United States Patent [191
`Makino et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,985,244
`Jan. 15, 1991
`
`[54] STABILIZED LIVE ATTENUATED VACCINE
`AND ITS PRODUCTION
`[75] Inventors: Satoshi Makino; Keiko Sasaki, both
`of Kanagawa; Masaharu Nakagawa,
`Tokyo, all of Japan
`[73] Assignee: The Kitasato Institute, Tokyo, Japan
`[21] Appl. No.: 203,411
`[22] Filed:
`Jun. 8, 1988
`[30]
`Foreign Application Priority Data
`Jun. 8, 1987 [JP]
`Japan .............................. .. 62-142465
`
`[51] Int. Cl.5 ....................... .. A61K 39/12; C12N 7/00
`[52] US. Cl. .................................... .. 424/89; 435/235;
`435/236; 435/238; 435/239
`[58] Field of Search ................ .. 424/89; 435/235, 236,
`435/ 238, 239
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,337,242 6/1982 Markus et a1. ...................... .. 424/89
`4,338,335 7/1982 McAleer et al. .................... .. 424/89
`4,500,512 2/1985 Barme .
`
`FOREIGN PATENT DOCUMENTS
`
`67887/87 7/ 1987 Australia .
`0028563 5/1981 European Pat. Off. .
`2424031 1 1/1979 France .
`
`OTHER PUBLICATIONS
`“Stability on Storage at Various Temperatures of Live
`Measles, Mumps and Rubella Virus Vaccines in New
`Stabilizer”, Journal of Biological Standardization, vol. 8,
`No. 4, 1980, by William McAleer et al., pp. 281-287.
`“Behaviour of Five Commercial Measles Vaccines in an
`Accelerated Stability Test”, Journal of Biological Stan
`
`dardization, vol. 10, No. 3, 1982, by G. Colinet et al., pp.
`241-247.
`“Thermodegradation of Lyophilized Measles Vac
`cines”, Reviews of Infectious Diseases, vol. 5, No. 3,
`May-Jun., 1983, by F. E. Andre, pp. 532-534.
`“Thermostability of Live Freeze-Dried Measles Vac
`cine After Reconstitution”, Proc. Symposium on Stability
`and Effectiveness of Measles, Poliomyelitis and Pertussis
`Vaccines, by D. Ikic et al., pp. 103-112.
`“The In?uence of Storage at 37° C. on the Potency of
`Frezze-Dried and Reconstituted Live Measles Vac
`cine”, Proc. Symposium on Stability and E?‘ectiveness of
`Measles, Poliomyelitis and Pertussis Vaccines, by H.
`Cohen et al., pp. 95-101.
`“10th International Immunobiological Symposium”,
`Proceedings, Symposium on Stability and Effectiveness of
`Measles, Poliomyelitis and Pertussis Vaccines, Organized
`by Yugoslav Academy of Sciences and Arts and Insti
`tute of Immunology, Sep. 1976, pp. 87-94.
`
`Primary Examiner-Howard E. Schain
`Assistant Examiner-Abdel A. Mohamed
`Attorney, Agent, or Firm-Young & Thompson
`[57]
`ABSTRACT
`A stabilized live attenuated vaccine with improved
`thermal stability, which comprises a live attenuated
`plain vaccine consisting of measles, mumps or rubella
`virus grown in a medium-199 for cell culture, or a com
`bined live attenuated vaccine thereof, containing a sta
`bilizing agent at a ?nal concentration of lactose 2.5-5
`W/V %, saccharose 2.5-5 W/V %, D-sorbitol 1.8-2
`W/V %, sodium glutamate about 0.1 W/V % and gela
`tin hydrolyzate, M.W. approx. 35,000, 2-3 W/V %.
`
`3 Claims, 2 Drawing Sheets
`
`CFAD Exhibit 1026
`
`1
`
`

`

`US. Patent
`
`Jan. 15, 1991
`
`Sheet 1 of 2
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`4,985,244
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`

`1
`
`STABILIZED LIVE A'I'I‘ENUATED VACCINE AND
`ITS PRODUCTION
`
`4,985,244
`2
`conventional live attenuated plain vaccine which is
`measles, mumps or rubella virus grown in a medium-199
`for cell culture, or a combined live attenuated vaccine
`thereof, containing a stabilizing agent.
`A further object of the present invention is to provide
`a process for preparing live attenuated vaccines.
`
`25
`
`FIELD OF THE INVENTION
`This invention relates to a stabilized live attenuated
`vaccine and its production.
`BACKGROUND OF THE INVENTION
`Titers of liveattenuated vaccines are usually ther
`mally unstable. Vaccine preparations are therefore sup
`plied as low-temperature frozen products or lyophilized
`products. To stabilize them, a chemical stabilizing agent
`is added to the vaccine solution. Examples of chemical
`stabilizing agents hitherto known are human albumin,
`gelatin hydrolyzate, sugar alcohols, amino acids and
`other non-toxic substances. For example, for this pur
`pose it is known to use a preparation consisting of a
`basic amino acid such as arginine, lysine or histidine,
`each 5 W/V% or adding thereto various sugar alcohols
`such as saccharose, inositol or sorbitol, each 5 W/V%
`(Jap. Patent Appln. No. 45-1887), a preparation ob
`tained by mixing peptone 5-l0 W/V%, arginine or
`lysine 3 W/V% and saccharose 5 W/V% (Jap. Patent
`Unexam. Publ. No. 50.2225), a preparation obtained by
`adding lactose 4 W/V% and sorbitol 2 W/V% to a
`phosphate buffer solution containing Ca+ +and Mg+ ‘1',
`or adding at least one amino acid 0.005M-0.05M thereto
`(Jap. Patent Appln. No. 57-81338), a preparation con
`30
`sisting of lactose 5 W/V%, D-sorbitol 1.5 W/V%,
`dextran 7O O.3W/V%, potassium glutamate 0.048%,
`disodium phosphate 0.0625 W/V%, potassium phos
`phate 0.026 W/V%, gelatin 0.3 W/V% and human
`albumin 0.25 W/V% (Jap. Patent Appln. No.
`55-80465), and a preparation wherein a vaccine solution
`is acidi?ed to pH 6.0-6.5 prior to lyophilization by
`adding a phosphate, buffer solution containing a stabiliz
`ing agent comprising a gelatin partial hydrolyzate,
`M.W. approx. 3,000, sorbitol, saccharose, lactose, mal
`tose, L-glutamate and L-arginine (Jap. Patent Unexam.
`Publ. No. 57-114527).
`Nowadays, vaccines are distributed to tropical
`countries where there are no refrigerated distribution
`sytems. In these areas, the vaccine preparations sup
`plied must have heat stability at high temperature. The
`45
`above known stabilizing agents were not suitable in this
`regard.
`We have found that a live attenuated vaccine shows
`strong thermostability when lactose, D-sorbitol, saccha
`rose, gelatin hydrolyzate and sodium glutamate at spe
`ci?c ratios of concentration are added to a vaccine virus
`suspension comprising a medium-199 for cell culture
`(Morgan, et al., Proc. Soc. Exp. Biol. Med , 73:1-8,
`1950) (hereinafter designated as medium-199). Each
`component thereof has been known as a stabilizing
`agent for live attenuated vaccines; however, it has
`never been known that a combination thereof at a spe
`ci?c ratio of concentrations exhibits unexpected ther
`mostability.
`OBJECTS OF THE INVENTION
`An object of the present invention is to provide a
`stabilized live attenuated vaccine comprising an admix
`ture of a conventional live attenuated vaccine with
`lactose, D-sorbitol, saccharose, gelatin hydrolyzate and
`sodium glutamate at speci?c ratios of concentration.
`Another object of the present invention is to provide
`a stabilized live attenuated vaccine which comprises a
`
`SUMMARY OF THE INVENTION
`In the present invention, a vaccine solution is pre
`pared by growing a seed virus in a culture system using
`medium-199. Examples of live attenuated vaccine are
`plain or combined vaccines of measles, mumps or ru
`bella. A thermostable high titer vaccine can be obtained
`by adding the additives recited below, at speci?c con
`centrations and lyophilizing the vaccine solution. A
`further speci?c feature of the present invention is that it
`is not required to adjust the pH of the vaccine solution
`below neutral pH but only to add each sterilized com
`ponent prior to lyophilization. A still further advantage
`of the present invention is that it does not contain
`human albumin, which provides a superior stabilization
`effect, and so the present additives are safer chemical
`compounds. Therefore the adverse effect of an immu
`nological preparation caused by its carrier or vehicle
`can be reduced.
`The composition of the present stabilizing agent is
`illustrated as follows. The components and the ?nal
`concentration of the stabilizing agent in the ?nal bulk
`vaccine solution which is composed of an original cul
`tured vaccine using a medium-199 and a diluent for
`medium-199, are lactose 2.5-5 W/V%, saccharose
`2.5-5 W/V%, D-sorbitol 1.8-2 W/V%, sodium gluta
`mate about 0.1 W/V% and gelatin hydrolyzate, M.W.
`approx. 35,000, 2-3 W/V%. These substances are dis
`solved in medium-199 at preferred concentrations and
`are aseptically ?ltered.
`The thermostability of the vaccine of the present
`invention can be observed by virus suspension (Table
`l), and is more apparently in the lyophilized product
`(Table 2). The thermostability of the lyophilized live
`attenuated vaccine product containing a stabilizing
`agent of the present invention is superior to that of the
`known vaccine preparations containing conventional
`stabilizing agents. For example, when a measles vaccine
`preparation containing a conventional stabilizing agent
`consisting of lactose 5 W/V%, sodium glutamate 0.1
`W/V% and gelatin hydrolyzate 0.2 W/V% as its ?nal
`concentration, is kept at 37° C. for one week, its titer
`decreases to l/100-1/500 with corresponding loss of
`immunogenicity. On the contrary, a product containing
`a stabilizing agent of the present invention loses not
`more than one third of its titer under the same condi
`tions and so retains its immunogenicity as compared
`with the titer of the original vaccine preparation.
`As shown in FIGS. 1 and 2, the thermostability of
`mumps and rubella vaccines containing a stabilizing
`agent of the present invention is also superior to that of
`a commercial vaccine preparation containing conven
`tional stabilizing agents. Live attenuated measles vac
`cine containing the stabilizing agent of the present in
`vention can be used for the worldwide project for the
`eradication of measles sponsored by WHO, including
`the countries having no refrigerated distribution sys
`tems.
`
`40
`
`50
`
`55
`
`65
`
`4
`
`

`

`3
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIGS. 1 and 2 show the heat stability of vaccines
`containing a stabilizing agent of the present invention,
`and that of the commercially available vaccines.
`The following example illustrates the present inven
`tion but is not to be construed as limiting.
`
`EXAMPLE
`Lactose 5 W/V%, saccharose 5 W/V%, D-sorbitol
`1.8 W/V%, sodium glutamate 0.1 W/V% and gelatin
`hydrolyzate, M.W. approx. 35,000, 2 W/V%, ?nal con
`centration, (Behringwerke AG, West Germany, gelatin
`hydrolyzate powder dissolved in medium—199 “HEMA
`CEL" (trade name: Gelatin hydrolyzate for liquid
`transfusion)) were added to a measles vaccine virus
`
`4,985,244
`4
`hereinabove and with a commercial lyophilized vaccine
`product (four aliquots) was performed.
`Also a live attenuated mumps or rubella vaccine con
`taining the stabilizing agent of the present invention
`hereinabove was prepared, in two batches, and the
`vaccine solution was lyophilied to prepare four aliquots
`of the products. Comparative accelerated heat stability
`tests were run with the products hereinabove and with
`commercial lyophilized vaccine products (two aliquots
`each). As shown in Table 2 and FIGS. 1 and 2, the
`decrease in titer of the lyophilized live attenuated vac
`cine of measles, mumps and rubella containing a stabiliz
`ing agent of the present invention, after storage at 37° C.
`and at 45° C. for one week, respectively, is signi?cantly
`less than that of the commercially available conven
`tional vaccines.
`TABLE 1
`Survival ratio of vaccine virus in medium - 199 containing stabilizing
`agent of the present invention and in conventional liquid for vaccine,
`after heatijg at 37° C.
`
`vaccine virus
`measles (AIK-C strain)
`
`measles (AIK-C strain)
`
`mumps (HOSHINO strain)
`
`suspension
`medium-199 containing
`stabilizing agent
`conventional liquid
`for vaccine
`medium-199 containing mumps (HOSHINO strain)
`stabilizing agent
`conventional liquid
`for vaccine
`‘: logmTClDm/ml
`Q : infective titer after heating/before heating X 100% = survival ratio of virus (Survival ratio of virus is calculated by
`replacing a loglo-value of each infective titer to antilogarithmic number)
`
`Survival ratio of virus
`after heating at 37° C15’
`infectivity titer
`before heating ’ after 6 hrs.
`after 18 hrs.
`4.1
`1.0%
`§0.064%
`
`4.2
`
`4.4
`
`4.0
`
`0.2%
`
`5.7%
`
`3.8%
`
`§0.03l%
`
`0.70%
`
`20.10%
`
`TABLE 2
`Stability of lyophilized vaccine
`Lyophilized live attenuated measles vaccine, strain AlK-C
`Lyophilized live attenuated mumps vaccine, strain HOSHINO
`‘I rubella vaccine, strain TAKAHASHI
`Lyophilized live M
`Before heatim7
`37° C. 1 week
`45° C. 1 week
`No.
`No.
`Titer‘
`Decreased titer?
`Vaccine
`tested (M 1 SD)“ tested
`(M : SD)
`Lot#
`Stabilizing agent of the present invention
`Measles TV-Z
`5
`4.3 -l_- 0.11
`TV-3
`5
`4.3 t 0.22
`TV-4
`5
`4.6 i 0.27
`TV-S
`5
`4.4 i 0.25
`'I'V-6
`5
`4.3 i 0.11
`Mumps TV-l
`5
`5.2 i 0.35
`‘TV-2
`5
`5.1 i 0.18
`TV-3
`5
`5.2 t 0.14
`TV-4
`5
`5.0 i 0.20
`TV-l
`5
`4.5 t 0.22
`'l'V-Z
`5
`4.6 i 0.37
`"l‘V-3
`5
`4.5 :L- 0.16
`W4 5
`4.5 i 0.31
`
`No.
`tested
`
`Decreased titer
`
`5
`5
`5
`S
`5
`5
`5
`5
`5
`5
`5
`5
`5
`
`0.2 i 0.22
`0.3 i 0.20
`0.2 i- 014
`0.3 t 0.30
`0.2 i 0.11
`0.3 i 0.31
`0.2 i 0.18
`0.2 i 0.27
`0.1 t 0.16
`0.2 i‘. 0.11
`0.3 i 0.11
`0.3 i 0.17
`0.2 i 0.18
`
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`5
`
`1.1 i 0.14
`1.7 i 0.27
`1.8 + 0.25
`l 7 + 0.36
`1.7 t 0.38
`1.6 _+_ 0.16
`1.6 i 0.30
`1.4 i- 0.13
`1.3 i 0.21
`0.9 i 0.42
`0.8 i 0.27
`0.8 i 0.44
`0.9 i 0.35
`
`Rubella
`
`Measles
`
`MlO-l
`MID-27
`M11-7
`Mll-ll
`Mumps K01-l2
`K0l-l8
`823-11
`823-15
`
`Rubella
`
`5
`5
`5
`5
`5
`5
`5
`5
`
`4.5 i 0.18
`4.1 i 0.11
`4.4 -_l- 0.25
`4.6 i 0.13
`5.1 i 0.26
`4.3 i 0.26
`4.4 i 0.25
`4.5 i 0.13
`
`5
`5
`5
`5
`5
`5
`5
`5
`
`2.8 i 0.21
`2.3 i 0.25
`2.4 i 0.33
`2.2 -+_- 0.21
`1.4 i 0.29
`1.6 i 0.21
`0.8 t 0.16
`1.0 i 0.23
`
`5
`5
`5
`5
`5
`5
`5
`5
`
`4.4 i 0.31
`4.0 i- 0.38
`4.3 i 0.38
`4.1 i 0.31
`3.7 i 0.41
`3.6 i 0.42
`1.5 i 0.22
`1.8 i 0.29
`
`': logwTclDso/ml
`'°: mean i S. D.
`5y: (Titer before heating) — (titer after heating) = Decreased titer (lOgmTCIDm/ml)
`
`suspension in medium-199 to prepare a bulk vaccine (2
`batches). The vaccine is lyophilized and finally ?ve
`aliquots of vaccine preparation were produced. A com
`parative accelerated heat stability test with the product
`
`65 We claim:
`1. A stabilized live attenuated vaccine, which com
`prises a live attenuated plain vaccine consisting of mea
`sles, mumps or rubella virus grown in a medium-199 for
`
`5
`
`

`

`5
`cell culture, or a combined live attenuated vaccine
`thereof, in admixture with a stabilizing agent which is a
`combination consisting essentially of lactose 2.5-5
`W/V%, saccharose 2.5-5 W/V%, D-sorbitol 1.8-2
`W/V%, sodium glutamate about 0.1 W/V% and gela
`tin hydrolyzate, M.W. approx. 35,000, 2-3 W/V%.
`2. A stabilized live attenuated vaccine according to
`claim 1 wherein the said vaccine is a lyophilized vaccine
`produced by lyophilizing a live attenuated plain vaccine
`consisting of measles, mumps or rubella virus grown in
`a medium-199 for cell culture, or a combined live atten
`
`4,985,244
`6
`uated vaccine thereof, in admixture with said stabilizing
`agent.
`3. A process for preparing a live attenuated vaccine
`which comprises growing a seed virus in a medium-199
`for cell culture to produce a live attenuated plain vac
`cine of measles, mumps or rubella, or a combined live
`attenuated vaccine thereof, adding said stabilizing agent
`thereto to prepare a stabilized live attenuated vaccine
`solution, and lyophilizing the said solution.
`# i t i i
`
`10
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`6
`
`