U.S. patent application number 11/621306 was filed with the patent office on 2007-09-13 for methods for the production of 3-o-deactivated-4'-monophosphoryl lipid a (3d-mla).
This patent application is currently assigned to Conxa Corporation. Invention is credited to Kent R. Myers, D. Scott Snyder.
Application Number | 20070212758 11/621306 |
Document ID | / |
Family ID | 23071615 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070212758 |
Kind Code |
A1 |
Myers; Kent R. ; et
al. |
September 13, 2007 |
METHODS FOR THE PRODUCTION OF 3-O-DEACTIVATED-4'-MONOPHOSPHORYL
LIPID A (3D-MLA)
Abstract
Herein is disclosed a method for producing lipopolysaccharide
(LPS), comprising: (a) growing a culture of deep rough mutant
bacterial strain in a medium; (b) maintaining the culture in
stationary phase for at least about 5 hr; (c) harvesting cells from
the culture; and (d) extracting LPS from the cells. The method
allows for the production of an LPS which can be used to produce a
3-O-deacylated monophosphoryl lipid A (3D-MLA) having at least
about 20 mol % of the hexaacyl congener group. Also herein is
disclosed a method of extracting lipopolysaccharide (LPS) from a
culture of deep rough mutant bacterial strain cells, comprising:
(a) extracting the cells with a solution consisting essentially of
at least about 75 wt % of an aliphatic alcohol having from 1 to 4
carbon atoms and the balance water, thereby producing cells with
reduced phospholipid content; and (b) extracting the cells with
reduced phospholipid content with a solution comprising chloroform
and methanol, thereby yielding a solution of LPS in chloroform and
methanol (CM). This method provides LPS solutions in CM that have
reduced phospholipid content and are produced by relatively simple
and inexpensive process steps.
Inventors: |
Myers; Kent R.; (Hamilton,
MT) ; Snyder; D. Scott; (Hamilton, MT) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B475
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Assignee: |
Conxa Corporation
Seattle
WA
|
Family ID: |
23071615 |
Appl. No.: |
11/621306 |
Filed: |
January 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10099313 |
Mar 14, 2002 |
|
|
|
11621306 |
Jan 9, 2007 |
|
|
|
60280089 |
Mar 30, 2001 |
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Current U.S.
Class: |
435/85 ;
435/252.34; 536/53 |
Current CPC
Class: |
C12P 19/04 20130101;
A61P 37/04 20180101; C07H 13/06 20130101; A61K 2039/55572
20130101 |
Class at
Publication: |
435/085 ;
435/252.34; 536/053 |
International
Class: |
C12P 19/28 20060101
C12P019/28; C08B 37/00 20060101 C08B037/00; C12N 1/21 20060101
C12N001/21 |
Claims
1-25. (canceled)
26. A composition of 3D-MLA comprising at least about 20 mol % of
hexaacyl congener.
27. A composition according to claim 26 comprising between 20 and
50 mol % of hexaacyl congener
28. A composition according to claim 26 comprising at least 30 mol
% of hexaacyl congener.
29. A composition according to claim 26 comprising 21.5% hexaacyl
congener.
30. A composition of LPS comprising at least about 20 mol % of a
combination of heptaacyl congener and 3-O-deacylated hexaacyl
congener.
31. A composition according to claim 30 comprising at least 30 mol
% of a combination of heptaacyl congener and 3-O-deacylated
hexaacyl congener.
32. A composition according to claim 30 comprising 36% of a
combination of heptaacyl congener and 3-O-deacylated hexaacyl
congener.
33. A composition of MLA comprising at least about 20 mol % of a
combination of heptaacyl congener and 3-O-deacylated hexaacyl
congener.
34. A composition according to claim 33 comprising at least 30 mol
% of a combination of heptaacyl congener and 3-O-deacylated
hexaacyl congener.
35. A composition according to claim 33 comprising 32.7% of a
combination of heptaacyl congener and 3-O-deacylated hexaacyl
congener.
36. A composition according to claim 26 wherein said 3D-MLA is
extracted from a deep rough mutant strain of a gram-negative
bacterium.
37. A composition according to claim 30 wherein said LPS is
extracted from a deep rough mutant strain of a gram-negative
bacterium.
38. A composition according to claim 33 wherein said MLA is
extracted from a deep rough mutant strain of a gram-negative
bacterium.
39. A composition according to claim 36 wherein said 3D-MLA is
extracted from a Salmonella bacteria.
40. A composition according to claims 30 wherein said LPS is
extracted from a Salmonella bacteria.
41. A composition according to claims 33 wherein said 3D-MLA is
extracted from a Salmonella bacteria.
42. A composition according to claim 34 wherein said bacterium is
Salmonella Minnesota.
43. A composition according to claim 35 wherein said bacterium is
strain Salmonella Minnesota R595.
44. A plurality of compositions of 3D-MLA having a consistent
hexaacyl content.
45. A plurality of compositions according to claim 44 wherein said
hexaacyl content is consistently at least about 20 mol %.
46. A plurality of compositions according to claim 44 wherein said
hexaacyl content is consistently between 20 mol % and 50 mol %.
47. A plurality of compositions according to claim 44 wherein said
hexaacyl content is consistently at least about 50mol %.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a non-provisional of and claims the
benefit of U.S. Provisional Application No. 60/280,089, filed Mar.
30, 2001.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] NOT APPLICABLE
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
biosynthetic production of 3-O-deacylated-4'-monophosphoryl lipid A
(3D-MLA). More particularly, it concerns methods of improving the
yield of desired 3D-MLA congeners or minimizing the cost of
purifying lipopolysaccharide (LPS) precursors of 3D-MLA.
[0005] 2. Description of Related Art
[0006] It has long been recognized that enterobacterial
lipopolysaccharides (LPS) are potent stimulators of the immune
system. A variety of responses, both beneficial and harmful, can be
elicited by submicrogram amounts of LPS. The fact that some of the
responses are harmful, and some of these can be fatal, has
precluded clinical use of LPS per se. It has been observed that the
component of LPS most responsible for endotoxic activity is lipid
A.
[0007] Accordingly, much effort has been made towards attenuating
the toxic attributes of LPS or lipid A without diminishing the
immunostimulatory benefits of these compounds. Notable among these
efforts were those of Edgar Ribi and his associates, which resulted
in the production of the lipid A derivative
3-O-deacylated-4'-monophosphoryl lipid A (3D-MLA; compositions
comprising 3D-MLA are commercially available under the trade name
MPL.RTM. from Corixa Corporation (Seattle, Wash.)), 3D-MLA has been
shown to have essentially the same immunostimulatory properties as
lipid A but lower endotoxicity (Myers et al., U.S. Pat. No.
4,912,094). Myers et al. also reported a method for production of
3D-MLA, as follows. First LPS or lipid A obtained from a deep rough
mutant strain of a gram-negative bacterium (e.g. Salmonella
minnesota R595) is refluxed in mineral acid solutions of moderate
strength (e.g. 0.1 N HCl) for a period of approximately 30 min.
This leads to dephosphorylation at position 1 of the reducing-end
glucosamine and decarbohydration at the 6' position of the
non-reducing glucosamine of lipid A. Second, the dephosphorylated
decarbohydrated lipid A (a.k.a. monophosphoryl lipid A or MLA) is
subject to base hydrolysis by, for example, dissolving in an
organic solvent such as chloroform:methanol (CM) 2:1 (v/v),
saturating the solution an aqueous solution of 0.5 M
Na.sub.2CO.sub.3 at pH 10.5, and flash evaporating solvent. This
leads to selective removal of the .beta.-hydroxymyristic acid
moiety at position 3 of the lipid A, resulting in 3-O-deacylated
-4'-monophosphoryl lipid A (3D-MLA).
[0008] The quality of the 3D-MLA produced by the above method is
highly dependent on the purity and composition of the LPS obtained
from the gram-negative bacterium. For one example, the lipid A
component of LPS is a mixture of closely related species that
contain between about 5-7 fatty acid moieties. In the formation of
3D-MLA, as is clear from the above discussion, one fatty acid
moiety is removed, yielding 3D-MLA with between about 4-6 fatty
acid moieties. It is generally held that 3D-MLA with at least 6
fatty acid moieties is preferred in terms of the combination of
maintained or enhanced immunostimulatory benefits, reduced
toxicity, and other desirable properties (Qureshi and Takayama, in
"The Bacteria," Vol. XI (Iglewski and Clark, eds.), Academic Press,
1990, pp. 319-338).
[0009] For another example, commercial scale extraction of LPS from
gram-negative bacteria typically involves the Chen method (Chen et
al., J. Infect. Dis. 128:543 (1973)); namely, extraction with CM,
which leads to an LPS- and phospholipid-rich CM phase from which
LPS can later be purified. However, purification of LPS from the
LPS- and phospholipid-rich CM phase typically requires multiple
precipitation steps to obtain LPS of sufficient purity for use in
immunostimulatory applications such as, for example, use as a
vaccine adjuvant.
[0010] Therefore, it would be desirable to have methods for
conveniently preparing highly pure LPS compositions. Further, it
would be desirable to have methods for generating LPS compositions
which compositions have 3D-MLA with increased levels of hexaacyl
congeners.
[0011] Known fermentation techniques have been used to prepare
cultures of gram-negative bacteria comprising readily purifiable
LPS. These known techniques typically involve harvesting of
bacterial cultures at early stationary phase, in keeping with
standard bacteriological practices. However, it has been observed
that the degree of acylation of LPS produced according to known
conditions is variable. For example, the content of heptaacyl
species in the lipid A of S. minnesota R595 can vary from 20% to
80%, depending on the batch (Rietschel et al., Rev. Infect. Dis.
9:S527 (1987)). This variability in heptaacyl congener content
would result in the significant differences in the hexaacyl
congener content in the 3D-MLA prepared from these LPS batches.
SUMMARY OF THE INVENTION
[0012] In one embodiment, the present invention relates to a method
for producing lipopolysaccharide (LPS), comprising: [0013] (a)
growing a culture of a deep rough mutant bacterial strain in a
medium; [0014] (b) maintaining the culture in stationary phase for
at least about 2 hr; [0015] (c) harvesting cells from the culture;
and [0016] (d) extracting LPS from the cells.
[0017] The method allows for the production of an LPS that yields
3D-MLA with a relatively high proportion (i.e. at least about 20
mol %) of congeners comprising 6 fatty acid moieties.
[0018] In another embodiment, the present invention relates to a
method of extracting lipopolysaccharide (LPS) from a culture of
deep rough mutant bacterial strain cells, comprising:
[0019] (a) extracting the cells with a solution consisting
essentially of at least about 75 wt % of an aliphatic alcohol
having from 1 to 4 carbon atoms and the balance water, thereby
producing cells with reduced phospholipid content;
[0020] (b) extracting the cells with reduced phospholipid content
with a solution comprising chloroform and methanol (CM), thereby
yielding a solution of LPS in CM.
[0021] This method provides LPS solutions in CM that have reduced
phospholipid content and that are therefore well-suited to further
modification and purification to 3D-MLA. The method involves
relatively simple and inexpensive steps.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0023] FIG. 1 shows TLC plates of ethanol extracts and LPS samples
obtained with different temperatures during the ethanol
extractions. The plate on the left shows, going from left to right,
the ethanol extracts at temperatures of 22.degree. C., 37.degree.
C., and 50.degree. C. The sample at the far right of this plate is
an authentic LPS sample. The plate on the right shows the LPS
obtained from each preparation. The samples in lanes 3, 4, and 5
correspond to LPS from cells subjected to pre-extractions with
ethanol at 22.degree. C., 37.degree. C., and 50.degree. C.,
respectively. The heavy bands at R.sub.f.about.0.6 correspond to
phospholipid and fatty acid impurities. The levels of these
impurities are reduced by increasing the temperature of the ethanol
extractions, and are very low in the sample that was pre-extracted
at 50.degree. C.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0024] In one embodiment, the present invention relates to a method
for producing lipopolysaccharide (LPS), comprising:
[0025] (a) growing a culture of a deep rough mutant bacterial
strain in a medium;
[0026] (b) maintaining the culture in stationary phase for at least
about 2 hr;
[0027] (c) harvesting cells from the culture; and
[0028] (d) extracting LPS from the cells.
[0029] Lipopolysaccharides are the main lipid constituent in the
outer leaflet of the outer membrane of gram-negative bacteria. The
lipopolysaccharide fraction of a gram-negative bacterium comprises,
among other components, lipid A. As has been described, lipid A can
be decarbohydrated and partially dephosphorylated to yield
monophosphoryl lipid A (MLA), and MLA can be selectively deacylated
at position 3 to yield 3-O-deacylated-4'-monophosphoryl lipid A
(3D-MLA).
[0030] However, lipid A produced by gram-negative bacteria
typically comprises a number of species that have the same overall
lipid A structure but differ in the number of fatty acid moieties
they contain. Groups of lipid A species with the same number of
fatty acids are referred to herein as "congeners." Lipid A
congeners having from 4 to 7 fatty acid moieties are produced by
standard commercial-scale culturing of gram-negative bacteria such
as S. minnesota R595. As a result, the 3D-MLA produced from, e.g.,
S. minnesota R595 lipid A has a congener composition typically
ranging from 3 to 6 fatty acid moieties (because 3D-MLA has
undergone loss of one fatty acid moiety).
[0031] Heterogeneity in 3D-MLA (via lipid A and MLA) congener
composition is attributable to two sources: (1) biosynthetic
variability in the assembly of the lipid A and (2) loss of fatty
acid moieties from the lipid A backbone during processing to
3D-MLA. Though not to be bound by theory, biosynthetic variability
is believed to occur because of non-absolute substrate specificity
of the acyltransferases involved in the terminal steps of lipid A
biosynthesis, among other explanations. Loss of fatty acid moieties
from the lipid A backbone may also occur during the acid and
alkaline hydrolyses typically used in 3D-MLA production.
[0032] Surprisingly, it was discovered that 3D-MLA congener
composition can be altered by altering the parameters of a process
of culturing a deep rough mutant bacterial strain that produces
lipid A. Specifically, it was discovered that maintaining the
culture of the deep rough mutant bacterial strain at stationary
phase for at least about 5 hr prior to harvesting results in a
change in the proportions of lipid A congeners so produced such
that, typically, at least about 20 mol % of the 3D-MLA later
produced from the lipid A contains 6 fatty acids. Preferably, at
least about 50 mol % of the 3D-MLA contains 6 fatty acids. A
maintenance at stationary phase time of about 5.5 hr has been found
to be particularly effective. This is in distinction to the typical
culturing processes known in the art, wherein harvesting occurs
almost immediately after entry of the culture into the stationary
phase; in the known process, the congener content of the LPS is
highly variable and results in 3D-MLA with variable hexaacyl
congener content.
[0033] By "deep rough mutant bacterial strain" is meant a strain of
a gram-negative bacterium having a deep rough phenotype. A "deep
rough" phenotype means that the polysaccharide moiety attached to
the lipid A consists of only about 2-3 residues of
2-keto-3-deoxy-D-mannooctulonic acid (KDO). Preferably, the deep
rough mutant bacterial strain is selected from the genus
Salmonella. More preferably, if the deep rough mutant bacterial
strain is of genus Salmonella, it is of species Salmonella
minnesota, and even more preferably, it is strain Salmonella
minnesota R595. Other deep rough mutant bacterial strains, such as
Proteus mirabilis strains, among others, can be used.
[0034] Any technique appropriate for growing a deep rough mutant
bacterial strain can be used. Typically, this will involve the use
of at least one commercial-scale bioreactor. In one embodiment, the
technique involves inoculating a relatively small (e.g. 15 L)
bioreactor with cells of the deep rough mutant bacterial strain,
growing the deep rough mutant bacterial strain until a stationary
phase, followed by aseptic transfer of the 15-L cell broth to a
large (e.g. 750 L) bioreactor.
[0035] The growing can be performed on any medium known or
discovered to allow the growth of the deep rough mutant bacterial
strain. In one preferred embodiment, the medium is M9, a mixture of
inorganic salts supplemented with dextrose and casamino acids. The
composition of M9 is well-known to one of ordinary skill in the
art.
[0036] After the deep rough mutant bacterial strain has been
maintained at stationary phase for at least about 5 hr, the cells
can be harvested from the culture and LPS extracted from the cells.
Known techniques may be employed to harvest cells from the culture
and extract LPS from the cells, although a preferred technique for
extracting LPS from the cells is described below.
[0037] Harvesting can be performed by any known technique. In one
preferred embodiment, after the cell culture has been maintained at
stationary phase for at least about 5 hr, the contents of the
bioreactor are pumped to a tangential filtration apparatus to
separate spent medium from the cells.
[0038] The LPS is then extracted from the cells by any appropriate
technique. Known techniques include the Galanos method, which
involves extracting LPS with a mixture of phenol, chloroform, and
petroleum ether (PCP), followed by evaporation of the chloroform
and petroleum ether, addition of acetone and water to precipitate
LPS, and recovery of LPS by centrifugation or filtration (Galanos
et al., Eur. J. Biochem. 9:245 (1969)), and the Chen method, cited
above, which involves extracting LPS with a mixture of chloroform
and methanol (CM), followed by a series of methanol precipitation
steps.
[0039] An improvement of the Chen method is described below, and is
preferred for manufacture of LPS and its derivatives for commercial
applications.
[0040] Regardless of the extraction technique, the result is a
substantially pure dried LPS, which can be further processed by
sequential acid hydrolysis and base hydrolysis to form 3D-MLA, as
is taught by Ribi, U.S. Pat. No. 4,436,727, and Myers et al., U.S.
Pat. No. 4,912,094, which are hereby incorporated herein by
reference. To summarize the teachings of these references as a
preferred embodiment for the formation of 3D-MLA, the LPS is
reacted with an organic or inorganic acid, and then lyophilized to
produce MLA. The inorganic acid is preferably hydrochloric acid,
sulfuric acid, or phosphoric acid. The organic acid is preferably
toluene sulfonic acid or trichloroacetic acid. The reaction may be
performed at a temperature between about 90.degree. C. and about
130.degree. C. for a sufficient time to complete hydrolysis,
commonly between about 15 min and about 60 min. The MLA may be
treated with a solvent, preferably acetone, to dissolve fatty acids
and other impurities, and the impurity-rich fatty acid solvent is
removed.
[0041] Thereafter, the MLA is subjected to mild alkaline treatment
to selectively remove the .beta.-hydroxymyristic acid from position
3 of the MLA (under mild alkaline conditions, only the
.beta.-hydroxymyristic acid at position 3 in labile). The mild
alkaline treatment can be carried out in aqueous or organic media.
Appropriate organic solvents include methanol or other alcohols,
dimethyl sulfoxide (DMSO), dimethylformamide (DMF), chloroform,
dichloromethane, or mixtures thereof, among others. Combinations of
water and organic solvents miscible with water may also be
employed.
[0042] The Alkaline base used to perform the hydroloysis is
preferably selected from hydroxides, carbonates, phosphates, or
amines. Illustrative inorganic bases include sodium hydroxide,
potassium hydroxide, sodium carbonate, potassium bicarbonate,
sodium bicarbonate, and potassium bicarbonate, among others.
Illustrative organic bases include alkyl amines (such as
diethylamine and triethylamine, among others), among others.
[0043] In aqueous media, the pH is typically between about 10 and
about 14 , preferably between about 10 and about 12. The hydrolysis
reaction is typically performed at from about 20.degree. C. to
about 80.degree. C., preferably from about 50.degree. C. to about
60.degree. C., for a period of about 10 min to about 48 hr.
[0044] One preferred technique for alkaline hydrolysis involves
dissolving MLA in CM 2:1 (v/v), saturating the solution with an
aqueous buffer of 0.5 M Na.sub.2CO.sub.3 at pH 10.5, and then flash
evaporating the solvent at 45-50.degree. C. under a vacuum
aspirator (approximately 100 mm Hg).
[0045] In another embodiment, the present invention relates to a
method of extracting lipopolysaccharide (LPS) from a culture of
deep rough mutant bacterial strain cells, comprising:
[0046] (a) extracting the cells with a solution consisting
essentially of at least about 75 wt % of an aliphatic alcohol
having from 1 to 4 carbon atoms and the balance water, thereby
producing cells with reduced phospholipid content;
[0047] (b) extracting the cells with reduced phospholipid content
with a solution comprising chloroform and methanol, thereby
yielding a solution of LPS in chloroform and methanol.
[0048] The deep rough mutant bacterial strain cells, the culture
thereof, and methods of preparing the culture are as described
above. Preferably, the deep rough mutant bacterial strain is
selected from the genera Salmonella or Escherichia. More
preferably, if the deep rough mutant bacterial strain is of genus
Salmonella, it is of species Salmonella minnesota, and even more
preferably, it is strain Salmonella minnesota R595. If the deep
rough mutant bacterial strain is of genus Escherichia, more
preferably it is of species Escherichia coli, and more preferably
it is strain Escherichia coli D31m4.
[0049] The first extracting step can be performed with any short
chain aliphatic alcohol. The aliphatic alcohol can be linear,
branched, or cyclic. Preferably, the aliphatic alcohol has from 2
to 4 carbon atoms and is miscible with water. More preferably, the
aliphatic alcohol is ethanol.
[0050] The solution comprising the aliphatic alcohol can comprise
any proportion of aliphatic alcohol of 75 wt % or greater.
Preferably, the solution comprises between about 85 wt % and about
95 wt % aliphatic alcohol. Essentially, the balance of the solution
is water. Traces of other compounds may be present as a result of
incomplete purification or other contamination of the aliphatic
alcohol and water components of the solution.
[0051] The temperature at which the first extracting step is
performed can be any temperature which is effective in providing
sufficient extraction of phospholipid from the cultured cells.
Preferably, the temperature is between about 35.degree. C. and
about 65.degree. C. More preferably, the temperature is between
about 45.degree. C. and about 55.degree. C.
[0052] Other parameters of the first extracting step, such as rate
of addition of the aliphatic alcohol solution, duration of contact
of the solution and the cells, and agitation or lack thereof, among
others, can be routinely varied by one of ordinary skill in the
art.
[0053] The first extracting step results in (i) a phospholipid-rich
aliphatic alcohol solution phase and (ii) cells with a reduced
phospholipid content. The LPS component of the cell membranes
segregates substantially completely with the cells with a reduced
phospholipid content.
[0054] The second extracting step involves extracting the cells
with a reduced phospholipid content with a solution of
chloroform:methanol (CM).
[0055] Any proportion of chloroform and methanol known to be
suitable for use in extracting LPS from cell membranes (such as in
the Chen method) may be used in the second extracting step.
Typically, the proportion of chloroform to methanol is from about
2:1 to about 9:1. Solvent mixtures with properties equivalent to
those of CM may also be used to obtain LPS from the cells with a
reduced phospholipid content.
[0056] An advantage of the present method over the Chen method lies
in the removal of phospholipid in the first extracting step.
Whereas the CM extraction of the Chen method results in an LPS
solution that contains substantial levels of phospholipids, the
second extracting step of the present invention, being performed on
cells with a reduced phospholipid content, results in an LPS-rich
solution that is substantially devoid of phospholipid. Alternative
methods of producing LPS preparations that are relatively free of
phospholipids, such as the method of Galanos (see above), are less
desirable because they are not amenable to large scale production,
they use solvent mixtures that pose health and safety concerns
(e.g. phenol:chloroform:petroleum ether), or both.
[0057] Given the substantial absence of phospholipid from the LPS
solution, further purification of the LPS according to this method
is generally simpler and less expensive than under the Chen method.
It has been found that a dry LPS residue of sufficient purity can
be formed by evaporating the chloroform and methanol from the LPS
solution.
[0058] Optionally, the LPS can be further processed, such as by the
acid hydrolysis and base hydrolysis steps described above, to
produce MLA or 3D-MLA.
[0059] The 3D-MLA produced by following the methods described above
can be used for a variety of purposes. One preferred use is as an
immunostimulant or adjuvant for pharmaceutical compositions
comprising an immunogenic polynucleotide, polypeptide, antibody,
T-cell, or antigen-presenting cell (APC). An immunostimulant or
adjuvant refers to essentially any substance that enhances or
potentiates an immune response (antibody and/or cell-mediated) to
an exogenous antigen.
[0060] One immune response which the MLA or 3D-MLA produced
according to the present invention may stimulate is the Th1 type. A
combination of monophosphoryl lipid A (MLA), preferably
3-de-O-acylated monophosphoryl lipid A (3D-MLA), together with an
aluminum salt has been observed to be effective as an adjuvant for
eliciting a predominantly Th1-type response. High levels of
Th1-type cytokines (e.g., IFN-.gamma., TNF.alpha., IL-2 and IL-12)
tend to favor the induction of cell-mediated immune responses to an
administered antigen. In contrast, high levels of Th2-type
cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the
induction of humoral immune responses. Following application of a
pharmaceutical composition comprising MLA or 3-D-MLA, a patient
will support an immune response that includes Th1- and Th2-type
responses. When the response is predominantly Th1-type, the level
of Th1-type cytokines will increase to a greater extend than the
level of Th2-type cytokines. The levels of these cytokines may be
readily assessed using standard assays. For a review of the
families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol.
7:145-173, 1989.
[0061] In one preferred embodiment, the adjuvant system includes
the combination of a monophosphoryl lipid A (MLA), preferably
3D-MLA, with a saponin derivative (such as Quil A or derivatives
thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc.,
Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium
quinoa saponins), such as the combination of QS21 and 3D-MLA
adjuvant, as described in WO 94/00153, or a less reactogenic
composition where the QS21 is quenched with cholesterol, as
described in WO 96/33739. One preferred formulations comprise an
oil-in-water emulsion and tocopherol. Another particularly
preferred adjuvant formulation employing QS21, 3D-MLA, and
tocopherol in an oil-in-water emulsion is described in WO
95/17210.
[0062] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE 1
General Methods
[0063] A. Media Preparation
[0064] Cell growth was conducted in M9 medium, which is prepared by
combining sterile solutions of inorganic salts, casamino acids, and
dextrose. The M9 salt solution is typically prepared in the
fermentor and contains the following salts: 2.0 g/L NaCl, 0.2 g/L
MgSO.sub.4.7H.sub.2O, 3.0 g/L KH.sub.2PO.sub.4, 6.0 g/L
Na.sub.2HPO.sub.4, and 1.0 g/L NH.sub.4Cl. Sterile solutions of 20%
(w/v) casamino acids (20 mL/L) and 50% (w/v) dextrose (32 mL/L) and
then added aseptically to the fermentor to yield the completed
medium.
[0065] B. Seed Growth
[0066] Typically, a sterile 250 mL Erlenmeyer flask was charged
with 50 mL sterile M9 medium. A seed vial of Salmonella minnesota
R595 (ca. 10.sup.8 cfu) was thawed and added to the flask, which
was then stoppered with a gauze plug. The culture was incubated at
37.degree. C. for 6-8 h, until robust growth is evident.
[0067] C. Cell Growth
[0068] Cultures of Salmonella minnesota R595 were grown in a BioFlo
III fermentor (New Brunswick Scientific, Inc.) equipped with a 2.5
L glass vessel. In a typical run, the vessel was charged with 2.0 L
of M9 salts solution, autoclaved, and sterile solutions of casamino
acids and dextrose were then added aseptically. The fermentor was
equipped with feedlines for antifoam (0.1% SAG-471, Witco Corp.)
and NH.sub.4OH (30%) as well as probes for pH, dO.sub.2, and foam.
The medium was adjusted to pH 6.9 using the NH.sub.4OH feed. The
fermentor was then inoculated with the entire seed culture and was
incubated at 37.degree. C. with air sparging (typically 2.0 Lpm)
and stirring (typically 50 rpm). The growth phase of the culture
was monitored by measuring optical density at 590 nm. Cells were
harvested by either centrifugation or tangential flow filtration,
washed with water, and lyophilized.
[0069] D. Extraction of Lipopolysaccharide (LPS)
[0070] LPS was isolated according to the procedure of Qureshi et
al. (1986) with minor modifications. In a typical run, the dried
cells were first stirred at a concentration of 20 mg/mL in 90%
ethanol (v/v) at room temperature for 1 h and were then recovered
by vacuum filtration. The cells were subjected to a second ethanol
extraction followed by sequential extractions with acetone and
diethyl ether (15 min each, both at 40 mg/mL based on initial
weight), and the resulting ether powder was allowed to air dry
overnight. Meanwhile, a solution of phenol
(89%):chloroform:petroleum ether 19:45:72 (v/v/v; abbreviated PCP)
was prepared and allowed to stand overnight. The ether powder was
suspended in PCP, which was decanted off of the excess water, at a
concentration of 70 mg/mL. The solution was stirred for 30 min and
then was centrifuged (3000.times.g, 15 min, 0-5.degree. C.). The
supernatant fraction was decanted into a round bottom flask and the
cell pellet was extracted a second time with PCP. The supernatant
fractions were combined and rotary evaporated at 40.degree. C.
until all volatile solvents were largely removed. The remaining
volume was then measured. Water was added dropwise until a
persistent turbidity was evident, and then 5 volumes acetone
followed by 1 volume diethyl ether (both chilled in an ice bath)
were added to the phenol solution with rapid mixing. The solution
was placed in an ice bath for 30 min and then the precipitated LPS
was recovered by centrifugation (5000.times.g, 15 min, 0-5.degree.
C.). It was generally necessary to gravity filter the supernatant
fraction to recover any LPS that did not remain in the pellet. The
LPS was washed one time in a minimal volume of cold acetone,
recovered by centrifugation/filtration, and was then dried under
vacuum. Typical yields were 4-5% based on the initial dry weight of
cells.
[0071] E. Preparation of 4'-monophosphoryl Lipid A (MLA)
[0072] LPS was suspended in water at a concentration of 10 mg/mL,
using bath sonication at 45-55.degree. C. to aid in dispersing the
solid material. The resulting solution should be slightly turbid
with no solid visible to the unaided eye. To this solution was
added 1 volume of 0.2 N HCl, and it was then placed in a boiling
water bath for 15 min. The reaction was quenched in an ice bath,
and then was extracted with 5 volumes (relative to the initial LPS
solution) of chloroform:methanol 2:1 (v/v). The biphasic solution
was vortexed and the phases were separated by low speed
centrifugation (500-1000.times.g). The lower phase was recovered
and evaporated under nitrogen, yielding crude MLA.
[0073] F. Preparation of 3-O-deacylated-4'-monophosphoryl Lipid A
(3D-MLA)
[0074] Crude MLA was dissolved in chloroform:methanol 2:1 (v/v) at
a concentration of between about 1-5 mg/mL, and 3.0 mL of this
solution was transferred to a 16.times.100 mm test tube. An
additional 0.4 mL of methanol was added to the tube, and it was
then placed in a water bath at 50.degree. C. for 10 min. The
reaction was initiated by addition of 40 .mu.L 0.5 M KHCO.sub.3, pH
10.5, and the solution was incubated at 50.degree. C. for 20 min.
At the end of this time, the tube was removed from the water bath
and the reaction was quenched by addition of 2.0 mL 0.1 N HCl
(chilled) followed by vortexing. 3D-MLA was recovered by addition
of 1.0 mL methanol, vortexing, centrifugation (500-1000.times.g),
and evaporation of the lower (organic) phase to dryness under
nitrogen.
EXAMPLE 2
Analytical Methods
[0075] A. Thin Layer Chromatography (TLC) of MLA and Related
Samples
[0076] All TLC analyses were carried out using 5.times.10 cm plates
coated with Silica Gel 60 (E Merck). Samples were generally applied
to the TLC plates as 10 mg/mL solutions in chloroform:methanol 4:1
(v/v), with 3 .mu.L solution (30 .mu.g sample) applied in small
spots to a 5 mm line using a capillary pipette. Plates were
developed with a solvent system comprising
chloroform/methanol/water/ammonium hydroxide 50:31:6:2 (v/v). Bands
on the developed plates were visualized by spraying with a solution
of 10% (w/v) phosphomolybdic acid in ethanol followed by charring
at 150-160.degree. C. In some cases, relative intensities of spots
were quantified by scanning densitometry with a Shimadzu CS9000U
Dual Wavelength Flying Spot Scanner (Shimadzu Corp.), using a
scanning wavelength of 520 nm.
[0077] B. Analysis of MLA/3D-MLA by High Performance Liquid
Chromatography (HPLC)
[0078] Samples to be analyzed were first converted to the free acid
form by washing a solution of 3-5 mg sample in 5 ml
chloroform:methanol (2:1 v:v) with 2 ml 0.1 N HCl. The biphasic
system was vortexed, centrifuged, and the lower (organic) phase was
transferred to a test tube and evaporated under a stream of
nitrogen. The residue was then methylated by treatment with
diazomethane. Briefly, an ethereal solution of diazomethane was
prepared by placing 60-100 mg 1-methyl-3-nitro-1-nitrososguanidine
(MNNG; Aldrich) in a 2 dram vial, adding 60 .mu.L diethyl ether per
mg MNNG, then adding 9 .mu.L 5 N NaOH per mg MNNG while stirring
the solution at <-10.degree. C. Following completion of the
reaction, the lemon yellow ether phase was dried by transferring it
to a second vial that contained several pellets of NaOH and
swirling, all while at <-10.degree. C. The acid-washed sample
was dissolved in 1 ml chloroform:methanol 4:1 (v:v), placed in a
bath at <-10.degree. C., and diazomethane solution was added
dropwise with stirring until a faint yellow tint persists. The
solvent was then evaporated at ambient temperature under a stream
of nitrogen and was further dried under vacuum for at least 30
min.
[0079] Chromatographic analyses were conducted on a C.sub.18
reverse phase column (Nova-Pak, 4 .mu.m particle size, 8
mm.times.10 cm [Waters]). Methylated samples were dissolved in
chloroform:methanol 4:1 (v/v) at a concentration of 100 .mu.g/mL
and passed through a 0.45 .mu.m PTFE syringe filter. An injection
volume of 20-25 .mu.L was typically used, followed by elution with
a linear gradient of 20 to 80% isopropanol in acetonitrile over 60
min at a flow rate of 2 ml/min with monitoring at 210 nm.
[0080] C. Analysis of LPS Congener Content by HPLC
[0081] LPS tends to be a highly heterogeneous material due to
variability in 1) the number of sugar residues in the O-antigen and
core regions, 2) polar substitutions in the core region and on the
phosphates in the lipid A, and 3) the number and location of fatty
acids attached to the lipid A backbone. It is this latter source of
variability that is of interest relative to the congener content of
3D-MLA (MPL.RTM.). Hydrolysis of LPS to MLA and 3D-MLA removes
variability in the O-antigen and core regions, however it also
introduces additional heterogeneity due to controlled loss of
O-linked fatty acids. This prevents the acylation pattern in the
intact LPS from being accurately known. As a way around this, a
method was developed wherein the phosphates and the core region are
removed under mild conditions that do not result in loss of
O-linked fatty acids. The resulting dephosphorylated lipid A (zero
phosphoryl lipid A, or ZPL) can then be analyzed by HPLC, yielding
an accurate reflection of the acylation pattern in the parent
LPS.
[0082] The method was typically carried out as follows. Between
0.5-5.0 mg of LPS sample was hydrolyzed in 200 .mu.L concentrated
hydrofluoric acid for 3-4 h at 27.degree. C. This reaction must be
done in a tightly capped Teflon tube and in a well-ventilated fume
hood. The HF was removed by evaporation under a stream of nitrogen
at ambient temperature, and the hydrolysate was then dissolved in
chloroform:methanol 4:1 (v/v) and transferred to a 16.times.100 mm
glass test tube, and solvent was evaporated under a stream of
nitrogen. The residue was suspended in 1.0 mL 0.1% triethylamine
using bath sonication, 1.0 mL 40 mM NaOAc was added, and the tube
was suspended in a boiling water bath for 30-45 min. The reaction
was quenched by cooling in an ice bath and the ZPL was recovered by
extraction with 5 mL chloroform:methanol 2:1 (v/v). The organic
phase was transferred to a small screw cap vial and solvent was
evaporated under nitrogen. The ZPL was derivatized by adding 200
.mu.L of 10 mg/mL O-(3,5-dinitrobenzyl)hydroxylamine HCl (Regis
Technologies, Inc.) in pyridine, tightly capping the vial, then
incubating at 60.degree. C. for 3 h. Pyridine was evaporated under
nitrogen and the residue was further dried under vacuum for >30
min. The residue was then suspended in 500 .mu.L
chloroform:methanol 2:1 (v/v) and loaded onto a 0.5-1.0 mL bed of
Accell-QMA (acetate form; Waters) that had been pre-equilibrated in
the same solvent. The column was rinsed with a total of 5.0 mL
chloroform:methanol 2:1 (v/v) in several small portions and the
eluate was collected in a 16.times.100 mm test tube. 2.0 mL 0.1 N
HCl was added to the eluate, the biphasic system was vortexed,
centrifuged briefly at 500-1000.times.g, and the lower (organic)
phase was transferred to a another test tube and evaporated under
nitrogen. The residue was dissolved in 100-300 .mu.L
chloroform:methanol 4:1 (v/v) and filtered through a 0.45 .mu.M
PTFE syringe filter. The filter was rinsed twice with
chloroform:methanol 4:1 (v/v) and the filtrate was evaporated under
nitrogen. The filtrate was finally taken up in 50-150 .mu.L
chloroform:methanol 4:1 (v/v) and transferred to an autoinjector
vial for HPLC analysis. HPLC conditions were as follows: C.sub.18
reverse phase column (e.g., Waters), 10 .mu.L injection volume,
linear gradient 20 to 80% isopropanol in acetonitrile over 60 min
at a flow rate of 2 ml/min, monitor at 254 nm.
EXAMPLE 3
Comparison of Congener Composition of MLA/3D-MLA from Cultures
Harvested at Different Times
[0083] A series of fermentor runs was conducted with the following
parameters: 2.0 L M9 medium (initial pH 6.84-6.87), 2 Lpm air flow,
stirring at 50 rpm, 37.degree. C., no pH control. Cultures were
monitored by measuring optical density at 590 nm and were stopped
when the desired growth stage was attained. Cells were processed
and extracted as described above to yield LPS samples, which were
then hydrolyzed to MLA and 3D-MLA and analyzed by HPLC (see
Examples 1 and 2). Results are summarized in Table 1.
TABLE-US-00001 TABLE 1 Congener composition of MLA and 3D-MLA from
cells harvested at different ages. MLA 3D-MLA 3-O- 3-O- Culture age
Time in deacylated deacylated Run Description at harvest stationary
phase hexaacyl heptaacyl hexaacyl A Late exponential 6.75 h N/A
12.4% 12.2% 9.9% B Early stationary 9.5 h .about.0.5 h 9.2% 6.8%
9.2% phase C Late stationary 15 h .about.6 h 19.5% 13.2% 21.5%
phase
[0084] The data show that cultures of S. minnesota R595 alter the
acylation pattern of their LPS during stationary phase, resulting
in an increase in the overall content of 3-O-deacylated hexaacyl
plus heptaacyl species in MLA derived from this LPS, and this in
turn gives rise to increased content of 3-O-deacylated hexaacyl
species in 3D-MLA prepared from this MLA.
EXAMPLE 4
Comparison of Congener Composition of LPS from Cultures Harvested
at Different Times
[0085] A series of fermentor runs was conducted with the following
parameters: 2.0 L M9 medium (initial pH 6.84-6.87), 2 Lpm air flow,
stirring at 225 rpm, 37.degree. C., no pH control. The growth stage
of the cultures was monitored by measuring optical density at 590
nm. Cells were processed and extracted as described in Example 1 to
yield LPS samples. LPS samples were hydrolyzed to ZPL and analyzed
by HPLC as described in Example 2. Results are summarized in Table
2. TABLE-US-00002 TABLE 2 Congener composition of LPS from cells
harvested at different ages. Congener content Culture Time in 3-O-
age at time stationary 3-O-acyl deacylated Run Description of
harvest phase hexaacyl hexaacyl heptaacyl A Early 9 h .about.0.5 h
76% 0% 24% stationary phase B Late 15 h .about.6 h 48% 17% 19%
stationary phase
[0086] No 3-O-deacylated hexaacyl component was detected in the LPS
from the early stationary phase cells (run A). Thus, the only
source of hexaacylated congeners in 3D-MLA prepared from this LPS
would be the heptaacylated material (24%). In contrast, LPS from
cells harvested at late stationary phase contained both heptaacyl
and 3-O-deacylated hexaacyl species (19% and 17%, respectively).
Both of these species would contribute to the hexaacyl content in
3D-MLA (MPL.RTM.) prepared from this LPS. It was unexpected to find
that cells produce 3-O-deacylated hexaacyl LPS species under
certain conditions.
EXAMPLE 5
Effect of Pre-extraction Temperature on Purity of S. minnesota R595
LPS
[0087] Cells of S. minnesota R595 were grown in an 80 L fermentor
(New Brunswick Scientific) using essentially the same conditions as
outlined in Example 1. The cells were concentrated by tangential
flow filtration but were not centrifuged, and the slurry contained
51.5 mg dry cell mass per mL. Three solutions were prepared in
which 150 mL aliquots of the cell suspension were each combined
with 600 mL ethanol. The ethanol solutions were stirred for 1 h at
22.degree. C., 37.degree. C., and 50.degree. C. and were filtered.
The cells were subjected to a second ethanol extraction under the
same conditions except using 95% ethanol. The cells were recovered
by suction filtration and were then extracted overnight in
chloroform:methanol 4:1 (v/v) at 50.degree. C. The solutions were
filtered and the filtrates were rotary evaporated to dryness,
yielding the LPS preparations. Samples of the first and second
ethanol extraction filtrates obtained at each temperature as well
as the LPS obtained from the pre-extracted cells were analyzed by
thin layer chromatography according to the method in Example 2.
Images of the TLC plates are shown in FIG. 1.
[0088] FIG. 1 shows TLC plates of ethanol extracts and LPS samples
obtained with different temperatures during the ethanol
extractions. The plate on the left shows, going from left to right,
the ethanol extracts at temperatures of 22.degree. C., 37.degree.
C., and 50.degree. C. The sample at the far right of this plate is
an authentic LPS sample. The plate on the right shows the LPS
obtained from each preparation. The samples in lanes 3, 4, and 5
correspond to LPS from cells subjected to pre-extractions with
ethanol at 22.degree. C., 37.degree. C., and 50.degree. C.,
respectively. The heavy bands at R.sub.f.about.0.6 correspond to
phospholipid and fatty acid impurities. The levels of these
impurities are reduced by increasing the temperature of the ethanol
extractions, and are very low in the sample that was pre-extracted
at 50.degree. C.
[0089] It is apparent from the TLC plates in FIG. 1 that
pre-extraction with ethanol at elevated temperatures is effective
at removing impurities that are otherwise co-extracted with
chloroform:methanol 4:1 (v/v). Pre-extraction at 50.degree. C.
results in LPS that is largely free of these impurities.
EXAMPLE 6
Comparison of LPS Obtained by with and without Pre-extraction with
Ethanol
[0090] Three batches of cells of S. minnesota R595 were grown in a
750 L fermentor (B. Braun) using essentially the same conditions as
outlined in Example 1. Cells were harvested by tangential flow
filtration, and a sample of the cell suspension was obtained from
each batch and lyophilized. The bulk of the cells were subjected to
two pre-extractions with 90% ethanol at 50.degree. C. for 1 hr.
Cells were recovered by tangential flow filtration between
extractions. The cells were then extracted overnight with
chloroform:methanol 4:1 (v/v) at reflux. The extract was recovered
by tangential flow filtration and evaporated to dryness. The
lyophilized cell samples were extracted overnight in refluxing
chloroform:methanol 4:1 (v/v), and the solutions were filtered and
the filtrates were evaporated to dryness. LPS samples obtained with
and without ethanol pre-extraction was analyzed by TLC essentially
as described in Example 2. TLC plates were scanned from about
R.sub.f=0.01 to 0.90, and the ratio of intensity in the LPS region
(R.sub.f=0.01 to 0.020) to total intensity was calculated for each
sample. The results are given in Table 3. TABLE-US-00003 TABLE 3
LPS purity from cells with and without pre-extraction with ethanol.
Percent of total intensity in LPS region.sup.1 without ethanol with
ethanol Run Lot Number pre-extraction pre-extraction A 48020-B2698C
7 86 B 48020-C0598A 11 83 C 48020-C0598B 14 88 Note: .sup.1Percent
of total intensity in LPS region = [(intensity in R.sub.f = 0.01 to
0.20)/(intensity in R.sub.f = 0.01 to 0.90)] .times. 100
[0091] The results in Table 3 demonstrate that the LPS obtained
following pre-extraction of S. minnesota R595 cells with 90%
ethanol at 50.degree. C. is substantially purer than material from
cells without pre-extraction.
[0092] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the steps or in the sequence of steps of the methods
described herein without departing from the concept, spirit and
scope of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.
* * * * *