Ex Parte Varadi et al

Board of Patent Appeals and Interferences

May 4, 2012

10816099 (B.P.A.I. May. 4, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte KATALIN VARADI, PETER TURECEK, BRIGITTE KEIL,
SYLVIA PEYRER-HEIMSTAETT, and HANS-PETER SCHWARZ
__________

Appeal 2011-006183
Application 10/816,099
Technology Center 1600
__________

Before FRANCISCO C. PRATS, MELANIE L. McCOLLUM, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

McCOLLUM, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a
thrombin generation measuring kit and method. The Examiner has rejected
the claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We
affirm.
STATEMENT OF THE CASE
Claims 1-8, 10-13, 22, and 23 are on appeal (App. Br. 3). Claims 1
and 22 are representative and read as follows:
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1. A kit for measuring the thrombin generation in a sample, said kit
comprising
(i) a lyophilized tissue factor (TF)/phospholipid (PL)-complex; and
(ii) a lyophilized mixture comprising CaCl2 and a thrombin substrate
that comprises a fluorescent label, where said thrombin substrate that
comprises the fluorescent label is Z-Gly-Gly-Arg-AMC; wherein the
lyophilized mixture is prepared from a solution comprising the substrate,
CaCl2 and DMSO and forms a clear solution when dissolved in water, and
further, wherein the amount of water that dissolves the lyophilized mixture
to form the clear solution provides a concentration of 1 mM thrombin
substrate and 15 mM CaCl2.
22. A method for measuring the thrombin generation in a whole
blood or plasma sample, comprising the steps of:
(a) providing a lyophilized tissue factor (TF)/phospholipid (PL)-
complex and a lyophilized mixture containing a thrombin-substrate that
comprises a fluorescent label, where said thrombin substrate that comprises
the fluorescent label is Z-Gly-Gly-Arg-AMC, and CaCl2, wherein the
lyophilized mixture is prepared from a solution comprising the thrombin
substrate, CaCl2 and DMSO and forms a clear solution when dissolved in
water, wherein the amount of water that dissolves the lyophilized mixture to
form the clear solution provides a concentration of 1 mM thrombin substrate
and 15 mM CaCl2;
(b) contacting the whole blood or plasma sample with said lyophilized
TF/PL-complex and said lyophilized mixture containing thrombin-substrate
and CaC12; and
(c) measuring the thrombin generation in said sample.
Claims 1-8, 10-13, 22, and 23 stand rejected under 35 U.S.C. § 103(a)
as obvious over Wöber
1
in view of Hawkins,
2
Váradi,
3
Chan,
4
Hogan,
5

Weinstein,
6
and Dubrow
7
(Ans. 4).

1
Wöber et al., US 6,124,110, Sep. 26, 2000.
2
Hawkins et al., US 5,625,036, Apr. 29, 1997.
3
Váradi et al., Monitoring the bioavailability of FEIBA with a thrombin
generation assay, 1 J THROMBOSIS & HAEMOSTASIS 2374-2380 (2003).
4
Chan, US 5,952,198, Sep. 14, 1999.
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The Examiner relies on Wöber for disclosing “reagents and an assay
for measuring thrombin generation in plasma samples” (id.). In particular,
the Examiner finds that Wöber discloses “natural tissue factor (TF) as a dry
powder and solutions of the phospholipids phosphatidylserine (PS) and
phosphatidylcholine (PC)”; “that these three reagents are combined to
prepare a solution of vesicles or liposomes containing TF, i.e., a TF/PL
complex”; and that this “solution may be frozen in assay portions” (id. at 4-
5).
The Examiner relies on Hawkins for disclosing “that the TF/PL
solution may be lyophilized” (id. at 5). The Examiner concludes that “[o]ne
of ordinary skill in the art at the time of the invention would have been
motivated to lyophilize the TF/PL preparation of Wöber . . . to reduce the
weight and volume for shipping purposes and to impart stability to the
reagent” (id.).
“Regarding the lyophilized thrombin substrate and CaCl2
preparation,” the Examiner finds that Wöber discloses “a dry chromogenic
thrombin substrate, S 2238 (Chromogenix, now Diapharma) that is soluble
in water and that the thrombin reaction is initiated by the addition of CaCl2
to the assay samples” (id. at 6). The Examiner concludes that “[o]ne of
ordinary skill in the art would have been motivated to prepare a lyophilized
reagent containing thrombin substrate and CaCl2, because Hawkins et al.

5
Hogan et al., US 6,074,826, Jun. 13, 2000.
6
Weinstein et al., US 6,576,422 B1, Jun. 10, 2003.
7
Dubrow et al., US 6,756,019 B1, Jun. 29, 2004.
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teach the advantages of lyophilized reagents in clinical assays” (id.). In
addition, the Examiner finds:
Combining the substrate and the cofactor reduces the number of
pipetting steps, thereby reducing the chance of assay errors due
to pipetting errors, and reduces the number of assay steps,
allowing the assay to be performed faster. Because the
thrombin substrate and CaCl2 are both soluble in water or
buffer, one of ordinary skill in the art would have recognized
that a solution containing both of these substances would have
been prepared and lyophilized.
(Id.) Moreover, the Examiner finds that Hogan teaches “that, in a diagnostic
kit, or when performing an assay with a diagnostic kit, the reagents may be
premixed before lyophilization so that, when reconstituted, a complete
mixture is formed with the reagents in the proper ratio and ready for use”
(id. at 6-7).
The Examiner relies on Váradi for disclosing “a thrombin substrate
for a thrombin generation assay that contains a fluorescent label, Z-Gly-Gly-
Arg-AMC” and that an “assay reagent comprising 1 mM thrombin substrate
and 15 mM calcium chloride was prepared” (id. at 7). The Examiner
concludes:
One of ordinary skill in the art would have been motivated to
use the thrombin substrate of Váradi et al. as the thrombin
substrate in the set of reagents disclosed by Wöber et al., i.e., a
fluorescent label instead of a colored label, because Váradi et
al. teach that their substrate is available as a dry powder that is
soluble in the buffers used in a thrombin generation assay.
(Id.)
The Examiner relies on Chan, Weinstein, and Dubrow to teach or
suggest features of dependent claims (id. at 7-9).
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ISSUE
Does the evidence support the Examiner‟s conclusion that the
lyophilized mixture comprising CaCl2 and thrombin substrate Z-Gly-Gly-
Arg-AMC would have been obvious?
FINDINGS OF FACT
1. The Specification discloses:
The present invention encompasses a process for preparing a
mixture containing a thrombin-substrate and CaCl2 resulting in
an easily water-dissolvable preparation. The thrombin-
substrate preparations known in the prior art require initial
dissolution in a suitable buffer, often containing DMSO,
followed by further dilution with water. The subsequent
addition of CaCl2 to prior art thrombin substrate preparations
results in a precipitate which is difficult to dissolve and thus
difficult to use.
(Spec. ¶ [028].)
2. The Specification also discloses that the process for preparing
the thrombin substrate/CaCl2 mixture comprises: “(a) dissolving the
thrombin-substrate in a suitable solvent; (b) adding CaCl2 and dissolving the
formed precipitate containing the thrombin substrate and CaCl2, particularly
to get a clear solution; and (c) lyophilizing the mixture containing the
thrombin-substrate and CaCl2” and that the “dissolution in step (b) is
preferably carried out at a temperature of about 37°C until a clear solution
appears” (id. at ¶¶ [029]-[030]).
3. Váradi discloses a thrombin generation assay having the
following steps: “We added 10 L of TF/PL solution to 50 L of 1-mM
thrombin substrate Z-Gly-Gly-Arg-AMC . . . and 15mM CaCl2. The
addition of 40 L FVIII inhibitor plasma started the reaction. The
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components were incubated at 37°C.” (Váradi 2375: Thrombin generation
assay.)
4. Váradi also discloses that “this thrombin generation assay
enables the bioavailability and pharmacokinetics of the in vivo thrombin-
generating capacity of FVIII-bypassing agent to be monitored” (id. at 2380).
5. Hawkins relates “to the field of Prothrombin Time reagents”
(Hawkins, col. 1, ll. 12-14).
6. Hawkins discloses: “Recombinant human tissue factor . . . was
combined with a mixture of purified bovine phosphatidyl serine (PS) and
purified phosphatidyl choline (PC). . . . The formulations were dispensed
into vials and freeze-dried.” (Id. at col. 8, ll. 8-20.)
7. Hawkins also discloses that it “is important for a PT
[Prothrombin Time] reagent to . . . be stable for storage in the freeze-dried
(lyophilized) state” (id. at col. 1, ll. 45-50).
8. Hogan discloses: “[T]he required enzymes, the nucleotide
triphosphates, the primers and probes may be provided as a lyophilized
reagent. These lyophilized reagents may be premixed before lyophilization
so that when reconstituted form a complete mixture with the proper ratio of
each of the components ready for use in the assay.” (Hogan, col. 37, ll. 15-
21.)
PRINCIPLES OF LAW
“Evidence of secondary considerations, including evidence of
unexpected results . . . , [is] but a part of the „totality of the evidence‟ that is
used to reach the ultimate conclusion of obviousness. . . . The existence of
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such evidence . . . does not control the obviousness determination.”
Richardson-Vicks Inc. v. Upjohn Co., 122 F.3d 1476, 1483 (Fed. Cir. 1997).
“[W]hen unexpected results are used as evidence of nonobviousness,
the results must be shown to be unexpected compared with the closest prior
art.” In re Baxter Travenol Labs., 952 F.2d 388, 392 (Fed. Cir. 1991).
“Mere recognition of latent properties in the prior art does not render
nonobvious an otherwise known invention.” Id.
ANALYSIS
Váradi
8
discloses a thrombin generation assay that uses a tissue
factor/phospholipid reagent and a thrombin substrate Z-Gly-Gly-Arg-
AMC/CaCl2 reagent (Finding of Fact (FF) 3). Hawkins discloses storing
assay components, specifically a tissue factor/phospholipid mixture, in a
lyophilized state (FF 5-7). Hogan discloses premixing assay reagents prior
to lyophilization (FF 8). Based on these disclosures, we agree with the
Examiner that it would have been prima facie obvious to lyophilize Váradi‟s
tissue factor/phospholipid reagent and thrombin substrate/CaCl2 reagent. In
addition, in the absence of evidence to the contrary, we conclude that the
resulting lyophilized thrombin substrate/CaCl2 reagent would have the
claimed solubility in water (see FF 1-2).

8
According to the Appeal Brief, “Appellants provided a Declaration under
37 C.F.R. § 1.132 by all of the inventors stating that to the extent that the
invention is disclosed in Váradi, it is their invention” (App. Br. 6).
However, given that “Appellants have not included arguments relating to
this inventors‟ declaration in the [Appeal] brief” (id.) or in the Reply Brief,
for the purpose of this appeal, we are assuming that Váradi is prior art
properly relied upon in this obviousness rejection.
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Appellants argue, however, that the “fluorescent substrates of the
claimed invention are not soluble in aqueous solutions” and that “the
addition of CaCl2 to a fluorescent substrate in solution leads to the formation
of a precipitate” (App. Br. 8).
[G]iven the insolubility of the recited fluorescent substrate in
the absence of an organic solvent, [Appellants argue that] the
rejection fails to provide a clear articulation of why one of skill
would have made the proposed modification to substitute an
insoluble, fluorescent thrombin substrate for a soluble
chromogenic thrombin substrate, prepare a mixture with CaCl2
and then lyophilize it with the expectation of obtaining a
mixture that is dissolvable in water.
(Id. at 9-10.) We are not persuaded.
Váradi teaches a reagent containing 1 mM thrombin substrate Z-Gly-
Gly-Arg-AMC and 15mM CaCl2 (FF 3). Thus, even assuming that the
substrate was reconstituted in a DMSO-containing buffer as alleged in the
Appeal Brief (App Br. 8), Váradi specifically teaches mixing the substrate
with CaCl2 in an aqueous solution. In addition, to establish prima facie
obviousness, we do not agree that the Examiner needs to establish that there
would have been an expectation that the mixture would be dissolvable in
water alone. On the contrary, a prima facie case only requires a reasonable
expectation that lyophilizing Váradi‟s reagent would provide a lyophilized
reagent capable of being used in a thrombin generation assay. Given that
Váradi teaches a successful assay that uses an aqueous solution containing
the thrombin substrate and CaCl2 (FF 3-4) and Hawkins and Hogan teach
lyophilizing a variety of assay reagents (FF 5-8), we agree with the
Examiner that there would have been a reasonable expectation of success
(Ans. 12).
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Appellants also argue that “the CaCl2/fluorescent thrombin
lyophilized mixture . . . has the unexpected property of being dissolvable in
water” (App. Br. 4). We are not persuaded.
Appellants have provided evidence tending to show that mixing CaCl2
with the claimed thrombin substrate prior to lyophilization provides a
lyophilized product that it more easily dissolved in water than when the
claimed thrombin substrate is not mixed with CaCl2 prior to lyophilization
(Dec.
9
¶¶ 4-16). However, given that Váradi specifically teaches a reagent
containing both the thrombin substrate and CaCl2 (FF 3) and Hawkins and
Hogan both provide motivation to lyophilize assay reagents (FF 5-8), we
conclude that, on balance, the evidence of obviousness outweighs the
evidence of nonobviousness.
In addition, Appellants argue that claim 22 is “separately patentable
because the rejection does not establish evidence or reasoning that one of
skill could actually successfully perform the claimed method” (App. Br. 16-
17). In particular, Appellants argue that “one of skill would not have
expected the lyophilized CaCl2/thrombin-substrate mixture to be soluble
when an aqueous solution that does not contain an organic solvent is added”
(id. at 17). We are not persuaded.
We note initially that Appellants have not pointed to any teaching in
Váradi suggesting that an organic solvent is needed to solubilize the
thrombin substrate in water. However, to the extent that one of ordinary
skill in the art would have understood that Váradi‟s thrombin substrate

9
Declaration Under 37 C.F.R. § 1.132 by Dr. Peter Turecek dated
November 7, 2008.
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reagent contains an organic solvent, Appellants have not established that
lyophilization would be expected to remove the organic solvent. Moreover,
we do not interpret claim 22 to exclude the addition of an organic solvent
needed to solubilize the thrombin substrate. As a result, given that Váradi
discloses a successful thrombin generation assay (FF 4), we agree with the
Examiner that there would have been a reasonable expectation that a
lyophilized CaCl2/thrombin substrate reagent could also be used in a
thrombin generation assay.
CONCLUSION
The evidence supports the Examiner‟s conclusion that the lyophilized
mixture comprising CaCl2 and thrombin substrate Z-Gly-Gly-Arg-AMC
would have been obvious. We therefore affirm the obviousness rejection of
claims 1 and 22. Claims 2-8 and 10-13, which are not separately argued, fall
with claim 1 and claim 23, which is argued with claim 22, falls with
claim 22. 37 C.F.R. § 41.37(c)(1)(vii).
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

AFFIRMED

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