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Vaccine 32 (2014) 2931–2938

Contents lists available at ScienceDirect

Vaccine

j our na l ho me page: www.elsev ier .com/ locate /vacc ine

se of adenovirus as a model system to illustrate a simple methodsing standard equipment and inexpensive excipients to remove liveirus dependence on the cold-chain

. Stewart, S.J. Ward, J. Drew ∗

tabilitech Ltd, Unit 6 Sovereign Business Park, Albert Drive, Burgess Hill. RH15 9TY, UK

r t i c l e i n f o

rticle history:vailable online 21 March 2014

a b s t r a c t

Thermolability of complex biological molecules is a major consideration for the long-term maintenanceof titer during periods of storage.

The development of a simple, cost effective method for long term storage of virus samples, whichmaintains viral titer would prove useful for a wide variety of applications including the preservation ofviral vaccines, and is paramount for alleviating the reliance upon the cold chain.

We have investigated the potential use of a method adapted for this purpose originating from a naturalmechanism used by plants which helps to maintain the integrity of seeds, enabling them to overcomeextensive periods of temperature elevation and desiccation.

As maturation of a seed progresses, many complex biological macromolecules are laid down whichmaintain the germination potential. Sucrose and raffinose (in addition to other oligosaccharides) arecommonly found to accumulate. In addition highly charged protein molecules accumulate, Late Embryo-genesis Abundant (LEA) proteins, reaching their maximal level when the seed is most desiccation andthermally tolerant, and indeed are among the first molecules to be lost when germination is initiated.

We have examined the potential use of sucrose and raffinose in concert with chemical replacementsfor the LEA, which when dried with the active product forms an amorphous solid able to maintain thetiter of infectious Adenovirus at elevated temperatures for extended periods, in the case of lyophilizedpresentations several months at 37 ◦C, or as liquid, stability for several weeks at 37 ◦C was achieved.

We demonstrate that after embedding the active product in the matrix, the viral titer is preserved evenat temperatures for relatively extended periods at temperatures significantly greater than ambient.

In addition we believe that these results could open the way for a new type of vaccine which we refer toas a hybrid stability vaccine, whereby for the first time the same excipient components are used to conferstability in both liquid and solid forms (albeit at different concentrations) which may ultimately resultin a stable vaccine which has a very high stability index whilst dry, whereas upon reconstitution usingnothing more than WFI at proscribed volumes, the vaccine would benefit from having much improved

stability during the administration procedures.

This paper describes the use of Adenovirus (itself fast becoming a vector of choice for a new generationof vaccines) as a model system, and identifies non-toxic, inexpensive excipients which are compatiblewith current manufacturing processes which could be instrumental in removing the dependence uponthe cold chain.

. Introduction

The inherent thermolability of complex biological moleculess well documented. Indeed, this has been exploited in some

∗ Corresponding author.E-mail address: jeff@stabilitech.com (J. Drew).

ttp://dx.doi.org/10.1016/j.vaccine.2014.02.033264-410X/© 2014 Elsevier Ltd. All rights reserved.

© 2014 Elsevier Ltd. All rights reserved.

methodologies to remove infectious virus selectively [1]. Howevermaintenance of viral titre for research purposes and vaccines isoften necessary and is achieved by cryogenic storage or by main-taining a strict “cold-chain”.

The introduction of thermostability to viral particles, with the

concomitant release from the constraints of the “cold chain” holdsmany potential benefits, including significantly simplifying thelogistics for transporting biopharmaceuticals, viral delivery vectorsand vaccines to all areas of the world.

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Improving the stability of vaccines is an important goal formproving the impact of vaccines on world health. One of the keyactors contributing to the successful eradication of smallpox in980 was the availability of heat-stable vaccine [2]. The campaigno eradicate polio by vaccination has been seriously held back byhe lack of thermo stable polio vaccine.

Maximizing stability of new vaccines should be one of the majorbjectives of formulation development.

Recombinant adenovirus vectors are becoming increasinglymportant for use in vaccine development and gene therapy appli-ations but poor stability of adenovirus has limited its potential byestricting storage conditions almost exclusively to the frozen state.owever, stable vaccine formulations will be critical for maximiz-

ng the potential of recombinant adenovirus vectors, particularlyor use in the developing world where maintaining a cold chain isften challenging.

The optimal design of vaccine formulations for developing worldpplications requires several criteria such as (a) stability throughultiple heating/cooling cycles, (b) sufficient stability at to allow

or medium term temperature increases above ambient (typicallyonsidered to be 25 ◦C), (c) sufficient stability in a liquid state tollow for short-term storage and handling in the field, withoutefrigeration in order that they can be used effectively, (d) Wheniquid have an 18–36-month shelf-life when stored at 2–8 ◦C, (e) beasily sourced, inexpensive and non-toxic excipients. There haveeen several reports in the literature on development of more sta-le adenovirus formulations [3], however, none of the reporteddenovirus formulations meet the above criteria, clearly indicatinghe need for additional adenovirus vaccine formulation develop-

ent.Our development efforts have been to design formulations that

eet all of the stability and compatibility criteria outlined above.Design of Experiment approaches, were used to screen a panel

f excipients, then to optimize the mixes. Several different liquidormulations were evaluated for their ability to stabilize live virus,nder accelerated storage conditions at −20, 4, 25, or 40 ◦C, overeveral months.

In order to identify thermostability to biological preparations weook the approach of examining natural systems in which complexiological molecules are capable of withstanding relatively adversehermal conditions. Plants use one applicable process, during seedevelopment.

After maturation, seeds are capable of withstanding extensiveeriods of desiccation and thermal variation with little if any effectpon their ability to germinate.

The development of seeds is a complex biological phenomenon.ith many compounds either found solely or at levels only seen

n the seed are produced and laid down during maturation. Thisuggests that these compounds must play a pivotal role in the main-enance of germination potential, by preserving the integrity ofecessary enzymes and proteins necessary for subsequent growth.

Compounds frequently observed to accumulate in developingeeds, include the so called Late Embryogenic Abundant proteinsLEAs). These comprise a complex set of robust hydrophilic pro-eins [4] associated with acquisition of desiccation tolerance prioro maturation drying in orthodox seeds (reviewed by [5,6]). Addi-ionally, sugars have also been implicated in the preservation ofermination potential, and include sucrose, oligosaccharides oralactosyl cyclitols.

The accumulation of non-reducing sugars, particularly those ofhe raffinose series [7–9] and/or galactosyl cyclitols [10,11] haveeen associated with desiccation tolerance.

Two functional explanations have been proposed for the pro-ective effects afforded by the respective compounds. The first,he so-called “water replacement hypothesis” [12,13] suggests thathe sugars displace water from amongst other things membrane

2 (2014) 2931–2938

surfaces hence maintaining the lipid bi-layer. Although Trehalosehas been suggested to be the most effective sugar to replace water inthis manner [12–14] studies on angiosperm seeds indicate sucroseis the most abundant disaccharide.

Indeed other molecules may also have an influence in this modeof protection, for example the LEA antigens, which accumulate asthe seeds approach maturity, reaching peak concentrations whenthe seeds are at their most thermo- or lyo-stable, and are amongstthe first molecules to be lost during germination, suggesting a roleduring the period when the seed is at its most thermal/desiccationtolerant.

The LEA proteins are a group of highly charged proteins presentin both plants and animals. Various functions have been attributedto these proteins, including their being associated with toleranceto water stress resulting from desiccation and cold shock and maybehave as molecular chaperones. However, although various func-tions of LEA proteins have been proposed, their precise role has notbeen defined.

There are at least three major classes of LEA proteins whichhave been defined on the basis of expression pattern; Group 1LEA are only found in plants; Group 2 proteins (two superfami-lies) are characterized by up to three sequence motifs, K-domain(lysine rich), Y-domain (DEYGNP) and the S-segment (poly-serinestutter); Group 3 LEA proteins (two superfamilies) are character-ized by an 11-mer amino acid repeat motif broadly defined as��E/QX�KE/QK�XE/D/Q (� represents a hydrophobic residue).Interest in this Group has been increased by the discovery of homo-logues in organisms other than plants, including the nematodesCaenorhabditis elegans, Steinernema feltiae and A. avenae, andthe prokaryotes D. radiodurans, Bacillus subtilis and Haemophilusinfluenzae.

Several investigators have suggested that sucrose, in the pres-ence of other oligosaccharides ((e.g. raffinose) which prevents itscrystallization) and now feasibly protein such as LEA has a role indehydration and desiccation-tolerance of some seeds and pollens(e.g. [14–17]).

The second proposed mechanism involves the aqueous phasevitrification which generates what is commonly termed the“glassy state” [7,11,18–20]. This proposed mechanism relies onthe assumptions that upon loss of water, sucrose and associatedoligosaccharides (or galactosyl cycitols) form high viscosity, amor-phous super saturated solutions. Due to the high viscosity of theglass, compounds trapped within it are held in a form of “stasis”,with all cellular or enzymatic activity (deleterious or otherwise)largely prevented from proceeding. Indeed it has been postulatedthat in the seed the role of glasses is not to confer desiccation toler-ance, but to maintain viability for extended periods in the dry state[20].

We have investigated the possibility that the protective mech-anisms afforded by molecules in cooperation with sugars in plantseeds could be replicated using simple chemical mixtures for thepreservation of complex biological molecules during similar varia-tions in storage conditions. As indicated previously, two of the mostabundant sugars found within many seeds are sucrose and raffi-nose. These two saccharides have been found at ratios approaching85% and 15% of the total sugar content of the seed (dry weight). Wehave identified less obvious excipients which when combined atnovel ratios with sugar(s) are able to stabilize biological molecules.In the following study, we used a Design of Experiment approach tooptimize the mixtures used in our stability studies, which includedthe concentrations of sugars and other chemical constituents of themixes.

For the first time we are able to demonstrate a set of excipi-ents, which can stabilize fragile biological molecules in both liquidand solid settings, possible opening the way for a completely novelpresentation of vaccine, a so called hybrid stability vaccine.

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. Materials and methods

.1. Adenovirus culture

.1.1. AdenovirusStocks of Adenovirus (Ad5) were obtained from Vector Biolabs.

his is a recombinant Adenovirus 5 expressing enhanced GFP under CMV promoter. Ad5 had a titre (pre-freeze) of 6.7 × 105 pfu/ml inSC and was stored at −80 ◦C. Virus was cultured in human embry-nic kidney 293 (HEK293) cells supplemented with DMEM and 10%etal bovine serum (FBS).

.2. Adenovirus assay

.2.1. Assay of AdenovirusHEK 293 cells were prepared in 96 well flat bottomed cell cul-

ure dishes for inoculation by seeding at 105 cells per ml (100 mler well) and maintained at 37 ◦C with 5% CO2. After 2 h cells were

noculated. Virus samples were diluted 1 in 10, 1 in 30 and 1 in00 in DMEM and 10% FBS. 100 �l of each of the resulting dilutedirus samples were then added to triplicate wells of the assaylate.

As a positive control, an aliquot of the original Adenovirus in SSCas thawed from −80 ◦C and a 10 fold dilution series (from 1 in

0–1 in 10,000) was also prepared in DMEM and 10% FBS. Dilutions in 1000 and 1 in 10,000 of this positive control dilution seriesere inoculated in triplicate at the top and bottom of each 96 welllate used. Plates were incubated at 37 ◦C with 5% CO2. After a 48 hhe number of GFP cells per well were counted using fluorescent

icroscopy and the titre calculated.

.3. Excipient screening

.3.1. ScreeningA DOE screening design was adopted to rapidly assess which

andidate novel excipients would maintain the highest infectiv-ty of Ad5 over long term storage. MODDE 9.0 (Umetrics) wassed to generate a Doehlert experimental design. Doehlert designsre response surface modelling designs constructed from regu-ar simplexes. They are easily extendable in different directionsnd new factors can be added to an existing design. Unlike reg-lar formulation designs, non-significant factors can be eliminatedrom the analysis and so do not become a confounding fac-or. Furthermore, different factors within the design are testedt a different number of levels, so it is possible to allocateore test levels to factors that we suspect are of greater impor-

ance.

.4. Excipient optimization

Optimized formulations in the solid and liquid state were cre-ted using further iterations of the method outlined in 2.3.

Prior to use, formulations were sterilized by a 0.22-mm filtra-ion. 50 �l aliquots of Ad5 virus were added to 2 ml glass vials. Toach vial 250 �l of an optimized excipient blend, control or Merck195 Gold Standard [3] was admixed. Formulations are described

n the appendix.Vials for liquid setting were stoppered and crimped before being

laced at +37 ◦C for one week of thermochallenge.Vials for lyophilization had rubber bungs partially inserted, and

ere loaded onto a Virtis advantage freeze-dryer and lyophilized.fter lyophilization samples were immediately capped underacuum, removed, crimped and placed at 37 ◦C for 1 week of ther-ochallenge.

2 (2014) 2931–2938 2933

2.5. Long term studies

2.5.1. Long term studyThe lead optimized formulations, were taken into a long term

study. Vials were set up for 2 years testing, with time points at1 week, 2 week, 6 weeks, 3 months, 6 months, 9 months, 1 year,18 months and 2 years. Additional vials of the formulations wereset up to allow for post lyophilization testing and repeats. Freeze-dried samples were reconstituted in 300 �l SSC immediately priorto assay.

2.6. Statistical analysis

A one way Analysis of Variance (ANOVA) test followed by a Bon-ferroni post test was performed to analyze significance betweendifferent excipients using PRISM Graphpad software version 4.00.The p value summaries are *p < 0.05; **p < 0.01; ***p < 0.001.

3. Results

3.1. Identification of optimal excipient formulations

Over 100 excipient blends were tested for efficacy (data notshown); of these the optimal formulations for both solid and liq-uid state were screened to determine which were most suitable forlong-term stability. Adenovirus 5 was used as the model systemand infectivity as the measure of viral integrity.

3.2. Temporal analysis of the effect of temperature on virusstability in the presence of target excipients

In order to ascertain the efficacy of the various excipients, Ad5was lypophilized as described in the presence of the selected for-mulations and A13, AB1 and AB9. In addition the sugar only controlspreviously outlined were included (Fig. 1). The dried productswere then exposed to a range of temperatures for varying short(1–6 weeks) and a longer 6–12 months time periods (Fig. 2). Viralintegrity was measured by infectivity. At 4 ◦C, at all timepoints upto 6 weeks, the optimized excipients were superior to the sugarsalone. This was also true for temperatures up to 25 ◦C. Of particularnote, even at extreme temperatures of 40 ◦C the loss of Ad5 infec-tivity was minimal (c. 0.3 log). Based upon these data, an extendedstudy was conducted over 12 months (Fig. 3). As the sugar combina-tions provided no recoverable viral infectivity they were excludedfrom the analysis. Whilst all three excipients provided high levelsof recoverable virus after storage at the 4 ◦C or 25 ◦C, only one ofthe tested excipient blends provided significant protection to thevirus after storage at 37 ◦C.

3.3. Comparison of A13, AB1 and AB9 with the current best drystate stabilization excipients Croyle 1 and 2

The three selected formulations A13, AB1 and AB9 (Fig. 4)were compared for efficacy versus Croyle 1 and 2 [21], which waspreviously shown to be superior for freeze dry stabilization ofadenovirus. These were tested post lyophilization, and after twomonths thermo-challenge at 4, 25 and 40 ◦C. In this setting thesucrose and raffinose control were added. Analysis of Ad5 infectiv-ity showed that sucrose (A) and Croyle 1 both showed the greatest

loss of infectivity, whilst, Croyle 2 appeared to afford similar pro-tection to the sucrose:raffinose (AB) combination. Strikingly underall test conditions excipients A13, AB1 and AB9 were shown to besignificantly superior.

2934 M. Stewart et al. / Vaccine 32 (2014) 2931–2938

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ig. 1. Effect of excipient mixes on the viability of the Ad5 construct during lyophilas measured by infectivity) complete mixes need to be used. The sugars alone or m

.4. Analysis of liquid stability

In a similar set of studies to those outlined above, we havelso explored the ability of our excipient mixtures to conserveiral infectivity in a liquid state. In this instance following initialcreening, two blends were taken forward for further study (A13nd AB1) and these were compared with sucrose alone control (A)r sucrose in conjunction with raffinose (AB) control.

In the case of liquid presentations, overall there was a somewhatower level of thermal stability. However there was good stabilityt 25 ◦C for at least 6 months (Fig. 5) whereas at 40 ◦C 1 month sta-ility was achieved (Fig. 6). When these optimally blended viral

ig. 2. The lyophilized samples were subjected to thermal challenge at a variety of temnstances where the complete excipient formulation was used and some recovery in sugp to 6 weeks, little loss of virus infectivity was seen.

n. We can clearly see that in order to maintain the maximal levels of viral integrityf the same are insufficient to maintain maximal virus recovery post lyophilization.

mixes were compared to the currently accepted best stabilizer(A195 buffer Evans et al) it was clearly demonstrated that theywere able to improve on the impressive stability seen from thisstabilizing mixture quite dramatically (Fig. 7).

4. Discussion

Previous investigations into the causes of viral inactivation upon

storage implicate surface adsorption, freeze–thaw damage, andfree-radical oxidation as the major inactivation pathways, and spe-cific formulations designed to inhibit these pathways significantlystabilize Ad5 during storage at 4 ◦C and at elevated temperatures.

peratures for different times. We observed good stability at 4 ◦C and 25 ◦C in allar mixes. However, of particular note is the fact that at the higher temperature for

M. Stewart et al. / Vaccine 32 (2014) 2931–2938 2935

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ig. 3. Longer term studies of the viral stability under thermal challenge for 6 or 1aw relatively modest losses in infectivity at 4 ◦C or 25 ◦C and one formulation demoted that after 12 months no infectivity could be recovered from any of the mixes

ptimization of the formulation composition and pH allowed thedentification of a formulation (A195) with an estimated t1/2 (time

or 50% loss of infectivity) of 7 years at 4 ◦C. Moreover, Ad5 formu-ated in A195 has a t1/2 of 13 days at 37 ◦C and is stable throughreeze–thaw cycling. Our results have dramatically improved uponhese earlier findings providing a t1/2 of approximately 93 days at

ig. 4. The final study in this series included a 2 month comparison of the current best lyo

xtent as Sucrose Raffinose mix) and those stabilizers developed at Stabilitech. In theseirus can be seen when stabilizers developed by Croyle et al. are used which is in stark co

nths. In these studies the long term effects of thermal challenge were studies. Weted high levels of recovered virus after storage at 37 ◦C for 12 months. It should beining only sugars or sugar mixes.

40 ◦C in a lyophilized form, and a t1/2 in a liquid format of approxi-mately the same duration but at the elevated temperature of 40 ◦C.

Preservation of viral titer is an important consideration for sev-eral medical and research fields. The method presented in thisshort communication offers a simple, cost-effective alternativemeans of long-term preservation for many non-enveloped complex

stabilizer as developed by Croyle et al. (which appeared to offer stability to a similar examples we can clearly see that after 2 months challenge at 37 ◦C no detectablentrast to the infectivity observed when Stabilitech stabilizers are used.

2936 M. Stewart et al. / Vaccine 32 (2014) 2931–2938

Fig. 5. Stabilizing effect of Stabilitech formulations on infectious Adenovirus type 5 when maintained in a liquid setting. We can clearly see that the Stabilitech formulationsoffer significant stabilizing effects upon the virus even at 25 ◦C for up to 6 months.

M. Stewart et al. / Vaccine 32 (2014) 2931–2938 2937

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ig. 6. Higher temperature stability of virus in a liquid state. When the preservatioigher temperatures (40 ◦C) we still observed a surprisingly high level of recoverab

iological molecules. It also has the added benefit of relieving theurden of maintaining cryostorage for valuable viral stocks. Indeed,he method could theoretically be applied to the transport of viralaccines (e.g. Poliovirus) to areas where the maintenance of a coldhain poses particular problems.

Live viruses, as complex biological macromolecular assemblies,re susceptible to inactivation during storage and handling. Theirnherent labile nature usually requires that they be stored as cryo-enically frozen preparations, to limit the loss of infectivity duringanufacturing, storage, and administration. However, if formula-

ions could be devised which can afford very high levels of stabilityt elevated temperatures for extended periods of time, with min-mal loss during manufacture and which offer enhanced stabilityt refrigeration temperatures for extended times post reconstitu-ion, there would be enormous cost, time and complexity savings.n terms of cost, much less vaccine would be wasted and ease of

dministration of the liquid vaccine would save time of thawingnd maintaining on ice, and complexity of the storage would beignificantly simplified by removing the need for such stringentold chain maintenance and access to cryogenic freezers.

ig. 7. The final study in the series involved a head to head study with the leading liquid suffer as developed by Merck Ltd. In this instance we could clearly see that the Stabilitec

fectivity of the virus in a liquid state by Stabilitech formulations was looked at, ats.

Significant progress in identifying factors that affect Ad5 sta-bility and have designed both liquid and lyophilized formulations[3,21,22] with enhanced storage stability but in the best cases onlylonger term storage at 4 ◦C was achievable. Similarly, Blanche et al.[23] developed the liquid Ad5 formulation and reported their Ad5was stable for >18 months but once again this is only at 4 ◦C.

Evans et al. [3] reported from their work that Ad5 formulatedin their proprietary A195 buffer has a projected half-life of almost7 years at 4 ◦C (approximately 0.1 logs of infectivity lost after 24months). Ad5 in A195 is also stable (<5% loss of infectivity) for 1week at 30 ◦C and for 1 day at 37 ◦C, showing that it has at leastsome stability when out of the cold chain.

In summary, we have conducted a systematic investigationusing Design of Experiment approaches to initially screen, andthen to optimize mixes of inert excipients into final formulationsthat positively affect adenovirus storage stability. Using the same

individual components, we have developed optimized liquid for-mulations and solid formulations which when compared to the“gold standard” preparations for those formats shows very clearlythat our formulations are far superior to any currently available

tate stabilizer in the form of a buffer made to the specifications set out for the A195h formulation was able to significantly enhance the liquid stability of the virus.

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938 M. Stewart et al. / Vac

ormulation for the stabilization of Ad5. Due to the use of the sameonstituents in the formulation of both liquid and solid, the only dif-erences being their concentrations, it may be possible to engineern novel type of vaccine, one which is transported as a classicalry lyophilized cake which is extremely stable and easily able toithstand the commonly seen variations in the vaccine distributionetworks, and upon arrival at its final destination can be recon-tituted and take on a new presentation as a liquid vaccine withnhanced stability at 4–8 ◦C (or ambient e.g. 25 ◦C).

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