Tatiana Egorova-Zachernyuk

Patent Trials and Appeals Board

Sep 2, 2020

2019006193 (P.T.A.B. Sep. 2, 2020)

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.
10/564,012 08/11/2006 Tatiana A. Egorova-Zachernyuk P206037PCT1/US 3752
132452 7590 09/02/2020
N.V. Nederlandsch Octrooibureau
New Babylon City Offices
Anna van Buerenplein 21a
The Hague, 2595 DA
NETHERLANDS
EXAMINER
BARRON, SEAN C
ART UNIT PAPER NUMBER
1653
NOTIFICATION DATE DELIVERY MODE
09/02/2020 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.
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PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte TATIANA A. EGOROVA-ZACHERNYUK

Appeal 2019-006193
Application 10/564,012
Technology Center 1600
Before HUBERT C. LORIN, FRANCISCO C. PRATS, and
JAMIE T. WISZ, Administrative Patent Judges.

WISZ, Administrative Patent Judge.

DECISION ON APPEAL

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STATEMENT OF THE CASE
Pursuant to 35 U.S.C. § 134(a), Appellant1 appeals from the
Examiner’s decision to reject claims 13–15, 39 and 40. We have jurisdiction
under 35 U.S.C. § 6(b).
We AFFIRM.
CLAIMED SUBJECT MATTER
The Specification describes “the labelling of biological compounds
with stable isotopes.” Spec. 1. Uniform labelling of biological compounds
“with stable isotopes allows the determination of the three-dimensional
structure by NMR spectroscopy of the biological compound.” Id. Claim 13,
the only independent claim, is illustrative of the claimed subject matter and
is reproduced below:
1. A method for producing a biomolecule in mammalian or
insect cells, whereby at least 95% of the atoms in the
biomolecule, for at least one of H, C or N, are isotopically
labelled, the method consisting of the steps of:
a) growing an organism selected from the group consisting
of Pichia pastoris, Hansenula polymorpha, Cyanidium
caldarium, Galdieria sulpuraria, Scenedesmus obliquus,
and Methylobacillus flagellatus on a chemically-defined
or mineral medium which supports growth of the
organism, whereby in the medium contains at least one
of:
i) a sole carbon source which is 13C-glucose or
NaH[13C]O3, wherein at least 95% of the atoms in the
carbon source is isotopically labelled, and

1 We use the word “Appellant” to refer to “applicant” as defined in
37 C.F.R. § 1.42. Appellant identifies the real party in interest as Tatiana
Egorova-Zachernyuk. Appeal Br. 2.
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ii) a sole nitrogen source which is 15NH4Cl,
wherein at least 95% of the N atoms in the nitrogen
source is isotopically labelled, to produce labelled
biomass;
(b) autolysing the biomass of the organism
grown as in (a) to produce an autolysate;
(c) composing a nutrient medium for
mammalian or insect cells by combining the autolysate as
obtained in (b) with further components necessary for
growth of the mammalian or insect cells;
(d) growing a culture of the mammalian or
insect cells producing the biomolecule under conditions
conducive to the production of the biomolecule, in the
nutrient medium ; and
(e) recovery of the biomolecule,
whereby the biomolecule is a mammalian polypeptide
produced as a result of genetic engineering of the
mammalian or insect cells.
Appeal Br. 13 (Claims App.).
REJECTIONS
The Examiner rejected claims 13 and 40 under 35 U.S.C. § 103(a) as
obvious over Hansen,2 in view of Skladnev.3
The Examiner rejected claims 14 and 15 under 35 U.S.C. § 103(a) as
obvious over Hansen and Skladnev, and further in view of Werten.4

2 Andrew P. Hansen et al., A Practical Method for Uniform Isotopic
Labeling of Recombinant Proteins in Mammalian Cells, 31 Biochemistry
12713–12718 (1992) (“Hansen”).
3 D.A. Skladnev et al., Preparation of 13C+15N-Modified Peptide Antibiotic
Zervamycin II, 5 Biotechnology in Russia, 41–50 (2002) (“Skladnev”).
4 Paul J.L. Werten et al., Large-scale purification of functional recombinant
human aquaporin-2, FEBS Letters 504, 200–205 (2001) (“Werten”).
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The Examiner rejected claim 39 under 35 U.S.C. § 103(a) as obvious
over Hansen and Skladnev, and further in view of Iding5 and Castro6 and as
evidenced by Chung.7
ISSUES AND ANALYSIS
Rejection of claims 13 and 40 under 35 U.S.C. § 103(a) as obvious over
Hansen in view of Skladnev
The Examiner finds that “Hansen teaches a method of growing
biomass (E. coli) in a medium consisting of only [15N] ammonium chloride
as the nitrogen source . . . so 100% of the available nitrogen would be
labeled.” Final Act. 6 (citing Hansen 12714). The Examiner also finds that
“Hansen teaches 15N/13C labeled amino acids obtained from lyophilized
algae” and “enzymatic and acid hydrolysis of bacterial and algal biomass to
produce a[n] isotopically-labeled protein lysate.” Id. (citing Hansen 12714).
The Examiner further finds that “Hansen teaches a nutrient medium
comprising the isotopically-labeled hydrolyzed protein” and “teaches
culturing Sp2/0 mammalian hybridoma cells transfected with a urokinase-
expressing construct . . . in a media containing acid-hydrolyzed bacterial and
algal extracts that have been labeled with 13C and 15N, then recovering the
labeled urokinase.” Id. (citing Hansen 12713–12715). According to the

5 K. Iding et al., An Automatic System for the Assessment of Complex
Medium Additives under Cultivation Conditions, 73 Biotech. and
Bioeng. (2), 442–448 (2001) (“Iding”).
6 Paula Maria Lima e Castro, Optimisation of CHO Cell Growth and
Recombinant Interferon-γ Production, Ph.D. Thesis, University College
London (1993) (“Castro”).
7 John D. Chung et al., Extension of Sp2/0 Hybridoma Cell Viability
Through Interleukin-6 Supplementation, 55 Biotech. and Bioeng. (2), 439–
446 (1997) (“Chung”).
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Examiner, Hansen also “teaches isolation of isotopically-labeled urokinase”
and that the “urokinase produced using Hansen’s method allows for study
with NMR techniques.” Id. (citing Hansen 12715, 12717).
The Examiner acknowledges that Hansen does not teach
Methylobacillus flagellates as claimed, but finds that this deficiency is cured
by Skladnev. Final Act. 7. According to the Examiner, Skladnev, which
“teaches methods of making 13C and 15N-labeled Zervamycin II,” also
teaches growing Methylobacillus flagellates on 13C-methanol
and 15N-ammonium chloride as exclusive carbon and nitrogen
sources respectively on a chemically-defined (e.g. minimal
medium) . . . preparing an autolysate of the 13C and 15N-labeled
M. flagellates . . . adding the 13C and 15N-labeled M. flagellates
autolysate to a E. salmosynnemata nutrient medium . . . growing
E. salmosynnemata with the nutrient medium comprising 13C and
15N-labeled M. flagellates autolysate to yield 13C and 15N labeled-
Zervamycin II.
Id. (citing Skladnev 43–45).
The Examiner further finds that “it would have been obvious before
the invention was made to substitute the 13C and 15N M. flagellatus
autolysate of Skladnev for the 13C and 15N hydrolyzed bacterial and algal
biomass of Hansen.” Final Act. 7. The Examiner also finds that a person of
ordinary skill in the art would have had a reasonable expectation of success
in doing so “because Hansen and Skladnev are both directed towards
methods of making radiolabeled proteins by growing cells with a 13C and
15N radiolabeled nutrient source such that 13C and 15N are incorporated into
the protein of interest.” Id. The Examiner concludes:
The skilled artisan would have been motivated to [do so]
because Hansen teaches there are known limitations of
radiolabeled hydrolyzed biomass (e.g. acid hydrolysis of
glutamine, asparagine, cysteine and tryptophan) and so the
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substitution of the autolyzed biomass of Skladnev would
improve Hansen’s methods as the lack of any acid hydrolysis
step would preserve those radiolabeled acid-labile amino acids
for incorporation into the downstream mammalian cell culture
and radiolabeled protein production methods of Hansen.
Id. at 7–8.
Appellant argues that Skladnev does not teach autolysing as defined in
the Specification and as commonly used in the field despite Skladnev’s use
of the term. Appeal Br. 6–7. According to Appellant, “[a]utolysis is a term
that is commonly used in the field of biology, it is also known as
self-digestion and refers to the destruction of a cell through the action of its
own enzymes.” Id. at 6. Appellant further asserts that “[a]utolysis of cells
usually comprises an incubation of the cells at an elevated temperature (30-
50°C) for a prolonged period of time (3-18 hours) in the presence of a
plasmolysing agent, such as e.g. NaCl, ethanol, ethyl acetate, chloroform or
dextrose” and that “[d]uring the incubation cellular components are
hydrolysed by the cell’s endogenous hydrolytic enzymes, the cell wall
breaks and disintegrates and releases the proteinaceous content into the
aqueous environment.” Id. (citing Spec. 20:19–31).
Appellant further contends that the Specification “demonstrates that
the process of autolysis is not carried out at an acidic pH” and cites to the
Specification which states, “‘[s]ubsequently the yeast cells are allowed to
autolyse at a pH of 6.5-10.0, preferably a pH of 7.5-8.0 and at 10% w/v
NaCl’” and to “Example 1 wherein the pH during autolysis is pH 8.0.” Id.
at 7 (quoting Spec. 21–22) (emphasis omitted). According to Appellant,
“Skladnev clearly teaches to use acid hydrolysis to prepare isotopically
labelled amino acids for mammalian cell growth” because, in Skladnev, “the
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cells were subjected to a mild autolysis with a solution of 0,3 M HCl.” Id.
(quoting Skladnev 43). Appellant contends that “[a]utolysis of cells does
not comprise adding a strong acid such [as] HCl to the cells as this would
interfere with the process of autolysing.” Id. Therefore, according to
Appellant, Skladnev “teaches that the cells are lysed through acidic
hydrolysis” which is “similar to the process used by Hansen who correctly
refers to this process as the acid hydrolysis of bacterial and algal cells.” Id.
at 7–8.
Appellant also asserts that the combined teaching of Skladnev and
Hansen do not yield the claimed method. Appeal Br. 8. According to
Appellant, Skladnev does not teach autolysis and Hansen does not remedy
this deficiency and, in fact, teaches away from using acid hydrolysis. Id.
Specifically, Appellant asserts that Hansen teaches that acid hydrolysis
causes detrimental side effects to the amino acids in the medium because
hydrolysis destroys amino acids such as glutamine, asparagine, cysteine, and
tryptophan and that cell growth is stunted because acid hydrolysis was used
to lyse the biomass. Id. (citing Hansen 12715). Therefore, according to
Appellant, the skilled person would have to look for other ways to lyse the
biomass in order to prepare autolysates of the bacteria but would not find
that solution in Skladnev because Skladnev teaches to use acid hydrolysis.
Id.
Having considered Appellant’s arguments in support of claim 13, we
are not persuaded of any reversible error in the Examiner’s rejection of this
claim. We find that the Examiner has presented a prima facie case of
obviousness and Appellant has not sufficiently rebutted this prima facie case.
First, although Appellant asserts that Skladnev does not teach an autolytic
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process, as that process is commonly defined in the field of biology,
Appellant does not provide any evidence of how this term is defined in the
field nor provide evidence that autolysis excludes the use of hydrochloric
acid as taught in Skladnev. Without such evidence, Appellant merely relies
on attorney argument regarding how the term “autolysis” is used in the field
of biology. “[S]tatements of counsel in a brief cannot take the place of
evidence.” In re Walters, 168 F.2d 79, 80 (CCPA 1948).
Next, we agree with the Examiner that Appellant is relying on
unclaimed features to establish patentability because claim 13 does not recite
a particular pH nor does it exclude the use of hydrochloric acid for
autolysing the biomass. “[T]he claims define the invention. . . .
[L]imitations from the specification are not to be read into the claims.”
Sjolund v. Musland, 847 F.2d 1573, 1582 (Fed. Cir. 1988). In addition,
although Appellant asserts that the Specification indicates that the process of
autolysis is not carried out in an acidic pH, the Specification does not
explicitly define autolysis and includes an example in which the yeast cells
are allowed to autolyze at a pH of 6.5–10.0, which would include acidic pH
ranges of 6.5–6.9. See Spec. 21–22. Furthermore, these pH ranges are
provided in the context of exemplary conditions and there is no explicit
definition of autolysis in the Specification which includes particular pH
ranges or excludes the use of 0.3 M HCl as taught in Skladnev. “[D]uring
examination proceedings, claims are given their broadest reasonable
interpretation consistent with the specification.” In re Hyatt, 211 F.3d 1367,
1372 (Fed. Cir. 2000). Therefore, we find that Skladnev teaches autolysis as
recited in claim 13.
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We are also not persuaded by Appellant’s argument that Hansen
teaches away from the invention, which is premised on Appellant’s assertion
that Skladnev does not disclose autolysis. As discussed above, we find that
Skladnev does disclose autolysis and we are not persuaded by Appellant’s
attorney arguments that the autolysis disclosed in Skladnev, which uses 0.3
M HCl, is the same as the acid hydrolysis disclosed in Hansen, which uses 4
M methanesulfonic acid. Furthermore, one of ordinary skill in the art would
not have been dissuaded from autolysing the biomass using the teachings in
Skladnev based on the disclosure of Hansen. As discussed by the Examiner,
because Hansen teaches that there are known limitations to acid hydrolysis,
one of skill in the art would have been motivated to use the autolytic process
of Skladnev instead.
Appellant further contends that, even if Skladnev and Hansen were
combined, there would be no reasonable expectation of success. Appeal Br.
9. First, Appellant contends that because Skladnev fails to disclose
autolysing the biomass of the organism to produce an autolysate and because
Hansen does not cure this deficiency, the “reasonable expectation of
success” rationale cannot be applied to this situation. Id. Second, Appellant
contends that, even if the rationale could be applied, there would be no
reasonable expectation of success because of the different types of cells
used. Id. Specifically, according to Appellant, Skladnev teaches the use of
the “acid hydrolysed biomass of M. flagellates for the preparation of a
medium to grow E. salmosynnemata to yield 13C and 15N
labeled-Zervamycin II” and that “E. salmosynnemata is a mycelial fungus.”
Id. (citing Skaldnev Introduction). Appellant further argues:
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Thus Skaldnev teaches a method of incorporating 13C and 15N
labeled amino acids into fungal cells, i.e. microbial cells. In
contrast, claim 13 relates to a method for producing a
biomolecule in mammalian or insect cells. A skilled person
would have no reasonable expectation of success that a method
to grow microbial cells would also work to grow mammalian or
insect cells since Hansen teaches that “Mammalian cells [. . .]
are more sensitive to toxic substances than bacteria (Thomas,
1990; Eagle, 1955)” (top right hand column page 12713 of
Hansen).
Id. Appellant further argues:
Hansen further teaches that when algal proteins are
enzymatically hydrolyzed, the media prepared with the resulting
amino acids was unable to support the growth of mammalian
cells. Hansen states that “endotoxins typically found in algal cell
extracts[. . .] are known to be toxic to mammalian cells.” (See
first paragraph of the right hand column at page 12715 of
Hansen).
Id.
Therefore, Appellant asserts that “one of ordinary skill in the art
would have recognized that it is unpredictable whether an autolysate of a
particular microorganism can successfully be used as a nutrient for culturing
mammalian or insect cells” and “would not have reasonably expected to
successfully arrive at the claimed invention by using the autolysate of the M
flagellateus of [Skladnev] to grow mammalian or insect cells as recited in
claim 13.” Appeal Br. at 9–10.
We are not persuaded by Appellant’s arguments. First, as discussed
above, we find that Skladnev does disclose autolysing the biomass of the
organism to produce an autolysate. Second, we note that “[o]bviousness
does not require absolute predictability of success. . . . For obviousness
under § 103, all that is required is a reasonable expectation of success.” In
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re O’Farrell, 853 F.2d 894, 903–04 (Fed. Cir. 1988). None of Appellant’s
arguments suggests that Skladnev’s radiolabeled autolysate from the
bacteria, M. flagellatus, could not be predictably added to Hansen’s cell
culture medium, which is also used for radiolabeled amino acids obtained
from bacteria (E. coli). Therefore, there is no suggestion that the
mammalian cells disclosed in Hansen would not be able to take up the
radiolabeled carbon and nitrogen atoms from the autolyzed biomass of M.
flagellates as disclosed in Skladnev as they took up the radiolabeled atoms
from hydrolyzed E. coli. Also, because Skladnev’s radiolabeled autolysate
is obtained from the bacteria M. flagellates, there is no indication of, and
Appellant has not pointed towards any, alleged toxic substance found in the
M. flagellates’ autolysate that could support Applicant’s argument of
unpredictability. Id.
Appellant also contends that claim 13 uses the phrase “consisting of”
and, therefore, excludes steps that are not specifically recited in the claim.
Appeal Br. 10–11. We agree with Appellant that claim 13 recites the phrase
“consisting of” in the preamble such that the claim excludes additional
unrecited steps. However, Appellant has not pointed to a specific step from
the combination of Hansen and Skladnev that is excluded by the claim.
Furthermore, since the claim does not recite the specific conditions for
autolysis, Skladnev teaches this step for the reasons discussed above.
For the reasons described herein and those already of record, we
sustain the Examiner’s rejection of independent claim 13 under 35 U.S.C.
§ 103(a) as being obvious over Hansen in view of Skladnev. Claims 40 is
not argued separately, and, therefore, falls with claim 13. See 37 C.F.R.
§ 41.37(c)(1)(iv).
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Rejection of claims 14 and 15 under 35 U.S.C. § 103(a) as obvious over
Hansen and Skladnev, and further in view of Werten
The Examiner’s findings with respect to Hansen and Skladnev are
discussed above. Regarding claims 14 and 15, the Examiner acknowledges
that Hansen and Skladnev do not teach expressing a membrane protein but
finds that Werten cures this deficiency. Final Act. 8. Specifically, the
Examiner finds that “Werten teaches a method of overexpressing the
membrane receptor aquaporin-2 using recombinant DNA constructs in insect
cells and purification of aquaporin-2.” Id. (citing Werten Abstract, 201).
The Examiner also finds that “[a] person of ordinary skill in the art would
have had a reasonable expectation of success in substituting Werten's
aquaporin-2 and insect cells for Hansen's urokinase and CHO cells.” Id. at
9.
In response to this rejection, Appellant states that, “[a]s outlined here
above, a person skilled in the art reading Hansen and Skladnev would not
arrive at the present invention” and that Werten does not remedy these
deficiencies. Appeal Br. 11. Therefore, for the reasons explained above, we
also sustain the Examiner’s obviousness rejection of claims 14 and 15.
Rejection of claim 39 under 35 U.S.C. § 103(a) as obvious over Hansen and
Skladnev, and further in view of Iding and Castro and as evidenced by
Chung
The Examiner’s findings with respect to Hansen and Skladnev are
discussed above. The Examiner also finds that Hansen further teaches
culturing Sp2/0 cells in Gibco® Hybridoma-SFM culture media. Final Act.
10 (citing Hansen 12714, Fig. 1). The Examiner also relies on Chung to
show that Sp2/0 cells are hybridoma cells and relies on Iding for its teaching
of methods of culturing hybridoma cells with media comprising yeast extract
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and glucose. Id. (citing Chung Abstract; Iding 442). The Examiner also
finds that Castro teaches that glucose and glutamine are typical carbon
sources in method of mammalian cell culture. Id. (citing Castro 36).
Regarding claim 39, the Examiner finds that “absence evidence to the
contrary the Gibco® Hybridoma-SFM culture media meets the limitations of
[this claim].” Id.
In response to this rejection, Appellant states that, “[a]s outlined here
above, a person skilled in the art reading Hansen and Skladnev would
not . . . arrive at the present invention” and that the teachings of Iding and
Castro as evidenced by Chung do not remedy these deficiencies. Appeal Br.
11. Therefore, for the reasons explained above, we also sustain the
Examiner’s obviousness rejection of claim 39.
CONCLUSION
For the reasons described herein and those already of record, we
affirm the Examiner’s rejection of claims 13–15, 39 and 40.

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DECISION SUMMARY
In summary:
Claims
Rejected
35 U.S.C.
§
Reference(s)/Basis Affirmed Reversed
13, 40 103 Hansen, Skladnev 13, 40
14, 15 103 Hansen, Skladnev,
Werten
14, 15
39 103 Hansen, Skladnev,
Iding, Castro,
Chung
39
Overall
Outcome
13–15, 39,
40

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). See 37 C.F.R.
§ 1.136(a)(1)(iv).
AFFIRMED