Note: Descriptions are shown in the official language in which they were submitted.
- 2181875
- BEHRINGWERKE AKTIENGESELLSCHAFT 1995/B017 - Ma 1073
Dr.Lp/hg
Processes for quantifying activated factors
_ _ _ _______________________
The invention relates to sensitive processes for the
selective determination of single factors (activated
coagulation proteases) for example in factor
concentrates such as PPSB, i.e. in mixtures of
activated and non-activated factors.
Congenital and acquired deficiencies of plasma proteins
or blood cells are currently substituted by infusing
plasma, cell concentrates as well as proteins and
factor concentrates which have been purified by plasma
pool~. In addition to the potential hazard of virus
transmissions, which are m;n;m; zed by single-donor
testing and suitable virus reduction and inactivation
processes, undesirable reactions, such as, for example,
drop in blood pressure, and thrombogenic or hemagogic
effects, are observed. These side effects can be caused
by the presence of so-called activated factors. Even
though these products are purified under the mildest
possible conditions and are subject to suitable quality
checks and licensing criteria, the abovementioned side
effects have been reported in individual cases.
Retrospectively, analyses have frequently confirmed the
suspected presence of activated factors in these
products.
The concentrates prepared from blood plasma can contain
traces of other plasma proteins which usually do not
noticeably affect efficacy and safety of the product.
If these proteins, i.e. enzymes/proteases, are present
in the activated state, this may have fatal
consequences for the patient post-application. In
addition to the activated form of prothrombin, i.e.
thrombin, traces of the activated coagulation factors
VII, IX, X, XI, XII or of prekallikrein or of protein C
(F VIIa, F IXa, F Xa, F XIa, F XIIa, kallikrein, APC)
- 2 1 8-1 8 75
or of the complement system may also be present.
~owever, even the purified proteins themselves, whether
they have been prepared by conventional precipitation
and chromatographic methods from plasma or other
starting materials or by me~n~ of recombinant
technology, may be partially activated. Of equal
importance is the detection of these activated factors.
The fact that these are already active in minute
concentrations and may cause triggering or reinforcing
in-vivo effects requires tests which are as sensitive
and specific as possible.
Currently used methods record for example the enzyma-
tic/proteolytic activities of these factors, while
their zymogens are, accordingly, inactive. It is
conventionally attempted to record these activities
with the aid of chromogenic substrates, the amidolytic
liberation of, for example~ p-nitrophenylanilide or
fluorogenic groups being recorded and evaluated
photometrically. Even though these substrates are
optimized for specific proteases or activated factors,
they are also cleaved by other proteases to a lesser or
greater extent. This is why differentiation and
quantification of single components - and therefore
also more or less "harmful" activities - is
particularly difficult in complex protein (protease)
mixtures such as, for example, prothrombin complex
concentrates. It is only made possible when a
comparison measurement i8 carried out in the presence
of a highly specific inhibitor, as in the case of
thrombin +/- hirudin, with which the specific activity
can be eliminated. In the case of the other proteases,
this has not been possible to date since the inhibitors
available are not sufficiently specific, albeit highly
potent in some cases.
Immunochemical detection systems such as, for example,
RIA, E~ISA etc. are highly selective and sensitive
methods. Such test systems, which could specifically
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quantify the activated protease, would~ be suitable.
However, obtaining antibodies which, on~the one hand,
exclusively recognize the activated form of a factor is
frequently difficult or, in many cases,` has not been
carried out successfully to date. However, this is
required for the products described, since a mixture of
zymogen and activated form is present. On the other
hand, protease inhibitor complexes which are present
must not be measured simultaneously since they would
falsify the results of a quantification of the free,
blologically active form. These complexes can be formed
during protein purification or by deliberately added
inhibitors. Accordingly, the aim ia to provide suitable
assay systems to quantify these active
factoræ/proteaæeæ, in particular the factors VIIa, F
IXa, F Xa, F XIa, F XIIa, kallikrein and APC as well as
proteases of the complement system. A homogeneous assay
method in the liquid phase has been described for free
thrombin with the aid of the thrombin/antithrombin III
complex in coagulation products (Kohler et al., Thromb.
Res. 1990; 60: 63-70).
In the present invention, the product or concentrate is
treated with an enzyme/protease binding partner, pre-
ferably an inhibitor, which can be bound by anantibody, the mixture is incubated as required, and the
enzyme-/protease inhibitor complexes generated are
quantified by means of solid-phase ELISA (enzyme-linked
;mmllnosorbent assay).
Suitable protease inhibitors are known binding
partners, such as antithrombin III (AT III), a2-
macroglobulin, al-antitrypsin, a2-antiplasmin, inter-a-
trypsin inhibitor, Cl-inhibitor, protein C inhibitor
and the like, preferably AT III, optionally in the
presence of heparin, and al-antitrypsin. AT III is the
most important physiological regulator/inhibitor of the
coagulation system which reacts with activated factors
with a different affinity/rate and forms a complex.
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While, for example, the reaction with thrombin proceeds
relatively rapidly and is accelerated greatly by
heparin, as is well known, an interaction with, for
example, F VIIa proceedæ merely very slowly at room
temperature or 37C or only when F VIIa is present as a
complex with thromboplastin. Another possibility was
described by Godal et al. (Thromb. Res. 1974; 5: 773-
775), where F VIIa is incubated with ATIII/heparin for
several hours under cold conditions and the remaining
coagulation-promoting F VIIa is subsequently determined
in a suitable activity assay.
In contrast, specificity and sensitivity are increased
when the protease inhibitor complexes formed are quan-
tified with the aid of an ;mmllnological assay. Accord-
ingly, polyclonal (Pab) or monoclonal (Mab) antibodies
or their F(ab') or F(ab) fragments, preferably a
monoclorlal ab or its Fab, are coupled to, or adsorbed
onto, the solid phase by known methodq, the st~n~rd or
sample solution is incubated therewith, the solid phase
is washed, and the bound complexes are incubated with a
polyclonal or monoclonal ab, or Fab against the compiex
partner (protease or inhibitor). This ab is conjugated
for example with an enzyme, such as POD. After a
suitable enzyme substrate has been added, the substrate
conversion rate is measured and the concentration of
the bound complex is determined by means of a standard
curve. The concentration of the protease which exists
in free form in the product can be calculated
accordingly.
If a limited amount of protease inhibitor complexes
(for example for preparation reasons) exists already
prior to the addition of an adequately high
concentration of the inhibitor (fully to convert the
free protease into complexes), then the control value
(product dilution without additionally added inhibitor)
is subtracted from the sample value whereby the
concentration of the free, activated protease is
calculated.
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In a preferred embodiment (A), known methods are used
for directly coupling the inhibitor to the solid phase
or binding it by means of an antibody (to the
inhibitor) which is coupled to the solid phase, and
thus exposed to the free proteases in solution. After
incubation and formation of the now immobilized
inhibitor protease complexes, the solid phase is washed
and the complexes are detected by means of a suitable
(conjugated) antibody to the prot~ase or to a
neoepitope of the complex (or by means of a conjugated
antibody to these antibodies).
If appropriate, the detecting antibody/Fab may already
be pre ent while the solid phase i~ incubated with the
sample solution.
In a further preferred ~mhodiment (B), an antibody/Fab
suitable for the enzyme/protease to be detected is
bound to the solid phase (or an antibody to this
antibody, for example goat-anti-mouse, which presents
the specific antibody). The solution which comprises
the free enzyme/protease is brought into contact with
the solid phase and incubated. After the free,
activated protease (as well as non-activated
enzymes/proteases in solution) have bound, the solid
phase is washed and incubated with a solution
comprising the corresponding inhibitor. After the solid
phase has been washed, the complexes are detected with
the aid of a conjugated antibody which binds to the
inhibitor or to a neoepitope of the complex. Equally,
the solution which comprises the activated, free
protease may be treated with the inhibitor before (and
incubation) or during the incubation with the contact
phase.
If appropriate, the detecting antibody/Fab may already
be present during incubation of the solid phase with
the inhibitor.
The abovementioned embodiments are used depending on
the intended aim: while, in embodiment (A), the
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immobilized binding partner (here the inhibitor) binds
exclusively activated proteases, allowing traces of
these non-complex molecules to be detected, embodiment
(B) `additionally creates the possibility for the
limited enzyme/protease inhibitor complex which may be
present to be measured even before the inhibitor is
added. A parallel ~Y~m; n~ tion of the sample solution
(+/- inhibitor) followed by subtraction ((+) inhibitor
minus (-) inhibitor) allows both pieces of information
to be obtained.
Preferred combinations of binding partners for
determining activated factorR are
AT III, if appropriate, F VII a, F IX a, F X a,
plus heparin: F XI a, F XII a
C1 inactivator: F XI a, kallikrein, Clr
a1 antitrypsin: activated protein C,
elastase, other serine
proteases
Protein C inhibitor: activated protein C
Example:
Quantification of FIXa-AT III complex (cx)
Materials:
Microtiter plates were coated with monoclonal
antibodies (Mab, 10 ~g/ml) to FIX/FIXa and washed.
Peroxidase (POD)-conjugated Mab to ATIII were used to
detect FIXa-ATIII-cx.
AT III (Kybernin~), Mab to FIX and to ATIII were
obtained from Behringwerke AG (Germany). Heparin
(Liquemin~) was obtained from Hoffmann-La-Roche GmbH
(Germany). Factors IX and IXa were acquired from
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Calbiochem (USA) and Serbio (France). The chromogenic ~~
substrate S-2765 was from Chromogenix AB (Sweden).
Preparation of an FIXa-ATIII-cx st~n~rd:
500 ~l of a buffer solution comprising FIXa (2~g/ml)
were incubated with an equal volume of an ATIII/heparin
solution comprising a 10-fold molar excess (dilution
from 10 I.U./ml of ATIII/20 I.U./ml of heparin) for
90 minutes at room temperature (RT). The complete com-
plexing of FIXa with ATIII was monitored by photometric
determination of the amidolytic FIXa-activity by means
of S-2765 in comparison with the control batch (without
ATIII). After the abovementioned incubation time,
activity in the batch comprising FIXa/ATIII/heparin was
no longer detectable - FIXa had been converted fully
into the FIXa-ATIII-cx.
Establishing an FIXa-ATIII-cx standard curve:
Aliquots of the FIXa-ATIII-cx standard solution
(1.0 ~ g/ml) were diluted stepwise down to a factor of
1250 and the dilutions used for establishing a standard
curve. In each case 50 ~l of a sample buffer were
introduced into the wells of the microtiter plates
coated with anti-FIX-Mab, and in each case 50 ~l of the
cx solutions were added.
After incubation for 1 hour at 37C, the solid phase
was washed three times, and in each case 100 ~l of the
solution comprising anti-ATIII-Mab-POD were added.
After reincubation for 1 hour at 37C, the plates were
washed three times. In each case 100 ~l of POD-
substrate solution (O-phenylenediamine hydrochloride)
were pipetted into each well, the plates were incubated
for 20 minutes at RT (in the dark), the reaction was
stopped, and the OD4g2 was measured:
21&1875
FIXa-ATIII-cx (ng/ml) OD4g2 (mean value of
- duplicate tests)
0.8 0.027
1.6 0.028
5-3.1 0.043
6.25 0.09
12.5 0.13
0.24
0.42
10100 0.71
125 0.86
250 1.34
500 1.98
Accordingly, this gave a linear measurement range of at
least 1.5 to 150 ng/ml (when plotting log concentration
cx versus log OD4g2)-
Quantification of FIXa-ATIII-cx in FIX/FIXa mixtures:
The following mixtures of FIX, FIXa and ATIII/heparin
were prepared, incubated for 90 minutes at RT and
treated as sample as described above:
End concentration in
the batch
1. 40 ~l FIX (50 ng/ml) 20 ng/ml
40 ~l FIXa (150 ng/ml) 60 ng/ml
20 ~l ATIII/Hep (5 U/ml) 1 U/ml
2. 40 ~l FIX (100 ng/ml) 40 ng/ml
40 ~l FIXa (50 ng/ml) 20 ng/ml
20 ~l ATIII/Hep (5 U/ml) 1 U/ml
3. 40 ~1 FIX (450 ng/ml) 180 ng/ml
40 ~l FIXa (50 ng/ml) 20 ng/ml
20 ~l ATIII/Hep (5 U/ml) 1 U/ml
Control batches comprising the factors IX, IXa and
ATIII/Hep in each case separately were also prepared at
the concentrations shown from 1. to 3.. Neither FIX,
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g
nor IXa nor ATIII/Hep showed a reaction in this assay
system. This equally applied to the mixture FIX/FIXa
(without subsequent incubation with ATIII/Hep, which
guaranteed selectivity for the FIXa-ATIII-cx.
FIXa-ATIII-cx formed was quantified using the above-
mentioned standard curve. The table shows the mean
values of the results of test dilutions.
Batch No. FIXa-ATIII-cx Recovery rate Dilutions
(factor of (ng/ml) (%)
x)
1 58.6 98 2/4/8/16/32
2 25 125 4
3 24 120 1/2
