US 20120091026Al
`
`(19) United States
`(12;) Patent Application Publication (10) Pub. No.: US 2012/0091026 A1
`
`Chacornac et a1.
`(43) Pub. Date:
`Apr. 19, 2012
`
`(54) METHOD OF STORIN GA VACCINE
`CONTAINING AN ALUMINUM ADJUVANT
`
`(30)
`
`Foreign Application Priority Data
`
`(75)
`
`Inventors:
`
`
`Isabelle Chacornac, Tupin 3t
`Semons (FR); Nabila
`
`{gfigeilcl301%”; “ioitmmaagg
`3
`3 “ ‘9“ “e “r ., you
`Sandrine Lentsch Graf, Sainte Foy
`Les Lyon (FR)
`
`(73) Assignee:
`
`SANOFI PASTEUR, Lyon cedex
`(FR)
`
`,
`\
`(2]) Appl. No“
`\
`.
`(22,)
`Flledi
`
`13/274’714
`
`0613- 179 2011
`
`Related US. Application Data
`
`(60) Provisional application No. 61/454,248, filed on Mar.
`18, 2011~
`
`Oct. 18, 2010
`
`(FR) ....................................... 1058464
`
`Publication Classification
`
`(51)
`
`Int Cl
`
`(2006.01)
`B65B 7/28
`52 US. Cl. ........................................ 206/524.3- 53 '471
`)
`5
`I
`(
`ABSTRACT
`(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—
`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 TeflonTM; and/or (b) loading the
`composition into a container wherein the inner surface of
`which is coated with polymerized silicone. The use of fluo-
`
`ropolymer or polymerized silicone optimizes the adsorbed
`antigen stability upon storage. In a particular embodiment,
`the antigen is the hepatitis B surface antigen and the alumi-
`num adjuvant is aluminum oxy hydroxide.
`
`Regeneron Exhibit 1014.001
`
`

`

`US 2012/0091026 A1
`
`Apr. 19,2012
`
`METHOD OF STORING A VACCINE
`CONTAINING AN ALUMINUM ADJUVANT
`
`CROSS—REFERENCE TO RELATED
`APPLICATIONS
`
`[0009] Whatever the container used for storage and the
`device used for closing the container, administration at the
`time of injection consists of using a syringe and sliding the
`plunger or the stopper/plunger combination so that the vac—
`cine is delivered.
`
`[0001] This application claims the benefit of U.S. provi-
`sional application 61/454,248,
`filed Mar. 18, 201 l, and
`French patent application no. 10 58464, filed Oct. 18, 2011.
`The entire contents ofboth of these applications are incorpo-
`rated by reference.
`
`BACKGROUND Of TI IE INVENTION
`
`1. Field of the Invention
`[0002]
`[0003] The present invention relates to a method for reduc-
`ing and/or decelerating the desorption of an antigen that has
`been adsorbed on an aluminum adjuvant as well as to the
`product thereofinamely the combination of a vaccine com-
`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, inpar‘ticular at neutral pH or at a pH close
`to neutrality, which is the pH naturally required for compo-
`sitions that have to be administered to mammals, including
`humans.
`
`Provided that the amount of adjuvant is such that the
`[0006]
`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-
`tions, the percentage of adsorbed antigen (adsorption rate)
`may decrease, and this desorption may constitute an instabil-
`ity factor.
`[0007] Knoan environmental conditions that can affect the
`percentage of adsorbed antigen (adsorption rate) include, for
`example, variations in pH (even slight variations), and the
`addition ofone 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-
`position may be loaded in a mono—dose vial or a ready—for—
`injection syringe consisting, in a standard manner, of a reser-
`voir containing the vaccine, a plunger that closes the reservoir
`at its distal end, and a device for administration, such as a
`needle attached at its proximal end. According to an alterna-
`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 pleger or the stopper is simply made of
`plastic, such as a chlorobutyl or bromobutyl polymer, without
`particular lamination. Standard glass or plastic syringes are
`sold, for example, by Becton-Dickinson; GerrcsheimcrA G,
`Schott A G, Germany; Nuova Ompi srl, Italy; and West
`Pharma/Daykio. In order to facilitate sliding, the plunger or
`stopper may have been i1rm1ersed in a silicone-in—water emul-
`sion so that a silicone film is formed at its surface. Standard
`plungers/stoppers are sold by Helvoet. Stelmi and West
`Pharma, for example; some ofthem already being sold coated
`with a silicone film (ref. B2 from West Pharma).
`
`SUMMARY OF THE INVENTION
`
`[0010] We have now found that the material ofthe container
`itselfas well as that ofthe device for closing the container can
`affect the adsorption rate.
`[0011] We observed that a vaccine composition containing
`the hepatitis B surface antigen fliBsAg) adjuvanted with an
`aluminum adjuvant and loaded as a single dose 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-
`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] Aftera certain period oftime, the level ofadsorption
`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 or to use a stopper made of a material that does not
`contribute to decreased adsorption. In one embodiment, the
`invention comprises a method and apparatus using a stopper
`coated with a film of a fiuoropolymer, such as TeflonTM or a
`TeflonTMilike sub stance (such as OmnifiexTM from Helvoet
`Pharma or PluorotecTM from West Pharma), which decrease
`or eliminate desorption caused by the stopper.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`In one aspect, the invention comprises a first method
`[0014]
`for reducing and/or slowing down the desorption of an anti-
`gen initially adsorbed on an alulninum adj uvant during stor-
`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-
`tactable with the composition being coated with a fiuoropoly—
`mer.
`
`In other words. the invention relates to a first method
`[0015]
`for filling and storing a composition containing the antigen
`adsorbed on the aluminum adjuvant which comprises (i) fill-
`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 fiuoropolymer.
`[0016]
`I11 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 ofthe device
`contactable with the composition being coated with a fluo-
`ropolymer.
`
`Regeneron Exhibit 1014.002
`
`

`

`US 2012/0091026 A1
`
`Apr. 19,2012
`
`[0017] The container may be, for example, a vial or the
`reservoir ofa syringe. This also applies to all the otheraspects
`of the invention described hereinafter.
`
`
`
`[0018] The vaccine composition may be liquid or solid, e.g.
`lyophilized. A lyophilized composition may have the appear-
`ance of a powder. At the time of injection to a patient, the
`lyophilized composition is reconstituted with an appropriate
`oharmaceutical solution. This also applies to all the other
`aspects of the invention described hereinafter.
`[0019] According to conventional practice in the art and for
`he purposes herein, it is understood that “antigen adsorbed”
`or “initially adsorbed” is not intended to mean that 100% of
`he antigen amount is actually adsorbed. These terms simply
`mean that a substantial amount of antigen is adsorbed. This
`also applies to all the other aspects ofthe invention described
`iereinafter.
`[0020] As mentioned above, the device may be a plastic
`device made out of, for example, a chlorobutyl or bromobutyl
`Jolymer. This standard device is treated with a fiuoropoly-
`mer; in particular, it may be submitted to a laminar flow
`reatment with a fluoropolymer, this laminar flow treatment
`jeing carried out on the entire device or, at the very least, on
`he surface of the device contactable with the composition.
`The laminar flow treatment makes it possibleto deposit avery
`hin layer (e.g., film) of the lluoropolymer. As will be appre-
`ciated, the area of the coated surface may exceed the surface
`contactable with the composition. Indeed, in one embodi—
`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—
`etrafluoropropylenc (PTFP), fluorinated ethylene propylene
`PEP, a copolymer ofhexafluoropropylene and tetrafluoroet-
`1ylene), polychlorotrifluoroethylene (PCTFE), perfiuoro-
`alkoxy co-polymer (PFA), poly(ethylene-co-tetrafluoroeth-
`ylene)
`(ETFE),
`po1y(ethylenechlorotrifluoroethylene)
`
`ECTFE), polyvinyl fluoride (PVF) or polyvinylidene fluo—
`ride (PVPF).
`[0022] The method/use disclosed herein makes it possible
`0 reduce the desorption speed of the antigen adsorbed on the
`aluminum adjuvant and/or the desorption percentage (or des—
`orption rate) after a defined storage time at a given tempera-
`ure. The desorption rate may be expressed as follows:
`amount of non-adsorbed antigen)/(total antigen amount
`firesent in the composition). Typically, the desorption rate can
`3e assessed by centrifuging the vaccine composition
`samples at T (time)*0 and at the end of the experiment);
`recovering the supernatants which contain the desorbed anti-
`gen; and then quantifying the desorbed fraction by assaying
`the antigen in the supernatants and in the whole vaccine using
`a suitable method chosen according to the nature of the anti-
`gen. The desorption percentage (or desorption rate) can vary
`from one antigen to another according to the strength/weak—
`ness of the antigen-adjuvant interaction. Nevertheless, it is
`considered that the desorption percentage (or desorption rate)
`can be reduced by 10 to 15 or 20% compared with a standard
`loading method using standard stoppersisaid 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 desorption rate).
`[0023] When the device is used not only to close the con-
`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—
`tion rate ob served in compositions stored in syringes conven-
`tionally siliconized by mere surface-treatment with a sili-
`cone-in—water emulsion may be higher than the desorption
`rate observed in compositions stored in non-siliconized con-
`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-
`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-
`tainer, for example at a temperature 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-
`neous that a simple standard siliconizing operation; and (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 pg 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 amount of silicone offrom about 50
`to 125 ug/cmz), whereas from 40 to 100 pg of silicone are
`sufficient for the same syringe (about 5 to 12 ug/cmz) if
`silicone is deposited on the inner surfaces ofthe container and
`then polymerized, for example by heating. The fact that the
`inner surface of the syringe is coated with a low amount of
`polymerized silicone in a more homogenous maimer than
`with a low amount of free silicone allows non—siliconized
`plungers to slide smoothly, whereas such plungers are inop—
`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—
`orption of an antigen adsorbed on an aluminum adjuvant, the
`method comprising filling a container with a vaccine compo-
`sition comprising the antigen adsorbed on the aluminum
`adj uvant, wherein the inner surface of the container is coated
`with polymerized silicone.
`[0028]
`In other words, the invention also comprises a sec—
`ond method for loading and storing a vaccine composition
`containing an antigen adsorbed on the aluminum adjuvant,
`the method comprising filling a container with the composi-
`tion wherein the inner surface of the container is coated with
`polymerized silicone.
`[0029]
`lna similar manner, the invention also comprises the
`use of a container having an inner surface coated with poly—
`merized silicone for storing a vaccine composition compris-
`ing an antigen adsorbed on an aluminum adjuvant.
`
`Regeneron Exhibit 1014.003
`
`

`

`US 2012/0091026 A1
`
`LN
`
`Apr. 19, 2012
`
`the container having inner sur-
`[0030] Advanta geou sly,
`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 ug/cng advan—
`tageously from 5 to 20 ug/cm2; preferably from 5 to 15
`ug/cmz.
`[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] AiA container (i) which contains a vaccine com—
`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] BiA container (i) having im1er surfaces coated
`with polymerized silicone; and (ii) containing a vaccine com-
`position comprising an antigen adsorbed on an aluminum
`adjuvant; and
`[0036] CiA container (i) having im1er surfaces coated
`with polymerized silicone; (ii) containing a vaccine compo—
`sition comprising an antigen adsorbed on an aluminum adju-
`vant; and (iii) which is closed by a device acting as a stopper,
`wherein the surface of the device contactable with the com-
`position is coated with a fluoropolymer.
`[0037]
`In other words, the invention relates to 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 lea st the surface ofthe device contactable with the
`composition is coated with a fluoropolymer.
`[0038] Vaccine compositions stored in containers accord-
`ing to 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 adj uvant;
`[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 lea st:
`
`(a) 65-70% ofthe total antigen amount present
`[0041]
`in the composition, when, immediately after loading,
`the composition is stored at (i) 513° C. for 2-3 years;
`or (ii) 25:3° C. for 2-3 months; or
`[0042]
`(b) 80-90%, of the total antigen amount present
`in the composition, when, immediately after loading,
`the composition is stored at 523° C. for 18 months;
`and
`
`[0043] A vaccine composition comprising an aluminum
`adjuvant and an antigen able to adsorb onto the alumi-
`num adjuvant, wherein the percentage of the antigen
`adsorbed on the aluminum adjuvant is at lea st 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
`with non-polymerized silicone, the comparison between
`the antigen adsorption percentages being carried out
`
`after storage of the vaccine-containing containers at 250
`C. for 2 months, starting from the date of filling the
`containers .
`
`For the purposes of the present invention, the con-
`[0044]
`tainer may be any type ofreservoir, such as vials or syringes,
`and may contain multiple doses (multidose container) or a
`single dose (single—dose container). As an example, the con—
`tainer may be a syringe or a part ofa 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 plunger). 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—
`flexTM technology) and by West Pharma (FluorotecTM tech-
`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
`OBaked—on technology).
`[0046] The antigen and the aluminle adjuvant may be any
`antigen and any aluminum adjuvant provided, of course, that
`they are both capable of interacting with each other. Further—
`more, it may easily be understood that the present invention is
`of particular interest for an antigen-alumimlm adjuvant pair
`having a relatively weak interaction force; the interaction
`force possibly depending on the environment. This interac-
`tion force can be assessed according to a variety of tests. For
`example. an aluminum adjuvant may be used to form 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 to compete with the antigen for the
`interaction with the adjuvant is added. The various prepara-
`tions are centrifuged and the supematants recovered. Finally,
`the amount ofantigen desorbed is assayed in the supernatants,
`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
`affected 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-
`plex.
`[0048] By “adsorption” it is generally meant any phenom-
`enon aimed at fonning an antigen-a djuvant complex involv-
`ing i.a. electrostatic interaction forces, hydrophobic interac-
`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.
`For use in the present invention, an aluminum adju-
`[0049]
`vant may be aluminum oxy hydroxide (A1001 1), such as the
`product sold by BrermtagAG (Superfos) or Reheis Corp; and
`aluminum hydroxy phosphate (AlOHPO4), such as the prod-
`uct sold by Alphos.
`[0050]
`For a vaccine composition to be effective, the mini-
`mal antigen amount required for adsorption onto the alumi-
`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 (HBsAg). It is particularly advan-
`tageous to adsorb HBsAg onto AlOOH as HBsAg exhibits an
`
`Regeneron Exhibit 1014.004
`
`

`

`US 2012/0091026 A1
`
`Apr. 19, 2012
`
`iso electric point (lEP) less than 7 (about 4 to 5) and AlOOH
`exhibits a point of zero charge (PZC) greater than 7 (about 9
`to 1 1 ).
`[0052] The vaccine composition for use in the present
`invention can contain one or more antigen(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
`AlOOH (AlOOH—HbsAg complex) and a second antigen,
`which is polyribosylribitol phosphate (PRP) ofHaemop/zilus
`influenzae UiiR valence), preferably in a form conjugated to
`a carrier protein (C) which rrray be i.a. Dt or Tt.
`[0054] According to another particular embodiment, when
`the antigen 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-
`theria valence); tetanus toxoid (Tt) (tetanus valence); Borde-
`Iella pertussis detoxified toxin (Ptdx), limbriae, filamentous
`haemagglutinin 0:HA) and/or per’tactin (69 kD antigen) (per-
`tussis valence); inactivated poliovirus serotype 1, 2, or 3
`(polio valence); and polyribosylribitol phosphate (PRP) of
`Haemophilus influenzae (HiB valence), preferably in a form
`conjugated to a carrier protein (C) which may be i.a. Dt or Tt.
`[0055] As a matter of example, the composition may com-
`prise HBsAg, Dt, Tt, Pt and FHA adsorbed on AlOOH (the
`AlOOH—HbsAg—Dt—Tt—Pt—FHA complex), the polio valence,
`and PRP-C substantially non-adsorbed on AlOOH.
`[0056]
`In a general manner, the invention is also particu-
`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 critical is the phenomenon of destabilization by the
`container. The antigen-adjuvant interaction force often dif-
`fers from one antigen to another and, in a composition con—
`taining several antigens, the antigen with the weakest inter—
`action force exhibits the highest tendency to desorb under
`adverse conditions.
`
`Examples and Experimental Results
`
`[0057] AiA 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 and non—adsorbed Haemop/zi-
`[us 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 stan-
`dard stopper/plunger made of non—laminated plastic;
`[0059]
`(2) standard siliconized glass syringes with fluo—
`ropolymer-coated stopper/plunger (West Pharma; Fluo-
`rotechTM technology); and
`[0060]
`(3) syringes, the imrer surface of which is coated
`with polymerized silicone (Baked—on syringe system
`LucrconcTM from Gerreshcimer) with fluoropolymer-
`coated stopper/plunger (West Pharma; FluorotechTM
`technology).
`[0061] The bulk was distributed in 0.5 mL single doses,
`each dose containing 10 ug of HBsAg, 30 Lf of Dt. 10 Lf of
`Tt, 25 ug of Pt, 25 ug of FHA, 40 DU (Antigen D Unit) of
`IPV1,8 DUoflPV2, 32 DUoflPV3, 12 ug ofPRP (in PRP—Tt
`
`conjugate form), 0.6 111g ofAl, 55 mM of phosphate ions, 20
`mM of carbonate 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 250 C. for two months (accelerated ageing).
`The HBsAg desorption was measured in each of the three
`categories at T:0 (just after loading the syringes) and then
`after two months.
`
`[0063] Desorption was evaluated by centrifuging the con-
`tent of the syringes and then measuring the amount of des-
`orbed HBsAg present in the supematant by ELI SA (sandwich
`ELISA, involving a mouse anti—HB sAg monoclonal antibody
`(lgM) for coating and capture, a second mouse anti-HBsAg
`monoclonal antibody (IgG) and a third anti-mouse lgG poly-
`clonal antibody coupled to peroxydase (Sigma, Ref A3673)
`which is revealed by adding tetramethyl benzidine).
`[0064] At T (tirnc):0, the HbsAg adsorptionlcvel was iden-
`tical in the three categories (98% of the total HBsAg was
`adsorbed). At T72 rrronths, desorption was observed in all the
`categories, but the desorption percentage differed depending
`upon the category. The highest desorption percentage was
`found in category (1) (At T’l and 2 months, 55 and 50% of
`the total HBsAg was adsorbed, respectively), whereas the
`lowest percentage was found in category (3) (At T:1 and 2
`months, 72 and 69% of the total HBsAg was adsorbed,
`respectively).
`[0065] BiA bulk of the vaccine composition described in
`Aiwas distributed into two categories of single-dose 1 n1L
`syringes, the characteristics of which were as follows:
`[0066]
`(1) standard siliconized glass syringes (free sili-
`cone); and
`[0067]
`(2) non-siliconized syringes.
`[0068] The bulk was distributed in 0.5 mL single doses,
`each dose containing 10 ug of HBsAg, 30 Lf of Dt, 10 Lf of
`Tt, 25 ug of Pt, 25 ug of FHA, 40 DU (Antigen D Unit) of
`ll’Vl, 8 DU ofll’V2, 32 DU ofMW3, 12 ug ofPRP (in l’Rl’-'l't
`conjugate form), 0.6 111g ofAl, 55 mM of phosphate ions, 20
`mM of carbonate 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 at 25° C. for two months (accelerated ageing). The
`HBsAg desorption was measured in each of the two catego-
`ries at T:0 (just after filling syringes) and then after two
`months, as described in Aiabovc.
`[0070] At T (time):0, the HbsAg adsorptionlevel was iden-
`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 differed depending
`upon the category. The highest desorption percentage was
`found in category (1) (At T:1 and 2 months, 69 and 68% 0f
`the total HBsAg was adsorbed, respectively), whereas the
`lowest desorption percentage was found in category (2) (At
`”:1 and 2 months, 73% of the total HBsAg was adsorbed).
`
`This clearly indicates that the antigen adsorption onto an
`aluminum adjuvant is sensitive to free silicone.
`[0071] CiA bulk of the vaccine composition described in
`Aiwas distributed into three types of single-dose 1 ml,
`syringes, the characteristics of which were as follows:
`[0072]
`(1) low siliconized glass syringes (50-100 ug frce
`silicone/syringe);
`[0073]
`(2) standard highly siliconized glass syringes
`((RTF syringe LucrconcTM from Gerrcshcimcr): 800 ug
`to 1 mg free silicone/syringe); and
`
`Regeneron Exhibit 1014.005
`
`

`

`US 2012/0091026 A1
`
`U}
`
`Apr. 19, 2012
`
`(3) syringes having imier surface coated with
`[0074]
`polymerized silicone (50-100 ug/syringe).
`[0075]
`Syringes of category ( 1) 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 of non-siliconized stopper.
`[0077] The bulk vaccine was distributed in 0.5 mL single
`doses, each dose being as described inA and B.
`[0078] All the syringes of the three types were stored ver—
`tically at 250 C. for two months (accelerated ageing). The
`HB sAg desorption was measured in each ofthe three types at
`T:0 (just after filling syringes) and then after two months, as
`described in A above.
`[0079] At T (time):0, the HbsAg adsorption level was iden—
`tical
`in the three types (94% of the total HBsAg was
`adsorbed). At '1':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 Ti] and 2 months, 60 and 58% of the total
`HBsAg was adsorbed, respectively), whereas the desorption
`percentage was found similar in types (1) and (3) and defini-
`tively much lower than in type (2): In type (1), at T:1 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.
`[0081] DiA stability study has been conducted at 513“ C.
`for 18 months with the filled syringes described inAi(3).At
`least 80-90% ofthe total IIBsAg 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 container with a device acting as a stopper,
`wherein the surface of the device contactable with the com-
`position is coated with a fluoropolymer.
`2. The method as claimed in claim 1, wherein the vaccine
`composition is in liquid or lyophilized form,
`3. The method as claimed in claim 2, wherein the fluo-
`ropolymer is selected from the group consisting of polytet-
`rafluoroethylene (PTFE), polytetrafluoropropylene (PTFP),
`fluorinated ethylene propylene (FEP,
`a copolymer of
`hexafluoropropylene and tetra fluoroethylene), polychlorotri-
`
`fluoroethylene (PCTFE), perfluoroalkoxy co-polyrner (PFA),
`poly(ethylene-co—tetrafluoroet 1ylene)
`(ETFE), poly(ethyl-
`
`enechlorotrifluoroethylene)
`(ECTFE), polyvinyl
`fluoride
`(PVF) and polyvinylidene fluoride (PVPF).
`4. The method as claimed in claim 3, wherein the container
`is the reservoir of a syringe and the device acting as a stopper
`is a plunger.
`5. The method as claimed in claim 4, wherein the inner
`surface of the container is coated with polymerized silicone.
`6. A method for storing a vaccine composition containing
`an antigen adsorbed on an aluminum adjuvant, the method
`comprising loading the composition into a container wherein
`the inner surface of the container is coated with polymerized
`silicone.
`7. The method as claimed in claim 6, wherein the vaccine
`composition is in liquid or lyophilized form.
`8. The method as claimed in claim 1, wherein the antigen
`contained in the composition is hepatitis B surface antigen
`and the aluminum adjuvant is aluminum oxy hydroxide.
`
`
`
`9. A container