CA1205012A - Method for the production of therapeutically administrable plasma derivatives filled in final containers - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:

There is disclosed a method for the production of thera-peutically administrable plasma derivatives filled in final containers, which are free of prekallikrein activa-tor, hypotensively active constituents and other undesired pharmacologically active substances, of plasma or plasma crude fractions by stepwise enrichment of the plasma pro-teins, sterile filtration and optionally virus inactiva-tion. In order to make available such plasma fractions that do not develop pharmacologically negative side ef-fects when applied and are producible with an improved activity and yield, C1-esterase inhibitor is added during the production process of the plasma derivative, yet prior to its filling into final containers.

Description

The invention relates to a method for the productlon of therapeutically adminis-trable plasma derivatives fllled in final containers, which are free of prekallikrein ac-tivator, hypotensively active const:Ltuents and other unde-sired pharmacologically active substances, in particular for the production of albumin, globulin and coagulation-factor preparations, of plasma or plasma crude frac-tions by stepwise enrichment of the plasma protelns, sterile filtration as well as, if desired, by virus inactivation.
The administration of blood derivatives of ~he -type mentioned may lead to undesired side reactions in pa-tien-ts, in particular to a spontaneous blood pressure decrease, which may reach dangerous extents. This hypotensive acti-vi-ty of the prepara-tions is assumed to go back to a content of proteins that can be elimlnated durlng plasma fractlon-action only partially or with high losses of the desired ac-tive subs-tances (cf. e.g. The New England Journal of Medicinel "Hypotension Associated with Prekallikrein Acti-vator (Hagemann-Factor Fragments~ in Plasma Pro-tein Frac-20 tion", Vol. 299, Alving e-t al., July 1978, pp. 6~ to 70).
The lndesired subs-tances with hypotensive effec-ts have been classified as fragments of the coagula-tion fac-tor XII (F XIIf~ on the one hand, their activi-ty having been denoted as "prekallikrein activator activity" (PKKA); and by substances which, as impurities in the preparations, may cause pharmacologically undesired side reac-tions and lead to reactions irrespective of PKKA activity, on -the other hand. The in vi-tro assayiny of PKKA ac-tivi-ty usually is realized via -the determina-tion of kallikrein (KK), 30 which is genera-ted from an inac-tive pres-tage (prekalli 5~2 krein) a~ter addition of E' XIIf. Kallikrein cleaves tri-peptide-based chromogen:ic substrate. The chromophore group libera-ted is photome-trically measured (cf~ Develop.
biol. Standard, ~ol. 44, pp~ 115 to 120, "The Assay of Prekallikrein-Activator in Human Blood Products", T.J.
Snape et al.). The content of PKKA in the test material is indirectly concluded and related to an In-ternational Stand-ard (Reference PKA Standard Lot 2 of the Bureau of Bio-logics, Bethesda, ~S~Ao) ~
Since side reactions depend not only from -the PKKA
content, further test methods suggesting in vivo reactions have been applied -to investigate blood derivatives. One of these methods is the kallikrein burst test, with which the sample is added to human plasma, the spontaneously occur-ring kallikrein liberation in the plasma being photo-metrically measured with chromogenic substrate S 2302 (Kabi) via the libera-tion of p-ni-troanilid from the chro-mogenic subs-trate. This kallikrein 1ibera-tion from plasma, as opposed to PKKA-induced kallikrein liberation from the inactive pre-stage, is to be assessed difierently~ since no kinetics is involved, bu-t it occurs spontaneously with-in the first minutes, disappearing again.
A further possibility to investigate into pharmaco-logical side reactions resides in the contraction a-t the isolated ileum of guineapigs. By the samples -to be tes-ted, substances are liberated in human plasma tha-t lead to a contraction at the isolated ileum.
It is known that -the ac-tivi-ty of the prekallikrein activator may be reduced or suppressed, if an inhibitor, i.e. C1-es-terase inhibitor (C1INA) is present. This C1-esterase inhibitor has been described in literature alsoas F XIIf inhibi-tor (cf. The Journal of Clinical Investiga-tion, Vol. 52, June 1973, pp. 1~02 to 1409, A.D. Schreiber et al.). The inhibitor is prepared from human plasma according to known modes of operation (Vox~ Sang. 26: 118 to 127 (1974), "Contributions to the Optimal Use of Human Blood"l ~.F. Vogelhaar et al.~.
It has already been proposed to add C1-esterase inhi-bitor to a stable plasma protein solution (PPL) immediate-ly before being applied to the patient. This ready-made applicable pxeparation has an ionic streng-th of 130 to 160 mequ/l. A disadvantage of this method, however, is that the C1-esteraae inhibitor is only insufficiently utilized.
The invention aims at avoiding this disadvantaye and moreover, has as its objec-t to make available not only al-bumin fractions (PPL) -that are free of prekallikrein act-vator activity, but also other plasma fractions that, in addition to being free of prekallikrein activator activity, are free of prekallikrein buxst active and ileum contract-ing substances, too; thus, they are no-t to develop pharma~
cologically negative side efects when applied and, more-over, are to be producible with an improved ac-tivity and yield.
This object is achieved according to -the inven-tion in that C1~esterase inhibi-tor is added during the production process of the plasma deriva-tive, yet prior to its filling into final con-tainers.
According to a preferred embodiment o~ the invention the addition of C1-esterase inhibitor is carried out at a ~2~

salt concentration of 5 to 200 Na~ mequ~l, preferably 10 to 50 Na~ mequ/l, and in a p~ ranye of 5 to 9. It has proved that the addition of Cl-esterase inhibitor enables a be-tter utilization of the inhibitor at the formation of the complex P~KA~C I~A with a lower sal-t concentration. A
further preferred embodiment of the production of albumin preparations consists in that the final containers, after having been filled with the produc-t, are subjected to a thermal virus inactivation for 10 hours at 60 C. This em-10 bodiment is based on the surprising finding that - al-though the C1-esterase inhibitor is thermally unstable -the complex compound of prekallikrein activator with C1-esterase inhibitor is thermally stable and resists thermal inactiva-tion, usually a thermal inactivation during 10 hours at 60 C. A further preferred embodiment consists in that the added C1-esterase inhibitor itself is virus-inactivated.
The assay methods wi-th respect to the freedom from side effects of the plasma derivatives produced according to the invention are to be performed as follows:
Determination of prekallikrein activator:
10 Method:
From a purified prekallikrein preparation (PKK) kal-likrein (KK) is generated by means of a prekallikrein ac-tivator (PKKA). Kallikrein amidolytically cleaves p-nitro anilid (pNA) from a specific chromogenic substrate. The concentra-tion of pNA is measured photome-trically at a wave length of ~05 nm~

2. Reagents:
Buffer I: 6.0 g TRIS and 23.38 g ~aCl are dissolved 30 in about 500 ml H20 dist. and adjusted to a pH of 8.0 by a~ --dilute ~ICl and filled up to 1,000 ml with H20 dis-t.
Buffer II: 1.81 g TRIS, 1.02 y ilnidazole and 6.43 g NaCl are dissolved in about 500 ml H20 dist. and adjusted to a pH of 7.9 by dilute HCl and filled up -to 1,000 ml with H20 dist.
Chromogenic substrate: S 2302 (Kabi) H-D-prolyl-1--phenylalanyl-L-arginin-p-nitroanilid-dihydrochloride. A
10 m molar aqueous solution is prepared. 25 mg S 2302 in 4.1 ml H20 dist.
Prekallikrein preparation: The produc-tion of the pre-paration is carried out according -to a prescription by Har-pel, modified by M.S. Horowitz (New York Blood Cen-ter).
Human citrated plasma is treated with the help of a DEAE
cellulose. The fraction that has not been bound to DEAE
cellulose contains the prekallikrein.
Positive control (standard): As standard (= reference value), an albumin preparation of the Bureau of Biologics (BoB) of the Food and Drug Administration, Bethesda, Mary-land 20205, U.S.A.,is used. This preparation contains a pre-kallikrein activator. The genera-tion of kallikrein by this BoB standard constitutes the reference value 1 and is equated to 100 %.
Sample: If necessary, the sample is used in the assay in a dissolved or dilute state.
Assay: In a water bath at a temperature of 37 C
100 ~l prekallikrein prepara-tion 50 jul buffer I
25 ~l sample are pipetted into a plastic tube. After an incubation -time of 15 min at 37 C

300 ,ul buffer II
50 ~l S 2302 substra-te are pipe-tted. This mxture is in-troduced into a photometer brought to a temperature of 37 C~ and the increase in -the optical density per minute (~OD/min) at a wave lenght o~
405 nm wi-th a layer thickness of 10 mm was measured. The activity of a sample (aOD/min) is expressed factorially-relative to the BoB standard having the number 1 - or in per cent of the BoB standard.
Determination of spontaneous kalllkrein liberation in plasma (burst reaction):
1. MethodO
From human plasma kallikrein (KK) is spontaneously (within the first minute) liberated by means of the added sample. Kallikrein amidolytically cleaves pNA from the specific chromogenic substrate S 2302. pNA is measured photometrically at 405 nm.
2. Reagents:
a) Human plasma from a normal donator collective b) Buffer II, chromogenic substrate as described in "De-termination of PKKA"
c) Sample is added to test mixture undilute or in precise-ly determined dilu-tions.
In order to be able to better detect the spontaneous~
ly generated kallikrein activities, i.e. to lower the de-tection limits, C1INA antlserum may be added to human plasma, which neu-tralizes the main inhibitor of -the kalli-krein burst activity. Thereby it is achieved that the ex-tent of activity oE the burst test is increased. Further-more, the added sample may contain enzymes that react wi-th ~ZI;1~2 the chromogenic substrate itself. This can be opposed by lowering this autoamudolytic activi-ty by certain inhibi tors, wherein not even the kallikrein burst activity is affected (e.g. aprotinin, trasylene).

3. Assayo At 37 C
0.25 ml human plasma 0.025 ml sample are pipetted into a plastic -tube and incubated for 1 min at 37 C, whereupon 0.25 ml buffer II
0.05 ml chromogenic su~strate S 2302 are immediately added, this mixture is introduced into a photometer brought to 37 C and the increase in the optic-al density per minute (~OD/min) at a wave length of ~05 nm with a 10 mm layer thickness is measured.
Determination of hypotensively active substances at the isolated guineapig ileum:
1. Method:
From human plasma, substances that produce reactions on the smooth muscles are liberated by means of sample to be tested (Handbook of Experimental Pharmacology, Ed. E.G.
Erdos, ~ol. XXV, Springer 1970). In order that the liber-ated reactive substances will not be degraded, thus evading being tested at the ileum, a suitable kininase inhibitor (e.g. D-3-mercap-to-2 methylpropionyl-L-prolin) is added to the reaction mixture.
2. Assay:
An isolated guineapig ileurn having a length of 20 rnm and a dia~leter of 5 mrn is introduced into a 10 ml organ ~ 7 ~

~f~ L2 bath (modified according to Schul~-Dahle) and calibrated as to its contractability by means of about ten subsequen-t histamine additions of 5 ng histamine each. Thereafter, the ileum is washed free from histamine.
In a water bath at 37 C a reaction mixture according to the following pipetting sequence is prepared:
100 ~l human plasma 250 ~l D-3-mercapto-2-methylpropionyl-L-prolin correspond-ing to 2 ~lg 440 Jul sample to be tested ar~ mixed and incubated for 10 min at 37 C. Subsequently, this reaction mixture is applied onto the ileum in the or-gan bath. If reactive substances are liberated by the ad-ded sample, the ileum will contract. As the measure for the contraction, the ampli-tude that is reached within 30 s after the addition of the reaction mixture onto the ileum is measured in millimeters, the calibration being fixed at 0.315 mN per mm of aplitude with a measurement amplifica-tion of 5 mV over a scaie length of 250 mm.
The method according to the invention is going to be explained by the following examples.
Example 1:
Preparation of an albumin fraction:
8 % ethanol are added to 10 ml human blood plasma at a pH of 7.0 and a temperature of -2 C, a precipitate con-taining fibrinogen depositing. After separa-tion of this precipitate the ethanol concentration was raised to 25 %
and the temperature was lowered to -6 C. The precipita-te depositing, which contains immune globulin, is separated and the ethanol concentration of the superna-tant is raised to 40 ~ at a p~ of 6.5 and a temperature of -8 C.
The formed precipitate is separated and discarded.
The pH of the supernatant is adjusted to 5.4 at the same temperature, with al~umin precipitatiny. The latter is se-para-ted by centrifuga-tion and subjected to a further step of purification: the precipitate is dissolved in water and the ethanol concentration is adjusted to 10 % at a pH of

4.8 and a temperature of -2 C. The precipitated globulin is separated and discarded. The ethanol concentration of the supernatant is increased to 40 %, the temperature is lowered to --~ C and the pH is adjusted to 5.1.
The albumin precipitate is collected by centrifuga-tion and, if desired after intermediate lyophilization, is further treated according to the invention in the follo-wing manner:
The albumin concentrate is dissolved in NaCl solution, a 4.3 % protein solution having a pH of 6.9 and a salt con-centration of 140 mequ Na /l being obtained. Portions of the solution are admixed with C1INA in order to obtain a concentration series of 1. 0.01 U C1INA per g protein 2. 0.1 U C1INA per g protein 3. 1.0 U C1INA per g protein 4. 10.0 U C1INA per g protein.
The mixtures remain standing at room temperature for a-bout 24 hours. Thereafter, stabilizing agents (Na-capry-late and Na-acetyl tryp-tophanate) are added -to the solu-tions in a conventional manner, they are sterile fil-tered and heated at 60 C for 10 hours. A parallelly treated al~
bumin sample of -the same batch withou-t addi-tion of C1INA
g serves as reference.
One unit of C1 inhibitor (U) corresponds to the amount of C1 inhibitors contained in 1 ml of fresh plasma.

o s~ a o o ~
a~ r-l ~ ~
R~ ~ `1 ~ ~ O O
O ~ ~
~ F:
a) ~ ~
~ O
H ~ .

O
10 'J.
o t~ ~
a) o O ~ o~ ~r O O O
U~ O ~ ~_

5~ 0 m ~

a) td o ~ ~
O ~ ~ ~ ~ f'~ ~ O
U~ , ~ ~9 ~ 0~o ~
m o ~ ri m a ,~
,~ o ~ ~ a~
a) ~ ~, ,~
a) ~ 1 ~ a) ~ o -r~ .0 rl a 0 3 3 ~ 3 ~o) 3 ~0 3 Q
~ ~ ~ R~ R~
O O ~: O O Q~ 0 0 rl rl O ~1 ~ rl -rl ~ ~1 ~¢
~) ~ rl ~ ~ ~ tn l ) ~ ~ Z
rl ~ Z, ~ ~ ~ Z
O O ~ O H O ~; O H O C,) U~ H U~ U~

~ ~ Z~ O
,~ ,~ H Q O Q ~ n o Q o 'C O ~ O ~C ~ ~C
Heated at 60C :Eor 10 hours _x m~ 2:
Preparation of an al.bum.in fraction-An albumin fraction resultiny from the alcohol frac-tionation - as described in Example 1 - is dissolved in an aqueous solution, a solution of 5 % pro-tein conten-t and a Na concentration of about 10 mequ/l being obtained. The pH of the solution amounts to 7.2.
This solution is admixed with C1INA in order to ob-tai.n a concentration series of 1. 0.01 U C1INA/g protein 2. 0.1 U C1INA/g protein 3. 1.0 U C1INA/g protein 4. 10.0 U C1INA/g protein.
The mixtures remain standing at 37 C for about 24 hours, are then admixed with stabilizing .agents as in Example 1, brought to a Na~ concentration of 130 -to 160 mequ/l, steri].e filtered and heated at 60 C for 10 hours.

:~z~

o ~ ~
~ o ~
o o r Q
o ~ o o o o o c .,~ ,1 v aJ o 1-- ~ ~ O
~ o m s~
4~ ~ ~D
~ o ~
O ~O ~r o o 0 0\o ~i ~
~, ,1 m ~4 o m 3 o 3 ~ 3 ~
~, o o o o o ~ o Z ~ ~i ~ ~
O O H O æ o z U~ Ui ~ U~ H U~ H
O ~ ,.
r~
1 ~ o ,q ~ .q o ~i ~i O ~i O ~i ~
E~eated at 60C :Eor 1 0 hours ~2~5Q~2 Example 3:
Preparation of an irnmune globulin :fraction:
8 % ethanol is added to 10 l of h~an ~lood plasma at a pH of 7.0 and a temperature of -2 C, a prec:ipitate con-taining fibrinogen depositing. After separation of this precipitate, the ethanol concentration is raised -to 25 %
and the temperature is lowered to -6 C. The precipitate depositing, substantially comprised of immune globulin,is suspended in a phosphate acetate buffer and is admixed with 12 % ethanol at a pH of 5.3 and a temperature of -2 C. The precipitate depositing, which contains ~ and ~
globulin, is discarded; then, the ethanol concentration of the supernatant is increased to 25 % at a pH of 7.0 and a temperature of -6 C, whereby immune globulin is precipi-tated. The thus obtained immune globulin is collected, if desired lyophilized, and further treated according to the invention in the following manner:
The solution, which has a protein content of 12.5 %
under physiologic saline and pH conditions, is admixed with C1INA so that a ratio of 1. 0.01 U C1INA/g protein 2. 0.1 U C1INA/g protein 3. 1.0 U C1INA/g protein 4. 10.0 U C1INA/g protein will result. The mixtures are maintained a-t +4 C for a-bout 24 hours, are sterile filtered and filled into con-tainers.

~2~

~, o .
o .~
~, ~, ...
,~
Ql o~ Ln O ~ oo ~3 0 0 0 oo a) o H rl ~) g ~
rl rl ~i ~
a) o 10 S~ ~
.~ O ~ ~ , ~ O
In O O In Ln 5~ 0 ~ ~ ~_ m ~

~d ~, o o .~
c) 0~0 .~, o o o o o o o o ~9 r~
~1 ~ ~ ~ ~
,~ o ~ ~ ~
m o ~
O ~ o 3 :~0~ 3 ~ 3 Q
r~ O r l ~ r-l ~) r~ ~ r~ ~, ')-rl Q ~ ~ 1 Z Q
Orl O H O Z O Z O H
r-l ~ r-l ~ r-l H r-l H r-l ~ O
HC_~ H O H O H ~ H ~

~ 15 -ExampIe 4:
Preparation of an immune globulin fraction:
An immune globulin fraction prepared as in Example 3 and having a protein content of 5.03 % is adjusted to 1. 0.01 U C1INA/g protein 2. 0.1 U C1INA/g protein 3. 1.0 U C1INA/g protein 4. 10.0 U C1INA~g protein, sterile fil-tered, filled into containers and incubated a-t 37 C for 24 hours.

o ~ ~
~ o ~
(~ ~
a) ~ ~ ~ .,. Lr) 00 o ~ ~ ~ o ~r o H ~1 O S ~
.,~ rl V
a Q) O
5~ ~
-1' 1- Ln o O O
U~ o s~ O
m s~
.~.
o o\O ~ O O ~ O
U~ 00 ~9 OD
~, ~
m m IJ
'~
~1 ~ O ~1 ~ rl ~ rl O
3 3 ~ 3 ~0 :~ ~ 3 s~
rl o ~ ~ ~ ~ ~ ~, Q ~ ~q æ ~ ~ ~ ~ Q æ
Orl O H O Z O Z O H
~1 ~ r-l H r--l H ~
r~ tnC) ~ ~ ~ r_ t~ t ) rd O C~
a~ (L) ~ (1) a) a) o ~æ ~ o ~ ~ ~ O H O H ~ 1--1 ~ 17 -Example 5_ Preparation of a factor VIII fraction:
46 l of fresh -frozen plasma are -tha~led a-t 0 C to t 4 C. The cryoprecipita-te formed is separated by cen-tri-fugation and dissolved in 960 ml 0.1 % -trisodiumcitrate a-t 37 C0 8 % polyethyleneglycol 2000 are added a-t a pH of

6~3. Thus, a precipitate forms, which is separated by centrifug~tion and discarded. By the addition of 12 %
ethanol to the supernatant at ~3 C -the enriched factor VIII is precipitated. After separation it is dissolved in a physiologic buffer and, if desired after intermediate lyophilization, is further treated according to the invention:
From the solution, which has a protein concentration of 1.8 % protein, a concentration series of 1. 0.01 U C1INA/y protein 2. 0.1 U C1INA/g protein 3. 1.0 U C1INA/g protein is prepared by the addition of C1INA. The mixtures remain standing a-t t4 C for about 24 hoursî thereafter, it is sterile filtered and a ready-made product is prepared~

5~

a o o ~
~, ~, ...
~ In S~ O ~ In 1-- I

a) o ,~ ~ .,, H

O
~ I

~, ~ ~ ~ ~.
O
U~ o _~ O~
m a~ ~
F~ O ~:
o 0\o ~
~r ~ ~ O
~C ~ ~D ~ ~
m o P~ m .
a) ~ ~: o o ~ o ~ o o o o r O ~ ~ r h -r h t~ ~ (d ~ (~ ~ (~ ~
~,r~ ~Q, Z Ql ~ Ql '¢
a)~d a) H a)Z; O Z
~ I H ~ I H
QlIz¢ Ql t,) H H H ~ H I--I
H ~ 1--1 H ::) H ~) H C~ ~ ~o ~ o .~ . .
O ~1 0 O O O O O
~ ~ O ~ C~ ~ O ~
1~ d rl 1~rl rci rl ~ 3 ~4 ~ ILI 3 ~l 3 ~L2~

In the further examples it is illustrated that virus-es other than hepatitis virus also are inactivatable during the production of albumin preparations within the scope of the method according to the invention.
Example 6:
From an albumin fraction, a 5 % plasma protein solu-tion is prepared. The Na+ concentra-tion of this solution is 140 mequ Na /l, the pH being 6.9. This solution is ad-mixed with stabilizing agen-ts (Na-caprylate and Na-acetyl-tryptophanate). At the same time, C1-esterase inhibitox is added in such an amount that a content of 13 units of C1INA/g protein will result. The thus prepared mixture is sterile filtered and subsequently is admixed with polio virus type I. Then, a thermal inactlvation takes place at 60 C for 10 hours. For control purposes, a plasma protein solution admixed with polio virus type I remains at +4 C
for 10 hours. The control sample and the thermally treated solution are subjected -to a virus titer determination. The values given in the following table are decadic logarithms of TCID50 per 0.1 ml, TCID50 indicating tha-t 50 % of the tissue culture preparations exhibit a cytopathogenic ef-fect.
Table:
Virus titer of control ~ 9 Virus titer of sample treated at 60 C for 10 hours c;1 Example 7:
From an albumin fraction a 5 % and a 20 % protein solution are prepared. The Na concentra-tion of both solu-tions is adjusted to 150 mequ/l, the pI-I amourlting to 7Ø

~s~

As described in Example 6, the solutions are admixed w.ith stabilizing agents and C1INA and subjected to sterlle fil-tration. Thereafter, the 5 % and the 20 % protein solu-tions are admixed wi-th polio virus type I, ro-tavirus, can-ine hepatitis virus and Coxsackie virus and are suhjected to a thermal inactivation a-t 60 C for 16.5 hours. For con-trol purposes, samples admixed with virus remain at -~4 C
for the same time. The assessment of virus destruction is effected as in Example 6.
Table-Polio Rota Canine Coxsackie Type I virus hepati-tis Control 6.6 5.6 5.4 6.6 5 % protein solution C 1 C 1 c~1 C 1 rt 20 ~ protei.n O solution C 1 G~1 C 1 ~1

Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a method for the production of therapeutically ad-ministrable plasma derivatives filled in final con-tainers, which are free of prekallikrein activator, hypo-tensively active constituents and other undesired pharmacologically active substances, such as for the production of albumin, globulin and coagulation-factor preparations, of plasma or plasma crude fractions by stepwise enrichment of plasma proteins, sterile fil-tration and optionally virus inactivation, the improve-ment which is characterized by the addition of C1-esterase inhibitor during the production process of said plasma derivatives, yet prior to its filling into said final containers.

2. A method as set forth in claim 1, wherein said ad-dition of C1-esterase inhibitor is carried out at a Na+ concentration of 5 to 200 mequ/l and in a pH range of 5 to 9.

3. A method as set forth in claim 2, wherein said Na+
concentration is 10 to 50 mequ/l.

4. A method as set forth in claim 1 for the production of an albumin preparation, further comprising the step of subjecting said final containers to a thermal virus inactivation at 60° C for 10 hours after filling said albumin preparation into said final containers.

5. A method as set forth in claim 1, wherein said C1-esterase inhibitor added itself is virus inactivated.