Note: Descriptions are shown in the official language in which they were submitted.
~ ~5"`3~3~7
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IMPROVED COMPLEXES
,
5PROCESSE~ FO~ OBTA~NING THEI~' AND FORMULAll IONS
CONTAIN~NG SUCH C~:)MPLEXE~i
__ __ _
The present invention relates to novel complexes comprising polysaccharide and
metal constituents and which are suitable for use in vaccines to provide
10protection against bacterial infections.
Certain bacteria, in addition to possessing an outer membrane structure which
forms the boundary of cell envelope, also have an additional layer outside the
membrane known as the capsule. An example of a gram negative bacterium
with both of these features, is Neisseria meningitidis (N. meningitidis). The
outer membrane of the cell envelope, contains a number of substances, including
lipopolysaccharides, pili (surface protrusions), major (high molecular weight)
proteins, minor (lo~ molecular weight) proteins, lipids, and lipoproteins. Of
these, the first three have been identi Fied as principal antigens. Another
important class of antigens comprises the constituents of the capsule, known as
capsular polysaccharides. Capsular polysaccharides and outer-membrane
proteins are believed to exist as a non-covalent complex on the exterior of the
bacterium. During growth, such bacteria continuously shed capsular polysac-
charides and outer-membrane prsteins, in their free and complexed forms. The
polysaccharides9 in both forms, can be precipitated from the culture by addition
of a suitable electrolyte, which also precipitates most negatively charged
polymers which are present.
The serology of N. meningitidis enables the associated capsular polysaccharide
and outer-membrane proteins to be classified according to a recognised nomen-
clature. There are nine recognised serogroups A, B, C, L, X, Y, Z, W135 and
29E, further characterised into serotypes numbered 1-15 (see for example
L.Weinstein and B.N. Fields (Eds), Seminars in Infectious Disease, Stratton
Intercontinental Medical Book Corp, 1979: Chapter 10; C.E. Frasch, Noncapsu-
lar Surface Antigens of N. ~, pp. 308-310). Different strains within a
group may possess the same serotype protein, and a number of ungroupable
strains have also been discovered. The capsular polysaccharides are specific to
particular serogroups (group specific polysaccharides) and the major outer
membrane proteins are specific to particular serotypes (type specific proteins).However, the determinants of serotypes 4, 5 and 8 are
RMW/CB/26th October 1984
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lipopolysaccharides rather than outer-membrane proteins. Thus, the capsular
polysaccharides may be referred to, merely by their serogroup specicifity (eg.
Group B polysaccharide) and the outer membrane proteins by their serotype
specificity, (e.g. type 2 protein). This convention is generally recognised, forexample, as referred to by: C.E. Frasch supra, C-M.Tsai et al in ~ournal of
Bacteriology Vol. 146 (1981) pp.69-78, C.E. Frasch et al in Journal of
Bacteriology, Vol 127 (1976) 973-981, N.A. Vendros in T.Bergan and J.R. Norris
(Eds), Methods in Microbiology, Vol 10, Academic Press, London: Chapter Xl,
serology of the Meningococcus, or one of the following references: C.E. Frasch
and S.S. Chapman, Infection and Immunity, Vol 6 (1972) pp. 674~681; J.T.
Poolman, C.T.P. Hopman and H.C. Zanen, F-EMS Microbiology Letters, Vol 3
(1982) pp. 339-34B. For convenience, meningococcal capsular polysaccharides
(MPS) of a particular serotype, say A or C, will be referred to by the
abbreviation MPS (A), MPS (C) etc, and meningococcal outer-membrane proteins
of a particular serotype say 2 or 6, will be referred to by the abbreviation T(2),
T(6) etc.
The Escherichia coli (E.coli) strain conventionally designated K1, contains a
...... ~ _ _ _
capsular polysaccharide known as colominic acid. This is substantially identicalin structure to MPS (B).
N.meningitidis normally inhabits the human nasopharynx and can cause the
serious and often fatal disease, cerebrospinal meningitis to which infants are
particularly vulnerable. _.coli K1 is also responsible for some cases of
meningitis in the new-born. Previous attempts to identify and isolate meningo-
coccal antigens, have concentrated on capsular polysaccharides and the principal
outer-membrane antigens referred to above, namely lipopolysaccharides, pili and
major proteins. The free capsular polysaccharides, MPS (A) and MPS (C) are
reasonably successful in conferring immunity against meningococcal strains
belonging to serogroups A and C respectively. Serogroup B has recently been
identified with increasing infantile meningococcal infection and the lack of a
vaccine effective against infection by group B meningococcal strains has createda growing demand for such a vaccine from international health authorities, eg
the World Health Organisation. Some immunity to Group B strains may result
from vaccination with MPS (A) or MPS (C) vaccines, but the protection provided
is generally not sufficient in infants, and vaccines based on MPS (B) alone do not
confer viable immunity against infection by group B strains.
RMW/CB/26th October 1984
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Previously proposed vaccines based on -Free MPS(B) suffer from the additional
disadvantage that they tend to be unstable. It is believed that this is because
capsular polysaccharides which include sialic acid, for example MPS(B) and
colominic acid, are prone to intra-molecular esterification between the polysac-charide residues (see R.Lifely, et al Carbohydrate Research, Vol 94 (1981)
193 -203).
In Canadian patent application S.N. 487,~48, filed July 25, 1985,
C. Moreno et al, there are described immunogenic,
substantially pyrogen -free compositions containing complexes o~ MPS(B) and
certain outer membrane proteins which are less prone to the aforementioned
instability oE free MPS(B).
We have now discovered that the stability of polysaccharides which contain sialic
acid, whether in the free form or in complex with bacterial outer-membrane
protein, may be enhanced by complexing with a metal. It is known that some
metals can complex with the capsular polysaccharides exuded by certain bacteria
of the genera Xanthomonas, Achromobacl:er and Pseudomonas (for example
Xanthomonas fuscans) when they inhabit the rhizosphers of plants. The
pharmacokinetics of complexes of iron and non-bacterial polysaccharides (used
for the treatment of anaemia) are described by Robuste et al in Cien. and Ind.
Farm., 9 (1977) 159-63. Belgian patent specification no. 889 979 discloses a
process for isolation of meningococcal and other capsular polysaccharide
antigens wherein prior to separation of the polysaccharide ~ se it is extracted
from culture as an intermediate complex with an organic quaternary ammonium
salt in the presence of an inert porous support such as kieselguhr. The above
references giYe no indication that sialic acid-containing polysaccharides may bestabilised by complexing with a metal.
Thus in one aspect the present invention provides a complex comprising a metal
con~:tituent and a bacterial capsular polysaccharide constituent, wherein the said
bacterial capsular polysaccharide constituent contains sialic acid.
The sialic acld-containing bacterial capsular polysaccharide may for example be
colominic acid or capsular polysaccharide specific to serogroup B of N.
tidis.
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Desirably, the said bacterial polysaccharide constituent is complexed with the
said metal constituent and also complexed with a further constituent comprising
bacterial (eg. meningococcal) outer-membrane protein. Such a complex of
metal, polysaccharide and protein is hereinafter termed a triple complex.
The metal constituent may for example be selected from metals in groups IIA,
IB, IIB and IIIB of the periodic table, as well as the transition metals, especially
those of group VIII. Preferred metals include aluminium, ruthenium, zinc, iron,
nickel and calcium. Other suitable metals may also include the following in all
of their various oxidation states: lithium,, sodium, magnesium, potassium,
scandium, titanium, vanadium, chromium, manganese, cobalt, copper, gallium,
strontium, niobium, molybdenum, palladium, silver, indium, tin, tungsten,
rhenium, platinum, gold and gadolinium. The metals are preferably provided in
ionic form, (preferably derived from an appropriate metal compound) for
example the A13+, Ru3+, Zn2+, Fe3+, Ni2+ and Ca2+ ions. Especially preferred
metals are aluminium and ruthenium, particularly in the form of the A13+ and
Ru3~ ions respectively. Such metal ions may be present in the complex alone or
with other inorganic ions, eg-originating from metal salts from which the
complex according to the invention is prepared (as further described
hereinbelow). Thus for example, ruthenium ions may be present in the complex
with one or more types of ion such as hydroxy, oxychloride and ammonium ions.
When present, the outer-membrane protein constituent may for example be
selected from any of the proteins found in the outer-membrane of any strain of
_. meningitl , especially those specific to serotypes 1-3, 6, 7 and 9-15 and
immunological equivalents thereof.
Outer-membrane proteins derived from mutants of N. meninqitidis strains may
be regarded as "immunologically equivalent" to those isolated from a particular
meningococcal serotype when the gel electrophoresis characteristic patterns of
the outer-membrane proteins of such mutants do not necessitate classification
of the mutant organisms in another recognised serntype, and their immunological
properties are not substantially altered. Such mutants may be found in nature ashas been found with mutants of serotype 2 (per Poolman et al supra) or may be
produced by techniques known to those skilled in the art.
Particularly preferred outer-membrane proteins are those specific to serotypes 6and 2 of N. rneningtidis, especially the serotype 2 protein having a single
dominant component having a molecular weight of 42,000 + 3,000.
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For convenience, reFerences hereinafter to particular complexes or classes of
complex according to the invention are referred to by an abbreviation of their
constituents. For example, complexes lackin, a protein constituent are termed
metal-B, aluminium-B, aluminium-Col etc where 'B' refers to MPS(B) and 'Col' to
colominic acid. Triple complexes according to the invention are termed for
example metal-B2, aluminium-B6 etc where the number refers to the
meningococcal serotype specificity of a particular polysaccharide constituent,
and includes immunological equivalents as hereinbefore defined. The number '2'
includes meningococcal serotype 2 protein having a single dominant component
having a molecular weight of 42,000 + 3,000. Complexes of capsular polysac-
charide and outer-membrane protein (which are starting materials in the
preparation of certain complexes according to the present invention) are
designated simply as 32, B6 etc.
A particularly preferred class of complexes according to the present invention
are antigenic complexes, that is to say those complexes capable of raising
antibodies in a mammal inoculated therewith. When intended for therapeutic
use, such an antigenic complex may be provided in the form of an antigenic
composition, substantially free from bacterial cells and from lipopolysaccharide,
and wherein the metal constituent comprises a pharmacologically acceptable
metal, preferably as an ion of the metal. Such a composition is substantially
free from lipopolysaccharide in that it contains a weight percentage of lipopoly-
saccharide insufficient to produce significant toxic effects when a given amountof the composition is administered to the human or animal body. For normal
administration doses of the composition, the weight percentage will be generally1% or less. The lipopolysaccharide may be present in the free form or associatedwith another constituent of the composition, for example the polysaccharide
protein complex.
The pharmacologically acceptable metal referred to above as a constituent of a
complex contained in an antigenic compostion must be pharmacologically accep-
table in the sense that its identity, chemical form (e.g. ion) and amount must be
tolerated by the recipient for as long as it remains in the body thereof.
We have found that when experimental animals are immunised with an antigenic
composition containing an antigenic triple complex according to the invention,
antibody levels with respect to both serogroup-and serotype-specific antigens are
increased, as compared with antibody levels achieved by immunisation with only
a polysaccharide-protein complex. This contrasts with the well-known adjuvant
RMW/OB/26th October 1984
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effect oF certain metal salts (including hydroxides), which would be expected toenhance antibody level only with respect to protein carriers such as serogroup-
specific antigens. This observation provides evidence that the metal constituento-f the triple complexes is actually compl0xed with the polysaccharide and
protein constituents. We have also obtained an indication of complexing of
metals with free capsular polysaccharides which contain sialic acid, by NMR and
electrochernical measurements on an aluminium~Col complex, and by counter
immuno-electrophoresis measurements of MPS(B) concentrations in solution as a
function of time, with and without the presence of aluminium.
We have found that complexes according to the present invention usually have an
apparent molecular weight of at least 2 x 107 when determined by
chromatography on Sepharose CL-2B (Trade Mark). We have also observed that
in complexes according to the invention which do not have a protein constituent,and wherein the metal constituent is aluminium, the ratio of sialic acid to
aluminium is generally greater than 1:0.3 w/w.
Complexes according to the invention which have a protein constituent may be
subjected to gel electrophoresis, particularly sodium dodecyl sulphate-polyacry-
lamide gel electrophoresis (SDS-PAGE) to provide patterns characteristic of the
components of the said protein constituent. For example, one may employ the
SDS-PAGE procedure described by U.K. Laemrmli in Nature (London~ Vol 227
(1970) pp.680-685. The serotype specificity of meningococcal proteins in a
complex according to the invention may for example be verified by comparlson
of its pattern with those given in the above literature references concerned with
meningococcal serology and its experimental determination eg. C.E. Frasch et
_ in Journal of Bacteriology supra.
The serogroup specicifity of the capsular polysaccharide component of a complex
according to the invention may be verified by any appropriate method of
compositional analysis known to those skilled in the art. The sialic acid content
of the polysaccharide consituent may be determined by colorimetric methods.
The polysaccharide component may also be determined by nuclear rnagnetic
resonance, or immunologically with specific antisera.
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~7~ A700
The. antigenic compositions according to the present invention may optionally
include one or more other antigenic components e.g. free MPS(A) and MPS(C) as
well as one or more other complexes of men;ngococcal capsular polysaccharide
and outer-membrane protein such as MPS(B)/T(7), MPS(C)/T(2) or MPS(A)/T(2) in
order to provide a broader spectrum of immunity. The antigenic compositions
may simultaneously contain more than one complex according to the present
invention, for example aluminium-B2 and aluminium-B6.
lû
In another aspect, the present invention also provides a process for preparing acomplex according to the present invention, comprising bringing into association,
bacterial capsular polysaccharide which contains sialic acid and, a metal.
Where the desired product is a triple complex, the capsular polysaccharide
starting material may be provided in the form of a complex of the polysaccha-
ride with bacterial (eg. rneningococcal) outer-membrane protein, for example
any of the proteins referred to above. The metal may for example be any of
those indicated previously, and is generally employed as a compound thereof (eg
as an ion of a metal salt). It is desirable that the metal and polysaccharide are
brought into association in solution. This may entail admixture of a solution
containing the polysaccharide and a solution containing the metal (eg. as a metal
ion derived from a salt therof), and optionally if desired, incubating the resulting
mixture. Alternatively the two solutions may be brought into association by
dialysis of one against the other. Preferably the solvent in each case is water,optionally containing one or more solubilising agents. Suitable metal salts
include water soluble salts such as those derived from inorganic and organic acid
anions, for example halides (ie. fluorides, chlorides, bromides and iodides)
sulphites, sulphates, nitrites, nitrates, phosphates, alkanoates (eg acetates),
3U fumarates, benzoates, succinates, phthalates and oxalates. When the metal is
ruthenium, the salt conveniently may be "ruthenium red'l that is ruthenium
oxychloride, either in its normal or ammoniated form.
The polysaccharide or polysaccharide-protein complex used as a starting
material in the process according to the invention, may be prepared by any
method known to those skilled in the art, for example as described in the
aforementioned references, or as disclosed in the aforementioned Canadian
patent application S.N. 487,448. The latter application describes
a process for the isolation of a complex of a
RMW/CB/26th October 1984
,9~7
bacterial capsular polysaccharide and a bacterial outer~rnembrane protein, the
process comprising the steps:
(i) culturing in a medium, a bacterium which possesses an outer-membrane
and capsule and obtaining an aqueous phase which includes a complex of a
bacterial capsular polysaccharide and a bacterlal outer-membrane protein;
(ii) admixing the aqueous phase obtained in step (i) with a quaternary
ammonium salt, to effect precipitation oF a precipitate containing the said
complex;
(iii) admixing the precipitate obtained in step (ii) with a water-soluble salt of
calcium or magnesium in an aqueous rnedium to form an aqueous solution
which includes said complex as a solute;
(iv) admixing the aqueous solution obtained in step (iii) with a lower alkanol, to
effect precipitation of a precipitate containing the said complex; and
(v) separating the complex from any other components present in the precipi-
tate resulting from step (iv).
The latter process provides the desired complex in a relatively pure form,
substantially free, for example, From complexes other than that it is intended to
isolate, lipopolysaccharides, lipids, nucleic acids and other impurities, and
enable one to obtain the complexes dlrectly.
3~ In performing the latter process, the aqueous phase is desirably obtained before
the culture has reached a stationary phase, in order to minimise the release of
lipopolysaccharide. It is also advantageous to remove the bacterial cells and cell
debris from the aqueous phase, for example by centrifugation, before performing
the precipitation of step (ii), although such removal may be effected at any
appropriate stage of the process. In step (ii), the quaternary ammonium salt is
desirably cetyl-trimethylammonium bromide, ie Cetavlon (Trade Mark) or cetyl-
pyridinium chloride. Such salts are used to form an insoluble complex salt with
the free capsular polysaccharide which is present~ and thus they are readily
eliminated in steps (iii) and (iv). As well as the desired complex, the precipitate
formed in this step of the process also contains most negatively charged
RMW/CB/26th October 1984
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polymers which are present in the aqueous phase, ie proteins (including those
from the outer-membrane), lipopolysaccharide and partially degraded nucleic
acids. When Cetavlon is used, the step (ii) p~ecipitation is desirably per-formed
at a temperature in the range from 12 to 25C, optirnally at or about 18C.
When the salt is cetylpyridinium chloride, the precipitation may be performed ata temperature in the range 0-10C, optimally at or about 4C. In either case,
the salt is preferably present at around 1% w/v.
The water-soluble salt referred to in step (iii) is preferably c alcium chloride or
aluminium chloride . As well as the complex, the solution in step (iii) also
contains, as a solute, the other components present in the precipitate formed instep (ii), but not the capsular polysaccharide complex salt, which is thus
eliminated.
In performing the latter process, the alkanol is used in step (iv) in an amount and
at a concentration so that there occurs subtantially no dissociation of any
polysaccharide/protein complex present. Preferably, the alkanol is used to
provide a concentration in the range of 50-95% v/v when in admixture with the
aqueous solution. A particularly preferred concentration is about 75%. As used
herein, the term "lower alkanol" denotes an alkanol containing 1 to 4 carbon
atoms, for example ethanol or methanol. In addition to the complex, the
precipitate formed in step (iv) also contains some low molecular weight
impurities such as non-outer-membrane protein, low molecular weight nucleic
acid fragments and a little lipopolysaccharide. These impurities are
substantially eliminated in the separation in step (v), which is preferably
performed by gel filtration, eg using Sepharose CL-2B or any other system
having similar properties.
3û
Further purification, if required, may be carried out on the product of step (v) in
order to minimise the presence of contaminating materials such as degraded
nucleic acids and uncomplexed polysaccharides and proteins.
The present invention further provides a vaccine formulation comprising an
antigenic composition according to the present invention and at least one
adjuvant and/or carrier therefor. In such formulations, the composition may
include further antigenic componer~ts described above, such as one or more free
meningococcal capsular polysaccharides. A vaccine intended to provide protec-
tion against meningococci oF serogroups B and A and/or C can contain respec
RMW/CB/26th October 1984
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tively a mixture oF one or more complexes according to the invention, together
with MPS (A) and/or MPS (C). Additionally or alternatively, such formulations
may contain, if desired, complexes according to the invention, and one or more
other complexes of meningococcal capsular polysaccharide and outer-membrane
protein such as MPS(B)/T(7), MPS(C)/T(2) or MPS (A)/T(2), as well as more than
one complex according to the present invention, for example alurninium-B2 and
aluminium-B6.
The vaccine formulations according to the present invention may be presented in
a sterile form, such as is suitable for administration to humans. In these
formulations, the carrier may for example be water. Such a formulation may
additionally or alternatively contain one or more appropriate non-metal consti-
tuents such as one or more metal salts (provided as acljuvants and not in the form
of a complex), lactose as an antigen stabiliser, or one or more salts such as
sodium chloride, to render the vaccine isotonic with blood. Preferably, an
appropriate bufFer is also included. In such vaccines, the complex according to
the invention may conveniently be administered in a dosage of from 0.1 ~9 to 3
mg, desirably 1.0 to 300 ug, preferably about 50 1l9.
The invention further provides a process for preparation of a vaccine formula-
tion, the process comprising admixture of an antigenic composition according to
the invention and at least one adjuvant or carrier therefor. In the process, thevaccine may be rendered sterile, eg. by detergent-assisted filtration.
In another aspect, the invention provides an antigenic complex according to the
present invention for use in a method of prophylaxis or treatment of a bacterial(eg meningococcal or E. coli) disease of a mammal such as man. For example,
the antigenic complex may be used for the prophylaxis or treatment of
cerebrospinal meningitis. In such use, the antigenic complex may be presented inany formulation described herein. The antigenic complex may be administered in
one or more doses. If more than one dose is administered, it is desirable that the
administration is spaced over a suitable time scale to take advantage of
secondary immunisation. The antigenic complex may for example be adminis-
tered as two or three doses, For example over an interval one to three weeks.
Where appropriate, doses subse~uent to the first, may contain amounts of the
antigenic complex which are less than the amount contained in the primary dose.
The antigenic complex may be administered by any convenient route such as the
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parçnteral (e.g. sub-cutaneous, intravenous, intra-peritoneal), oral, or as may be
desirable -for infants, intra-nasal route. The invention also provides a method o~
prophylaxis or treatrnent of a bacterial (eg mcningococcal or E. coli) disease o-f a
mammal such as man, comprising administration of an effective amount of
antigenic complex according to the present invention to said mammal. The
antigenic complex is advantageously employed in the form of an antigenic
composition or a vaccine formulation according to the invention. The invention
includes complexes produced by the process of the invention, as defined above.
The following examples illustrate the present invention, reference being made tothe accompanying drawings, in which:
Figure 1 shows the elution profile of a typical crude B6 complex from a
Sepharose CL-2B chromatographic column by monitoring absorbance of the
fractions at 260 and 2S0 nm, and by sialic acid determination by the
resorcinol-HCl colorimetric method.
Figure 2 shows the elution profile of a typical crude B2 complex from a
Sepharose CL-2B chromatographic column by monitoring absorbance of the
fractions at 280 nm, and by sialic acid determination by the resorcinol-HCl
colorimetric method.
Figure 3 shows the elution profile upon Sepharose CL-2B re-chromatography of
the purified typical B6 complex by monitoring absorbance of the eluent at 250
nm, and by sialic acid determination by the resorcinol-BlCl colorimetric method.
Figure 4 shows the elution profile upon Sepharose CL-2B re-chromatography of
the purified typical B2 complex by monitoring absorbance of the eluent at 280
nm, and by sialic acid determination by the resorcinol-~lCl colorimetric method.
Figure 5 shows patterns obtained from SDS-PAGE of B2 and B6 complexes, and
molecular weight marker standards.
Figure 6 shows the elution profile upon Sepharoso CL-2B rechromatography of an
aluminium B-complex and pure MPS(B) as monitored for aluminium and sialic acid.
Figure 7 shows a polarographic DPP plot of peak current as a function at A13+
4û concentration in solution, with and without the presence of colominic acid.
RMW/CB/26th October l9S4
:1~5~`3~3~
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Figure 8 shows rounter immuno-electropho- esis measurement of the time-
dependent degradation of MPS(B) in solution, with and without various concen-
trations of A13+.
Example 1_Isolation and Purification of a B6 Complex
A culture medium was prepared as an aqueous solution of the following
ingredients, in the amounts stated, and the resultant solution was rnade up to
volume of 16 litres with added water.
per 16 litres
K2HPO4 3 . 68 9
L-Glutamic acid 20.8 9
Cysteine HCI 0.48 9
Na H CO3 13 . 47 9
Tricine, N tris
(hydroxymethyl)
methyl glycine11.46 9
Fe 5047H~O 0 045 9
NH4C1 8 . 0 9
254 0 . 77 9
Ca Cl22H2O solution,
7.4mg/100 ml 16 ml
Mg C126H2 1.71 9
Casein Hydrolysate320 9
RMW/CB/26th October 1984
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The~ pH of the solution was adjusted to p~l 7.2 and two 400 ml aliquots were
transFerred to two, one litre conical flasks. The remainder of the solution was
transferred to a 15 litre bottle. A siphon was connected to the bottle and both
flasks and the bottle were autoclaved for 15 minutes at a pressure of 15 lb/sq
inch. The contents of the bottle were then transferred to a 20 litre fermenter
and 300 ml of sterile 50% aqueous glucose was added, to form the medium. An
inoculum was prepared by adding one drop of a culture containlng a serogroup
B, serotype 6 strain of N.meningitidis, to both flasks containing 400 ml of the
solution.
The flasks were incubated at 37C, with shaking, for 12 hours, to form 800 ml
of seed culture, the purity of which was checked by the Grams stain technique.
All of the seed culture was then used to inoculate the medium in the fermenter.
The whole culture medium was then grown under the following experimental
conditions.
Temperature constant at 37C
pH constant at 7.2 with steril~e 2N HCl and 2N NaOH
Stirring speed 100-700 rpm automatically adjusted.
Dissolved oxygen minimum 10% saturation.
Air flow 1-5 litre per minute adjusted manually to
maintain dissolved oxygen concentration.
Antifoam Polypropylene glycol added manually when required.
The culture was allowed to grow until an optical density of approximately 9.0
was reached. This O.[~. might normally be expected to be achieved after about
6.5 hours.
The culture was then transferred from the fermenter and passed through a
continuous flow centrifuge at a flow rate sufficient to produce a substantially
clear supernatant. Following the centrifugation, to the supernatant was added,
10% by volume of 10% w/v Cetavlon in water, to produce a precipitate in
suspension. The suspension was centrifuged at 2000 rpm at 4C. The supernatant
was then discarded, and the precipitate was suspended in 600 ml of water.
600 ml of 2M aqueous calcium chloride was then added and the mixture stirred
for 1 hour at ~C, then centrifuged for 20 minutes at 5000 rpm.
RMW/CB/26th October 1984
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Io the supernatant was added one drop of caprylic alcohol as an anti-foaming
agent and the supernatant was de-gassed under vacuum, with stirring, for 10
minutes. The de-gassed supernatant was st aod in an ice bath, and to it was
addsd 3 volumes of absolute ethanol at 0C with continuous stirring. The
mixture was then stood in the bath for two hours before being centrifuged at
5000 rpm for 15 minutes, at a temperature of 4C. The supernatant was
discarded and the precipitate resuspended in 75% ethanol and centrifuged to
form a pellet which may be stored at - 20C or treated as below.
The pellet was suspended at a rate of 20mg/ml in 0.1M ammonium acetate
containing 0.01% thiomersalate. The suspension was subjected to ultrasonic
agitation fqr 10 minutes, and passed through a 311m membrane to remove
insoluble material. The supernatant was then passed through a 5 litre column of
Sepharose CL-2B equilibrated with buffer at 4C. The column was washed
through with buffer, and the effluent monitored by UV absorption at the
wavelength 260 and 280 nm and resorcinol-HC1 sialic acid determination. The
void volume fractions exhibiting absorption at 280 nm and containing sialic acid(resorcinol-HC1 determination) were pooled. Sodium deoxycholate was added up
to a final concentration of 0.1% w/v buffered to within pH 8-9. (Other
concentrations and pH values are also possible, eg a final concentration of 1%
w/v buffered to pH 11 has been found to reduce the amount of material which
adheres to the membrane). The resultant solution was immediately filtered
through a Sartobran 0.22 capsule with a 0.45 prefilter. The filtrate was
precipitated with 3 volumes of absolute ethanol (concentration after solution
about 75%). After this filtration, all procedures were performed under sterile
conditions. After centrifugation, the supernatant was then discarded, and the
precipitate suspended in 200 ml of cold ethanol to wash it. The suspension was
then centrifuged, the supernatent discarded, and the precipitate (which
comprised substantially only the B6 complex) was, again subjected to the same
washing process before being redissolved in 200 ml of cold water. (Alternatively,
injectable solutions may be prepared eg. with 5% lactose and 0.01 M Na3 PO4;
pH 7.3).5
Example 2: Isolation and Purification of a B2 Complex
A B2 complex was prepared by the process of Example 1, starting from a culture
containing a serogroup B, serotype 2 strain of N. meningitidis.
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Example 3: Isolation and P_rification of a B6 Complex:
Small-Scale Process
Three litres of diluted culture medium were prepared by a method analogous to
that used in Example 1, using the same ingredients. A portion of about 200-500
ml of the medium was transferred to a non-baffled shaped-flask and inoculated
with 1 drop of a culture containing a serogroup B, serotype 6 strain of
N.meningitidis. The inoculum was incubated as specified in Example 1, then
added to the remainder of the culture medium, which was incubated in a manner
analogous to that described in Example 1, for 6.75 hours. The culture was then
transferred as 6 x 500 ml to a centrifuge and centrifuged for 20 minutes at
7000 rpm.
To the supernatant was added 10% by volume of 10% w/v Cetavlon. The
suspension was leFt to settle at 4C for 16 hours, after which, the major part of
the supernatant was syphoned off7 and the remainder, containing the precipitate,was centrifuged at 2000 rpm for 20 minutes at 4C. The precipitate was
resuspended in 20 ml of distilled water, 20 ml of 2M CaCl2 was added, and the
mixture stirred for 1 hour at 4C, then centrifuged for 20 minutes at 5000 rpm.
The crude precipitate was redissolved and part-purified by chromatography on a
500 ml column of Sepharose CL-2B, as described in Example 1. The void volume
fractions containing sialic acid and material absorbing at 280 nm were pooled,
precipitated by addition of ethanol to a final volume of 75% v/v, then collectedby centrifugation at 15,000 9 for 20 minutes at 4C. The precipitate was washed
with absolute ethanol and freeze-dried in 5% lactose and 0.01M Na3PO4; pH 7.3.
Example 4: Isolation and Purification of a B2 Complex: Small Scale Process
A B2 complex was prepared by the process of example 3, starting from a culture
containing a serogroup B, serotype 2 strain of N. meningitidis.
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Example 5: Characterisation of B2 and B6 Cc,mplexes
The complexes -formed in Examples 1-4 were identified in each case as the peak
eluting in the void volume on Sepharose CL-2B (MW 20 x 106) at 4C as assayed
-For sialic acid by the resorcinol-HCl method and for absorbance at 260 and 280
1t) nm in the case of B6 complexes (see Figure 1) and at 280 nm only in the case of
B2 complexes (see Figure 2). The latter plot was obtained from a freeze-dried
sample of a B2 complex having a sialic acid content of 48% and a protein contentof 36.1% (ratio 1.33:1). The complexes were found not to dissociate (ie. proteinand polysaccharide remain in the void volume) upon re-chromatography on
Sephrose CL-2B (Figure 3 For the complex used in Figure 1 and Figure 4 for the
complex used in Figure 2) at 4C. SDS-PAGE of the complexes gave the protein
component pattern characteristic of serotype 6 -for the products of Examples 1
and 3 and serotype 2 for the products of Examples 2 and 4; the samples were
boiled for 3 minutes in SDS at 95C then subjected to the procedure of Laemmli
~e~. 10 ug of sample per track, staining with Coomassie blue. In Figure 5,
track 1 shows molecular weight marker bands. Track 2 was obtained from a B2
complex. The pattern of track 2 shows a major band due to a dominant protein
component having a molecular weight in the region of 42,000. A minor band also
appears around 32,000. Track 3 was obtained from a crude precipitate including
B6 complex obtained by precipitation with 1% Cetavlon and before purification]
Tracks 4 and 5 were obtained from samples o-F freeze-dried B6 vaccine
compositions obtained from isolation processes including precipitation with
cetylpyridinium chloride, the tracks being from samples, respectively before andafter sterile filtration. In both tracks 4 and 5, the two bands have apparent
molecular weights of around 38,000 and 43,000. Chemical composition of the
complexss was determined by colorimetric methods, for sialic acid
(resorcinol-HC1), and for protein (Lowry method using a standard of bovine
serum albumin).
Example 6: Preparation of an Aluminium-B6 complex
(i) A B6 complex, isolated by the method of Example 1 was complexed with
aluminium by preparation of a solution as 0.5 ml aliquots, each containing:
100~9 B6 complex in 5% lactose; 0.01M sodium phosphate to pH 7.4;
0.001M A12 (5~)3
RMW/CB/26th October 1984
~ ~ rj~3~3~L~7
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(ii) A B6 cnmplex, isolated by the method of Example 1 was cornplexed with
aluminium by preparation of an aqueous solution of the B6 complex (3ml) at
0C, to which was added 2x10 2M AlC13 to giYe a molar ratio of sialic
acid: aluminium of between 1:0.1 and l:3. Following incubation for 10
min. at 0C, the solution was made to 0.1M w.r.t. NaCl and 3 volumes of
absolute ethanol were added. The precipitate was collected by
centrifugation, suspended in absolute ethanol and recentrifuged, and finally
redissolved or resuspended in water (3ml). An equal volume of 0.02M
Na3P04 pH 7.3 and 10~/o lactose buffer was added, and aliquots were freeze
dried.
In a manner analogous to that described in Example 6, the title cornplex was
prepared using 100~9 of B6 complex in 5% lactose; 0.01M sodium phosphate to
pH 7.2; 0.001M ruthenium red (ammoniated ruthenium oxychloride).
Example 8: Isolation and purification of MPS(B)
Three 15 litre tanks of diluted culture medium, were prepared by a method
analogous to that used in Example 1, using the same ingredients. Each tank was
seeded with 800ml of fresh medium, the seed being prepared from a culture
(containing a serogroup B strain o-F Neisseria meningitidis. The tanks of mediumwere incubated in a manner analogous to that described in Example 1, for about
5.5 hours, and harvested into 15 litre bottles. The contents of these bottles was
then spun-down by centrifuging at 40-50,000 rpm, the speed varying according to
the density of the harvest. Immediately after harvest, 150ml of 10% Cetavlon
was added to each partially-clarified batch of supernatent, which was then left
at 4C to settle.
After two days, mest of the supernatent was syphoned off from the 15 litre
bottles and the remainder was combined and spun down in 4 one-litre portions.
The spun-down precipitates were stored at minus 20C, and were later suspended
in 300ml of 1/10 saturated sodium acetate, pH 7Ø 300ml of 2M CaC12 was
added to the suspension, and the mixture was stored for 1 hour at 4C. Absolute
ethanol was then added to form a concentration of 25% v/v, the total volume
added being 200ml. The precipitate was spun-down and the supernatent
retained. The supernatent was filtered through a No. 50 filter paper to clarify
it. The thus filtered supernatent was perfectly clear to the eye.
-
RM~AI/rR/~th nrtnh~r 19~
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The etl-anol concentration in the supernatent was then increased to 75% v/v by
the acldition of a furlher :L600ml oF absolute etharlol.
The precipitate was then spun-down in 4 one litre portions. The supernatent was
discarded and the precipitate washed twice in ethanol and twice in acetone. The
wet weight yield was 2.59. After storage at -20C, the precipitate was dissolved
in 350ml of 1/10 saturated neutral sodium acetate, to yield an opalescent
solution.
The precipitate solution was thawed and extracted twice with two 0.5 volumes of
cold phenol (lOOg crystalline phenol plus 40ml 1/10 saturated neutral sodium
acetate). The extraction was achieved by shaking in the hand for approximately
1 minute. Phase separation was effected by centrifugation at 7,000 rpm (9,400G)
in polypropylene pots. During this process, the temperature was raised to 10C,
to give better phase separation. The phenol phase was discarded, and the
aqueous phase was extracted with chloroform/butanol 5:1 by homogenisation.
Phase separation was achievecl as above, but using glass pots with spinning at 580
rpm (4,800G) for 30 minutes. The aqueous solution was removed and stored at
- 20C-
The slightly opalescent material obtained above, was thawed and dialysed for 18
hours agains 0.1M CaC12 (5 litres). The volume after dialysis was 250ml. This
?5 was distributed amongst 50ml centrifuge tubes and spun for 3 hours at 100,000G.
The clear supernatent was poured-of f of the gelatenous precepitate produced
(which was discarded).
750ml of absolute ethanol was added to precipitate the meningococcal group B
polysaccharide after 1 hour to allow for complete precipitation. The polysaccha-ride precipitate was washed twice with ethanol and twice with acetone and then
dried. The dry weight yield was 0.983.
Exam le 9 Isolation and urification of MPS(B) small-scale rocess
~ P
The methodology of Examples 1-4 and 8 was followed, using a seed from a
culture of serogroup B Neisseria mening tidis with a culture time of 6.5 hours,
to produce 1800ml nf culture which was spun-down to yield slightly cloudy
supernatent. 18ml of 10% Cetavlon was added and left overnight at 4C.
RMW/CB/26th October 1984
5~ 17
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The! precipitate thus formed, was spun-down and resuspended in approximately
lûOml of distilled water and stored at -20C. Further addition of Cetavlon to
the consequent supernatent did not produce any Further precipitation. The
Cetavlon precipitate was dissociated with 100ml of 2M CaC12 (stirred at 4C for
1 hour).
A second batch of culture was similarly prepared, using a culture time of 6.25
hours. Most of the harvest was extracted and spun-down as before, to yield
1500ml of supernatent. This was concentrated by ultrafiltration, choosing a
1û0,ûûûMW cuto-ff. Final volume was approximately 150ml and the filter was
back-washed to produce final volume oF 162ml. This opalescent liquid was spun
down at 8,û00G to achieve clarification. 0.9ml was taken and 0.1ml of 10%
Cetavlon was adoed. Precipitation was immediate. A further 1.2ml was taken
and 0.4ml of absolute ethanol was added, to achieve a total ethanol concentra-
tion of 25% v/v. There was no increase in precipitation and a further 3.2ml of
absolute ethanol was added to 75% v/v, which produced only a very slight
cloudiness.
The culture was spun-down as for the first batch. 15Dûml of opalescent
supernatent were stored overnight at 4C and then diafiltered using as
100,000MW cut-off membrane to produce 162ml of retentate. This material was
spun-down at 8,000G to clariFy, the pellets being retained. A precipitate was
obtained from the resultant liquid by addition of 0.1% Cetavlon. 1.2ml of
supernatent taken and û 4ml of absolute ethanol were added to 25% v/v. No
precipitation occured, indicating very little Iysed proteinaceous material in the
culture. Increase in the ethanol concentration to 75% v/v~ by addition of 3.2ml
of absolute ethanol, produced only slight cloudiness, indicating either much
release of polysaccharide below 100,000MW or low polysaccharide yield.
The first possibility was checked by adding Cetavlon to the dialysate from the
ultrafiltration apparatus. No precipitation occured, indicating the second of the
options above. The remainder of the retentate (159.8ml) was further concentra-
ted overnight by vacuum dialysis to 44ml, lml of which was removed. The
concentrate was readily precipitated with 0.1% Cetavlon. The remaining 42ml
was precipitated by addition of 126ml of absolute ethanol to 75% v/v (no
discernable precipitation occuring at 25%). The precipitate increased on
standing, but no observable increase was produced by addition of 1.269 o f
ammonium acetate. The precipitate gradually aglommerated.
RMW/CB/26th October 1984
3~ 7
-20- A70LI
The p~l of the solution was found to be 7.4. The total quantity of precipitate
obtained, was washed twice with ethanol and twice with acetone, then dried; the
dry weight yield was 40mg.
Example 10: Preparation of an Alumin!um-B Complex (Dialysis method)
Meningococcal group B polysaccharide (20~mol sialic acid) was dialysed against
AlC13 solution (2011mol A13+ in two litres of distilled water) at 4C for 24
hours. The solution inside the dialysis sac, was precipitated with 75% v/v EtOH,the precipitate centrifuged, and the pellet freeze-dried. Analysis of the B
polysaccharide-aluminium complex for sialic acid, Ca2+ and A13+ ions yave a
molar ratio of 1.00:0.01:0.34. Analysis of metal ions was afforded by atomic
absorption spectroscopy.
Example 11: Preparation of an A uminium-B (Incubation Method)
Group B Polysaccharide (2011mol sialic acid) was incubated with AlC13
(2-20~mol A13+) in distilled water (2 ml) at room temperature for 10 minutes.
1M ammonium acetate (0.2ml) was added, and the solution precipitated with 75%
v/v EtOH. The precipitate was centrifuged, and the pellets freeze-dried. At the
higher A13+ molar ratios (i.e. sialic acid: A13+1:1 and 1:0.5) a water-insoluble
complex was obtained, whereas at lower A13+ molar ratios (i.e. sialic acid: A13+1:0.3, 1:0.2 and 1:0.1) the complex was water-soluble.
Example 12: Characterisation of Aluminium-B Come~es
The molecular weight of the metal complexes formed by the methods of
Examples 10 and 11 was investigated by Sepharosae CL-4B Chromatography. In
both cases, the void volume peak (VO) contained sialic acid and aluminium. The
complex obtained from the method of the latter example, however, had an
increased molecular weight (see Figure 6).
Example 13: In-situ preparation of Aluminium-Col and Aluminium-B Complexes
and their Characterisat on
.
NMR and electrochemical measurements on aluminium-colominic acid systems
were performed to provide characterisation data and evidence of complex
formation. Counter immuno-electrophoresis measurements of time-dependant
degradation of MPS(B) in solution, with and without the presence of aluminium,
RMW/CB/26th October 1984
~S'~9~
-21- A700
were also used. In each of these experiments, the polysaccharide and alurninium
are brought into association during preparation of the various experimental testsolutions. The corresponding complexes were thus formed in situ.
(a) Electrochemical measurements
Two (0.125 mM to 1 mM) A1lIl concentration series were polarographed,
one in the absence of the polysaccharide, and the other in its presence at
constant concentration 1 rnM wrt. the monomer (NANA) or 1011M (8-16~1M)
wrt. the polymer. As indicated by the DPP plot shown in Figure 7, ~ 10
mM colominic acid in 0.137 M-NaC104 were found to bind on the order of
100 IIM-A1 so that up to this aluminium concentration, no free metal ions
are found to be present in the mixture. Since the colominic acid dilution
data are somewhat divergent with respect to the free A1 data, secondary
binding of aluminium is suggested) the extent of which is given by the
difference of the slopes (derivatives) of the two curves.
In the unbuffered solutions, pH was controlled by hydrolysis of aluminium
and by proton transfer equilibria of colominic acid. Whilst the pH of the
background solution was 8.721, alurninium sulphate at 1 mM reduced the pH
to 4.191. The pH then increased with dilution exponentially to 4.857 at 125
nM-A12 (SO4)3. Colominic acid gave pH 6.739 at 1 mM (wrt. NANA),
which increased very little with dilution, to 6.784 at 125 nM (wrt. NANA).
Equimolar mixture of 1 mM each (polysaccharide (wrt. NANA) plus
aluminium III) gave p~l 3.927. This suggests that increased proton dissocia-
tion by colominic acid results from aluminium binding (e.g. release of
hydroxylic protons to form metal-oxygen bonds) or that the acidification of
the unbuffered mixture results from hydrolysis of aluminium as a result of
binding the minority species of the aluminium.
(b) NM_ measurements
13C n.rn.r. showed that A1(III) complexed primarily at the carboxylate
group but that other interactions were present and that several slowly
exchanging species were present in aqueous solutions.
RMW/CB/26th October 1984
~,~5q3~317
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(c) Counter immunoelectrophoresis measurements
The persistence in solution of MPS(B) as a Function of time, with and
without the presence of A13+ (10-3, 10-4 and 10-5M) was monitored by
counter immunoelectrophoresis (pH 4.0 antiserum: Rabbit a Meningo B
ES.77.1). Results are shown in Figure 8. The stabilisation of MPS(B)
observed is the presence o-f AI3+ is evidence of complexing of the metal ion
with the sialic acid repeat unit.
Example A: Immunisation of Mice with metal-B6 complexes
.
(a) Aluminium-B6
The level of antibodies produced against group B and type 6 meningococcal
antigens respectively, was measured in mice at 7 days after secondary
immunisation with an aluminium B6 complex prepared by the method of Example
6. Mice were injected intaperitioneally with the test and reference preparationsindicated below.
Individual bleedings were taken -from the mice at 7 days after immunisation. Theantibody level was detemined as ~g/ml of serum, by solid phase
radioimmunoassay in plate sensitised with purified group B meningococcal
polysaccharide. In this method, microtiter soft plates were pre-treated with
poly L-lysine (Sigma, 10û 119 per ml) and sensitised with the polysaccharide.
After incubation with the complex, wells were counted and a linear correlation
between the counts and 1O92 of the serum dilution was obtained. Extrapolated
values at 1/50 dilution of serum were compared with linear correlations of a
standard.
Test and reference preparations in each case wers prepared as follows:
Preparation No. Injection Solution
1. 10119 B6 complex, 5% lactose, 0.01M
Na3 PO4 pH 7.4 (0.5ml)i.p.
2. 5% lactose, 0.01M Na3PO4 pH 7.4
(0.5 ml)i.p.
RMW/CB/26th October 1984
~.~5~;1 7
-23- A7t)0
3- Solution of Example 6 (0.5 ml)i.p.
4. 5% lactose, 0.0lM Na3PO4 pH 7.4,
0.001M A12 (S04)3 (0.5 ml) i.p.
The antibody responses to each preparation for group B antigens are shown in thefollowing Table.
Antibody Responses follow ng
Immunisation of CBA Mice
Preparation No. Anti-B Re_ponse ~Ig/ml antibody in
serum (standard error)
1. 1.6 (1.38)
2. 0.49 (1.04)
3. 3.g2 (1.16)
4. 0.65 (1.20)
(b) Ruthenium-B6
The complex produced in Example 7 above was tested in an analogous manner to
that described above, in female CBA mice, 5 per group. Individual bleedings
were taken 7 days after immunisation and anti-B antibody serum levels
determined by solid phase radioimmunoassay.
Test and reference preparations in each case were prepared as follows:
RMW/CB/26th October 1984
.
~ ~5'3~
-24- A700
Preparation No. Injection Solution
1 lù~lg B6 complex, 5% lactose, 0.01M
Na3P04 pH 7.2 (0.2ml) i.p.
2 Solution of Example 6 (0.2ml) i.p.
1û 3 Solution of Example 7 (0.2ml) i.p.
Results
Preparation No. Antibody Response Statistical
(IJg/ml)-Geometric Significance
Average (Standard (Students Test)
Error*)
.
2û
4.33 (1.16)
2 7.29 (1.24) p< 0.05
3 7.76 (1.27) p<O.025
* ie. multiplied or divided by same.
Example B: Preparation of Vaccine Formulation
The purified complexes obtained from Examples 6, 9 and 10 were each dispersed
as 1.0 mg per ml of aqueous sterile sodium phosphate (O.OlM, pH 7.2). To the
resulting solutions, was added 50 mg/ml of lactose with mixing. The solutions
were then freeze-dried and stored at -20C until used. Reconstitution was
achieved after thawing, by solution to the original volume, in sterile, pyrogen-free water.
RMW/CB/26th October 1984
