Process for removing bacterial endotoxin from gram-negative polysaccharides

A process for removing endotoxin from Gram-negative polysaccharides such as H. influenzae polyribosylribitol phosphate by adding alcohol incrementally until substantially all lipopolysaccharide precipitates.

BACKGROUND OF THE INVENTION 
The invention is a process for removing bacterial endotoxin from gram 
negative polysaccharides. 
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 with 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 phenomena. 
U.S. Pat. No. 4,695,624 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 than 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 
(Parental 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., Proceedings of the 
Society for Experimental Biology and Medicine, Vol. 149, pp. 766-770 
(1975), describe endotoxin binding by charged and uncharged resins. 
It is a purpose of the present invention to provide an effective, accurate 
method for obtaining Gram-negative polysaccharide mixtures having low or 
negligable levels of endotoxin. 
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 ethanol 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 counterion solution; and 
(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. 
Preferably, the initial addition of alcohol, and temperature after cooling 
in step (d), result 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 corresponds 
to the percentage of alcohol present 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 the 
precipitation of most of the endotoxin with some polysaccharide and a 
negligible amount of endotoxin remaining in solution. 
The counter ion is preferably divalent, although a monovalent counter ion 
may be used. 
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. 
The following abbreviations are used in the description of the present 
invention: 
PRP - polyribosylribitol phosphate, an 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 
The process of the present invention removes impurities such as lipids, 
lipopolysaccharides, proteins and nucleic acids by selective ethanol 
fractionation from fermentation products such as those including 
Gram-negative bacteria polysaccharides. 
Polysaccharide solutions from which endotoxin is removed in accordance with 
the present invention may be those having 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). 
In one embodiment of the present invention, polyribosylribitol phosphate, 
shown below, in the protonated form, 
##STR1## 
is prepared having low or negligable amounts of endotoxin. 
Polyribosylribotol phosphate is a polysaccharide useful for preparation of 
protein-polysaccharide conjugates such as those described in Marburg et 
al., U.S. Pat. No. 4,695,624. 
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. Cells 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 precipitate is 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. 
Incremental alcohol addition 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. When the cloud point 
is reached, lipopolysaccharide and polyribosylribitol phosphate begin to 
precipitate. Lipopolysaccharide is precipitated along with some 
polysaccharide, leaving polysaccharide in solution essentially 
unaccompanied by lipopolysaccharide. The low ethanol precipitate, which 
contains large quantities of lipopolysaccharides, is removed by 
centrifugation and discarded. Additional ethanol is added and the 
polysaccharide precipitate is collected by centrifugation. By following 
the procedure of the present invention, most lipopolysaccharide can be 
removed from solution before experiencing intolerable losses of 
polysaccharide. The resulting product is then suitable for efficient 
preparation of protein-polysaccharide conjugates. 
Protein-polysaccharide conjugates useful for vaccination of patients 
against infections such as those caused by H. influenzae type b bacterium 
may be prepared using the process of the invention. 
Endotoxin reduction resulting from the process of the invention is 
typically 30-100 fold between starting and final powder. 
Polyribosylribitol phosphate yield is typically at least 35% of the level 
in the starting material. 
EXAMPLE 
H. Influenzae Polysaccharide Isolation Process With Selective Ethanol 
Fractionation 
A schematic representation of the process followed in this example is shown 
FIG. 1. 
H. influenzae type b was grown in an 800 L fermentor (640 L working 
volume). A sample for culture purity was obtained and the culture 
transferred to a kill tank where it was treated with Thimerosal. At the 
completion of the kill cycle (10 hours at 37.degree. C.), the temperature 
was reduced and the broth held until released by the culture viability 
test (30 hours). The inactivated whole broth was then transferred out of 
the containment area, the cells and other debris removed by Sharples 
centrifugation, and the clarified broth stored at 2.degree.-8.degree. C. 
Since the PRP was released into the culture media, the collected cells 
were discarded after weighing. The dilute cell-free broth is concentrated 
and a first ethanol fractionation is performed to remove contaminating 
protein, nucleic acid and endotoxin. A second ethanol fractionation is 
then performed to further purify the concentrated broth, followed by a 
series of phenol extractions to remove residual protein, endotoxin and 
pigments. These fractionations and extractions result in material which 
contains undesirable amounts of endotoxin. 
In the selective ethanol fractionation step, the lipopolysaccharide was 
precipitated as alcohol concentration increased, along with some 
polysaccharide, leaving polysaccharide in solution which was lower in 
lipopolysaccharide level. Preciptate containing substantial quantities of 
lipopolysaccharide with polysaccharide is known as the "low-cut". 
Thus, the solution from which endotoxin was to be removed was cooled and a 
salt such as CaCl.sub.2 or NaCl was added. Chilled denatured alcohol was 
added to achieve a concentration slightly below (about 0.5-1.0% below) the 
cloud point (see Graph 1). Sequential addition thereafter of about 0.2% 
alcohol at a time was performed until a two-fold increase in turbidity 
occurred, at which point the cloud point was reached. 
Products obtained from Tests a, c, d, and e in Table 1 show dramatic 
reductions of endotoxin levels following the process of the invention. 
Reduction of endotoxin level is measured by measuring limulus ameobocyte 
lysate (LAL) test values. The test is described in "Guideline on 
validation of the LAL test as an end product endotoxin test for human and 
animal parenteral drugs, biological products, and medicinal devices". U.S. 
Department of Health and Human Services, December 1987. Product from 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 
the product from Test f. 
TABLE 1 
______________________________________ 
Endotoxin Level Reduction 
By Selective Ethanol Fractionation (Endotoxin Units/mcg) 
Test 
Process Stage a b c d e f 
______________________________________ 
Pre-phenol Powder 
750 650 530 600 780 -- 
Post-phenol Powder 
45 140 60 60 135 -- 
Low cut Powder 
30 600 340 30 300 -- 
Post Selective Ethanol 
1.5 0.9 1.4 0.4 2.8 0.09 
Fractionation Powder 
______________________________________ 
To accomplish the selective ethanol 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 
achieve a level of 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 began 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. Historical data was used to decide how much additional alcohol should 
be added after the cloud point was reached in order to reduce the 
lipopolysaccharide to meet the specification. 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. 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 using 1.2 to 2% above 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 
product from test e, 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. 
The polysaccharide product resulting from the endotoxin removal procedure 
of the invention is especially useful where endotoxin-free polysaccharide 
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.