the numerous of the tumor tissue;
test and risk evaluations of the cell lines employed
by the manufacturers can be found, for example, n Assessment of the tumorigenic potential of cell
in the publicly available reports of the Vaccines components (cell lysates) and cellular DNA in
and Related Biological Products Advisory experimental animals with a normal, but
Committee of the US FDA, Center for Biologics immature immune system (newborn mice,
Evaluation and Research. To date, two influenza rats and hamsters);
vaccines produced from MDCK cell culture n Quantification of the elimination of intact
technology have been licensed (Solvay-Abbott cells, cell components and DNA at the rele-
had a national license for a split-vaccine in the vant steps in the vaccine production process;
Netherlands, and Novartis has an EU-wide EMA
license for the subunit vaccine Optaflu��������� ��������
and sev- n Calculation of the probability of a tumor
eral national licenses for Celtura®). Vaccines and developing in the vaccine on the basis of points
Related Biological Products Advisory Committee one to three above.
Meeting reports for these two vaccines and their Studies by both manufacturers (Novartis
related cell lines are available [102,103] . In addition, and Solvay) demonstrated that only intact
the European Public Assessment Report of the MDCK cells possessing the ability to divide
EMA for the Novartis vaccine is publicly available are tumorigenic in immunodeficient nude mice
[104] . Apart from the mentioned licensed prod- (i.e., these cells may continue to grow and form
ucts or development projects for novel inactivated tumors). Both manufacturers demonstrated
influenza virus vaccines, MDCK cells have also that intact MDCK cells are completely elimi-
been considered for the development of a live nated during the production process of the
attenuated vaccine and relevant safety evaluations vaccine. Many MDCK cells die off on infec-
have been made [2,17] . tion with the influenza virus, while surviving
cells are removed, inactivated or destroyed by
Tumorigenicity & oncogenicity numerous consecutive steps during purifica-
For the evaluation of the theoretical risk of tion of the vaccine. These steps include cen-
tumorigenicity and oncogenicity of permanent trifugation, filtration through 0.2  µm filters
cell lines, various studies and investigations (free floating MDCK cells are ~15 µm in diam-
were conducted to prove the absence of risks eter), chromatography column purifications,

future science group www.futuremedicine.com 147


Special Report Gregersen, Schmitt, Trusheim & Bröker

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

148 Future Microbiol. (2011) 6(2) future science group


Safety of MDCK cell culture-based influenza vaccines Special Report

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

future science group www.futuremedicine.com 149


Special Report Gregersen, Schmitt, Trusheim & Bröker

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

reduced to less than 1 millionth of a human infectious dose.


n No evidence of replication or transmission of transmissible spongiform encephalopathy agents was found in MDCK cells inoculated with

high doses of scrapie prions or with Creutzfeldt-Jakob disease material.


Clinical safety, immunogenicity & efficacy
n When tested in comparison with an egg-derived subunit vaccine in more than 12,000 test subjects, the cell-based Optaflu vaccine was
equally well tolerated and demonstrated a comparable immunogenicity and safety.
n An observer-blinded and placebo-controlled efficacy study involving more than 11,000 test subjects, showed a clinical efficacy

(endpoint: laboratory-confirmed influenza) of the cell-based vaccine of 83.8%, compared with placebo.

150 Future Microbiol. (2011) 6(2) future science group


Safety of MDCK cell culture-based influenza vaccines Special Report

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.

Bibliography n References [6–8] describe the mutant 16. Onions D, Egan W, Jarrett R, Novicki D,
Papers of special note have been highlighted as: selection induced by passages of influenza Gregersen JP: Validation of the safety of
n of interest viruses in embryonated eggs and assess the MDCK cells as a substrate for the production
1. Devuyst O, Beauvens R, Denef JF, Crabbe J, implications. of a cell-derived influenza vaccine. Biologicals
Abramov M: Subtypes of Madin–Darby 38, 544–551 (2010).
9. Katz JM, Webster RG: Efficacy of inactivated
canine kidney (MDCK) cells defined by influenza A virus (H3N2) vaccine grown in n Detailed description of methods and
immunocytochemistry: further evidence for mammalian cells or embryonated eggs. discussion of results of safety studies for
properties of renal collecting ducts. Cell Tissue J. Infect. Dis. 160, 191–198 (1989). MDCK 33016 PF cells.
Res. 277, 231–237 (1994).
n Animal studies demonstrating higher 17. Liu J, Shi X, Schwartz R, Kemble G: Use of
2. Liu J, Mani S, Schwartz R, Richman L, vaccine protection with influenza virus MDCK cells for production of live attenuated
Tabor DE: Cloning and assessment of grown in MDCK cells compared with influenza vaccines. Vaccine 27, 6460–6463
tumorigenicity and oncogenicity of a egg-passage virus. (2009).
Madin–Darby canine kidney (MDCK) cell
10. Committee for medical products for human use 18. Yang H, Zhang L, Galinski M: A
line for influenza vaccine production. Vaccine
(CHMP): Concept paper on the need to update probabilistic model for risk assessment of
28, 1285–1293 (2010).
the current annex guideline on cell culture residual host cell DNA in biological products.
n Basic safety studies for a specific Madin inactivated influenza vaccines with respect to Vaccine 28, 3308–3311 (2010).
Darby canine kidney (MDCK) cell clone the derivation of cell-isolated influenza vaccine 19. Gregersen JP: A risk-assessment model to
intended for the development of a live viruses. EMEA/CHMP/BWP/481473/2008, rate the occurrence and relevance of
attenuated vaccine. EMEA, London, 22 January 2009. adventitious agents in the production of
3. Medema JK, Meijer J, Kersten AJ, Horton R: 11. European Medicines Agency: Guideline on influenza vaccines. Vaccine 26, 3297–3304
Safety assessment of Madin Darby Canine quality aspects on the isolation of candidate (2008).
Kidney Cells as vaccines substrate. Dev. Biol. influenza vaccine viruses in cell culture. Draft n Risk assessment algorithm to rate and
(Basel) 123, 243–250 (2006). released for consultation. EMA/CHMP/ compare virus safety risks of conventional
n Basic safety studies for adherent MDCK BWP/68803/2010. Available online: see [105] . and cell-derived influenza vaccines for
cells used for the development of an 12. ICH International Conference on numerous adventitious viruses.
inactivated influenza vaccine. Harmonization of Technical Requirements for 20. Polymenidou M, Trusheim H, Stallmach L
4. Zambon M: Cell culture for surveillance of the Registration of Pharmaceuticals for Human et al.: Canine MDCK cell lines are refractory
influenza. Dev. Biol. Stand. 98, 65–71 Use: Harmonized Tripartite Guideline on viral to infection with human and mouse prions.
(1999). safety evaluations of biotechnology products”. Vaccine 26, 2601–2614 (2008).
ICH Q5A, Geneva 1997. See also CPMP/
5. Minor PD, Engelhardt OG, Wood JM et al.: 21. Ambrozaitis A, Groth N, Bugarini R,
ICH/295/95: Note for guidance on quality of
Current challenges in implementing Sparacio V, Podda A, Lattanzi M: A novel
biotechnology products derived from cell lines
cell-derived influenza vaccines: implications mammalian cell-culture technique for
of human or animal origin. CPMP/
for production and regulation. Vaccine 27, consistent production of a well-tolerated and
ICH/295/95 (2007). Available online: see [105].
2907–2912 (2009). immunogenic trivalent subunit influenza
13. Food and Drug Administration: Guidance for vaccine. Vaccine 27, 6022–6029 (2009).
n Describes basic influenza manufacturing Industry. Characterization and qualification of
issues, such as virus strain derivation and 22. Symczakiewicz-Multanowska A, Groth N,
cell substrates and other biological starting
selection, and generation of potency test Bugarini R et al.: Safety and immunogenicity
materials used in the production of viral vaccines
reagents. Discusses implications of the of a novel influenza subunit vaccine produced
for the prevention and treatment of infectious
introduction of cell culture systems. in mammalian cell culture. J. Infect. Dis. 200,
diseases. US Food and Drug Administration
841–848 (2009).
6. Schild GC, Oxford JS, DeJong JC, (2006). Available online: see [106] .
Webster RG: Evidence for host-cell selection 23. Reisinger KS, Block SL, Izu A, Groth N,
14. Trusheim H, Roth B, Wilms R et al.: The
of influenza virus variants. Nature 303, Holmes SJ: Subunit influenza vaccines
MDCK 33016-PF cell line is not only suitable
706–709 (1983). produced from cell culture or in embryonated
for the production of cell-based influenza
chicken eggs: comparison of safety,
7. Oxford JS, Cororan T, Knott R et al.: vaccine but is also an ideal substrate for
reactogenicity, and immunogenicity. J. Infect.
Serological studies with influenza A (H1N1) influenza virus isolation. Presented at: Options
Dis. 200, 849–857 (2009).
viruses cultivated in eggs or in canine kidney for the Control of Influenza VI, Toronto,
cell line (MDCK). Bull. WHO 65, 181–187 Canada, 17–23 June 2007. 24. Frey S, Vesikari T, Szymczakiewicz-
(1987). Multanowska A et al.: Clinical efficacy of cell
15. Gregersen JP: A quantitative risk assessment
culture-derived and egg-derived inactivated
8. Robertson JS: Clinical influenza virus and the of exposure to adventitious agents in a cell
influenza vaccines in healthy adults. Clin.
embryonated hen’s egg. Med. Virol. 3, 97–106 culture-derived subunit influenza vaccine.
Infect. Dis. 51, 997–1004 (2009).
(1993). Vaccine 26, 3332–3340 (2008).

future science group www.futuremedicine.com 151


Special Report Gregersen, Schmitt, Trusheim & Bröker

n Multicenter clinical efficacy studies www.who.int/biologicals/publications/trs/ regulatory considerations. Geneva: World


comparing conventional and cell-based areas/vaccines/cells/WHO_TRS_878_ Health Organization, April 10, 2007
influenza subunit vaccines. A1Animalcells.pdf www.who.int/vaccine_research/diseases/
102. Influenza Cell Culture Subunit Vaccine, influenza/ WHO_Flu_Cell_Substrate_
25. Collin N, de Radiguès X: Vaccine production
Vaccine and Related Biological Products Version3.pdf
capacity for seasonal and pandemic (H1N1)
2009 influenza. Vaccine 27, 5184–5186 (2009). Advisory Committee (VRBPAC), Briefing 105. ICH and EMA Guidelines
Document Chiron, Bethesda, 16 www.ema.europa.eu
26. Schmitt HJ, Gregersen JP, Trusheim H,
November, 2005 106. FDA Guidelines
Bröker M: [Safety of cell culture-based
www.fda.gov/ohrms/dockets/AC/05/ www.fda.gov/cber/guidelines.htm
influenza vaccines]. Med Monatsschr Pharm.
briefing/5–4188B1_18.pdf
33(1), 4–10 (2010). 107. EPAR (European Public Assessment Report)
103. Vaccine and Related Biological Products Optaflu®, EMEA 2007
27. Rappuoli R. Cell-culture-based vaccine
Advisory Committee (VRBPAC), November www.emea.europa.eu/humandocs/PDFs/
production. Technological Options. The
2005, Madin Darby Canine Kidney EPAR/optaflu/H-758-en6.pdf
Bridge 36(3), 25–30 (2006).
Continuous Cell Line, Briefing
108. Centers for Disease Control and Prevention
Document Solvay
Websites www.fda.gov/ohrms/dockets/AC/05/
www.cdc.gov/vaccines/vpd-vac/rotavirus/
default.htm
101. World Health Organization: Requirements briefing/5–4188B1_19a.pdf
for the use of animal cells as in vitro 104. Patriarca PA. Use of cell lines for the
substrates for the production of biologicals. production of influenza virus vaccines: an
WHO Technical Report Series No. 878, (1998) appraisal of technical, manufacturing, and

152 Future Microbiol. (2011) 6(2) future science group

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