Mutual Citation

Chromatography, Vol.23 No.2 (2002)

Mutual Citation

Endotoxin Removal in Some Medicines andHuman Serum Albumin Solution by Affinity Membranes
Wei Gui−lin, Liu Xue−liang*, Li Jing−hua, Liu Ying and Shang Zhen−hua
Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116012, China

Abstract
Three kinds of affinity membranes utilized for endotoxin removal, namely, chitosan affinity membrane (KFCC 517), hydrophobic and cationic charged membrane (KFCG 316) and metal chelate affinity membrane (KFCM 402) were prepared by using cellulose membrane as support. The adsorbability and applications of the membranes in endotoxin removal were investigated. The results showed that all of these affinity membranes could be utilized for endotoxin removal in several kinds of solutions. The factors affecting endotoxin removal such as ionicstrength, pH value and flow rate were investigated, and the results were discussed. Three cartridges of membranes KFCC 517, KFCG 316 andKFCM 402 were utilized for endotoxin removal in human serum albumin solution. The cartridge of membrane KFCC 517 also was utilized in
medicine injections, such as hydrocortisone, puerarin, tetracaine hydrochloride and dextran 40 glucose. Under the optimal conditions, the removal efficiencies of endotoxin were over 80% and the recoveries of desirable substances were over 95%.
Key words: endotoxin, removal, affinity membrane, human serum albumin, medicine

were prepared with cellulose−based filter paper as support. All of

1. Introduction
Endotoxin, the ingredient of the cell wall of gramnegativethese membranes prepared can be utilized for endotoxin removal inbacteria, possesses potential biological toxicity that causes pyro-

solutions, and the removal efficiencies were over 80%. The endo-

genic and shock reactions in mammals when intravenously injected

toxin adsorption capacity of the membrane KFCC 517 is higher

even in a very small amount [1]. Its main toxic moiety is lipid A,

than 2.4×106 EU/g. These affinity membranes can be utilized towhich is composed of a diglucoamine that is highly substituted

treat several kinds of sample solutions.

with amide− and ester−linked long−chain fatty acids and negatively charged phosphate groups [2,3].

2. Experimental

A number of methods, such as decomposition by acids, alkalis

2.1. Material and equipmentor oxidizing agents [4], ultrafiltration and ion exchange [5], have

Cellulose−based filter paper (initial membrane) was purchased

been extensively utilized to remove endotoxin, but these methods

from Hangzhou Xinhua Paper Manufactory (Hangzhou, China).

display lower selectivities to the target components. In the last dec-

Glycidylmethacrylate (GMA), 1−vinylimidazole, N , N , N’ , N’ −te-ade, the adsorbents with immobilized affinity ligands such as his-tramethylethylenediamine and 1,6−dichlorohexane were fromtidine and polymyxin have been investigated for endotoxin re-

Fluka (Buchs, Switzerland). The endotoxin standard sample wasmoval [5−11], but these methods still show some limitations.

from National Institute for the Control of Pharmaceutical and Bio-

This work mainly deals with the preparation, adsorbability

logical Products (Beijing, China). A Tachypleus Amebocyte Lysate

and applications of affinity membranes utilized for endotoxin re-(TAL) test kit for endotoxin determination was from Marine Or-

moval. Three kinds of membranes, namely, chitosan affinity mem-

ganism Product Factory (Zhanjiang, China). Human serum albumin

brane (KFCC 517), hydrophobic and cationic charged membrane(HSA) and all the medicine injections were kindly supplied by The(KFCG 316) and metal chelate affinity membrane (KFCM 402)

First Affiliated Hospital of Dalian Medical University (Dalian,*Corresponding author: e−mail: lxl 99@mail.dlptt.ln.cn.
―７９―Chromatography, Vol.23 No.2 (2002)

with 1000 mL deionized (D.I.) water.

China). All other chemicals were of analytical grade.
A BET−16 bacterial endotoxin test apparatus was from Elec-

Secondly, the oxidized membranes obtained from the last step

tronic Instrument Factory of Tianjin University (Tianjin, China). A

were immersed in a solution of 0.9 g of chitosan in 100 mL of so-

BT 01−100 peristaltic pump was from Lange Constant Flow Pump

dium acetate buffer (pH 6.0). The mixture was placed to react for

Corporation (Baoding, China).

12 h at room temperature with occasional stirring. After that, themembranes were washed with 100 mL of 1% (V/V) acetic acid solution and 1000 mL D.I. water.

2.2. Membrane preparation

Finally, the membranes grafted with chitosan were immersed
in a solution of 0.3 g potassium tetrahydroborate in 100 mL of 0.1

2.2.1. Chitosan affinity membraneFirst, cellulose−based filter paper was cut to round pieces with

mol/L sodium carbonate buffer (pH 9.0) and placed to react for 12

diameter of 47 mm. Then 40 sheets of this round cellulose mem-h at room temperature with occasional stirring. The final membrane

branes were immersed in a solution of 1.5 g of potassium periodate

was taken out from the reaction solution, washed with 1000 mL of

in 100 mL of 10% (V/V) sulfuric acid solution. The mixture ob-

D.I. water, and dried by air. The chitosan affinity membranes ob-

tained was placed to react for 12 h at room temperature with occa-

tained were marked as KFCC 517 (see Table 1).

sional stirring. After that, the oxidized membranes were washed
Table 1 Label and composition of membranes
MembraneMatrix

LigandKFCC 517

cellulose−chitosan

KFCG 316

cellulose−GMA−vinylimidazole−dichlorohexane−tetramethylethyldiamine

KFCM 402

cellulose−GMA−IDA−Zn2+
scribed in section 2.2.2. were immersed in the solution and allowed

2.2.2. Hydrophobic and cationic charged affinity membrane

to stand at 50℃−60℃ for 6 h. Finally, the membranes were taken

For grafting GMA onto start cellulose membrane, the mem-out and washed with 1000 mL of D.I. water and air−dried.

brane was first immersed in 100 mL of D. I. water and heated to 70℃with occasional stirring. Then 8 mL of GMA, 0.4 g of ammo-

The membranes obtained above were loaded into a membranenium persulfate (APS) and 0.6 g of sodium thiosulfate (STS) were

cartridge designed in our laboratory [12]. A volume of 250 mL ofadded successively into the reaction mixture and the reaction was

0.05 mol/L zinc chloride solution was pumped through the mem-allowed to proceed for 1 h. Then the mixture was cooled to room

brane cartridge at a flow rate of 0.5 mL/min. Then the cartridge

temperature. The supernatant was decanted and the membrane was

was washed with 1000 mL of D. I. water. So we obtained the car-washed with 1000 mL of D.I. water and air−dried.

tridge of the membrane KFCM 402 (see Table 1).

After grafted, the membranes were immersed in a solution of
2.3. Endotoxin adsorption

8 mL of 1−vinylimidazole and 100 mL of D.I. water in a reactor.
The reaction was allowed to proceed at 70℃ for 1 h−3 h. After the

Sample solutions containing endotoxin were prepared by put-membranes were taken out and washed with D.I. water, they wereting a certain amount of endotoxin standard into the sample solu-

immersed in 100 mL of D.I. water (70℃）again. Ten mL of N, N,

tions and shaking to make uniformity.

N’, N’ −tetramethylethyl−enediamine and 10 mL of 1, 6−dichloro-

Dynamic test: 20 sheets (about 3 g) of membranes preparedhexane were added into the reactor and the reaction was allowed toabove were packed into a cartridge as described elsewhere [12]. A

proceed overnight with occasional stirring. Then the reactor was

sample solution containing endotoxin was pumped through the car-cooled to room temperature. The supernatant was decanted and thetridge at a flow rate of 2 mL/min at room temperature for endo-

membranes were washed with D.I. water and air−dried. The mem-

toxin removal. The filtrate was collected in fractions and the endo-

branes marked KFCG 316 were obtained (see Table 1).

toxin content of each fraction was determined by the BET−16 apparatus.
Static test: the weighed amounts of membranes were dispersed

2.2.3. Metal chelate affinity membrane
A solution of 100 mL of 0.1 mol/L sodium carbonate (pH 8.0)

into the solutions of endotoxin in sterile tubes at room temperature

containing 4.0 g of iminodiacetic acid (IDA) was heated to 50℃.

while agitating occasionally for 2.5 h. The endotoxin content in the

The cellulose membranes (20 sheets) grafted with GMA as de-supernatant was assayed after the membranes were settled down.

―８０―Chromatography, Vol.23 No.2 (2002)

Wei gui−lin, Liu Xue−liang, Li Jing−hua, Liu Ying and Shang Zhen−hua

Regeneration of the membrane cartridges: the membrane cartridges were regenerated by flushing successively with 100 mL ofdepyrogen water and the following solutions, 20 mL of 0.2 mol/L
sodium hydroxide, 50 mL of 5 g/L deoxycholic sodium, 160 mL of
0.2 mol/L sodium hydroxide, and 250 mL of 1.5 mol/L sodium
chloride, finally 250 mL of depyrogen water.
2.4. Substance assay
Endotoxin content was assayed by a kinetic−turbidimetricmethod. The turbidity produced by reaction of TAL with endotoxin

Figure 1. Endotoxin removal by three type membranes
Endotoxin initial concentration: 1.0 EU/mL; ionicstrength: 0.15 mol/L, pH 6.0; flow rate: 0.5 ml/min.

can be inspected by the BET−16 apparatus at 660 nm. The data obtained were processed by a software automatically. The minimum
detection limit of the BET−16 apparatus was 0.001 EU/mL. The
endotoxin standards were utilized for making a calibration curve.

brane KFCG 316. Compared with the membranes KFCC 517 and

The endotoxin contents of each fraction were obtained by compar-

KFCG 316, the metal chelate affinity membrane KFCM 402 is lessing with the calibration curve.

efficient. This is probably because either chitosan grafted on the

HSA was determined by the absorbance measurement at 280

membrane KFCC 517 or lipid A of endotoxin has the similar struc-nm by a spectrophotometer. According to the Beer’s Law (A=abC),

ture of amino glucose and it causes good biological compatibility

the recovery (C i /C0) was obtained by calculating Ai /A0.

between them. Otherwise, the partially positively charged aminogroup of chitosan may attract the negatively charged phosphate es-

Hydrocortisone was determined by the absorbance measure-ter groups of endotoxin, so the affinity membrane KFCC 517 canment at 242 nm by a spectrophotometer.
Tetracaine hydrochloride was assayed by titration with silver

effectively remove endotoxin. The membrane KFCG 316 also cannitrate solution. Then the recovery of tetracaine hydrochloride wasremove endotoxin because both of its two parts, i.e. hydrophobic

calculated.

and cationic charged parts can interact with the long−chain fatty
acids and negatively charged phosphate groups of endotoxin re-

The recovery of dextran 40 glucose was determined by po-

spectively. However, as a general ligand, the metal chelate affinity

larimetric analysis with an automatic polarimeter.

ligand is often utilized for the separation of many biological mole-

Puerarin content was determined by the absorbance measure-cules, but the membrane KFCM 402 has weak cationic charge andment at 250 nm by a spectrophotometer.

short alkyl chains, so it is not good enough to adsorb much endo3. Results and discussion

toxin.

3.1. Results of endotoxin removal with the affinity membranes

3.1.2. Adsorption capacity

3.1.1. Efficiencies of three types of membranes in endotoxin re-investigated with 2.0 mg of membrane KFCC 517 by static test.

The adsorption of endotoxin onto the affinity membranes was
moval
Three types of membranes were packed into three membrane
cartridges (20 sheets of the membrane for each cartridge), NaCl solution containing endotoxin was prepared by putting 120 EU endotoxin standard into 120 mL normal saline.
Then the solution was pumped through the cartridge at a flow−rate of 0.5 mL/min. The filtrate from the outlet of the cartridge
was collected at 30 min, 90 min, 150 min and 210 min respectively
and 2 mL for each time was collected. The Endotoxin content ofeach fraction was determined by the BET−16 apparatus.
Fig.1 shows that the removal efficiencies of endotoxin in

Figure 2. Equilibrium isotherms of endotoxin onto the membrane
KFCC 517
NaCl solutions, room temperature; ionic strength: 0.15
mol/kg, pH 6.0; adsorption time: 2 h.

NaCl solutions are all over 92% by three kinds of membranes. The
best one is the affinity membrane KFCC 517 modified with chitosan, and the second is the hydrophobic and cationic charged mem-

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Chromatography, Vol.23 No.2 (2002)

The initial solution was prepared by putting different amounts ofso the ionic interaction between the affinity membrane and endo-

endotoxin standard into normal saline. The results are shown in

toxin was reduced. However, on the other hand, the hydrophobic

Fig.2. As the high concentration of endotoxin was not available,interaction could make up the decrease of ionic force since there

the adsorption capacity of the membrane did not reach saturation

was hydrophobic action between the membrane and endotoxin.

and saturated capacity was not calculated owing to the lack of data.

Therefore, the membrane still maintained its adsorption to endo-

However, the adsorption capacity calculated could be higher than

toxin.

6

2.4×10 EU/g according to the content of endotoxin in the superna-

It also can be seen that the removal efficiency of the mem-tant of the original standard with high concentration in this test.

brane KFCC 517 was affected a little more severely than that of the

The value was similar to some other adsorbents reported in litera-membrane KFCG 316. The highest adsorption occurred at pH val-ture [2,9,10].

ues between 6−8. The removal efficiency decreased obviously at
pH 10. This is probably because, after pH value greater than 8, theincrease of the concentration of hydroxide ion inhibited the ioniza-

3.2. Effects of experimental conditions
Affinity interaction mechanism is a complicated process, sotion of the amino group of chitosan. Therefore the cationic charge

the experimental conditions, such as ionic strength, pH value andof the affinity ligand was cut down, and the interaction between theflow rate can all affect the removal efficiency of endotoxin. The ef-

affinity membrane and endotoxin fell down. Thus, the removal ef-

fects of some factors were investigated as follows.

ficiency reduced considerably.

3.2.1. Effect of ionic strength
The effect of ionic strength on the efficiency of endotoxin removal are shown in Fig.3. It can be seen that ionic strength has
only minimal influence on the removal of endotoxin in the range of
0.05 mol/kg−0.6 mol/kg. As endotoxin is negatively charged in addition to hydrophobic character, normally, there are both ionicforce and hydrophobic interaction existed between endotoxin andthe affinity membrane. Such interactions lead to opposite effects
with the change of ionic strength, at the synergic interaction ofionic force and hydrophobic force; the change of the removal effi-

Figure 4. Effect of pH on endotoxin removal
Affinity membrane: 5.0 mg; room temperature; endotoxin initial concentration: 5.0 EU/mL; ionic strength:
1.5 mol/kg: adsorption time: 2 h.

ciency is minimized.

3.2.3. Effect of flow rate
The cartridge with 20 sheets of the membrane KFCG 316 was
utilized to remove endotoxin from endotoxin−containing water(2.24 EU/mL) at different flow rates in dynamic tests. The results
are displayed in Fig.5. They show clearly that the removal efficiency of the cartridge decreases not much with the increase of
Figure 3. Effect of ionic strength on endotoxin removal
Affinity membrane: 5.0 mg; room temperature; endotoxin initial concentration: 5.0 EU/mL; pH 6.0; adsorption time: 2 h.

3.2.2. Effect of pH
The effect of pH values on the efficiency of endotoxin removal are shown in Fig.4. It can be seen that the membrane KFCG
316 is not severely affected by pH value. On the one hand, with the
Figure 5. Effect of flow rate on endotoxin removal

increase of pH the positive charge of the membrane was decreased,―８２―Chromatography, Vol.23 No.2 (2002)

Wei gui−lin, Liu Xue−liang, Li Jing−hua, Liu Ying and Shang Zhen−hua

flow rates.
3.3. Applications of affinity membranes
Endotoxin in medicine injections is harmful to human’s
health. Thus, the membranes prepared were utilized for endotoxin
removal in several medicine injections. The results are shown as
follows.
3.3.1. Endotoxin removal in some medicine injections
The sample solutions were prepared by adding 60 EU of endotoxin standard into 60 mL of the medicine injections andpumped all the solutions through the membrane cartridges. The endotoxin contents in the filtrates from the cartridges were determined.
The results of the membrane KFCC 517 cartridge utilized forendotoxin removal in hydrocortisone, puerarin, tetracaine hydrochloride, dextran 40 glucose injections are shown in Table 2.
Figure 6. Structures of medicines
a. hydrocortisone; b. puerarin; c. tetracaine hydrochloride

Table 2 The results of the membrane KFCC 517 used for endotoxin removal in several medicine injections
Injections

Endotoxin
Endotoxin
Main
concentration
removalcomponent(EU/mL)efficiency (%) recovery (%)

caine hydrochloride were positively charged, the removal efficiencies by both the membrances KFCC 517 and KFCG 316 were increased to 81.15%, and the recovery of tetracaine hydrochloride

sample filtrate

still remained at 100%. The results are shown in Table 3 in detail.

Hydrocortisone

1.0

0.059

94.1

100

Dextran 40 glucose

1.0

0.080

92.0

97.8

Puerarin

1.0

0.031

96.9

97.2

Tetracainehydrochloride

1.0

0.420

58.0

100

Table 3 Effect of pH on endotoxin removal in tetracaine hydrochloride

Membrane

It can be seen from Table 2 that the recoveries of hydrocorti-

pH

Endotoxin concentration (EU/ml) Removal
samplefiltrate

efficiency

61.00.4358

56.42

7

1.0

0.1885

81.15

6

1.0

0.3442

65.58

7

1.0

0.1885

81.15

KFCC 517

sone, puerarin, dextran 40 glucose injections are all over 95% andthe removal efficiencies of endotoxin are over 92%. However, for
KFCG 316

tetracaine hydrochloride injection, the recovery of tetracaine hydrochloride was 100% while endotoxin removal efficiency was only
58%. This is reasonable if we think over the interaction between

3.3.2. Endotoxin removal in HSA solutions

the medicines (see Fig.6) and endotoxin. For hydrocortisone, puerarin and dextran 40 glucose, there is little interaction between them

The membrane cartridges of KFCC 517, KFCG 316 andand endotoxin because there are not so many cationic charges in

KFCM 402 were utilized for endotoxin removal in HSA solutions

their structures. However, for tetracaine hydrochloride it was posi-

and the results are shown in Table 4.

tively charged at pH 6 because there are two amino groups in its

HSA solutions containing endotoxin were prepared by adding

structure so it can react with the negatively charged phosphate

60 EU endotoxin standard into 100 mL HSA solutions with HSA

group of endotoxin molecule. Such interaction disturbs the interac-concentration of 2.0 g/L.

tion between the membrane and endotoxin. Therefore, its effi-

Table 4 shows that the membranes KFCC 517 and KFCG 316

ciency of endotoxin removal was lower than those of other medi-

can remove endotoxin effectively while KFCM 402 only can re-cine injections.

move 83.3% of endotoxin in HSA solutions. By comparing the removal efficiencies of endotoxin in Table 4 and Fig.1, it can be seen

When pH was adjusted from 6 to 7, less amino groups in tetra-―８３―Chromatography, Vol.23 No.2 (2002)

Table 4 Removal efficiencies of Endotoxin and HSA recoveries in
HSA solutions
MembraneRemoval efficiency (%)

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KFCC 517

94.8

99.2

KFCG 316

94.8

98.9

KFCM 402

83.3

98.4

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745.

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4. Conclusion[10] Sakata M, Sueda T, Ihara H, et al, Chem Pharm Bull, 1996,

All three kinds of membranes prepared in this study could be

44, 328.

utilized for endotoxin removal in solutions. The adsorption capacity for the membrane KFCC 517 was higher than 2.4×106 EU/g.

[11] Wakita M, Adackhi T, Ida J, et al, Bull Chem Soc Jpn, 1996,
69, 1017.

The Effects of ionic strength, pH value and flow rate on the effi-[12] Guo W, Shang Z H, Yu Y N, et al, Biomed Chromatogr,

ciency of endotoxin removal were not severe. These affinity mem-

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branes were utilized to treat HSA solutions and some medicine in-[13] Wei G L, Shang Z H, Pan M C, et al, Chin Chem Lett, 2000,

jections such as hydrocortisone, puerarin, dextran 40 glucose and4, 357.

tetracaine hydrochloride. Under the optimal conditions, the removal efficiencies of endotoxin were over 80% and the recoveries[Originally published in SEPU, 2002, Vol. 20, No.2: 108−114]

of target substances were over 95%.

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