Ex Parte Tsuji et al

Patent Trial and Appeal Board

Feb 23, 2016

11427482 (P.T.A.B. Feb. 23, 2016)

UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE
111427,482 0612912006
38108 7590 02/25/2016
CERMAK NAKAJIMA MCGOWAN LLP
127 S. Peyton Street
Suite 210
ALEXANDRIA, VA 22314
FIRST NAMED INVENTOR
Yuichiro Tsuji
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
ATTORNEY DOCKET NO. CONFIRMATION NO.
US-236 2511
EXAMINER
RAMIREZ, DELIA M
ART UNIT PAPER NUMBER
1652
NOTIFICATION DATE DELIVERY MODE
02/25/2016 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):
cgoode@cnmiplaw.com
ip@cnmiplaw.com
scermak@cnmiplaw.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte YUICHIRO TSUJI, NA OTO KA TO,
NAOTO KOY AMA, and YUJI JOE
Appeal2013-007048
Application 11/427,482
Technology Center 1600
Before JEFFREY N. FREDMAN, JOHN G. NEW, and
ULRIKE W. JENKS, Administrative Patent Judges.
FREDMAN, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal 1 under 35 U.S.C. § 134 involving claims to a method
for producing L-threonine. The Examiner rejected the claims as obvious.
We have jurisdiction under 35 U.S.C. § 6(b).
We affirm.
1 Appellants identify the Real Party in Interest as Ajinomoto Co.,
Inc. (see App. Br. 3).
Appeal2013-007048
Application 11/427,482
Statement of the Case
Background
"Sulfur is an essential factor for bacterial cell growth and is usually
included in the medium for L-threonine fermentation in the form of
ammonium sulfate. With regard to L-threonine production by fermentation;
however, regulation of the sulfur concentration in the fermentation medium
and the effect of lowering the sulfur concentration are unknown" (Spec. i-f 9).
The Claims
Claims 1, 4, 6-10, 13, 15-18, 20, and 21 are on appeal. 2 Independent
claims 1 and 10 are representative and reads as follows:
1. A method for producing L-threonine comprising:
A) culturing an Escherichia coli that has an ability to
produce 50g/[L] or higher of L-threonine, in a fermentation
medium containing a carbon source, a nitrogen source, and a
sulfur source,
B) regulating the sulfur concentration in the medium so
that it is 0.35 g/L or lower during said culturing of step A); and
C) collecting L-threonine from the medium.
10. A method for producing a composition comprising a
dried crude fermentation broth, comprising:
A) culturing an Escherichia coli that has an ability to
produce 50g/[L] or higher of L-threonine, in a fermentation
medium containing a carbon source, a nitrogen source, and a
sulfur source,
B) maintaining the sulfur concentration in the medium
during fermentation so that it is 0.35 g/L or lower,
C) drying the crude fermentation broth so that the water
content is 10% or less by weight.
2 Claims 2, 3, 5, 11, 12, 14, and 19 were cancelled (see Response
to Non-final Action 10/27/2008).
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Application 11/427,482
The Issues
A. The Examiner rejected claims 1, 4 and 6-9 under 35 U.S.C. § 103(a)
as obvious over Kruse3, De Hollander4, and Debabov5 (Ans. 3--4).
B. TheExaminerrejectedclaims 10, 13, 15-18,20,and21 under35
U.S.C. § 103(a) as obvious over Kruse, De Hollander, Debabov, and Binder6
(Ans. 4--5).
A. 35 U.S.C. § 103(a) over Kruse, De Hollander, and Debabov
The Examiner finds that Kruse teaches "a method for the production
of L-threonine" comprising "culturing E. coli BKIIM B-3996 ... in a fed-
batch culture" medium that includes a carbon source and a nitrogen/sulfur
source (ammonium sulfate), and collecting the produced L-threonine (Ans.
2). The Examiner finds that the specification of the instant application
teaches E. coli BKIIM B-3996 (also called E. coli VKPM B-3996), the same
L-threonine producing strain taught by Kruse (ii .. ns. 2). The Examiner finds
that, as evidenced by Debabov, the E. coli strain of Kruse (E. coli BKIIM B-
3996) has been shown to produce 85 g/L in a batch culture (Ans. 3).
The Examiner acknowledges that neither Kruse nor Debabov "teach a
fed-batch culture where sulfur concentration is regulated" (Ans. 3). The
Examiner finds that De Hollander teaches that "in fed-batch cultures one
could achieve sustained [microbial] growth by providing essential nutrients
which are limiting and that the limiting nutrient will be present in the culture
3 Kruse et al., WO 2005/014840 Al, published Feb. 17, 2005.
4 De Hollander et al., US 5,763,230, issued June 9, 1998.
5 Debabov et al., US 5,538,873, issued July 23, 1996.
6 Binder et al., US 5,431,933, issued July 11, 1995.
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Appeal2013-007048
Application 11/427,482
fluid at a very low concentration" (Ans. 3). The Examiner finds that De
Hollander teaches that "there are different types of nutrient limitation
strategies that can be used, such as limitation by carbon, by sulfur, or by
phosphorous" (Ans. 3). The Examiner finds that De Hollander teaches "a
working example which shows that a phosphorous limiting nutrient strategy
using an L-lysine producing C. glutamicum strain resulted in improved
production of L-lysine" (Ans. 3).
The Examiner finds that it would have been obvious to one of
ordinary skill in the art at the time the invention was made to "use a sulfur
limiting nutrient strategy by supplying sulfur at a rate which avoids
accumulation, thus maintaining sulfur at negligible levels, in view of the
teachings of De Hollander ... regarding the increase in L-amino acid
production when a phosphorous limiting nutrient strategy or a
phosphorous/carbon source limiting nutrient strategy was used in a microbial
culture" (Ans. 4).
The issues with respect to this rejection are:
(i) Does the evidence of record support the Examiner's conclusion
that Kruse, De Hollander, and Debabov render claims 1, 4, and 6-9 obvious?
(ii) If so, have Appellants presented evidence of secondary
considerations, that when weighed with the evidence of obviousness, is
sufficient to support a conclusion of non-obviousness?
Findings of Fact
1. The Specification teaches that " [ s ]ulfur is an essential factor for
bacterial cell growth and is usually included in the medium for L-threonine
fermentation in the form of ammonium sulfate" (Spec. i-f 9).
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Application 11/427,482
2. Table 1 of the Specification is reproduced below:
1.H 0.54 3{:.f.•
~ .i) 0. ?.9 3~ .-6
{LS: {f125 ~>9"9
o.,; O . .l5 40.S
0,1: t\10 -t3 .6
(Spec. i-f 119). Table 1 provides results of effect of regulation of initially
added sulfur on L-threonine fermentation yield in fed-batch culture (Spec. i-fi-1
110, 118).
3. Table 2 of the Specification is reproduced below:
(LO
(Spec. i-f 125). Table 2 provides results of effect of regulation of sulfur in the
feed medium on L-threonine fermentation yield in fed-batch culture (Spec.
,-r,-r 120, 124).
4. Kruse teaches a fermentation process, characterized in that: a
bacterium of the Enterobacteriaceae family (e.g., E. coli BKIIM B-3996) is
cultured in a first nutrient medium, and a further nutrient medium
comprising at least one carbon source, at least one nitrogen source (such as
ammonium sulfate), at least one phosphorous source; and removing the
formed L-threonine, wherein the concentration of the carbon source during
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Appeal2013-007048
Application 11/427,482
culturing is adjusted to not more than 30 g/L (Kruse 4, 1. 14 to 5, 1. 14; 20, 1.
25; 13, 11. 5---6).
5. Kruse teaches that the "first nutrient medium comprises as the
source of carbon one or more of the compounds ... in concentrations of 1 to
50 g/kg"; that the "sources of nitrogen can be used individually or as a
mixture in concentrations of 1 to 40 g/kg"; that the "source of phosphorus in
the first nutrient medium [is] in concentrations of 0 .1 to 5 g/kg" and that
"magnesium sulfate or iron sulfate ... are present in concentrations of 0.003
to 3 g/kg" (Kruse 11, 1. 29 to 12, 1. 28).
6. Debabov teaches a process for producing L-threonine using E.
coli BKIIM B-3996, capable in producing 85 g/L L-threonine for a 36-hour
fermentation period, in a fermentation medium, which contains a source of
carbon and nitrogen (Debabov col. 4, 11. 9-15; col. 1, 11. 12-15; col. 2, 11. 9-
10).
7. Example 1 ofDebabov, which teaches a nutrient medium for E.
coli BKIIM B-3996, wherein saccharose is a carbon source, and ammonium
sulfate is a nitrogen/sulfur source, is reproduced below:
~aci;;b;p;~-s~
{1\;"H.:;b,S04
KHJl04
MgSO.o..7H20
FeS04.~0
Mg$04.5~0
yewit autolyz;ate
water ·
4_Q
o.~
0,2
0.,04
0.002
0~002
0~2
to ma..\e 100 percent
(Debabov, col. 4, 11. 49---65; col. 3, 11. 57---62).
8. De Hollander teaches that
In a batch fermentation an organism grows until one of the
essential nutrients in the medium becomes exhausted .. .In
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Appeal2013-007048
Application 11/427,482
fed-batch fermentations ... The microorganism will
continue to grow, at a growth rate dictated by the rate of
nutrient feed. Generally a single nutrient, very often the
carbon source, will become limiting for growth ....
Different types of nutrient limitation can be employed.
Carbon source limitation is most often used. Other examples
are limitation by the nitrogen source ... limitation by
sulphur and limitation by phosphorous. The latter two types
of limitation are less common employed, because most
organisms require only small amounts of these compounds
for growth, and it can be difficult to achieve such a
limitation during an industrial scale fermentation, especially
when raw materials of a complex or partly unknown
composition are used.
(col. 1, 1. 50 to col. 2, 1. 1).
9. Example 2 of De Hollander teaches testing of different ratios of
phosphorous and carbon and of a phosphorus limitation for a lysine over-
producing Corynebacterium glutamicum strain, determining the conditions
of optimum phosphorous/ carbon ratio, and further determining as a result
that relative concentration of lysine and biomass in the culture are strongly
dependent on the degree of phosphorous limitation (col. 6, 11. 25-5 6).
10. Table 1 of the Ishikawa Declaration7 discloses production of
lysine by E. coli WC196LC/pCABD2 under sulfur-limited condition.
7 Declaration of Kohei Ishikawa, dated Mar. 24, 2010.
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Appeal2013-007048
Application 11/427,482
Table 1. Experimenbl condition and results
MgS04•7H20 (g/l) MgCl2·6II;~O (gil)
----"------"--"----"""---· ·~··-----------------
LOOO 0.000
0.750 0.206
0.500 0.412
0.250 0.619
0.100 0.742
0.075 0.763
0.050 0.784
0.025 0.804
0.010 0.816
0 0.825
(Ishikawa Declaration 2).
lysine production (gll)
5.440
5.955
6.615
6.580
5.670
4.690
5.625
4.345
4.445
3.900
11. Table 1 of the Iida Declaration8 discloses production of L-
threonine by E. coli VKPM B-5318 under phosphorous-limited condition.
(Iida Declaration 2).
Principles of Law
l''--·~!s~~~~~;~;:;;i::~····-··f ·-~~~~~~~1
~ t~ .... ,....,.1:,.~-=--~""-L.~ \·Z-" J ... ,.!,,~ ~ f :.~;~.-~ ~
~-.......... ,.....-.................... ........, ......................... ,... ....... ...,_,,_._,_._..,~ .. -.. ..._ .... ;,..::::~2..- .... {
~ {"!: ~ _.·~-c {'!- ;
~--......... _· ........ ...:.,·.· .... ~ _.\'? ...... .:.· .. · ....... ; .. ,,.~-- ... ~>) ... :~--- ~
l -~ l ~:i··< ' i
i::: :::: :1: :::~ ::: =il!ti]
Although the record may establish evidence of secondary
considerations which are indicia of nonobviousness, the record may also
establish such a strong case of obviousness that the objective evidence of
nonobviousness is not sufficient to outweigh the evidence of obviousness.
8 Declaration of Yohei Iida, dated Feb. 17, 2011.
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Application 11/427,482
Newell Cos. v. Kenney Mfg. Co., 864 F.2d 757, 769 (Fed. Cir. 1988), cert.
denied, 493 U.S. 814 (1989). See also Pfizer Inc. v. Apotex Inc., 480 F.3d
1348, 1372 (Fed. Cir. 2007) (unexpected results not sufficient to outweigh a
strong showing of obviousness).
Analysis
We adopt the Examiner's findings of fact and reasoning regarding the
scope and content of the prior art (Ans. 2-5; FF 4--9) and agree that the
claims are rendered obvious by Kruse, De Hollander, and Debabov. We
address Appellants' arguments below.
Prima F acie Obviousness
Appellants argue that "in Kruse and DeHollander, there is absolutely
no description of limiting the sulfur concentration to 0.35 g/L or lower in the
fermentation process to produce L-threonine" (App. Br. 6).
We are not persuaded. Kruse teaches that the amounts of medium
components may vary over a wide range (FF 5) while De Hollander teaches
optimizing the amounts of medium components (FF 9). "When the general
conditions of a claim are disclosed in the prior art, it is not inventive to
discover the optimal or workable ranges by routine experimentation." In re
Aller, 220 F.2d 454, 456 (CCPA 1955).
De Hollander expressly suggests, as a part of this routine
experimentation, to assess limitation conditions of carbon, nitrogen, sulfur
and phosphorous in the nutrient bacterial feed (FF 7-9). De Hollander
discloses the general principle of limitation by these ingredients, as well as
teaches a specific example of optimizing the ratio of phosphorous to carbon
in a lysine producing bacterium strand, and of determining as a result that
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Appeal2013-007048
Application 11/427,482
relative concentration of lysine and biomass in the culture are strongly
dependent on the degree of phosphorous limitation (FF 9).
Appellants argue that De Hollander teaches away from limiting sulfur
in the fermentation because it discloses that "limiting sulfur is far less
common than limiting other ingredients, and is actually difficult to achieve",
and that "limiting sulfur and phosphorous is less commonly employed
because most organisms require only small amounts of sulfur and
phosphorous, and it is difficult to achieve such limitation during industrial
scale fermentation" (App. Br. 6). Appellants admit that De Hollander has
shown the effect of limiting phosphorous, or limiting both phosphorous and
carbon in combination, on lysine production by Corynebacterium
glutamicum, but contend that "there are no statements or indications about
limiting sulfur, nor the effect of such, other than those as mentioned above"
(Id.).
We find the teaching away arguments unpersuasive. A teaching away
requires a reference to actually criticize, discredit, or otherwise discourage
the claimed solution. See In re Fulton, 391F.3d1195, 1201 (Fed. Cir. 2004)
("The prior art's mere disclosure of more than one alternative does not
constitute a teaching away from any of these alternatives because such
disclosure does not criticize, discredit, or otherwise discourage the solution
claimed"). De Hollander's teaching that limiting sulfur may be difficult in
industrial fermentation using unknown medium sources (FF 8) represents a
nonpreferred embodiment, but does not rise to the level of discrediting such
sulfur limitation. De Hollander merely discloses an alternative in an
example (limiting phosphorous and/or carbon) and a successful one in favor
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Application 11/427,482
of establishing the principle of using nutrient limitation, which is not, by
itself, sufficient to demonstrate a teaching away. Disclosed examples and
preferred embodiments do not constitute a teaching away from a broader
disclosure or non-preferred embodiments. In re Susi, 440 F .2d 442, 446 n.3
(CCPA 1971).
Secondary Considerations
Applicants contend that the Ishikawa Declaration presents proof that
"the sulfur-limiting conditions in the fermentation of threonine-producing
bacteria is novel, unobvious, and completely unexpected from the prior art
teachings" (App. Br. 7). "More specifically, the data shows that less and
less lysine was produced as the sulfur concentration in the medium was
decreased" (Id.).
We are not persuaded. The Ishikawa Declaration does not
conclusively establish that "less and less lysine was produced as the sulfur
concentration in the medium was decreased" (id.), and in fact, for most of
the data points, the data points to the contrary conclusion (FF9). Thus, at the
highest MgSQ4.7H20 concentration of 1.000 g/l, the data shows lysine
production of 5 .440 g/l, which on dropping the MgSQ4. 7H20 concentration
to 0.750 g/l and 0.500 g/l, in fact increases, not decreases the lysine
production- to 5.955 g/l, and 6.615 g/l, respectively (FF9). The production
is further higher than 5.440 g/l, respectively- 6.580 g/l, 5.670 g/l and 5.625
g/l, for three MgSQ4.7H20 concentrations lower than 1.000 g/l, specifically-
0.250 g/l, 0.100 g/l and 0.050 g/l.
While for three concentrations on the very low end (0.075 g/l, 0.025
g/l and 0.010 g/l) Appellants show a higher lysine production as compared to
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Application 11/427,482
zero MgSQ4. 7H20 g/l, it is unclear, given the rest of the data, whether this
shows an effect, as asserted by Appellants, of decreasing lysine production
with decreasing MgS04. 7H20 concentration, or simply some other effect,
i.e. too critically low MgSQ4. 7H20 concentrations for bacterial growth (Id.).
In this regard, we also note a comparison between the data Appellants
presented in the specification as to their own invention (FF 2, 3), and the
three lower concentrations in the Ishikawa Declaration for which Appellants
purport an effect of sulfur limiting condition giving a positive effect on
lysine fermentation (0.075 g/l, 0.025 g/l and 0.010 g/l) (FF9). Thus, in
comparison the lowest sulfur concentration, which Appellants tested and
reported in their own specification is 0.2 g/l of (NH4)2S04 in Table 1, and
1.0 g/l of (NH4)2S04 in Table 2. Appellants provide no explanation of the
discrepancies of the higher tested concentrations in Appellants'
specification, as compared to the lower tested concentrations in Appellants'
data in the Ishikawa Declaration.
The burden of showing unexpected results rests on "he who asserts
them." See In re Klosak, 455 F.2d 1077, 1080 (CCPA 1972). Given the
above discussed data, Appellants have not met this burden with the Ishikawa
Declaration. Therefore, we do not address any further arguments between
the Examiner and Appellants as to additional differences between the
Ishikawa Declaration and the prior art, such as Appellants' having used in
the Ishikawa Declaration a batch culture, as opposed to a batch-fed culture
as in De Hollander (App. Br. 7-9; Ans. 10-11).
We do note, however, that Appellants did not compare their data to
the closest prior art of Example 1 of Debabov, where an E.coli within the
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Application 11/427,482
scope of claim 1 is cultured in a medium containing carbon, nitrogen and
0.544 mass percent sulfur containing compounds to obtain 85 g/L of L-
threonine (see FF 7). See In re Baxter Travenol Labs., 952 F.2d 388, 392
(Fed. Cir. 1991) ("when unexpected results are used as evidence of
nonobviousness, the results must be shown to be unexpected compared with
the closest prior art.").
Appellants further present data from the Iida Declaration.
In the experiment shown in the Iida Declaration, fed-
batch cultures were performed by adding the feed medium
containing various concentrations of KH2P04. As shown in
the data, when adding the feed medium containing 0, 1, or 2
g/F of KH2P04, the phosphorous concentration in the
fermentation medium decreased to approximately 0 in 13
hours after the start of cultivation, and did not increase until
the end of the cultivation (Figure. 1 ). That is, phosphorous
was limited and the production of L-threonine was
unimproved. By contrast, when adding the feed medium
containing 3, 4, or 5 g/F ofKH2P04, the phosphorous
concentration in the fermentation medium gradually
increased at a latter half of the cultivation (Figured). That is,
phosphorous was not limited.
(App. Br. 10; FFlO; see also Reply Br. 1).
We agree with Appellants that the data in the Iida Declaration
establishes that when phosphorous was limited the production of L-threonine
was unimproved. However, as noted above, the data was not compared with
the closest prior art of Debabov at the particular concentration of sulfur in
Debabov (FF 7).
We conclude that even if the evidence provides a weak indication of a
secondary consideration, the showing is insufficient to overcome the strong
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Application 11/427,482
showing of obviousness in this case. See Pfizer, Inc. v. Apotex, Inc., 480
F.3d 1348, 1372 (Fed. Cir. 2007) ("[W]e hold that even if Pfizer showed that
amlodipine besylate exhibits unexpectedly superior results, this secondary
consideration does not overcome the strong showing of obviousness in this
case. Although secondary considerations must be taken into account, they
do not necessarily control the obviousness conclusion.) This conclusion is
reasonable because Iida does not directly demonstrate that the claimed level
of sulfur is unobvious over the prior art, only that limiting a different
nutrient in a different particular situation with a different particular strain
reduced yield of L-threonine.
Conclusion of Law
(i) The evidence of record supports the Examiner's conclusion that
Kruse, De Hollander, and Debabov render claims 1, 4, and 6-9 obvious.
(ii) Appellants have not presented evidence of secondary
considerations, that when weighed with the evidence of obviousness, is
sufficient to support a conclusion of non-obviousness.
B. 35 U.S.C. § 103(a) over Kruse, De Hollander, Debabov, and Binder
Appellants do not argue separately this obviousness rejection, instead
relying upon their arguments to overcome the combination of Kruse,
Debabov, and De Hollander. The Examiner provides sound fact-based
reasoning for combining Kruse, De Hollander, Debabov, and Binder (see
Ans. 5---6). Having affirmed the obviousness rejection of claims 1, 4, and 6-
9 over Kruse, De Hollander, and Debabov for the reasons given above, we
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Application 11/427,482
also find that the further obvious combination with Binder renders the
rejected claims obvious for the reasons given by the Examiner.
SUMMARY
In summary, we affirm the rejection of claims 1, 4, and 6-9 under 35
U.S.C. § 103(a) as obvious over Kruse, De Hollander, and Debabov.
We affirm the rejection of claims 10, 13, 15-18, 20, and 21under35
U.S.C. § 103(a) as obvious over Kruse, De Hollander, Debabov, and Binder.
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a)(l).
AFFIRMED
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