U.S. patent application number 17/170036 was filed with the patent office on 2021-06-17 for media and fermentation methods for producing polysaccharides in bacterial cell culture.
This patent application is currently assigned to Pfizer Inc.. The applicant listed for this patent is Pfizer Inc.. Invention is credited to Sunil Gururao Desai, Michael Allen Hanson, Jonathan Patrick Kinross, Daniel R. Lasko, Scott Ellis Lomberk, Jason Arnold Lotvin, Sujata Kaushikbhai Patel-Brown, Weiqiang Sun, Peter Anthony Tomasello.
Application Number | 20210180008 17/170036 |
Document ID | / |
Family ID | 1000005417453 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210180008 |
Kind Code |
A1 |
Desai; Sunil Gururao ; et
al. |
June 17, 2021 |
MEDIA AND FERMENTATION METHODS FOR PRODUCING POLYSACCHARIDES IN
BACTERIAL CELL CULTURE
Abstract
The present invention relates to media and fermentation methods
for producing polysaccharides in bacterial cell culture. In one
aspect, the invention relates to a complex culture medium
comprising a vegetable hydrolysate, a yeast extract, and a carbon
source. In another aspect, the invention relates to a defined media
having a total amino acid concentration greater than about 50 mM. A
further aspect of the invention relates to the use of fed batch and
perfusion fermentation methods for cultivating
polysaccharide-producing bacteria.
Inventors: |
Desai; Sunil Gururao;
(Andover, MA) ; Hanson; Michael Allen; (Pearl
River, NY) ; Kinross; Jonathan Patrick; (Reading,
MA) ; Lasko; Daniel R.; (Medford, MA) ;
Lomberk; Scott Ellis; (Suffern, NY) ; Lotvin; Jason
Arnold; (West Nyack, NY) ; Patel-Brown; Sujata
Kaushikbhai; (Valley Cottage, NY) ; Sun;
Weiqiang; (Morristown, NJ) ; Tomasello; Peter
Anthony; (Methuen, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc. |
New York |
NY |
US |
|
|
Assignee: |
Pfizer Inc.
New York
NY
|
Family ID: |
1000005417453 |
Appl. No.: |
17/170036 |
Filed: |
February 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15775172 |
May 10, 2018 |
10947494 |
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PCT/IB2016/056780 |
Nov 10, 2016 |
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17170036 |
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62413051 |
Oct 26, 2016 |
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62256347 |
Nov 17, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12R 2001/46 20210501;
C12N 1/20 20130101; C12N 1/205 20210501; C12P 19/04 20130101 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12P 19/04 20060101 C12P019/04 |
Claims
1. A polysaccharide-producing bacterial cell culture medium
comprising a vegetable hydrolysate, a yeast extract, and a carbon
source.
2. The medium of claim 1, wherein the vegetable hydrolysate is a
soy hydrolysate.
3. The medium of claim 2, wherein the soy hydrolysate is selected
from the group consisting of HYPEP 1510 (Kerry Group Services
Ltd.), HYPEP 4601 (Kerry Group Services Ltd.), HYPEP 5603 (Kerry
Group Services Ltd.), HY-SOY (Kerry Group Services Ltd.), AMI-SOY
(Kerry Group Services Ltd.), N-Z-SOY (Kerry Group Services Ltd.),
N-Z-SOY BL4 (Kerry Group Services Ltd.), N-Z-SOY BL7 (Kerry Group
Services Ltd.), SHEFTONE D (Kerry Group Services Ltd.), SE50M,
SE50MK, soy peptone, BACTO soytone (Difco Laboratories Inc.),
NUTRISOY 2207 (ADM), NUTRISOY (ADM), NUTRISOY flour (ADM), and
soybean meal.
4. The medium of claim 3, wherein the soy hydrolysate is HYPEP 1510
(Kerry Group Services Ltd.).
5. The medium of any one of claims 1-4, wherein the concentration
of the vegetable hydrolysate is between about 5 g/L and about 75
g/L.
6. The medium of claim 5, wherein the concentration of the
vegetable hydrolysate is between about 10 g/L and about 50 g/L.
7. The medium of claim 6, wherein the concentration of the
vegetable hydrolysate is about 28 g/L.
8. The medium of any one of claims 1-7, wherein the yeast extract
is a yeast autolysate, an ultrafiltered yeast extract, or a
synthetic yeast extract.
9. The medium of claim 8, wherein the yeast extract is an
ultrafiltered yeast extract.
10. The medium of claim 9, wherein the ultrafiltered yeast extract
is AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD
Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE
(Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services
Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry
Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or
HY-YEST 504 (Kerry Group Services Ltd.).
11. The medium of any one of claims 1-10, wherein the concentration
of yeast extract is between about 1 g/L to about 50 g/L.
12. The medium of claim 11, wherein the concentration of yeast
extract is between about 5 g/L to about 25 g/L.
13. The medium of claim 12, wherein the concentration of yeast
extract is about 10 g/L.
14. The medium of any one of claims 1-13, wherein the carbon source
is selected from the group consisting of glucose, dextrose,
mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose,
and mannose.
15. The medium of claim 14, wherein the carbon source is
glucose.
16. The medium of any one of claims 1-15, wherein the concentration
of the carbon source is between about 25 g/L to about 100 g/L.
17. The medium of claim 16, wherein the concentration of the carbon
source is between about 50 g/L to about 90 g/L.
18. The medium of claim 17, wherein the concentration of the carbon
source is about 80 g/L.
19. The medium of any one of claims 1-18, wherein the medium
comprises soy hydrolysate, an ultrafiltered yeast extract, and
glucose.
20. The medium of any one of claims 1-19, wherein the medium
further comprises a phosphate-containing ingredient.
21. The medium of claim 20, wherein the phosphate-containing
ingredient is Na.sub.2HPO.sub.4, K.sub.2HPO.sub.4, or
KH.sub.2PO.sub.4.
22. The medium of any one of claims 1-21, wherein the medium
further comprises at least one amino acid, vitamin, nucleoside, or
inorganic salt.
23. A polysaccharide-producing bacterial cell culture medium having
a total amino acid concentration greater than about 50 mM.
24. The medium of claim 23, wherein the medium comprises a total
glycine concentration of between about 1.5 mM and about 60.0
mM.
25. The medium of claim 24, wherein the total glycine concentration
is between about 5.0 mM and about 15.0 mM.
26. The medium of claim 25, wherein the total glycine concentration
is about 7.5 mM.
27. The medium of any one of claims 23-26, wherein the medium
comprises a total arginine concentration of between about 1.0 mM
and about 30.0 mM.
28. The medium of claim 27, wherein the total arginine
concentration is between about 1.0 mM and about 20.0 mM.
29. The medium of claim 28, wherein the total arginine
concentration is about 4.0 mM.
30. The medium of any one of claims 23-29, wherein the medium
comprises a total cysteine concentration of between about 0.1 mM
and about 5.0 mM.
31. The medium of claim 30, wherein the total cysteine
concentration is between about 0.1 mM and about 3.5 mM.
32. The medium of claim 31, wherein the total cysteine
concentration is about 0.4 mM.
33. The medium of any one of claims 23-32, wherein the medium
comprises a total serine concentration of between about 5.0 mM and
about 75.0 mM.
34. The medium of claim 33, wherein the total serine concentration
is between about 5.0 mM and about 15.0 mM.
35. The medium of claim 34, wherein the total serine concentration
is about 7.5 mM, or about 10 mM.
36. The medium of any one of claims 23-35, wherein the medium
comprises a total glutamine concentration of between about 1.0 mM
and about 30.0 mM.
37. The medium of claim 36, wherein the total glutamine
concentration is between about 1.0 mM and about 20.0 mM.
38. The medium of claim 37, wherein the total glutamine
concentration is about 4.0 mM.
39. The medium of any one of claims 23-38, wherein the medium
comprises a total concentration of tyrosine of between about 0.1 mM
and about 5.0 mM.
40. The medium of claim 39, wherein the total tyrosine
concentration is between about 1.0 mM and about 3.5 mM.
41. The medium of claim 40, wherein the total tyrosine
concentration is about 2.9 mM or about 3.0 mM.
42. The medium of any one of claims 23-41, wherein the medium
comprises a total concentration of asparagine of between about 5.0
mM and about 50.0 mM.
43. The medium of claim 42, wherein the total asparagine
concentration is between about 10.0 mM and about 30.0 mM.
44. The medium of claim 43, wherein the total asparagine
concentration is about 20.0 mM.
45. The medium of any one of claims 23-41, wherein the medium does
not contain asparagine.
46. The medium of any one of claims 23-45, wherein the medium
further comprises a potassium salt.
47. The medium of claim 46, wherein the potassium salt is potassium
chloride or potassium sulfate.
48. The medium of claim 46 or claim 47, wherein the total
concentration of potassium salt is between about 0.1 g/L and about
25 g/L.
49. The medium of claim 48, wherein the total potassium salt
concentration is between about 0.2 g/L and about 1.25 g/L.
50. The medium of claim 49, wherein the total potassium salt
concentration is about 0.9 g/L.
51. The medium of any one of claims 23-50, wherein the medium
further comprises a carbon source.
52. The medium of claim 51, wherein the carbon sources is selected
from the group consisting of glucose, dextrose, mannitol, lactose,
sucrose, fructose, galactose, raffinose, xylose, and mannose.
53. The medium of claim 52, wherein the carbon sources is
glucose.
54. The medium of any one of claims 51-53, wherein medium comprises
a total concentration of the carbon source of between about 25 g/L
and about 100 g/L.
55. The medium of claim 54, wherein the total concentration of the
carbon source is between about 25 g/L and about 80 g/L.
56. The medium of claim 55, wherein the total concentration of the
carbon source is about 50 g/L.
57. The medium of any one of claims 23-56, wherein the medium
further comprises sodium bicarbonate.
58. The medium of claim 57, wherein the medium comprises a
concentration of sodium bicarbonate of between about 0.1 g/L and
about 20 g/L.
59. The medium of claim 58, wherein the concentration of sodium
bicarbonate is between about 0.5 g/L and about 1.0 g/L.
60. The medium of claim 59, wherein the concentration of sodium
bicarbonate is about 0.84 g/L.
61. The medium of any one of claims 23-60, wherein the medium
further comprises a yeast extract.
62. The medium of claim 61, wherein the yeast extract is selected
from the group consisting of a yeast autolysate, an ultrafiltered
yeast extract, and a synthetic yeast extract.
63. The medium of claim 62, wherein the yeast extract is an
ultrafiltered yeast extract.
64. The medium of claim 63, wherein the ultrafiltered yeast extract
is AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD
Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE
(Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services
Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry
Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or
HY-YEST 504 (Kerry Group Services Ltd.).
65. The medium of any one of claims 61-64, wherein the
concentration of yeast extract is between about 1 g/L to about 50
g/L.
66. The medium of claim 65, wherein the concentration of yeast
extract is between about 5 g/L to about 25 g/L.
67. The medium of claim 66, wherein the concentration of yeast
extract is about 10 g/L.
68. The medium of any one of claims 23-67, wherein the medium
comprises at least about 50 mM of amino acids, a potassium salt, a
carbon source, and optionally, a yeast extract.
69. The medium of claim 68, wherein the medium comprises at least
about 50 mM of amino acids, between about 5.0 mM and about 15.0 mM
of glycine, between about 0.2 g/L and about 1.25 g/L of a potassium
salt, between about 25 g/L and about 80 g/L of a carbon source, and
between about 5 g/L to about 25 g/L of a yeast extract.
70. The medium of claim 69, wherein the medium comprises at least
about 60 mM of amino acids, about 7.5 mM of glycine, about 0.9 g/L
of potassium chloride, 50 g/L of glucose, and about 10 g/L of an
ultrafiltered yeast extract.
71. A method of cultivating a polysaccharide-producing bacteria
comprising a) adding a medium of any one of claims 1-70 to a
bioreactor, b) seeding the medium with a polysaccharide-producing
bacteria, and c) cultivating the bacteria by fermentation, wherein
said cultivation comprises the addition of a nutrient at a constant
rate to the medium.
72. The cultivation method of claim 71, wherein the nutrient is a
carbon source.
73. The cultivation method of claim 72, wherein the carbon source
is glucose.
74. The cultivation method of any one of claims 71-73, wherein the
cultivated bacteria have a cell density of at least 9.0.
75. The cultivation method of any one of claims 71-74, wherein the
cultivated bacteria have a polysaccharide concentration of at least
about 250 mg/L.
76. The cultivation method of any one of claims 71-75, wherein the
polysaccharide-producing bacteria is selected from the group
consisting of Streptococcus agalactiae, Streptococcus pneumoniae,
Staphylococcus aureus, Neisseria meningitidis, Escherichia coli,
Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae,
Enterococcus faecium, and Enterococcus faecalis.
77. A method of cultivating a polysaccharide-producing bacteria
comprising a) adding a medium of any one of claims 1-70 to a
bioreactor, b) seeding the medium with a polysaccharide-producing
bacteria, and c) cultivating the bacteria by perfusion, wherein the
cultivation comprises (i) removing spent medium from the culture,
(ii) adding fresh medium, and (iii) retaining the bacteria.
78. The cultivation method of claim 77, wherein the rate of
perfusion is between about 0.07 VVH to about 2.00 VVH.
79. The cultivation method of claim 78, wherein the rate of
perfusion is between about 0.67 VVH to about 1.33 VVH.
80. The cultivation method of claim 79, wherein the rate of
perfusion is about 1.20 VVH.
81. The cultivation method of claim 77, wherein the rate of
perfusion is varied.
82. The cultivation method of claim 81, wherein the perfusion
starts at a first rate and the rate is increased to a second
rate.
83. The cultivation method of claim 81, wherein the perfusion
starts at a first rate and the rate is decreased to a second
rate.
84. The cultivation method of any one of claims 77-83, wherein the
duration of perfusion is between about 1 hour and about 15
hours.
85. The cultivation method of claim 84, wherein the duration of
perfusion is between about 1 hour and about 10 hours.
86. The cultivation method of claim 85, wherein the duration of
perfusion is about 7 hours.
87. The cultivation method of any one of claims 77-86, wherein the
cell growth of the cultivated bacteria is at least 2-fold greater
than the cell growth in a batch fermentation system.
88. The cultivation method of any one of claims 77-87, wherein the
cultivated bacteria have reached a cell density of at least
20.0.
89. The cultivation method of any one of claims 77-88, wherein the
cultivated bacteria have reached a polysaccharide concentration of
at least about 600 mg/L.
90. The cultivation method of any one of claims 77-89, wherein
wherein the polysaccharide-producing bacteria is selected from the
group consisting of Streptococcus agalactiae, Streptococcus
pneumoniae, Staphylococcus aureus, Neisseria meningitidis,
Escherichia coli, Salmonella typhi, Haemophilus influenzae,
Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus
faecalis.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to media and fermentation
methods for producing polysaccharides in bacterial cell culture. In
one aspect, the invention relates to a complex culture medium
comprising a vegetable hydrolysate, a yeast extract, and a carbon
source. In another aspect, the invention relates to a defined media
having a total amino acid concentration greater than about 50 mM. A
further aspect of the invention relates to the use of fed batch and
perfusion fermentation methods for cultivating
polysaccharide-producing bacteria.
BACKGROUND OF THE INVENTION
[0002] A cell surface polysaccharide refers to a polysaccharide
having at least a portion located on the outermost bacterial cell
membrane or bacterial cell surface, including the peptidoglycan
layer, cell wall, and capsule. Cell surface polysaccharides,
particularly capsular polysaccharides, have become increasingly
important as therapeutic agents. Typically, a cell surface
polysaccharide is associated with inducing an immune response in
vivo. Some examples of polysaccharide vaccines include
PNEUMOVAX.RTM. 23, which is a 23-valent vaccine for the prevention
of invasive disease, such as pneumonia, febrile bacteraemia, and
meningitis, caused by Streptococcus pneumoniae; MENCEVAX.RTM.,
which is a quadrivalent vaccine for the prevention of invasive
disease caused by Neisseria meningitidis; TYPHERIX.RTM. and TYPHIM
VI.RTM., both of which prevent typhoid fever caused by Salmonella
typhi Vi.
[0003] Although polysaccharides are immunogenic on their own,
conjugation of polysaccharides to protein carriers has been used to
improve immunogenicity, particularly in infants and the elderly.
The chemical bonding of the polysaccharide and protein carrier
induces an immune response against bacteria displaying the
polysaccharide contained within the vaccine on their surface, thus
preventing disease. Accordingly, vaccination using polysaccharides
from pathogenic bacteria is a potential strategy for boosting host
immunity.
[0004] There are several polysaccharide-protein conjugate vaccines
currently available and several more under development to address
unmet therapeutic areas in need. For instance, there are three
pneumococcal conjugate vaccines used to protect against invasive
pneumococcal disease available on the global market: PREVNAR.RTM.
(called PREVENAR.RTM. in some countries) (heptavalent vaccine),
SYNFLORIX.RTM. (a decavalent vaccine), and PREVNAR 13.RTM.
(tridecavalent vaccine). MENINGITEC.RTM., MENJUGATE.RTM., and
NEISVAC-C.RTM. are meningococcal serogroup C conjugate vaccines
while MENVEO.RTM., MENACTRA.RTM., and NIMENRIX.RTM. are
quadrivalent meningococcal conjugate vaccines that protect against
N. meningitidis serogroups A, C, Y, and W-135. HIBERIX.RTM.
prevents against disease caused by Haemophilus influenzae type
b.
[0005] Individual monovalent polysaccharide-protein conjugates of
Streptococcus agalactiae, also known as Group B Streptococcus
(GBS), serotypes Ia, Ib, II, III, and V have been evaluated in
phase 1 and 2 clinical trials in non-pregnant adults (Brigtsen, A.
K., et al., Journal of Infectious Diseases, 185(9):1277-1284
(2002); Baker, C. J., et al., J. Infect. Dis., 188(1):66-73 (2003);
Baker, C. J., et al., J. Infect. Dis., 189(6):1103-1112 (2004);
Baker, C. J., et al., Vaccine, 25(1):55-63 (2007)). Bivalent II-TT
and III-TT glycoconjugate vaccines and a trivalent vaccine
comprising Ia-CRM.sub.197, Ib-CRM.sub.197 and III-CRM.sub.197
glycoconjugates have also been studied (Baker JID 2003;
Clicaltrials.gov NCT01193920, NCT01412801, and NCT01446289).
However, no GBS vaccines have yet been approved.
[0006] A vaccine comprising capsular polysaccharide-protein
conjugates is also being developed to prevent surgical site
infections caused by Staphylococcus aureus (Anderson, A. S., et
al., Hum. Vaccin. Immunother., 8(11):1585-1594 (2012)).
[0007] Accordingly, there is a need for the development of improved
systems for producing polysaccharides by bacterial cell
culture.
SUMMARY OF THE INVENTION
[0008] To meet these and other needs, the present invention relates
to media and fermentation methods for producing polysaccharides in
bacterial cell culture and includes the invention disclosed in U.S.
Provisional Application No. 62/256,347, filed Nov. 17, 2015, the
entirety of which is hereby incorporated by reference. The
following clauses describe some aspects and embodiments of the
invention.
[0009] One aspect of the invention relates to a
polysaccharide-producing bacterial cell culture medium comprising a
vegetable hydrolysate, a yeast extract, and a carbon source. In one
embodiment, the vegetable hydrolysate may be a soy hydrolysate,
such as HYPEP 1510 (Kerry Group Services Ltd.), HYPEP 4601 (Kerry
Group Services Ltd.), HYPEP 5603 (Kerry Group Services Ltd.),
HY-SOY (Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services
Ltd.), N-Z-SOY (Kerry Group Services Ltd.), N-Z-SOY BL4 (Kerry
Group Services Ltd.), N-Z-SOY BL7 (Kerry Group Services Ltd.),
SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK, soy peptone,
BACTO Soytone (Difco Laboratories Inc.), NUTRISOY 2207 (Archer
Daniels Midland Company (ADM)), NUTRISOY (ADM), NUTRISOY FLOUR
(ADM), or soybean meal. In another embodiment, the concentration of
the soy hydrolysate may be between about 5 g/L and about 75 g/L,
such as between about 10 g/L and about 50 g/L, or about 28 g/L.
[0010] In a further embodiment, the yeast extract may be a yeast
autolysate, an ultrafiltered yeast extract, or a synthetic yeast
extract. In a particular embodiment, the yeast extract is an
ultrafiltered yeast extract, such as AMBERFERM 5902 (Sensient
Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry
Group Services Ltd.), HY-YEST 412 (Kerry Group Services Ltd.),
HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry Group
Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), HY-YEST
504 (Kerry Group Services Ltd.), or ULTRAPEP YE (Kerry Group
Services Ltd.). In yet another embodiment, the concentration of
yeast extract is between about 1 g/L to about 50 g/L, such as
between about 5 g/L to about 25 g/L, or about 10 g/L.
[0011] In one embodiment, the carbon source may be glucose,
dextrose, mannitol, lactose, sucrose, fructose, galactose,
raffinose, xylose, or mannose. In a particular embodiment, the
carbon source is glucose. In a further embodiment, the
concentration of the carbon source is between about 25 g/L to about
100 g/L, such as between about 50 g/L to about 90 g/L, or about 80
g/L.
[0012] In one embodiment, the medium further comprises a
phosphate-containing ingredient, such as Na.sub.2HPO.sub.4,
K.sub.2HPO.sub.4 or KH.sub.2PO.sub.4.
[0013] In another embodiment, the medium further comprises at least
one amino acid, vitamin, nucleoside, or inorganic salt.
[0014] Another aspect of the invention relates to a
chemically-defined polysaccharide-producing bacterial cell culture
medium having a total amino acid concentration greater than about
50 mM. In one embodiment, the medium comprises a total glycine
concentration of between about 1.5 mM and about 60.0 mM, such as
between about 5.0 mM and about 15.0 mM, or about 7.5 mM. In another
embodiment, the medium comprises a total arginine concentration of
between about 1.0 mM and about 30.0 mM, such as between about 1.0
mM and about 20.0 mM, or about 4.0 mM. In a further embodiment, the
medium comprises a total cysteine concentration of between about
0.1 mM and about 5.0 mM, such as between about 0.1 mM and about 3.5
mM, or about 0.4 mM. In yet another embodiment, the medium
comprises a total serine concentration of between about 5.0 mM and
about 75.0 mM, such as between about 5.0 mM and about 15.0 mM, or
about 7.5 mM. In another embodiment, the medium comprises a total
glutamine concentration of between about 1.0 mM and about 30.0 mM,
such as between about 1.0 mM and about 20.0 mM, or about 4.0 mM. In
a further embodiment, the medium comprises a total concentration of
tyrosine of between about 0.1 mM and about 5.0 mM, such as between
about 1.0 mM and about 3.5 mM, or between about 2.9 mM and about
3.0 mM. In yet another embodiment, the medium comprises a total
concentration of asparagine of between about 5.0 mM and about 50.0
mM, such as between about 10.0 mM and about 30.0 mM, or about 20.0
mM. In a particular embodiment, the medium does not contain
asparagine.
[0015] In one embodiment, the medium further comprises a potassium
salt, such as potassium chloride or potassium sulfate. In an
embodiment, the total concentration of potassium salt is between
about 0.1 g/L and about 25 g/L, such as between about 0.2 g/L and
about 1.25 g/L, or about 0.9 g/L.
[0016] In one embodiment, the medium further comprises a carbon
source, such as glucose, dextrose, mannitol, lactose, sucrose,
fructose, galactose, raffinose, xylose, or mannose. In a particular
embodiment, the carbon source is glucose. In an embodiment, the
total concentration of the carbon source may be between about 25
g/L and about 100 g/L, such as between about 25 g/L and about 80
g/L, or about 50 g/L.
[0017] In one embodiment, the medium further comprises sodium
bicarbonate. In an embodiment, the concentration of sodium
bicarbonate may be between about 0.1 g/L and about 20 g/L, such as
between about 0.5 g/L and about 1.0 g/L, or about 0.84 g/L.
[0018] In one embodiment, the medium further comprises a yeast
extract, such as a yeast autolysate, an ultrafiltered yeast
extract, or a synthetic yeast extract. In a particular embodiment,
the yeast extract is an ultrafiltered yeast extract, such as
AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD
Biosciences), HYPEP YE (Kerry Group Services Ltd.), HY-YEST 412
(Kerry Group Services Ltd.), HY-YEST 441 (Kerry Group Services
Ltd.), HY-YEST 444 (Kerry Group Services Ltd.), HY-YEST 455 (Kerry
Group Services Ltd.), HY-YEST 504 (Kerry Group Services Ltd.), or
ULTRAPEP YE (Kerry Group Services Ltd.). In a further embodiment,
the concentration of yeast extract is between about 1 g/L to about
50 g/L, such as between about 5 g/L to about 25 g/L, or about 10
g/L.
[0019] In a particular embodiment, the medium comprises at least
about 50 mM of amino acids, a potassium salt, a carbon source, and
optionally, a yeast extract.
[0020] In another embodiment, the medium comprises at least about
50 mM of amino acids, between about 5.0 mM and about 15.0 mM of
glycine, between about 0.2 g/L and about 1.25 g/L of a potassium
salt, between about 25 g/L and about 100 g/L of a carbon source,
and between about 5 g/L to about 25 g/L of a yeast extract.
[0021] In a further embodiment, the medium comprises at least about
60 mM of amino acids, about 7.5 mM of glycine, about 0.9 g/L of
potassium chloride, 50 g/L of glucose, and about 10 g/L of an
ultrafiltered yeast extract.
[0022] A further aspect of the invention relates to a method of
cultivating a polysaccharide-producing bacteria comprising a)
adding a medium of the invention to a bioreactor, b) seeding the
medium with a polysaccharide-producing bacteria, and c) cultivating
the bacteria by fermentation, wherein said cultivation comprises
the addition of a nutrient at a constant rate to the medium. In one
embodiment, the nutrient is a carbon source, such as glucose. In
one embodiment, the cultivation is carried out until the bacteria
have a cell density, as determined by optical density (OD) at 600
nm, of at least 9.0. In another embodiment, the cultivated bacteria
have a cell density, as determined by OD at 600 nm, of at least
9.0. In another embodiment, the cultivation is carried out until
the bacteria have a polysaccharide concentration of at least about
250 mg/L. In another embodiment, the cultivated bacteria have a
polysaccharide concentration of at least about 250 mg/L. In a
further embodiment, the polysaccharide-producing bacteria is
selected from the group consisting of Streptococcus agalactiae,
Streptococcus pneumoniae, Staphylococcus aureus, Neisseria
meningitidis, Escherichia coli, Salmonella typhi, Haemophilus
influenzae, Klebsiella pneumoniae, Enterococcus faecium, and
Enterococcus faecalis.
[0023] Yet another aspect of the invention relates to a method of
cultivating a polysaccharide-producing bacteria comprising a)
adding a medium as described above to a bioreactor, b) seeding the
medium with a polysaccharide-producing bacteria, and c) cultivating
the bacteria by perfusion, wherein the cultivation comprises (i)
removing spent medium from the culture, (ii) adding fresh medium,
and (iii) retaining the bacteria. In one embodiment, the rate of
perfusion is between about 0.07 volumes of feed per starting
culture volume per hour (VVH) to about 2.00 VVH, such as between
about 0.67 VVH to about 1.33 VVH, or about 1.20 VVH. In another
embodiment, the rate of perfusion is varied. For instance, in one
embodiment the perfusion starts at a first rate and the rate is
increased to a second rate. In another embodiment, the perfusion
starts at a first rate and the rate is decreased to a second
rate.
[0024] In one embodiment, the duration of perfusion is between
about 1 hour and about 15 hours, such as between about 1 hour and
about 10 hours, or about 7 hours.
[0025] In another embodiment, the cell growth of the cultivated
bacteria is at least 2-fold greater than the cell growth in a batch
fermentation system. In one embodiment, the cultivation is carried
out until the bacteria have a cell density, as determined by OD at
600 nm, of at least 20.0. In a further embodiment, the cultivated
bacteria have a cell density, as determined by OD at 600 nm, of at
least 20.0. In yet another embodiment, the cultivation is carried
out until the bacteria have a polysaccharide concentration of at
least about 600 mg/L. In yet another embodiment, the cultivated
bacteria have a polysaccharide concentration of at least about 600
mg/L.
[0026] In a further embodiment, the polysaccharide-producing
bacteria is selected from the group consisting of Streptococcus
agalactiae, Streptococcus pneumoniae, Staphylococcus aureus,
Neisseria meningitidis, Escherichia coli, Salmonella typhi,
Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus
faecium, and Enterococcus faecalis.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention provides media and methods for
producing polysaccharides by bacterial cell culture. In particular,
the invention provides systems that maximize capsular
polysaccharide production of encapsulated bacteria.
[0028] Before the present composition and methods are described, it
is to be understood that this invention is not limited to
particular methods and experimental conditions described, as such
methods and conditions may vary. It is also to be understood that
the terminology used herein is for purposes of describing
particular embodiments only and is not intended to be limiting.
[0029] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the invention, the preferred methods and materials are now
described. All publications mentioned herein are incorporated by
reference in their entirety.
[0030] The terms used herein have the meanings recognized and known
to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth
below and throughout the specification.
[0031] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0032] The term "about" or "approximately" means within a
statistically meaningful range of a value. Such a range can be
within an order of magnitude, typically within 20%, more typically
still within 10%, and even more typically within 5% of a given
value or range. The allowable variation encompassed by the term
"about" or "approximately" depends on the particular system under
study, and can be readily appreciated by one of ordinary skill in
the art. Whenever a range is recited within this application, every
whole number integer within the range is also contemplated as an
embodiment of the invention.
[0033] The term "batch culture" as used herein refers to a method
of culturing cells in which all the components that will ultimately
be used in culturing the cells, including the medium as well as the
cells themselves, are provided at the beginning of the culturing
process. A batch culture is typically stopped at some point and the
cells and/or components in the medium are harvested and optionally
purified.
[0034] The term "bioreactor" as used herein refers to any vessel
used for the growth of a bacterial cell culture. The bioreactor can
be of any size so long as it is useful for the culturing of
bacterial cells. Typically, the bioreactor will be at least 1 liter
and may be 10; 50; 100; 250; 500; 1,000; 2,500; 5,000; 8,000;
10,000; 12,000 liters or more, or any volume in between. The
internal conditions of the bioreactor, including, but not limited
to pH and temperature, are typically controlled during the
culturing period. The bioreactor can be composed of any material
that is suitable for holding bacterial cell cultures suspended in
media under the culture conditions of the present invention,
including glass, plastic or metal. The term "production bioreactor"
as used herein refers to the final bioreactor used in the
production of the polysaccharide of interest. The volume of the
large-scale cell culture production bioreactor is typically at
least 500 liters and may be 1,000; 2,500; 5,000; 8,000; 10,000;
12,0000 liters or more, or any volume in between. One of ordinary
skill in the art will be aware of and will be able to choose
suitable bioreactors for use in practicing the present
invention.
[0035] The term "capsular polysaccharide" or "capsule
polysaccharide" refers to a glycopolymer that includes repeating
units of one or more monosaccharides joined by glycosidic linkages.
A capsular polysaccharide typically forms a capsule-like layer
around a bacterial cell.
[0036] The term "cell density" as used herein refers to that number
of cells present in a given volume of medium.
[0037] The term "cell viability" as used herein refers to the
ability of cells in culture to survive under a given set of culture
conditions or experimental variations. The term as used herein also
refers to that portion of cells which are alive at a particular
time in relation to the total number of cells, living and dead, in
the culture at that time.
[0038] Terms such as "comprises", "comprised", "comprising",
"contains", "containing" and the like can have the meaning
attributed to them in U.S. patent law; e.g., they can mean
"includes", "included", "including" and the like. Such terms refer
to the inclusion of a particular ingredients or set of ingredients
without excluding any other ingredients.
[0039] The terms "consists of" and "consisting of" have the meaning
ascribed to them in U.S. patent law; namely, that these terms are
close-ended. Accordingly, these terms refer to the inclusion of a
particular ingredient or set of ingredients and the exclusion of
all other ingredients.
[0040] Terms such as "consisting essentially of" and "consists
essentially of" have the meaning attributed to them in U.S. patent
law, e.g., they allow for the inclusion of additional ingredients
or steps that do not detract from the novel or basic
characteristics of the invention, i.e., they exclude additional
unrecited ingredients or steps that detract from novel or basic
characteristics of the invention, and they exclude ingredients or
steps of the prior art, such as documents in the art that are cited
herein or are incorporated by reference herein, especially as it is
a goal of this document to define embodiments that are patentable,
e.g., novel, non-obvious, inventive, over the prior art, e.g., over
documents cited herein or incorporated by reference herein.
[0041] The terms "culture", "cell culture" and "bacterial cell
culture" as used herein refer to a bacterial cell population that
is suspended in a medium under conditions suitable to survival
and/or growth of the cell population. As will be clear to those of
ordinary skill in the art, these terms as used herein may refer to
the combination comprising the bacterial cell population and the
medium in which the population is suspended.
[0042] The term "disaccharide" as used herein refers to a
polysaccharide composed of two monosaccharide units or moieties
linked together by a glycosidic bond.
[0043] The term "fed-batch culture" as used herein refers to a
method of culturing cells in which additional components are
provided to the culture at some time subsequent to the beginning of
the culture process. The provided components typically comprise
nutritional supplements for the cells which have been depleted
during the culturing process. A fed-batch culture is typically
stopped at some point and the cells and/or components in the medium
are harvested and optionally purified.
[0044] These terms "medium", "cell culture medium", "bacterial
culture medium", and "culture medium" as used herein refer to a
solution containing nutrients which nourish growing bacterial
cells. Typically, these solutions provide essential and
non-essential amino acids, vitamins, energy sources, lipids, and
trace elements required by the cell for minimal growth and/or
survival. The solution may also contain components that enhance
growth and/or survival above the minimal rate, including hormones
and growth factors. The solution is preferably formulated to a pH
and salt concentration optimal for cell survival and proliferation.
The medium may also be a "defined media"--a serum-free media that
contains no proteins, hydrolysates or components of unknown
composition. Defined media are free of animal-derived components
and all components have a known chemical structure.
[0045] The term "metabolic waste product" as used herein refers to
compounds produced by the cell culture as a result of normal or
non-normal metabolic processes that are in some way detrimental to
the cell culture, particularly in relation to the production of the
capsular polysaccharide. For example, the metabolic waste products
may be detrimental to the growth or viability of the cell culture
or may decrease the amount of capsular polysaccharide produced.
Exemplary metabolic waste products include lactate, which is
produced as a result of glucose metabolism, and ammonium, which is
produced as a result of glutamine metabolism. One goal of the
present invention is to slow production of, reduce or even
eliminate metabolic waste products in bacterial cell cultures.
[0046] A "monosaccharide" as used herein refers to a single sugar
residue in an oligosaccharide.
[0047] An "oligosaccharide" as used herein refers to a compound
containing two or more monosaccharide units or moieties. Within the
context of an oligosaccharide, an individual monomer unit or moiety
is a monosaccharide which is, or can be, bound through a hydroxyl
group to another monosaccharide unit or moiety. Oligosaccharides
can be prepared by either chemical synthesis from protected single
residue sugars or by chemical degradation of biologically produced
polysaccharides. Alternatively, oligosaccharides may be prepared by
in vitro enzymatic methods.
[0048] The term "perfusion culture" as used herein refers to a
method of culturing cells in which additional components are
provided continuously or semi-continuously to the culture
subsequent to the beginning of the culture process. The provided
components typically comprise nutritional supplements for the cells
which have been depleted during the culturing process. A portion of
the cells and/or components in the medium, such as metabolic waste
products, are typically harvested on a continuous or
semi-continuous basis and are optionally purified.
[0049] The term "polysaccharide" (PS) refers to a linear or
branched polymer of at least 5 monosaccharide units or moieties.
For clarity, larger number of repeating units, wherein n is greater
than about 5, such as greater than about 10, will be referred to
herein as a polysaccharide.
[0050] As used herein, the term "saccharide" refers to a single
sugar moiety or monosaccharide unit as well as combinations of two
or more single sugar moieties or monosaccharide units covalently
linked to form disaccharides, oligosaccharides, and
polysaccharides. The term "saccharide" may be used interchangeably
with the term "carbohydrate."
[0051] The term "seeding" as used herein refers to the process of
providing a cell culture to a bioreactor or another vessel. The
cells may have been propagated previously in another bioreactor or
vessel. Alternatively, the cells may have been frozen and thawed
immediately prior to providing them to the bioreactor or vessel.
The term refers to any number of cells, including a single
cell.
[0052] The term "titer" as used herein refers to the total amount
of polysaccharide produced by a bacterial cell culture divided by a
given amount of medium volume. Titer is typically expressed in
units of milligrams of polysaccharide per liter of medium.
[0053] The terms "vaccine" or "vaccine composition", which are used
interchangeably, refer to pharmaceutical compositions comprising at
least one immunogenic composition that induces an immune response
in an animal.
Bacteria
[0054] Any bacteria having a cell wall polysaccharide may be
utilized in accordance with the present invention. In a preferred
embodiment, the bacteria are encapsulated bacteria. Non-limiting
examples of encapsulated bacteria that may be used in accordance
with the present invention include Streptococcus species, such as
S. agalactiae and S. pneumoniae, Staphylococcus aureus, Neisseria
meningitidis, Escherichia coli, Salmonella typhi, Haemophilus
influenzae, Klebsiella pneumoniae, Enterococcus faecium, and
Enterococcus faecalis. In a more preferred embodiment, the bacteria
have fastidious growth requirements. Fastidious bacteria include,
but are not limited to, Streptococcus species (e.g. S. agalactiae
and S. pneumoniae).
[0055] There are ten different serotypes of S. agalactiae, also
known as Group B Streptococcus (GBS), any of which may be used in
the present invention. Those serotypes include Ia, Ib, II, III, IV,
V, VI, VII, VIII, and IX. All GBS capsular polysaccharides have a
branched repeat structure with a terminal .alpha.2-3-linked sialic
acid that is required for bacterial virulence. Some examples of GBS
strains contemplated for use in the present invention include, but
are not limited to, 090, A909 (ATCC Accession No. BAA-1138), 515
(ATCC Accession No. BAA-1177), B523, CJB524, MB 4052 (ATCC
Accession No. 31574), H36B (ATCC Accession No. 12401), S40, S42, MB
4053 (ATCC Accession No. 31575), M709, 133, 7357, PFEGBST0267, MB
4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No.
BAA-1175), S16, S20, V8 (ATCC Accession No. 12973), DK21, DK23,
UAB, 5401, PFEGBST0708, MB 4082 (ATCC Accession No. 31577), M132,
110, M781 (ATCC Accession No. BAA-22), D136C(3) (ATCC Accession No.
12403), M782, S23, 120, MB 4316 (M-732; ATCC Accession No. 31475),
M132, K79, COH1 (ATCC Accession No. BAA-1176), PFEGBST0563, 3139
(ATCC Accession No. 49446), CZ-NI-016, PFEGBST0961, 1169-NT1,
CJB111(ATCC Accession No. BAA-23), CJB112, 2603 V/R (ATCC Accession
No. BAA-611), NCTC 10/81, CJ11, PFEGBST0837, 118754, 114852,
114862, 114866, 118775, B 4589, B 4645, SS1214, CZ-PW-119, 7271,
CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62, and
IT-PW-64.
[0056] There are more than 90 different serotypes of S. pneumoniae,
any of which are contemplated for use in the present invention.
Examples include, but are not limited to, serotypes 1, 2, 3, 4, 5,
6A, 6B, 7A, 7C, 7F, 8, 9N, 9L, 9V, 10A, 10B, 11A, 11F, 12A, 12F,
14, 15A, 15B, 15C, 17A, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F,
24F, 33F 35, 38, 39, 40, and 42. For example in one embodiment, S.
pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F or 33F may be used
in the present invention. In another embodiment, S. pneumoniae
serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F may
be used in the present invention.
[0057] Similarly, any encapsulated strain of S. aureus may be used
in the present invention. Preferably, S. aureus strains producing
serotype 5 or 8 capsular polysaccharides, such as Reynolds, Becker,
Newman, PS80, JL278, and JL812, are contemplated.
[0058] Any strain of N. meningitidis serogroups A, C, Y, and W-135
may be used in the present invention.
[0059] Any strain of E. coli may be used in the present
invention.
[0060] Any strain of S. typhi Vi may be used in the present
invention.
[0061] Any strain of H. influenza type b may be used in the present
invention.
[0062] Any strain of K. pneumoniae may be used in the present
invention.
[0063] E. faecalis may be used in the present invention.
[0064] Exemplary strains of E. faecium that may be used in the
present invention include those listed in Table 1.
TABLE-US-00001 TABLE Strains of E. faecium Strain Strain E1162
(Genome GenBank U0317 (Genome GenBank accession number accession
number ABQJ00000000) ABSW00000000) E0510 E1728 E1760 E1731 E1679
(Genome GenBank E1794 (DO strain; accession number TX0016 strain)
ABSC00000000) (Genome GenBank accession number ACIY00000000) E1644
E1360 E1716 E1674 E1717 E1675 E1441 E1643 E1435 E1850 E0734 E0005
E1652 E0321 E0745 E0322 E0470 E0027 E1340 E1149 E0013 E1147 E0300
E0802 E0155 E0849 E0161 E1316 E1132 E1554 E1263 E1133 E1250 E1764
E1283 E1766 E1284 E1485 E1734 E1590 E1467 E0060 E1500 E0128 E1737
E0135 E1463 E1002 E1499 E1039 (Genome GenBank accession number
ACOS00000000) E1735 E0980 (Genome GenBank accession number
ABQA00000000) E0380 E1071 (Genome GenBank accession number
ABQI00000000) E1391 E1759 E1403 E1628 E1421 E1630 E1423 E1573 E0333
E0172 E1292 E0211 E1620 E0466 E1621 E1574 E1623 E0463 E1625 E1607
E1636 (Genome GenBank E1619 accession number ABRY00000000) E0073
E1576 E0125 E1781 E0772 E0685 E1172 E0144 E1302 E0045 E1307 E0429
E1308 E1622 E1721
[0065] Additionally, any number of commercially and
non-commercially available bacteria having cell wall
polysaccharides may be utilized in accordance with the present
invention. One skilled in the art will appreciate that some
bacteria have different nutrition requirements and/or might require
different culture conditions for optimal growth and will be able to
modify conditions as needed.
[0066] In many instances, the strains of bacteria will be selected
or engineered to produce high levels of polysaccharide. In some
embodiments, the bacterial cells are genetically engineered to
produce high levels of polysaccharide.
Cell Culture Media
[0067] The present invention provides a variety of media
formulations that maximize polysaccharide production in bacterial
cell cultures.
Complex Media
[0068] Bacterial cell cultures, particularly for fastidious
bacteria and/or those bacteria producing cell wall polysaccharides,
are often grown in complex media such as Columbia broth,
Luria-Bertani (LB) broth, Todd-Hewitt broth, GC medium, blood
broth, or brain-heart infusion broth. Accordingly, a complex media
was developed to maximize bacterial growth and polysaccharide
production. In one aspect, the invention relates to a complex
culture medium comprising a vegetable hydrolysate, a yeast extract,
and a carbon source.
[0069] Suitable vegetable hydrolysates include, but are not limited
to, HYPEP 1510 (Kerry Group Services Ltd.), HYPEP 4601 (Kerry Group
Services Ltd.), HYPEP 5603 (Kerry Group Services Ltd.), HY-SOY
(Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services Ltd.),
N-Z-SOY (Quest), N-Z-SOY BL4 (Kerry Group Services Ltd.), N-Z-SOY
BL7 (Quest), SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK,
soy peptone, BACTO soytone (Difco Laboratories Inc.), NUTRISOY 2207
(ADM), NUTRISOY (ADM), NUTRISOY flour (ADM), and soybean meal. In a
preferred embodiment, the vegetable hydrolysate is soy hydrolysate.
Preferably, the soy hydrolysate is HYPEP 1510 (Kerry Group Services
Ltd.).
[0070] Concentrations of the vegetable hydrolysate in the culture
medium can range between about 5 g/L and about 75 g/L, such as
between about 5 g/L and about 65 g/L, between about 5 g/L and about
55 g/L, between about 5 g/L and about 45 g/L, between about 5 g/L
and about 35 g/L, between about 10 g/L and about 70 g/L, between
about 10 g/L and about 60 g/L, between about 10 g/L and about 50
g/L, between about 10 g/L and about 40 g/L, between about 15 g/L
and about 75 g/L, between about 15 g/L and about 65 g/L, between
about 15 g/L and about 55 g/L, between about 15 g/L and about 45
g/L, between about 20 g/L and about 70 g/L, between about 20 g/L
and about 60 g/L, or between about 20 g/L and about 50 g/L. In a
preferred embodiment, the concentration of vegetable hydrolysate in
the culture medium is between about 10 g/L and about 50 g/L, most
preferably about 28 g/L.
[0071] Yeast extracts suitable for use in the present invention may
include yeast autolysate, ultrafiltered yeast extracts, and
synthetic yeast extracts. In one aspect, the yeast extract is BD
BBL (BD Biosciences), BD BACTO (BD Biosciences), HY YEST 412 (Kerry
Group Services Ltd.), HY YEST 444 (Kerry Group Services Ltd.),
HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 455 (Kerry Group
Services Ltd.), or HY YEST 504 (Kerry Group Services Ltd.). In
another aspect, the yeast extract is an ultrafiltered yeast
extract, such as AMBERFERM 5902 (Sensient Technologies Corp.), BD
DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), or
ULTRAPEP YE (Kerry Group Services Ltd.). In a further aspect, the
yeast extract is a synthetic yeast extract, such as BD RECHARGE (BD
Biosciences). Most preferably, the yeast extract is an
ultrafiltered yeast extract, such as AMBERFERM 5902 (Sensient
Technologies Corp.).
[0072] Concentrations of the yeast extract in the culture medium
can range from about 1 g/L to about 50 g/L, such as between about 1
g/L and about 40 g/L, between about 1 g/L and about 30 g/L, between
about 1 g/L and about 25 g/L, between about 1 g/L and about 20 g/L,
between about 1 g/L and about 15 g/L, between about 1 g/L and about
10 g/L, between about 5 g/L and about 50 g/L, between about 5 g/L
and about 40 g/L, between about 5 g/L and about 30 g/L, between
about 5 g/L and about 25 g/L, between about 5 g/L and about 20 g/L,
between about 5 g/L and about 15 g/L, between about 10 g/L and
about 50 g/L, between about 10 g/L and about 40 g/L, between about
10 g/L and about 30 g/L, between about 10 g/L and about 35 g/L,
between about 10 g/L and about 30 g/L, between about 10 g/L and
about 25 g/L, between about 10 g/L and about 20 g/L, between about
15 g/L and about 50 g/L, between about 15 g/L and about 40 g/L,
between about 15 g/L and about 30 g/L, or between about 15 g/L and
about 25 g/L. In a preferred embodiment, the concentration of yeast
extract in the culture medium is between about 5 g/L to about 25
g/L, most preferably about 10 g/L.
[0073] Any carbon source may be used in the culture medium of the
present invention. Suitable carbon sources include glucose,
dextrose, mannitol, lactose, sucrose, fructose, galactose,
raffinose, xylose, and/or mannose. Preferably, the carbon source in
the culture medium is glucose.
[0074] Concentrations of the carbon source in the culture medium
can range from about 25 g/L to about 100 g/L, such as between about
25 g/L and about 90 g/L, between about 25 g/L and about 80 g/L,
between about 25 g/L and about 70 g/L, between about 25 g/L and
about 60 g/L, between about 25 g/L and about 50 g/L, between about
50 g/L and about 100 g/L, between about 50 g/L and about 90 g/L,
between about 50 g/L and about 80 g/L, between about 50 g/L and
about 70 g/L, between about 60 g/L and about 100 g/L, between about
60 g/L and about 90 g/L, between about 60 g/L and about 80 g/L,
between about 70 g/L and about 100 g/L, or between about 70 g/L and
about 90 g/L. In a preferred embodiment, the concentration of the
carbon source in the culture medium is between about 50 g/L to
about 90 g/L, most preferably about 80 g/L.
[0075] Accordingly, the inventors discovered that a combination of
a vegetable hydrolysate, a yeast extract, and a carbon source helps
to support maximal bacterial cell growth and polysaccharide
production. In one aspect, the invention relates to a culture
medium including a vegetable hydrolysate, a yeast extract, and a
carbon source. The vegetable hydrolysate can be any suitable
vegetable hydrolysate known in the art, such as those described
above. Preferably, the hydrolysate is soy hydrolysate. More
preferably, the soy hydrolysate is HYPEP 1510 (Kerry Group Services
Ltd.). Any yeast extract known in the art, such as those described
above, may be used. In a preferred embodiment, the yeast extract is
AMBERFERM 5902 (Sensient Technologies Corp.).
[0076] In one aspect, the complex culture medium of the present
invention may include phosphate-containing ingredients such as
Na.sub.2HPO.sub.4, K.sub.2HPO.sub.4, or KH.sub.2PO.sub.4.
[0077] In another aspect, the culture media may include various
other factors known in the art to enhance growth, such as amino
acids, vitamins, nucleosides, and inorganic salts.
[0078] In yet another aspect, the cultivation is carried out using
any of the methods disclosed herein until the cell density, as
determined by optical density (OD) at 600 nm, of the bacterial cell
culture using the complex media of the invention is at least 15.0,
such as at least 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0,
24.0, 25.0, 26.0, 27.0, 28.0, 29.0 or 30.0. In a preferred
embodiment, the cultivation is carried out until the cell density
is at least 15.0. In yet another aspect, the cell density, as
determined by optical density (OD) at 600 nm, of the bacterial cell
culture using the complex media of the invention may be at least
15.0, such as at least 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0,
23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0 or 30.0. In a preferred
embodiment, the cell density is at least 15.0.
[0079] GBS polysaccharide yield may be determined by measuring
sialic acid concentration. Sialic acid is released from cell bound
polysaccharide by digesting pelleted cells by methods well-known in
the art. The digest can be assayed by anion exchange chromatography
(AEX) via high performance liquid chromatography (HPLC).
Polysaccharide concentration is then determined by multiplying the
sialic acid value times a repeat unit weight conversion factor. For
example, the conversion factor for each GBS serotype is as follows:
Ia, Ib, and III=3.24; II and V=4.29; and IV=3.77. Polysaccharide
quantification for S. pneumoniae or other encapsulated bacteria is
achieved by first releasing the capsular polysaccharide from the
cell wall by treatment with a detergent, such as sodium deoxycholic
acid (DOC) or sodium N-lauryl-sarcosine (NLS); acid treatment at
high temperature; base treatment; and/or mechanical lysis. The
released polysaccharide in the crude lysate is then assayed against
an authentic standard using size exclusion chromatography (SEC)
HPLC.
[0080] In one aspect, the cultivation is carried out using any of
the methods disclosed herein until the polysaccharide
concentration, as determined by sialic acid concentration, of the
bacterial cell culture using the complex media of the invention is
at least about 200 mg/L, such as at least about 250 mg/L; 300 mg/L;
350 mg/L; 400 mg/L, 450 mg/L; 500 mg/L; 550 mg/L; 600 mg/L; 650
mg/L; or 700 mg/L. In a preferred embodiment, the culviation is
carried out until the polysaccharide concentration is at least
about 600 mg/L. In one aspect, the polysaccharide concentration, as
determined by sialic acid concentration, of the bacterial cell
culture using the complex media of the invention may be at least
about 200 mg/L, such as at least about 250 mg/L; 300 mg/L; 350
mg/L; 400 mg/L, 450 mg/L; 500 mg/L; 550 mg/L; 600 mg/L; 650 mg/L;
or 700 mg/L. In a preferred embodiment, the polysaccharide
concentration is at least about 600 mg/L.
Defined Media
[0081] In view of the potential inconsistency of complex media,
however, a chemically defined media was also investigated to
maximize bacterial growth and polysaccharide production. It was
surprisingly discovered that Applicant's proprietary mammalian cell
culture media disclosed in U.S. Pat. No. 7,294,484, which is
incorporated by reference herein in its entirety, provided both
unexpectedly high cell growth and polysaccharide production.
Specifically, the present inventors found that a defined media
having a total amino acid concentration greater than about 50 mM
provided both unexpectedly high cell growth and polysaccharide
production. An exemplary mammalian cell culture media is shown in
Table 2 below. Traditional media formulations begin with a
relatively low level of total amino acids in comparison with the
media formulations of the present invention. For example, the
traditional cell culture medium known as DME-F12 (a 50:50 mixture
of Dulbecco's Modified Eagle's medium and Ham's F12 medium) has a
total amino acid content of 7.29 mM, and the traditional cell
culture medium known as RPMI-1640 has a total amino acid content of
6.44 mM (See e.g., H. J. Morton, In Vitro, 6:89-108 (1970), R. G.
Ham, Proc. Nat. Assoc. Sci. (USA), 53:288-293 (1965), G. E. Moore
et al., J. Am. Medical Assn., 199:519-24 (1967), all incorporated
herein by reference).
TABLE-US-00002 TABLE 2 Exemplary Mammalian Cell Culture Media Amino
Acids mg/L mM alanine 17.80 0.20 arginine 696.00 4.00
asparagine.cndot.H.sub.2O 3000.00 20.00 aspartic acid 219.45 1.65
cysteine.cndot.HCl.cndot.H.sub.2O 70.40 0.40 cysteine.cndot.2HCl
468.75 1.50 monosodium 33.80 0.20 glutamate glutamine 584.00 4.00
glycine 115.50 1.54 histidine.cndot.HCl.cndot.H.sub.2O 474.60 2.26
isoleucine 570.73 4.36 leucine 1030.70 7.87 lysine.cndot.HCl
1401.40 7.70 methionine 387.40 2.60 phenylalanine 507.00 3.07
proline 539.50 4.69 serine 1052.00 10.02 threonine 564.80 4.75
tryptophan 274.16 1.34 tyrosine.cndot.2Na.cndot.2H.sub.2O 745.75
2.86 valine 749.00 6.40 Vitamins mg/L mM biotin 2.68 0.01 calcium
pantothenate 21.92 0.05 choline chloride 158.46 1.14 folic acid
25.93 0.06 inositol 163.98 0.91 nicotinamide 26.23 0.22
pyridoxal.cndot.HCl 2.03 0.01 pyridoxine.cndot.HCl 36.13 0.18
riboflavin 2.41 0.01 thiamine.cndot.HCl 39.43 0.12 vitamin B12
21.17 0.02 Inorganic Salts mg/L mM CaCl.sub.2 116.55 1.05 KCl
312.90 4.19 Na.sub.2HPO.sub.4 56.60 0.40 NaCl 1100.00 18.80
NaH.sub.2PO.sub.4.cndot.H.sub.2O 645.84 4.68 MgSO.sub.4 138.00 1.15
MgCl.sub.2 28.50 0.30 NaHCO.sub.3 2000.00 23.81 Trace Elements
.mu.g/L nM Sodium Selenite 69.16 400.00
Fe(NO.sub.3).sub.3.cndot.9H.sub.2O 50.00 123.76 CuSO.sub.4 10.24
64.00 CuSO.sub.4.cndot.5H.sub.2O 99.88 400.00
FeSO.sub.4.cndot.7H.sub.2O 4170 15000 ZnSO.sub.4.cndot.7H.sub.2O
2640 9200 MnSO.sub.4.cndot.H.sub.2O 33.80 200.00
Na.sub.2SiO.sub.3.cndot.9H.sub.2O 284.07 1000
(NH.sub.4).sub.6Mo.sub.7O.sub.24.cndot.4H.sub.2O 247.20 200.00
NH.sub.4VO.sub.3 2.34 20.00 NiSO.sub.4.cndot.6H.sub.2O 5.26 20.00
SnCl.sub.2.cndot.2H.sub.2O 0.90 4.00 AlCl.sub.3.cndot.6H.sub.2O
0.97 4.00 KBr 0.48 4.00 CrCl.sub.3 15.83 100.00 NaF 0.17 4.00
GeO.sub.2 0.42 4.00 Kl 33.20 200.00 RbCl 0.48 4.00 H.sub.3BO.sub.3
12.37 200.00 LiCl 0.17 4.00 Other Components .mu.g/L nM
Hydrocortisone 540.00 1.49 Putrescine.cndot.2HCl 15000 93.11
linoleic acid 290.00 1.04 thioctic acid 716.00 3.48 Other
Components mg/L mM D-glucose (Dextrose) 15000.00 83.33 PVA 2560.00
Nucellin .TM. 50.00 Sodium Pyruvate 55.00 0.50
[0082] Accordingly, the present invention relates to a cell culture
media having a total amino acid concentration of at least about 50
mM, such as at least about 55 mM, at least 60 mM, at least 70 mM,
and at least 75 mM. In a preferred embodiment, the total amino acid
concentration is at least 60 mM.
[0083] In an aspect of the invention, the total glycine
concentration in the bacterial cell culture media can range between
about 1.5 mM and about 60.0 mM, such as between about 1.5 mM and
about 50.0 mM, between about 1.5 mM and about 40.0 mM, between
about 1.5 mM and about 30.0 mM, between about 1.5 mM and about 20.0
mM, between about 1.5 mM and about 15.0 mM, between about 1.5 mM
and about 10.0 mM, between about 1.5 mM and about 7.5 mM, between
about 1.5 mM and about 5.0 mM, between about 5.0 mM and about 60.0
mM, between about 5.0 mM and about 50.0 mM, between about 5.0 mM
and about 40.0 mM, between about 5.0 mM and about 30.0 mM, between
about 5.0 mM and about 20.0 mM, between about 5.0 mM and about 15.0
mM, between about 5.0 mM and about 10.0 mM, between about 5.0 mM
and about 7.5 mM, between about 7.5 mM and about 60.0 mM, between
about 7.5 mM and about 50.0 mM, between about 7.5 mM and about 40.0
mM, between about 7.5 mM and about 30.0 mM, between about 7.5 mM
and about 20.0 mM, between about 7.5 mM and about 15.0 mM, or
between about 7.5 mM and about 10.0 mM. In a preferred embodiment,
the total concentration of glycine in the bacterial cell culture
media is between about 5.0 mM and about 15.0 mM, most preferably
about 7.5 mM.
[0084] In an aspect of the invention, the total arginine
concentration in the bacterial cell culture media can range between
about 1.0 mM and about 30.0 mM, such as between about 1.0 mM and
about 20.0 mM, between about 1.0 mM and about 15.0 mM, between
about 1.0 mM and about 10.0 mM, between about 1.0 mM and about 7.5
mM, between about 1.0 mM and about 5.0 mM, between about 4.0 mM and
about 20.0 mM, between about 4.0 mM and about 15.0 mM, between
about 4.0 mM and about 10.0 mM, between about 4.0 mM and about 7.5
mM, between about 10.0 mM and about 30.0 mM, between about 10.0 mM
and about 25.0 mM, between about 10.0 mM and about 20.0 mM, between
about 10.0 mM and about 15.0 mM, between about 15.0 mM and about
30.0 mM, between about 15.0 mM and about 25.0 mM, or between about
15.0 mM and about 20.0 mM. In a preferred embodiment, the total
concentration of arginine in the bacterial cell culture media is
between about 1.0 mM and about 20.0 mM, most preferably about 4.0
mM.
[0085] In an aspect of the invention, the total cysteine
concentration in the bacterial cell culture media may be between
about 0.1 mM and about 5.0 mM, such as between about 0.1 mM and
about 4.5 mM, between about 0.1 mM and about 4.0 mM, between about
0.1 mM and about 3.5 mM, between about 0.1 mM and about 3.0 mM,
between about 0.1 mM and about 2.5 mM, between about 0.4 mM and
about 5.0 mM, between about 0.4 mM and about 4.5 mM, between about
0.4 mM and about 4.0 mM, between about 0.4 mM and about 3.5 mM,
between about 0.4 mM and about 3.0 mM, between about 0.4 mM and
about 2.5 mM, or between about 0.4 mM and about 2.0 mM. In a
preferred embodiment, the total concentration of cysteine in the
bacterial cell culture media is between about 0.1 mM and about 3.5
mM, most preferably about 0.4 mM.
[0086] In an aspect of the invention, the total serine
concentration in the bacterial cell culture media may be between
about 5.0 mM and about 75.0 mM, such as between about 5.0 mM and
about 50.0 mM, between about 5.0 mM and about 40.0 mM, between
about 5.0 mM and about 30.0 mM, between about 5.0 mM and about 20.0
mM, between about 5.0 mM and about 20.0 mM, between about 5.0 mM
and about 15.0 mM, between about 10.0 mM and about 75.0 mM, between
about 10.0 mM and about 50.0 mM, between about 10.0 mM and about
40.0 mM, between about 10.0 mM and about 30.0 mM, between about
10.0 mM and about 20.0 mM, between about 15.0 mM and about 75.0 mM,
between about 15.0 mM and about 50.0 mM, between about 15.0 mM and
about 40.0 mM, between about 15.0 mM and about 30.0 mM, or between
about 20.0 mM and about 50.0 mM. In a preferred embodiment, the
total concentration of serine in the bacterial cell culture media
is between about 5.0 mM and about 15.0 mM, most preferably about
7.5 mM.
[0087] In an aspect of the invention, the total glutamine
concentration in the bacterial cell culture media may range between
about 1.0 mM and about 30.0 mM, such as between about 1.0 mM and
about 20.0 mM, between about 1.0 mM and about 15.0 mM, between
about 1.0 mM and about 10.0 mM, between about 1.0 mM and about 7.5
mM, between about 1.0 mM and about 5.0 mM, between about 4.0 mM and
about 20.0 mM, between about 4.0 mM and about 15.0 mM, between
about 4.0 mM and about 10.0 mM, between about 4.0 mM and about 7.5
mM, between about 10.0 mM and about 30.0 mM, between about 10.0 mM
and about 25.0 mM, between about 10.0 mM and about 20.0 mM, between
about 10.0 mM and about 15.0 mM, between about 15.0 mM and about
30.0 mM, between about 15.0 mM and about 25.0 mM, or between about
15.0 mM and about 20.0 mM. In a preferred embodiment, the total
concentration of glutamine in the bacterial cell culture media is
between about 1.0 mM and about 20.0 mM, most preferably about 4.0
mM.
[0088] In an aspect of the invention, the total tyrosine
concentration in the bacterial cell culture media can range between
about 0.1 mM and about 5.0 mM, such as between about 0.1 mM and
about 4.5 mM, between about 0.1 mM and about 4.0 mM, between about
0.1 mM and about 3.5 mM, between about 0.1 mM and about 3.0 mM,
between about 0.1 mM and about 2.5 mM, between about 1.0 mM and
about 5.0 mM, between about 1.0 mM and about 4.5 mM, between about
1.0 mM and about 4.0 mM, between about 1.0 mM and about 3.5 mM,
between about 1.0 mM and about 3.0 mM, between about 1.0 mM and
about 2.5 mM, or between about 1.0 mM and about 2.0 mM. In a
preferred embodiment, the total concentration of tyrosine in the
bacterial cell culture media is between about 1.0 mM and about 3.5
mM, most preferably about 2.9 mM or about 3.0 mM.
[0089] In an aspect of the invention, the total asparagine
concentration in the bacterial cell culture media may be between
about 5.0 mM and about 50.0 mM, such as between about 5.0 mM and
about 40.0 mM, between about 5.0 mM and about 30.0 mM, between
about 5.0 mM and about 25.0 mM, between about 5.0 mM and about 20.0
mM, between about 5.0 mM and about 15.0 mM, between about 5.0 mM
and about 10.0 mM, between about 10.0 mM and about 50.0 mM, between
about 10.0 mM and about 40.0 mM, between about 10.0 mM and about
30.0 mM, between about 10.0 mM and about 25.0 mM, between about
10.0 mM and about 20.0 mM, between about 15.0 mM and about 50.0 mM,
between about 15.0 mM and about 40.0 mM, between about 15.0 mM and
about 30.0 mM, between about 15.0 mM and about 25.0 mM, or between
about 15.0 mM and about 20.0 mM. In a preferred embodiment, the
total concentration of asparagine in the bacterial cell culture
media is between about 10.0 mM and about 30.0 mM, most preferably
about 20.0 mM.
[0090] In another aspect of the invention, the cell culture media
does not contain asparagine.
[0091] The present inventors also found that potassium was a
beneficial salt for the production of polysaccharides, which was
independent of growth. Accordingly, in one aspect of the invention,
the cell culture media comprises a potassium salt, such as
potassium chloride or potassium sulfate.
[0092] In one embodiment, the concentration of potassium salt in
the cell culture media may be between about 0.1 g/L and about 25
g/L, such as between about 0.1 g/L and about 20 g/L, between about
0.1 g/L and about 10 g/L, between about 0.1 g/L and about 5 g/L,
between about 0.1 g/L and about 1.5 g/L, between about 0.1 g/L and
about 1.25 g/L, between about 0.1 g/L and about 1.0 g/L, between
about 0.1 g/L and about 0.9 g/L, between about 0.1 g/L and about
0.8 g/L, between about 0.1 g/L and about 0.7 g/L, between about 0.1
g/L and about 0.6 g/L, between about 0.1 g/L and about 0.5 g/L,
between about 0.2 g/L and about 1.5 g/L, between about 0.2 g/L and
about 1.25 g/L, between about 0.2 g/L and about 1.0 g/L, between
about 0.2 g/L and about 0.9 g/L, between about 0.2 g/L and about
0.8 g/L, between about 0.2 g/L and about 0.7 g/L, between about 0.2
g/L and about 0.6 g/L, between about 0.2 g/L and about 0.5 g/L,
between about 0.3 g/L and about 1.5 g/L, between about 0.3 g/L and
about 1.25 g/L, between about 0.3 g/L and about 1.0 g/L, between
about 0.3 g/L and about 0.9 g/L, between about 0.3 g/L and about
0.8 g/L, between about 0.3 g/L and about 0.7 g/L, between about 0.3
g/L and about 0.6 g/L, between about 0.3 g/L and about 0.5 g/L,
between about 0.5 g/L and about 1.5 g/L, between about 0.5 g/L and
about 1.25 g/L, or between about 0.5 g/L and about 1.0 g/L. In a
preferred embodiment, the total concentration of potassium salt in
the bacterial cell culture media is between about 0.2 g/L and about
1.25 g/L, most preferably about 0.9 g/L.
[0093] In one aspect of the invention, the cell culture media of
the present invention contains a carbon source. Suitable carbon
sources include glucose, dextrose, mannitol, lactose, sucrose,
fructose, galactose, raffinose, xylose, and/or mannose. In a
preferred embodiment, the carbon source is glucose.
[0094] The total concentration of the carbon source in the
bacterial cell culture media can range between about 25 g/L to
about 100 g/L, such as between about 25 g/L and about 90 g/L,
between about 25 g/L and about 80 g/L, between about 25 g/L and
about 70 g/L, between about 25 g/L and about 60 g/L, between about
25 g/L and about 50 g/L, between about 50 g/L and about 100 g/L,
between about 50 g/L and about 90 g/L, between about 50 g/L and
about 80 g/L, between about 50 g/L and about 70 g/L, between about
60 g/L and about 100 g/L, between about 60 g/L and about 90 g/L,
between about 60 g/L and about 80 g/L, between about 70 g/L and
about 100 g/L, or between about 70 g/L and about 90 g/L. In a
preferred embodiment, the concentration of the carbon source in the
culture medium is between about 25 g/L and about 80 g/L, most
preferably about 50 g/L.
[0095] In another aspect, the cell culture media is modified to
accommodate the sodium bicarbonate requirement of bacteria grown
anaerobically. Some examples of polysaccharide-producing bacteria
that are grown anaerobically include S. agalactiae and S.
pneumoniae. In one embodiment, about 0.1 g/L to about 20 g/L of
sodium bicarbonate is added to the media. For example, the sodium
bicarbonate concentration may be between about 0.1 g/L and about 15
g/L, between about 0.1 g/L and about 10 g/L, between about 0.1 g/L
and about 5.0 g/L, between about 0.1 g/L and about 3.0 g/L, between
about 0.1 g/L and about 2.0 g/L, between about 0.1 g/L and about
1.25 g/L, between about 0.1 g/L and about 1.0 g/L, between about
0.1 g/L and about 0.9 g/L, between about 0.1 g/L and about 0.8 g/L,
between about 0.1 g/L and about 0.7 g/L, between about 0.1 g/L and
about 0.6 g/L, between about 0.1 g/L and about 0.5 g/L, between
about 0.5 g/L and about 20 g/L, between about 0.5 g/L and about 15
g/L, between about 0.5 g/L and about 10 g/L, between about 0.5 g/L
and about 5.0 g/L, between about 0.5 g/L and about 3.0 g/L, between
about 0.5 g/L and about 2.0 g/L, between about 0.5 g/L and about
1.25 g/L, between about 0.5 g/L and about 1.0 g/L, between about
0.5 g/L and about 0.9 g/L, between about 0.5 g/L and about 0.8 g/L,
between about 0.5 g/L and about 0.7 g/L, between about 0.75 g/L and
about 20 g/L, between about 0.75 g/L and about 15 g/L, between
about 0.75 g/L and about 10 g/L, between about 0.75 g/L and about
5.0 g/L, between about 0.75 g/L and about 3.0 g/L, between about
0.75 g/L and about 2.0 g/L, between about 0.75 g/L and about 1.25
g/L, between about 0.75 g/L and about 1.0 g/L, or between about
0.75 g/L and about 0.9 g/L. Preferably, the sodium bicarbonate
concentration is about 0.84 g/L, or between about 1.8 g/L and about
2.4 g/L.
[0096] In one aspect of the invention, the defined bacterial cell
culture media comprises yeast extract. Yeast extracts suitable for
use in the present invention may include yeast autolysate,
ultrafiltered yeast extracts, and synthetic yeast extracts. In one
aspect, the yeast extract is BD BBL (BD Biosciences), BD BACTO (BD
Biosciences), HY YEST 412 (Kerry Group Services Ltd.), Y YEST 441
(Kerry, Inc. Kerry Group Services Ltd.), HY YEST 444 (Kerry Group
Services Ltd.), or HY YEST 504 (Kerry Group Services Ltd.). In
another aspect, the yeast extract is an ultrafiltered yeast
extract, such as AMBERFERM 5902 (Sensient Technologies Corp.), BD
DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), or
ULTRAPEP YE (Kerry Group Services Ltd.). In a further aspect, the
yeast extract is a synthetic yeast extract, such as BD RECHARGE (BD
Biosciences). Most preferably, the yeast extract is an
ultrafiltered yeast extract, such as AMBERFERM 5902 (Sensient
Technologies Corp.).
[0097] Concentrations of the yeast extract in the culture medium
can be between about 1 g/L to about 50 g/L, such as between about 1
g/L and about 40 g/L, between about 1 g/L and about 30 g/L, between
about 1 g/L and about 25 g/L, between about 1 g/L and about 20 g/L,
between about 1 g/L and about 15 g/L, between about 1 g/L and about
10 g/L, between about 5 g/L and about 50 g/L, between about 5 g/L
and about 40 g/L, between about 5 g/L and about 30 g/L, between
about 5 g/L and about 25 g/L, between about 5 g/L and about 20 g/L,
between about 5 g/L and about 15 g/L, between about 10 g/L and
about 50 g/L, between about 10 g/L and about 40 g/L, between about
10 g/L and about 30 g/L, between about 10 g/L and about 35 g/L,
between about 10 g/L and about 30 g/L, between about 10 g/L and
about 25 g/L, between about 10 g/L and about 20 g/L, between about
15 g/L and about 50 g/L, between about 15 g/L and about 40 g/L,
between about 15 g/L and about 30 g/L, or between about 15 g/L and
about 25 g/L. In a preferred embodiment, the concentration of yeast
extract in the culture medium is between about 5 g/L to about 25
g/L, most preferably about 10 g/L.
[0098] One aspect of the invention relates to a defined cell
culture media comprising at least about 50 mM of amino acids, a
potassium salt, a carbon source, and optionally, a yeast
extract.
[0099] In one embodiment, the cell culture media comprises at least
about 50 mM of amino acids, between about 5.0 mM and about 15.0 mM
of glycine, between about 0.2 g/L and about 1.25 g/L of a potassium
salt, between about 25 g/L and about 80 g/L of a carbon source, and
between about 5 g/L to about 25 g/L of a yeast extract.
[0100] In a preferred embodiment, the cell culture media comprises
at least about 60 mM of amino acids, about 7.5 mM of glycine, about
0.9 g/L of potassium chloride, 50 g/L of glucose, and about 10 g/L
of an ultrafiltered yeast extract.
[0101] Furthermore, one of ordinary skill in the art will recognize
that any of the conditions listed above may be used either singly
or in various combinations with one another. By utilizing media
formulation which exhibit one, some or all of the above
characteristics, one of ordinary skill in the art will be able to
optimize cell growth and/or viability and to maximize the
production of polysaccharide.
[0102] Any of these media formulations disclosed in the present
invention may optionally be supplemented as necessary with
particular ions (such as sodium, chloride, calcium, magnesium, and
phosphate), buffers, vitamins, trace elements (inorganic compounds
usually present at very low final concentrations), amino acids,
lipids, protein hydrolysates, or glucose or other energy source.
These optional supplements may be added at the beginning of the
culture or may be added at a later point in order to replenish
depleted nutrients or for another reason. One of ordinary skill in
the art will be aware of any desirable or necessary supplements
that may be included in the disclosed media formulations.
[0103] In another aspect, the cultivation is carried out by any of
the methods disclosed herein until the cell density, as determined
by optical density (OD) at 600 nm, of the bacterial cell culture
using the defined media of the invention is at least 9.0, such as
at least 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0,
14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5,
or 20.0. In a preferred embodiment, the cultivation is carried out
by any of the methods disclosed herein until the cell density is at
least 9.0.
[0104] For polysaccharides that contain sialic acid such as GBS,
polysaccharide yield may be determined by measuring sialic acid
concentration. Sialic acid is released from cell bound
polysaccharide by digesting pelleted cells by methods well-known in
the art. The digest is assayed by anion exchange chromatogrphay
(AEX) via high performance liquid chromatography (HPLC).
Polysaccharide concentration is then determined by multiplying the
sialic acid value times a repeat unit weight conversion factor. For
example, the conversion factor for each GBS serotype is as follows:
Ia, Ib, and III=3.24; II and V=4.29; and IV=3.77.
[0105] In one aspect, the cultivation is carried out using any of
the methods disclosed herein until the polysaccharide
concentration, as determined by sialic acid concentration, of the
bacterial cell culture using the defined media of the invention is
at least about 250 mg/L, such as at least about 300 mg/L, 350 mg/L,
400 mg/L, 450 mg/L, 500 mg/L, 550 mg/L, 600 mg/L, 650 mg/L, 700
mg/L, 750 mg/L, 800 mg/L, 900 mg/L, 1000 mg/L, 1200 mg/L, 1500 mg/L
or 2000 mg/L. In a preferred embodiment, the cultivation is carried
out using any of the methods disclosed herein until the
polysaccharide concentration is at least about 250 mg/L. In one
aspect, the polysaccharide concentration, as determined by sialic
acid concentration, of the bacterial cell culture using the defined
media of the invention may be at least about 250 mg/L, such as at
least about 300 mg/L, 350 mg/L, 400 mg/L, 450 mg/L, 500 mg/L, 550
mg/L, 600 mg/L, 650 mg/L, 700 mg/L, 750 mg/L, 800 mg/L, 900 mg/L,
1000 mg/L, 1200 mg/L, 1500 mg/L or 2000 mg/L. In a preferred
embodiment, the polysaccharide concentration is at least about 250
mg/L.
Fermentation Methods
[0106] The present invention provides fermentation methods for
cultivating polysaccharide-producing bacteria. In one aspect, the
cultivation methods of the present invention are used in
combination with the complex and defined media described herein to
maximize polysaccharide production.
Seed Growth
[0107] In one embodiment, growth of polysaccharide-producing
bacteria in the methods of the invention proceeds in at least two
phases: seed growth and fermentation. A seed culture is first grown
by inoculation from a stock culture, e.g., a working cell bank. The
seed is used either to inoculate a second seed culture or to
inoculate a relatively large fermentation culture. As is understood
in the art, the number of seed cultures used may depend, for
example, on the size and volume of the fermentation step.
[0108] Accordingly, in one aspect, the invention relates to a
method of culturing polysaccharide-producing bacteria. The method
includes culturing a polysaccharide-producing bacterial cell in a
first culture medium under conditions that facilitate growth of the
cell; inoculating a second culture medium with all or a portion of
said first medium after said first culturing; culturing said
inoculated second medium under conditions that facilitate cell
growth and/or polysaccharide production. The method may further
include isolating a polysaccharide from said second medium. In one
embodiment, the polysaccharide-producing bacteria are grown in a
first culture medium referred to as a seed culture. In one
embodiment, the seed culture includes a culture medium as described
above and an inoculation from a stock culture that was grown in the
medium. In one embodiment, the first and second culture media are
the same. In another embodiment, the first and second culture media
are different.
[0109] The seed growth phase (or phases) is generally carried out
to scale-up the quantity of the microorganism from a stored
culture, so that it can be used as an inoculant for the
fermentation phase. The volume and quantity of viable cells used to
inoculate the fermentation culture can be controlled more
accurately if taken from an actively growing culture (e.g., a seed
culture), rather than if taken from a stored culture.
[0110] In addition, more than one (e.g., two or three) seed growth
phases can be used to scale-up the quantity of
polysaccharide-producing bacteria for inoculation of the
fermentation medium. Alternatively, growth of
polysaccharide-producing bacteria in the fermentation phase can
proceed directly from the stored culture by direct inoculation, if
desired.
[0111] To start the fermentation phase, a portion or all of a seed
culture containing the polysaccharide-producing bacteria may be
used to inoculate a fermentation culture medium. An appropriate
concentration of seed culture to use to inoculate fermentation
media can be determined by those of skill in this art.
[0112] Fermentation may be used to produce the maximum cell growth
and/or polysaccharide production in a large-scale environment. In
one embodiment, the polysaccharide-producing bacteria are grown as
a fermentation culture. In one embodiment, the fermentation culture
was inoculated from a seed culture that was grown in the first
medium and the fermentation culture is carried out in a second
medium. In one embodiment, the second medium may be the complex or
defined media as described above. In another embodiment, the first
medium and the second medium are the same.
Fed Batch Fermentation Process
[0113] In one embodiment, the polysaccharide-producing bacterial
cell is cultured in a fed batch culture system using the complex
and defined media described above. In a fed batch system, the
culture is initiated with an inoculation of cells, supplemented
with at least one nutrient added during the culture, and terminated
with a single harvest of cells. In one embodiment, the nutrient is
added at a constant rate.
[0114] In one aspect, the carbon source is the nutrient added
during the culture. The carbon source may be any carbon source
described above for the complex and/or defined media. In a
preferred embodiment, the carbon source is glucose.
[0115] In an aspect of the invention, the amount of batched carbon
source/amount of fed carbon source may be about 10%/90%, 15%/85%,
20%/80%, 25%/75%, or 30%/70%. For instance, in a preferred
embodiment, 20% of the total concentration of the carbon source is
batched and the remaining 80% is fed at a constant rate over the
course of the culture. In another embodiment, 20% of the total
concentration of the carbon source is batched and the remaining
carbon source may also be fed at a non-constant rate over the
course of the culture.
[0116] In yet another aspect, the fed batch fermentation process is
carried out until the cell density, as determined by optical
density (OD) at 600 nm, of the bacterial cell culture is at least
9.0, such as at least 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,
13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,
18.5, 19.0, 19.5, or 20.0. In a preferred embodiment, the fed batch
fermentation process is carried out until the cell density is at
least 9.0.
[0117] In yet another aspect, the cell density, as determined by
optical density (OD) at 600 nm, of the bacterial cell culture by
the fed batch culture system of the invention may be at least 9.0,
such as at least 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0,
13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5,
19.0, 19.5, or 20.0. In a preferred embodiment, the cell density is
at least 9.0.
[0118] For polysaccharides that contain sialic acid such as GBS,
polysaccharide yield may be determined by measuring sialic acid
concentration. Sialic acid is released from cell bound
polysaccharide by digesting pelleted cells by methods well-known in
the art. The digest is assayed by anion exchange chromatogrphay
(AEX) via high performance liquid chromatography (HPLC).
Polysaccharide concentration is then determined by multiplying the
sialic acid value times a repeat unit weight conversion factor. For
example, the conversion factor for each GBS serotype is as follows:
Ia, Ib, and III=3.24; II and V=4.29; and IV=3.77. Polysaccharide
yield for S. pneumoniae or other encapsulated bacteria may be
quantified by first releasing the capsular polysaccharide from the
cell wall by treatment with a detergent, such as sodium deoxycholic
acid (DOC) or sodium N-lauryl-sarcosine (NLS); acid treatment at
high temperature; base treatment; and/or mechanical lysis. The
released polysaccharide in the crude lysate is then assayed against
an authentic standard using size exclusion chromatography (SEC)
HPLC.
[0119] In one aspect, the fed batch fermentation process is carried
out until the polysaccharide concentration, as determined by sialic
acid concentration, of the bacterial cell culture is at least about
250 mg/L, such as at least about 300 mg/L, 350 mg/L, 400 mg/L, 450
mg/L, 500 mg/L, 550 mg/L, 600 mg/L, 650 mg/L, 700 mg/L, 750 mg/L,
800 mg/L, 900 mg/L, 1000 mg/L, 1200 mg/L, 1500 mg/L or 2000 mg/L.
In a preferred embodiment, the fed batch fermentation process is
carried out until the polysaccharide concentration is at least
about 250 mg/L.
[0120] In one aspect, the polysaccharide concentration, as
determined by SEC HPLC, of the bacterial cell culture by the fed
batch culture system of the invention may be at least about 250
mg/L, such as at least about 300 mg/L, 350 mg/L, 400 mg/L, 450
mg/L, 500 mg/L, 550 mg/L, 600 mg/L, 650 mg/L, 700 mg/L, 750 mg/L,
800 mg/L, 900 mg/L, 1000 mg/L, 1200 mg/L, 1500 mg/L or 2000 mg/L.
In a preferred embodiment, the polysaccharide concentration is at
least about 250 mg/L.
Perfusion Fermentation Process
[0121] In one embodiment, the polysaccharide-producing bacterial
cell is cultured in a perfusion culture system. The inventors
discovered that maximal polysaccharide production may be obtained
in a perfusion culture using the complex and defined media
described above. An advantage of a perfusion system is that fresh
media may be added continuously. In addition, metabolic waste
products may be removed during production while maintaining cell
viability in the system.
[0122] The perfusion culture system may include providing fresh
medium to the cells while simultaneously removing spent medium that
is substantially free of cells or includes a substantially lower
cell concentration than that in the bioreactor. In a perfusion
culture, cells can be retained by, for example, filtration,
ultrasonic filtration, centrifugation, or sedimentation.
[0123] In one embodiment, the spent media is separated from the
cells and removed, while retaining the cells in or returning the
cells to the bioreactor. The separation step may be a normal flow
filter and/or a tangential flow filter. In one embodiment, said
filtration system comprises a hollow fiber filter. In another
embodiment, said filtration system comprises a flat-sheet cassette.
In another embodiment, the cells are separated from the spent
medium by a centrifugation step. In another embodiment, the cells
are separated from the spent medium by an ultrasonic separation
step. In another embodiment, the cells are separated from the spent
medium via a sedimentation system.
[0124] In one embodiment the rate of perfusion may be between about
0.07 VVH to about 2.00 VVH, such as between about between about
0.07 VVH to about 1.33 VVH, between about 0.07 VVH to about 1.20
VVH, between about 0.07 VVH to about 1.07 VVH, between about 0.07
VVH to about 0.93 VVH, between about 0.07 VVH to about 0.80 VVH,
between about 0.07 VVH to about 0.67 VVH, between about 0.07 VVH to
about 0.53 VVH, between about 0.07 VVH to about 0.40 VVH, between
about 0.07 VVH to about 0.27 VVH, between about 0.13 VVH to about
2.00 VVH, between about 0.13 VVH to about 1.33 VVH, between about
0.13 VVH to about 1.20 VVH, between about 0.13 VVH to about 1.07
VVH, between about 0.13 VVH to about 0.93 VVH, between about 0.13
VVH to about 0.80 VVH, between about 0.13 VVH to about 0.67 VVH,
between about 0.13 VVH to about 0.53 VVH, between about 0.13 VVH to
about 0.40 VVH, between about 0.13 VVH to about 0.27 VVH, between
about 0.27 VVH to about 2.00 VVH, between about 0.27 VVH to about
1.33 VVH, between about 0.27 VVH to about 1.20 VVH, between about
0.27 VVH to about 1.07 VVH, between about 0.27 VVH to about 0.93
VVH, between about 0.27 VVH to about 0.80 VVH, between about 0.27
VVH to about 0.67 VVH, between about 0.27 VVH to about 0.53 VVH,
between about 0.27 VVH to about 0.40 VVH, between about 0.40 VVH to
about 2.00 VVH, between about 0.40 VVH to about 1.33 VVH, between
about 0.40 VVH to about 1.20 VVH, between about 0.40 VVH to about
1.07 VVH, between about 0.40 VVH to about 0.93 VVH, between about
0.40 VVH to about 0.80 VVH, between about 0.40 VVH to about 0.67
VVH, between about 0.53 VVH to about 2.00 VVH, between about 0.53
VVH to about 1.33 VVH, between about 0.53 VVH to about 1.20 VVH,
between about 0.53 VVH to about 1.07 VVH, between about 0.53 VVH to
about 0.93 VVH, between about 0.53 VVH to about 0.80 VVH, between
about 0.53 VVH to about 0.67 VVH, between about 0.67 VVH to about
2.00 VVH, between about 0.67 VVH to about 1.33 VVH, between about
0.67 VVH to about 1.20 VVH, between about 0.67 VVH to about 1.07
VVH, between about 0.67 VVH to about 0.93 VVH, or between about
0.67 VVH to about 0.80 VVH. In one embodiment, the rate of
perfusion is between about 0.67 VVH to about 1.33 VVH, preferably
about 1.20 VVH.
[0125] In one aspect, the duration of the perfusion culture may be
between about 1 hour and about 15 hours, such as between about 1
hour and about 14 hours, between about 1 hour and about 13 hours,
between about 1 hour and about 12 hours, between about 1 hour and
about 11 hours, between about 1 hour and about 10 hours, between
about 1 hour and about 9 hours, between about 1 hour and about 8
hours, between about 1 hour and about 7 hours, between about 1 hour
and about 6 hours, between about 1 hour and about 5 hours, between
about 5 hours and about 15 hours, between about 5 hours and about
14 hours, between about 5 hours and about 13 hours, between about 5
hours and about 12 hours, between about 5 hours and about 11 hours,
between about 5 hours and about 10 hours, between about 5 hours and
about 9 hours, between about 5 hours and about 8 hours, or between
about 5 hours and about 7 hours. In one embodiment, the duration of
the perfusion culture is between about 1 hour and about 10 hours,
preferably about 7 hours.
[0126] In one particular aspect, the rate of perfusion may be
varied (increased or decreased) for the duration of the culture. In
one embodiment, the perfusion system starts at a first rate and the
rate is increased to a second rate. In another embodiment, the
perfusion system starts at a first rate and the rate is decreased
to a second rate. In an additional embodiment, the rate of
perfusion may be changed multiple times.
[0127] In one aspect, the rate of perfusion is kept constant for
the duration of the culture.
[0128] In another aspect of the invention, the cell growth in the
perfusion system may be at least 1.1-fold, such 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5-fold, greater
than in a batch fermentation system. In a preferred embodiment, the
cell growth in the perfusion system is at least 2-fold greater than
in a batch fermentation system.
[0129] In yet another aspect, the perfusion fermentation process is
carried out until the cell density, as determined by optical
density (OD) at 600 nm, of the bacterial cell culture is at least
20.0, such as at least 25.0, 30.0, 35.0, 40.0, 45.0 50.0, 55.0, or
60.0. In a preferred embodiment, the perfusion fermentation process
is carried out until the cell density is at least 20.0.
[0130] In yet another aspect, the cell density, as determined by
optical density (OD) at 600 nm, of the bacterial cell culture by
the perfusion system of the invention may be at least 20.0, such as
at least 25.0, 30.0, 35.0, 40.0, 45.0 50.0, 55.0, or 60.0. In a
preferred embodiment, the cell density is at least 20.0.
[0131] In another aspect of the invention, the polysaccharide
concentration in the perfusion system is at least 1.5-fold, such as
at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5-fold, greater than
in a batch fermentation system. In a preferred embodiment, the
polysaccharide concentration in the perfusion system is at least
2-fold greater than in a batch fermentation system.
[0132] For polysaccharides that contain sialic acid, such as GBS,
polysaccharide yield may be determined by measuring sialic acid
concentration. Sialic acid is released from cell bound
polysaccharide by digesting pelleted cells by methods well-known in
the art. The digest is assayed by anion exchange chromatogrphay
(AEX) via high performance liquid chromatography (HPLC).
Polysaccharide concentration is then determined by multiplying the
sialic acid value times a repeat unit weight conversion factor. For
example, the conversion factor for each GBS serotype is as follows:
Ia, Ib, and III=3.24; II and V=4.29; and IV=3.77. Polysaccharide
yield for S. pneumoniae or other encapsulated bacteria may be
quantified by first releasing the capsular polysaccharide from the
cell wall by treatment with a detergent, such as sodium deoxycholic
acid (DOC) or sodium N-lauryl-sarcosine (NLS); acid treatment at
high temperature; base treatment; and/or mechanical lysis. The
released polysaccharide in the crude lysate is then assayed against
an authentic standard using size exclusion chromatography (SEC)
HPLC.
[0133] In one aspect, the perfusion fermentation process is carried
out until the polysaccharide concentration, as determined by sialic
acid concentration, of the bacterial cell culture is at least about
600 mg/L, such as at least about 650 mg/L; 700 mg/L; 750 mg/L; 800
mg/L, 850 mg/L; 900 mg/L; 950 mg/L; 1,000 mg/L; 1,500 mg/L; or
2,000 mg/L. In a preferred embodiment, perfusion fermentation
process is carried out until the polysaccharide concentration is at
least about 600 mg/L.
[0134] In one aspect, the polysaccharide concentration, as
determined by SEC HPLC, of the bacterial cell culture by the
perfusion system of the invention may be at least about 600 mg/L,
such as at least about 650 mg/L; 700 mg/L; 750 mg/L; 800 mg/L, 850
mg/L; 900 mg/L; 950 mg/L; 1,000 mg/L; 1,500 mg/L; or 2,000 mg/L. In
a preferred embodiment, the polysaccharide concentration is at
least about 600 mg/L.
EXAMPLES
[0135] The following examples demonstrate some embodiments of the
present invention. However, it is to be understood that these
examples are for illustration only and do not purport to be wholly
definitive as to conditions and scope of this invention. It should
be appreciated that when typical reaction conditions (e.g.,
temperature, reaction times, etc.) have been given, the conditions
both above and below the specified ranges can also be used, though
generally less conveniently. All parts and percents referred to
herein are on a weight basis and all temperatures are expressed in
degrees centigrade unless otherwise specified.
[0136] Furthermore, the following examples were carried out using
standard techniques, which are well known and routine to those of
skill in the art, except where otherwise described in detail. As
noted above, the following examples are presented for illustrative
purpose, and should not be construed in any way limiting the scope
of this invention.
Example 1: A Defined Medium of the Invention
[0137] Applicant's proprietary, mammalian defined cell culture
medium ("R17") was modified for growth of Streptococcus pneumoniae
to create "Modified AS3" medium (also referred to as "mAS3"). The
Modified AS3 medium was formulated with the components of Table 3
below. The media prepared for serotypes 4, 5, 6A, 6B, 14, and 23F
was formulated with 30 g/L of dextrose and 0.6 g/L of magnesium
sulfate. In the case of serotypes 1, 3, 6B, 7F, 9V, 18C, 19A, and
19F, 30 g/L dextrose was added to the media in the reactor.
TABLE-US-00003 TABLE 3 Modified AS3 Medium Component Concentration
(g/L) R-17 dry powder 15.56 g/L L-tyrosine disodium salt, dihydrate
0.643 g/L Dextrose anhydrous 25 g/L Sodium chloride 1.1 g/L 300 mM
acidic cystine stock 3.75 mL/L L-Asparagine monohydrate 2.25 g/L
L-Glutamine 1.17 g/L 1 mM Ferrous sulfate stock 15 mL/L Trace
elements E 1 mL/L Magnesium sulfate heptahydrate 1.23 g/L
[0138] A full accounting of the composition of the amino acids,
vitamins and salts in the R17 powder is provided in Table 4
below.
TABLE-US-00004 TABLE 4 Composition of R17 Powder Amino Acids g/L mM
alanine 0.02 0.20 arginine 0.70 4.00 aspartic acid 0.22 1.65
cysteine.cndot.HCl.cndot.H.sub.2O 0.07 0.40 monosodium 0.03 0.20
glutamate glycine 0.12 1.54 histidine.cndot.HCl.cndot.H.sub.2O 0.47
2.26 isoleucine 0.57 4.36 leucine 1.03 7.87 lysine.cndot.HCl 1.40
7.70 methionine 0.39 2.60 phenylalanine 0.51 3.07 proline 0.54 4.69
serine 1.05 10.02 threonine 0.56 4.75 tryptophan 0.27 1.34 valine
0.75 6.40 Other Components g/L nM linoleic acid 0.0003 1.04
thioctic acid 0.0007 3.48 D-glucose (Dextrose) 5.00 83.33 Sodium
pyruvate 0.06 0.50 Inorganic Salts g/L mM CaCl.sub.2 0.12 1.05 KCl
0.31 4.19 Na.sub.2HPO.sub.4 0.06 0.40
NaH.sub.2PO.sub.4.cndot.H.sub.2O 0.65 4.68 MgCl.sub.2 0.03 0.30
MgSO.sub.4 0.14 1.15 Trace Elements .mu.g/L nM Sodium Selenite
69.16 400.00 CuSO.sub.4 10.24 64.00 CuSO.sub.4.cndot.5H.sub.2O
99.88 400.00 FeSO.sub.4.cndot.7H.sub.2O 4170 15000
MnSO.sub.4.cndot.H.sub.2O 33.80 200.00
Na.sub.2SiO.sub.3.cndot.9H.sub.2O 284.07 1000
(NH.sub.4).sub.6Mo.sub.7O.sub.24.cndot.4H.sub.2O 247.20 200.00
NH.sub.4VO.sub.3 2.34 20.00 NiSO.sub.4.cndot.6H.sub.2O 5.26 20.00
SnCl.sub.2.cndot.2H.sub.2O 0.90 4.00 AlCl.sub.3.cndot.6H.sub.2O
0.97 4.00 KBr 0.48 4.00 CrCl.sub.3 15.83 100.00 NaF 0.17 4.00
GeO.sub.2 0.42 4.00 Kl 33.20 200.00 RbCl 0.48 4.00 H.sub.3BO.sub.3
12.37 200.00 LiCl 0.17 4.00 Vitamins g/L mM biotin 0.003 0.01
calcium pantothenate 0.02 0.05 choline chloride 0.16 1.14 folic
acid 0.03 0.06 inositol 0.16 0.91 nicotinamide 0.03 0.22
pyridoxal.cndot.HCl 0.002 0.01 pyridoxine.cndot.HCl 0.004 0.18
riboflavin 0.002 0.01 thiamine.cndot.HCl 0.04 0.12 vitamin B12 0.02
0.02
[0139] Batch fermentation was performed in a 2 L bioreactor with
temperature control at 36.degree. C. and pH control at 7.0 with
NaOH used as base titrant. The fermentor was inserted with N2
overlay for serotypes 1, 3, 4, 6A, 6B, 7F, 9V, 18C, 19A, and 19F
and with air for serotypes 6B(2), 14 and 23F; no overlay was used
for serotype 5. The fermentation was stirred at 200 RPM. Results
are shown in Table 5 below.
TABLE-US-00005 TABLE 5 S. pneumoniae Growth and Polysaccharide
Production in Modified AS3 Medium Growth Polysaccharide Serotype
OD600 (g/L) 1 9.1 1.38 3 11.5 3.19 4 9.5 0.50 5 7.5 0.40 6A 6.6
1.10 6B 6.0 2.00 6B(2) 6.3 1.20 7F 8.0 0.70 9V 7.4 0.54 14 6.8 1.10
18C 7.0 0.97 19A 5.0 2.40 19F 5.5 0.86 23F 6.5 1.10
[0140] The thirteen S. pneumoniae serotypes were successfully grown
in a batch culture using the chemically defined Modified AS3
medium.
Example 2: Comparison of Commercially Available Defined Medium to a
Defined Medium of the Invention
[0141] Eagle's minimum essential medium (EMEM), a commercially
available defined cell culture medium, was tested in comparison to
R17 using various serotypes of GBS. The formulation of each was
modified from the label instructions to accommodate the sodium
bicarbonate requirement of GBS grown anaerobically. Each medium was
also supplemented with a high concentration of glucose to support
the higher cell density achievable in bacterial cultures. The
formulation for EMEM modified as a bacterial medium ("bacterial
EMEM") was as follows: 20.2 g/L EMEM powder, 1.17 g/L L-glutamine,
0.84 g/L sodium bicarbonate and 80 g/L glucose. The composition of
GBS mAS3mAS3 (as described in Example 1) was customized for GBS
growth (hereinafter "GBS mAS3") as follows: 0.21 g/L L-cysteine HCl
(instead of 300 mM acidic cystine stock), 2 mL/L (instead of 1
mL/L)Trace Element E 1000.times., 0.84 g/L sodium bicarbonate and
80 g/L glucose (instead of 25 g/L dextrose anhydrous).
[0142] Fermentation was performed at 10 L bioreactor scale with
temperature control at 37.degree. C. and pH control at 7.0 with
NaOH used as base titrant. The fermentor was inserted with N2
overlay at 0.1 vvm with respect to the batch volume; the
fermentation was stirred with agitation sufficient to achieve a kLa
of 1 hr.sup.-1. Results are shown in Table 6 below.
TABLE-US-00006 TABLE 6 Comparison of Bacterial EMEM and GBS mAS3
Growth Polysaccharide GBS OD.sub.600 (mg/L) Sero- Bacterial GBS
Bacterial GBS type EMEM mAS3 EMEM mAS3 Ia 3.9 8.9 70 750 Ib 3.6 8.3
90 270 II 3.1 9.7 60 190 III 3.7 5.1 210 270 IV 3.6 8.2 70 260 V
3.8 9.6 50 220
[0143] GBS mAS3 medium showed surprising superiority to the
bacterial EMEM medium in both growth and polysaccharide
concentration.
Example 3: Comparison of a Complex Medium to a Defined Medium of
the Invention
[0144] Modified AS3 medium as described in Example 1 and a soy
hydrolysate-based complex medium (BPDv3) were compared for various
serotypes of S. pneumoniae. BPDv3 was composed of 28 g/L HYPEP1510
(Kerry Group Services Ltd.), 54 g/L glucose, 3.5 g/L NaCl, 0.7 g/L
KH.sub.2PO.sub.4, 0.0182 g/L CaCl.sub.2.2H.sub.2O, 1 g/L
MgSO.sub.4.7H.sub.2O, 0.84 g/L NaHCO.sub.3, 3 g/L ammonium
chloride, 0.25 g/L uridine, 0.25 g/L adenosine, 0.03 g/L
niacinamide, 0.03 g/L pyridoxine HCl, 0.0075 g/L pantothenic acid
and 0.003 g/L PABA. Medium for serotype 12F was supplemented with 1
g/L monosodium glutamate, and medium for serotype 8 was modified to
contain 0.5 g/L ammonium chloride and 36 g/L glucose. Although
polysaccharide titer was not consistently improved in the Modified
AS3medium as compared to the complex medium, the Modified AS3
medium showed improved growth in almost all serotypes (see Table
7).
TABLE-US-00007 TABLE 7 Comparison of Modified R17 Medium and
Complex Medium Growth Polysaccharide (OD.sub.600) (mg/L) S.
pneumoniae Modified Modified Serotype AS3 BPDv3 AS3 BPDv3 8 9.2 5.2
2630 2310 10A 12.9 8.1 790 890 11A 14.8 7.3 1210 1140 12F 9.4 10.0
910 2130 15B 12.5 7.8 1170 1900 22F 14.4 6.4 1740 1350 33F 14.4
11.5 2580 3430
Example 4: Amino Acid Consumption
[0145] An analysis of amino acid consumption during the course of
GBS serotype Ill fermentation was performed to determine if amino
acids were depleted. An analysis of the concentration of amino
acids in the GBS mAS3 (as in Example 2) prior to inoculation and at
harvest is presented in Table 8.
TABLE-US-00008 TABLE 8 Amino Acid Consumption Initial Harvest Amino
Acid (mM) (mM) Alanine 0 0.6 Arginine 3.6 0 Aspartic acid 1.8 1.8
Asparagine 16.7 14.9 Cysteine < < Glutamic acid 0.3 1.5
Glutamine 9.8 6.2 Glycine 1.4 0.2 Histidine 2.3 2.2 Isoleucine 4.6
4 Leucine 8.6 8 Lysine 9.3 6.2 Methionine 2.9 2.6 Phenylalanine 3.4
3.1 Proline 5.1 5.1 Serine 10.8 0 Threonine 5.8 6.2 Tryptophan 1.5
1.5 Tyrosine 1.8 1.9 Valine 6.9 6.1
[0146] Although predicted required amino acids were not depleted,
four amino acids for which S. agalactiae is presumably prototrophic
were. Arginine, glycine, and serine were depleted to less than the
limit of quantification. Cysteine, which is difficult to measure by
the HPLC method, was not detected at either sample time. All other
amino acids were still in excess at harvest.
[0147] The four depleted amino acids were then supplemented to GBS
mAS3 medium in fermentation of various GBS serotypes at 4.times.
concentration with respect to the basic powder R17 formulation (16
mM arg, 1.6 mM cys, 6 mM gly, and 40 mM ser). Results are shown in
Table 9 below.
TABLE-US-00009 TABLE 9 Comparison of GBS mAS3 and GBS mAS3
Supplemented with Depleted Amino Acids Growth Polysaccharide
(OD.sub.600) (mg/L) GBS Supple- Supple- Sero- GBS mented GBS mented
type mAS3 CGRS mAS3 CGRS Ia 9.6 10.0 670 430 Ib 9.0 17.8 230 360 II
8.6 13.9 110 140 III 5.1 10.8 260 340 IV 7.4 10.7 330 270 V 9.4
16.4 140 210
[0148] A significant improvement in growth was observed in all six
serotypes tested with supplemented CGRS medium. Although the
polysaccharide titers did not increase for all serotypes, the
growth improvement encouraged further testing.
Example 5: Further Analysis of Depleted Amino Acids
[0149] The importance of each of the depleted amino acid to the
improvement in growth was assessed in an experiment in which each
of the four was sequentially deleted from the medium using GBS
serotype V as a model. Glycine was unexpectedly found to be the
sole contributor to improved growth (see Table 10).
TABLE-US-00010 TABLE 10 Sequential Deletion of Supplemented Amino
Acids Amino Acids Growth Polysaccharide Added (OD600) (mg/L) None
9.7 240 CGRS 15.2 330 GRS 15.6 330 CRS 9.6 200 CGS 14.4 350 CGRS
15.1 360
[0150] This was confirmed in a follow up study in which each of the
four amino acids were supplemented individually, again using GBS
serotype V as a model. The study confirmed that glycine was the
only amino acid of the four depleted amino acids that improved
growth and polysaccharide production (see Table 11).
TABLE-US-00011 TABLE 11 Supplementation of Individual Amino Acids
Amino Acid(s) Growth Polysaccharide Added (OD600) (mg/L) None 9.7
240 CGRS 15.2 330 G only 13.1 440 A only 8.8 280 C only 9.0 310 S
only 9.3 280
[0151] The importance of the added glycine as a sole supplement was
then tested in several GBS serotypes. Performance in GBS mAS3, GBS
mAS3 supplemented with all four amino acids, and GBS mAS3
supplemented with only glycine was compared. Results are shown in
Table 12 below.
TABLE-US-00012 TABLE 12 Comparison of GBS mAS3, GBS mAS3
Supplemented with All Four Amino Acids, and GBS mAS3 Supplemented
with Only Glycine Growth Polysaccharide (OD.sub.600) (mg/L) GBS
Supple- Supple- Sero- GBS mented GBS mented type mAS3 CGRS Glycine
mAS3 CGRS Glycine Ia 9.6 10.0 12.1 670 430 850 Ib 9.0 17.8 11.1 230
360 480 II 8.6 13.9 12.1 110 140 403 III 5.1 10.8 10.1 260 340 569
IV 7.4 10.7 10.9 330 270 530 V 9.4 16.4 15.7 140 210 290
[0152] In general, sole supplementation with glycine was sufficient
to improve growth in a manner about equivalent to supplementation
with all four amino acids. However, sole supplementation with
glycine surprisingly produced higher polysaccharide titer than GBS
mAS3 and supplementation with all four amino acids.
Example 6: Comparison of Glycine Concentrations
[0153] In view of the unexpectedly high production of
polysaccharides with the addition of glycine alone, an experiment
was conducted to determine if the maximal growth and polysaccharide
titer was obtained with the addition of 6 mM glycine to the GBS
mAS3 formulation. The experiment compared the addition of from 0.15
mM to 123.2 mM glycine using GBS serotype V as a model. The data in
Table 13 below show the addition of as little as 1.5 mM glycine or
as much as 61.6 mM supports the same improvement in polysaccharide
titer as seen with the addition of 6 mM glycine.
TABLE-US-00013 TABLE 13 Comparison of Glycine Concentrations
Glycine Concentration Growth Polysaccharide (mM) (OD600) (mg/L) 0
11.6 334 0.15 11.9 356 1.5 16.1 446 3.1 18.5 521 6.2 19.6 455 15.4
18.4 421 30.8 18.5 396 61.6 19.5 381 123.2 0 0
Example 7: Determining Nonessential Components of the GBS mAS3
Medium
[0154] The GBS mAS3 formulation and its glycine containing
derivatives in Example 56 contained 80 g/L glucose to assure that
the carbon source would be in excess throughout and at the end of
the fermentation. In general, when growth and polysaccharide
production had ceased, about 30 g/L glucose remained unconsumed
(data not shown). Therefore, an experiment was conducted to
determine if a more efficient medium could be achieved.
Glycine-supplemented GBS mAS3 media with glucose concentrations of
80 g/L, 70 g/L, 60 g/L, and 50 g/L were tested with GBS serotype V
as a model. The data in Table 14 below show that a glucose
concentration of 50 g/L leaves no residual glucose but also does
not compromise polysaccharide titer.
TABLE-US-00014 TABLE 14 Comparison of Glucose Concentrations
Batched Growth Polysaccharide Residual Glucose Glucose (g/L)
(OD600) (mg/L) (g/L) 80 17.6 410 28 70 18.2 420 17 60 18.8 430 10
50 19.4 420 0
[0155] Similarly, the importance of all amino acids and salts added
to the R17 powder was examined by omitting each, one at a time, in
a drop out experiment. The work was performed with GBS serotype V
in glycine-supplemented GBS mAS3 medium. The data in Table 15 below
indicate that tyrosine, glutamine, and cysteine are essential for
growth, whereas asparagine is not, and all salts are
nonessential.
TABLE-US-00015 TABLE 15 Drop Out of Amino Acids and Salts Component
Growth Polysaccharide Deleted (OD600) (mg/L) None 12.7 570 Asn 12.5
600 Gln 1.0 40 Tyr 0.2 10 Cys 6.2 290 None 13.2 510 Magnesium 13.5
540 sulfate Ferrous sulfate 12.3 510 Trace elements 13.1 500 E
Sodium chloride 13.1 520
Example 8: Consumption of Vitamins and Salts/Trace Elements
[0156] An assessment of residual vitamins and salts/trace elements
versus starting concentrations was done in glycine-supplemented GBS
mAS3 media using GBS serotype III as a model. A total of 13
vitamins were examined: biotin, choline cyanocobalamin, folic acid,
niacin, niacinamide, nicotinamide, p-aminobenzoic acid, panthotenic
acid, pyridoxal, pyridoxamine, pyridoxine, riboflavin, and
thiamine. Twelve showed no significant change in concentration
during fermentation. Niacinamide was found to be depleted to zero
during the course of the fermentation, but an accompanying
accumulation of niacin would indicate that this vitamin family is
not depleted (data not shown).
[0157] Thirty-two salts and trace elements were analyzed. Eighteen
of these were below the limits of detection. Those 18 are as
follows: silver, aluminum, arsenic, beryllium, cadmium, chromium,
copper, mercury, lithium, manganese, nickel, lead, rubidium,
selenium, tin, titanium, thallium, and vanadium. Of the 14
detectable salts and trace elements, 12 showed no substantial
decline in concentration from initial inoculation of the medium to
after harvest (see Table 16). Phosphorous and potassium were the
two that showed a decline in concentration. The decline in
phosphorous concentration was expected as it is consumed for cell
growth, but it was not growth limiting since it remained in excess
at harvest. The decline in potassium concentration, however, was
unexpected.
TABLE-US-00016 TABLE 16 Salts and Trace Elements Consumption
Initial Harvest (mg/L) (mg/L) Boron 1.5 1.6 Barium 4.4 4.0 Calcium
36.1 23.4 Cobalt 0.9 0.8 Iron 0.8 0.7 Potassium 292.0 22.0
Magnesium 138.0 121.0 Molybdenum 0.1 0.1 Sodium 1134.0 10851.0
Phosphorous 159.0 44.0 Sulfur 303.0 349.0 Silicon 0.0 1.9 Strontium
0.1 0.1 Zinc 4.1 4.4
[0158] The data provoked two studies in which the effect of adding
an additional 2-fold amount of potassium chloride (0.6 g/L
additional to the 0.31 g/L R17 powder) to the glycine-supplemented
GBS mAS3 media in the fermentation of GBS serotype III was
examined. The results shown in Table 17 indicate that the increase
in KCl concentration was beneficial for polysaccharide titer in a
growth independent fashion.
TABLE-US-00017 TABLE 17 Supplementation with KCl KCl concentration
Growth Polysaccharide Study # (g/L) (OD600) (mg/L) 1 0.31 11.4 434
0.91 11.2 594 2 0.31 10.6 612 0.91 12.2 755
[0159] An additional study examined a fuller range of KCl
concentrations (from 0.03 g/L to 24 g/L) additional to the 0.31 g/I
KCl contained in the R17 basal powder) for their effect on growth
and polysaccharide synthesis. The results are shown in Table 18
which indicate KCl concentrations of from 0.3 to 24 g/L additional
to R17 powder confer improved growth and polysaccharide
production.
TABLE-US-00018 TABLE 18 Supplementation of Glycine-Containing R17
with a Range of KCl Concentrations Additional KCl Growth
Polysaccharide added (g/L) (OD600) (mg/L) 0 12.3 308 0.03 15.3 347
0.3 17.8 382 0.6 18.2 387 1.2 19.5 406 3.0 19.7 402 17.0 19.7 432
12.0 17.1 370 24.0 17.9 382
Example 9: Formulation of mAS3opt50 Medium
[0160] A medium was configured to incorporate the increases in
glycine and KCl, decrease in glucose concentration, and omission of
magnesium, asparagine, and NaCl ("mAS3opt50") in GBS mAS3 medium.
The new formulation was tested in comparison to GBS mAS3 medium for
six GBS serotypes. As shown in Table 19, the reformulated medium
affords substantially improved growth and concomitant
polysaccharide titers.
TABLE-US-00019 TABLE 19 Comparison of GBS mAS3 and mAS3opt50
Polysaccharide Growth (OD.sub.600) (mg/L) GBS GBS GBS Serotype mAS3
mAS3opt50 mAS3 mAS3opt50 Ia 8.9 18.6 750 930 Ib 8.3 19.3 270 560 II
9.7 17.0 190 440 III 5.1 13.9 270 750 IV 8.2 10.4 260 360 V 9.6
18.0 220 390
Example 10: Contribution of Glycine and KCl to Polysaccharide Yield
in mAS3opt50
[0161] A drop out approach was used to demonstrate the importance
of the glycine and KCl supplementation to polysaccharide yield in
the mAS3opt50 medium. The data shown in Table 20 clearly indicates
that each is important in supporting high yield.
TABLE-US-00020 TABLE 20 Drop Out of Glycine and KCl in mAS3opt50
GBS Growth Polysaccharide Sero- (OD.sub.600) (mg/L) type mAS3opt50
-glycine -KCl mAS3opt50 -glycine -KCl Ia 18.6 13.2 15.6 930 780 840
Ib 17.5 8.4 12.2 580 300 440 II 12.9 8.8 12.3 375 206 311 III 13.5
10.5 12.1 770 400 550 IV 9.3 8.7 11.5 375 305 331 V 19.3 10.4 13.9
428 265 328
Example 11: Comparison of mAS3opt50 to Complex Medium and Complex
Medium Supplemented with Yeast Extract
[0162] The starting complex medium ("HP") was a soy
hydrolysate-based formulation: 28 g/L HYPEP 1510 (Kerry Group
Services Ltd.), 3.5 g/L NaCl, 0.7 g/L KH.sub.2PO.sub.4, 0.0182 g/L
CaCl.sub.2.2H.sub.2O, 1 g/L MgSO.sub.4.7H.sub.2O, 0.84 g/L
NaHCO.sub.3, and 80 g/L glucose. A fermentation of six GBS
serotypes in this medium gave growth and titers that were
substantially lower than in mAS3opt50. Therefore, HP was
supplemented with 10 g/L AMBERFERM 5902 (Sensient Technologies
Corp.) ("HPYE"), an ultrafiltered yeast extract. The HPYE medium
had substantially improved cell density and polysaccharide titers
compared to the HP medium. While HPYE showed increased growth in
almost all serotypes compared to mAS3opt50, the polysaccharide
titers were somewhat less in the HPYE media. All data are shown in
Table 21 below.
TABLE-US-00021 TABLE 21 Comparison of mAS3opt50 to Complex Medium
and Complex Medium Supplemented with Yeast GBS Growth
Polysaccharide Sero- (OD.sub.600) (mg/L) type mAS3opt50 HP HPYE
mAS3opt50 HP HPYE Ia 18.6 9.8 17.9 930 410 770 Ib 19.3 8.1 22.7 560
120 460 II 17.0 13.1 19.1 440 150 230 III 13.9 11.4 19.3 750 440
630 IV 10.4 18.4 16.5 360 120 280 V 18.0 11.6 20.5 390 170 390
Example 12: Titration of Yeast Extract in Complex Medium
[0163] A titration was performed to determine the concentration of
yeast extract supplementation to confer optimal growth and
polysaccharide production. GBS serotype V was used as a model to
gauge the effect of yeast extract supplementation with AMBERFERM
5902 (Sensient Technologies Corp.) at 0 g/L, 2.5 g/L, 5 g/L, 10
g/L, 20 g/L and 40 g/L. As shown in Table 22, the data indicated
that supplementation with as little as 2.5 g/L yeast was sufficient
to stimulate growth and production of capsular polysaccharide.
However, the optimum concentration of yeast extract supplementation
was 10 g/L because the addition of greater amounts conferred no
additional benefit.
TABLE-US-00022 TABLE 22 Effect of Yeast Extract Titration on Growth
and Polysaccharide Production in Complex Medium Yeast extract
Growth Polysaccharide concentration (g/L) (OD.sub.600) (mg/L) 0
10.9 64 2.5 18.0 259 5.0 19.6 291 10.0 20.4 320 20.0 20.7 282 40.0
23.5 279
Example 13: Yeast Extract Supplementation of Defined Media
[0164] Given the positive impact of supplementation with yeast
extract on polysaccharide titer in the complex medium, experiments
that examined supplementation of GBS mAS3 with AMBERFERM 5902
(Sensient Technologies Corp.) was performed. GBS mAS3 supplemented
with the ultrafiltered yeast extract ("R17YE") was compared to GBS
mAS3 and mAS3opt50. Yeast extract supplementation of R17
dramatically improved polysaccharide titer compared to both GBS
mAS3 and mAS3opt50 (see Table 23).
TABLE-US-00023 TABLE 23 Comparison of Yeast Extract-Supplemented
GBS mAS3, GBS mAS3, and mAS3opt50 Growth Polysaccharide GBS
(OD.sub.600) (mg/L) Sero- GBS GBS type mAS3 R17YE mAS3opt50 mAS3
R17YE mAS3opt50 Ia 8.9 20.0 18.6 750 750 930 Ib 8.3 18.8 19.3 270
690 560 II 9.7 18.2 17.0 190 350 440 III 5.1 16.5 13.9 270 750 750
IV 8.2 16.2 10.4 260 560 360 V 9.6 18.8 18.0 220 470 390
[0165] A study was then performed to compare supplementation with
varying concentrations of ultrafiltered yeast extract to
supplementation with varying concentrations of a commercially
available "synthetic" yeast extract from BD Biosciences (BD
RECHARGE). GBS serotype V was used as a model. The data shown in
Table 24 indicates that, although 20 g/L yeast extract (either
ultrafiltered or synthetic) confers an improvement in growth, there
is no corresponding increase in polysaccharide titer.
Supplementation with the synthetic yeast extract improves growth
over GBS mAS3 control, but does not confer the maximum titer that
is achieved with the ultrafiltered yeast extract.
TABLE-US-00024 TABLE 24 Comparison of Varying Concentrations of
Ultrafiltered Yeast Extract and Synthetic Yeast Extract GBS mAS3
Growth Polysaccharide Supplementation (OD.sub.600) (mg/L) None 9.8
240 5 g/L AMBERFERM 15.5 440 5902 10 g/L AMBERFERM 18.1 470 5902 20
g/L AMBERFERM 26.2 480 5902 10 g/L BD 18.0 320 RECHARGE 20 g/L BD
19.2 320 RECHARGE
Example 14: Analysis of Constant Glucose Feed Fermentation
[0166] A constant glucose feed was examined for its effect in
supporting polysaccharide titers with the mAS3opt50 media using
various GBS serotypes as a model. A comparison of batching 50 g/L
glucose and glucose-fed fermentations (10 g/L glucose batched and
the remaining 40 g/L fed at a constant rate over the course of 7
hours beginning at 3-4 hours of EFT) indicated that comparable
growth and polysaccharide titers were achieved across all serotypes
(see Table 25). The fermentation control parameters were otherwise
those presented in Example 2.
TABLE-US-00025 TABLE 25 Glucose Fed-Batch Fermentation with
mAS3opt50 Media Polysaccharide GBS Growth OD.sub.600 (mg/L)
Serotype Batch Feed Batch Feed Ia 11.0 13.5 610 790 Ib 15.3 17.6
530 460 II 15.1 14.4 270 300 III 11.7 13.2 590 660 IV 9.7 9.3 330
340 V 15.2 15.0 280 360
Example 15: Glucose Fed-Batch Fermentation with HPYE and GBS mAS3
Media
[0167] Fed-batch fermentation was also examined for HPYE and GBS
mAS3 media using GBS serotype V as a model. The fermentation was
initiated with 10 g/L glucose batched, and then 70 g/L glucose was
fed over the course of 5 hours after 3-4 hours of EFT. The
fermentation was otherwise formulated as in Example 2. The data
presented in Table 26 indicates that the fed-batch approach
gives.about.equivalent productivity for GBS mAS3 versus the batch
approach. The fed approach supports polysaccharide production in
HPYE, but at a somewhat lower productivity than batch.
TABLE-US-00026 TABLE 26 Comparison of Growth and Polysaccharide
Production of Serotype V in Batch and Glucose Fed-Batch in
mAS3opt50, HPYE and GBS mAS3 Media Growth Polysaccharide
(OD.sub.600) (mg/L) Basal medium Batch Fed Batch Fed mAS3opt50 15.1
15.0 280 360 HPYE 19.8 23.4 390 334 GBS mAS3 9.7 10.1 175 182
Example 16: Perfusion Fermentation
[0168] Perfusion experiments using the Modified AS3 medium
described in Example 1 were performed on 13 serotypes of S.
pneumoniae. The medium was inoculated and run in batch mode in a 2
L bioreactor for about 4-10 hours until the OD.sub.600 reached 3-7.
The culture was then circulated through a perfusion system where
spent medium and waste products were removed and the culture volume
was maintained by the introduction of fresh medium. The perfusion
began at an initial rate of 0.13 VVH and gradually ramped to 0.80
VVH over the course of 3-5 hours, at which point the perfusion
batch was ended. The data, shown in Table 27 below, indicates a
significant increase in biomass levels with a corresponding
increase in polysaccharide produced compared to batch fermentation
in Example 1.
TABLE-US-00027 TABLE 27 S. pneumoniae Growth and Polysaccharide
Production in Perfusion Fermentation Compared to Batch Fermentation
Perfusion Batch Growth Polysaccharide Growth Polysaccharide
Serotype OD600 (g/L) OD600 (g/L) 1 36 3.4 9.1 1.38 3 20 2.4 11.5
3.19 4 51 2.4 9.5 0.50 5 32 2.7 7.5 0.40 6A 42 5.3 6.6 1.10 6B 30
3.5 6.0 2.00 7F 51 5.6 8.0 0.70 9V 37 5.1 7.4 0.54 14 30 2.2 6.8
1.10 18C 35.5 5.5 7.0 0.97 19A 26 4.9 5.0 2.40 19F 55 5.4 5.5 0.86
23F 37 4.4 6.5 1.10
Example 17: Comparison of Perfusion and Batch Fermentation Methods
in Three Different Media
[0169] Perfusion experiments using GBS mAS3 or HPYE as the basal
media were performed. A 1.times. medium (containing 0.5.times.
glucose) was inoculated and run in batch mode (5L working volume)
for about 3 hours. When the OD approached 1-5 OD, perfusion with
0.5.times. medium began at an initial rate of 0.13 VVH for
approximately one hour. The rate was ramped to 1.20 VVH over the
course of 6-7 hours at which point the perfusion batch was ended.
The data for GBS mAS3 perfusion, shown in Table 28 below, indicates
an approximate 1.4-2 fold increase in cell density over batch mode,
with a concomitant increase in polysaccharide titer.
TABLE-US-00028 TABLE 28 Polysaccharide GBS Growth OD.sub.600 (mg/L)
Serotype Batch Perfusion Batch Perfusion Ia 18.6 25.6 930 1300 Ib
19.3 41.6 560 740 II 17.0 27.7 440 860 III 13.9 32.3 750 1770 IV
8.2 ND 260 ND V 18.0 28.1 390 730 *ND = Test not done.
[0170] The data for perfusion based on the HPYE complex medium is
shown in Table 29 below. In general, perfusion resulted in a
greater than 2-fold increase in cell density over batch and a 2 to
3.5-fold improvement in polysaccharide titer.
TABLE-US-00029 TABLE 29 Polysaccharide GBS Growth OD.sub.600 (mg/L)
Serotype Batch Perfusion Batch Perfusion Ia 17.9 50.6 770 1940 Ib
22.7 27.9 460 1100 II 19.1 44.0 230 820 III 19.3 39.7 630 1150 IV
16.5 ND 280 ND V 20.5 50.0 390 680 *ND = Test not done.
[0171] A perfusion in a mAS3opt50-based medium was similarly
performed using serotype IV as a model. The harvest OD600 obtained
was 16.7; polysaccharide production was 667 mg/L. By comparison,
mAS3opt50 batch fermentation presented in Example 8 gave a cell
density of 10.4 and a polysaccharide production value of 360 mg/L,
evidencing a .about.1.9-fold productivity improvement.
[0172] In summary, the perfusion fermentation resulted in about a
2-fold or better polysaccharide productivity increase over batch
performance in all three media employed.
Aspects of the Invention
[0173] The following clauses describe additional embodiments of the
invention:
C1. A polysaccharide-producing bacterial cell culture medium
comprising a vegetable hydrolysate, a yeast extract, and a carbon
source. C2. The medium of C1, wherein the vegetable hydrolysate is
a soy hydrolysate. C3. The medium of C2, wherein the soy
hydrolysate is selected from the group consisting of HYPEP 1510
(Kerry Group Services Ltd.), HYPEP 4601 (Kerry Group Services
Ltd.), HYPEP 5603 (Kerry Group Services Ltd.), HY-SOY (Kerry Group
Services Ltd.), AMI-SOY (Kerry Group Services Ltd.), N-Z-SOY (Kerry
Group Services Ltd.), N-Z-SOY BL4 (Kerry Group Services Ltd.),
N-Z-SOY BL7 (Kerry Group Services Ltd.), SHEFTONE D (Kerry Group
Services Ltd.), SE50M, SE50MK, soy peptone, BACTO soytone (Difco
Laboratories Inc.), NUTRISOY 2207 (ADM), NUTRISOY (ADM), NUTRISOY
flour (ADM), and soybean meal. C4. The medium of C3, wherein the
soy hydrolysate is HYPEP 1510 (Kerry Group Services Ltd.). C5. The
medium of any one of C1-C4, wherein the concentration of the
vegetable hydrolysate is between about 5 g/L and about 75 g/L. C6.
The medium of C5, wherein the concentration of the vegetable
hydrolysate is between about 10 g/L and about 50 g/L. C7. The
medium of C6, wherein the concentration of the vegetable
hydrolysate is about 28 g/L. C8. The medium of any one of C1-C7,
wherein the yeast extract is a yeast autolysate, an ultrafiltered
yeast extract, or a synthetic yeast extract. C9. The medium of C8,
wherein the yeast extract is an ultrafiltered yeast extract. C10.
The medium of C9, wherein the ultrafiltered yeast extract is
AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD
Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE
(Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services
Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry
Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or
HY-YEST 504 (Kerry Group Services Ltd.). C11. The medium of any one
of C1-C10, wherein the concentration of yeast extract is between
about 1 g/L to about 50 g/L. C12. The medium of C11, wherein the
concentration of yeast extract is between about 5 g/L to about 25
g/L. C13. The medium of C12, wherein the concentration of yeast
extract is about 10 g/L. C14. The medium of any one of C1-C13,
wherein the carbon source is selected from the group consisting of
glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose,
raffinose, xylose, and mannose. C15. The medium of C14, wherein the
carbon source is glucose. C16. The medium of any one of C1-C15,
wherein the concentration of the carbon source is between about 25
g/L to about 100 g/L. C17. The medium of C16, wherein the
concentration of the carbon source is between about 50 g/L to about
90 g/L. C18. The medium of C17, wherein the concentration of the
carbon source is about 80 g/L. C19. The medium of any one of
C1-C18, wherein the medium comprises soy hydrolysate, an
ultrafiltered yeast extract, and glucose. C20. The medium of any
one of C1-C19, wherein the medium further comprises a
phosphate-containing ingredient. C21. The medium of C20, wherein
the phosphate-containing ingredient is Na.sub.2HPO.sub.4,
K.sub.2HPO.sub.4, or KH.sub.2PO.sub.4. C22. The medium of any one
of C1-C21, wherein the medium further comprises at least one amino
acid, vitamin, nucleoside, or inorganic salt. C23. A
polysaccharide-producing bacterial cell culture medium having a
total amino acid concentration greater than about 50 mM. C24. The
medium of C23, wherein the medium comprises a total glycine
concentration of between about 1.5 mM and about 60.0 mM. C25. The
medium of C24, wherein the total glycine concentration is between
about 5.0 mM and about 15.0 mM. C26. The medium of C25, wherein the
total glycine concentration is about 7.5 mM. C27. The medium of any
one of C23-26, wherein the medium comprises a total arginine
concentration of between about 1.0 mM and about 30.0 mM. C28. The
medium of C27, wherein the total arginine concentration is between
about 1.0 mM and about 20.0 mM. C29. The medium of C28, wherein the
total arginine concentration is about 4.0 mM. C30. The medium of
any one of C23-C29, wherein the medium comprises a total cysteine
concentration of between about 0.1 mM and about 5.0 mM. C31. The
medium of C30, wherein the total cysteine concentration is between
about 0.1 mM and about 3.5 mM. C32. The medium of C31, wherein the
total cysteine concentration is about 0.4 mM. C33. The medium of
any one of C23-C32, wherein the medium comprises a total serine
concentration of between about 5.0 mM and about 75.0 mM. C34. The
medium of C33, wherein the total serine concentration is between
about 5.0 mM and about 15.0 mM. C35. The medium of C34, wherein the
total serine concentration is about 7.5 mM, or about 10 mM. C36.
The medium of any one of C23-C35, wherein the medium comprises a
total glutamine concentration of between about 1.0 mM and about
30.0 mM. C37. The medium of C36, wherein the total glutamine
concentration is between about 1.0 mM and about 20.0 mM. C38. The
medium of C37, wherein the total glutamine concentration is about
4.0 mM. C39. The medium of any one of C23-C38, wherein the medium
comprises a total concentration of tyrosine of between about 0.1 mM
and about 5.0 mM. C40. The medium of C39, wherein the total
tyrosine concentration is between about 1.0 mM and about 3.5 mM.
C41. The medium of C40, wherein the total tyrosine concentration is
about 2.9 mM or about 3.0 mM. C42. The medium of any one of
C23-C41, wherein the medium comprises a total concentration of
asparagine of between about 5.0 mM and about 50.0 mM. C43. The
medium of C42, wherein the total asparagine concentration is
between about 10.0 mM and about 30.0 mM. C44. The medium of C43,
wherein the total asparagine concentration is about 20.0 mM. C45.
The medium of any one of C23-C41, wherein the medium does not
contain asparagine. C46. The medium of any one of C23-C45, wherein
the medium further comprises a potassium salt. C47. The medium of
C46, wherein the potassium salt is potassium chloride or potassium
sulfate. C48. The medium of C46 or C47, wherein the total
concentration of potassium salt is between about 0.1 g/L and about
25 g/L. C49. The medium of C48, wherein the total potassium salt
concentration is between about 0.2 g/L and about 1.25 g/L. C50. The
medium of C49, wherein the total potassium salt concentration is
about 0.9 g/L. C51. The medium of any one of C23-050, wherein the
medium further comprises a carbon source. C52. The medium of C51,
wherein the carbon sources is selected from the group consisting of
glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose,
raffinose, xylose, and mannose. C53. The medium of C52, wherein the
carbon sources is glucose. C54. The medium of any one of C51-053,
wherein medium comprises a total concentration of the carbon source
of between about 25 g/L and about 100 g/L. C55. The medium of C54,
wherein the total concentration of the carbon source is between
about 25 g/L and about 80 g/L. C56. The medium of C55, wherein the
total concentration of the carbon source is about 50 g/L. C57. The
medium of any one of C23-056, wherein the medium further comprises
sodium bicarbonate. C58. The medium of C57, wherein the medium
comprises a concentration of sodium bicarbonate of between about
0.1 g/L and about 20 g/L. C59. The medium of C58, wherein the
concentration of sodium bicarbonate is between about 0.5 g/L and
about 1.0 g/L. C60. The medium of C59, wherein the concentration of
sodium bicarbonate is about 0.84 g/L. C61. The medium of any one of
C23-C60, wherein the medium further comprises a yeast extract. C62.
The medium of C61, wherein the yeast extract is selected from the
group consisting of a yeast autolysate, an ultrafiltered yeast
extract, and a synthetic yeast extract. C63. The medium of C62,
wherein the yeast extract is an ultrafiltered yeast extract. C64.
The medium of C63, wherein the ultrafiltered yeast extract is
AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD
Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE
(Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services
Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry
Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or
HY-YEST 504 (Kerry Group Services Ltd.). C65. The medium of any one
of C61-C64, wherein the concentration of yeast extract is between
about 1 g/L to about 50 g/L. C66. The medium of C65, wherein the
concentration of yeast extract is between about 5 g/L to about 25
g/L. C67. The medium of C66, wherein the concentration of yeast
extract is about 10 g/L. C68. The medium of any one of C23-C67,
wherein the medium comprises at least about 50 mM of amino acids, a
potassium salt, a carbon source, and optionally, a yeast extract.
C69. The medium of C68, wherein the medium comprises at least about
50 mM of amino acids, between about 5.0 mM and about 15.0 mM of
glycine, between about 0.2 g/L and about 1.25 g/L of a potassium
salt, between about 25 g/L and about 80 g/L of a carbon source, and
between about 5 g/L to about 25 g/L of a yeast extract. C70. The
medium of C69, wherein the medium comprises at least about 60 mM of
amino acids, about 7.5 mM of glycine, about 0.9 g/L of potassium
chloride, 50 g/L of glucose, and about 10 g/L of an ultrafiltered
yeast extract. C71. A method of cultivating a
polysaccharide-producing bacteria comprising a) adding a medium of
any one of C1-C70 to a bioreactor, b) seeding the medium with a
polysaccharide-producing bacteria, and c) cultivating the bacteria
by fermentation, wherein said cultivation comprises the addition of
a nutrient at a constant rate to the medium. C72. The cultivation
method of C71, wherein the nutrient is a carbon source. C73. The
cultivation method of C72, wherein the carbon source is glucose.
C74. The cultivation method of any one of C71-C73, wherein the
cultivated bacteria have a cell density of at least 9.0. C75. The
cultivation method of any one of C71-C74, wherein the cultivated
bacteria have a polysaccharide concentration of at least about 250
mg/L. C76. The cultivation method of any one of C71-C75, wherein
the polysaccharide-producing bacteria is selected from the group
consisting of Streptococcus agalactiae, Streptococcus pneumoniae,
Staphylococcus aureus, Neisseria meningitidis, Escherichia coli,
Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae,
Enterococcus faecium, and Enterococcus faecalis. C77. A method of
cultivating a polysaccharide-producing bacteria comprising a)
adding a medium of any one of C1-C70 to a bioreactor, b) seeding
the medium with a polysaccharide-producing bacteria, and c)
cultivating the bacteria by perfusion, wherein the cultivation
comprises (i) removing spent medium from the culture, (ii) adding
fresh medium, and (iii) retaining the bacteria. C78. The
cultivation method of C77, wherein the rate of perfusion is between
about 0.07 VVH to about 2.00 VVH. C79. The cultivation method of
C78, wherein the rate of perfusion is between about 0.67 VVH to
about 1.33 VVH. C80. The cultivation method of C79, wherein the
rate of perfusion is about 1.20 VVH. C81. The cultivation method of
C77, wherein the rate of perfusion is varied. C82. The cultivation
method of C81, wherein the perfusion starts at a first rate and the
rate is increased to a second rate. C83. The cultivation method of
C81, wherein the perfusion starts at a first rate and the rate is
decreased to a second rate. C84. The cultivation method of any one
of C77-C83, wherein the duration of perfusion is between about 1
hour and about 15 hours. C85. The cultivation method of C84,
wherein the duration of perfusion is between about 1 hour and about
10 hours. C86. The cultivation method of C85, wherein the duration
of perfusion is about 7 hours. C87. The cultivation method of any
one of C77-C86, wherein the cell growth of the cultivated bacteria
is at least 2-fold greater than the cell growth in a batch
fermentation system. C88. The cultivation method of any one of
C77-C87, wherein the cultivated bacteria have reached a cell
density of at least 20.0. C89. The cultivation method of any one of
C77-C88, wherein the cultivated bacteria have reached a
polysaccharide concentration of at least about 600 mg/L. C90. The
cultivation method of any one of C77-C89, wherein wherein the
polysaccharide-producing bacteria is selected from the group
consisting of Streptococcus agalactiae, Streptococcus pneumoniae,
Staphylococcus aureus, Neisseria meningitidis, Escherichia coli,
Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae,
Enterococcus faecium, and Enterococcus faecalis.
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