OA16951A - Method for formulating a vaccine containing at least two antigens capable of adsorbing onto aluminium oxyhydroxide. - Google Patents
Method for formulating a vaccine containing at least two antigens capable of adsorbing onto aluminium oxyhydroxide. Download PDFInfo
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- OA16951A OA16951A OA1201400304 OA16951A OA 16951 A OA16951 A OA 16951A OA 1201400304 OA1201400304 OA 1201400304 OA 16951 A OA16951 A OA 16951A
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- Prior art keywords
- hbsag
- antigen
- aiooh
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- 239000000427 antigen Substances 0.000 title claims abstract description 135
- 102000038129 antigens Human genes 0.000 title claims abstract description 135
- 108091007172 antigens Proteins 0.000 title claims abstract description 135
- 229960005486 vaccines Drugs 0.000 title claims abstract description 45
- MKGYEFNKQXAIGY-UHFFFAOYSA-N [Al].OOO Chemical compound [Al].OOO MKGYEFNKQXAIGY-UHFFFAOYSA-N 0.000 title abstract description 3
- 239000000203 mixture Substances 0.000 claims abstract description 82
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- 229910002706 AlOOH Inorganic materials 0.000 claims description 25
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminum Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 15
- 239000000725 suspension Substances 0.000 claims description 15
- 229910052782 aluminium Inorganic materials 0.000 claims description 14
- 238000007792 addition Methods 0.000 claims description 11
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- VXAUWWUXCIMFIM-UHFFFAOYSA-M aluminum;oxygen(2-);hydroxide Chemical compound [OH-].[O-2].[Al+3] VXAUWWUXCIMFIM-UHFFFAOYSA-M 0.000 claims description 9
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- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 4
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- ZUVPWWASXUUVFX-UHFFFAOYSA-M 4-hydroxybutyl(trimethyl)azanium;iodide Chemical compound [I-].C[N+](C)(C)CCCCO ZUVPWWASXUUVFX-UHFFFAOYSA-M 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K Aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- OMLWNBVRVJYMBQ-YUMQZZPRSA-N Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OMLWNBVRVJYMBQ-YUMQZZPRSA-N 0.000 description 1
- 229960003589 Arginine hydrochloride Drugs 0.000 description 1
- BNODVYXZAAXSHW-UHFFFAOYSA-N Arginyl-Histidine Chemical compound NC(=N)NCCCC(N)C(=O)NC(C(O)=O)CC1=CN=CN1 BNODVYXZAAXSHW-UHFFFAOYSA-N 0.000 description 1
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- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 1
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- IGRMTQMIDNDFAA-UHFFFAOYSA-N Lysyl-Histidine Chemical compound NCCCCC(N)C(=O)NC(C(O)=O)CC1=CN=CN1 IGRMTQMIDNDFAA-UHFFFAOYSA-N 0.000 description 1
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Abstract
The subject matter of the invention is a method for preparing a vaccine composition comprising at least aluminium oxyhydroxide (AIOOH), and at least the hepatitis B surface antigen and the Haemophilus influenzae type b antigen. According to the invention, the hepatitis B surface antigen is kept adsorbed on the AIOOH, whereas the Hib antigen is kept nonadsorbed. To this end: the hepatitis B surface antigen is adsorbed onto AIOOH in order to obtain an AIOOH/HBsAg complex, then - said AIOOH/HBsAg complex is mixed with the Hib antigen in the presence of cationic amino acids at a concentration of at least 100 mg/l, and of phosphate ions at a concentration of 35 to 45 mMol/l.
Description
Method for formulating a vaccine containing at least two antigens capable of adsorbing onto aluminium oxyhydroxide
The invention relates to the field of vaccine combinations comprising both the hepatitis B valence consisting of the hepatitis B virus surface antigen (HBsAg) and the Haemophilus influenzae type b valence, consisting of its capsular polysaccharide, called polyribosytribitol phosphate (PRP), which is, in order to be effective in children under the âge of two, conjugated to a carrier protein, for example the tetanus protein.
Such combinations, which are intended for administration in young children, generally comprise other antigens that make it possible to vaccinate against several diseases in a single operation, and also an aluminum-based adjuvant.
Thus, patent application WO 99/13906 discloses a vaccine composition comprising, as is described on page 13, antigens against diphtheria, tetanus, polio, whooping cough, hepatitis B and Haemophilus influenzae type b infections. Some of these antigens need to be adsorbed onto an aluminum sait in order to be immunogenic. This is in particular the case with the hepatitis B surface antigen or HBsAg.
However, as is indicated on page 12 of application WO 99/13906, the Hib valence, consisting of the capsular polysaccharide conjugated to the tetanus protein, has a tendency to lose its Immunogenicity over time when it is adsorbed onto aluminum salts. In order to avoid this drawback, the solution proposed in this prior art is, as had already been recommended in the prior application PCT/FR96/00791, to add anions and in particular phosphate, carbonate or citrate ions.
However, the authors of the présent invention hâve noted that, while the addition of ions, in particular of phosphates or of carbonates, actually makes it possible to prevent the adsorption of PRP-T onto aluminum oxide hydroxide (AIOOH), and therefore to maintain its immunogenicity over time, this addition also has the drawback of desorblng the hepatitis B surface antigen when the latter has, itself, also been adsorbed onto aluminum oxide hydroxide.
It is therefore necessary to find a method for preparing a vaccine combination comprising aluminum oxide hydroxide, in which the hepatitis B surface antigen is kept adsorbed on AIOOH, whereas the Hib antigen is kept nonadsorbed.
To this end, the subject of the présent Invention is a method for preparing a liquid vaccine combination comprising at least:
~ one hepatitis B surface antigen (HBsAg),
-2- one Haemophiïus influenzae type b (Hib) antigen consisting of capsular polysaccharide conjugated to a carrier protein,
- aluminum oxide hydroxide (AIOOH), in which the hepatitis B surface antigen Is kept adsorbed on AIOOH, whereas the Hib antigen Is kept nonadsorbed, wherein:
- the hepatitis B surface antigen Is adsorbed onto AIOOH In order to obtain an AlOOH/HBsAg complex,
- said AlOOH/HBsAg complex Is mîxed with the Hib antigen in the presence of cationic amino acids at a concentration of at least 100 mg/l and of phosphate ions at a concentration of 35 to 45 mMol/l.
By virtue of the method according to the invention, it is possible to achieve the desired balance between keeping the adsorptlon of the hepatitis B surface antigen on the aluminum oxide hydroxide and keeping the nonadsorption ofthe Hib antigen.
According to one particular embodiment of the invention, the HBsAg antigen is adsorbed onto the aluminum by mixing a suspension of AIOOH with a suspension of HBsAg with stirring for at least 4 hours, preferably at least 12 hours, preferably between 20 and 24 hours.
According to one particular embodiment ofthe invention, the cationic amino acids are added to said AlOOH/HBsAg complex before the mixing with the Hib antigen.
According to an alternative embodiment of the invention, the cationic amino acids are added to said Hib antigen before the mixing with the AlOOH/HBsAg complex.
According to one embodiment of the invention, the phosphate Ions are added to said AlOOH/HBsAg complex before the mixing with the Hib antigen.
According to one embodiment of the invention, the pH ofthe préparation comprising the AlOOH/HBsAg complex is adjusted to 7.1 + 0.1 before the mixing with the Hib antigen.
The subject of the Invention is also a vaccine combination obtained according to the method claimed and comprising at least:
• the hepatitis B surface antigen,
- the diphtheria antigen In the form of diphtheria toxin D,
- the tetanus antigen in the form of tetanus toxin T,
- the whooping cough antigens in the form of Purified Toxin (PTxd) and of Filamentous
Hemagglutinin (FHA),
-3- the Haemophilus influenzae type b antigen, in the form of polyribosylribitol phosphate conjugated to the tetanus protein (PRP-T),
- the polio antigens In the form of inactivated type 1,2 and 3 viruses.
Such a vaccine composition has the advantage of being In liquid form, thereby avoiding operations to take up the lyophiiisate; it has proven to be sufficiently stable to remain immunogenic until the day It Is used, even 36 months after its date of manufacture.
According to the Invention, the vaccine composition comprises a hepatitis B surface antigen (HBsAg). This antigen may in particular be a hepatitis B surface antigen such as the one présent In the Recombivax HB™ vaccine, or in any other hepatitis B vaccine, it may in particular be the recombinant antigen obtained by fermentation of a Hansenula polymorphe yeast which has been modified, according to the technoiogy developed by Crucell, such as the one présent In the Hepavax-Gene™ vaccine. For the purposes ofthe invention, the amount of HBsAg présent in a 0.5 ml dose is advantageously between 5 and 15 pg, and In particular 10 pg.
According to the Invention, the vaccine composition comprises a Haemophilus Influenzae type b (Hib) antigen . This antigen consists of the capsular polysaccharide of the bacterium or PoiyribosylRibitoi Phosphate (PRP) which is conjugated to a carrier protein. The carrier protein may be any protein used In this respect in the vaccine field. It may be, for example, diphtheria toxin D, tetanus toxin T, Haemophilus Influenzae lipoprotein D, CRM197 or the N. meningitidis outer membrane protein (OMP). Use is preferably made of the tetanus protein in order to obtain the PRPT conjugate. For the purposes of the présent invention, the PRP-T conjugate may be présent in a proportion of from 1 to 30 pg of PRP per 0.5 ml dose; advantageously from 5 to 25 pg of PRP per dose; preferably from 10to 15 pg of PRP per dose; entireiy preferably from 10 to 12 pg of PRP per dose, and more particularly 12 pg of PRP, conjugated to 22-36 pg of tetanus protein.
According to the Invention, the vaccine composition comprises aluminum oxide hydroxide AIOOH. This aluminum sait is very commonly incorrectly called aluminum hydroxide. The AIOOH which can be used for the purposes of the présent Invention may be, for example, the AIOOH sait sold by Brenntag AG, or Rehydragel HPA from Reheis Corp. (Berkeley Heights, NJ), although the method of production of each of the two adjuvants diffère. The amount of AIOOH used Is calculated so as to allow a satisfactory Immune response to be achîeved; it dépends in particular on the number and on the nature of the antigens présent In the composition and also on the amount of each of these antigens.
Purely by way of information, the maximum adsorption of HBsAg onto AIOOH is about 780 pg of protein per mg of aluminum (conventionally, the amount of AIOOH is expressed as amount of aluminum Al3*). For a vaccine dose comprising 10 pg of HBsAg, without any other additional
-4antigens, 13 pg of aluminum would thus suffice, which Is an amount that Is however Insufficient to obtain the desired efficacy when other antigens are added. Thus, depending on the addition of one or more additional antigens, 10 pg of HBsAg may be brought into contact with from 0.01 mg to 1.25 mg of aluminum, which Is the maximum amount recommended by the European pharmacopeia.
For exampie, a 0.5 ml dose of a pédiatrie vaccine comprising diphtheria, tetanus, whooping cough and hepatitis B antigens may conventîonally contain from 0.5 to 0.7 mg of aluminum, preferably approximately 0.6 mg of aluminum.
According to the invention, the hepatitis B surface antigen is kept adsorbed on ΑΙΟΟΗ, which means that at ieast 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% of the total amount of this antigen présent in the composition is présent in adsorbed form.
According to the invention, the Hib antigen is kept nonadsorbed on AIOOH, which means that at least 65%, preferably at least 70%, preferably at least 75% of the total amount of this antigen présent in the composition is présent in nonadsorbed form.
According to the invention, the period of time during which the hepatitis B surface antigen is kept adsorbed on the AIOOH and the Hib antigen is kept nonadsorbed is at least 3 months, preferably at ieast 6 months, preferably at least 12 months, preferably at least 18 months, preferably at least 24 months, more preferably at least 36 months starting from the date of manufacture of the composition, when the storage température is 5 + 3*C. Preferably, the amount of adsorbed or nonadsorbed antigens is stable over time, but it may also vary, provided that It remains within acceptable limits. Thus, when at ieast 85% of the total amount of the HBsAg présent In the composition Is adsorbed on the AIOOH for 3 months starting from the date of manufacture of the composition stored at a température of 5 + 3’C, it is entirely possible that, after one year and still under the same storage conditions, 80% of the total amount of the HBsAg présent In the composition is kept adsorbed.
In order to assess the amount of antigen adsorbed, those skilled In the art may use any known method.
With regard to the détermination of the percentage adsorption of the HBsAg, it Is possible to use a sandwich ELISA method according to the rules defined by the European pharmacopeia 2,7.1.
Briefly, the HBsAg is captured by an anti-HBsAg primary monoclonai antibody of IgM type, in wells of a 96-weli plate. The HBsAg thus bound is coated with an anti-HBsAg secondary monoclonal antibody, of IgG type, which Is itself detected by means of a peroxidase-coupled anti-IgG polyclonal antibody. A chromogenic substrate for peroxtdase, tetramethylbenzidine (TMB), serves as a developing agent. When it Is added, a color develops, the Intensity of which Is proportional to the
-5amount of HBsAg captured in the well. The results are analyzed according to the parallel line method described in the European pharmacopeia 5.3.3. The percentage adsorption is obtained from the détermination of the total HBsAg content and of the nonadsorbed HBsAg content.
With regard to the amount of nonadsorbed PRP-T, the évaluation can be carried out by HPAECPAD (high-performance ion exchange chromatography - pulsed amperometric détection).
According to the method of the invention, the hepatitis B surface antigen is adsorbed onto AIOOH. This step can be carried out by bringing the hepatitis B surface antigen and the AIOOH into contact in the absence of any other antigen and allowing the hepatitis B surface antigen to adsorb onto the AIOOH for at least 4 hours, preferably at least 12 hours, entirely preferably between 20 and 24 hours, so as to obtain a préparation containing an AlOOH/HBsAg complex. This adsorption can be carried out, according to the invention, in the absence of phosphate ions. The objective pursued by means of a prolonged contact time between the hepatitis B surface antigen and the AIOOH consists in maximizing electrostatïc interactions and in promotîng stable interactions, which can thus resuit in adsorption by ligand exchange. This contact is advantageously continued with stirring. According to the method of the invention, the AlOOH/HBsAg complex is mixed with the Hib antigen in the presence of cationic amino acids and of phosphate ions.
For the purposes of the invention, the tenn cationic amino acids is intended to mean amino acids of which the pHi is higher than the pH of the vaccine composition and which will therefore be in cationic form at the pH of the vaccine; these are In particular Lysine (Lys), Arginine (Arg) or Histidine (His); each of these amino acids can be used alone, as a mixture either in pairs (Lys + Arg; Lys + His; Arg + His) or ail 3 together (Lys + Arg + His). According to one particular embodiment, the cationic amino acids may be associated in dipeptide form. Mention may particularly be made of the dipeptides Lys-Lys, Lys-Arg, Lys-His, Arg-Arg, Arg-Lys, Arg-His, HisHis, His-Lys and His-Arg. Altematively, a dipeptide of use for the purposes of the présent invention may be composed of a cationic amino add and of an uncharged amino add selected from Ala, Val, Leu, Iso, Pro, Met. Phe, Trp, Gly, Ser, Thr, Cys, Tyr, Asp and Gin. Thus, in practice, a préparation containing one or more cationic amino acid(s) in free and/or dipeptide form may be used. It is also possible to use amino acid préparations comprising cationic amino acids in desired amount, as a mixture with other amino acids. In order not to produce too great a drop in the pH during the addition of the amino acids, it is possible to envision increasing the pH of the préparation comprising the amino acids before adding it to the AlOOH/HBsAg complex, by means of a base, in particular sodium hydroxide.
- 6 According to the invention, the amount of cationic amino acids ultimately présent in the vaccine composition must be at least 100 mg/l, advantageously at least 300 mg/l, advantageously at least 400 mg/l, entirely preferably at least 500 mg/l. There is no critical maximum dose. However, it is préférable for the maximum amount to be at most 2 mg/ml, more preferably at most 1 mg/ml, more preferably at most 800 pg/ml, entirely preferably at most 700 pg/ml. When calculating the amount of cationic amino acids to be added, the cationic amino acids that may be introduced by the media in which the antigens other than HBsAg and the Hib antigen présent should be taken Into account.
According to one alternative of the method, it is possible to détermine the amount of cationic amino acids relative to the weight of the Hib capsular polysaccharide and to provide for a Hib polysaccharide/cationic amino acid weight ratio of 1: 4 to 1:100, advantageously of 1:10 to 1: 80, preferably of 1:15 to 1:60, or particularly preferably of 1:20 to 1: 30 or 40.
According to the method of the Invention, the AlOOH/HBsAg complex is mixed with the Hib antigen In the presence of cationic amino acids, but also in the presence of phosphate Ions. According to one embodiment of the Invention, the phosphate ions are added to the AlOOH/HBsAg complex before it is brought into contact with the Hib antigen. The phosphate ions can, for example, be Introduced by adding sodium hydrogen phosphate or potassium hydrogen phosphate, or else a mixture of the two. The amount of phosphate ions is calcuîated so that the maximum amount of hepatitis B surface antigens remains adsorbed on the AIOOH while at the same time avoiding the adsorption of the Hib antigen. This amount varies according to the nature and the number of the antigens présent, and in particular according to the antigens other than the HBsAg and Hib antigens.
Thus, for a vaccine combination comprising the antigens normally used in pédiatrie vaccines, that is to say, in addition to the HBsAg and the Hib antigen, the dlphtheria, tetanus and whooping cough antigens, It may be advantageous to add phosphate ions such that the phosphate Ion concentration In the vaccine composition ultimately obtained Is at least equal to 35 mMol/l, and more particularly is between 35 and 45 mMol/l, limite Included; preferably between 38 and 44 mMol/l, limite Included; entirely preferably between 38 and 42 mMol/l, limits Included. According to one preferred embodiment, the phosphate Ion concentration in the vaccine composition finaliy obtained is 40 mMol/l.
According to an alternative embodiment, the phosphate ions are added in an amount such that they are présent in the vaccine composition at a final concentration of between 35 and 38 mMol/l, limits included. This Is completed by also adding carbonate Ions, but In a limited amount however, since it has in fact been noted that an excessive amount of carbonate ions is unfavorable. Advantageously,
-7they may be added in an amount such that they are présent in the vaccine composition at a final concentration of less than or equal to 10 mMol/l.
ln order to avoid an excessive ionic shock which could déstabilisé the HBsAg and promote desorption thereof, It is recommended to add the phosphate Ions In several (example In 2) distinct operations (steps). Thus, the phosphate Ions may be added in a first operation, in an amount which makes it possible to achieve a final concentration of between 20 and 30 mMol/i, limits included; then, ln a second operation, in an amount which makes it possible to achieve a final concentration as specified above.
According to one preferred embodiment of the method according to the invention, the pH of the préparation obtained Is in addition adjusted to 7.1 + 0.1, before mixing the Hib antigen with the AlOOH/HBsAg complex. It has in fact been noted that such a pH value has a positive effect on keeping the Hib antigen in the nonadsorbed state.
According to one particular embodiment, the pH Is in addition adjusted to 7.1 + 0.1 after the mixing phase.
Thus, by virtue of the method according to the Invention, it is possible to obtain a vaccine composition in which:
(i) at least 60% or 80%, preferably at least 85%, of the total amount of the hepatitis B surface antigen présent in the composition is adsorbed on the AIOOH for at least 3 months starting from the date of manufacture of the composition stored at a température of 5 + 3*C,· and (ii) at least 65%, 70% or 75% of the total amount of the Hib antigen présent in the composition Is not adsorbed on the AIOOH.
The expression AlOOH/HBsAg complex should be Interpreted as meaning that the complex comprises at least the HBsAg antigen adsorbed on AIOOH. The complex may contain other antigens, whether that is specified or not.
One or more additional antigène may In addition corne to form the complex. They may ln particular be diphtheria toxoid (D), tetanus toxoid (T), whooping cough acellular antigens such as: Bordetella pertussis detoxified toxin (PTxd), filamentous hemagglutinin (FHA), pertactin (69 kDa antigen) and agglutinogens (fimbriae) of this same bacterium. According to one particularly advantageous embodiment, D, T, PTxd and FHA antigens may be added in order to form the complex.
The additional antigens may be added In various ways. They may be added sequentialiy following the hepatitis B surface antigen preadsorbed (i) either onto the total amount of AIOOH that needs to be présent in the vaccine composition; (ii) or onto a partial amount, subsequently added to ln order
-8to reach the total amount. Alternatively, the additional antigens may be adsorbed separately, each onto a partial amount of AIOOH just like the HBsAg. A mixed adsorption process may also be provided for - some antigens being adsorbed separately, others being adsorbed sequentially.
According to one particular embodiment of the method according to the Invention, the composition obtained Is stirred after the addition of each antigen.
According to one advantageous embodiment, given only by way of example, the HBsAg is adsorbed separately onto a partial amount of AIOOH corresponding to approximately 30% (one third) of the total amount of AIOOH présent in the final composition. In parallel, the D and T antigens are sequentially adsorbed onto the additional part of the AIOOH. The PTxd and FHA whooplng cough antigens are then added to the préparation containing the AIOOH-D-T complex, each of these two whooplng cough antigens themselves having been Indivldually preadsorbed onto AIOOH. Finally, the two préparations (AIOOH - HBsAg complex and AIOOH - D-T-PTxd-FHA complex) are combined so as to form a préparation comprising the AIOOH - HBsAg-D-T-PTxd-FHA In which the amounts of each of the éléments hâve been chosen so to obtain vaccine doses of 0.5 ml comprising, conventionally:
- from 5 to 15 pg of HBsAg / dose; preferably 10 pg / dose,
- from 20 to 40 Lf of D / dose; preferably from 25 to 35 Lf / dose; entirely preferably 30 Lf / dose (Lf = llmit of flocculation) (expressed In another way, the amount of D Is greater than or equal to 20 IU / dose),
- from 5 to 25 Lf of T / dose; preferably from 10 to 15 Lf / dose; entirely preferably 10 Lf / dose (expressed In another way, the amount of T is greater than or equal to 40 III / dose),
- from 20 to 30 pg of FHA / dose; preferably 25 pg / dose,
- from 20 to 30 pg of PTxd / dose; preferably 25 pg / dose.
According to one particular embodiment of the invention, polio antigens, which conventionally consist of inactivating viruses, are also added. It can be envisioned to add polioviruses of the 3 types usually présent in pédiatrie vaccines, I.e. types 1, 2 or 3, or else, In the case where it would not be necessary to vaccinate against the 3 types, to Introduce only the types against which protection Is sought. The amounts of poliovirus per dose may In particular be:
- between 20 and 43 DU (D antigen units), In particular 40 for type 1,
- between 5 and 9 DU, In particular 8, for type 2,
- between 17 and 36 DU, In particular 32, for type 3.
-9These antigens are not necessarily preadsorbed onto an aluminum sait before being added to the vaccine préparation.
According to one particular embodiment, the method according to the invention is a method wherein:
(i) (a) the HBsAg and the AIOOH are brought into contact ln the absence of any other antigen, and the HBsAg is allowed to adsorb onto the AIOOH for at least 4 hours, preferably at least 12 hours, entirely preferably approximately 24 hours, so as to obtain a préparation containing an AlOOH/HBsAg complex;
(i) (b) a préparation comprising the D, T, PTxd and FHA antigens, preadsorbed onto AIOOH, and optionally additional Bordetella pertussls antigens such as pertactin and agglutinogens, is added to the préparation obtained ln point (i) (a);
(ii) phosphate Ions are added to the préparation obtained in point (i) (b) in order to obtain a final concentration ln the vaccine of 40 mMol/l;
(iii) polio antigens are optionally added to the préparation obtained ln point (ii);
(iv) a préparation containing the Hib antigen ls added to the préparation obtained in point (ii) or (iii);
(v) the pH is adjusted to 7.1 + 0.1; and (vi) (a) at least one catlonrc amino add is added so as to complété the préparation obtained in point (il) or (iii) before the addition of the Hib antigen, or (b) at least one cationlc amino add is added to the Hib antigen;
said cationic amino acid being added in an amount suffident to obtain a final concentration ln the vaccine of at least 100 mg/l.
According to one particular embodiment, the vaccine composition according to the Invention is a composition comprising HBsAg, diphtheria toxin D, tetanus toxin T, the PTxd and FHA whooping cough antigens having been preadsorbed onto AIOOH, the Hib antigen and, optionally, the polio valence, in which:
(i) at least 85%, preferably at least 90%, of the total amount of the HBsAg présent ln the composition is kept adsorbed on the AIOOH for at least 3 months starting from the date of manufacture of the composition stored at a température of 5 + 3“C; and (ii) at least 65%, 70% or preferably 75% of the total amount of the Hib antigen présent in the composition is not adsorbed on the AIOOH, this amount remaining relatively stable over time.
-ιοIn addition, by virtue of the method according to the invention, the antigens other than HBsAg which showed that It was advantageous for them to be adsorbed onto aluminum oxide hydroxide In order to be immunogenic, are also kept adsorbed.
Thus, it is indicated, by way of example, that a composition according to the invention may 5 comprise:
- from 10 to 30 pg of HBsAg /ml; preferably 20 pg/ml;
- from 40 to 80 Lf of D/ml, preferably from 50 to 70 Lf /ml;
- from 10 to 50 Lf of T/ml, preferably from 10 to 30 Lf /ml;
- from 40 to 60 pg of FHA /ml; preferably 50 pg/ml;
- from 40 to 60 pg of PTxd/ml; preferably 50 pg/ml;
- from 2 to 60 pg of PRP/ml; preferably 20-24 pg/ml;
- from 1 to 2 mg of AI00H/ml; preferably 1.2 mg of AlOOH/ml;
- phosphate Ions at a concentration of 35 to 45 mMol/l, preferably from 38 to 42 mMol/l of phosphate Ions;
- from 100 to 1000 pg/ml of cationic amino acids, preferably from 400 to 800 pg/ml; and optionaily
- poliovirus types 1, 2 and 3 in inactivated form, In a respective amount of 80,16 and 64 DU/ml. As previously Indicated, a composition according to the Invention may also comprise additional Bordetella pertussis antigens, such as pertactin (of 69 kDa) or agglutinogens.
Description of the figure: Figure 1 Is a scheme of a prior art method wherein mixing is carried out after the addition of each component.
EXAMPLE - Industrlal-scale préparation of a bulk (250 I) of a HepB-Dt-Tt-Pertuss/s-polio-HiB hexavalent vaccine
This préparation is carried out under stérile conditions and with continuous stirring.
A - Préparation of HBsAg adsorbed onto AIOOH
A homogeneous suspension of aluminum oxide hydroxide (AIOOH) gel sold by Brenntag AG, at 8 g 30 of aluminum/l, Is Introduced aseptically Into a 501 tank.
- n After filtration through a 0.22 pm filter, the volume of HBsAg required to obtain a concentration of 20 pg/ml in the final vaccine Is continuously added to the tank already containing the AIOOH.
The mixture is left to stir for 20 to 24 hours at ambient température so as to obtain a homogeneous suspension.
B * Préparation of D + T + PTxd + FHA adsorbed onto alumlnum gel ln parallel, a mixture of aiuminum gel, diphtheria toxoid (D), tetanus toxoid (T), Bordetella pertussis toxoid (PTxd) and Bordetella pertussis fîlamentous hemagglutinin (FHA) is prepared in the following way:
A homogeneous suspension of AIOOH gel sold by Brenntag AG, at 8 g of aluminum/1, is introduced asepticaliy into a 2501 tank.
The solutions of D and then, after homogenization, of T are successïvely introduced, after filtration through a 0.22 pm filter, into the 250 I tank already containing the AIOOH, in order to obtain respective D and T concentrations ln the final vaccine of 60 Lf (timit of flocculation)/ml and 20 Lf/ml.
Once the homogeneous mixture has been obtained, the suspension of PTxd preadsorbed onto AIOOH and then the suspension of FHA preadsorbed onto AIOOH are successïvely added asepticaliy into this tank, in order to obtain PTxd and FHA concentrations in the final vaccine of 25 pg/ml.
Finally, the volume of 500 mM of phosphate buffer required to obtain a phosphate ion concentration of 20 to 30 mMol/l is added after filtration through a 0.22 pm filter.
The resulting D-T-PTxd-FHA-AlOOH suspension is left to stir for at least 14 hours at a température of5 + 3’C.
C - Préparation of the mixture of HBsAg + D + T + PTxd + FHA adsorbed onto aiuminum gel
The préparation obtained in point A is added asepticaliy to the préparation obtained in point B.
This mixture is left to stir so as to obtain a homogeneous suspension.
The volume of 500 mM phosphate buffer required to obtain a phosphate ion concentration of 40 mMol/l in the final composition Is then added after filtration through a 0.22 pm filter.
D - Saturation of the eiectrostatlc sites of the alumlnum gel / HBsAg + D + T + PTxd + FHA complex with a solution of amino acids ,
- 12A solution of amino acids containing 12 essential amino acids, having the following composition, Is prepared:
| - Arginine hydrochloride...... - Cystine........................ | 2.1 g/l, I.e. 1.73 g/l of arginine 1.2 g/l | |
| - Histidine..................... | ... 0.8 g/l | |
| - Isoleucine................... | 2.6 g/l | |
| - Leucine...................... | ... 2.6g/l | |
| - Lysine hydrochloride.. | 3.65 g/l, i.e. 2.91 g/l of lysine | |
| - Méthionine................. | 0.75 g/l | |
| - Phenylalanine.............. | 1.65 g/l | |
| - Threonine................... | 2.4 g/l | |
| -Tryptophan................. | 0.4 g/l | |
| -Tyrosine..................... | .. 1.8 g/l | |
| - Vallne........................ | .. 2.35 g/l |
That is to say 21.2 g/l of amino acids, including 5.44 g/l of cationic amino acids (His - Arg - Lys). 450 ml of 2.5 N sodium hydroxide (NaOH) are added (0.5 l/min). The homogenization is allowed to continue with stirring for 10 min.
This amino acid solution, filtered through a 0.22 pm filter, is continuously added to the mixture obtained in C, so as to obtain a concentration of 572 pg/ml of cationic amino acids in the final composition.
E - pH Adjustment
The pH of the suspension obtained în point D is adjusted to pH 7.1 (7.0 - 7.2) using a filtered stock solution of sodium hydroxide at 2.5 N.
F - Addition of the polio antigens
A préparation containing poliovirus serotypes 1, 2 and 3 in inactivated form (Mahoney, MEF-1 and
Saukett strains, respectively). filtered through a 0.22 pm filter, is then introduced into the tank containing the suspension obtained in E.
G- Addition ofPRP-T
-13An Intermediate solution of PRP-T Is first of ail prepared In the following way: Tris-sucrose buffer, filtered beforehand through a 0.22 pm filter, is added to a préparation of PRP-T filtered through a 0.22 pm filter, so as to constitute an Intermediate mixture.
This mixture 1s introduced aseptically Into the mixture obtained In F.
H - Final adjustment phase
After homogenization of the suspension obtained in G, an amount of prefiltered injection-grade water sufficlent to reach the target volume of 250 I Is added. Then, if necessary, the pH of the mixture Is adjusted to pH 7.1 +0.1 by adding a prefiltered 2.5 N sodium hydroxide or 10% acetic add solution.
The mixture Is stored at 5 + 3eC , then distributed Into syringes or bottles In a proportion of 0.5 ml/dose.
One 0.5 ml dose thus contains 600 pg of Al3*. 10 pg of HBsAg, no less than 20 IU of D, no less than 40 IU of T, 25 pg of Pt, 25 pg of FHA, between 20 and 43 DU (D antigen units) of polio type 1, between 5 and 9 DU of polio type 2, between 17 and 36 DU of polio type 3, 12 pg of PRP (in the form of PRP-T), phosphate Ions at a concentration of 40 mMol/l, Tris-sucrose buffer at a concentration of 2.5 mMol/l of Tris and of 2.125% of sucrose, and also 286 pg of cationic amino adds (His - Arg - Lys).
Cilnlcal trial
The vacdne composition prepared according to the example above was tested in a dinical trial, compared to a hexavalent vacdne already présent on the market, called Infanrix Hexa™, which makes It possible to vaccinale children against the same diseases as the vacdne prepared according lo the Invention (diphtheria, tetanus, whooping cough, polio, Hib Infections and hepatitis B), but which has In particular the drawback that a part of it is iyophiiized and therefore requires an operation to take up the lyophilisate prior to the administration procedure.
During the dinical trial, the 2 types of vaccine compositions were administered to children, In a vacdne scheme comprising 3 doses administered at 2, 4 and 6 months. The liquid vacdne prepared according to the method of the invention proved to be very well tolerated, and as immunogenic as the vacdne présent on the market.
EXPERIMENTAL DATA
Percentage adsorptlon of HBsAg/amount of nonadsorbed PRP-T
- 14Three batches of final bulk product (PFV39-41-42) and also three batches distributed Into bottes (S12-13-14) - ail the batches having been obtained according to the example provided - were stored at + 5’C and analyzed at various times over a period of 9 and 22 months, respectively. The analysis related to the percentage adsorptîon of HBsAg and the amount of nonadsorbed PRP-T.
The percentage adsorptîon of the HBsAg was determined, as was previously Indicated, from the total HBsAg content and the nonadsorbed HBsAg content, the HBsAg détermination being carried out using a sandwich ELISA method according to the rules defined by the European pharmacopeia
2.7.1. Briefly, the HBsAg was captured by an anti-HBsAg primary monoclonal antibody of IgM type, in wells of a 96-well plate. The HBsAg thus bound was coated with an anti-HBsAg secondary monoclonai antibody, of IgG type, which was itself detected by means of a peroxidase-coupled antiIgG polyclonal antibody. A chromogenic substrate for peroxidase, tetramethylbenzidine (TMB), served as a developing agent, When it was added, a color developed, the intensity of which was proportionai to the amount of HBsAg captured in the well. The results were analyzed according to the parallel line method described In the European pharmacopeia 5.3.3.
In order to détermine the percentage adsorptîon of the HBsAg, the vaccine was subjected to centrifugation (8800 g; 5 min; 20“C), which made it possible to recover the supematant containing the nonadsorbed HBsAg. The samples of supematants to be tested were diluted In ELISA buffer comprising a desorption buffer, In 2-fold serial dilutions In a range Included, for example, between 1/400 and 1/12 800.
The total-vaccine and standard-rangé samples were diluted In ELISA buffer comprising a desorption buffer, in 2-fold sériai dilutions in a range induded, for example, between 1/8 and 1/25 600.
A 96-well plate coated with the primary monoclonal antibody was incubated for 12 h at 5’C and then washed with a PBS-Tween 20 solution. The dilutions of the supematants, of the total vaccine and of the standard range were distributed Into the wells. The secondary antibody was then added and the deveioping was carried out with the peroxidase-conjugated antibody and TMB (tetramethylbenzidine). The reaction was stopped by adding 1 N HCl. After each step, the plate was Incubated for 30 min at 25’C and then subsequently washed with the PBS-Tween 20 solution. A blank (dilution buffer) was added to the available wells and underwent the same treatments. The plate was read at OD 450 and 630 nm.
The amount of nonadsorbed PRP-T was evaluated by HPAEC-PAD (high performance anion exchange chromatography - puised amperometric détection) in the following way:
A standard range of reference PRP-T of 0.5 to 12.5 pg/ml was first of ail prepared. ~
-15The samples to be tested and also the standard-rangé samples were centrifuged at 5000 g for 5 min at ambient température. The supematants were collected and then hydrolyzed with a 1.5 N NaCl solution containing glucosamine-1 -phosphate as Internai standard. A blank (0.9% NaCl; 1.5 N NaOH + Internai standard) was added.
The chromatography was carried out with a mobile phase composed of 35 mM NaOH and 114 mM of sodium acetate, Injected into the column at a rate of 1.2 mi/min.
The concentration of nonadsorbed PRP (pg/ml) was calculated using the following equality: (Surface area of the PRP peak / surface area of the internai standard peak) = a x [PRP concentration] + b in which a Is the slope and b is the intercept on the y-axis, a and b having 10 been determined from the régression line.
The results are given in the 4 tables below:
| Percentage adsorption of HBsAg in the formulation of the final bulk product (PVF 39-41-42) at +5’C | |||
| Time elapsed after the formulating operation | PFV39 | PFV41 | PFV42 |
| 0 | 95 | 97 | 98 |
| 1 month | 92 | 93 | 95 |
| 2 months | 92 | 92 | 93 |
| 3 months | 91 | 90 | 91 |
| 4 months | 89 | 90 | 92 |
| 5 months | 85 | 88 | 91 |
| 6 months | 89 | 88 | 89 |
| 9 months | 88 | 86 | 91 |
| Percentage adsorption of HBsAg in the formulation stored in bottles (batches S12-S13-S14) at +5’C | |||
| Time elapsed after | S12 | S13 | S14 |
| the formulating operation | |||
| 0 | 95 | 97 | 98 |
| 4 months | 88 | 88 | 90 |
| 5 months | 85 | 86 | 88 |
| 7 months | 85 | 80 | 86 |
| 10 months | 83 | 84 | 87 |
| 13 months | 82 | 86 | 84 |
| 16 months | 81 | 83 | 85 |
| 22 months | 83 | 78 | 86 |
| Nonadsorbed PRP-T (pg/ml) in the formulation of the final bulk product (PVF 39-41-42) at +5’C | |||
| Time elapsed after the formulating operation | PFV39 | PFV41 | PFV42 |
| 0 | 22.6 | 20.0 | 21.0 |
| 1 month | 23.1 | 19.5 | 20.4 |
| 2 months | 23.3 | 21.6 | 22.0 |
| 3 months | 24.9 | 22.6 | 23.1 |
| 4 months | 24.2 | 22.0 | 20.7 |
| 5 months | 22.2 | 22.7 | 24.5 |
| 6 months | 27.7 | 22.9 | 21.6 |
| 9 months | 27.0 | 24.8 | 23 |
Nonadsorbed PRP-T (pg/ml) in the formulation stored in bottles
- I7-
| (batches S12-S13-S14) at +5°C | |||
| Time elapsed after the formulating operation | S12 | S13 | S14 |
| 0 | 22.6 | 20.0 | 21.0 |
| 4 months | 21.1 | 21.1 | 19.8 |
| 5 months | 23.0 | 19.6 | 18.6 |
| 7 months | 23.1 | 21.0 | 19.5 |
| 10 months | 25.0 | 23.4 | 21.8 |
| 13 months | 24.6 | 22.6 | 20.8 |
| 16 months | 23.5 | 22.3 | 20.8 |
| 22 months | 23.9 | 22.0 | 19.9 |
For comparison, the adsorption over time of the HBsAg contained in three batches of final bulk product (FDN5-6-7) and also in three batches distributed into bottles (S 44-45-46), of a liquid préparation composed of the same antigens but obtained according to a linear formulation method 5 described in figure 1 and therefore different than that described in the example above, was also tested. This préparation contained, in 0.5 ml: 10 pg of HBsAg, 30 Lf of Dt, 10 Lf of Tt, 25 pg of Pt, 25 pg of FHA, 40 DU of poliovirus type 1, 8 DU of poliovirus type 2, 32 DU of poliovirus type 3, 12 pg of PRP (in the form of PRP-T), 0.6 mg of Al, phosphate ions at a concentration of 55 mMol/l, carbonate ions at a concentration of 20 mMol/l, Tris-sucrose buffer at a concentration of 2.5 mMol/l 10 with respect to Tris and 2.125% with respect to sucrose, and 14 pg of cationlc amino acids (His Arg - Lys) originatïng from the M199 medium (polio valence), pH 6.8 - 7.2 .
The results obtained were the following:
| Percentage adsorption of HBsAg in the formulation ofthe final bulk product (FDN5-6-7) at +5’C | |||
| Time elapsed after the formulating operation | FDN5 | FDN6 | FDN7 |
| 0 | 81 | 85 | 81 |
| 1 month | 78 | 82 | 62 |
| 2 months | 74 | 78 | 63 |
| 3 months | 66 | 84 | 62 |
| 6 months | 61 | 77 | 61 |
| Percentage adsorption of HBsAg in the formulation stored ln bottles (batches S44-S45-S46) at +5*C | |||
| Time elapsed after the formulating operation | S44 | S45 | S46 |
| 0 | 81 | 85 | 81 |
| 7 months | 63 | 64 | 77 |
| 10 months | 57 | 65 | 51 |
| 13 months | 47 | 54 | 53 |
| 16 months | 33 | 56 | 38 |
| 22 months | 44 | 47 | 36 |
| 28 months | 44 | 51 | 44 |
| 34 months | 45 | 54 | 42 |
| 40 months | 49 | 52 | 48 |
The amounts of nonadsorbed PRP-T measured over the same period showed that there was little 5 variation compared with time 0, and were therefore satisfactory. However, these results show that, ln this case, which does not correspond to a formulation obtained by vlrtue of a method according to the invention, the hepatitis B surface antigen did not remain adsorbed on the alumînum oxide hydroxide.
Experimental data relatlng to the catlonic amino acids
-19A composition according to the Invention resulting directly from the experimental protocol carried out In the example provided and a composition obtained by virtue of a protocol modified In that a composition containing only the three cationic amino acids (Arg - Lys - His) had been substituted for the composition of the 12 essential amino acids were compared with regard to the nonadsorbed 5 amount of PRP-T at the end. No différence In the amount of nonadsorbed PRP-T was observed, thereby showing that only the cationic amino acids are Important.
Claims (14)
- Claims1. A method for preparing a liquid vaccine combination comprising at least:- alumlnum oxide hydroxide (AIOOH),- one hepatitis B surface antigen (HBsAg),- one Haemophilus Influenzae type b (Hib) antigen consisting of capsular polysaccharide conjugated to a carrier protein, in which the hepatitis B surface antigen is kept adsorbed on AIOOH, whereas the Hib antigen Is kept nonadsorbed, wherein:- the hepatitis B surface antigen is adsorbed onto AIOOH in order to obtain anAlOOH/HBsAg complex,- said AlOOH/HBsAg complex is mixed with the Hib antigen in the presence of cationic amino acids at a concentration of at least 100 mg/l and of phosphate ions at a concentration of 35 to 45 mMol/l.
- 2. The method as claimed în claim 1, wherein the HBsAg antigen is adsorbed onto the aluminum by mixing a suspension of AIOOH with a suspension of HBsAg with stirring for at least 4 hours, preferably at least 12 hours, preferably between 20 and 24 hours.
- 3. The method as claimed in either of the preceding claims, characterized In that the cationic amino acids are added to said AlOOH/HBsAg complex before the mixing with the Hib antigen.
- 4. The method as daimed in either of daims 1 and 2, charaderized in that the cationic amino adds are added to said Hib antigen before the mixing with the AlOOH/HBsAg complex.
- 5. The method as daimed in one of the preceding daims, charaderized In that the phosphate ions are added to said AlOOH/HBsAg complex before the mixing with the Hib antigen.
- 6. The method as daimed In one of the preceding daims, characterized in that the pH of the préparation comprising the AlOOH/HBsAg complex Is adjusted to 7.1 + 0.1 before the mixing with the Hib antigen.
- 7. The method as daimed in claim 1, characterized in that it also consiste in:- preparing a composition comprising at least one antigen chosen from diphtheria, tetanus, polio and whooping cough antigens, and also aluminum oxide hydroxide, and- mixing said AlOOH/HBsAg complex with said composition, before carrying out the mixing with the Hib antigen.
- 8. The method as daimed In claim 7, characterized in that it consists in preparing said composition by adding each of the antigens successively to a suspension of aluminum oxide hydroxide and by stirring between each addition of antigens.
- 9. The method as daimed in claim 1, charaderized in that:- the HBsAg Is adsorbed onto a partial amount of AIOOH representing one third of the total AIOOH présent in the final composition, fora period of 20 to 24 hours, in order to form an AlOOH/HBsAg complex,- in parallel, the following are successively adsorbed onto an additional amount ofAIOOH: diphtheria toxin D, tetanus toxin T, Bordetella pertussis purified toxin PTxd, itself preadsorbed onto AIOOH, and Bordetella pertussis filamentous hemagglutinin FHA, Itself preadsorbed onto AIOOH, then phosphate ions are added thereto, then the AlOOH/HBsAg complex is added thereto,- phosphate Ions are again added in an amount which makes it possible to achieve a concentration of 40 mMol/1 In the final composition,- at least one cationic amino add Is added in an amount which makes it possible to achieve a concentration of at least 100 mg/l in the final composition,- the pH is adjusted to 7.1 + 0.1,- polio antigens in the form of inadivated type 1 and/or type 2 and/or type 3 viruses are added,- the Hib antigen is added,-22- the pH Is adjusted to 7.1 + 0.1,- the composition obtained Is distributed Into syringes or into bottles.
- 10. The method as daimed in one of the preceding daims, characterized in that at least 2501 of vaccine composition are prepared on an Industrial scale.
- 11. A vacdne composition obtained according to the method of any one of the preceding claims, and comprising at least the hepatitis B surface antigen (HBsAg) and the Hib antigen which consists of polyribosylri bitol phosphate conjugated to the tetanus protein (PRP-T).
- 12. The vacdne composition as daimed in the preceding daim, charaderized in that it also comprises diphtheria, tetanus, polio and whooping cough antigens.
- 13. The vaccine composition as daimed in either of claims 11 and 12, charaderized in that it comprises at least:- the hepatitis B surface antigen, HBsAg,- the diphtheria antigen In the form of diphtheria toxln D,- the tetanus antigen in the form of tetanus toxin T,- the whooping cough antigens in the form of Purified Toxin (PTxd) and of FilamentousHemagglutinin (FHA),- the Haemophilus influenzae type b antigen, in the form of polyribosylribitol phosphate conjugated to the tetanus protein (PRP-T),- the polio antigens In the form of inadivated viruses chosen from types 1,2 and 3.
- 14. The vacdne composition as daimed in daim 13, charaderized in that it comprises at least:- from 10 to 30 pg of HBsAg /ml, preferably 20 pg/ml;- from 40 to 80 Lf of D/ml, preferably from 50 to 70 Lf /ml;- from 10 to 50 Lf of T/ml, preferably from 10 to 30 Lf /ml;- from 40 to 60 pg of FHA /ml, preferably 50 pg/ml;-23- from 40 to 60 pg of PTxd/ml, preferably 50 pg/ml;- from 2 to 60 pg of PRP/ml, preferably 20-24 pg/ml, in PRP-T conjugate form;- from 1 to 2 mg of AI00H/ml, preferably 1.2 mg of AI00H/ml;- from 35 to 45 mMol/l, preferably from 38 to 42 mMol/l, of phosphate ions;5 - from 100 to 1000 mg/l of cationic amino acids, preferably from 400 to 800 mg/l;- poliovirus types 1, 2 and 3 in inactivated form, ln a respective amount of 80,16 and 64 DU/ml.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1250464 | 2012-01-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA16951A true OA16951A (en) | 2016-01-25 |
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