Denis Drapeau et al.

Patent Trials and Appeals Board

Aug 25, 2021

2020005928 (P.T.A.B. Aug. 25, 2021)

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.
11/585,607 10/24/2006 Denis Drapeau PC60105A 7317
25291 7590 08/25/2021
Pfizer Inc.
Attn:Legal Patent Department, Chief IP Counsel
235 East 42nd Street
New York, NY 10017
EXAMINER
POPA, ILEANA
ART UNIT PAPER NUMBER
1633
NOTIFICATION DATE DELIVERY MODE
08/25/2021 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):
PfizerPatentDocketing@pfizer.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte DENIS DRAPEAU, YEN-TUNG LUAN,
PAUL THODAY, WENGE WANG, and JUDY CHOU
__________

Appeal 2020-005928
Application 11/585,607
Technology Center 1600
__________

Before JEFFREY N. FREDMAN, TAWEN CHANG, and
TIMOTHY G. MAJORS, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal1 under 35 U.S.C. § 134 involving claims to a method
of producing a protein of interest in cell culture. The Examiner rejected the
claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We affirm.

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 Pfizer Inc.
(see Appeal Br. 3). We refer to the Specification of Oct. 24, 2006 (“Spec.”);
Non-Final Action of Apr. 18, 2019 (“Non-Final Act.”); Appeal Brief of Mar.
13, 2020 (“Appeal Br.”); Examiner’s Answer of June 18, 2020 (“Ans.”); and
Reply Brief of Aug. 17, 2020 (“Reply Br.”).
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Statement of the Case
Background
“Culturing cells has produced many protein products. These products,
such as hybridoma-produced monoclonal antibodies, can be used for
therapeutic, research or other applications” (Spec. ¶ 2). “Adding a chemical
agent to a cell culture medium can increase cell productivity by inducing
cells to produce a product . . . . The addition of an agent can increase the
specific productivity of the cells, but also have cytotoxic effects and can
inhibit cell growth and viability” (id. ¶ 3). “Therefore, what is needed is a
chemical agent that is added to the cell culture medium that can enhance the
expression of a protein of interest while maintaining high cell viability” (id.
¶ 5).
The Claims
Claims 1, 5–7, 9, 17–19, 21, and 24–33 are pending. Independent
claim 1 is representative and reads as follows:
1. A method of producing a protein of interest in cell
culture comprising steps of:
culturing mammalian cells that are genetically
manipulated to express or activate expression of a gene
encoding the protein of interest, which gene is expressed
under conditions of cell culture, in a cell culture medium
comprising an anti-senescence compound, wherein the
anti-senescence compound is selected from the group
consisting of carnosine, acetyl-carnosine, homo-
carnosine, anserine, and β-alanine and combinations
thereof; and
maintaining the culture under conditions and for a time
sufficient to permit expression of the protein;
wherein the cell culture exhibits an improved cell culture
characteristic that differs from a corresponding cell
culture characteristic that would be observed under
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otherwise identical conditions in an otherwise identical
medium that lacks the anti-senescence compound;
wherein the improved culture characteristic is selected
from the group consisting of: increased titer, increased
cell specific productivity, decreased accumulation of high
molecular weight aggregates, decreased accumulation of
acidic species, and combinations thereof; and wherein the
mammalian cells are Chinese hamster ovary (CHO) cells.
The Issues
A. The Examiner rejected claims 1, 5–7, 9, 17–19, 21, 24, and 26–33
under 35 U.S.C. § 103(a) as obvious over Schatz,2 Chaplen,3 Grigg,4 and
Hipkiss5 (Non-Final Act. 3–5).
B. The Examiner rejected claims 1, 5–7, 9, 17–19, 21, and 24–33 under
35 U.S.C. § 103(a) as obvious over Schatz, Chaplen, Grigg, Hipkiss, and
Veldman6 (Non-Final Act. 5).

A. 35 U.S.C. § 103(a) over Schatz, Chaplen, Grigg, and Hipkiss
The Examiner finds Schatz teaches
method of producing Fab antibodies fragments in a CHO cell
line, the method comprising: (i) a growth or exponential phase
wherein the Fab-expressing CHO cells are expanded in cell
culture medium . . . (ii) a temperature shift from 37°C to 28°C
wherein the medium is replaced with fresh medium; and (iii) a

2 Schatz et al., Higher Expression of Fab Antibody Fragments in a CHU Cell
Line at Reduced Temperature, 84 Biotechnology & Bioengineering 433–8
(2003).
3 Chaplen et al., Evidence of high levels of methylglyoxal in cultured Chinese
hamster ovary cells, 95 Proc. Nat’l Acad. Sci. USA 5533–8 (1998).
4 Grigg et al., WO 92/09298 A1, published June 11, 1992.
5 Hipkiss et al., A possible new role for the anti-ageing peptide carnosine, 57
Cell Mol. Life Sci. 747–53 (2000).
6 Veldman et al., WO 2004/037861 A2, published May 6, 2004.
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production phase wherein the Fab-expressing CHO cells are
cultured at 28°C to maximize Fab production.
(Non-Final Act. 3). The Examiner acknowledges that Schatz doesn’t teach
“supplementing their culture medium with carnosine” (id.). The Examiner
finds Chaplen teaches “methylglyoxal is produced during CHO cell cultures
and . . . causes growth inhibition” and that Hipkiss teaches “carnosine as an
anti-senescence compound . . . [that] protects the cells from deleterious
agents such as methylglyoxal” (id.). The Examiner finds Grigg teaches
“carnosine in the culture medium promotes cell growth . . . slows down
senescence of late passage cells in culture, and rejuvenates senescent cells”
(id. at 4). The Examiner finds it obvious to add carnosine to cell growth
media “in order to promote cell growth, slow down senescence of late
passage cells in culture, and rejuvenate[] senescent cells” (id.).
Appellant contends
there is nothing in the combined teachings of Chaplen, Grigg,
and Hipkiss that would suggest to a skilled person that an
obvious way to improve a method of producing a protein of
interest would be to add an anti-senescence compound to a cell
culture method as presently claimed[.] This is for at least the
reasons that:
i) The cited references collectively provide very little
information regarding the effect of anti-senescence compounds
on CHO cells;
ii) The cited references collectively provide no
information regarding the effect of anti-senescence compounds
on the production of a protein of interest in cell culture;
iii) A person of skill in the art would not have had a
reasonable expectation of success of the claimed method based
on the teachings in the cited references.
(Appeal Br. 9).
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The issue with respect to this rejection is: Does a preponderance of
the evidence of record support the Examiner’s conclusion that the prior art
suggests, with a reasonable expectation of success, that the addition of
carnosine to cell growth media would improve protein production?
Findings of Fact
1. Schatz teaches “Chinese hamster ovary (CHO) cells . . . are
most widely used for establishing stable mammalian cell lines for protein
production” (Schatz 433, col. 1).
2. Schatz teaches “Fab expression vectors for establishing stable
CHO transfectant cells were constructed by standard recombinant DNA
technology” (Schatz 434, col. 1).
3. Schatz teaches “plasmids were co-transfected into CHO dhfr-
cells and stable cell lines were established . . . . Secretion of functional Fab
fragments was confirmed by ELISA and Western blotting . . . . A stable Fab-
expressing clone designated, EDHProl98AB2Fab, was used for the
subsequent temperature shift experiment without prior gene amplification”
(Schatz 435, col. 2).
4. Schatz teaches “cells were propagated in SM and cells were
split and distributed to tissue culture flasks . . . . [M]easurement of the Fab
concentrations showed that 9 μg Fab had been produced per flask during the
growth period” (Schatz 435, col. 2).
5. Schatz teaches:
We harvested three flasks from each
temperature/medium combination after 24, 48, 72, 120, 144,
and finally 216 h. In each flask the cells were counted and Fab
concentrations measured. At 37°C cells continued to grow in
SM, leading to a cell density twofold higher after 216 h than
that under the other conditions. Accordingly, production
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continued and the total amount of Fab increased threefold
during this time period, yielding 12 μg Fab per flask.
(Schatz 435, col. 2).
6. Schatz teaches a “simple shift to 28ºC enabled the cells to
increase their specific productivity whereby growth was arrested, and a
much more efficient expression during the entire 9-day production period
was achieved” (Schatz 437, col. 1).
7. Schatz teaches “the potential for gaining the far higher yield we
have shown to be possible is of extraordinary interest for any protein
production, whether on the laboratory scale or in an industrialized process”
(Schatz 437, col. 2).
8. Chaplen teaches: “By using the assay reported herein, up to 310
μM methylglyoxal was detected in CHO cells. This represented 100- to
l,000-fold more methylglyoxal than previously reported in biological
systems” (Chaplen 5537, col. 2). Chaplen teaches “the level of free
methylglyoxal in the extracellular growth medium can be expected to
accurately reflect the levels found in the cell” (id.).
9. Chaplen teaches a “growing body of evidence supporting a
possible role for endogenously produced methylglyoxal in AGE formation,
mutagenesis, apoptosis, and growth inhibition” (Chaplen 5538, col. 1;
citations omitted).
10. Grigg teaches “the senescence of cells, particularly human
fibroblast cells, may be delayed, prevented and/or reversed by maintaining
the cells in the presence of an effective amount of a compound having
chemical properties similar to carnosine” (Grigg 2:34 to 3:3). Grigg teaches
“that the active compound is present in the composition in a concentration
range of 10mM to 100mM” (Grigg 6:19–20).
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11. Grigg teaches an “active compound is carnosine” that “may be
administered to the cells in a culture medium in the case of cells being
cultured in vitro” (Grigg 6:11–26).
12. Figure 8 of Grigg is reproduced below:

“Figure 8 shows the growth of MRC-5 cells after transfer from normal
medium to medium containing 20mM (0-0) or 30mM (x-x) carnosine at PD
level 55. The control cells (0-0) ceased growth at PD level 61 (dashed line)”
(Grigg 8:22–25).
13. Hipkiss teaches “carnosine can react with low-molecular-
weight aldehydes and ketones indicating that it might also act as a naturally
occurring anti-glycating agent” (Hipkiss 747, col. 2).
14. Hipkiss teaches carnosine “can react with many potential
glycating agents thereby inhibiting their ability to react with and modify
polypeptides, as shown by our studies of protein cross-linking in the
presence of . . . methylglyoxal” (Hipkiss 748, col. 1).
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15. Hipkiss teaches “carnosine inhibited the reaction of [14C]-
lysine with the methylglyoxal-treated protein . . . not only can carnosine
react with protein carbonyls but it can also moderate their deleterious action
towards other molecules” (Hipkiss 748, col. 2 to 749, col. 1).
16. Hipkiss teaches “[c]arnosine has been shown to protect cells
(cultured human fibroblasts and lymphocytes, cultured rat brain endothelial
cells and Chinese hamster ovary cells) against the deleterious effects of
acetaldehyde and malondialdehyde” (Hipkiss 748, col. 1).
17. Hipkiss teaches “a model system also showed that carnosine
inhibited modification of a protein by the AGE formed from the reaction of
lysine with methylglyoxal” (Hipkiss 748, col. 1).
Principles of Law
A prima facie case for obviousness “requires a suggestion of all
limitations in a claim,” CFMT, Inc. v. Yieldup Int’l Corp., 349 F.3d 1333,
1342 (Fed. Cir. 2003), and “a reason that would have prompted a person of
ordinary skill in the relevant field to combine the elements in the way the
claimed new invention does.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398,
418 (2007).
Analysis
We adopt the Examiner’s findings of fact and conclusion of law (see
Non-Final Act. 3–5, FF 1–16) and agree that the combination of prior art
renders the claims obvious. We address Appellant’s arguments below.
Appellant contends “the totality of information in the cited references
directly relating to the effect of carnosine on CHO cells is that carnosine has
been shown to protect CHO cells against the deleterious effects of
acetaldehyde and malondialdehyde” and also contends “this information
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would be of unclear significance to a person of skill in the art interested in
producing a protein of interest in CHO cell culture” (Appeal Br. 9–10).
Appellant also contends “Hipkiss does not state that carnosine protects cells
from methylglyoxal. While Hipkiss does mention that carnosine can react
with glycating agents such as methylglyoxal . . . this is not a teaching that
carnosine can protect cells from methylglyoxal” (id.).
We find these arguments unpersuasive as failing to address the
references in combination and especially the teachings of Hipkiss. As found
above, Schatz teaches culturing CHO cells to express proteins of interest and
maintaining the culture to permit protein expression (FF 1–6). Schatz
motivates improvements in the protein production process by teaching “the
potential for gaining the far higher yield we have shown to be possible is of
extraordinary interest for any protein production” (FF 7). Chaplen teaches
that naturally occurring methylglyoxal is present in cells during cell culture
and is associated with cell death (apoptosis) and growth inhibition (FF 8–9).
Hipkiss not only teaches “[c]arnosine has been shown to protect . . .
Chinese hamster ovary cells [CHO] . . . against” aldehyde compounds (FF
16) as noted by Appellant, but immediately preceding that statement Hipkiss
also teaches that carnosine can react with glycating agents “thereby
inhibiting their ability to react with and modify polypeptides, as shown by
our studies of protein cross-linking in the presence of . . . methylglyoxal”
(FF 14). And Hipkiss teaches “a model system also showed that carnosine
inhibited modification of a protein by the AGE [advanced glycation
endproducts] formed from the reaction of lysine with methylglyoxal” (FF
17).
Thus, the ordinary artisan would reasonably be motivated to
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incorporate carnosine into Schatz’s CHO cell growth medium in order to
protect the CHO cells from methylglyoxal caused cross-linking of
polypeptides, particularly when Schatz is interested in producing
polypeptides (FF 7–9, 14, 15).7 The ordinary artisan would also recognize
and reasonably expect that by limiting the ability of methylglyoxyl to
crosslink polypeptides in CHO cells or crosslink with AGE to form
aggregates, there would be a decreased accumulation of high molecular
weight aggregates composed of such methylglyoxyl cross-linked
polypeptides (FF 14). That is, as “carnosine inhibited the reaction of [14C]-
lysine with the methylglyoxal-treated protein . . . not only can carnosine
react with protein carbonyls but it can also moderate their deleterious action
towards other molecules” (FF 15). Thus, Hipkiss does teach that carnosine
can protect proteins within cells from deleterious actions due to
methylglyoxal, an additional reason to incorporate carnosine into CHO cell
growth medium (see FF 15).
Appellant more specifically contends that
Chaplen just uses CHO cells as a model system for mammalian
cells, and is not focused understanding CHO cells in order to
improve methods of CHO cell culture. Thus, a person of skill
in the art would not conclude from Chaplen that methylglyoxal
was clearly a significant factor negatively affecting CHO
cultures for the production of recombinant proteins.
(Appeal Br. 10).
We find this argument unpersuasive because the ordinary artisan,
informed by Chaplen that methylglyoxal found in CHO cells (FF 8) inhibits

7 We also find unpersuasive Appellant’s argument contending that the
motivation could not be strong because the invention was not anticipated
(see Reply Br. 4) because attorney assumptions are not evidence.
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cell growth, induces cells to apoptose, and causes cross-linking between
AGE and polypeptides (FF 9), would have had reason to reduce the
methylglyoxal inhibitory compound to achieve Schatz’s goal of increased
polypeptide yield (FF 7). See In re Kemps, 97 F.3d 1427, 1430 (Fed. Cir.
1996) (“[T]he motivation in the prior art to combine the references does not
have to be identical to that of the applicant to establish obviousness.”).
Appellant contends
none of the cited references provide any information regarding
the effect of an anti-senescence compound on the production of
a protein of interest in cell culture. Furthermore, as described
further below, the two cited references that relate to anti-
senescence compounds (Hipkiss and Grigg) do not provide cell
culture-related information that is of clear relevance to a
method for producing a protein of interest.
(Appeal Br. 11). Appellant further contends “methods for producing a
protein of interest in cell culture as provided in the present application occur
over a much shorter time frame than the experiments provided in Grigg” (id.
at 12). Appellant contends “a potential benefit that appears to only manifest
late in the cell culture process (as provided in the data of Grigg) would be of
low interest to a person of skill in the art seeking to develop improved
method of producing a protein of interest in cell culture” (id.).
We find this argument unpersuasive for several reasons. First, both
Grigg and Hipkiss teach that carnosine makes cells healthier (FF 10–17) and
Hipkiss specifically states that carnosine prevents cross-linking of proteins
and modification by AGE (FF 15, 17). And Grigg teaches a fairly linear
effect of carnosine on cell growth over time and doublings, suggesting that
carnosine is beneficial to cell growth generally, not just at late times (see FF
12). We agree with the Examiner that the ordinary artisan would have
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recognized “protein production is correlated to cell viability and growth and
thus, would have readily concluded that increased cell viability and growth
would translate into improved protein production” (Ans. 9). Dead cells
produce no protein. We therefore also find that addition of carnosine would
have been expected to result in improved protein production by CHO cells
based on the teachings in the prior art (FF 8–17).
Second, while Appellant is correct that example 1 of the Specification
completed protein production in 14 days (see Spec. ¶ 104), the claims
include no limitation regarding the length of time in cell culture for which
protein production may occur and Appellant does not identify any disclosure
in the Specification suggesting a limit on the time period for cell culture.
Instead, the Specification states the “production phase continues until a
desired endpoint is achieved” (Spec. ¶ 39). See In re Self, 671 F.2d 1344,
1348 (CCPA 1982) (“[A]ppellant’s arguments fail from the outset because
. . . they are not based on limitations appearing in the claims.”)
Third, Appellant’s entire line of reasoning represents attorney
argument without any evidentiary support. Appellant provides no evidence
that benefits occurring later in the protein culture process are of low interest,
but simply asserts that statement as a fact. However, “attorney argument [is]
not the kind of factual evidence that is required to rebut a prima facie case of
obviousness.” In re Geisler, 116 F.3d 1465, 1470 (Fed. Cir. 1997).
Appellant contends that:
To the extent these references teach that carnosine can react
with glycating agents in certain circumstances (Hipkiss) or
delay cell senescence (Grigg), Appellants emphasize that these
generalized teachings still do not provide a reasonable
expectation that addition of carnosine to a culture of CHO cells
that express a protein of interest would result in an improved
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cell culture characteristic selected from the group consisting of
“increased titer, increased cell specific productivity, decreased
accumulation of molecular weight aggregates, decreased
accumulation of acidic species, and combinations thereof” as
provided in independent claims 1 and 9.
(Appeal Br. 12–13). Appellant contends “[i]n the context of this uncertainly
and limited data provided in the cited references, a person of skill in the art
would not have had even a reasonable expectation of success of the claimed
invention” (id.).
We find this argument unpersuasive for several reasons. First, the
Examiner reasonably finds that the result of adding carnosine to the protein
production method of Schatz for the benefits disclosed in Grigg and Hipkiss
would inherently and necessarily result in the improved properties recited in
the claims (see Non-Final Act. 4). “[A]fter the PTO establishes a prima
facie case of anticipation based on inherency, the burden shifts to appellant
to ‘prove that the subject matter shown to be in the prior art does not possess
the characteristic relied on.’” In re King, 801 F.2d 1324, 1327 (Fed. Cir.
1986). Appellant has provided no evidence that the Examiner erred or that
the “wherein clause” results would not be inherent.
Second, the “wherein clause” recites a Markush group of improved
culture cell characteristics including “decreased accumulation of high
molecular weight aggregates.” Hipkiss directly teaches that carnosine
inhibits glycating agents from reacting with polypeptides (FF 14) and
demonstrates “carnosine inhibited the reaction of [14C]- lysine with the
methylglyoxal-treated protein . . . not only can carnosine react with protein
carbonyls but it can also moderate their deleterious action towards other
molecules” (FF 15). Hipkiss teaches “a model system also showed that
carnosine inhibited modification of a protein by the AGE formed from the
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reaction of lysine with methylglyoxal” (FF 17). Thus, Hipkiss directly
demonstrates that carnosine inhibits at least some forms of cross-linking and
therefore reduces aggregation due to agents including methylglyoxal (FF 14,
15, 17). And so Hipkiss evidences that carnosine reduces aggregation of
molecules including polypeptides consistent with the requirement of claim 1.
Third, while the prior art may not absolutely predict success using
carnosine, “[o]bviousness does not require absolute predictability of success
. . . all that is required is a reasonable expectation of success.” In re Kubin,
561 F.3d 1351, 1360 (Fed. Cir. 2009). The evidence in Chaplen, Grigg, and
Hipkiss provides a reasonable expectation that carnosine would be expected
to reduce the negative effects of methylglyoxal in CHO cells and therefore
would be expected to reduce cross-linking and aggregation (FF 15, 17) and
would be expected to reduce methylglyoxal caused AGE formation,
apoptosis (cell death) and growth inhibition (FF 9). We agree with the
Examiner that “the combination of the cited prior art provides enough
information for one of skill in the art to reasonably expect success in
improving protein production by adding carnosine to the culture medium.
There is no evidence to the contrary of record” (Ans. 9).
Conclusion of Law
A preponderance of the evidence of record supports the Examiner’s
conclusion that the prior art suggests, with a reasonable expectation of
success, that the addition of carnosine to cell growth media would improve
protein production.

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DECISION SUMMARY
In summary:
Claims
Rejected
35 U.S.C.
§
References/Basis Affirmed Reversed
1, 5–7, 9, 17–
19, 21, 24,
26–33
103(a) Schatz, Chaplen,
Grigg, Hipkiss
1, 5–7, 9,
17–19, 21,
24, 26–33

1, 5–7, 9, 17–
19, 21, 24–33
103(a) Schatz, Chaplen,
Grigg, Hipkiss,
Veldman
1, 5–7, 9,
17–19, 21,
24–33

Overall
Outcome
1, 5–7, 9,
17–19, 21,
24–33

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