Hydrophobic adsorbents and their use for the adsorption of lipoproteins

Adsorbents composed of a polysaccharide support which is suitable for chromatography and is preferably based on agarose which has undergone a chemical reaction with the glycidyl ethers of nonionic polyoxyethylene detergents of the type HO--(CH.sub.2 CH.sub.2 O).sub.n --O--R to give a compound of the formula I ##STR1## where n is an integer from 2 to 30 and PA1 R is an alkyl radical having 4-20 or a phenyl radical or a phenylalkyl radical, where the alkyl radical has 1-16 carbon atoms, to a process for their preparation and to their use for removing lipoproteins from human or animal body fluids are described.

The invention relates to adsorbents composed of a polysaccharide support 
which is suitable for chromatography, and is preferably based on agarose 
which is subjected to a chemical reaction with the glycidyl ethers of 
nonionic polyoxyethylene detergents of the type HO--(CH.sub.2 CH.sub.2 
O).sub.n --O--R, to a process for their preparation and to their use for 
removing lipoproteins from human or animal body fluids. 
Storage of body fluids such as, for example, serum, plasma, cerebrospinal 
fluid, pleural exudate or ascites of human or animal origin may result in 
deposits or turbidities which considerably impair the quality of these 
products. The lipoprotein macromolecules occurring in body fluids are 
regarded as partly responsible for these instabilities, and they display a 
tendency to aggregation per se because of their content of phospholipids 
which are insoluble in water. 
Removal of the lipoproteins from the abovementioned fluids as a rule leads 
to good storage stability. 
Furthermore, a number of medical diagnostic tests are susceptible to 
interference on use of patients' samples with a high lipoprotein content. 
The presence of lipoproteins may also impede or prevent the processing of 
biological materials to pharmaceuticals or diagnostic test components. 
There has been no lack of attempts in the past to develop methods suitable 
for removing lipoproteins, especially from sera or plasmas. However, these 
known methods have disadvantages which restrict or render impossible their 
industrial use: 
a) Extraction with liquid halohydrocarbons (for example Lipoclean.RTM.) is 
objectionable for environmental-protection reasons. 
b) Absorption with dextran sulfate is not quantitative for all 
lipoproteins. 
c) Octyl- or phenyl-Sepharose.RTM. has a low capacity and can be 
regenerated only in an elaborate procedure. 
d) The method described in EP 137221 using a polyhydroxymethylene 
derivative is suitable for the process in chromatography columns only with 
restrictions; the material can be regenerated only with difficulty. 
e) Methods which operate with silicon dioxide adsorbents are unsuitable for 
plasma because coagulant proteins such as, for example, fibrinogen are 
also bound. 
f) The method of flotation of lipoproteins in an ultracentrifuge requires 
very costly equipment and provides only a low throughput. 
The aim of the present invention was to prepare an adsorbent which permits 
the lipoproteins to be removed quantitatively from untreated biological 
fluids with a high throughput. It was moreover intended that the adsorbent 
be easy to regenerate and display high and reproducible capacities. 
It was intended that it be possible to obtain, without damage, the 
lipoproteins desorbed from the adsorbent in the regeneration process so 
that it would be possible to use them as raw materials for producing 
diagnostic tests or therapeutic products. 
The present invention relates to adsorbents for the removal of lipoproteins 
from human or animal body fluids, composed of a polysaccharide support 
which is suitable for chromatography and is preferably based on agarose 
which has undergone a chemical reaction with the glycidyl ethers of 
nonionic polyoxyethylene detergents of the type HO--(CH.sub.2 CH.sub.2 
O).sub.n --O--R. 
Adsorbents of this type can be represented in particular by the formula I 
##STR2## 
where n is an integer from 2 to 30, preferably 6 to 20, and 
R is an alkyl radical having 4-20, preferably having 10-16, carbon atoms or 
a phenyl radical or a phenylalkyl radical, where the alkyl radical has 
1-16 carbon atoms and is preferably tert-octyl. 
The support polysaccharide is preferably a macroporous agarose gel with an 
agarose content of 1-6%. It is advantageous to use Sepharose.RTM. of the 
type CL-2B. 
Examples of suitable polyoxyethylene detergents are known under the 
proprietary names Brij.RTM., Genapol.RTM., Nonidet.RTM., Tergitol.RTM. or 
Triton.RTM.. These proprietary products are not homogeneous and comprise a 
mixture of ethers in which the polyoxyethylene chain length differs. The 
manufacturers indicate in each case the main component or a chain-length 
range. The structure and composition of polyoxyethylene detergents of 
these types is described in Methods in Enzymology, LVI, 734-749. 
The glycidyl ethers of the detergents have the formula II 
##STR3## 
The invention also relates to a process for the preparation of the 
adsorbents according to the invention. 
The reaction of alcohols or phenols with epichlorohydrin to give the alkyl 
or phenyl glycidyl ethers is described in the Journal of Chromatography, 
101 (1974) 281-284. This process can be used to prepare the glycidyl 
ethers of the nonionic detergents described above. This entails the 
nonionic detergent being heated with an epihalohydrin such as 
epichlorohydrin in the presence of a Lewis acid such as BF.sub.3 etherate, 
and subsequently reacted in the presence of NaOH at room temperature to 
give the glycidyl ether derivative. The reaction scheme is generally 
described below (HO--R.sup.1 =detergent): 
##STR4## 
The process for alkyl or phenyl glycidyl ethers which is described in 
Journal of Chromatography, 101 (1974) 281-288 can be used for the reaction 
of the glycidyl ethers of the nonionic detergents with a polysaccharide. 
This entails the dehydrated polysaccharide being suspended in an anhydrous 
inert solvent, for example dioxane, and reacted with the glycidyl ether of 
a nonionic detergent in the presence of BF.sub.3 etherate: 
##STR5## 
The polysaccharide derivative (adsorbent) according to the invention is 
transferred into an aqueous medium and is ready for use. 
It is advantageous to pack the adsorbents according to the invention in a 
chromatography column or in a cartridge for the adsorption of lipoproteins 
from biological fluids. 
However, it is also possible to carry out the adsorption of the 
lipoproteins by direct addition of the adsorbents to biological fluids. 
The adsorbent can also be added in dry form for this purpose. 
After the biological fluids have been contacted with an adsorbent according 
to the invention in one of the abovementioned methods, they are obtained 
as clear lipoprotein-free fluids. 
After adsorption onto an adsorbent, the lipoproteins can be eluted with 
solutions of substances which split hydrophobic linkages. Examples of 
these are solutions of ionic or nonionic detergents, of urea, KSCN or 
guanidine. HCl or solutions of alcohols such as ethylene glycol. 
The substances preferably chosen are those which cause no denaturation of 
the lipoproteins. Rapid and quantitative elution is achieved on use of a 
solution of the nonionic detergent whose glycidyl ether was employed for 
the derivatization of the polysaccharide.

EXAMPLES 
1. Preparation of the glycidyl ethers of Genapol.RTM. T250 
10 g of Genapol.RTM. T250 were dissolved in 40 ml of anhydrous dioxane, and 
1 ml of BF.sub.3 etherate was added. The mixture was heated to 50.degree. 
C. in a round-bottom flask with drying tube attachment and magnetic 
stirrer. 2 ml of epichlorohydrin were added, and the mixture was stirred 
at 50.degree. C. for 2 h. 
The mixture was subsequently cooled to room temperature, and 4 g of NaOH 
pellets were added. The mixture was stirred at room temperature for 1.5 h. 
After centrifugation of the mixture in a bench centrifuge it was possible 
to obtain the solution of the glycidyl ether of Genapol.RTM. T250 in 
dioxane as the supernatant. 
2. Dehydration of Sepharose.RTM. CL-2B 
150 ml of an aqueous suspension of Sepharose.RTM. CL-2B were packed in a 
glass chromatography column and subsequently washed with the following 
solutions under gravity: 
a) 1 column volume of water 
b) then 1 column volume of 25% ethanol in water 
c) then 1 column volume of 50% ethanol in water 
d) then 1 column volume of 96% ethanol in water 
e) finally 2 column volumes of anhydrous dioxane. 
The dehydrated Sepharose.RTM. CL-2B from the column was placed in a vessel 
and stored with exclusion of moisture until used. 
3. Coupling of the glycidyl ether of Triton.RTM. X-100 to Sepharose.RTM. 
CL-2B 
100 g of Sepharose.RTM. dehydrated as in Example 2 were suspended in 100 ml 
of anhydrous dioxane in a closed stirring vessel and 2 ml of BF.sub.3 
etherate and 10 ml of a 20% strength solution of the glycidyl ether of 
Triton.RTM. X-100 in dioxane were added, and the mixture was slowly 
stirred at room temperature for 2 h. The derivatized gel was then packed 
into a glass chromatography column and washed with the following 
solutions: 
a) one column volume of dioxane one column volume of 96% ethanol in water 
one column volume of 50% ethanol in water one column volume of 25% ethanol 
in water. 
The derivatized gel was stored in 25% ethanol at 4.degree. C. until used. 
4. Adsorption of the lipoproteins from human citrated plasma 
A chromatography column (4.times.30 cm) was packed with 120 ml of a 
Triton.RTM. X-100 derivative of Sepharose.RTM. CL-2B and equilibrated with 
a buffer composed of 0.9% NaCl, 10 mM sodium citrate, pH 7.0, called 
buffer A hereinafter. 
The column was charged with 360 ml of a fresh human citrated plasma at a 
flow rate of 6 ml/min. It was then washed with buffer A. 
Fractions with maximum absorption were collected. It was possible to obtain 
320 ml of a clear human plasma in which neither cholesterol (Monotest.RTM. 
cholesterol, Boehringer: &lt;2.5 mg/dl) nor apolipoprotein A-I and 
apolipoprotein B (nephelometric tests, Behringwerke AG: ApoAl &lt;5 mg/dl; 
ApoB &lt;7.3 mg/dl) were detectable. 
5. Delipidation of rabbit serum 
400 ml of rabbit serum were passed through a column as described in Example 
4, maintaining a flow rate of 5 ml/min. Fractions with maximum protein 
concentration were collected. The column was then washed with buffer A. It 
was possible to obtain 360 ml of a clear rabbit serum in which cholesterol 
was no longer detectable (&lt;2.5 mg/dl). 
6. Elution of the lipoproteins 
A column as described in Example 4 and loaded with human lipoproteins was 
washed with buffer A until the eluate reached the base line absorption. 
The lipoproteins were then eluted from the column in a buffer composed of 
buffer A with 1% Triton.RTM. X-100. Fractions with maximum absorption were 
collected. It was possible to obtain a lipoprotein concentrate which 
contained about 80% of the loaded lipoproteins in a concentration which 
was about twice that in the loaded plasma.