Design of a new plasma separation membrane by graft copolymerization

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Pergamon

Radiat. Phys. Chem, Vol. 46, No. 2, pp. 21%223, 1995 Copyright © 1995ElsevierScienceLtd 0969-806X(95)OOO16..X Printed in Great Britain.All rights reserved 0969-806X/95 $9.50+ 0.00

DESIGN OF A NEW PLASMA SEPARATION MEMBRANE BY GRAFT COPOLYMERIZATION M. ONISHI, K. SHIMURA, Y. SEITA and S. YAMASHITA R&D Center, Terumo Corp., lnokuchi 1500, Nakai-machi, Kanagawa 259-01, Japan Abstraet--A new type of hydrophilic membrane for blood plasma separation has been successfully developed by vapor-phase glow discharge-initiated graft copolymerization. After exposing microporous polypropylene (PP) membrane to argon glowdischarge,it was allowed to react with 2-methoxyethylacrylate (MEA) vapor to produce graft polymers. The polyMEA-grafted PP (PP-g-PMEA)membrane has the novel property of not causing hemolysis when blood first comes into contact with it in a dry state. It is thought that hydrophilic microporous membranes in a dry state cause hemolysiswhen they are initially exposed to blood, because plasma immediatelypenetrates into their pores by capillary attraction and erythrocytes are trapped rapidly on the micropores and lysed. Since PP-g-PMEA membranes have a weakly hydrophilic character, plasma penetrates into the micropores only slowly and hemolysis does not occur. Therefore, priming with physiological saline prior to use is not required, and consequently plasma separation procedures are simplified and shortened. A disk-type plasma separator equipped with a PP-g-PMEA membrane had good hemo-compatibility and an excellent separation capacity, enabling high recovery of plasma components. A decreased adsorption of plasma proteins due to the PMEA-grafted layer may be the reason for the performance of the membrane.

INTRODUCTION The collection of sufficient plasma for therapeutic use as fresh frozen plasma and as a source of plasma derivatives, such as albumin, globulin and antihemophilic factor, has been an important issue in many countries. Most plasma is collected by centrifugation. However, plasma collected by this method contains cellular components, such as leucocytes, which may carry various viruses. To overcome this problem, membrane technology is increasingly being applied to the collection of plasma by donor plasmapheresis, a procedure in which plasma is separated from whole blood and the cellular components are reinfused to the donor (Stromberg and Friedman, 1989). In order to completely reject cellular components and to transmit nearly all proteins, we had previously prepared a porous polypropylene (PP) membrane having a 0.45 #m effective pore size, and employed it in the disk-type membrane plasma separator of the constant pressure apheresis system II (CPAS-II) (Takahashi et al., 1992). Microporous PP is a hydrophobic material that has an excellent dimensional stability upon swelling; however, it is non-wettable by water, and thus plasma does not permeate the membrane in a dry state. Therefore, the plasma separator was hydrophilized to be a wet-type module filled with water by an ethanol/water two-step priming method, and it was necessary to replace the water with physiological saline prior to use. Glow discharge generates radicals on polymer substrates which can act as sites for initiating graft copolymerization (Bradley and Fales, 1971; Masuoka

et al., 1989). In a previous study, we demonstrated that the interior of a microporous substrate could be modified by glow discharge-initiated graft copolymerization, and prepared an extremely hydrophilic membrane for blood plasma separation by grafting N,N-dimethylacrylamide onto the PP membrane (Onishi et al., 1992). This membrane exhibited excellent plasma separation capability and hemo-compatibility. However, it is necessary to prime the membrane, or to fill up the microporous spaces inside it with physiological saline prior to use, because hydrophilic membranes in a dry state cause hemolysis when exposed to blood. In the present work, we have prepared a novel hydrophilic membrane that does not cause hemolysis when blood first comes to contact with it in a dry state, in order to eliminate the priming step and prevent the dilution of plasma components with saline. We report the features of this new membrane in this paper and comment on the possible mechanism by which it does not cause hemolysis.

EXPERIMENTAL Membrane preparation

A microporous PP membrane with an effective pore size of 0.45 #m and a thickness of 80 ,urn was prepared by the thermally-induced phase separation method from a mixture of PP, liquid paraffin and a nucleating agent (Seita and Onishi, 1988). The PP membrane was modified by vapor-phase glow discharge-initiated graft copolymerization (Onishi et al., 1992). The monomers for grafting were purified by vacuum 219

220

M. Onishi et al.

distillation and degassed by freeze-thaw cycles under reduced pressure. The apparatus used is schematically illustrated in Fig. 1. PP substrate, supported by a stainless steel mesh, was placed in the reaction vessel, and exposed to argon glow discharge which was excited by a 13.56 MHz radio frequency at a power of 50 W. After glow discharge treatment for 10-15 s at 13 Pa, the pressure in the vessel was immediately reduced to 0.2 Pa, and monomer vapor was introduced to initiate graft copolymerization. The resulting membranes were washed with an azeotropic mixture of dichloromethane and methanol using Soxhlet extracting apparatus for 16 h to remove unreacted monomer and non-grafted homopolymer. PP membrane coated with poly(ethylene-vinyl alcohol) copolymer (EVAL) was prepared by soaking the membrane in a 2% solution of EVAL (ethylene content 32 wt%) in 70/30 (v/v) methanol/water mixture at 607C, and drying it at 60°C for 16 h.

the weights of grafted membrane (Wg) and non-grafted original membrane (Wo) by the following equation: % G = 100 × ( W , -

Wo)/Wo

Hemolysis caused by hydrophilic membranes was assessed by the following plasma separation procedure using a small test module (effective area, 24 cm 2; blood flow path thickness, 350 pro). Fresh bovine blood anticoagulated with acid-citrate-dextrose formula A (ACD-A) was run through the module with a dry membrane without prior priming by physiological saline. Blood flow rate and wall shear rate were kept at 20 ml/min and 400 s-~, respectively. Transmembrane pressure was kept below 25mmHg. The concentration of free-hemoglobin in preblood used was 38-46 mg/dl. Hemolysis ratio was defined by the following equation after measuring free hemoglobin concentration (CHb) in plasma before and after the separation: Hemolysis ratio

Membrane characterization procedures

and plasma separation

Wetting time, an indicator of the hydrophilicity of porous membranes, was determined according to ASTM D4199 method B procedures as follows. The membrane was placed on the surface of water. Wetting time is defined as the time required for water to travel through the pores and reach the opposite side of the membrane until it is completely wetted. The morphological details of the grafted membranes were observed using scanning electron microscopy (JEOL JSM-840). The degree of grafting (%G) was calculated from

i f . . . . . .f~ I ~ Matchir~gNetwork H ! I

RF Generat°r I

Pressure

CHb in filtered plasma CHb in preblood plasma by centrifugation An in vitro evaluation of the PMEA-grafted PP (PP-g-PMEA) membrane was performed by the use of a disk-type plasma separator with 0.16 m 2 effective area and a constant pressure plasma separation system without a priming step. The blood bag containing about 450ml of fresh human blood anticoagulated with ACD-A, was subjected to a pressure of 120 mmHg. The sieving coefficients (SC) of various plasma components were calculated from their concentration in preblood plasma (Cp,O and filtered plasma (C~) by the following equation: SC-

fill

RESULTSAND DISCUSSION

Gauge

Membrane preparation The PP membrane was successfully converted to a hydrophilic membrane by graft copolymerization of N,N-dimethylacrylamide (DMAA) or 2Substrate I t 1 I _.j methoxyethylacrylate (MEA), without using any i._. I V wetting agent such as hydrophilic chemicals or I ..',;- ,',,,".',"."I I surfactants. The amount of PMEA and PDMAA branches grafted onto the PP membrane increased ! " M Monomer 1 linearly with increasing polymerization time (Fig. 2). nomer 2 The rate of grafting was very high even in vapor-solid phase reaction under reduced pressure. The degree of grafting reached 10% within 3 min polymerization and the grafted membranes became hydrophilic. This indicates that hydrophilic polymers are graft copolymerized onto all surface areas of the membrane including pore walls, and consequently water is able to Fig. I. The apparatus for vapor-phase plasma-initiated graft penetrate freely into the micropores and pass through copolymerization onto microporous membranes. the membrane without the application of any pressure. Electrodes

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Ar

A new plasma separation membrane 50

221

Table I. Hemolysis ratios of modified PP membranes

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ol "6

30-

o

20-

PP-g-PMEA . _ . . _ . . ~ . f , , O

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PP-g-PDMAA

Membranes

%G

PP-g-P(MEA-EA) PP-g-P(DMAA-EA) PP-g-PPMEA PP-g-P(DMAA-MEA) PP-g-PDMAA PP coated with EVAL

24 21 17 22 14 --

Wetting time (s) Hemolysis ratio 60 30 I0 5 < 1 < 1

1.0 1.0 1.0 3.5 7.8 7.8

PP, polypropylene; DMAA, N,N-dimethylacrylamide; EA, ethylacrylate; MEA, 2-methoxyethylacrylate; EVAL, ethylene-vinylalcohol copolymer.

10-

0 0

I 5

I 10

Graft copolymerization time (min)

Fig. 2. Relationship between graft copolymerization time and the degree of grafting (%G):/"q, DMAA, 1.3 × 102Pa, 298 K; O, MEA, 1.6 x 102 Pa, 298 K. Grafted polymers did not leach out from the membrane matrix, even when washed with solvent, because they are chemically bound to the PP substrate. PDMAA is a water-soluble polymer. Therefore PDMAA-grafted PP (PP-g-PDMAA) membranes become extremely hydrophilic and their wetting time was <1 s. On the other hand, MEA is insoluble in water, so that the hydrophilicity of polyMEA (PMEA) is less than that of PDMAA. Consequently, the wetting time of PMEA-grafted PP (PP-g-PMEA) membrane (%G = !7) was longer than that of PP-g-PDMAA membane (%G = 14), being 10s. Figure 3 shows SEM pictures of the PP substrate and the PP-g-PMEA membrane ( % G = 17). No significant morphological changes were observed between the two membrane surfaces; the pores of the PP-g-PMEA membane are not plugged by grafted PMEA. In fact, the PP-g-PMEA membrane had almost the same pore size distribution and water flux as the PP membrane.

Original PP

Hemolysis measurement Hemolysis, which leads to the release of hemoglobin contained in erythrocytes into the plasma, is undesirable since free hemoglobin can be toxic. We prepared various hydrophilic membranes possessing different wetting times, ranging from 1 to 60 s, by grafting hydrophilic and hydrophobic monomers, and assessed the hemolysis that is observed when hydrophilic membranes in a dry state are exposed to blood. The hemolysis ratios were calculated from the concentration of free-hemoglobin in the plasma filtered during the first 5 rain of plasma separation. No significant differences in plasma filtration rate among tested membranes were observed in these experiments, probably because of the concentration polarization effects of blood cells which dominate filtration kinetics in membrane plasma separation (Zydney and Colton, 1982). The relationship between the wetting time and the hemolysis ratio is summarized in Table 1. The highly hydrophilic membranes with a short wetting time caused extensive hemolysis. Figure 4 demonstrates that the hemolysis occurred at the beginning of plasma separation. The concentration of free hemoglobin in filtrate plasma significantly decreased with increasing separation time. In the case of hydrophilic membranes with a short wetting time, such as those of PP-g-PDMAA and PP coated with EVAL, plasma penetrates into pores immediately by capillary attraction, and erythrocytes are trapped rapidly on the micropores of the porous

PP-g-PMEA

Fig. 3. Scanning electron micrographs of original PP and PP-g-PMEA membranes (%G = 17).

M. Onishi et al.

222

Table 3, Release of free-hemoglobin and //-thromboglobulin, and complement activation by PP-g-PMEA membrane

30

o

~ 25 .~_

o E

I I"1

[] PP coated with EVAL (Wetting time = 1 s)

20

0 PP-g-PMEA (Wetting time - 10 s)

15 []

Mesurements

Preblood

Postblood

Free hemoglobin (mg/dl) fl-Thromboglobulin (ng/ml) C3a (ng/ml) CSa (ng/ml)

0.6 78 506 9

4.2 ± 160 + 581 ± 14 ±

± ± + ±

0.5 23 61 3

Filter plasma

1.1 0.5 + 14 87 + 182 858 + 7 9±

0.4 19 393 3

n = 3, mean ± SD. 5 1

"a.ck :a.o.~

.-0 0 - 0 - 0 ~ 0 ....

5 ....

,O-El 1'0 . . . .

15

Plasma separation time (rain)

Fig. 4. Changes of the hemolysisratio in the early stages of plasma separation. Membranes were used without priming by physiological saline: wall shear rate, 400 s-'; TMP < 25 mmHg. membrane and lysed. On the other hand, weakly hydrophilic membranes with a long wetting time, such as those of PP-g-PMEA and PP-g-P(MEA-EA), do not cause hemolysis. This can be explained by the fact that plasma penetrates into the pores only slowly because of the less hydrophilic nature of the membrane, and consequently erythrocytes are not trapped in the micropores to the same extent. These data indicate that dry-type plasma separators equipped with the PP-g-PMEA membrane, which has a wetting time of longer than 10 s, do not require priming with physiological saline prior to use. Thus, separation procedures are simplified and shortened by eliminating the priming step. Another advantage is that the collected plasma is not diluted by saline. Plasma separation experiments In vitro plasma separation experiments with the PP-g-PMEA membrane (%G = 16 + 5) were conducted without a priming step, using a disk-type plasma separator and CPAS-II. The concentration of free hemoglobin before and after the separation was below 5 mg/dl, indicating that hemolysis did not occur during the plasma separation, as expected. The plasma obtained was not diluted by saline, so that the sieving coefficients of various plasma components were approx. 1.0 (Table 2). Another reason for the excellent recovery may be due to the PMEA-grafted layer which could prevent proteins from adhering to the membrane surface. The original PP membrane has lower recovery rates for coagulation factors such as Table 2. Sieving coefficients of plasma components by PP-g-PMEA membrane Plasma components Total protein Albumin Total cholesterol Fibrinogen Factor VIII:C n = 3, mean.

Sieving coefficient 1.00 1.02 1.00 1.00 0.99

fibrinogen than the PP-g-PMEA membrane (Takahashi et al., 1990; Takahashi et al., 1993). This can be explained by the fact that hydrophobic polymers such as polyethylene and polypropylene adsorb these proteins through strong hydrophobic interactions (Baszkin and Lyman, 1980). In order to test the hemo-compatibility of PP-g-PMEA membranes, the amount ofC3a, C5a and /Lthromboglobulin (fl-TG) in plasma before and after separation were measured (Table 3). fl-TG is released when platelets are activated. About a 2-fold increase of fl-TG was observed in the post-separated blood; however, the actual level is relatively low compared with other membranes that have been widely used (Rock et al., 1986), and therefore it may be not a very serious problem. One of the important problems in membrane plasmapheresis is the activation of complement during the passage of plasma through the membrane, and the production of anaphylatoxins (C3a, C5a) that can function as mediators of the acute inflammatory response and serve as immunoregulatory factors (Chenoweth, 1988). Although the C3a concentration was higher after plasma separation, activation of complement factors by PP-g-PMEA membranes was found to be relatively mild compared with other hydrophilic membranes that have already been used clinically (Takaoka et al., 1984). Since PP-g-PMEA does not contain reactive groups such as hydroxy groups, it does not activate C3 through the mechanism involving covalent attachment of C3 to the reactive groups (Law, 1983). It is reported that anaphylatoxins are rapidly inactivated in vivo by carboxypeptidase (Goldstein, 1988), and that more than 103 ng/ml of C3a and C4a can be found in blood stored for a prolonged period of time to be utilized for transfusion (Sekiguchi et al., 1990). Therefore, the activation of complement factors by PP-g-PMEA membranes may not present a serious problem. Clinical evaluation

The clinical evaluation of the disk-type plasma separator equipped with a PP-g-PMEA membrane was performed for more than 100 healthy donors by the Japan Red Cross, Tokyo Women's Medical College and Tohoku University School of Medicine, using a special compact device (CPAS-II) that is designed to be utilized for donor plasmapheresis under various circumstances, such as mobile collection sites and at the bedside. This device functioned well to collect cell-free plasma and none of the donors

A new plasma separation membrane experienced adverse effects. The PP-g-PMEA membrane had an excellent plasma separation capability and good hemo-compatibility, and exhibited sieving coefficients of approx, 1.0 for proteins and coagulation factors, as in our in vitro experiments. The details have been described elsewhere. (Osada et al., 1993; Takahashi et al., 1993) CONCLUSIONS A new type of hydrophilic membrane for plasma separation, a PP-g-PMEA membrane, was successfully produced by graft copolymerization of M E A onto microporous PP membrane. The main advantage of this membrane is that it does not require priming by physiological saline prior to use although it is in a dry state. A disk-type plasma separator with a PP-gP M E A membrane had good hemo-compatibility and an excellent plasma separation capacity, enabling a high recovery of plasma components. REFERENCES

Baszkin A. and Lyman D. L (1980) The interaction of plasma proteins with polymers. I. Relationship between polymer surface energy and protein adsorption/desorption. J. Biomed. Mater. Res. 14, 393 Bradley A. and Fales J. D. (1971) Prospects for industrial applications of electrical discharge. Chem. Technol. 1,232 Chenoweth D. E. (1988) Complement activation produced by biomaterials. Artif. Organs 12, 502 Goldstein I. M. (1988) Complement: biologically active products. In Inflammation-basic Principles and Clinical Correlates (Edited by Gallin J.I., Goldstein I.M. and Snyderman R.), p. 55. Raven Press, New York. Law S. A. (1983) Non-enzymic activation of the covalent binding reaction of the complement protein C3. Biochem. J. 211, 381. Masuoka T., Hirasa O., Suda Y. and Onishi M. (1989) Plasma surface graft of N,N-dimethylacrylamide onto porous polypropylene membrane. Radiat. Phys. Chem. 33, 421.

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Onishi M., Shimura K., Seita Y., Yamasita S., Takahashi A. and Masuoka T. (1992) Preparation and properties of plasma-initiated graft copolymerized membranes for blood plasma separation. Radiat. Phys. Chem. 39, 569. Osada K., Fujii H. and Shimizu M. (1993) Clinical evaluation of a novel membrane plasma separator for donor plasmapheresis. Jpn. J. Transf. Med. 39, 959. Rock G., Tiffley P. and McCombie N. (1986) Plasma collection using an automated membrane device. Transfusion 26, 269. Seita Y. and Onishi M. (1988) A flat permeable membrane. U.S.Patent No. 4,743,375. Sekiguchi S., Takahashi T. A., Yamamoto S., Hasegawa H., Takenaka Y., Uemitsu J. and Fukumi H. (1990) A new type of blood component collector: plasma separation using gravity without any electrical devices. Vox Sang. 58, 182. Stromberg R. R. and Friedman L.I. (1989) Membrane technology applied to donor plasmapheresis. J. Membrane Sci. 44, 131 Takahashi T. A., Nakase T., Yokoyama M., Kodama H., Yagishita H., Sado M. and Sekiguchi S. (1993) Clinical study of a disk-type membrane plasmapheresis system (CPAS-II) with a hydrophilic membrane. In Therapeutic Plasmapheresis (XII) (Edited by Agishi T., Kawamura A. and Mineshima M.), p. 825. VSP, Utrecht, The Netherlands. Takahashi T. A., Hosoda M., Yamamoto S., Nakai K., Sekiguchi S., Yagishita A., Fujii T. and Takahashi A. (1990) A new donor plasmapheresis system with disk type membrane separator. Jpn. J. Transf. Med. 36, 418. Takahashi T. A., Nakase T., Yokoyama M., Kodama H., Hosoda M., Keino H., Fujii T., Yagishita H., Shimane H., Sado M. and Sekiguchi S. (1992) Clinical evaluation of the constant apheresis system type I1 (CPAS-11). In Therapeutic' Plasmapheresis (X) (Edited by Yamagawa J., Inoue N. and Nakagawa S.), p. 401. ICAOT Press, Cleveland. Takaoka T., Goldcamp J. B., Abe Y., Matsugane T., Blasutig E., Smith J. W., Malchesky P. S. and Nose Y. (1984) Biocompatibility of membrane plasma separation. Trans. Am. Soc. Artif. Intern. Organs 30, 347. Zydney A. L. and Colton C. K. (1982) Continuous flow membrane plasmapheresis: theoretical model for flux and hemolysis prediction. Trans. Am. Soc. Artif. Intern. Organs 28, 408