Ex Parte Huang et al

Patent Trial and Appeal Board

Apr 11, 2017

13222697 (P.T.A.B. Apr. 11, 2017)

United States Patent and Trademark Office
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O.Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
13/222,697 08/31/2011 Fen Huang 016026-9526-US01 5923
91007 7590 04/13/2017
Michael Best & Friedrich LLP (Promega)
100 East Wisconsin Avenue
Suite 3300
Milwaukee, WI 53202
EXAMINER
LIU, SAMUEL W
ART UNIT PAPER NUMBER
1656
NOTIFICATION DATE DELIVERY MODE
04/13/2017 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
madipdocket@michaelbest.com
heather.gerard@promega.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte FEN HUANG, DIETER KLAUBERT, JOHN SHULTZ, and
WENHUI ZHOU
Appeal 2017-004152
Application 13/222,6971
Technology Center 1600
Before JEFFREY N. FREDMAN, RACHEL H. TOWNSEND, and
DEVON ZASTROW NEWMAN, Administrative Patent Judges.
NEWMAN, Administrative Patent Judge.
DECISION ON APPEAL
This appeal under 35 U.S.C. § 134 involves claims to an assay for
detection of oxidized glutathione. The Examiner entered final rejections for
obviousness.
We have jurisdiction under 35 U.S.C. § 6(b). We REVERSE.
1 Appellants identify the Real Party in Interest as Promega Corporation.
App. Br. 3.
Appeal 2017-004152
Application 13/222,697
STATEMENT OF THE CASE
Background
The Specification discloses that
[t]o remain healthy, cells, in particular mammalian cells, need
to maintain a balance between oxidizing and reducing
conditions sometimes referred to as redox state/potential. One
of the most important mechanisms used to set and preserve the
redox state/potential occurs through maintaining the relative
amounts of oxidized and reduced glutathione in the cell.
Spec. 13. The Specification discloses “an assay for detection of oxidized
glutathione (GSSG) and the determination of the ratio of GSH[, the reduced
form of glutathione,] and GSSG in a cell.” Id. at 12. “The method of the
present invention uses an enzymatic reaction for the measurement of the
amount of GSH in a sample which requires no processing steps and prevents
GSH loss.” Id. atl 10.
The Claims
Claims 1—4, 6—8, 13, 14, 28—30, 32—34, 39, 40, and 42—53 are on
appeal. Claim 1 is illustrative and reads as follows:
1. A method for detecting GSSG comprising:
a. contacting a sample with a sulfhydryl alkylating agent;
b. contacting the sample with an excess of a reducing
agent, glutathione-S-transferase, and a substrate for glutathione-
S-transferase which is converted to a signal generating
compound in the presence of GSH and glutathione-S-
transferase, wherein the reducing agent inactivates the
sulfhydryl alkylating agent and reduces any GSSG in the
sample to GSH; and
c. detecting the signal generated from step (b), thereby
confirming the presence of GSSG in the sample, wherein
neither the inactivated sulfhydryl alkylating agent nor the
reducing agent is removed from the sample.
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Application 13/222,697
App. Br. 21 (Claims Appendix) (emphasis added).
The Issues
The following rejections are before us to review:
I. Claims 1—4, 6—8, 13, 14, 28—30, 32—34, 39, and 40 are rejected
under 35 U.S.C. § 103 (a) as being unpatentable over Wendell,2 Promega,3
Mistry,4 and Glatz.5 Ans. 2.
II. Claims 42—53 are rejected under 35 U.S.C. § 103 (a) as being
unpatentable over Wendell, Mistry, Glatz, Sigma,6 Sigma-Aldrich,7 and
Perry.8 Ans. 7.
I
The Examiner has rejected claims 1—4, 6—8, 13, 14, 28—30, 32—34, 39,
and 40 as obvious based on Wendell, Promega, Mistry, and Glatz. The
Examiner finds that Wendell discloses an assay for measuring GSSG in
2 P.L. Wendell, Measurement of Oxidized Glutathione and Total Glutathione
in the Perfused Rat Heart, 117: J. Biochem 661—665 (1970).
3 GSH-Glo™ Glutathione Assay, Technical Bulletin www.promeya.com, 1—
20 (2008).
4 Sharad C. Mistry et al. Evidence that rat liver pyruvate dehydrogenase
kinase activator protein is a pyruvate dehydrogenase kinase 275 :J. Biochem
775-779 (1991).
5 Z. Glatz et al, Use of thiopropyl Sepharose for the synthesis of an
adsorbent for the affinity chromatography of glutathione S-transferase 688:
J. Chromatog. B 239-243 (1997).
6 Glutathione Assay Kit, Fluorimetric, Sigma Product Information \-A
(2009).
7 Glutathione Assay Kit, Fluorimetric, Sigma-Aldrich Material Safety Data
Sheet 1 (2009).
8 Perry et al., US 2002/0016284 Al, published Feb. 2, 2002
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Application 13/222,697
heart tissue, which the Examiner finds meets the claim limitation “detecting”
of claim 1. Ans. 2. The Examiner finds the assay
first treat[s] the sample with N-ethylmaleimide (NEM) [] to
prevent oxidation of GSH to GSSG in the sample by making it
react with NEM [], wherein said oxidation can cause large error
for measuring GSSG []. Here, reaction of NEM with GSH
produces a form that cannot contribute to [a] “signal” [] for
measuring GSSG. Thus, pre-treating the sample with NEM is
necessary for said measurement.
Id. The Examiner finds that excess NEM must be removed “because the
excess NEM inactivates/inhibits GR enzyme.” Id. at 2—3. The Examiner
finds excess NEM is removed by chromatography, and GSSG is measured
by “‘gluathione reductase” (GR) catalyzed reduction of ‘GSSG’ to ‘GSH.’”
Id. at 2.
The Examiner finds Promega teaches a luminescence-based “method
of detecting and quantifying” GSH called “GSH-Glo glutathione assay.” Id.
at 3. The Examiner finds the
assay is based on the reaction where GSH-dependent
conversion of a GSH probe, Luciferin-NT [the GST substrate]
to luciferin by a glutathione-S-transferase (GST) enzyme is
coupled to a firefly luciferase reaction. Light from luciferase is
dependent on the amount of luciferin formed, which is in turn
dependent on the amount of GSH present. This makes the
luminescent signal directly proportional to the amount of GSH.
Id. (emphasis original).
The Examiner concludes that Wendell’s method could be “improved
or modified” to overcome the disadvantages of “less sensitivity” and lower
efficiency by using the Luciferin-NT and reaction buffer of Promega in the
Wendell assay. Id. at 4.
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Application 13/222,697
The Examiner finds that Mistry teaches “use of 5 mM DTT [] to
rapidly remove (i.e., chemically inactivate) the excess NEM” as an
“[alternative to using the column to remove excess of NEM in the sample.”
Id. (emphasis original). The Examiner concludes that the NEM treatment
“must completely inhibit all NEM (i.e., completely chemical[ly] remov[e]
all of [the] excess NEM compounds) otherwise there would be some of the
enzyme inactivation.” Id. (emphasis original).
The Examiner finds Glatz teaches “use of DTT with concentration of
20 mM for reductive elution and purification of GST enzyme” resulting in
full recovery of GST activity. The Examiner concludes “DTT has no
inhibitory effect on GST and [is] very suitable for GST required assay.” Id.
(emphasis original).
Based on the above teachings, the Examiner concludes it would be
obvious “to use DTT as reducing agent in Promega’s ‘GSH-Glo glutathione
Assay’ system. When used, DTT would have rapidly and completely
reduce[d] all GSSG to GSHs in the sample [with] the reduced GSHs in turn
[becoming the] substrate of GST enzyme in Promega’s ‘GSH-Glo
glutathione assay’.” Id. at 5. The Examiner concludes the skilled artisan
“would have still kept Wendell’s sample preparation part, i.e., NEM pre­
treatment to remove ... all [of the] GSHs in original sample . . . followed
by DTT mediated removal of the excess NEM in the sample (Mistry); this is
to ensure that the measured ‘GSSG’ will not be interfered by the oxidization
ofGSH. . .to ‘GSSG.’” Id. at 7.
Among other points, Appellants argue that the Examiner has misstated
Mistry’s teachings regarding removal of excess NEM:
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Mistry discloses “rapid removal of NEM before assay of KAP
... by addition of DTT.” Appellant submits that, as would be
appreciated by one skilled in the art, “removal” in this context
is through “inactivation” by covalent modification of NEM
with DTT, to prevent further NEM reaction with any free thiol
groups. Thus, “removal” in this context is not physical
removal, but rather chemical “removal” of NEM by
inactivation. But Mistry also states that after treatment with
NEM, DTT was added and then “NEM was removed
(Sephadex G25).” In this context, Appellant submits that one
skilled in the art would have recognized that Sephadex® G25 is
a gel filtration medium that is used for column chromatography
to separate components by size, and as a method for buffer
exchange and small molecule removal. In this context,
“removal” clearly refers to physical removal. Therefore,
Mistry uses the word “removed” to indicate two different
things: both chemical inactivation and physical removal.
App. Br. 11 (emphasis original).
Appellants argue the skilled artisan would understand that Mistry discloses
chemical inactivation by reaction with DTT, “then the product of the
inactivation (the NEM that has reacted with the DTT) was physically
removed from the protein preparation using the Sephadex® G25 column. . . .
neither Wendell nor Mistry discloses or suggests that the inactivated NEM
can remain in the sample during an assay of a protein.” Id. at 12. According
to Appellants, “the use of the term ‘removed’; in the pending claims clearly
refers to the fact that the indicated components are not physically removed
from the sample” and because the cited references do not teach the required
claim element “wherein neither the inactivated sulfhydryl alkylating agent
nor the reducing agent is removed from the sample,” the rejection should be
reversed. Id.
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Application 13/222,697
The Examiner responds:
Mistry discloses rapid removal [of] excess of NEM without
need of the physical removal of NEM using Sephadex G25
column as asserted by Appellants, because Mistry clearly
indicates that “freed of DTT (Sephadex G25)” in line 1 of Table
2 legend. This indicates that when the “Sephadex G25” is used
for physically removing excess NEM, the sample is NOT
treated of DTT due to said “freed of DTT”. Furthermore, since
there is no inactivation of KAP enzyme with 5 mM DTT plus 2
mM NEM within 10 min, and since, in said 10 min time course,
the KAP enzyme completely loses activity (p.778, right col.,
lines 3-8, Mistry), DTT must completely inhibit all NEM by
completely removing all NEM compounds, otherwise there
would be some of the enzyme inactivation. . . . The Mistry’s
reference nowhere teaches physical removal of NEM or/and
DTT with Sephadex G25 column........ Instead, Mistry
provides the teaching and motivation to use DTT to rapidly and
completely remove all of excess NEM in a sample/solution
without need of physical removal of the inactivated NEM
or/and DTT.
Ans. 12-13.
We agree with Appellants that the Examiner has not shown that the
combination of Wendell, Promega, Mistry, and Glatz teaches or suggests a
method for detecting GSSG “wherein neither the inactivated sulfhydryl
alkylating agent nor the reducing agent is removed from the sample.”
The relevant teachings of Mistry are:
Table 2. Inactivation of rat liver KAP by PCMB and NEM
Rat liver KAP prepared by method C and freed of DTT
(Sephadex G25) was incubated (2 min, 30 -C) either with 0.2
mM-PCMB, and after removal of PCMB (Sephadex G25) was
concentrated with Centriflo cones, or with 2 mM-NEM, after
which DTT was added to 5 mM and NEM was removed
(Sephadex G25).[] Incubated with 2 mM-DTT for 5 min at 30
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Application 13/222,697
-C. The activity of KAP was assayed through its effect on the
activity of pig heart PDH kinase (measured as the apparent
first-order rate constant for ATP-dependent inactivation).
Rat liver KAP . . . was inactivated > 90% by 2 min of
incubation at 30 -C with 0.2 mM-NEM (Table 2, column 4). A
study of the time course showed that > 90% inactivation
occurred within 1 min, with complete loss of activity occurring
between 5 and 10 min (results not shown). In this time course,
rapid removal of excess of NEM before assay of KAP was
achieved by addition of DTT to 5 mM; no inactivation of KAP
was observed on incubation for up to 10 min with 2 mM-NEM
plus 5 mM-DTT (results not shown).
Mistry 778 (column 1, lines 1—10; column 2, lines 1—8). Appellants
persuade us that “freed of DTT (Sephadex G25)” means that Mistry ran the
assay components over a Sephadex G25 column to remove residual DTT by
chromatography. The chromatography step is completed on two separate
occasions in the disclosed text to purify the assay components and Mistry
contains no explicit teaching of leaving DTT in the reaction with NEM
without subsequent chromatography. While we agree with the Examiner
that Mistry teaches use of DTT to chemically inactivate NEM, we are not
persuaded that the skilled artisan would read Mistry to teach that the DTT
can be added to the assay without subsequently purifying the solution by gel
chromatography. Rather, we read the passage above to mean that 5 mM
DTT was sufficient to chemically react with, and functionally remove
residual NEM in the composition, but that Mistry purified the so treated
KAP composition by gel chromatography. Mistry 778. At best, the
meaning of the Mistry passage above is ambiguous and therefore insufficient
to establish obviousness by a preponderance of the evidence. Accordingly,
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Application 13/222,697
Appellants have persuaded us that the Examiner has not established that the
claim element “the inactivated sulfhydryl alkylating agent nor the reducing
agent is removed from the sample” is taught by the cited references or that
there are other reasons that would render this claim element obvious.
“An examiner bears the initial burden of presenting a prima facie case
of obviousness.” In re Huai-Hung Kao, 639 F.3d 1057, 1066 (Fed. Cir.
2011). Because that burden has not been carried here, we reverse the
rejection of claims 1—4, 6—8, 13, 14, 28—30, 32—34, 39 and 40 under 35
U.S.C. § 103(a).
II
The Examiner has rejected claims 42—53 under 35 U.S.C. § 103 (a) as
being unpatentable over Wendell, Mistry, Glatz, Sigma, Sigma-Aldrich, and
Perry. Independent claims 42 and 48 also contain the limitation “the
inactivated sulfhydryl alkylating agent nor the reducing agent is removed
from the sample”. The Examiner has not established that Sigma or Sigma-
Aldrich cure the deficiency of Mistry, as discussed above. Accordingly, we
find the preponderance of evidence on this record does not support the
Examiner’s finding on this issue.
Conclusion of Law
The preponderance of the evidence relied upon by the Examiner fails
to support a finding of obviousness.
SUMMARY
We reverse the rejection of all claims.
REVERSED
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