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`us 20120091026Al
`
`c19) United States
`c12J Patent Application Publication
`Chacornac et al.
`
`c10J Pub. No.: US 2012/0091026 Al
`Apr. 19, 2012
`(43) Pub. Date:
`
`(54) METHOD OF STORI~G A VACCINE
`CO'IJTAINING AN ALUMINUM ADJUVANT
`
`(75)
`
`Inventors:
`
`Isabelle Chacornac, Tupin Et
`Semons (FR); Nabila
`Ikhclcf-Gribi, Francheville (FR);
`Frederic Ronzon, Montromant
`(FR); Julien Tirefort, Lyon (FR):
`Sandrine Lentsch Graf Sainte Foy
`Les Lyon (FR)
`
`(73) Assignee:
`
`SANOFI PASTEUR, Lyon cedex
`(FR)
`
`(21) Appl. No ..
`
`13/274,714
`
`(22) Filed:
`
`Oct. 17, 2011
`
`Related U.S. Application Data
`
`(60) Provisional application No. 61/454,248, filed on Mar.
`18, 2011.
`
`(30)
`
`Foreign Application Priority Data
`
`(FR) ....................................... 1058464
`Oct. 18, 2010
`Publication Classification
`
`(51)
`
`Int.Cl.
`B65D 85/00
`(2006.01)
`B65B 7128
`(2006.01)
`(52) U.S. Cl . ........................................ 206/524.3; 53/471
`ABSTR,\CT
`(57)
`
`The invention relates to a method for loading and storing a
`vaccine composition, containing the antigen adsorbed on the
`aluminum adjuvant which (a) comprises (i) loading the com(cid:173)
`position into a container; and (ii) closing the container with a
`device in particular acting as a stopper, the surface of the
`device getting into contact with the composition being coated
`with a fluoropolymcr such as Teflon™; and/or (b) loading the
`composition into a container wherein the inner surface of
`which is coated with polymerized silicone. The use of fluo(cid:173)
`ropolymer or polymerized silicone optimizes the adsorbed
`antigen stability upon storage. ln a particular embodiment,
`the antigen is the hepatitis B surface antigen and the alumi(cid:173)
`num adjuvant is aluminum oxy hydroxide.
`
`Regeneron Exhibit 1014.001
`
`

`

`US 2012/0091026 Al
`
`Apr. 19, 2012
`
`METHOD OF STORING A VACCINE
`CONTAINING AN ALUMINU.\'I ADJUVA~T
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[UUUlJ This application claims the benefit of U.S. provi(cid:173)
`sional application 61/454,248, filed Mar. 18, 2011, and
`French patent application no. 10 58464, filed Oct. 18, 2011.
`The entire contents ofboth of these applications are incorpo(cid:173)
`rated by reference.
`BACKGROUND or nm INVENTION
`1. Pield of the Invention
`[0002]
`[0003] The present invention relates to a method for reduc(cid:173)
`ing and/or decelerating the desorption of an antigen that has
`been adsorbed on an aluminum adjuvant as well as to the
`product thereof-namely the combination of a vaccine com(cid:173)
`position comprising the antigen adsorbed on the aluminum
`adjuvant and a container for the composition, said container
`having particular characteristics.
`[0004] 2. Summary of the Related Art
`[0005] A large number of antigens are able to adsorb on an
`aluminum adjuvant in particular at neutral pH or at a pH close
`to neutrality, which is the pH naturally required for compo(cid:173)
`sitions that have to be administered to mammals, including
`humans.
`[0006] Provided that the amount of adjuvant is such that the
`antigen can actually adsorb on the adjuvant in an optimum
`amount when the two compounds are mixed together, the
`maximum degree of adsorption is very frequently achieved.
`However, over time, depending on the environmental condi(cid:173)
`tions, the percentage of adsorbed antigen (adsorption rate)
`may decrease, and this desorption may constitute an instabil(cid:173)
`ity factor.
`[0007] Known environmental conditions that can affect the
`percentage of adsorbed antigen ( adsorption rate) include, for
`example, variations in pH ( even slight variations), and the
`ac.lc.li lion of one or more medium component( s) or one or more
`additional antigen(s) that may compete with the first antigen
`for the adsorption sites on the adjuvant.
`[0008] Conventionally, a ready-to-use multi-dose vaccine
`composition is loaded into vials, e.g., glass vials closed with
`a plastic stopper. Similarly, a single dose of a vaccine com(cid:173)
`position may be loaded in a mono-dose vial or a ready-for(cid:173)
`inj ection syringe consisting, in a standard manner, ofa reser(cid:173)
`voir containing the vaccine, a plunger that closes the reservoir
`al its distal encl, and a device for ac.lminislralion, such as a
`needle attached at its proximal end. According to an alterna(cid:173)
`tive standard filling mode. the vaccine dose may also be
`loaded in a needleless syringe to which the practitioner adds
`a separately packaged needle at the time of the injection. The
`reservoir of the syringe is generally made of either glass or
`plastic and the plm1ger or the stopper is simply mac.le of
`plastic, such as a chlorobutyl or bromobutyl polymer, without
`particular lamination. Standard glass or plastic syringes are
`sold, for example, by Becton-Dickinson; Gcrrcshcimcr AG,
`Schott A G, Germany; Nuova Ompi srl, Italy; and West
`Pharma/Daykio. In order to facilitate sliding, the plunger or
`stopper may have been immersed in a silicone-in-waler emul(cid:173)
`sion so that a silicone film is formed at its surface. Standard
`plungers/stoppers are sold by Helvoet, Stelmi and West
`Pharma, for cxaniplc; some of them already being sold coated
`with a silicone film (ref. 82 from West Pharma).
`
`[0009] Whatever the container used for storage and the
`device used for closing the container, ac.lminislralion al the
`time of injection consists of using a syringe and sliding the
`plunger or the stopper/plunger combination so that the vac(cid:173)
`cine is delivered.
`
`SUMMARY OF THE INVENTION
`
`[001 OJ We have now found that lhemalerial oflhe container
`itself as well as that of the device for closing the container can
`affect the adsorption rate.
`[UUllJ We observed that a vaccine composition containing
`the hepatitis B surface antigen (HBsAg) adjuvanted with an
`aluminum ac.ljuvanl anc.l loac.lec.l as a single close in standard
`syringe closed with a standard stopper underwent different
`adsorption rates when stored under identical conditions and
`for the same period of time ( a few days to several months)
`depending on whether the syringe was stored in the vertical or
`horizontal position. The same phenomenon has also been
`seen with vials. The practical consequence of the different
`storage positions was that the vaccine contained in the reser(cid:173)
`voirs stored vertically was not in contact with the stopper,
`whereas there was contact between the composition and the
`stopper in the horizontally stored reservoirs.
`[0012] After a certain period of time, the level of adsorption
`of HBsAg was measured, and we observed that the level of
`adsorption was much lower for the HBsAg contained in the
`horizontally-stored reservoirs. This indicated to us that the
`material of the standard stopper (chlorobutyl or bromobutyl
`polymer) was responsible for the adsorption decrease.
`[0013] The solution to this problem is either to prevent
`contact between the adjuvanted vaccine composition and a
`stopper made of a material that contributes to decreased
`adsorption orto use a stopper made of a material that does not
`contribute lo decreased adsorption. In one embodiment, the
`invention comprises a method and apparatus using a stopper
`coated with a film of a fluoropolymer, such as Teflon™ or a
`Teflon™-like substance (such as Omniflex™ from Helvoet
`Pharma or PluorotecT" from West Pharma), which decrease
`or eliminate desorption caused by the stopper.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0014]
`In one aspect, the invention comprises a first method
`for reducing and/or slowing down the desorption of an anti(cid:173)
`gen initially adsorbed on an aluminum ac.ljuvanl during stor(cid:173)
`age, the method comprising (i) loading a container with a
`vaccine composition containing the antigen initially adsorbed
`on the aluminum adjuvant: and (ii) closing the container with
`a device acting as a stopper, the surface of the device con(cid:173)
`tactable with the composition being coated with a fluoropoly(cid:173)
`mer.
`[0015]
`In other words, the invention relates to a first method
`for filling and storing a composition containing the antigen
`adsorbed on the aluminum adjuvant which comprises (i) fill(cid:173)
`ing a container with the vaccine composition; and (ii) closing
`the container with a device acting as a stopper, the surface of
`the device contactable with the composition being coated
`with a fluoropolymer.
`[0016]
`In a similar manner, the invention also relates to the
`use of a device acting as a stopper for closing a container
`containing a vaccine composition comprising the antigen
`adsorbed on an aluminum adjuvant, the surface of the device
`contactable with the composition being coated with a fluo(cid:173)
`ropolymer.
`
`Regeneron Exhibit 1014.002
`
`

`

`US 2012/0091026 Al
`
`Apr. 19, 2012
`
`2
`
`[0017] The container may be, for example, a vial or the
`reservoir of a syringe. This also applies lo all lhe other aspects
`of the invention described hereinafter.
`[ UU18J The vaccine composition may be liquid or solid, e.g.
`lyophilized. A lyophilized composition may have the appear(cid:173)
`ance of a powder. At the time of injection to a patient, the
`lyophilized composition is reconstituted with an appropriate
`pharmaceutical solution. This also applies to all the other
`aspects of the invention described hereinafter.
`[0019] According to conventional practice in the art and for
`the purposes herein, it is understood that "antigen adsorbed"
`or "initially adsorbed" is not intended to mean that 100% of
`the antigen amollllt is actually adsorbed. These terms simply
`mean that a substantial amount of antigen is adsorbed. This
`also applies to all the other aspects of the invention described
`hereinafter.
`[0020] As mentioned above, the device may be a plastic
`device made out of, for example, a chlorobutyl or bromobutyl
`polymer. This standard device is treated with a fluoropoly(cid:173)
`mer; in particular, it may be submitted to a laminar flow
`treatment with a fluoropolymer, this laminar flow treatment
`being carried out on the entire device or, at the very least, on
`the surface of the device contactable with the composition.
`The laminar flow treatment makes itpossibleto deposit a very
`thin layer (e.g., Glm) of the 1luorupolymer. As will be appre(cid:173)
`ciated, the area of the coated surface may exceed the surface
`contactable with the composition. Indeed, in one embodi(cid:173)
`ment, the whole surface of the device is coated with the
`fluoropolymer.
`[0021] For use in the present invention, the fluoropolymer
`may be, for example, polytetrafluoroethylene (PTFE), poly(cid:173)
`tctrafluoropropy lcne (PTFP ), fluorinated ethy lcnc propy lcnc
`(PDP, a copolymer ofhexafluoropropylene and tetrafluoroet(cid:173)
`hylene ), polychlorotrifluoroethylene (PCTFE), perfluoro(cid:173)
`alkoxy co-polymer (PFA), poly(ethylene-co-tetrafluoroeth(cid:173)
`y Jene)
`(ETFE),
`poly( ethy lenechlorotrifluoroethy Jene)
`(ECTFE), polyvinyl fluoride (PVF) or polyvinylidene fluo(cid:173)
`ride (PVPF).
`[0022] The method/use disclosed herein makes il possible
`to reduce the desorption speed of the antigen adsorbed on the
`aluminum adjuvant and/or the desorption percentage ( or des(cid:173)
`orption rate) after a defined storage time at a given tempera(cid:173)
`ture. The desorption rate may be expressed as follows:
`(ammmt of non-adsorbed antigen)l(total antigen ammmt
`present in the composition). Typically, the desorption rate can
`be assessed by centrifuging the vaccine composition
`(samples at T (timeJ-O and at the end of the experiment);
`recovering the supernatants which contain the desorbed anti(cid:173)
`gen; and then quantifying the desorbed fraction by assaying
`lhe antigen in the supemalanls and in the whole vaccine using
`a suitable method chosen according to the nature of the anti(cid:173)
`gen. The desorption percentage ( or desorption rate) can vary
`from one antigen to another according to the strength/weak(cid:173)
`ness of the antigen-adjuvant interaction. Nevertheless, it is
`considered that the desorption percentage ( or desorption rate)
`can be reduced by 10 lo 15 or 20% compared with a standard
`loading method using standard stoppers-said reduction
`being measured 1 or 2 months after the date of loading.
`During this period of time, the storage is carried out at a
`temperature of +5 to 25° C. As may be easily appreciated, the
`adsorption percentage (adsorption rate) may be easily
`deduced from the desorption percentage (or desorplionrale).
`[0023] When the device is used not only to close the con(cid:173)
`tainer but also to deliver the composition contained in the
`
`container, such as by sliding the plunger of a syringe, it is
`recommended to siliconize the inner surface of the container.
`[0024] However, it has been observed that silicone may in
`some cases be detrimental to adsorption. Indeed, the desorp(cid:173)
`tion rate observed in compositions stored in syringes conven(cid:173)
`tionally siliconized by mere surface-treatment with a sili(cid:173)
`cone-in-water emulsion may be higher than the desorption
`rate observed in compositions stored in non-siliconized con(cid:173)
`tainers. We postulate that although the silicone adheres to the
`inner surface of the container, it remains in free form and,
`upon shaking or stirring, can flow away from the inner surface
`and pass into the container's content (the vaccine composi(cid:173)
`tion).
`[0025] We have now found that this latter problem can be
`solved by using a container wherein the inner surfaces are
`coated with polymerized silicone. Such a container can be
`obtained by treating the inner surface of the container with a
`silicone-in-water emulsion, followed by heating the con(cid:173)
`tainer, for example at a temperamre of 270 to 330° C. for 30
`min. Upon heating, the silicone polymerizes on the inner
`surface of the container and is therefore no longer capable of
`mixing with the composition. Polymerizing the silicone
`makes it possible to reduce the surface energy of the silicone
`to which the vaccine composition may be sensitive.
`[0026] Additionally, the siliconizing operation comprising
`a polymerization step (i) is more precise and more homoge(cid:173)
`neous that a simple standard siliconizing operation; mid (ii)
`makes it possible to reduce the amount of silicone that is used
`(that is, loaded on the inner surface of the container) by about
`a factor of 10 without any loss of lubricating effect. For
`example, according to a standard siliconizing process, from
`400 to 1000 µg of silicone are deposited in a syringe intended
`to contain doses of 0.5-1 ml (the total inner surface of the
`0.5-1 ml syringe reservoir is about 8 cm2
`; in this example this
`surface corresponds to an amollllt of silicone of from about 50
`to 125 µg/cm 2
`), whereas from 40 to 100 ~tg of silicone are
`sufficient for the smne syringe ( about 5 to 12 µg/cm 2
`) if
`silicone is deposited on the inner surfaces of the container and
`then polymerized, for example by heating. T11e fact that the
`inner surface of the syringe is coated with a low amount of
`polymerized silicone in a more homogenous manner than
`with a low amollllt of free silicone allows non-siliconized
`plungers to slide smoothly, whereas such plungers are inop(cid:173)
`erative with syringes coated with low amount of free silicone.
`[0027] This is the reason why the invention also comprises
`a second method for reducing and/or slowing down the des(cid:173)
`orption of an antigen adsorbed on an aluminum adjuvant, the
`method comprising filling a container with a vaccine compo(cid:173)
`sition comprising the antigen adsorbed on the aluminum
`adjuvant, wherein the inner surface of the container is coated
`with polymerized silicone.
`[0028]
`In other words, the invention also comprises a sec(cid:173)
`ond method for loading mid storing a vaccine composition
`containing an antigen adsorbed on the aluminum adjuvant,
`the method comprising filling a container with the composi(cid:173)
`tion wherein the inner surface of the container is coated with
`polymerized silicone.
`[0029]
`Ina similar manner, the invention also comprises the
`use of a container having an inner surface coated with poly(cid:173)
`merized silicone for storing a vaccine composition compris(cid:173)
`ing an antigen adsorbed on an aluminum adjuvant.
`
`Regeneron Exhibit 1014.003
`
`

`

`US 2012/0091026 Al
`
`Apr. 19, 2012
`
`3
`
`[0030] Advantageously, the container lmving im1er sur(cid:173)
`faces coated with polymerized silicone is made of plastic or
`glass. Advantageously, the container is the reservoir of a
`syringe.
`[0031] The amount of polymerized silicone coated on the
`inner surface of the container is from 3 to 25 µg/cm 2
`; advan(cid:173)
`tageously from 5 to 20 µg/cm 2
`; preferably from 5 to 15
`µg/cni2.
`[0032] As may be easily understood, the container used in
`the first methods of the invention may be advantageously
`coated with polymerized silicone as described above.
`[0033]
`In another aspect, the invention comprises:
`[0034] A-A container (i) which contains a vaccine com(cid:173)
`position comprising an antigen adsorbed on an aluminum
`adjuvant; and (ii) which is closed by a device acting as a
`stopper, wherein the surface of the device contactable with
`the composition is coated with a fluoropolymer;
`[0035] B-A container (i) having inner surfaces coated
`with polymerized silicone; and (ii) containing a vaccine com(cid:173)
`position comprising an antigen adsorbed on an aluminum
`adjuvant; and
`[0036] C-A container (i) having im1er surfaces coated
`with polymerized silicone; (ii) containing a vaccine compo(cid:173)
`sition comprising an antigen adsorbed on an aluminum adju(cid:173)
`vant; and (iii) which is closed by a device acting as a stopper,
`wherein the surface of the device contactable with the com(cid:173)
`position is coated with a fluoropolymer.
`[0037]
`In other words, the invention relates lo a vaccine
`composition comprising an antigen adsorbed on an aluminum
`adjuvant which is loaded into and stored in a container (i)
`having the inner surface coated with polymerized silicone;
`and/or (ii) which is closed by a device acting as a stopper,
`wherein at least the surface of the device contactable with the
`composition is coated with a fluoropolymer.
`[0038] Vaccine compositions stored in containers accord(cid:173)
`ing lo the invention include:
`[0039] A vaccine composition comprising an antigen,
`wherein the minimal antigen amount required for
`intended use ( e.g., as a dose for administration to a
`human) is adsorbed on an aluminum adjuvant;
`[0040] A vaccine composition comprising an antigen
`adsorbed on an aluminum adjuvant, wherein the vaccine
`composition when loaded in a container for use in the
`present invention, exhibits an adsorption percentage of
`at least:
`[0041]
`(a) 65-70% of the total antigen amount present
`in the composition, when, immediately after loading,
`the composition is stored al (i) 5:t3° C. for 2-3 years;
`or (ii) 25:t3° C. for 2-3 months; or
`[0042]
`(b) 80-90%, ofthe total antigen amount present
`in the composition, when, inmlediately after loading,
`the composition is stored at 5:t3° C. for 18 months;
`and
`[0043] A vaccine composition comprising an aluminum
`adjuvant and an antigen able to adsorb onto the alumi(cid:173)
`num adjuvant, wherein the percentage of the antigen
`adsorbed on the aluminum adjuvant is at least 5 or 10%
`higher than the percentage observed when the same
`vaccine composition is contained in a standard container
`having uncoated inner surfaces or inner surfaces coated
`withnon-polymcrizcd silicone, the comparison between
`the antigen adsorption percentages being carried out
`
`after storage of the vaccine-containing containers at 25°
`C. for 2 months, starling from the date of filling the
`containers.
`[0044] For the purposes of the present invention, the con(cid:173)
`tainer may be any type of reservoir, such as vials or syringes,
`and may contain multiple doses (multidose container) or a
`single dose (single-dose container). As an example, the con(cid:173)
`tainer may be a syringe or a part of a syringe comprising the
`reservoir containing the vaccine closed by a device acting as
`a stopper and as a system for releasing the vaccine at the time
`ofadministration (e.g., using a phmger). The device acting as
`a stopper may be a plunger.
`[0045] Stoppers and/or plungers for use in the present
`invention are sold. for example, by Helvoet Pharma (Omni(cid:173)
`flcx™ technology) and by West Pharma (Fluorotcc™ tech(cid:173)
`nology). Glass syringe reservoirs coated with polymerized
`silicone for use in the present invention are sold, for example,
`by Nuova Ompi srl, Becton-Dickinson and Gerresheimer
`(Baked-on technology).
`[0046] The antigen and the aluminum adjuvanl may be any
`antigen and any aluminum adjuvant provided, of course, that
`they are both capable of interacting with each other. Further(cid:173)
`more, it may easily be understood that the present invention 1s
`of particular interest for an antigen-aluminum adjuvant pair
`having a relatively weak interaction force; the interaction
`force possibly depending on the environment. This interac(cid:173)
`tion force can be assessed according to a variety of tests. For
`example, an aluminum adjuvant may be used to fonn various
`antigen-adjuvant pairs ( the antigen varies from one pair to the
`other, the adjuvant remaining the same). Then a large amount
`of a compound able lo compete with the antigen for the
`interaction with the adjuvant is added. The various prepara(cid:173)
`tions are centrifuged and the supematants recovered. Finally,
`the amount of antigen des orbed is assayed in the supcrnatants,
`and as a result antigens may be compared for their interaction
`force with the adjuvant.
`[0047] A relatively weak interaction force is an interaction
`force that leads to an adsorption that may be detrimentally
`a1Tected by a standard filling with the composition containing
`the antigen-aluminum adjuvant complex. Various elements
`involved in the manufacture of a container, such as, for
`example, latex, antioxidants, silicone and metal ions (e.g.,
`zinc and tungsten), can destabilize the antigen-adjuvant com(cid:173)
`plex.
`[0048] By "adsorption" it is generally meant any phenom(cid:173)
`enon aimed at fanning an antigen-adjuvant complex involv(cid:173)
`ing i.a. electrostatic interaction forces, hydrophobic interac(cid:173)
`tions or ligand exchange. Thus, the antigen may be attached at
`the surface of the network of the aluminum adjuvant or
`embedded inside after co-precipitation with the aluminum
`adjuvant.
`[0049] For use in the present invention, an aluminum adju(cid:173)
`vant may be aluminum oxy hydroxide (AlOOII), such as the
`product sold by BrenntagAG (Superfos) or Reheis Corp.; and
`aluminum hydroxy phosphate (AlOHP04 ), such as the prod(cid:173)
`uct sold by Alphas.
`[0050] For a vaccine composition to be effective, the mini(cid:173)
`mal antigen amount required for adsorption onto the alumi(cid:173)
`num adjuvant essentially depends upon the antigen itself, and
`is readily determinable by those of ordinary skill in the art.
`[0051]
`In a particular embodiment, the antigen can be the
`hepatitis B surface antigen (HBsAgJ. It is particularly advan(cid:173)
`tageous to adsorb HRsAg onto Al OOH as HRsAg exhibits an
`
`Regeneron Exhibit 1014.004
`
`

`

`US 2012/0091026 Al
`
`Apr. 19, 2012
`
`4
`
`iso electric point (IEP) less than 7 (about 4 to 5) and AlOOH
`exhibils a poinl of zero charge (PZC) grealer than 7 (aboul 9
`to 11 ).
`[0052] The vaccine composition for use in the present
`invention can contain one or more antigcn(s), at least one of
`them being adsorbed on the aluminum adjuvant and it being
`possible for the others to be adsorbed as well or not.
`[0053] According to one embodiment, the composition for
`use in the present invention comprises HBsAg adsorbed on
`AIOOH (AIOOH-HbsAg complex) and a second m1tigen,
`which is polyribosylribitol phosphate (PRP) of Haemophilus
`influenzae (HiR valence), preferably in a fom1 conjugated to
`a carrier protein (C) which may be i.a. Dt or Tt.
`[0054] According to another particular embodiment, when
`the mitigen adsorbed on the aluminum adjuvant is HBsAg, the
`vaccine composition may also contain, as additional antigens,
`one or more of the following: diphtheria toxoid (Dt) (diph(cid:173)
`theria valence); tetanus toxoid (Tt) (tetanus valence); !1orde(cid:173)
`tella pertussis detoxified loxin (Ptdx), fonbriae, filamentous
`haemagglutinin (FHA) and/orpenactin (69 kD antigen) (per(cid:173)
`tussis valence); inactivated poliovirus serotype 1, 2, or 3
`(polio valence); and polyribosylribitol phosphate (PRPJ of
`Haemophilus influenzae (IIil3 valence), preferably in a form
`conjugated to a carrier protein (C) which may be i.a. Dt orTt.
`[0055] As a maller of example, the composilion may com(cid:173)
`prise HBsAg, Dt, Tt, Pt and FHA adsorbed on AIOOH (the
`AIOOH-HbsAg-Dt-Tt-Pt-FHA complex), the polio valence,
`and PRP-C substantially non-adsorbed on AIOOH.
`[0056]
`ln a general manner, the invention is also particu(cid:173)
`larly advantageous when the vaccine composition comprises
`several valences. for example 2, 3, 4, 5, 6 or more, each
`represented by one or more antigens (2, 3, 4 or 5), several
`antigens being adsorbed on the aluminum adjuvant. Indeed,
`under the standard filling mode, the higher the number of
`antigens/valences adsorbed on the aluminum adjuvant, the
`more crilical is lhe phenomenon of deslabilization by the
`container. The antigen-adjuvant interaction force often dif(cid:173)
`fers from one antigen to another and, in a composition con(cid:173)
`taining several antigens, the antigen with the weakest inter(cid:173)
`action force exhibits the highest tendency to desorb under
`adverse conditions.
`
`Examples and Experimenlal Resulls
`
`[0057] A-A bulk of a vaccine composition containing the
`hepatitis B surface antigen, diphtheria toxoid, tetanus toxoid,
`and pertussis valence, each adsorbed on aluminum oxy
`hydroxyde, as well as the polio andnon-adsorbedHaemophi(cid:173)
`lus influenzae B (HiB) valences was distributed into three
`categories of single-dose syringes, the characteristics of
`which were as follows:
`[0058]
`(1) standard siliconized glass syringes with stmi(cid:173)
`dard stopper/plunger made of non-laminated plastic;
`[0059]
`(2) standard siliconized glass syringes with fluo(cid:173)
`ropolymer-coated stopper/plunger (West Pharma; fluo(cid:173)
`rotechT"' technology); and
`[0060]
`(3) syringes, the im1er surface of which is coated
`with polymerized silicone (Baked-on syringe system
`lucrconc™ from Gcrrcshcimcr) with fluoropolymcr(cid:173)
`coated stopper/plunger (West Pharma; fluorotechrn
`technology).
`[0061] The bulk was distributed in 0.5 ml single doses,
`each dose containing 10 µg of HBsAg, 30 Lf of Dt. 10 Lf of
`Tt, 25 µg of Pt, 25 ~tg of FHA, 40 DU (Antigen D Unit) of
`lPVI, 8 DU of1PV2, 32 DU of1PV3, 12 µg ofPRP (in PRP-Tt
`
`conjugate form), 0.6 mg of Al, 55 mM of phosphate ions, 20
`mM of carbonale ions, and Tris sucrose buffer, 2.5 mM,
`2.125%, at pH 6.8-7.2.
`[0062] All the syringes of the three categories were stored
`horizontally at 25° C. for two months (accelerated ageing).
`The HBsAg desorption was measured in each of the three
`categories at T=O (just after loading the syringes) and then
`after two months.
`[0063] Desorption was evaluated by centrifuging the con(cid:173)
`tent of the syringes mid then measuring the amount of des(cid:173)
`orbed HBsAg present in the supernatant by ELISA ( sandwich
`ELISA, involving a mouse m1ti-HBsAg monoclonal antibody
`(lgM) for coating and capture, a second mouse anti-HBsAg
`monoclonal antibody (IgG) and a third anti-mouse IgG poly(cid:173)
`clonal m1tibody coupled to peroxydase (Sigma, Ref. A3673)
`which is revealed by adding tetrmnethyl benzidine ).
`[0064] At T (time )=O, the HbsAg adsorption level was iden(cid:173)
`tical in the three categories (98% of the total HRsAg was
`adsorbed). At T-2 months, desorption was observed in all the
`categories, but the desorption percentage differed depending
`upon the category. The highest desorption percentage was
`found in category (I) (At T-J and 2 months, 55 and 50% of
`the total HBsAg was adsorbed, respectively), whereas the
`lowest percentage was found in category (3) (At T=l and 2
`months, 72 and 69% of the Iola! HBsAg was adsorbed,
`respectively).
`[0065] B-A bulk ofthe vaccine composition described in
`A-was distributed into two categories of single-dose 1 mL
`syringes, the characteristics of which were as follows:
`[ 0066 J
`(I) standard siliconized glass syringes (free sili(cid:173)
`cone); and
`[0067]
`(2) non-siliconizcd syringes.
`[0068] The bulk was distributed in 0.5 ml single doses,
`each dose containing 10 µg of HBsAg. 30 Lf of Dt, 10 lf of
`Tt, 25 µg of Pt, 25 µg of FHA, 40 DU (Antigen D Unit) of
`ll'Vl, 8 DU ofll'V2, 32 DU ofll'V3, 12 µgofl'Rl' (inl'Rl'-Tt
`conjugate form), 0.6 mg of Al, 55 mM of phosphate ions, 20
`mM of carbonale ions, and Tris sucrose buffer, 2.5 mM,
`2.125%, at pH 6.8-7.2.
`[0069] All the syringes of the two categories were stored
`vertically al 25° C. for two months (accelernted ageing). The
`HBsAg desorption was measured in each of the two catego(cid:173)
`ries at T=O Gust after filling syringes) and then after two
`months, as described in A-above.
`[0070] At T (time )=O, the HbsAg adsorption level was iden(cid:173)
`tical in the two categories (98% of the total HBsAg was
`adsorbed). At T=2 months, desorption was observed in all the
`categories, but the desorption percentage diffored depending
`upon the category. The highest desorption percentage was
`found in category (1) (At T=l and 2 months, 69 and 68% of
`the total HBsAg was adsorbed, respectively), whereas the
`lowest desorption percentage was found in category (2) (At
`T=l and 2 months, 73% of the total HBsAg was adsorbed).
`This clearly indicates that the m1tigen adsorption onto an
`aluminum adjuvant is sensitive to free silicone.
`[0071] C-A bulk of the vaccine composition described in
`A-was distributed into three types of single-dose 1 mL
`syringes, the characteristics of which were as follows:
`(1) low sihcomzed glass synngcs (50-100 ,ug free
`[ 0072J
`silicone/syringe);
`[0073]
`(2) standard highly siliconized glass syringes
`((RTF syringe lucrconc™ from Gerrcshcimcr): 800 µg
`to I mg free silicone/syringe); and
`
`Regeneron Exhibit 1014.005
`
`

`

`US 2012/0091026 Al
`
`Apr. 19, 2012
`
`5
`
`[0074]
`(3) syringes having im1er surface coated with
`polymeri,:ed silicone (50-100 ,LLg/syringe).
`[0075] Syringes of category (I) are operative only if the
`plunger used for injection is also siliconized, because the
`amount of silicone coating the inner surface of the syringe is
`too low to allow sliding on its own.
`[0076] All the syringes (types 1 to 3) were closed with the
`same type ofnon-siliconized stopper.
`[0077] The bulk vaccine was distributed in 0.5 mL single
`doses, each dose being as described in A and B.
`[0078] All the syringes of the three types were stored ver(cid:173)
`tically at 25° C. for two months (accelerated ageing). The
`HBsAg desorption was measured in each of the three types at
`T=O Gust after filling syringes) and then after two months, as
`described in A above.
`[ 0079] At T ( time )=O, the HbsAg adsorption level was iden(cid:173)
`tical in the three types (94% of the total HBsAg was
`adsorbed). At T=2 months, desorption was observed in all
`syringes, but the desorption percentage differed depending
`upon the type. The highest desorption percentage was found
`in type (2) (At T-1 and 2 months, 60 and 58% of the total
`HBsAg was adsorbed, respectively), whereas the desorption
`percentage was found similar in types ( l) and (3) and defini(cid:173)
`tively much lower than in type (2): In type (1), at T=l and 2
`months, 73% and 68% of the total HBsAg was adsorbed,
`respectively. In type (3 ), at T = 1 and 2 months, 69% and 66%
`of the total HBsAg was adsorbed, respectively.
`[0080] Again, this clearly indicates that (i) the antigen
`adsorption onto an aluminum adjuvant is sensitive to free
`silicone loaded in an amount necessary for sliding and (ii)
`polymerization of silicone allows overcoming this issue.
`[UU81J D-A stability study has been conducted at 5±3° C.
`for 18 months with the tilled syringes described inA-(3 ).At
`least 80-90% ofthe total III3sAg was still adsorbed at the end
`of the 18-month period.
`1. A method for storing a vaccine composition containing
`an antigen adsorbed on an aluminum adjuvant, the method
`comprising (i) loading the composition into a container and
`(ii) closing the contai