Note: Descriptions are shown in the official language in which they were submitted.
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BEHFdIIdGWERICB ARTIENGESF~~.LSCHAE'T 92/B 013J - Ma 924
Dr. Ha/hg
Process for the inactivation of viruses
in preparations Of proteins
The invention relates to a process for the inactivation
of viruses in preparations of proteins. To do this, a
solution of the preparation of a protein is briefly
heated.
Proteins within the meaning of the invention are placen-
tal proteins, gleams proteins, proteins from cell cul-
tures or from microbial fermentations. An example of a
protein of this type is tissue thromboplastin.
The reagents employed for the Quick determination of the
prothrombin time contain tissue thromboplastin as active
constituent. The prothrombin time is an important screen
ing test in the diagnosis of coagulation disorders.
In addition, the therapeutic use of tissue thromboplastin
as FVIII bypassing agent has been suggested for the
treatment of hemophiliacs with inhibitors. The virus
safety of the preparation is an indispensable pre-
requisite for this use. However, virus safety is also
required on use of tissue thromboplastin as diagnostic
aid, having regard to the preservation of the health of
the user.
At present, tissue thromboplastins for preparing pro-
thrombin time reagents are normally obtained from brain
or placenta of mammals. Contaminations by viruses such as
HBV, HCV, HIV fox preparations of human origin or by the
agent causing BSE in the case of cattle cannot in prin-
ciple be ruled out with preparations of this type. This
means that a process for virus inactivation in tissue
thromboplastin preparations has great importance.
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To date, attempts at virus inactivation by established
processes (detergents, hypochlorite, UV/gamma irradiation
etc.) have failed because of the great sensitivity of the
preparations. In particular, all attempts at pasteur
ization or dry heating have been unsuccessful.
It has emerged, surprisingly, that a brief heating, for
example in an apparatus disclosed in Chem.-Ing.-Tech. 62
(1990), 486~487 (German Patent Application 39 05 066),
has no adverse effect on the properties of tissue
thromboplastin but, on the other hand, completely inacti-
vates viruses. In the known processes for virus inacti-
vation (pasteurization, "dry heating"), heating is
customarily carried out for at least one minutes, but
usually for several hours.
The invention relates to a process for the inactivation
of viruses in a preparation of a protein from the group
of placental proteins, plasma proteins or proteins
prepared in cell culture or microbially, which comprises
heating a solution of this preparation for a short time.
Heating is carried out with indirect heating in a heat
exchanger. Suitable heat exchangers have any desired type
of construction, such as plate exchangers or tubular
exchangers.
A heat exchanger as disclosed in German Patent Appli-
ration 39 05 066 is particularly suitable because, while
the heat transfer coefficient is high, a short residence
time and a lower wall temperature is possible.
This heat exchanger is a heat exchange module composed of
stacked metal foils with spacers arranged between them,
3U the metal foils being composed of metal cards which are
provided with at least 2 openings on each of the opposite
sides, the spacers being composed of fabric cards with
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openings which are coincident with the metal cards so
that the openings form tubular channels when the cards
are stacked, the encircling rim of the fabric cards and
an annular area of the fabric which encloses some of the
openings being filled with sealing agent, there being
alternation of the openings with and without sealing
agent in a row of a fabric card and in the case of the
tubular channels.
The heating temperature can be between +45°C and +95°C,
but preferably between +65°C and ~0°C.
In order that the fed-in liquid can be heated in the
minimum time to the particular heating temperature and
subsequently cooled down again in the same time, the
apparatus in which heating is carried out is expediently
equipped with a connector each for heating medium and
cooling medium.
The constructional design of the heat exchangers ought to
be such that the difference between the heating temper-
ature for the solution and the temperature of the heating
medium is a minimum, which results in minimum damage to
the product because of the low wall temperature resulting
therefrom.
The heating and/or cooling time should be less than
seconds, but preferably less than 5 seconds. The dwell
25 time can be between 0.1 and 20 s, but preferably between
0.5 and 5 seconds.
The protein, preparation can be, for example, a therapeu
tic agent containing tissue thromboplastin, or a diag
nostic agent containing tissue thromboplastin, for
30 example a prothrombin time reagent.
In the following example, the virus inactivation by brief
heating is shown, without limiting the invention, by the
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example of RThromborel S, a prothrombin time reagent con-
taining tissue thromboplastin from human placenta of
Behringwerke AG. The success of the inactivation was
examined. Complete virus inactivation was found at a
heating temperature above 45°C, preferably at least 65°C.
The diagnostic properties of the briefly heated reagent
were compared with those of untreated aThromborel S. No
disadvantageous changes in these properties were observed
at least up to a heating temperature of 75°C.
Example
Procedure for the brief heating
For this, two heat exchangers (W 1.1 and W 1.2, Fig. 1)
were mounted in a common fixing device which essentially
corresponds to commercially available ultrafiltration
cassette holders. The heat exchangers had a modular '
construction and were separated from one another by a
specially constructed spacer plate.
This made it possible to connect the heat exchangers in
such a way that the fed-in liquid was heated to the
particular heating temperature within 2 seconds and
subsequently cooled down again in the same time. For this
purpose, the apparatus was equipped with a connector each
for heating medium and cooling medium. The dwell time at
the said heating temperature was about 1.5 seconds.
Before the process started, the entire system on the
product side was flushed with water and heated to the
operating temperature. For this, the pump P1 (Fig. 1)
transported heating medium through the system until the
required heating temperature was reached. It was then
changed from water to the solution to be heated, and in
control tests the pump P2 was subsequently switched on
to meter the virus suspension. Samples were taken before
heating and after the experimentally determined minimum
test time of 20 or 48 seconds.
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Results of the virus iaaactivatioa (Table 1j
As a control, a solution of tissue thromboplastin was
passed at room temperature through the system. The
results show that a virus inactivation of more than 5
powers of ten was achieved in all tests on the coat-free
and heat-resistant poliovirus. The inactivation was
likewise eomplete in the case of the herpesvirus BSA-1.
The inactivation was examined for 2 different initial
titers (about 3.5 and 5.0) and was complete in both case.
By contrast, in the control experiment, virus inacti-
vation was undetectable or only low.
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Comparison of briefly heated with untreated tissue
thromboplastin
RThromborel S was briefly heated at various temperatures
in the above system without metering in virus suspension.
Untreated material served as control. All the prepar
ations were freeze-dried and reconstituted before testing
in the same volume of distilled water. The results which
are presented below were obtained with a representative
batch.
To construct the reference plots (k'ig. 2 ) , standard human
plasma from Uehringwerke AG was employed undiluted and
diluted with isotonic sodium chloride solution as sample.
Sample (100 ~l) and reagent (200 ~1) were mixed and the
clotting time was measured in a Schnitger & Gross
coagulometer. The reference plots for heated and
untreated RThromborel S are essentially identical.
The sensitivity of prothrombin time reagents is expressed
by ISI (international sensitivity index). The ISI values
for heated and untreated RThromborel S, determined on
plasma from healthy subjects and subjects undergoing oral
anticoagulation, compared with a reference thromboplastin
are indistinguishable (Tab. 2).
Table 2
International
Sensitivity
Index
Determination
1 Determination
2
Control 1.09 1.09
65C 1.12 1.09
75C 1.10 1.12
80C I 1.10 1.10
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To characterise the sensitivity of heated and untreated
RThromborel S to the coagulation factors II, V, VTT and
X, standard human plasma was mixed with the appropriate
coagulation factor-deficient plasma from Behringwerke AG
to adjust the activities of the relevant factor to
between 5 and 100 of normal. The clotting times of the
mixtures (100 ~cl) were determined after additions of
200 ~cl of heated or untreated ~Thromborel S in a Schnitger
& Gross coagulometer. The clotting time is plotted
against the content of factor II and F VII, respectively,
as a percentage of normal in Fig. 3 and 4p this revealed
that the sensitivity to the tested factors was as good as
for the control. The sensitivities for factor V and
factor X (snot shown) were indistinguishable within the
accuracy of measurement.
