Process for removing bacterial endotoxin from gram-negative polysaccharides

A process for removing endotoxin from Gram-negative polysaccharides such as polyribosylribitol phosphate by solubilizing polysaccharide-containing powder derived from Gram-negative bacteria fermentation broth to provide a divalent counter ion for endotoxin and adding alcohol incrementally to induce lipopolysaccharide precipition, and mixing material resulting from the alcohol addition with a nonionic resin, a detergent and a chelating agent.

BACKGROUND OF THE INVENTION 
The invention is a process for removing bacterial endotoxin from 
Gram-negative polysaccharides without incurring substantial loss of 
polysaccharide. 
Bacterial endotoxin is a potent pyrogen that can often produce fever 
reactions when administered to patients. Endotoxin is an integral 
component of the outer cell surface of Gram-negative bacteria. It exists 
in its natural state as a complex of lipid, carbohydrate and protein. When 
highly purified, endotoxin does not contain protein, and by its chemical 
composition is referred to as a lipopolysaccharide (see Weary and Pearson, 
Bio. Pharm. April (1988) pp. 22-29). 
The outer-wall layer of Gram-negative bacteria serves as an outer barrier 
through which materials must penetrate if they are to reach the cell. It 
is selectively permeable. Generally, endotoxin is released in large 
amounts only when the cell wall is lysed. 
Removal of contaminating endotoxin from Gram-negative polysaccharides is 
important when the polysaccharide is to be administered to humans. 
Endotoxins in large quantities can cause shock, severe diarrhea, fever and 
leukopenia followed by leukocytosis, and can elicit the Shwartzman and 
Sanarelli-Shwartzman and phenomena. 
U.S. Pat. No. 4,695,624, issued to Marburg et al., describes 
covalently-modified polyanionic bacterial polysaccharides, stable covalent 
conjugates of these polysaccharides with immunogenic proteins, and methods 
of preparing the polysaccharides and conjugates and of confirming 
covalency. The patent describes purification of the polysaccharide in 
Example 1, beginning in column 14. After fermentation, inactivation and 
cell removal, the resulting product undergoes a series of cold ethanol 
fractionations. Following phenol extraction are diafiltration, ethanol 
precipitation, ultracentrifugation in ethanol, and collection of the 
finished product. 
Frequently, the amount of contaminating endotoxin remaining after the 
above-described procedure is higher then desired. 
Methods for removing endotoxin which are known in the art are described by 
Weary and Pearson (ibid): rinsing with nonpyrogenic solution (Feldstine et 
al., J. Parenter. Drug Assoc., 33, p. 125 (1979) and Berman et al., J. 
Parenter. Sci. Technol., 41, p. 158 (1987); distillation; ultrafiltration 
using membranes rated by molecular weight exclusion (Sweadner et al., 
Appl. Environ. Microbiol., 34, p. 382 (1977) and Henderson et al., Kidney 
Int., 14, p. 522 (1978); reverse osmosis using thin cellulose acetate or 
polyamide materials (Nelson, Pharm. Technol., 2, p. 46 (1978); 
electrostatic attraction (Gerba et al., Pharm Technol., 4, p. 83 (1980) 
and Hou et al., Appl. Environ. Microbiol., 40, p. 892 (1980); hydrophobic 
attraction using aliphatic polymers (Robinson et al., in Depyrogenation 
(Parenteral Drug Association, Philadelphia (1985), pp. 54-69); adsorption 
using activated carbon (Berger et al., Adv. Chem. Ser., 16, p. 169 (1956), 
Gemmell et al., Pharm J., 154, p. 126 (1945), and Brindle et al., Pharm. 
J., 157, p. 85 (1946); and affinity chromatography (Soter, Bio/Technology, 
12, p. 1035 (1984). 
Sawada et al., Applied and Environmental Microbiology, April 1986, pp. 
813-820, describe removal of endotoxin from water by microfiltration 
through a microporous polyethylene hollow-fiber membrane. Gerba et al., 
Applied and Environmental Microbiology, December 1985, pp. 1375-1377, 
describe endotoxin removal from various solutions using charged nylon and 
cellulose-diatomaceous earth filters. Nolan et al., Proceeding of the 
Society for Experimental Biology and Medicine, vol. 149, pp. 766-770 
(1975), describe endotoxin binding by charged and uncharged resins. 
Sweadner, K. et al., Applied and Environmental Microbiology, Vol. 34, pp. 
382-385 (1977) explain that lipopolysaccharide often exist in an 
aggregated state, and that dissociating the lipopolysaccharide with 
detergent or chelating agents can facilitate its removal from aqueous 
solutions by filtration. Shands, J. et al., J. Biological Chemistry, Vol. 
255, pp. 1221-1226 (1980), show that lipopolysaccharide is associated with 
divalent cations, and that dispersion of Gram-negative lipopolysaccharides 
can be achieved using deoxycholate. 
McIntire, et al, Biochemistry, Vol. 8, No. 10, pp. 4063-4066 (1969) 
describes reversible inactivation, by sodium deoxycholate, of Escherichia 
coli lipopolysaccharide. Ribi, et al., Journal of Bacteriology, Vol. 92, 
No. 5, pp. 1493-1509 (1966) described physical and biological properties 
of endotoxin treated with sodium deoxycholate. 
It is a purpose of the present invention to provide an effective method of 
obtaining Gram-negative polysaccharide mixtures having low or negligable 
levels of endotoxin, without suffering substantial loss of polysaccharide. 
It is also a purpose of the present invention to provide a process for 
eliminating endotoxin from a solution of bacterial polysaccharide while 
minimizing the removal of bacterial polysaccharides and other desired 
species. 
SUMMARY OF THE INVENTION 
The invention is a process for removing endotoxin from Gram-negative 
polysaccharides such as polyribosylribitol phosphate (PRP) which 
comprises: 
(a) growing Gram-negative bacteria in fermentation broth, releasing 
polysaccharide into the broth, and adding alcohol to the broth to remove 
impurities by precipitation; 
(b) isolating the remaining high molecular weight species and 
resolubilizing them in phenol and extracting other impurities; 
(c) centrifuging remaining high molecular weight species and resolubilizing 
in a counter ion solution; 
(d) adding alcohol to the solution, cooling the solution and thereafter 
incrementally adding alcohol to achieve lipopolysaccharide precipitation 
and lipopolysaccharide/polysaccharide precipitation by selective alcohol 
fractionation; and 
(e) mixing lipopolysaccharide-and polysaccharide-containing material 
resulting from the alcohol addition with a nonionic resin, a detergent and 
a chelating agent, to remove lipopolysaccharide by resin elimination. 
Preferably, the initial addition of alcohol and the temperature after 
cooling in step (d) results in an alcohol concentration which is up to 2%, 
preferably between 0.5-1% below the alcohol concentration at the cloud 
point. Incremental alcohol addition is preferably a sequential addition of 
about 0.2% at a time until a two-fold increase in turbidity occurs, at 
which time the cloud point has been reached. The cloud point is the 
percentage of alcohol when endotoxin and polysaccharide start to 
precipitate, causing turbidity. After the cloud point has been reached, an 
additional amount of alcohol is added which results in precipitation of 
most of the endotoxin with some polysaccharide. 
The counter ion is preferably divalent, but may be monovalent. 
Various alcohols may be successfully used during endotoxin removal. 
Suitable alcohols include denatured ethanol (SDA3A, which is 4.7% MeOH, 
88.1% EtOH, 7.2% H.sub.2 O), 95% EtOH, absolute EtOH, isopropanol, and 
other alcohols having 1 to 4 carbons which precipitate endotoxin. 
Material mixed with resin, detergent and chelating agent may be powder 
derived from the operation in step (d). Such powder is obtained by drying 
the precipitate resulting from step (d). It is comprised of polysaccharide 
and lipopolysaccharide. 
Material mixed with resin, detergent and chelating agent may also be a 
solution obtained from the operation in step (d) which comprises 
polysaccharide and lipopolysaccharide. 
Subjecting these materials to treatment with resin, detergent and chelating 
agent removes substantially all lipopolysaccharide and improves the 
overall yield of purified polysaccharide which would otherwise be obtained 
using only incremental alcohol fractionation. Furthermore, use of the 
resin elimination methodology allows for manipulation of the amounts of 
non-polysaccharide and nonlipopolysaccharide species in the finished 
product, while achieving endotoxin removal. 
After treatment of the material in step (e), the resin is removed and the 
polysaccharide precipitated from solution with alcohol. The precipitate is 
centrifuged, the pellet triturated with alcohol and the resulting product 
dried to form a powder. 
Removal of lipopolysaccharides by the process of selective alcohol 
fractionation in combination with resin elimination is useful for 
manipulating the amounts of materials other than polysaccharides and 
lipopolysaccharides in the final product. Selective alcohol fractionation 
removes materials primarily on the basis of molecular weight. Increasing 
concentrations of alcohol result in elimination of species of decreasing 
molecular weight. Removal of lipopolysaccharides using resin depends on 
the ability of the resin to recognize lipopolysaccharide structures and 
eliminate species of that nature from solution. Therefore, the process of 
the present invention is particularly advantageous for minimizing 
undesirable eliminations of components other than polysaccharides and 
lipopolysaccharides. Amounts of components other than polysaccharides and 
lipoplysacchardes can be regulated by utilizing either the selective 
alcohol fractionation or resin elimination to a greater extent while 
obtaining the desirable result of essentially eliminating endotoxin from 
the final product. 
The following abbreviations are used in the description of the present 
invention: 
PRP--polyribosylribitol phosphate, and H. influenzae type b capsular 
polysaccharide. 
LAL test value--limulus ameobocyte lysate test value, which is an 
indication of endotoxin level in the end-product. 
LPS--lipopolysaccharide, which is the general structure of endotoxin when 
it is apart from the outer cell surface of Gram-negative bacteria. 
EU/mcg--Endotoxin units (a measure of LPS) per microgram PRP.

DETAILED DESCRIPTION OF THE INVENTION 
In one embodiment of the invention, a production fermenter containing 
complete Haemophilus medium with an antifoaming agent is inoculated with 
the seed culture. The fermenter is maintained at 37.degree..+-.3.degree. 
C. for a minimum of twelve hours with moderate aeration and agitation. The 
H. influenzae type b culture is inactivated after the fermentation is 
completed by addition of thimerosal under agitation. Cell debris, media 
components and other impurities are removed by centrifugation or 
filtration and discarded. The culture supernatant is concentrated by 
ultrafiltration and additional impurities are removed by alcohol 
fractionation. 
The high molecular weight species are dissolved in calcium chloride 
solution and a minimum of one additional alcohol fractionation is 
completed as described above to remove additional impurities. The second 
alcohol precipitate is collected by centrifugation and a dry powder is 
obtained by resuspending the precipitate in absolute ethanol followed by 
filtration, acetone wash and drying. 
The powder is dissolved in sodium acetate solution and extracted several 
times with phenol to remove impurities. The aqueous solution containing 
polysaccharide is diafiltered with water to remove phenol. Calcium 
chloride solution is added to the solution and high molecular weight 
species are precipitated with alcohol and collected by centrifugation. The 
post phenol powder is resolubilized in calcium chloride solution and is 
then subjected to selective alcohol fractionation. 
Selective alcohol fractionation is an effective process for reducing the 
level of endotoxin to the point where it meets product specification, 
while minimizing the loss of polysaccharide from solution. By changing the 
alcohol concentration, different molecular weight species become insoluble 
and precipitate out of solution. Increasing alcohol concentration 
precipitates species of decreasing molecular weight. Ethanol is 
incrementally added, thereby increasing ethanol concentration towards the 
cloud point. When the cloud point is reached, polysaccharide and LPS 
precipitate. Because it can be advantageous to recover the polysaccharide 
precipitating with the lipopolysaccharide, the combination is dried to a 
"low cut" powder and later treated by the resin elimination method. 
Lipopolysaccharide is precipitated along with some polysaccharide, leaving 
polysaccharide in solution essentially unaccompanied by 
lipopolysaccharide. 
The precipitating material which contains lipopolysaccharides, 
polysaccharides and other species, such as proteins and lipids, is 
combined with a resin, a detergent and a chelating agent to remove 
polysaccharide. The material is first combined with the detergent and 
chelating agent under basic pH, and resin beads are then added to and 
mixed with the solution in an orbital shaker for several hours below room 
temperature. The beads are then removed from solution, and the filtrate is 
diafiltered using hollow fiber membranes to remove detergent and chelating 
agent. Retentate is recovered and calcium chloride added. The 
polysaccharide is precipitated from solution with alcohol. The precipitate 
is centrifuged and the pellet is triturated with alcohol and acetone. The 
resulting product is vacuum dried. This process reduces endotoxin level 
without significant loss of polysaccharide. 
The process of the present invention, therefore, accomplishes removal of 
impurities such as lipopolysaccharides from fermentation products of 
Gram-negative bacteria by selective alcohol fractionation followed by 
treatment with resin, a detergent and a chelating agent. 
Although sodium citrate is a preferred chelating agent, other chelating 
agents which are capable of acting on divalent calcium ions present in the 
solution, and which are capable of serving as a buffer for maintaining 
basic pH are suitable. Other suitable chelating agents include 
ethylenediaminetetraacetic acids such as disodium 
ethylenediaminetetraacetic acid. Preferably, the amount of chelating agent 
is between about 1% and about 10%, more preferably between about 2% and 
about 7% and even more preferably about 6%. 
Although deoxycholate is a preferred detergent, other detergents which are 
capable of breaking aggregated lipopolysaccharide are suitable. Suitable 
detergents include Triton X-100, CHAPS, sodium dodecyl sulfate, and sodium 
lauryl sulfate. Preferably, the amount of detergent is between about 0.1% 
and about 2.0%, more preferably between about 0.2% and about 1.0% and even 
more preferably about 0.75%. 
Nonionic resins which bind to lipopolysaccharide, which do not bind to 
polysaccharides, and which are useful in the present invention include but 
are not limited to Borate Avidgel (Amicon), Amberlite XAD and Amberchrome 
(Rohm & Haas), Octyl Cellulose (Phoenix Chem.), Silicon C8 (Baker), SP207 
and HP20 (Mitsubishi Chem.). Of these resins, HP20 is preferred because of 
lipopolysaccharide reduction, ease of use, availability, cost, and its 
propensity to avoid binding to polysaccharides. Preferably the resin is 
washed prior to use with pyrogen free water. More preferably, the resin is 
washed prior to use with acid solution, an alkali solution, or a polar 
solvent (e.g. ethanol or methanol) and then with pyrogen free water. 
In a preferred embodiment of the invention, powder obtained by drying the 
precipitate resulting from step (d), which comprises H. influenzae, type b 
bacterium polyribosylribitol phosphate, lipopolysacchride and various 
lipids and proteins, is mixed with HP20 (highly porous styrene and 
divinylbenzene copolymer) resin, sodium citrate, and deoxycholate, under 
suitable conditions. The lipopolysaccharide binds to the resin which is 
thereafter removed. The filtrate is diafiltered, the retentate recovered, 
and polyribosylribitol phosphate precipitated from solution with alcohol. 
The precipitate is centrifuged, the resulting pellet triturated with 
ethanol and acetone, and resulting solution vacuum dried. In the process, 
the detergent breaks the association of the aggregated lipopolysaccharide. 
The chelating agent ties up the divalent calcium ions so the vesicular 
structure of the lipopolysaccharide cannot be maintained, and also serves 
as a buffer to maintain the pH above 8, mainly to prevent detergent 
gelation. The lipopolysaccharide is then able to bind hydrophobically to 
the resin. The PRP, which does not bind to the resin, remains free in 
solution and can be recovered in the filtrate. The membrane diafiltrations 
which follow remove the detergent and chelating agent from the solution, 
and the PRP is then precipitated and dried in a typical manner. 
Polysaccharide solutions from which endotoxin is removed in accordance with 
the present invention may contain any bacterial polysaccharides with acid 
groups, but are not intended to be limited to any particular types. 
Examples of such bacterial polysaccharides include Haemophilus influenzae 
(H. flu) type b polysaccharide; Neisseria meningitidis (meningococcal) 
groups A, B, C, X, Y, W135 and 29E polysaccharides; and Escherichia coli 
K1, K12, K13, K92 and K100 polysaccharides. Particularly preferred 
polysaccharides, however, are those capsular polysaccharides selected from 
the group consisting of H. flu b polysaccharide, such as described in 
Rosenberg et al., J. Biol. Chem., 236, pp. 2845-2849 (1961) and Zamenhof 
et al., J. Biol. Chem., 203, pp. 695-704 (1953). 
H. influenzae type b polyribosylribitol phosphate, shown below, 
##STR1## 
may be prepared for use in protein-polysaccharide conjugates such as those 
described in Marburg et al., U.S. Pat. No. 4,695,624. 
The limulus ameobocyte lysate (LAL) test, described in "Guideline on 
validation on the LAL test as an end-product endotoxin test for human and 
animal parenteral drugs, biological products, and medical devices." U.S. 
Department of Health and Human Services, December 1987, is used to 
determine endotoxin levels. 
EXAMPLE 1 
Endotoxin Removing Using Alcohol Fractionation and Resin Elimination 
A schematic representation of the process followed in this example is shown 
in FIG. 1. 
In the selective ethanol fractionation step, the lipopolysaccharide was 
precipitated as alcohol concentration increased, along with some 
polysaccharide, leaving polysaccharide in solution with reduced 
lipopolysaccharide. Precipitate containing lipopolysaccharide along with 
polysaccharide is known as the "low-cut" or "pre-cut". The pre-cut 
material is subjected to further endotoxin removal using resin, detergent, 
and chelating agent (as described later). 
Incremental addition of alcohol is an effective process for reducing the 
level of endotoxin to the point where it meets product specification, 
while minimizing the loss of polysaccharide from solution. By changing the 
alcohol concentration, different molecular weight species become insoluble 
and precipitate out of solution. Increasing alcohol concentration 
precipitates species of decreasing molecular weight. 
Thus, the solution from which endotoxin is to be removed is cooled and a 
salt such as CaCl.sub.2 or NaCl is added. Chilled alcohol, such as SDA3A, 
is added to achieve a concentration slightly below (about 0.5-1.0%) less 
than the cloud point (see Graph 2). Sequential addition thereafter of 
about 0.2% alcohol at a time is performed until a two-fold increase in 
turbidity occurs, at which point the cloud point has been reached. 
Products obtained from Tests a, c, d, and e in Table 1 show dramatic 
reductions of endotoxin level following the process of the invention. Test 
e, which had an unacceptably high level of endotoxin, was treated a second 
time by selective ethanol fractionation, the results of which are shown in 
test f. 
TABLE 1 
______________________________________ 
Endotoxin Reduction Using 
Selective Alcohol Fractionation 
Test (EU/mcg) 
a b c d e f 
______________________________________ 
Pre-phenol 750 650 530 600 780 -- 
Powder 
Post-phenol 
45 140 60 60 135 -- 
Powder 
Low cut 30 600 340 30 300 -- 
Powder 
Powder After 
1.5 0.9 1.4 0.4 2.8 0.09 
Selective 
Alcohol 
Fractionation 
______________________________________ 
To accomplish the selective alcohol fractionation, the post-phenol powder 
was solubilized at 2.5 g/L in a 0.05M CaCl.sub.2 solution to provide a 
divalent counter ion for both endotoxin and PRP. Alcohol was then added to 
26% (v/v). After the temperature equilibrated to a constant value in the 
2.degree. to 4.degree. C. range, alcohol was added incrementally until the 
PRP begins to precipitate (cloud point), causing turbidity as monitored by 
a turbidity probe. 
Graph 1 is a plot of % alcohol at the cloud point versus the temperature of 
a PRP powder solution. The % alcohol needed to reach the cloud point at 
6.degree. C. was 27.4% but for the 4.degree. C. only 26.7% was required. 
This seemingly small increase corresponded to 700 ml for a 100 L scale 
run. The final powder yield decreased as the difference between Low Cut 
Alcohol percent and Cloud Point percent increased. Graph 2 shows that an 
increase in alcohol content of 1% from the cloud point alcohol 
concentration removed 50% of the PRP. Endotoxin reduction, as measured by 
LAL, was about ten fold. Therefore, alcohol addition of 1% was not 
sufficient to reduce the endotoxin to a level of 3 EU/mcg when the 
starting LAL was greater than 30 EU/mcg. 
After the low cut alcohol was added, the solution was immediately 
centrifuged to remove low cut precipitate. Additional alcohol was added to 
the supernatant to 38% (v/v). The desired precipitate was collected via 
settling and/or centrifugation and dried to the final powder. Typical 
recoveries for this step at 1.2-2.0% above the cloud point were 30-40% of 
the post-phenol powder or 13-18% of the amount from the fermentor. 
The selective alcohol fractionation procedure can be repeated if the final 
powder does not meet the pyrogen specification. For reprocessing, the 
alcohol concentration was increased 0.2% above the low cut alcohol 
percentage. The yield was 78% and the endotoxin level was reduced from 2.8 
to 0.09 EU/mcg. 
Endotoxin Removal Using Resin, Detergent and Chelating Agent 
The pre-cut or low-cut material obtained after performing the selective 
ethanol fractionation step, containing precipitated lipopolysaccharide and 
polyribosylribitol phosphate, was further treated by mixing with HP20 
resin, deoxycholate and sodium citrate. This treatment removes substantial 
quantities of lipopolysaccharide without significantly affecting the level 
of desirable polyribosylribitol phosphate contained in the low cut 
material. The filtrate is diafiltered with a hollow fiber membrane, the 
retentate recovered, and polyribosylribtol phosphate precipitated from 
solution with ethanol. The precipitate is centrifuged and resulting pellet 
triturated with ethanol and acetone. The resulting solution is then vacuum 
dried, see FIG. 2. 
0.5% sodium deoxycholate and 3% sodium citrate were mixed with the 
lipopolysaccharide-polysaccharide mixture at pH 8-9. HP20 resin was added 
at 30 grams resin per gram polysaccharide (the resin was washed prior to 
use with pyrogen free water). The loose beads were mixed with the solution 
on an orbital shaker for 3 hours at 4.degree. C. After mixing, the beads 
are removed from the solution in a stainless steel filter funnel. Filtrate 
is then diafiltered in an Amicon H1P30-20 hollow fiber cartridge 
(0.06M.sup.2 surface area) vs. 5 vol. of 1.5% citrate followed by 10 vol. 
of pyrogen free water, maintaining an estimated polysaccharide 
concentration of .ltoreq.2.5 mg/ml, to remove detergent and chelating 
agent. The retentate is recovered and 2M calcium chloride is added to 
achieve a final calcium chloride concentration of 0.05M. Polysaccharide is 
precipitated from solution with excess 95% ethanol. The precipitate is 
centrifuged at 13,000 x g for 30 minutes, the pellet triturated with 
absolute ethanol and acetone, and then vacuum dried. The final powder is 
transferred to a sample container and frozen at -70.degree. C. 
Material treated with resin showed the following reductions of endotoxin 
levels and polysaccharide levels: 
______________________________________ 
LAL test value EU/mcg 
A B C 
______________________________________ 
initial 100 100 100 
final powder 0.06 0.03 0.06 
______________________________________ 
______________________________________ 
Polysaccharide level (%) 
______________________________________ 
initial 100 100 100 
final powder 90 100 92 
______________________________________ 
EXAMPLES 2, 3, 4, 5, 6, 7 AND 8 
Following the procedure for endotoxin removal described in Example 1, 
maintaining a concentration of sodium deoxycholate of 0.5%, and beginning 
with powder having LAL of 60 EU/mcg, we obtained considerable reduction in 
LPS with these varying amounts of sodium citrate: 
______________________________________ 
% Sodium LAL (EU/mcg) 
Citrate Start Finish 
______________________________________ 
Example 2 2 60 30 
Example 3 3 60 6 
Example 4 4 60 0.6 
Example 5 5 60 0.6 
Example 6 6 60 0.15 
Example 7 7 60 0.6 
Example 8 8 60 0.6 
______________________________________ 
EXAMPLES 9, 10 AND 11 
Following the procedure for endotoxin removal described in Example 1, 
maintaining a concentration of sodium citrate of 6%, and beginning with 
powder having LPS of 60 UE/mcg, we obtained considerable reduction in LPS 
with these varying amounts of deoxycholate: 
______________________________________ 
% Sodium LAL (EU/mcg) 
Deoxycholate Start Finish 
______________________________________ 
Example 9 0.25 60 15 
Example 6 0.5 60 0.15 
Example 10 
0.75 60 0.006 
Example 11 
1.0 60 0.6 
______________________________________ 
EXAMPLES 12, 13, 14, 15 AND 16 
Following the general procedures in Example 1, these examples include 
description or process variations within the invention. 
______________________________________ 
Process Variation 
______________________________________ 
Example 12 After treatment in accordance with 
Example 1, the procedure is 
repeated. 
Example 13 After treatment according to 
Example 3, filtrate is diafiltered 
with 3% sodium citrate, sodium 
deoxycholate is added, and the 
solution treated for three 
additional hours with the original 
HP-20 resin. 
Example 14 After three hours of HP-20 
treatment according to Example 3, 
an equal volume of 3% citrate with 
0.5% sodium deoxycholate was added 
to the mixture and resin treatment 
continued for another three hours. 
Example 15 After three hours according to 
Example 3, sodium citrate with 
0.5% sodium deoxycholate powder 
were added and the resin treatment 
continued for another three hours. 
Example 16 (a) Six percent sodium citrate 
a,b with 0.5% sodium deoxycholate or 
(b) 6% sodium citrate with 1% 
sodium deoxycholate was used for 
three hours. 
______________________________________ 
EXAMPLE 17 
All of the process steps of Example 1 are used, except that the resin is 
packed in a column, rather than mixed in batch with PRP. Thus, the PRP is 
dissolved in a solution of sodium citrate and sodium deoxycholate, and the 
resulting solution is passed through the column. The resulting product is 
similar to that obtained on Example 1. 
EXAMPLE 18 
All of the process steps of Example 1 are repeated, except that the resin 
is packed in a cartridge, rather than mixed in batch with PRP. Thus, the 
PRP is dissolved in a solution of sodium citrate and sodium deoxycholate, 
and the resulting solution is passed through the cartridge. The resulting 
product is similar to that obtained in Example 1. 
The polysaccharide product resulting from the endotoxin removal procedure 
of the invention is especially useful where endotoxin-free polysaccharide 
such as polyribosylribitol phosphate is desirable. It readily conjugates 
to proteins, e.g. immunogenic proteins, such as in the manner described in 
Marburg et al. (ibid). The conjugates are stable polysaccharide-protein 
conjugates, coupled through bigeneric spacers containing a thioether group 
and primary amine, which form hydrolytically-labile covalent bonds with 
the polysaccharide and the protein. Exemplary conjugates are those which 
may be represented by the formulae, Ps-A-E-S-B-Pro or Ps-A'-S-E'-B'-Pro, 
wherein Ps represents a polysaccharide; Pro represents a bacterial 
protein; and A-E-S-B and A'-S-E'-B' constitute bigeneric spaces which 
contain hydrolytically-stable covalent thioether bonds, and which form 
covalent bonds (such as hydrolytically-labile ester or amide bonds) with 
the macromolecules, Pro and Ps. The specific definitions of A,E,S,B,A',E' 
and B' are presented in Marburg et al. the contents of which are hereby 
incorporated by reference. Procedures for preparing polysaccharides and 
proteins for conjugation, performing conjugation, and determining 
conjugation are described in the patent.