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Vaccine 28 (2010) 1285–1293

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

loning and assessment of tumorigenicity and oncogenicity of a Madin–Darbyanine kidney (MDCK) cell line for influenza vaccine production

onathan Liu ∗, Sachin Mani, Richard Schwartz1, Laura Richman, David E. TaboredImmune, 3055 Patrick Henry Drive, Santa Clara, CA 95054, United States

r t i c l e i n f o

rticle history:eceived 6 September 2009eceived in revised form 3 November 2009ccepted 6 November 2009vailable online 25 November 2009

a b s t r a c t

An Madin–Darby canine kidney (MDCK) cell line, 9B9-1E4, was cloned by limit dilution from a heterolo-gous cell population and chosen as a potential production cell substrate for cell culture-based influenzavaccine manufacture. Since MDCK cells are transformed cells of canine origin, extensive characterization,including evaluation of tumorigenicity and oncogenicity, was performed to ensure the safety of this cell

eywords:DCK

ell cloningumorigenicityncogenicity

line for vaccine production. Injection of intact MDCK cells into adult and newborn athymic nude micedid not lead to progressive tumor formation in two separate tumorigenicity studies. In addition, neitherMDCK cell lysate nor cellular DNA induced tumors in newborn rodents (athymic nude mice, hamsters andrats) in six oncogenicity studies. Observations from these studies demonstrate the low tumorigenic andoncogenic potential of the MDCK cell clone 9B9-1E4. These observations coupled with other characteri-zation study results strongly suggest a high safety assurance level can be achieved through cell cloning

origen

nfluenza vaccine and selection of low tum

. Introduction

An influenza pandemic is a global disease outbreak caused bynfluenza virus often leading to the widespread morbidity and mor-ality with devastating social and economic costs. Vaccination is therimary and the most cost-effective strategy for reducing the mor-idity and mortality associated with annual influenza epidemics1]. Traditionally, vaccine manufacturing has relied on embry-nated chicken eggs for propagating influenza virus vaccine strains.lthough this is a well established process, it is relatively inflexible

n its ability to meet the surge in demand for vaccines, particu-arly in response to a pandemic. As a result, alternative strategiesuch as propagating influenza vaccine strains using mammalianell lines have been explored for pandemic preparedness [2]. Weave initiated a cell culture-based influenza vaccine program usingadin–Darby canine kidney (MDCK) cells as the production cell

ubstrate for live, attenuated influenza vaccine (LAIV) [3,4].MDCK cells were established from the kidney of a normal male

ocker spaniel by Madin and Darby [5]. Because of their suscep-ibility to many different influenza virus strains and their abilityo grow influenza viruses to relatively high titers, these cells haveeen used for world-wide influenza serosurveillance and determi-

∗ Corresponding author. Tel.: +1 650 603 2576; fax: +1 650 603 3576.E-mail address: liuj@medimmune.com (J. Liu).

1 Current address: Vaccine Research Center/NIAID/NIH, Building 40, Room 5502,0 Convent Drive, Bethesda, MD 20892-3005, United States.

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.11.023

ic and oncogenic cells for influenza vaccine production.© 2009 Elsevier Ltd. All rights reserved.

nation of influenza virus infectivity [3,4]. Originally isolated froma normal canine kidney, MDCK cells have been transformed into acontinuous cell line although the event or agent responsible for thistransformation remains unreported and undetected [6–8]. This lackof a documented transformation activity raises concerns relatedto the use of MDCK cells as a vaccine production cell substratethat some agent with transforming properties may still be associ-ated with these cells. Because of this concern, there is a significantrequirement to characterize the residual cellular components inthe vaccine product and demonstrate the elimination of adventi-tious agents to ensure that there is no element or agent presentwith potential oncogenic properties that pose a risk to the vaccinerecipient [9].

To address these concerns and ensure the safe use of MDCKcells for influenza vaccine production, a two-tier cell banking strat-egy was adopted for the generation of a serum-free (SF) MDCK cellline, designated MDCK cell clone 9B9-1E4. This strategy includedthe manufacture of a well characterized master cell bank (MCB)(Tier-1) followed by production of a manufacturer’s Working CellBank (WCB) (Tier-2), from which production campaigns will be ini-tiated. To evaluate the safety of the cell line, the MCB was expandedbeyond the expected end of production passage level (EOP) tomimic end-of-production cells for testing. These end-of-production

cells were evaluated in several in vivo systems in an attempt to mea-sure tumorigenic potential and detect any oncogenic elements ingenomic DNA and subcellular materials in lysed cells. The resultsof these studies demonstrated that up to 107 MDCK 9B9-1E4 cellsdid not form tumors in immunodeficient mice and neither FDA

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286 J. Liu et al. / Vaccin

pecified amounts of cellular DNA nor lysate were capable of induc-ng tumors in animals. These data combined with other cell bankelease test results that are normally required by FDA demonstratehigh level of assurance that the cells are safe as a substrate for

accine production.

. Materials and methods

.1. MDCK cells and cloning

The MDCK cells used in this study were originally establishedrom the kidney of a normal adult male cocker spaniel by Madin andarby in 1958, and deposited in the American Type Culture Collec-

ion (ATCC), Manassas, VA, at Passage 49 [5]. One vial of these cellsesignated as CCL34 was obtained from ATCC at Passage 54 andaintained in the serum-containing medium according to instruc-

ions provided by the supplier. At Passage 67 the cells were seededn 96-well plates at a density of 0.5 cells per well and cloned byimit dilution. Surviving clonal cells in individual wells were cul-ured and expanded at increasing scale in the order of 24-welllates, 6-well plates, T-25 flasks, T-75 and/or T-225 flasks. Duringell expansion, duplicate cell cultures were prepared in 24-welllates and one plate was infected with cold adapted (ca) A/Newaledonia/20/99 virus following the infection procedures describedelow. Several cell clones producing higher titers of virus wereelected for a subsequent round of cloning by repeating the aboveell cloning process. After the final selection, 7 highest producingDCK cell clones were adapted to serum-free growth conditions

y continuous passaging and selection in various commercial orn-house proprietary cell culture media for at least 25 passages.he cell clones that maintained the same growth rate and produc-ion titer of multiple ca influenza viruses were chosen for cell bankreparation and further analysis at Passage 85. A Master Cell BankMCB) and a Working Cell Bank (WCB) was prepared using MDCKell clone 9B9-1E4 and a proprietary serum-free cell culture mediaSFM), MediV-105 SFM under cGMP conditions at Passages 97 and01, respectively.

.2. Virus and virus infection

Multiple ca influenza virus strains were prepared and used tonfect MDCK cell clones at multiplicity of infection of 0.001 as pre-iously described [10,11]. Cell culture fluid was collected at 3 daysost-infection and the virus titer was determined either by TCID5012] or fluorescent focus assay [13]. The viruses used in this studyere ca A/New Caledonia/20/99 (H1N1), ca A/Hiroshima/52/05

H3N2), ca B/Malaysia/2506/04 (B) and ca A/Vietnam/1203/04H5N1).

.3. Evaluation of the tumorigenic and oncogenic potential ofDCK cells

.3.1. Tumorigenicity studiesTwo GLP studies were performed at BioReliance Corporation

Rockville, MD) to evaluate the tumorigenic potential of MDCK 9B9-E4 cells based on consultation with personnel from the Divisionf Vaccines and Related Products Applications (DVRPA) at CBER14,15]. The test article was MDCK 9B9-1E4 cells passaged to thestimated EOP passage level, Passage 113. Cells were diluted tohe desired concentration in Dulbecco’s phosphate buffered salinePBS). Cell viability and counts were confirmed 30 min prior to

njection to animals. All test articles were placed on wet ice untilse.

In the adult nude mouse study, 4-week-old female nude miceHarlan Sprague–Dawley: athymic nu-nu) were injected with00 �L of cell suspension subcutaneously between the scapulae

2010) 1285–1293

with each test animal receiving 101, 103, 105 or 107 MDCK cells.Two other groups of mice were injected with 107 HeLa cells orPBS, respectively. After injection, all animals were observed for aperiod of 6 months, during which time the animals were exam-ined for clinical symptoms and palpated twice a week to detectnodule development at the site of inoculation (SOI). At the endof the observation period, the surviving animals were euthanizedand necropsied. The site of injection, lungs, scapular lymph nodes,liver, kidney, spleen, brain and any gross lesions were harvestedand examined by an American College of Veterinary Pathologistsboard-certified pathologist.

In a separate study newborn (0–4 days old) athymic nude mice(Harlan Sprague–Dawley [Hsd]: athymic nude-Foxn1nu/Foxn1)were used, following the same experimental procedure asdescribed in the adult nude mouse study except that the inoc-ulation volume was reduced to 50 �L. Since only half of thenewborn mice were homozygous, twice as many animals wereacquired, randomized and inoculated with the MDCK cells. Twoweeks post-inoculation, heterozygous mice were identified bythe presence of hair and removed from the study. At 3 weeks ofage, the remaining homozygous nude mice were weaned, sexedand ear-tagged. Following ear-tagging, animals were randomlyselected within each test group, with an emphasis on equalizingthe number of animals/sex within each test group, to continuethroughout the duration of the study. All study animals wereobserved and examined as described for the adult mouse study fora period of 6 months. Table 1 outlines the study design and clinicalobservation criteria used in the tumorigenicity studies.

2.3.2. Oncogenicity studiesSix GLP newborn rodent studies were performed at BioReliance

Corporation to evaluate the oncogenic potential of MDCK cellu-lar components using two types of test articles (cell lysate andcell DNA) and three animal species (newborn nude mice, ham-sters and rats). These studies were conducted according to theregulatory guidelines on production cell substrate characterization[14,15] and after consultation with CBER. Table 2 summarizes theexperimental design and clinical observation criteria used in thesestudies.

MDCK cell lysate was prepared from MDCK MCB cells expandedto the EOP passage level by following the procedure described byLedwith et al. [16]. The cell lysate was measured for protein contentand dispensed into aliquots before storage at −80 ◦C. Total cellularDNA was extracted from MDCK MCB cells expanded to the EOPpassage level using an ABI PRISM® 6100 Nucleic Acid PrepStation(Applied Biosystems, Foster City, CA). The DNA concentration wasdetermined before lyophilization and subsequent storage at−80 ◦C.The lyophilized DNA was reconstituted to the desired concentrationat the time of injection.

In the mouse oncogenicity studies, newborn (0–4 days old)athymic nude mice (Harlan Sprague–Dawley [Hsd]: athymicnude-Foxn1nu/Foxn1) were subcutaneously injected between thescapulae with 50 �L of the test article or PBS. The test articleswere either MDCK cell lysate (prepared from 107 cells/animal) orMDCK cell DNA (100 �g/animal). In addition, a non-injected controlgroup was also maintained to monitor spontaneous tumor forma-tion. Again, twice as many animals were acquired and injected withthe test articles at the beginning of the study. The same proceduresas described in the tumorigenicity studies were followed to selectthe homozygous (nude) mice at 3 weeks of age. All study animals

were observed for 6 months and analyzed at the end of observationperiod as described in the tumorigenicity studies (Table 2).

In the hamster and rat oncogenicity studies, 0–4 days oldhamsters (HsdHan:AURA) or 0–4 days old rats (Hsd:SD) wereinjected subcutaneously between the scapulae with 100 �L of PBS

J. Liu et al. / Vaccine 28 (2010) 1285–1293 1287

Table 1Experimental study design of the tumorigenicity studies.

No. Groups Adult nude mousestudy (females)

Newborn nudemouse study(males/females)

Observations

1 Negative Control Group (Dulbecco’sPhosphate Buffered Saline)

33 43 (22/21) • Clinical signs—every working day• Palpations—bi-weekly• Body weightsmeasurements—weekly

2 101 MDCK cells 44 44 (21/23) • Lesion development—monitor andrecord lesions

3 103 MDCK Cells 44 44 (22/22) • Terminal endpoint—at the end of thestudy (6 months post-inoculation).Euthanize animals and perform grossnecropsy and histopathology

4 105 MDCK Cells 44 39 (16/23) • Unscheduled deaths—perform grossnecropsy and histopathology

5 107 MDCK Cells 44 44 (22/22) • Humane endpoint—euthanize animalif the lesion at site of injection exceedsmean tumor diameter exceeding10 mm, i.e., if the size of any lesionexceeds 10 mm in at least onedimension and/or if the tumorbecomes necrotic or ulcerates*

6 Positive Control Group (107 HeLa Cells) 41 44 (27/17) • Histopathology—primary tumor andthe site of inoculation, any gross

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r test article. The test articles were either MDCK cell lysate (pre-ared from 107 cells/animal) or MDCK cell DNA (100 �g/animal).t 3 weeks of age the pups were weaned, sexed and ear-

agged. Following ear-tagging, animals were randomly selectedithin each test group, with an emphasis on equalizing theumber of animals/sex within each test group, to continue

hroughout the duration of the study. All study animals werebserved and analyzed as described in the tumorigenicity studiesTable 2).

able 2xperimental study design for the oncogenicity studies of MDCK cell lysate and MDCK ce

Studies Groups Experimental animal

Newborn nudemouse study

Nh

MDCK Lysateoncogenicity studies

Uninjected SystemControl group

25 (12/13) 2

Negative Control group (PBS) 45 (23/22) 4

MDCK cell lysate (∼ of 1 × 107

cells/animal)a45 (22/23) 4

MDCK DNAoncogenicity studies

Uninjected System Controlgroup

25 (9/16) 2

Negative Control group (PBS) 44 (23/21) 4

MDCK cellular DNA(∼100 �g/animal)b

45 (21/24) 4

a 100 �L of MDCK lysate prepared from 1 × 108 cells/mL totaling to a dose equivalent tewborn hamster and newborn rat studies; whereas 50 �L of MDCK lysate prepared fro

njected into each test article group animal in newborn nude mice study.b 100 �L of 1 mg/mL genomic DNA (gDNA) totaling a dose equivalent to 100 �g/animewborn rat studies; whereas 50 �L of 2 mg/mL gDNA totaling a dose equivalent to 100tudy.

lesions, lungs, associated lymph nodes,liver, kidney, spleen and brain

2.4. Histopathological analyses

Histological analysis of formalin-fixed paraffin-embedded(FFPE) tissues was conducted according to previously reportedprocedure [17]. Tissues were sectioned at approximately 5 �mthickness, stained with hematoxylin and eosin (H&E) and exam-

ined for abnormalities. Histologically confirmed neoplasms werefurther characterized by immunohistochemistry (IHC) and/or PCRas described below.

llular DNA.

numbers (males/females) Observations

ewbornamster study

Newborn ratstudy

5 (14/11) 25 (11/14) • Clinical signs—every working day• Palpations—bi-weekly after weaning

5 (23/22) 45 (24/21) • Weekly body weights measurementsfor sudden weight gain or loss

5 (23/22) 45 (24/21) • Unscheduled deaths—gross necropsyand histopathology

5 (10/15) 25 (12/13) • Lesion development—euthanizeanimal if lesion at site of injectionexceeds mean tumor diameterexceeding 10 mm, i.e., if the size of anylesion exceeds 10 mm in at least onedimension and/or if the tumorbecomes necrotic or ulcerates

5 (23/22) 45 (29/16) • Terminal euthanasia—gross necropsyand histopathology

5 (23/22) 45 (23/22) • Histopathology—primary tumor andthe site of inoculation, any grosslesions, lungs, associated lymph nodes,liver, kidney, spleen and brain

o 1 × 107 viable cells/animal was injected into each test article group animal in them 2 × 108 cells/mL totaling to a dose equivalent to 1 × 107 viable cells/animal was

al was injected into each test article group animal in the newborn hamster and�g/animal was injected into each test article group animal in newborn nude mice

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serum-free media up to 25 passages, which was beyond the cal-culated EOP passage level (Fig. 2). In addition, these cell clonesmaintained their productivity when infected with ca A/New Cale-donia/20/99, ca A/Hiroshima/52/05, ca B/Malaysia/2506/04 andca A/Vietnam/1203/04 at both low (Passage 97, data not shown)

288 J. Liu et al. / Vaccin

.4.1. Tumor speciation using immunohistochemical analysis

.4.1.1. Immunohistochemical analysis. Immunohistochemicalssays were conducted to determine if the histologically con-rmed neoplasms were derived from MDCK cells and containedDCK cell antigens. Sections of the FFPE tumors were stained with

rimary antibodies, either purified mouse anti-canine Ezrin (BDiosciences, San Jose, CA) or goat anti-mouse Galectin-3 antibodyR&D Systems, Minneapolis, MN). The staining procedure followed

odifications of the pre-complexing immunohistochemical tech-ique of Tuson, Fung, Hierck and their colleagues [18–20], whichliminated the requirement for direct labeling of the primaryntibodies. In this method, the secondary antibody was allowedo bind the Fc� fragment of the unlabeled primary antibody toorm primary and secondary antibody complex prior to applyingo the tissue sections for 4 h to reduce the background staining.he secondary antibodies used in the study were biotinylated goatnti-mouse IgG (Dako, Carpinteria, CA) or Fc fragment specificabbit anti-goat IgG (Jackson ImmunoResearch, West Grove, PA).he primary antibody was used at the concentrations of 25 �g/mLnd mixed with secondary antibody at a ratio of 1:1.5 on the dayf staining. Thus, the final concentration of the secondary antibodyhat was applied to the test samples was 37.5 �g/mL. The testamples were incubated with pre-complexed antibodies at 2–8 ◦Cn a rocker platform for at least 4 h.

On the day of staining, endogenous peroxidase was quenchedy incubation of the slides with glucose oxidase (1 U/mL)/glucose10 mM) and sodium azide (1 mM) for 1 h. The slides were theninsed with the washing buffer (Tris-buffered saline, 0.15 M NaCl,H 7.6 and 0.01% Tween20). Next, the slides were blocked withvidin solution (Vector Laboratories, Burlingame, CA) for 15 min,insed with the washing buffer, followed by blocking with biotinolution (Vector Laboratories, Burlingame, CA) for 15 min at roomemperature, and rinsed again with the washing buffer. This wasollowed by application of a protein block designed to reduce non-pecific binding and incubation for 60 minutes. The protein blockontained phosphate-buffered saline, 0.15 M NaCl, pH 7.2; 0.5%asein (N-Z-amine), 1% BSA and 1.5% normal goat serum. Follow-ng the protein block, the pre-complexed primary and secondaryntibodies were applied to the slides and incubated for two hourst room temperature. Next, the slides were rinsed with the wash-ng buffer, treated with the ABC Elite reagent (Vector Laboratories,urlingame, CA) for 30 min, rinsed with the washing buffer andhen treated with DAB (Sigma–Aldrich, St. Louis, MO) for 4 min. Alllides were counterstained with hematoxylin, then dehydrated andoverslipped for interpretation by the study pathologist.

.4.1.2. Tumor speciation using SINE PCR analysis. Detection ofanine-specific or rodent-specific SINE DNA sequences in theistologically-confirmed tumors was performed using qualitativeaqMan® and SYBR® Green real-time PCR assays. Detection ofanine DNA, including DNA derived from Madin–Darby Canineidney (MDCK) cells, was accomplished through use of a qual-

tative TaqMan® real-time PCR assay. This assay utilizes canineINE-specific primers (forward primer, 5′-GGCCCAGGGCGTGATC-′ and reverse primer, 5′-GCAGGGAGCCCGATGTG-3′) and probe6FAM-TTCCGGGATCGAGTC-MGBNFQ) to amplify and detect

55 bp canine genomic DNA target. Detection of rodent DNAas accomplished with a qualitative SYBR® Green real-time

CR assay that was adapted from Walker et al. [21]. Thisssay utilized rodent SINE-specific primers (forward primer,′-AGATGGCTCAGTGGGTAAAGG-3′ and reserve primer, 5′-

TGGAGGTCAGAGGACAAACTT-3′) to amplify and detect a 118 bp

odent genomic DNA target. The PCR test samples were preparedrom FFPE sections using QIAGEN QIAamp DNA FFPE Tissue KitQIAGEN, Valencia, CA). Briefly, DNA was extracted in singlicaterom five, 10-micron FFPE tissue sections and was diluted 1:10 and

2010) 1285–1293

1:100 in nuclease-free water and tested in duplicate via TaqManand SYBR green assays on an Applied Biosystems 7500 real-timePCR instrument. Standard curve ranges of mouse, hamster, ratand MDCK genomic DNA were tested as positive controls in therodent and canine-specific PCR assays. No template controls wereperformed in duplicate.

3. Results

3.1. Identification of a high yield MDCK cell clone for coldadapted influenza vaccine production

To determine whether different MDCK cells in the starting pop-ulation had differing propensities for supporting influenza virusreplication, several cell clones were isolated and tested for virusproduction. MDCK cells were plated at a target concentration of 0.5cells per well and allowed to grow to confluency and approximately2500 MDCK cell clones were generated. Each clone was split intoreplicate plates upon passaging and one plate used for infectivitystudies. Infection of these clones with two ca influenza viruses, caA/Panama/2007/99 and ca A/New Caledonia/20/99 and comparisonof the virus titer produced by each clone, revealed large variationsin their ability to produce progeny viruses among these clones. Forexample, following infection with ca A/Panama/2007/99, the differ-ence in the output titer of virus among 1228 cell clones exceeded1.2 log10 fluorescent focus units (FFU)/mL (Fig. 1). The vast majorityof the cell clones produced viruses below 7.6 log10 FFU/mL and only3 out of 1228 clones were capable of producing the viruses withtiters equal to or higher than 8.5 log10 FFU/mL. These three clones,along with 51 other high producer clones, were chosen for an addi-tional round of cell cloning based on their high virus productivityafter infection with ca A/New Caledonia/20/99 and ca B/Jilin/20/03(data not shown). From the second round of cloning, 63 subcloneswere shown to produce a high titer of ca A/Panama/2007/99 virus.Of these subclones seven highest producing clones were selectedfor serum-free adaptation and additional productivity testing.

To transfer the MDCK cells to serum-free growth conditions,three commercially available serum-free media and one in-houseserum-free medium (MediV-105 SFM) were used to passage the 7selected MDCK cell clones. Most of the cell clones tested ceased togrow in serum-free media after 3 to 8 passages and had few viablecells by Passage 11 (data not shown). However, two cell clones,Clone 9B9-1E4 and Clone C, maintained their ability to grow in

Fig. 1. Virus titer produced by MDCK cell clones. Individual MDCK cell clones wereinfected with cold adapted A/Panama/2007/99 virus. Titer of progeny viruses weredetermined and used to group the cell clones. Most (1014 of 1228) cell clonesproduced low titer virus (<7.6 log10 fluorescent focus unit/mL).

J. Liu et al. / Vaccine 28 (2010) 1285–1293 1289

Table 3Virus productivity of two selected MDCK cell clones, Clone C and 9B9-1E4 at Passage 113.

Days Postinfection Day 3 Day 4

ca virus Clone C Clone 9B9-1E4 Clone C Clone 9B9-1E4

A/New Caledonia/20/99 8.1 ± 0.06 8.2 ± 0.10 8.0 ± 0.12 8.0 ± 0.068.1 ± 0.06 8.3 ± 0.10 8.0 ± 0.06 8.2 ± 0.06

A/Hiroshima/52/05 8.2 ± 0.06 8.1 ± 0.10 8.2 ± 0.00 8.0 ± 0.068.2 ± 0.10 8.2 ± 0.06 8.1 ± 0.06 7.0 ± 0.00

A/Vietnam/1203/04 8.3 ± 0.06 8.4 ± 0.06 8.3 ± 0.00 8.4 ± 0.068.4 ± 0.06 8.4 ± 0.06 8.3 ± 0.00 8.3 ± 0.06

B/Malaysia/2506/04 8.0 ± 0.00 8.0 ± 0.06 7.8 ± 0.06 7.8 ± 0.108.0 ±

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ote: Each cell clone was infected with the specified virus twice in T-75 flasks. Thssay results in the table.

nd high passage (Passage 113) levels (Table 3). There was min-mal difference in productivity between these two MDCK celllones. The virus titer was slightly lower for a B strain virus (ca/Malaysia/2506/04) compared to A strains (ca A/New Caledo-ia/20/99 and ca A/Hiroshima/52/05) and pandemic virus strainca A/Vietnam/1203/04) for both clones. There was no obvious dif-erence in virus titer between the cells infected for 3 and 4 daysTable 3). Based on the ability to produce high titer ca influenzairus and achieve sustainable cell growth in serum-free mediaDCK cell clone 9B9-1E4 was chosen to generate cGMP cell banks

nd characterized for tumorigenicity, oncogenicity and other anal-sis to assess its suitability as a production cell substrate for cellulture-based influenza vaccine manufacturing.

.2. Low tumorigenicity of MDCK 9B9-1E4 cells in nude mice

Two studies using athymic nude mice were performed; in eachtudy, groups of animals were inoculated with different numbers ofDCK 9B9-1E4 EOP cells, PBS as a negative control or HeLa cells aspositive control. In the adult nude mouse study, the positive con-

rol HeLa cells produced readily identifiable and palpable nodulest the site of inoculation in all 41 animals within 7 days of injec-ion (Table 4). The initial size of the nodules was approximately–8 mm which then progressed into larger neoplasms over time

n most animals (n = 35), requiring euthanasia on or before day 59f the study. Histopathological analyses confirmed that the nod-les formed in the skin at the site of inoculation corresponded toarcinomas arising from the injection and growth of HeLa cells.

In the athymic nude adult mice that received MDCK cells, palpa-le nodules were initially observed in the group that received theighest number of MDCK cells (107 cells); most of these nodulesegressed and disappeared by the end of the 6-month observationeriod. One mouse in the 107 MDCK group was sacrificed on day 84

ig. 2. Growth curve of MDCK cell clone 9B9-1E4 in serum free cell culture medium Medibility to grow in MediV-105 SFM for at least 25 passages. The cells were split and subcultuhe passage level is shown as P1 through P25 on the X-axis.

0.06 7.8 ± 0.06 7.9 ± 0.06

age virus titer (log10 FFU/mL) was listed along with standard errors of duplicated

due to a large progressing nodule. This nodule was subsequentlyconfirmed to be an abscess by histopathologic analysis. No addi-tional palpable nodules were observed at the end of the study atthe site of injection in any groups of mice that were inoculatedwith MDCK cells. Of the 176 animals injected with MDCK cells, oneanimal in the 105 MDCK group was observed to have a dark swollenabdominal area on day 112 and was sacrificed as moribund the nextday. Microscopic examination revealed histiocytic sarcoma in thelungs, liver and spleen of this animal. The histiocytic sarcoma tis-sue was tested for the presence of canine surface protein (Ezrin)and canine SINE DNA using immunohistochemistry and PCR anal-ysis. It was negative for the presence of canine Ezrin (Fig. 3) andcanine SINE DNA (data not shown), but stained positive for murineGalectin-3 surface protein (Fig. 3) and shown to contain rodentSINE sequences by PCR analysis. Thus the histiocytic sarcoma wasnot derived from the inoculated MDCK cells, rather was likely aspontaneous neoplastic event as commonly observed in athymicnude mice [22–24]. Two spontaneous neoplasms, a bronchiolo-alveolar adenoma in the lungs and malignant lymphoma in thelymph nodes, lungs, liver and kidneys, were also observed in twoseparate PBS-injected animals (Table 4).

In the newborn nude mouse study, all forty-four mice injectedwith HeLa cells developed nodules that rapidly progressed intolarge tumors (Table 4). Most of them (43 mice) had to be euthanizeddue to excessive tumor burden (>20 mm in dimension). Histolog-ically, all tumors were composed of sheets of HeLa cell carcinomacells at the SOI. Tumors were not observed in other organs or tis-sues.

Newborn athymic nude mice injected with the highest dose ofMDCK cells (107 MDCK cells) initially had palpable nodules in 36out of 44 mice. These nodules regressed over time with nearly allmice exhibiting complete regression by the end of the observationperiod. The only exception was Animal No. 4307 which developed

V-105 SFM. Two selected MDCK cell clones, 9B9-1E4 and Clone C, maintained theirred under the same growth conditions every 3 or 4 days as indicated in parenthesis.

1290 J. Liu et al. / Vaccine 28 (2010) 1285–1293

Table 4Summary of tumorigenicity study results.a.

Study Test sample Number of animalsinjected

Nodule at the injectionsite (at necropsy)

Confirmed tumors atinjection site(histology)

Tumors at otherlocations

Adult nude mousetumorigenicity study

Negative Control (PBS) 33 0 0 2/33c

Positive control (107 HeLa cells)c 41 38/41 37/41b 0101 MDCK cells 44 0 0 0103 MDCK cells 44 0 0 0105 MDCK cells 44 0 0 1/44d

107 MDCK cells 44 1 0 0

Newborn nude mousetumorigenicity study

Negative Control (PBS) 43 0 0 0Positive Control (107 HeLa cells)b 44 44/44 44/44 0101 MDCK cells 44 0 0 0103 MDCK cells 44 0 0 0105 MDCK cells 39 0 0 0107 MDCK cells 44 1 0 0

a Duration of the in-life phase of the study was 6 months.b 36 of 37 evaluable tumors at end of study; four mice died due to accidental drowning at day 49/50 of the study observation period.c A lymphoma and a bronchiolo-alveolar adenoma were observed in two mice belonging to the PBS (saline) control group. Both tumors were confirmed to be spontaneous

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odent tumors by location, incidence, and histopathological examination.d A Histiocytic sarcoma was observed in the 105 MDCK cells group. Although thistopathological examination, it was tested for and found to be negative for the prespectively.

small (1 mm × 1 mm × 1 mm) nodule that persisted through thentire 6-month observation period and was confirmed to be annflammatory cyst at the time of necropsy. No palpable nodules

ere observed at the SOI at the time of euthanasia in any otherewborn athymic nude mice injected with MDCK cells. Microscop-

cally identifiable neoplasms were not detected in any test articleroups.

.3. Lack of oncogenic potential of MDCK 9B9-1E4 cells

A total of 689 animals were examined for tumor formation andther related clinical signs in 6 oncogenicity studies. In spite ofnjecting large amount of MDCK cell lysate (equivalent to 107 cells)r DNA (100 �g), tumors were not observed at the SOI which is theost commonly reported tumors site associated with injection of

ell lysate or cellular DNA derived from tumorigenic cells [25,26].his was true for all 3 tested animal species: newborn nude mice,ewborn hamsters and newborn rats. The absence of tumors at theOI was observed for both MDCK cell lysate and MDCK cell DNAnjected animals (Table 5).

At the sites distal to the inoculation site, neoplastic lesions werebserved in one untreated animal, two PBS injected animals, oneDCK cell lysate injected animal and two MDCK cell DNA injected

nimals (Table 5). The neoplastic change in the lung of a non-njected control newborn nude mouse (Animal ID. 17801) consisted

f bronchiolo-alveolar adenoma, one PBS-treated rat developedepatocellular adenoma in the liver (Animal ID. 1256) and anotherad skin hemangiosarcoma (Animal ID. 1230). In animals injectedith MDCK subcellular components, an undifferentiated carcinomaas observed in the hind leg of a rat injected with MDCK cell lysate

able 5ummary of observations made in MDCK cell oncogenicity studies.

Groups MDCK lysate (107 cell equivalent)

Mice Hamster Rat

Non-injected (n = 25) 1a 0 0PBS (n = 45) 0 0 0Test (n = 45) 0 0 1d

a Bronchiolo-alveolar adenoma in the lung; spontaneous tumor/no canine DNA by SINEb Only 44 animals were available for post-weaning randomization in this group.c Hepatocellular adenoma and skin hemangiosarcoma; spontaneous tumors.d Hind leg carcinoma; no canine DNA by SINE; confirmed rodent origin.e Nephroblastoma; no canine DNA by SINE; confirmed rodent origin.

or was deemed as a spontaneous rodent tumor based on location, incidence, ande of canine protein and canine sequences by immunohistochemistry and SINE PCR

(No. 18046), bronchiolo-alveolar adenoma was confirmed in thelung of a newborn athymic nude mouse injected with MDCK cellu-lar DNA (Animal ID. 18551), and a nephroblastoma was observedin the kidney of a newborn hamster that was administered MDCKcellular DNA (Animal ID. 2298). The neoplasms observed in MDCKcell lysate or DNA treated animals were examined for their ori-gin. All three neoplasms tested negative for canine DNA, but werepositive for the presence of rodent SINE elements, indicating theywere of rodent origin and thus were highly likely to be spontaneousneoplasms as have been reported by others [23,27–30]. Under theexperimental conditions described here, neither MDCK cell lysatenor MDCK cellular DNA caused any local or systemic tumors in anyof the three animal species tested.

4. Discussion

Vaccination is a key protective mechanism for preventing mor-bidity and mortality associated with annual epidemics of influenza[1,31]. Delays in vaccine production or shortfalls in supplies canresult in significant social, economic and health consequences. Thisis particularly true during a global pandemic influenza outbreak.Traditionally, influenza vaccines are produced in embryonatedchicken eggs and production is limited by inflexibility in scale-up,difficulties in controlling both raw materials and the manufacturingprocesses and the inability to produce large quantities of vaccine

to vaccinate large populations in a short period of time such asin a pandemic outbreak [32]. A cell culture-based vaccine produc-tion platform provides significant advantages in all of these areasand thus becomes an attractive alternative to the conventional egg-based influenza vaccine production system, by rectifying vaccine

MDCK DNA (100 mg) Tumor incidence

Mice Hamster Rat

0 0 0 1/150 (0.6%)0b 0 2c 2/269 (0.7%)1a 1e 0 3/270 (1.1%)

; confirmed rodent origin.

J. Liu et al. / Vaccine 28 (

Fig. 3. Immunohistochemistry of the histiocytic sarcoma observed in the lungs,liver and spleen of a female nude mouse inoculated with 105 MDCK cell suspen-sion. Panel A shows the histiocytic sarcoma stained with Hematoxylin and Eosin.Panel B shows the tumor stained with the anti-canine Ezrin antibody. No stainingsmt

servliccwTtpt

ura[smcctfliaptTt

ignal was detected. Panel C shows the tumor stained with anti-Galectin-3 (murinearker). Intense staining by the anti-Galectin-3 antibody was detected indicating

he presence of murine Galectin-3 protein in this tumor tissue.

upply problems [32]. In spite of the progress made in the last sev-ral decades our knowledge about vaccine production substratesemains limited, especially in the use of novel cell substrates foraccine production. In fact, there are only three mammalian cellines that are currently used for producing licensed human vaccinesn United States: MRC-5, WI-38 and Vero cells [33–36]. The latterell line has been shown to be tumorigenic under specific growthonditions and used to produce LAIV that appeared to be safe,ell tolerated and immunogenic in your adult volunteers [37,38].

o develop new vaccine production substrates, we have exploredhe potential of a canine kidney-derived cell line, MDCK cells, forroduction of live attenuated influenza vaccine and evaluated itsumorigenicity and oncogenicity.

Although MDCK cells are extensively characterized and widelysed in public health surveillance, clinical virology, and a wideange of research areas [39–42], consensus regarding severalspects of these cells as they relate to safety has not been reached43–45]. This may be partly attributed to the heterogeneity of the-es cells and the highly variable experimental conditions used toaintain the cell lines. For example, MDCK cells are known to

ontain morphologically and biochemically distinct cell types andan be isolated as clonal cell lines [46–48]. Recently we reportedhat MDCK cells were identified as the highest producing cell lineor cold-adapted attenuated influenza vaccines [11]. From this celline, we also found that productivity varies significantly depend-ng on the specific vaccine strains tested. For some viruses such

s ca A/Texas/36/91, productivity is low rendering commercialroduction non-viable. To address this problem, we have set outo identify high producing cell clones from these MDCK cells.he experimental results described in this report demonstratehat biological cloning of MDCK cell lines is a useful approach

2010) 1285–1293 1291

to improve influenza vaccine productivity. The ATCC MDCK cellstock is heterogeneous with respect to virus production, vary-ing at least 20-fold among different clones. In addition, MDCKcell clones also show marked differences in their ability to adaptto serum-free growth conditions. Most MDCK cell clones ceasegrowing after 3–8 passages in serum-free cell culture media andare marginally viable after 11 passages. However, two individ-ual cell clones, including clone 9B9-1E4, continued to grow for 25passages.

MDCK cells have been used for commercial production in Europeand clinical investigation in United States for inactivated influenzavaccines [49–51]. However, their use for production of live attenu-ated influenza vaccines has yet to be fully explored [52]. One of themajor concerns is the potential of tumor formation by the intactcells. In this study we examined this property using a biologicallycloned MDCK cell line in 2 animal models (adult nude mice andnewborn nude mice). Our studies clearly show that MDCK cells con-tain cell populations that have low tumorigenic potential, definedin this study as no observed tumor formation at ≤107 cells peranimal. Due to technical and operational limitations, we are notable to test the cells at doses greater than 107 cells per animal.However, under the described test conditions, persistent tumorsdid not occur with MDCK cells. Among the 347 animals that wereinjected with various doses of MDCK cells (up to 107 cells) only oneanimal developed a tumor (histiocytic sarcoma). This tumor wasnot considered to be caused by the injected MDCK cells based onlack of either MDCK cell antigen or MDCK cell DNA in the tumorand similarity between rate of tumor events observed betweentest article-injected animals and negative control (PBS treated) ani-mals. Our study results are different from a previous report thatdescribes formation of tumors in newborn mice upon injection ofMDCK cells with as few as 10 cells [53]. This difference is not unex-pected as the MDCK cells described in that report were grown as asuspension culture, instead of adherent cells. Suspension cells gen-erally have a higher tumorigenic potential as reported by others[54,55]. In addition, that cell line may represent a different bio-logical population of MDCK cells because the original MDCK cellswere adherent cells. From the studies conducted in our laboratoryand others it is clear that different MDCK cell clones may behavedifferently and can have a widely varying degree of tumorigenicpotential. Weak tumorigenicity has been reported for MDCK cellsby others. Medema et al. found that MDCK cells do not inducetumor formation at lower dose (below 105 cells) [49]. At high doses(105 to 107 cells) injection of MDCK cells resulted in tumors inimmunodeficient mice and these tumors were benign in nature.These observations are similar to what we have seen in our study,although it is unclear if the MDCK cells reported by Medema areclonal cells [49]. Regardless of the similarities, we believe impor-tant differences exist between the described MDCK cells as we didnot observe any tumors at the highest injection dose (107 cells). Fur-thermore, our experiment results are similar to those reported by anumber of other laboratories. Stiles et al. screened a large numberof cell lines for their tumorigenic potential in nude mouse mod-els and found MDCK cells to be non-tumorigenic [45]. Boerner etal. also confirmed that MDCK cells are not tumorigenic withoutchemical treatment [56]. Percheson et al. confirmed that BV-5F1is a MDCK derived cell line and is non-tumorigenic in tests con-ducted in accordance with FDA guidelines [50]. These reports aresimilar to our observations that MDCK cell clone 9B9-1E4, whichwas obtained through two rounds of biological cloning and pro-duced under specific cGMP production conditions, are very low

in tumorigenic potential, at least up to the estimated EOP level,Passage 113.

A second set of studies were performed to determine whetherthe MDCK cell clone 9B9-1E4 had any oncogenic activities associ-ated with it. Since the original event that led to the transformation

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f MDCK cells is unknown, we could not develop specific and sensi-ive assays to detect this property in the cells. Rather, we evaluatedhe potential of MDCK subcellular components, cellular lysate andellular DNA, to induce tumor formation in three sensitive rodentpecies. The design of these experiments was constrained by thevailability of certain materials. For example, the appropriate pos-tive control would be the DNA of an MDCK cell clone with highlyncogenic potential. This control would enable determination ofhe sensitivity of the model and also enable evaluation of the effi-iency of individual manufacture processing steps, with respect toeduction of oncogenic activity. However, this type of control is noturrently available and therefore we chose to evaluate a very largexcess of material (cell lysate/DNA) to gain confidence that lowevel activities can be detected. The MDCK cell samples were testedt high concentration (107 cell equivalent cell lysate and 100 �gellular DNA) to maximize any potential effects of MDCK cell lysatend DNA. Our analysis of residual host cell DNA and proteins of mul-iple lots of purified vaccine products indicates the cell lysate andellular DNA tested at such high concentrations represent >100-old more total cell protein and >100,000-fold more cellular DNAhan that present a vaccine dose. Furthermore, this DNA does notccount for the impact of specific manufacture processes such asNA digestion which are designed to purify the vaccine strainsnd further reduce the effect of cellular DNA, but are beyond thecope of this communication. Additionally, although there was aow tumor incidence in all test and control groups with 1 of 1500.6%) non-injected animals developing tumors, 2 of 269 (0.7%) PBS-njected animals developing tumors and 3 of 270 (1.1%) for MDCKell lysate- or DNA-injected animals that developed tumors, nonef the tumors had evidence of MDCK cellular DNA based on SINECR analysis. We conclude that the MDCK cell clone 9B9-1E4 doesot contain oncogenic activities and these results are consistentith reports by others. In a 5-month oncogenicity study Medema

t al. confirmed that up to 100 micrograms of MDCK cell DNA didot cause tumor formation in 4-week-old nude mice, newborn ratsnd newborn hamsters [49].

Experimental results obtained from above 8 tumorigenicitynd oncogenicity studies demonstrate that MDCK cell clone 9B9-E4 is a cell line with very low tumorigenic potential and ison-oncogenic, but with high virus productivity. We have alsoonducted many other tests and demonstrated that the cell banksrepared from this clone are free of adventitious agents (data nothown). We have identified a potential production substrate thats a viable alternative for the currently used embryonated chickengg-based influenza vaccine production technology.

cknowledgements

This project is funded in whole or in part with Federal fundsrom the Office of the Assistant Secretary for Preparedness andesponse (ASPR), Biomedical Advanced Research and Develop-ent Authority, under Contract Nos. HHSO100200600010C andHSO100200700036C. The total federal program funding for theseontracts is $221,379,570, representing approximately 92% of theotal amount of the projects. The remaining 8% of the total amountor the projects is anticipated to be financed by nongovernmen-al sources. The authors thank the following MedImmune staffor excellent technical assistance in preparing MDCK cells andell lysate: Xiao Shi, Masiha Farooq, Thi Dang and Dai Quach. Were grateful to Samantha Mapes of University of California, Davis

or preparing MDCK cell DNA. We thank Joseph Madary, KarmaaCosta, Anmarie Boutrin, Martha Wester and Nancy Huddy for

heir histologic and immunohistochemisty expertise. We are inebt to Mark Galinski and John Finkbohner for the critical reviewf the manuscript.

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2010) 1285–1293

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