Inactivation of lipid-enveloped and non-lipid-enveloped model viruses in normal human plasma by crosslinked starch-iodine

F.A. HIGHSMITH, H. XUE, M. CAPLE, B. WALTHALL, W . N . DROHAN, AND E. SHANBROM

Background: The purpose of this study was to evaluate an iodine-based method of activating potential1 harmful viruses that might be found in normal human plasma. Study Design and kethods: A procedure has been developed for inactivating lipid- enveloped and non-lipid-enveloped model viruses in normal human plasma by us- ing iodine complexed to crosslinked potato starch. The established conditions are an iodine concentration of 1.05 mg per mL and 60 minutes' incubation. Results: Under these conditions, inactivation of more than 9 log,,, of vesicular stomatitis virus, a lipid-enveloped virus, and more than 7 loglo of encephalomyo- carditis virus, a non-lipid-enveloped virus, was achieved, with minimal losses of bio- lo ic activity of selected plasma proteins. Under these conditions, 70 percent of factor V f l activity, 77 percent of factor IX activity, and 100 percent of protein C activity in the plasma were retained. Conclusion: Crosslinked starch-iodine may be useful in the inactivation of viruses in sin le-donor plasma units and in pooled human plasma before fractionation. TRA8SFUSlON 1 99434322-327.

Abbreviations: EMCV=encephalomyocarditisvirus; F IX =factor IX; FVlll =factorVIII; VSV = vesicular stomatitis virus.

MANUFACTURERS OF THERAPEUTIC proteins from human plasma must be concerned about the presence of viruses in products. Current methods for the inactivation of vi- ruses in human plasma and plasma-derived products consist of treatment with one or a combination of the following methods: dry, wet, or steam heat;'.' pasteur- ization;'.3 solvenddetergent P-propiolactone treatment;' or ultraviolet irradiation.8 However, some of these methods are not effective against all of the viruses that might be present. Consequently, new methods are being evaluated that may lead to more effective inacti- vation of potentially harmful lipid- and non-lipid-envel- oped viruses that might be found in these products. In this article, we report an iodine-based method that inac- tivates vesicular stomatitis virus ( V S V ) and encephalo- myocarditis virus (EMCV) in normal human plasma with minimal losses of the biologic activity of selected plasma proteins.

We previously described the use of crosslinked polyvinylpyrrolidone iodine in inactivating lipid-envel- oped viruses and reported that VSV could be inactivated under conditions that allow for the recovery of more than 80 percent of the biologic activity of several plasma pro-

From the Plasma Derivatives Department, Holland Laboratory, American Red Cross, Rockville, Maryland. and lrvine Scientific Incorporated, Santa Ana, California.

Received for publication August 26, 1993; revision received November 16. 1993. and accepted November 22, 1993.

teins evaluated.Y Such methods have also been effective in inactivating non-lipid-enveloped viruses (data not shown), but crosslinked polyvinylpyrrolidone did not bind iodine as tightly as was considered necessary for commercial use.

Therefore, we decided to evaluate other carriers that were shown to complex with iodine more tightly than crosslinked polyvinylpyrrolidone (data not shown). Starch has been known to bind iodine tightly and, in fact, has often been used as a chemical test for free iodine, turning blue when brought in contact with solutions con- taining iodine. Consequently, we evaluated the ability of iodine, tightly bound to crosslinked starch, to inactivate lipid- and non-lipid-enveloped viruses while not signifi- cantly inactivating coagulation factor proteins found in human plasma.

On the average, unmodified potato starch is 20 per- cent amylose, a linear polymer of glucose subunits linked through a- 1,4-glycosidic bonds with a double helical crystalline structure, and 80 percent amylopectin, a highly branched polymer of glucose subunits 4 to 6 per- cent of which are linked by a-1,6-glycosidic bonds at branch points.I0 Unlike amylose, amylopectin lacks a double helical structure. According to Whistler et al.," x-ray diffraction suggests that amylose contains six D- glucose molecules per turn, with dimensions that enable an iodine molecule to be accommodated within the he- lix at every sixth carbon position, which gives rise to the

322

VSV AND EMCV INACTIVATION IN NORMAL HUMAN PLASMA 323 TRANSFUSION 19Y4-Vol. 34. No J

characteristic blue color of the starch-iodine complex. Because it lacks a helical structure, amylopectin does not readily form a blue color in the presence of iodine.,"

The selection of VSV and EMCV as model viruses for these studies was based on their common use as model viruses for monitoring the inactivation of lipid- and non-

though not a model for any particular human pathogen, is thought to be a very good model for the inactivation of lipid-enveloped viruses. Hollinger et al. l 4 suggested that EMCV is a good model for hepatitis A virus. The EMCV capsid is composed of a dense protein coat that is thought to be resistant to many chemical virucidal agents. Both hepatitis A virus and EMCV are non-lipid- enveloped viruses of the Picornaviridae family and ex- hibit similar biophysical characteristics.14 In these stud- ies, we have shown that inactivation of more than 9 log,,, of VSV and more than 7 log,, of EMCV can be obtained in plasma with minimal losses in the activity of selected plasma proteins.

lipid-enveloped viruses in biologic material^.^^^^'^^'^ vsv,

Materials and Methods Crosslinking of starch

We suspended 150 g of native potato starch (Sepafax, AVEBE America, Princeton, NJ) in 960 mL of absolute etha- nol (Fisher Scientific, Fairlawn, NJ) by shaking the mixture at 200 rpm for 30 minutes at ambient temperature (24C). Forty mL of deionized H,O, 90 mL of epichlorohydrin, and 210 mL of 5N NaOH was then added to the suspension. The complete reaction mixture was shaken at 200 rpm for 5 hours at 35C. At 5 hours the reaction was terminated with 600 mL of 10- percent acetic acid for 30 minutes at 35C. The crosslinked starch was placed in a sintered glass funnel (ASTM 40-6OC. Fisher Scientific) and washed once with 900 mL of ethanol, twice with 900 mL of H,O, two more times with 900 mL of ethanol, and once with 900 mL of acetone. We then dried the crosslinked starch at 60C for 2 hours. Afterward, the various granules were sized through a series of sieves (ASTM stan- dard sizes No. 200, 75 p; 140, 106 p; 60. 250 p; Fisher Scientific) and stored in polypropylene containers at ambient temperature (24C). In these experiments, only particle sizes between 106 and 250 p were used. Particle size played no role in the effectiveness of this method for virus inactivation. Additional details of the crosslinking reaction have been de- scribed previously. 11.15 .16

The method of crosslinking the starch is apparently critical to the preparation of crosslinked starch-iodine. If the washing and drying of the crosslinked starch is not performed properly, it may interfere with the subsequent immobilization of the iodine. We therefore caution other investigators to follow ex- actly the method described above. One of nine batches of crosslinked starch-iodine that we prepared was ineffective in inactivating viruses (for reasons still undetermined at this time). However, stability studies on some of the other batches have shown that their effectiveness is retained for 2 months after preparation. We have not tested material beyond that time.

Immobilization of iodine

A solution of 6 g of potassium iodide and 0.6 g of elemen- tal iodine (Aldrich Chemical Company, Milwaukee, WI) in 210

mL of H,O was added to 60 g of crosslinked starch. The re- sulting suspension (1% final iodine concentration) was mixed at 10 rpm for 2 hours at 24C. We then filtered the suspension through a 0 . 4 5 - p membrane to recover the crosslinked starch- iodine, dried it for 4 hours at 60C. and stored it in lightproof containers at ambient temperature (24C).

Plasma experiments

Ten units of human plasma were thawed in a 37C water bath, pooled, dispensed in 50-mL aliquots, and stored at -80C. For experimentation, the plasma aliquots were thawed at 37"C, pooled, and dispensed as 10-mL samples into polypropylene sample tubes. We treated the plasma samples with the crosslinked starch-iodine powder at the following concentra- tions: 0,0.35,0.65, 1.05, and 1 S O mg per mL of plasma. These concentrations were obtained by adding 0,470,910, 1470, and 2 100 mg of crosslinked starch-iodine to the plasma, and they represent the theoretically available iodine; that is, if all of the iodine complexed to the crosslinked starch were in solution, the final concentrations would be as described above. The so- lutions were then mixed for 0, 5, 20, 60, and 120 minutes at ambient temperature (24"C), filtered through 0 . 4 5 - p filters, snap-frozen in liquid nitrogen, and stored at -80C.

Protein studies

The activity of coagulation factor IX (F IX) in the treated samples was measured by a one-stage activated partial throm- boplastin time clotting assay (Pacific Hemostasis, Huntersville, NC) with a standard prepared from pooled normal human p1asma.I' Details of the assay procedure have been described by Menache et aI.'* Factor VIII (FVIII) activity was measured by an activated partial thromboplastin time clotting assay as described for F IX." For the FVIII assay, an international stan- dard (US Standard Antihemophilic Factor [Factor VIIII-MEGA I , Food and Drug Administration, Bethesda, MD) containing 10.2 IU per vial was reconstituted in 1 mL of deionized H,O, diluted 1 -in- 10 in FVIII-deficient plasma (Universal Reagents, Indianapolis, IN), and used to generate a standard curve.'"

A procedure developed by Guiglielmone et al.19 has been modified for determining the biologic activity of protein C in plasma. Protein C activity is measured by a one-stage activated partial thromboplastin time clotting assay as described for F IX with the following exceptions: 6 mL of activated partial thromboplastin time reagent (silica particulate) is added to one vial of the snake venom activator (Agkisrrodon contorrrix contortrix) (American Diagnostica, Greenwich, CT), and sample dilutions were made in buffer (0.10% [wt/vol] bovine serum albumin, 0.01% [vol/vol] Tween 20), 0.05 M (0.05 moV L) imidazole (Sigma Chemical, St. Louis, MO), and 0.10 M (0.10 mom) NaCI. We assayed samples on an automatic co- agulation timer (Electra 900, Medical Laboratory Automation, Pleasantville, NY).

Virus-inactivation experiments

Stocks of VSV and EMCV were diluted I-in-100 in sepa- rate aliquots of pooled normal human plasma to obtain final titers in plasma of approximately 1 o9.l tissue culture-infective dose units for VSV and lo7 tissue culture-infective dose units for EMCV. As a control, we incubated virus-spiked pooled human plasma with crosslinked starch, without the addition of iodine. In addition, an experiment that was equivalent to a stop control was performed, in which virus was spiked into plasma and titrated in the absence of crosslinked starch or starch-io- dine.

324 HIGHSMITH ET AL. TRANSFUSION Vol. 34. No. 4-1994

We treated the virus-inoculated plasma samples at 24C with varied concentrations of crosslinked starch-iodine as de- scribed above (including a control with no crosslinked starch- iodine). After 5, 20, 40, 60, and 120 minutes of treatment, the crosslinked starch-iodine was removed from each sample and the virus titer determined. One-in-five serial dilutions of the samples were prepared in quadruplicate in nonessential amino acid Minimum Essential Medium containing 10-percent fetal bovine serum (Irvine Scientific, Santa Ana, CA) and plated onto 96-well plates containing VERO cells (CRL 81, ATCC, Rockville, MD) at approximately 80-percent confluency. The lowest concentration used in this assay was a I-in-5 dilution of the original treated plasma. The cultures were incubated for 2 to 3 days at 37C in a 5-percent CO, atmosphere, and each well was examined for cytopathic effects. We then calculated the virus titer by the endpoint method.20.2' The log reduction (inactivation) for each virus was calculated as the difference between the titers of the treated and untreated plasma samples.

Results The effect of various concentrations of crosslinked starch-

iodine on the inactivation of VSV in normal human plasma is shown in Fig. 1. At all concentrations (final iodine concentra- tions of 0.35,0.65, and 1.05 mg/mL) and at all incubation times ( 5 , 20, 40, 60, and 120 min), complete inactivation of VSV was observed as compared to the control (no crosslinked starch- iodine added). As an additional control, we incubated plasma with crosslinked starch alone and observed no reduction of virus titer (data not shown).

The effect of various concentrations of crosslinked starch- iodine on the inactivation of EMCV in normal human plasma is shown in Fig. 2. At the lowest iodine concentration (0.35 mg/mL) and shortest incubation time (5 min), < I log,, of EMCV was inactivated. While the two lower iodine concen- trations failed to inactivate all virus added, we did observe that virus inactivation appeared to be directly proportional to incu- bation time. At the highest iodine concentration investigated, 1.05 mg per mL, all of the EMCV was inactivated at 60 min- utes' incubation. Crosslinked starch alone had no effect on the viability of EMCV (data not shown).

- 0 20 40 60 80 100 120

Time (min) FIG. I . Decrease in the titer of VSV in the presence of crosslinked

starch-iodine at 24OC (initial titer, approx. 9.1 * 0.4 log,,,) (n = 3). The plasma control (-+-) refers to virus-spiked plasma with no crosslinked starch or starch-iodine. The control of crosslinked starch in virus-spiked plasma provided no inactivation of virus over the incubation times studied (data not shown). Concentrations shown are 0.35 mg per mL. -8- ; 0.65 mg per mL, + ; and 1.05 mg per mL. -6- .

I T T 1

-1 1 -2 '

0 20 40 60 80 100 120 Time (min)

FIG. 2. Decrease in the titer of EMCV in the presence of crosslinked starch-iodine at 2 4 T (initial titer, approx. 7.0 f I .O log,,,) (n = 3). The plasma control (-+-) refers to virus-spiked plasma with no crosslinked starch or starch-iodine. The control of crosslinked starch in virus-spiked plasma provided no inactivation of virus over the incubation times studied (data not shown). Concentrations shown are 0.35 mg per mL. -G- ; 0.65 mg per mL, + ; and 1.05 mg per mL, 4- .

The percentage of biologic activity of FVIII remaining af- ter incubation of human plasma with crosslinked starch-iodine at 24C is shown in Fig. 3. At all iodine concentrations (0.35, 0.65, 1.05, and 1 S O mg/mL), more than 95 percent of the bio- logic activity of FVIII was retained after 5 minutes' incuba- tion. In fact, at 0.35 mg per mL of iodine, more than 80 per- cent of the FVIII activity was retained at all incubation times up to 120 minutes. At all iodine concentrations and times in- vestigated, except I S O mg per mL and 120 minutes, more than 70 percent of the FVIII biologic activity was maintained. In- cubation with crosslinked starch had no measurable impact on FVIII biologic activity (data not shown).

The percentage of biologic activity of F IX remaining after incubation of human plasma with crosslinked starch-iodine at 24C is shown in Fig. 4. At the highest iodine concentration evaluated ( I S O mg/mL), more than 70 percent of the F IX activity remained under all incubation conditions, except for

i i n .

c c a 2 0 iL--- 10 20 40 60 80 100 120 Time (min)

a " O

FIG. 3. The percentage of FVIIl activity remaining after the incubation of plasma with crosslinked starch-iodine at 24C ( n = 3). The plasma control (-+-)refers to virus-spiked plasma with no crosslinked starch or starch-iodine. The crosslinked starch control caused no changes in activity over the range of incubation times studied (data not shown). Concentrations shown are 0.35 mg per mL, : 0.65 mg per mL, -B- ; I .05 mg per mL, + ; and 1 S O mg per mL, + .

VSV AND EMCV INACTIVATION IN NORMAL HUMAN PLASMA TRANSFUSION 1994-Vol. 34. No. 4

- .- a60 .- c = c 40 0 3 0

s 10

c .-

c

325

a c 3 !2 c

al a "

0 20 40 60 80 100 120 Time (min)

FIG. 4. The percentage of F IX activity remaining after the incubation of plasma with crosslinked starch-iodine at 24C (n = 3). The plasma control refers to virus-spiked plasma with no crosslinked starch or starch- iodine. The crosslinked starch control caused no changes in activity over the range of incubation times studied (data not shown). Concentrations shown are 0.35 mg per mL, -8- ; 0.65 mg per mL, -f ; 1.05 mg per mL. -B- ; and 1 S O mg per mL, +.

the 120-minute incubation time. However, it can be seen that both increased iodine concentrations and increased incubation times lead to continued decreases in F IX biologic activity. In- cubation with crosslinked starch had no measurable impact on F IX biologic activity (data not shown).

To further evaluate the impact of incubating plasma with crosslinked starch-iodine, we evaluated the biologic activity of another vitamin K-dependent coagulation protein, protein C. As shown in Fig. 5, no more than 20 percent of the protein C activity was lost under any of the conditions tested. In fact, under many of the incubation conditions, there was no loss in the biologic activity of protein C. Crosslinked starch alone had no measurable effect on protein C activity (data not shown).

> > 130 0 120

110

CI .- .- c

y 100 .- c @ g o

I T

Discussion Although a number of methods have been developed

for the inactivation andor removal of viruses from plasma and plasma-derived products,"-6v8.'*12 recent reports on the transmission of hepatitis A22-24 and human B19 p a r ~ o v i r u s ~ ~ . ~ ~ by transfusion of coagulation factor con- centrates have accentuated the need for improved virus- inactivation methods that are capable of inactivating both lipid- and non-lipid-enveloped viruses. In this study on the use of crosslinked starch-iodine, we were able to in- activate >9 log,, of VSV, a model lipid-enveloped virus, under all of the conditions evaluated. We were surprised to see that as little as 0.35 mg per mL of iodine was ef- fective in inactivating the virus in as little as 5 minutes of incubation. EMCV, a non-lipid-enveloped model vi- rus, proved to be more resistant to inactivation by crosslinked starch-iodine. Only at the highest iodine con- centration employed (1.05 mg/mL) and at an incubation time of 1 hour or more was complete inactivation achieved.

The use of crosslinked starch-iodine can be tailored to meet a variety of applications including batch, filter, and column configurations, and, because of its large par- ticle size, it can easily be removed by filtration or cen- trifugation. It could be used alone to inactivate viruses in single-donor plasma units or in batches of pooled plasma intended for fractionation; it could also be used in combination with a solventldetergent treatment, to inactivate non-lipid-enveloped as well as lipid-enveloped viruses.

Omnipaque, a nonionic water-soluble contrast me- dium is administered intravascularly and used for com- puter tomographic imaging of the head and body.27 Some applications of computer tomographic imaging require the intravascular injection of 500 to 1500 mg of iodine per kg of body weight.27 At these concentrations, one fatality was reported in 1485 patients in a controlled clini- cal trial. In our procedure, if all the available iodine were to be released from the crosslinked starch-iodine, 87 to 375 mg of iodine per unit of plasma (250 mL) would be infused. This corresponds to 4 . 5 percent of that infused in the aforementioned computer tomographic imaging procedure and, thus, should diminish the already low possibility of an adverse reaction to infused iodinated plasma.

We had arbitrarily decided that, for this technology to be useful for inactivating viruses in human plasma, more than 70 percent of the activity of certain selected plasma proteins should be recovered. Therefore, we measured the survival of three coagulation proteins un- der the incubation conditions that led to virus inactiva- tion. FVIII was considered to be one of the most impor- tant proteins to evaluate, partly because of its clinical importance, but also because of its size and sensitivity

326 HIGHSMITH ET AL. TRANSNSION Vol. 34. No. 4-1994

to enzymatic We also evaluated the sta- bility of F IX and protein C, members of the vitamin K- dependent family of plasma proteins.22*23.30-33 After 1 hour of incubation, more than 70 percent of FVIII activity and more than 77 percent of F IX activity was retained at all crosslinked starch-iodine concentrations evaluated. Pro- tein C, a naturally occurring anticoagulant, was remark- ably resistant to inactivation by crosslinked starch-iodine; more than 80 percent of protein C activity was retained under all conditions evaluated. Thus, this method for the inactivation of lipid- and non-lipid-enveloped viruses meets our stated criteria. It should be noted, however, that increases in crosslinked starch-iodine concentrations and in incubation time beyond the range of these experiments led to increased coagulation FVIII and F IX inactivation (data not shown).

In summary, we have reported conditions under which lipid- and non-lipid-enveloped model viruses could be inactivated in normal human plasma, at the same time that more than 70 percent of the biologic activity of se- lected human plasma proteins was maintained. While the microbicidal action of iodophors is not completely un- derstood, it is believed that certain iodine species can rapidly penetrate the cell wall of microorganisms and interact with various reactive groups (oxidation of sulf- hydryl groups and reaction with phenol groups, carbon- carbon double bonds on unsaturated fatty acids, amino groups on amino acids, and bases on nucleotides) on pro- teins and ~iruses?~-~~Additional research is needed to de- termine the efficacy of crosslinked starch-iodine on hu- man pathogenic viruses, as are more detailed studies on the effects this material might have on plasma proteins. In addition, issues such as neoimmunogenicity, toxicol- ogy, tissue uptake mechanisms, pharrnacokinetics, and antigenicity should be addressed prior to the commercial- ization of this procedure.

Acknowledgment The authors are grateful to Craigenne A. Williams for

contributions to the preparation of the manuscript.

I .

2.

3.

4.

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Frank A. Highsmith, BS, Research and Development Engineer, Plasma Derivatives Department, Jerome Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way. Rockville. MD 20855. [Reprint requests]

Hao Xue, BS. Research Technologist, Plasma Derivatives Department, Jerome Holland Laboratory.

Matthew Caple. MS. Irvine Scientific Incorporated, 25 I 1 Daimler Street, Santa Ana, CA 92705.

Benjamin Walthall. PhD. Director, Research and Development. lrvine Scientific Incorporated.

William N. Drohan, PhD, Laboratory Head, Plasma Derivatives Department, Jerome Holland Laboratory.

Edward Shanbrom, MD, Independent Consultant, Santa Ana, CA (retired).