Original Paper Vox Sang 1992;62:12-20
Strinley E. Charma Steven Landau a Bolanle Williams’ Bernard Horowitz Alfred M . Prince’ Donna Pclscual
’
Charm Bioengineering, Inc., Malden, Mass., USA The New York Blood Center, New York, NY, USA
High-Temperature Short-Time Heat Inactivation of HIV and Other Viruses in Human Blood Plasma ................................................................................................. Abstract
An ultra-short-time heating system was used to process blood plasma spiked with various viruses (HIV, vesicular stomatitis virus, encephalomyocarditis virus). Virus reduction and recovery of plasma proteins were measured at various temperatures from 65 to 85°C. Processing at 77°C and 0.006 s resulted in a high level of virus kill, including 2 4.4 log,, HIV, while maintaining protein structure and activity essentially intact.
.....................
Introduction
With the exception of intramuscular immune globulin and albumin solutions [l], the transmission of viruses by plasma and plasma fractions has been a serious concern. While implementation of improved procedures for donor selection and donor blood screening can reduce viral risk, assurance of virus safety can only come from the introduction of virucidal procedures into manufacture; thus several methods of inactivation have come into routine use. With the best of these methods, the transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and non-A, non-B hepatitis viruses (NANBHV), including hepatitis C virus (HCV), has been greatly reduced and possibly eliminated [2-7, reviews]. When heating coagulation o r other labile factors at high temperatures, avoidance of loss of biological activity requires the prior addition of stabilizers such as sucrose and glycine [8], or the removal of water through lyophilization [9, lo]. Unfortunately, both approaches afford considerable protection to viruses.
Supported in part by NHLBI Grant No. HWI22I to The New York Blood Center Norrmber 16. 1990.
Received: Dec. 11. lPY0 Revised manuscript received: February 27. I W I Accepted: June 11. 1YYI
Advances in the application of high-temperature, short-time heating (HTST), both for sterilization and pasteurization, show that heat-sensitive materials including vitamins and heat-labile food flavors may be preserved as food is rendered safe from pathogens. A common HTST process for sterilization is 145°C for 2-4 s, and for pasteurization is 15 s at 65°C. The preservation of sensitive material and destruction of pathogens is based on their differences in rates of destruction. The rates of destruction vary with temperature. In order for the HTST process to be successful, the pathogen destruction rate must be much greater than the destruction rate of ‘sensitive’ material under the process conditions. When this is the case, it follows that the pathogen will suffer greater destruction than the heat-sensitive material. The HTST systems currently available are inadequate to achieve the time-temperature relationship that would permit the preservation of biological activity in blood products with the satisfactory reduction of contaminating viruses. Problems with the current systems include (a) the
Stanley E Charm Charm Bioengineenng. Inc. 36 Franklin Street Maldcn. MA 02148 (USA)
G 19Y2 S Karger AG Basel ~xu2-90(17/Y2/0b2I-l~1~
$ 2 730
relatively long time needed to achieve the process temperature, (b) the relatively long hold-up time at temperature, (c) the relatively long time to cool to nondestructive temperature. The commercially available Sterimedia-Mini, and Steri-Lab (Alfa Laval, Lund, Sweden) have hold times of 2-4 s or more [ll]. The currently available systems also have a large holdup volume, making it necessary to waste liters of product during processing. The Sterimedia-Mini has a hold-up volume of 1.5 liters, while the Thermalizer TL-10 can process samples of 10 ml with 80% recovery of sample volume. Chemical viral inactivation of blood fractions is routinely accomplished today using the organic solvent, tri(nbutyl) phosphate (TNBP)/detergent mixtures [12, 131. While excellent safety has been achieved with regard to HBV, HIV, and NANBHV [14,15], the major weakness of this solvent detergent approach is the inability of the process to inactivate non-lipid-membrane-coated viruses [QI. In our ongoing effort to explore processing methods which might further improve the virus safety of blood products, studies on HTST pasteurization have been initiated. The time fOr90% destruction of HIV has been found to be 24s at 60°C in a solution of A H F [16]. A prototype short-time system for processing plasma with a heating rate of 45"C/s and a hold time of 0.03 s caused 75% destruction of factor VIII [17]. This new work describes an improved ultra-short-time system developed to overcome the problems associated with earlier systems and presents our results on the heating of plasma. The inactivation kinetics of viruses and proteins may be expressed using the Arrhenius equation (1). However, in this case, the heat-up and cool-down timetemperature effects must be considered because they are in the same order of magnitude (see equation 2). Equation 2 can be used to estimate the destruction of both the proteins ands viruses for any pasteurization process.
I
0
where N/N, = active fraction at time 0,or virus H = energy of activation for destruction (cal/mol) R = ideal gas constant, = 1.987 cal/(mol."K) =
uc
T = temperature (OK) 0 = time during process (s) A = constant (mol)/s. "k) Equation 1.
By taking the ratio of equation 2 for two different holding temperatures, constant A is eliminated and,
+ Hold + Coo1)a (Heat + Hold + Cool)b
ln(N&J)a - (Heat ln(NJN)b
(3)
Using a trial and error method with equation 3, values for H can be determined, and equation 2 is then used to determine A . Methods Therrnalizer TL-10 HTST System Description The Thermalizer TL-10 (Charm Bioengineering, Inc. Malden, Mass., USA) is a HTST pasteurization system. Plasma, pumped continuously from a reservoir through the heatingsection, was heated to various temperatures between 60 and 90°C (fig. 1).held at the high temperature for a very short time, then immediately cooled in a heat exchanger. The TL-10 was engineered to minimize exposure time of the fluid to high temperatures, giving a fast spike of heat. It is also useful to be able to conduct this process on small samples, e.g. as little as 10 ml.
Equation 2.
13
Heating energy is produced by a 1.5-kW Air Cooled microwave generator. A Teflon tube is positioned inside the microwave field to achieve maximum energy transfer into the fluid. The final fluid temperature is controlled by adjusting the output power of the microwave generator. Fluid is held at processing temperature in the tube fitting between the microwave heater and the cooling section. Standard design of the holding tube gives a hold volume of 0.013 k 0.003 cm’. The hold time can be increased by coiling the Teflon heating tube between the heating and cooling systems. The plasma pasteurization experiments were all conducted with the system setup for minimum hold time. The product is cooled in a single stage shell and tube heat exchanger. Ice water is circulated throughout the outer shell while product flows through an inner stainless steel tube.
Fig. 1. Thermalizer, flow diagram.
Fig. 2. Thermalizer TL-10, time-temperature profile. Pasteurization process.
Pumps, valves, tubing and instrumentation are built on a mobile stainless steel frame with wheels. The Thermalizer is constructed usingo. 159-cmOD, 0.079-cmID316Lstainless tubing. Plastic tubing, PFA Teflon tubing, 0.159-cm OD, 0.079-cm ID is used in the heating section and injector loops. Rheodyne (Cotati, Calif., USA) stainless steel HPLC-type switchingvalves are used to control the flow through the injector loops and sample collection ports. HPLC-type stainless steel compression fittings (Swagelok. Highland Heights. Ohio, USA) 1/16 inch are used throughout the system. Two pressure vessels hold product and the rinse solution. These vesselsare pressurized to600 psi to forcesolution through the system. The pressure is provided by compressed dry nitrogen. Each tank is jacketed for recirculating water temperature control and equipped with pressure relief valves, level sensors, imput-output ports. and a temperature probe.
14
Processing Details Fresh-frozen human plasma obtained from New York Blood Center (Melville, N.Y., USA) was filtered through a 40-pm. then a 0.45pm membrane before entering the Thermalizer. Concentrated virus solutions were added to 200-ml batches of the filtered plasma to make virus-containing plasma samples. Virus-free plasma was first pumped through the TL-10 to achieve steady state at a flow rate of 170 mllmin. After 20 s of steady temperature and flow, a 10-ml bolusofplasma with viruswasinjected into the flowing stream using an injector loop. The exit time of the 10-ml sample from the TL-10 was determined by previously collected samples of injected dye or radioactive tracer. Automatic timed sampling valves collected the sample from the fluid stream. A small amount of “C-glucose wasadded to the HIV-contaminated plasma todetermine if any dilution of the sample occurred. When the peak heating temperature is too high, the heat exchanger may be progressively fouled by build-up of material inside the tubing. This is indicated by a reduced volume in the collected sample. When this happens, the Teflon heating tube is replaced, thermocouple cleaned, and the heat exchanger is rinsed with NaCI 0.9%. The hold time at temperature was 0.006 k 0.002 s (fig. 2). Hold time was calculated using the measured volume inside the fittings between the microwave heating section and heat exchanger inlet, and the measured flow rate. The range k 0.002 s was calculated by determining the uncertainties of the hold tube volume, and flow rate measurements. The Reynolds number in the heating, hold. and cooling sections was 4.500, indicating turbulent flow. A uniform residence time in the hold tube was assumed. It is not known whether any backmixing occurred. Temperature was measured using0.025-cm T-type thermocouples inserted into the fluid stream through a compression T fitting. The thermocouple accuracy was ? 1°C. Flow rate was measured by collecting samples in a graduated test tube for 2 s. The time-temperature profile was calculated knowing flow rate and the tubing volumes in each section of the process system. A typical temperature-time curve is shown in figure 2. Collected samples were frozen at -70°C in an alcohol/dry ice mixture and stored at -70°C until assayed. Protein Analysis Assay chemicals were reagent grade. The activity of coagulation factors VIII and IX was assessed by determining the degree of correction in activated partial thromboplastin time (General Diagnostics. Morris Plains, N.J.. USA) of the
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
appropriate factor-deficient plasma (George King, Overland Park, Kans., USA). The activity of factor V was assessed similarly except a nonactivated partial thromboplastin reagent (General Diagnostics) was used [18. 191. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of 1% dithiothreitol. essentially as described [20], and proteins were stained with Coomassie blue. Crossed immunoelectrophoresis was performed as described [21] using 1% agarose (Seakem, FMC Corp, Rockland Me., USA) in tricine buffer IV (BioRad. Rockville Center, N.Y.. USA) applied to Gelbond film (10 x 15 cm: FMC Corp.). Electrophoresis in the first dimension was at 20 mA per plate and proceeded until a bromophenol blue marking dye migrated two-thirds of the plate length. Agarose above the sample lane was removed and replaced with agarose containing rabbit anti-normal human serum antibody (Calbiochem, San Diego. Calif.. USA). An immunoglobulin preparation dissolved at a concentration of 2.5% (wlv) in a solution of 0.5% (wlv) human albumin, 2.5% (wlv) sucrose 10 mM NaH2P0, and 0.4% (wlv) NaCl at a pH of 5.5 was kindly provided by Dr. James McIver (Massachusetts Biologic Laboratories, Jamaica Plain, Mass., USA). IgG aggregation was assessed on a TSKG3000SW HPLC [22] by integrating the peak area of a known concentration of IgG monomer. Ailtibody A.~snys The samples were assayed for antibody to hepatitis B surface antigen (anti-HBs) reactivity by an Ausab solid-phase radioimmunoassay technique (Abbott Laboratories, Abbott Park, Ill., USA). Viriises mid Virits A.ssny.s Vesicular stomatitis virus (VSV) was obtained from Dr. William Stewart. 11. and cultured in mouse L929 cells. Encephalomyocarditis virus (EMC) was obtained from Dr. Emilio Emini. and stocks were prepared by culturing the virus in mouse L929 cells. Infectivity was assessed by endpoint dilution assay with tenfold serial dilutions in culture medium (MEM). Each dilution was used to inoculate eight replicate wells of human A549 cells in 96-well microtiter plates. Virus-induced cytopathology was scored after 72 h of incubation at 37°C in 5% CO,. The ID,"value was calculated using the Spearman-Karber method 123). High titer stocks of the HTLV IIIhstrain of HIV were purchased from Pharmacia (Piscataway, N. J.. USA). Titrations were carriedout with serial. tenfold dilutions in microtiter plates using RPMI 1640 containing 10% FCS, with CEM cells at a concentration of 8 x 10"lml. Each sample was assayed in quadruplicate. Before use, cells were conditioned by incubation for 1 h at 37°C in the above medium containing2pglml of polybrene. Virusin treatedsampleswasadsorbed to cellsfor2 hat37'Candthencultureswere washedtwiceinmediumby centrifuging plates for 10 min at 2,000 rpm and aspirating the supernatants. Cultures (IS0 $) were then fed with 25 pl of medium at 4.7, and 10 days. At 14 days, cultures were washed twice with PBS to remove viral antigens carried over from the inoculum. and the cells were lysed in 100 pl PBS containing0.5% Triton X-100. Lysateswere assayed for HIV p5S antigens by ELISA using plates coated with rabbit antiserum against recombinant pSS (kindly provided by Dr. Hardy Chan. Syntex Corp.. Palo Alto, Calif.. USA) and peroxidaselabeled rabbit anti-p55.
Table 1. Inactivation of HIV in plasma after treatment with the thermalizer (log TCID 5 0 ) Sample
HIV titer
Start Flow control 71 "C 71 "C 73"C 74°C 77 "C 78 "C 80°C Mean
4.8 4.75 3.2 3.0 2.9 1.8 50.04 50.04 10.04
U
HIV reduction
-
0.05 1.6 1.8 1.9 3.0 24.4 24.4 24.4
'I C tracer CPm
2,720 2,666 2.736 1,763 ' 2,677 2,723 2,SYY 2,744 2,644 2,456 57
'
This sample had 67% of the mean ''C cpm and 67% of the other 71°C sample HIV titer. It was not included in the Arrhenius calculations.
HIV Antigen Assay The HIV-inoculated plasma samples, along with positive and negative standard controls, were assayed for HIV p55 antigens by ELISAusingplatescoatedwithrabbit antiserum against recombinant p55. The assay is controlled by doing a 2-fold dilution of a standard HIV stock which has a concentration of 24 ng/ml. The titration of this standard must be between 1:64 and 1:256 (or 0.37 and 0.09 ng/ml). This sensitivity is equal to that of the Dupont p24 kit. Positive cut-off of the samples is twice the average of the negative control wells.
Results
Virus Inactivation Each of two viruses, a lipid enveloped virus, VSV, and a nonenveloped virus, EMC, was added to whole, filtered plasma and heated separately in the Thermalizer at varying temperatures. Total process duration was 0.250 s with 0.006 s at 'hold temperature'. The inactivation of detectable EMC was complete (210',' TCID'") at 72°C and above, and the inactivation of VSV was complete TCID'") at 75°C and above (fig. 3). HIV inactivation was assessed in a separate experiment. Complete inactivation (LlO'.' TCIDsO)of added HIV occurred at 77°C and higher; at 74°C only lo3 TCIDSowas inactivated (table 1). One of the 71°C HIV-inoculated plasma samples was found to contain 67% of the mean I4Ccpm and 67% of the HIV concentration of the other HIV 71°C sample. The
1s
a
Fig. 3.Inactivation of HIV, VSV and EMC virus. HIV, VSV and EMC virus, added to separate aliquots of human blood plasma, were processed with the Thermalizer at the indicated temperatures. Following treatment, viral titers were determined as described under methods.
data from this sample were not used for calculations. Other samples showed no evidence of dilution (see table 1). Constants H and A were calculated using equations 2 and 3 by trial and error employing two temperatures, 71 "C and 74°C. These temperatures were selected because no samples were taken below 71 "C and no virus was active above 74°C (fig. 3). For HIV, H = 6.76 X 10'' cal/ mol and A = 1.88 x lodsmol/(s."K). For EMC, temperatures of 66°C and 72°C were used to determine H = 2.19 x lo5 cal/mol and A = 1.26 x 10''' mol/(s."K). For VSV, temperatures of 69°C and 75°C were used to determineH = 1.2 X 105cal/molandA= 3.6X 107Xmol/(s~oK).
Recovery of Coagulation Factors The recovery of coagulation factors V, VIII and IX during two separate runs is given in figures 4a-c. The protein activity was 'Ompared against an untreated freshfrozen plasma sample. Of the factors measured, factor v was the most labile, with 60% of initial activity lost at 75-77"C (fig. 4a). It is interesting that treatment at ternperatures between 7 8 0 and ~ 84°C resulted in increased factor V activity. This increase was suggestive of activation. Excellent recovery of factor VIII and factor IX activities was observed at temperatures as high as 77°C and 89"C, respectively (fig. 4b,c). As with the virus, 75°C and 84°C was used to determine for factor VIII: H = 87.400 cal/mol and A 7.55 X
16
C
Fig. 4. Recovery of coagulation factors. Plasma processed with the Thermalizer at the indicated temperatures was assayed for factor V(a), factor VIII (b) and factor IX (c). Results are expressed as percent recovery ascompared with untreatedcontrols. Mock control is a sample of plasma processed in an unheated Thermalizer with no heat applied.
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
b
Fig. 6. Immunoelectrophoresis of heated'plasma. Plasma heated, with the Thermalizer was analyzed by using rabbit anti-human serum antibody. The samples were treated as indicated.
Fig. 5. SDS-PAGE of heated plasma. Plasma heated with the Thermalizer was subjected to SDS-PAGE and stained with Coomassie Blue. Lane 1, molecular weight standard; lane 2, untreated control; lane 3, mock control; lanes 4-7, heated at 69,75,81, and 86"C, respectively.
The same samples, when analyzed by crossed immunoelectrophoresis (fig. 6), showed some changes at 81"C and 86"C, and possibly some slight changes at 69°C and 75°C. For example, in comparison to the mock control, peak 2 appears unchanged when heated at 690c,but becomes progressively less distinct at higher temperatures; peak 3 starts sharp and ends diffuse; peak 6 is lost at 75 "C and peaks 8 and 9 are maintained at 81"C and not 86°C.
Gamma Globulins Cellulose acetate electrophoresis revealed no changes mol/(s."K). Samples taken at 71°C and 74°C determined in any of the samples, including heating at a temperature for factor V H = 1.42 x 10' cal/mol and A = 8.71 x lo9" up to 86°C. In each case, the percent protein in the gamma globulin region was 10.4-11.4% of the total. No pattern mol/(s .O K ) . was observed in these data. To assess IgG antibody reactivity, the titer of anti-HBs Electrophoretic Analysis Normal fresh-frozen plasma treated with the Thermal- antibody was determined in plasma treated at temperizer was subjected to SDS-PAGE (fig. 5). At temperatures atures between 69°C and 87°C (table2). The antibody as high as 75"C, little if any difference could be discerned. titer remained constant on heating at temperatures as high At 86°C a slow migrating form and loss of a band with as 80°C. A 20% decline in titer was observed on heating at 87°C. molecular weight above 200,000 could be discerned.
17
ZgG Concentrate and HPLC Analysis An IgG concentrate was processed in the Thermalizer TL-10 at temperatures between 65°C and 85°C. Samples subjected to 77°C for 0.006 s were found to have no significant aggregation when compared with a nontreated control (fig. 7). HIV Antigen Activity The pasteurized plasma samples were tested for the presence of HIV p55 antigen. All the samples tested positive for the antigen including those processed at 77°C where there was more than 4.4 log reduction of virus concentration (table 3). Fig. 7. Pasteurization of IgG concentrate. IgG concentrate heated by thermalizer was analyzed for aggregation by HPLC.
Discussion
Fig. 8. Calculation of HIV log reduction rate over full pasteurization process indicating effective hold time.
Table 2. Effect Sample of heating on ANTI-HBs reactivity Start 69°C 72°C 75 "C 80"C 87 "C
Table 3. ELISA tests on active and heat-inactivated HIV
18
Anti-HBsAg mIU/ml 41.2 43.1 38.9 40.9 42.2 33.4
Sample
ELISA p55 antigen titer
Start 71"C 74 "C 77°C
21:128 21:128 21:128 21:128
The data presented indicate that a high level of virus inactivation with modest to no changes in plasma components can be achieved with 0.006-second exposure times between 75°C and 78"C, while profound changes are observed at 81"C. All three viruses tested, including HIV, were reduced to less than the lowest detectable amount, i.e., a decrease of about 109 IDso, in this temperature range (table 1, fig. 3). This technique could be of special value in processing hyperimmune gamma globulin from high-risk plasma or in combination with other procedures such as solvent/detergent treatment. Although these 3 viruses were inactivated under the conditions used, other viruses may not. Porcine parvovirus requires 90°C 0.006 s for significant destruction (unpublished). This would not be a satisfactory condition for plasma proteins. However it is possible to operate the Thermalizer at less than 0.006 s so that plasma components may suffer even less destruction at higher temperatures. It is interesting that the H for factor VIII is in the range of activation energies reported for the heat denaturation of other proteins, e.g. ATPase is 70,000 callmol [24]. Energy of activation for factor VIII is 87,000 cal/mol. Equation 2, with H and A was used to check the experimental values of HIV and factor VIII for the process at 75°C and 0.006 s (fig. 8). The calculated values for HIV reduction and factor VIII recovery were found to compare favorably with experimental values, see tables 4 and 5. A range of process parameters was also used to calculate the estimated recovery of protein and inactivation of HIV. These
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
1 . 2 6 lo3 ~ 7.94 x 10 6 . 3 1 10' ~ l.loxloo
71°C 73"C 74°C 77°C
'
experimental
calculated
min.
max .
-1.65 -1.85 -2.95 4.71
-1.64 -2.38 -2.93 4.49
-1.33 -1.83 -2.21 -4.22
-2.34 -3.61 -4.56 -9.57
A = 1.88~10"mol/(s . "K). H = 1.71x1OJcal/mol. Using hold times of 0.004 s, 0.008 s and various temperatures f 1°C.
ranges account for uncertainty in measurement of hold time (k0.002 s) and temperature (k1"C). The terms in equation 2 can be evaluated separately to determine the amount of reduction due to each part of the process. A large portion of the HIV reduction occurs during the heat-up time (40% of all reduction), almost as much as occurs during the hold time (53% of all reduction). This is due to the shape of the time-temperature profile (fig. 2). The heat-up time is governed by equation 4 dT MC---=P dO
(4)
Table 5. Comparison of experimental and calculated values for factor VIII recovery
Temperature 69°C 75°C 80°C 84°C
I
*
Recovery, calculated, % '
Recovery, % experimental
calculated
min. / max.
84 90 51 13
97 90 59 13
96 83 38 2
-
97
- 93 - 71 -
27
A = 7.55xlO"moV(s O K ) . H = 8.74~10"cal/mol. Using hold times of 0.004 s, 0.008 s and various temperatures 1°C.
or
where P = power absorbed (gm.caYs) M = flow rate (cm3/s) T = temperature at time 0 ("C) To = initial temperature ("C) C = specific heat (g.cal/g."C) The data presented indicate that ultra-short-time heating at higher temperatures results in a high level of virus kill, including HIV, under conditions which appear to maintain protein structure and function. An antibody test suggested the antigen was still intact in the 77°C sample although there was about a 5-log reduction of HIV. Using the Arrhenius-derived constants H and A, the log reduction rate Of activity (LRR) as a tion of temperature was calculated for various viruses and factor VIII and factor V (fig. 9). 7
Figrn9. Idealized log rate of reduction of protein and virus using the Arrhenius equation and constants calculated from the experimental data.
19
The LRR expressed in seconds is equal to the Arrhenius constant for each component. Comparing LRR for viruses and plasma components, the differences in some cases become less as temperature increases. The optimum temperature is where the minimum reduction of plasma component activity occurs in the time for a prescribed reduction in viruses, e.g. 6 log cycles. In this case, ‘time’ refers to the ‘effective time’ at hold temperature, i.e., all the inactivation during ‘heat-up’ and ‘cool-down’ is converted to equivalent time at the ‘hold temperature’. In figure 8, the area enclosed by a full curve is equivalent to the area enclosed by the rectangle showing the effective hold time, graphically noted. For the viruses and components studies, 75-76OC, an effective time of 13 ms is the optimum range resulting in a 6 log cycle reduction of HIV and greater reduction for other viruses studied. A plot of log reduction rate vs. temperature for various viruses and plasma components shows inactivation rates vary considerably as temperature increases. At higher temperatures the rates of destruction of plasma components and some viruses approach each other (note fac-
tor V and HIV at 85OC). While at the lower temperatures (60°C), the rates of inactivation of VSV and factor VIII approach each other (fig. 9). About 50-90% of clotting factors were recovered after pasteurization. Variation in clotting factor assays presented uncertainties particularly about the 50% recovery associated with factor V. The activation of factor V at higher temperatures, e.g., 85”C, also introduces uncertainty in interpretation. Samples analyzed by SDS-PAGE showed some changes at the higher temperatures. Similar changes were observed by crossed immunoelectrophoresis analysis. Further studies are required including the validation of HBV and HCV inactivation, and the demonstration of the absence of neo-immunogens, and clinical studies assessing recovery, circulatory survival and efficacy, prior to routine use of HTST in the preparation of blood derivatives. Nonetheless, these preliminary results encourage the continued exploration of high temperature, short-duration heating technology.
..................................................................................................................................................... References Gerety RJ. Aronson DL: Plasma derivatives and viral hepatitis. Transfusion 1982:22:347351. Menache D, Aronson DL: Measures to inactivate viral contaminants of pooled plasma products: in Dodd RY, Barker LF (eds): Infection Immunity. and Blood Transfusion. New York. Liss, 1985. pp407423. Gomperts ED: Concise Review: Procedures for the inactivation of viruses in clotting factor concentrates. Am J Hematol 1986;23:295305. Prince AM. Horowitz B, Horowitz MS, Zang E: The development of virus-free labile blood derivatives - A review. Eur J Epidemiol 1987:3:103-1 18. Burnouf T, Martinache L, Goudemand M: L‘inactivation des virus dans les fractions plasmatiques a usage thirapeutique. Nouv Rev Fr Hematol 1987:29:93-96. Mannucci PM, Colombo M: Virucidal treatment of clotting factor concentrates. Lancet I988 :ii :782-78s. Horowitz B: Blood protein derivatives. A new era of safety. Yale J Biol Med, in press. Hilfenhaus J, Mauler R, Friis R. Bauer H: Safety of Human Blood Products. Inactivation of retroviruses by heat treatment at 60°C (42045). Proc SOCExp Biol Med 1985:178: 58c.584.
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9 Rubinstein A: Treatment of factor VIII concentrate to minimize the effect of undesirable micro organisms. US Patent No 4,556,558 ( 1985). 10 Hollinger B. D o h a G. Thomas W, Gyorkey F: Reduction in risk of hepatitis transmission by heat-treatment of a human factor VIII concentrate. J Infect Dis 1984:150:250-262. 11 Alfa Laval: Sterimedia Mini. Continuous Sterilization in Laboratory, Product Brochure, 1986. 12 Horowitz B. Wiebe M, Lippin A, Stryker M: Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri-(n-butyl) phosphate detergent combinations. Transfusion 1985:25:516522. 13 Edwards CA. Piet MPJ. Chin S , Horowitz B: Tri(n-butyl) phosphate detergent treatment of licensed therapeutic and experimental blood derivatives. Vox Sang 1987:52:53-59. 14 Horowitz MS, Horowitz B, Rooks C. Hilgartner M W Virus safety of solvent/detergent-treated antihaemophilic factor concentrate. Lancet 1988:ii:186-189. 15 Morgenthaler JJ (ed): Virus Inactivation in Plasma Products. Current Studies in Hematology and Blood Transfusion. Basel, Karger. 1989, VOI 56. pp 83-96.
16 McDougal JS, Martin LS, Cort SP, Mozen M. Heldebrant CM. Evatt BL: Thermal inactivation of the acquired immunodeficiency syndrome virus, human T lymphotropic virus-Ill/ lymphadenopathy-associated birus. with special reference to antihemophilic factor. J Clin Invest 1985:76:875-877. 17 Charm SE, Landau SH: Thermalizer: Hightemperature short-time steriliLation of heatsensitive biological materials. Biochem Eng 1987:5:608-612. 18 Lenahan JG. Phillips GE: Use of the activated partial thromboplastin time and the control of heparin administration. Clin Chem 1966;12: 269. 19 Miale JB: Laboratory Medicine: Hematology. ed 6. St Louis Mosby, 1982. 20 Laemmli UK: Cleavage of striictural proteins during the assembly of the head of bacteriophage T4. Nature 1970:227:680-685. 21 Laurel1 CB: Antigen-antibody crossed electrophoresis. Anal Biochem 1965: 10:358-361. 22 Kato Y, Komiya K, Sasaki H. Hasimoto T Separation range and separation efficiency in high-speed gel filtration on TSK-Gel SW columns. J Chromatogr 1980;190:297-303. 23 Spearman C: The method of right and wrong cases* (constant stimuli’) without Gauss’s formulae. Br J Psycho1 1908;2:227-242. 24 Laidler KJ. Bunting PS: The chemical kinetics of enzyme action, ed. 2. London. Oxford University Press 1973. p 430.
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
Strinley E. Charma Steven Landau a Bolanle Williams’ Bernard Horowitz Alfred M . Prince’ Donna Pclscual
’
Charm Bioengineering, Inc., Malden, Mass., USA The New York Blood Center, New York, NY, USA
High-Temperature Short-Time Heat Inactivation of HIV and Other Viruses in Human Blood Plasma ................................................................................................. Abstract
An ultra-short-time heating system was used to process blood plasma spiked with various viruses (HIV, vesicular stomatitis virus, encephalomyocarditis virus). Virus reduction and recovery of plasma proteins were measured at various temperatures from 65 to 85°C. Processing at 77°C and 0.006 s resulted in a high level of virus kill, including 2 4.4 log,, HIV, while maintaining protein structure and activity essentially intact.
.....................
Introduction
With the exception of intramuscular immune globulin and albumin solutions [l], the transmission of viruses by plasma and plasma fractions has been a serious concern. While implementation of improved procedures for donor selection and donor blood screening can reduce viral risk, assurance of virus safety can only come from the introduction of virucidal procedures into manufacture; thus several methods of inactivation have come into routine use. With the best of these methods, the transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and non-A, non-B hepatitis viruses (NANBHV), including hepatitis C virus (HCV), has been greatly reduced and possibly eliminated [2-7, reviews]. When heating coagulation o r other labile factors at high temperatures, avoidance of loss of biological activity requires the prior addition of stabilizers such as sucrose and glycine [8], or the removal of water through lyophilization [9, lo]. Unfortunately, both approaches afford considerable protection to viruses.
Supported in part by NHLBI Grant No. HWI22I to The New York Blood Center Norrmber 16. 1990.
Received: Dec. 11. lPY0 Revised manuscript received: February 27. I W I Accepted: June 11. 1YYI
Advances in the application of high-temperature, short-time heating (HTST), both for sterilization and pasteurization, show that heat-sensitive materials including vitamins and heat-labile food flavors may be preserved as food is rendered safe from pathogens. A common HTST process for sterilization is 145°C for 2-4 s, and for pasteurization is 15 s at 65°C. The preservation of sensitive material and destruction of pathogens is based on their differences in rates of destruction. The rates of destruction vary with temperature. In order for the HTST process to be successful, the pathogen destruction rate must be much greater than the destruction rate of ‘sensitive’ material under the process conditions. When this is the case, it follows that the pathogen will suffer greater destruction than the heat-sensitive material. The HTST systems currently available are inadequate to achieve the time-temperature relationship that would permit the preservation of biological activity in blood products with the satisfactory reduction of contaminating viruses. Problems with the current systems include (a) the
Stanley E Charm Charm Bioengineenng. Inc. 36 Franklin Street Maldcn. MA 02148 (USA)
G 19Y2 S Karger AG Basel ~xu2-90(17/Y2/0b2I-l~1~
$ 2 730
relatively long time needed to achieve the process temperature, (b) the relatively long hold-up time at temperature, (c) the relatively long time to cool to nondestructive temperature. The commercially available Sterimedia-Mini, and Steri-Lab (Alfa Laval, Lund, Sweden) have hold times of 2-4 s or more [ll]. The currently available systems also have a large holdup volume, making it necessary to waste liters of product during processing. The Sterimedia-Mini has a hold-up volume of 1.5 liters, while the Thermalizer TL-10 can process samples of 10 ml with 80% recovery of sample volume. Chemical viral inactivation of blood fractions is routinely accomplished today using the organic solvent, tri(nbutyl) phosphate (TNBP)/detergent mixtures [12, 131. While excellent safety has been achieved with regard to HBV, HIV, and NANBHV [14,15], the major weakness of this solvent detergent approach is the inability of the process to inactivate non-lipid-membrane-coated viruses [QI. In our ongoing effort to explore processing methods which might further improve the virus safety of blood products, studies on HTST pasteurization have been initiated. The time fOr90% destruction of HIV has been found to be 24s at 60°C in a solution of A H F [16]. A prototype short-time system for processing plasma with a heating rate of 45"C/s and a hold time of 0.03 s caused 75% destruction of factor VIII [17]. This new work describes an improved ultra-short-time system developed to overcome the problems associated with earlier systems and presents our results on the heating of plasma. The inactivation kinetics of viruses and proteins may be expressed using the Arrhenius equation (1). However, in this case, the heat-up and cool-down timetemperature effects must be considered because they are in the same order of magnitude (see equation 2). Equation 2 can be used to estimate the destruction of both the proteins ands viruses for any pasteurization process.
I
0
where N/N, = active fraction at time 0,or virus H = energy of activation for destruction (cal/mol) R = ideal gas constant, = 1.987 cal/(mol."K) =
uc
T = temperature (OK) 0 = time during process (s) A = constant (mol)/s. "k) Equation 1.
By taking the ratio of equation 2 for two different holding temperatures, constant A is eliminated and,
+ Hold + Coo1)a (Heat + Hold + Cool)b
ln(N&J)a - (Heat ln(NJN)b
(3)
Using a trial and error method with equation 3, values for H can be determined, and equation 2 is then used to determine A . Methods Therrnalizer TL-10 HTST System Description The Thermalizer TL-10 (Charm Bioengineering, Inc. Malden, Mass., USA) is a HTST pasteurization system. Plasma, pumped continuously from a reservoir through the heatingsection, was heated to various temperatures between 60 and 90°C (fig. 1).held at the high temperature for a very short time, then immediately cooled in a heat exchanger. The TL-10 was engineered to minimize exposure time of the fluid to high temperatures, giving a fast spike of heat. It is also useful to be able to conduct this process on small samples, e.g. as little as 10 ml.
Equation 2.
13
Heating energy is produced by a 1.5-kW Air Cooled microwave generator. A Teflon tube is positioned inside the microwave field to achieve maximum energy transfer into the fluid. The final fluid temperature is controlled by adjusting the output power of the microwave generator. Fluid is held at processing temperature in the tube fitting between the microwave heater and the cooling section. Standard design of the holding tube gives a hold volume of 0.013 k 0.003 cm’. The hold time can be increased by coiling the Teflon heating tube between the heating and cooling systems. The plasma pasteurization experiments were all conducted with the system setup for minimum hold time. The product is cooled in a single stage shell and tube heat exchanger. Ice water is circulated throughout the outer shell while product flows through an inner stainless steel tube.
Fig. 1. Thermalizer, flow diagram.
Fig. 2. Thermalizer TL-10, time-temperature profile. Pasteurization process.
Pumps, valves, tubing and instrumentation are built on a mobile stainless steel frame with wheels. The Thermalizer is constructed usingo. 159-cmOD, 0.079-cmID316Lstainless tubing. Plastic tubing, PFA Teflon tubing, 0.159-cm OD, 0.079-cm ID is used in the heating section and injector loops. Rheodyne (Cotati, Calif., USA) stainless steel HPLC-type switchingvalves are used to control the flow through the injector loops and sample collection ports. HPLC-type stainless steel compression fittings (Swagelok. Highland Heights. Ohio, USA) 1/16 inch are used throughout the system. Two pressure vessels hold product and the rinse solution. These vesselsare pressurized to600 psi to forcesolution through the system. The pressure is provided by compressed dry nitrogen. Each tank is jacketed for recirculating water temperature control and equipped with pressure relief valves, level sensors, imput-output ports. and a temperature probe.
14
Processing Details Fresh-frozen human plasma obtained from New York Blood Center (Melville, N.Y., USA) was filtered through a 40-pm. then a 0.45pm membrane before entering the Thermalizer. Concentrated virus solutions were added to 200-ml batches of the filtered plasma to make virus-containing plasma samples. Virus-free plasma was first pumped through the TL-10 to achieve steady state at a flow rate of 170 mllmin. After 20 s of steady temperature and flow, a 10-ml bolusofplasma with viruswasinjected into the flowing stream using an injector loop. The exit time of the 10-ml sample from the TL-10 was determined by previously collected samples of injected dye or radioactive tracer. Automatic timed sampling valves collected the sample from the fluid stream. A small amount of “C-glucose wasadded to the HIV-contaminated plasma todetermine if any dilution of the sample occurred. When the peak heating temperature is too high, the heat exchanger may be progressively fouled by build-up of material inside the tubing. This is indicated by a reduced volume in the collected sample. When this happens, the Teflon heating tube is replaced, thermocouple cleaned, and the heat exchanger is rinsed with NaCI 0.9%. The hold time at temperature was 0.006 k 0.002 s (fig. 2). Hold time was calculated using the measured volume inside the fittings between the microwave heating section and heat exchanger inlet, and the measured flow rate. The range k 0.002 s was calculated by determining the uncertainties of the hold tube volume, and flow rate measurements. The Reynolds number in the heating, hold. and cooling sections was 4.500, indicating turbulent flow. A uniform residence time in the hold tube was assumed. It is not known whether any backmixing occurred. Temperature was measured using0.025-cm T-type thermocouples inserted into the fluid stream through a compression T fitting. The thermocouple accuracy was ? 1°C. Flow rate was measured by collecting samples in a graduated test tube for 2 s. The time-temperature profile was calculated knowing flow rate and the tubing volumes in each section of the process system. A typical temperature-time curve is shown in figure 2. Collected samples were frozen at -70°C in an alcohol/dry ice mixture and stored at -70°C until assayed. Protein Analysis Assay chemicals were reagent grade. The activity of coagulation factors VIII and IX was assessed by determining the degree of correction in activated partial thromboplastin time (General Diagnostics. Morris Plains, N.J.. USA) of the
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
appropriate factor-deficient plasma (George King, Overland Park, Kans., USA). The activity of factor V was assessed similarly except a nonactivated partial thromboplastin reagent (General Diagnostics) was used [18. 191. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of 1% dithiothreitol. essentially as described [20], and proteins were stained with Coomassie blue. Crossed immunoelectrophoresis was performed as described [21] using 1% agarose (Seakem, FMC Corp, Rockland Me., USA) in tricine buffer IV (BioRad. Rockville Center, N.Y.. USA) applied to Gelbond film (10 x 15 cm: FMC Corp.). Electrophoresis in the first dimension was at 20 mA per plate and proceeded until a bromophenol blue marking dye migrated two-thirds of the plate length. Agarose above the sample lane was removed and replaced with agarose containing rabbit anti-normal human serum antibody (Calbiochem, San Diego. Calif.. USA). An immunoglobulin preparation dissolved at a concentration of 2.5% (wlv) in a solution of 0.5% (wlv) human albumin, 2.5% (wlv) sucrose 10 mM NaH2P0, and 0.4% (wlv) NaCl at a pH of 5.5 was kindly provided by Dr. James McIver (Massachusetts Biologic Laboratories, Jamaica Plain, Mass., USA). IgG aggregation was assessed on a TSKG3000SW HPLC [22] by integrating the peak area of a known concentration of IgG monomer. Ailtibody A.~snys The samples were assayed for antibody to hepatitis B surface antigen (anti-HBs) reactivity by an Ausab solid-phase radioimmunoassay technique (Abbott Laboratories, Abbott Park, Ill., USA). Viriises mid Virits A.ssny.s Vesicular stomatitis virus (VSV) was obtained from Dr. William Stewart. 11. and cultured in mouse L929 cells. Encephalomyocarditis virus (EMC) was obtained from Dr. Emilio Emini. and stocks were prepared by culturing the virus in mouse L929 cells. Infectivity was assessed by endpoint dilution assay with tenfold serial dilutions in culture medium (MEM). Each dilution was used to inoculate eight replicate wells of human A549 cells in 96-well microtiter plates. Virus-induced cytopathology was scored after 72 h of incubation at 37°C in 5% CO,. The ID,"value was calculated using the Spearman-Karber method 123). High titer stocks of the HTLV IIIhstrain of HIV were purchased from Pharmacia (Piscataway, N. J.. USA). Titrations were carriedout with serial. tenfold dilutions in microtiter plates using RPMI 1640 containing 10% FCS, with CEM cells at a concentration of 8 x 10"lml. Each sample was assayed in quadruplicate. Before use, cells were conditioned by incubation for 1 h at 37°C in the above medium containing2pglml of polybrene. Virusin treatedsampleswasadsorbed to cellsfor2 hat37'Candthencultureswere washedtwiceinmediumby centrifuging plates for 10 min at 2,000 rpm and aspirating the supernatants. Cultures (IS0 $) were then fed with 25 pl of medium at 4.7, and 10 days. At 14 days, cultures were washed twice with PBS to remove viral antigens carried over from the inoculum. and the cells were lysed in 100 pl PBS containing0.5% Triton X-100. Lysateswere assayed for HIV p5S antigens by ELISA using plates coated with rabbit antiserum against recombinant pSS (kindly provided by Dr. Hardy Chan. Syntex Corp.. Palo Alto, Calif.. USA) and peroxidaselabeled rabbit anti-p55.
Table 1. Inactivation of HIV in plasma after treatment with the thermalizer (log TCID 5 0 ) Sample
HIV titer
Start Flow control 71 "C 71 "C 73"C 74°C 77 "C 78 "C 80°C Mean
4.8 4.75 3.2 3.0 2.9 1.8 50.04 50.04 10.04
U
HIV reduction
-
0.05 1.6 1.8 1.9 3.0 24.4 24.4 24.4
'I C tracer CPm
2,720 2,666 2.736 1,763 ' 2,677 2,723 2,SYY 2,744 2,644 2,456 57
'
This sample had 67% of the mean ''C cpm and 67% of the other 71°C sample HIV titer. It was not included in the Arrhenius calculations.
HIV Antigen Assay The HIV-inoculated plasma samples, along with positive and negative standard controls, were assayed for HIV p55 antigens by ELISAusingplatescoatedwithrabbit antiserum against recombinant p55. The assay is controlled by doing a 2-fold dilution of a standard HIV stock which has a concentration of 24 ng/ml. The titration of this standard must be between 1:64 and 1:256 (or 0.37 and 0.09 ng/ml). This sensitivity is equal to that of the Dupont p24 kit. Positive cut-off of the samples is twice the average of the negative control wells.
Results
Virus Inactivation Each of two viruses, a lipid enveloped virus, VSV, and a nonenveloped virus, EMC, was added to whole, filtered plasma and heated separately in the Thermalizer at varying temperatures. Total process duration was 0.250 s with 0.006 s at 'hold temperature'. The inactivation of detectable EMC was complete (210',' TCID'") at 72°C and above, and the inactivation of VSV was complete TCID'") at 75°C and above (fig. 3). HIV inactivation was assessed in a separate experiment. Complete inactivation (LlO'.' TCIDsO)of added HIV occurred at 77°C and higher; at 74°C only lo3 TCIDSowas inactivated (table 1). One of the 71°C HIV-inoculated plasma samples was found to contain 67% of the mean I4Ccpm and 67% of the HIV concentration of the other HIV 71°C sample. The
1s
a
Fig. 3.Inactivation of HIV, VSV and EMC virus. HIV, VSV and EMC virus, added to separate aliquots of human blood plasma, were processed with the Thermalizer at the indicated temperatures. Following treatment, viral titers were determined as described under methods.
data from this sample were not used for calculations. Other samples showed no evidence of dilution (see table 1). Constants H and A were calculated using equations 2 and 3 by trial and error employing two temperatures, 71 "C and 74°C. These temperatures were selected because no samples were taken below 71 "C and no virus was active above 74°C (fig. 3). For HIV, H = 6.76 X 10'' cal/ mol and A = 1.88 x lodsmol/(s."K). For EMC, temperatures of 66°C and 72°C were used to determine H = 2.19 x lo5 cal/mol and A = 1.26 x 10''' mol/(s."K). For VSV, temperatures of 69°C and 75°C were used to determineH = 1.2 X 105cal/molandA= 3.6X 107Xmol/(s~oK).
Recovery of Coagulation Factors The recovery of coagulation factors V, VIII and IX during two separate runs is given in figures 4a-c. The protein activity was 'Ompared against an untreated freshfrozen plasma sample. Of the factors measured, factor v was the most labile, with 60% of initial activity lost at 75-77"C (fig. 4a). It is interesting that treatment at ternperatures between 7 8 0 and ~ 84°C resulted in increased factor V activity. This increase was suggestive of activation. Excellent recovery of factor VIII and factor IX activities was observed at temperatures as high as 77°C and 89"C, respectively (fig. 4b,c). As with the virus, 75°C and 84°C was used to determine for factor VIII: H = 87.400 cal/mol and A 7.55 X
16
C
Fig. 4. Recovery of coagulation factors. Plasma processed with the Thermalizer at the indicated temperatures was assayed for factor V(a), factor VIII (b) and factor IX (c). Results are expressed as percent recovery ascompared with untreatedcontrols. Mock control is a sample of plasma processed in an unheated Thermalizer with no heat applied.
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
b
Fig. 6. Immunoelectrophoresis of heated'plasma. Plasma heated, with the Thermalizer was analyzed by using rabbit anti-human serum antibody. The samples were treated as indicated.
Fig. 5. SDS-PAGE of heated plasma. Plasma heated with the Thermalizer was subjected to SDS-PAGE and stained with Coomassie Blue. Lane 1, molecular weight standard; lane 2, untreated control; lane 3, mock control; lanes 4-7, heated at 69,75,81, and 86"C, respectively.
The same samples, when analyzed by crossed immunoelectrophoresis (fig. 6), showed some changes at 81"C and 86"C, and possibly some slight changes at 69°C and 75°C. For example, in comparison to the mock control, peak 2 appears unchanged when heated at 690c,but becomes progressively less distinct at higher temperatures; peak 3 starts sharp and ends diffuse; peak 6 is lost at 75 "C and peaks 8 and 9 are maintained at 81"C and not 86°C.
Gamma Globulins Cellulose acetate electrophoresis revealed no changes mol/(s."K). Samples taken at 71°C and 74°C determined in any of the samples, including heating at a temperature for factor V H = 1.42 x 10' cal/mol and A = 8.71 x lo9" up to 86°C. In each case, the percent protein in the gamma globulin region was 10.4-11.4% of the total. No pattern mol/(s .O K ) . was observed in these data. To assess IgG antibody reactivity, the titer of anti-HBs Electrophoretic Analysis Normal fresh-frozen plasma treated with the Thermal- antibody was determined in plasma treated at temperizer was subjected to SDS-PAGE (fig. 5). At temperatures atures between 69°C and 87°C (table2). The antibody as high as 75"C, little if any difference could be discerned. titer remained constant on heating at temperatures as high At 86°C a slow migrating form and loss of a band with as 80°C. A 20% decline in titer was observed on heating at 87°C. molecular weight above 200,000 could be discerned.
17
ZgG Concentrate and HPLC Analysis An IgG concentrate was processed in the Thermalizer TL-10 at temperatures between 65°C and 85°C. Samples subjected to 77°C for 0.006 s were found to have no significant aggregation when compared with a nontreated control (fig. 7). HIV Antigen Activity The pasteurized plasma samples were tested for the presence of HIV p55 antigen. All the samples tested positive for the antigen including those processed at 77°C where there was more than 4.4 log reduction of virus concentration (table 3). Fig. 7. Pasteurization of IgG concentrate. IgG concentrate heated by thermalizer was analyzed for aggregation by HPLC.
Discussion
Fig. 8. Calculation of HIV log reduction rate over full pasteurization process indicating effective hold time.
Table 2. Effect Sample of heating on ANTI-HBs reactivity Start 69°C 72°C 75 "C 80"C 87 "C
Table 3. ELISA tests on active and heat-inactivated HIV
18
Anti-HBsAg mIU/ml 41.2 43.1 38.9 40.9 42.2 33.4
Sample
ELISA p55 antigen titer
Start 71"C 74 "C 77°C
21:128 21:128 21:128 21:128
The data presented indicate that a high level of virus inactivation with modest to no changes in plasma components can be achieved with 0.006-second exposure times between 75°C and 78"C, while profound changes are observed at 81"C. All three viruses tested, including HIV, were reduced to less than the lowest detectable amount, i.e., a decrease of about 109 IDso, in this temperature range (table 1, fig. 3). This technique could be of special value in processing hyperimmune gamma globulin from high-risk plasma or in combination with other procedures such as solvent/detergent treatment. Although these 3 viruses were inactivated under the conditions used, other viruses may not. Porcine parvovirus requires 90°C 0.006 s for significant destruction (unpublished). This would not be a satisfactory condition for plasma proteins. However it is possible to operate the Thermalizer at less than 0.006 s so that plasma components may suffer even less destruction at higher temperatures. It is interesting that the H for factor VIII is in the range of activation energies reported for the heat denaturation of other proteins, e.g. ATPase is 70,000 callmol [24]. Energy of activation for factor VIII is 87,000 cal/mol. Equation 2, with H and A was used to check the experimental values of HIV and factor VIII for the process at 75°C and 0.006 s (fig. 8). The calculated values for HIV reduction and factor VIII recovery were found to compare favorably with experimental values, see tables 4 and 5. A range of process parameters was also used to calculate the estimated recovery of protein and inactivation of HIV. These
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
1 . 2 6 lo3 ~ 7.94 x 10 6 . 3 1 10' ~ l.loxloo
71°C 73"C 74°C 77°C
'
experimental
calculated
min.
max .
-1.65 -1.85 -2.95 4.71
-1.64 -2.38 -2.93 4.49
-1.33 -1.83 -2.21 -4.22
-2.34 -3.61 -4.56 -9.57
A = 1.88~10"mol/(s . "K). H = 1.71x1OJcal/mol. Using hold times of 0.004 s, 0.008 s and various temperatures f 1°C.
ranges account for uncertainty in measurement of hold time (k0.002 s) and temperature (k1"C). The terms in equation 2 can be evaluated separately to determine the amount of reduction due to each part of the process. A large portion of the HIV reduction occurs during the heat-up time (40% of all reduction), almost as much as occurs during the hold time (53% of all reduction). This is due to the shape of the time-temperature profile (fig. 2). The heat-up time is governed by equation 4 dT MC---=P dO
(4)
Table 5. Comparison of experimental and calculated values for factor VIII recovery
Temperature 69°C 75°C 80°C 84°C
I
*
Recovery, calculated, % '
Recovery, % experimental
calculated
min. / max.
84 90 51 13
97 90 59 13
96 83 38 2
-
97
- 93 - 71 -
27
A = 7.55xlO"moV(s O K ) . H = 8.74~10"cal/mol. Using hold times of 0.004 s, 0.008 s and various temperatures 1°C.
or
where P = power absorbed (gm.caYs) M = flow rate (cm3/s) T = temperature at time 0 ("C) To = initial temperature ("C) C = specific heat (g.cal/g."C) The data presented indicate that ultra-short-time heating at higher temperatures results in a high level of virus kill, including HIV, under conditions which appear to maintain protein structure and function. An antibody test suggested the antigen was still intact in the 77°C sample although there was about a 5-log reduction of HIV. Using the Arrhenius-derived constants H and A, the log reduction rate Of activity (LRR) as a tion of temperature was calculated for various viruses and factor VIII and factor V (fig. 9). 7
Figrn9. Idealized log rate of reduction of protein and virus using the Arrhenius equation and constants calculated from the experimental data.
19
The LRR expressed in seconds is equal to the Arrhenius constant for each component. Comparing LRR for viruses and plasma components, the differences in some cases become less as temperature increases. The optimum temperature is where the minimum reduction of plasma component activity occurs in the time for a prescribed reduction in viruses, e.g. 6 log cycles. In this case, ‘time’ refers to the ‘effective time’ at hold temperature, i.e., all the inactivation during ‘heat-up’ and ‘cool-down’ is converted to equivalent time at the ‘hold temperature’. In figure 8, the area enclosed by a full curve is equivalent to the area enclosed by the rectangle showing the effective hold time, graphically noted. For the viruses and components studies, 75-76OC, an effective time of 13 ms is the optimum range resulting in a 6 log cycle reduction of HIV and greater reduction for other viruses studied. A plot of log reduction rate vs. temperature for various viruses and plasma components shows inactivation rates vary considerably as temperature increases. At higher temperatures the rates of destruction of plasma components and some viruses approach each other (note fac-
tor V and HIV at 85OC). While at the lower temperatures (60°C), the rates of inactivation of VSV and factor VIII approach each other (fig. 9). About 50-90% of clotting factors were recovered after pasteurization. Variation in clotting factor assays presented uncertainties particularly about the 50% recovery associated with factor V. The activation of factor V at higher temperatures, e.g., 85”C, also introduces uncertainty in interpretation. Samples analyzed by SDS-PAGE showed some changes at the higher temperatures. Similar changes were observed by crossed immunoelectrophoresis analysis. Further studies are required including the validation of HBV and HCV inactivation, and the demonstration of the absence of neo-immunogens, and clinical studies assessing recovery, circulatory survival and efficacy, prior to routine use of HTST in the preparation of blood derivatives. Nonetheless, these preliminary results encourage the continued exploration of high temperature, short-duration heating technology.
..................................................................................................................................................... References Gerety RJ. Aronson DL: Plasma derivatives and viral hepatitis. Transfusion 1982:22:347351. Menache D, Aronson DL: Measures to inactivate viral contaminants of pooled plasma products: in Dodd RY, Barker LF (eds): Infection Immunity. and Blood Transfusion. New York. Liss, 1985. pp407423. Gomperts ED: Concise Review: Procedures for the inactivation of viruses in clotting factor concentrates. Am J Hematol 1986;23:295305. Prince AM. Horowitz B, Horowitz MS, Zang E: The development of virus-free labile blood derivatives - A review. Eur J Epidemiol 1987:3:103-1 18. Burnouf T, Martinache L, Goudemand M: L‘inactivation des virus dans les fractions plasmatiques a usage thirapeutique. Nouv Rev Fr Hematol 1987:29:93-96. Mannucci PM, Colombo M: Virucidal treatment of clotting factor concentrates. Lancet I988 :ii :782-78s. Horowitz B: Blood protein derivatives. A new era of safety. Yale J Biol Med, in press. Hilfenhaus J, Mauler R, Friis R. Bauer H: Safety of Human Blood Products. Inactivation of retroviruses by heat treatment at 60°C (42045). Proc SOCExp Biol Med 1985:178: 58c.584.
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9 Rubinstein A: Treatment of factor VIII concentrate to minimize the effect of undesirable micro organisms. US Patent No 4,556,558 ( 1985). 10 Hollinger B. D o h a G. Thomas W, Gyorkey F: Reduction in risk of hepatitis transmission by heat-treatment of a human factor VIII concentrate. J Infect Dis 1984:150:250-262. 11 Alfa Laval: Sterimedia Mini. Continuous Sterilization in Laboratory, Product Brochure, 1986. 12 Horowitz B. Wiebe M, Lippin A, Stryker M: Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri-(n-butyl) phosphate detergent combinations. Transfusion 1985:25:516522. 13 Edwards CA. Piet MPJ. Chin S , Horowitz B: Tri(n-butyl) phosphate detergent treatment of licensed therapeutic and experimental blood derivatives. Vox Sang 1987:52:53-59. 14 Horowitz MS, Horowitz B, Rooks C. Hilgartner M W Virus safety of solvent/detergent-treated antihaemophilic factor concentrate. Lancet 1988:ii:186-189. 15 Morgenthaler JJ (ed): Virus Inactivation in Plasma Products. Current Studies in Hematology and Blood Transfusion. Basel, Karger. 1989, VOI 56. pp 83-96.
16 McDougal JS, Martin LS, Cort SP, Mozen M. Heldebrant CM. Evatt BL: Thermal inactivation of the acquired immunodeficiency syndrome virus, human T lymphotropic virus-Ill/ lymphadenopathy-associated birus. with special reference to antihemophilic factor. J Clin Invest 1985:76:875-877. 17 Charm SE, Landau SH: Thermalizer: Hightemperature short-time steriliLation of heatsensitive biological materials. Biochem Eng 1987:5:608-612. 18 Lenahan JG. Phillips GE: Use of the activated partial thromboplastin time and the control of heparin administration. Clin Chem 1966;12: 269. 19 Miale JB: Laboratory Medicine: Hematology. ed 6. St Louis Mosby, 1982. 20 Laemmli UK: Cleavage of striictural proteins during the assembly of the head of bacteriophage T4. Nature 1970:227:680-685. 21 Laurel1 CB: Antigen-antibody crossed electrophoresis. Anal Biochem 1965: 10:358-361. 22 Kato Y, Komiya K, Sasaki H. Hasimoto T Separation range and separation efficiency in high-speed gel filtration on TSK-Gel SW columns. J Chromatogr 1980;190:297-303. 23 Spearman C: The method of right and wrong cases* (constant stimuli’) without Gauss’s formulae. Br J Psycho1 1908;2:227-242. 24 Laidler KJ. Bunting PS: The chemical kinetics of enzyme action, ed. 2. London. Oxford University Press 1973. p 430.
Charm/Landau/Williams/Horowitz/Prince/ Heat Inactivation of Viruses Pascual
