Ex Parte Birch et al

Board of Patent Appeals and Interferences

Jan 5, 2012

10501777 (B.P.A.I. Jan. 5, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte JOHN ROBERT BIRCH, ROBERT CHARLES BORASTON,
and MARTYN SHAW
__________

Appeal 2011-005341
Application 10/501,777
Technology Center 1600
__________

Before ERIC GRIMES, FRANCISCO C. PRATS, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

GRIMES, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a method
of increasing glycosylation of a protein expressed by a human cell. The
Examiner has rejected the claims as obvious. We have jurisdiction under 35
U.S.C. § 6(b). We affirm.
STATEMENT OF THE CASE
The Specification discloses that “mammalian cell cultures are the
preferred source of a number of important proteins for use in human and
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animal medicine, especially those which are relatively large, complex and
glycosylated” (Spec. 1: 9-11). The Specification discloses that “when
glutamine is used as an energy substrate by cultured cells, ammonia is a
catabolite produced, which is cytotoxic and can inhibit … glycosylation of
proteins by its effect on pH within the Golgi of the cell” (id. at 1: 23-28).
The Specification discloses “a glutamine-auxotrophic human cell … which
has been transfected with a (first) exogenous DNA sequence encoding a
protein…, and further with a (second) exogenous DNA sequence encoding a
glutamine synthetase (GS) … said transfected cell [being] capable of
producing said protein and capable of growing in a glutamine-free medium”
(id. 2: 31 to 3: 5).
Claims 32-41 are on appeal. The claims have not been argued
separately and therefore stand or fall together. 37 C.F.R. § 41.37(c)(1)(vii).
Claim 32 is representative and reads as follows:
32. A method of increasing sialylation and/or N-glycan charge of a
glycosylated protein expressed by a glutamine auxotrophic human cell and
of extending the viability of said cell, said method comprising transfecting a
glutamine auxotrophic human cell with an exogenous DNA sequence
encoding a glutamine synthetase to produce a transfected human cell and
culturing said transfected human cell in a glutamine-free media such that
said sialylation and/or N-glycan charge of said glycosylated protein is
increased and the viability of said cell is extended.

Issues
The Examiner has rejected claims 32-41 under 35 U.S.C. § 103(a) as
being obvious in view of Wilson
1
or Bebbington,
2
in view of Brandt,
3

1
Wilson et al., WO 87/04462, published July 30, 1987.
2
Bebbington et al., US 5,891,693, issued Apr. 6, 1999.
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Schneider,
4
Gawlitzek,
5
Hermentin,
6
and Barsomian.
7
The Examiner finds
that Bebbington discloses “mouse and rat lymphoid cell lines … transformed
to glutamine independence by incorporating a gene encoding glutamine
synthetase (GS) so that the cells can grow in glutamine-free medium”
(Answer 4). The Examiner finds that Bebbington discloses that the cell also
“contains a gene coding for a heterologous protein” such as tPA (id. at 4-5)
and that Barsomian discloses that “tPA is a sialylated glycoprotein” (id. at
5).
The Examiner finds that Brandt discloses that it is advantageous to
use human cells to produce human pharmaceutical proteins in order to obtain
a “glycosylation pattern, especially a sialic acid profile[,] comparable to that
of the naturally occurring … protein” (id.). The Examiner finds that both
Gawlitzek and Schneider disclose that “ammonium is produced as a by-
product of glutamine metabolism and the thermal degradation of glutamine
… [and] that increased amounts of ammonium in cells leads to a decrease in
terminal galactosylation and sialylation of recombinant GCSF” (id. at 6-7),
and both suggest limiting glutamine in mammalian cell cultures (id. at 7).
The Examiner finds that Hermentin discloses “a process for characterizing
the glycosylation of glycoproteins” (id. at 8).

3
Brandt et al., US 6,395,484 B1, issued May 28, 2002.
4
Schneider et al., The importance of ammonia in mammalian cell culture, 46
J. BIOTECHNOL. 161-185 (1996).
5
Gawlitzek et al., Ammonium Alters N-Glycan Structures of Recombinant
TNFR-IgG: Degradative Versus Biosynthetic Mechanisms, 68 BIOTECHNOL.
BIOENG. 637-646 (2000).
6
Hermentin et al., US 6,096,555, issued Aug. 1, 2000.
7
Barsomian et al., US 5,238,821, issued Aug. 24, 1993.
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The Examiner concludes that one of ordinary skill in the art would
have been motivated to modify Bebbington’s method to use a human host
cell “to produce a human exogenous sialylated protein because Brandt et al
disclose that human cells are preferred for the preparation of human
proteins” (id. at 9). The Examiner further concludes that the
ordinary skilled artisan seeking to increase the sialylation
and/or N-glycan charge of a glycosylated protein, would have
been motivated to express the desired protein in a cell without
adding glutamine, since the prior art clearly teaches that
ammonium, a byproduct of glutamine metabolism has a direct
effect on the level of sialylation and/or N-glycan charge of a
glycosylated protein.
(Id. at 11-12.)
Appellants contend that “[t]here is no suggestion in the cited art or
reasonable expectation from the cited art that a glutamine auxotrophic cell
line … could grow in a glutamine-free media ... in such a manner to increase
sialylation and/or N-glycan charge and extend cell viability” (Appeal Br.
12). Appellants also argue that the method of claim 32 provides
“unexpected benefits in increasing sialylation and/or N-glycan charge of a
glycosylated protein and in allowing for a greater rate of protein synthesis
and increased maximum product concentration” (id. at 23).
The issues presented are: Does the evidence of record support the
Examiner’s conclusion that the method of claim 32 would have been prima
facie obvious in view of the cited references?
If so, have Appellants provided evidence of unexpected results that
outweighs the evidence supporting the prima facie case of obviousness?
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Findings of Fact
1. Schneider discloses that “[a]mmonia has been reported to be toxic
and inhibitory for mammalian cell cultures for many years” (Schneider,
abstract).
2. Schneider discloses that “high ammonia concentrations have been
shown to perturb processing and secretion of proteins as well as
glycosylation” (id. at 165).
3. Schneider discloses that the “main source of the ammonia which
accumulates in cell cultures is glutamine” (id. at abstract).
4. Schneider discloses that one strategy to reduce ammonia formation
in mammalian cell cultures is the adaptation of cells, including human cells,
in glutamine-free medium (id. at 174-175).
5. Schneider discloses that ammonia has been shown to have
deleterious effects on cell growth and viability (id. at 162-165, Table 1).
6. Gawlitzek discloses that “[a]mmonium … usually accumulates in
mammalian cell cultures…. Ammonium is mainly a byproduct of glutamine
metabolism and the thermal degradation of glutamine.” (Gawlitzek 637.)
7. Gawlitzek discloses that “[n]umerous studies reported negative
effects of elevated ammonium concentrations on cell growth and
productivity of different cell lines” (id.).
8. Gawlitzek “used tumor necrosis factor receptor-immunoglobulin G
(TNFR-IgG) as a model glycoprotein … to investigate the effect of
ammonium on protein glycosylation in CHO cell cultures.… Ammonium
decreased galactosylation and sialylation of TNFR-IgG N-glycans.” (Id. at
638.)
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9. Gawlitzek discloses that “[s]imilar mRNA levels (Fig. 6) and
enzyme activities (Fig. 7) were found when comparing cells cultivated under
low or high ammonium concentrations” (id. at 643).
10. Gawlitzek discloses that the results “strongly suggest that
ammonium inhibits galactosylation and sialylation of TNFR-IgG N-glycans
by pH-regulated mechanisms. We hypothesize that ammonium decreases
α2,3-sialyltransferase and β1,4-galactosyltransferase activities by increasing
the pH of the trans-Golgi compartment.” (Id. at 644.)
11. Bebbington discloses that “many lymphoid cell lines … cannot be
grown in vitro on media lacking in glutamine.… [I]t would be useful to be
able to transform lymphoid cell lines to glutamine independence, since this
may provide an advantageous method for selecting transformed cell lines.”
(Bebbington, col. 1, ll. 29-34.)
12. Bebbington discloses
a method for transforming a lymphoid cell line to glutamine
independence which comprises:
transforming the lymphoid cell line with a vector containing an
active glutamine synthetase (GS) gene;
growing the transformed cell line on a medium containing
glutamine; and
continuing the growth of the transformed cell line on a medium
in which the glutamine is progressively depleted or on a
medium lacking glutamine.
(Id. at col. 1, ll. 51-60.)
13. Bebbington discloses that preferably the GS-encoding vector
“also contains an active gene coding for a protein heterologous to the
lymphoid cell line. Alternatively, the lymphoid cell line may be co-
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transformed with a separate vector containing the active gene coding for the
heterologous protein.” (Id. at col. 2, ll. 25-30.)
14. Bebbington discloses that an example of a heterologous protein
product is tissue plasminogen activator (tPA) (id. at col. 2, ll. 31-35).
15. Barsomian discloses that tPA is a sialylated glycoprotein
(Barsomian, col. 2, ll. 49-51).
16. Brandt discloses a process for producing “human proteins … in a
form suitable for producing a pharmaceutical composition” (Brandt,
abstract).
17. Brandt discloses that a “human cell line is preferably used which
synthesizes the target protein with a glycosylation pattern, especially
regarding the number of the sialic acid moieties, comparable to that of the
naturally occurring target protein” (id. at col. 3, ll. 25-30).
Analysis
Claim 32 is directed to a method that comprises transfecting a
glutamine auxotrophic human cell with an exogenous DNA sequence
encoding a glutamine synthetase and culturing the cell in a glutamine-free
media such that the sialylation and/or N-glycan charge of a glycosylated
protein is increased and the viability of the cell is extended.
Schneider discloses that glutamine-associated ammonia accumulation
inhibits mammalian cell cultures and also interferes with protein
glycosylation. Schneider discloses that one strategy to reduce ammonia
formation is to adapt cells, including human cells, to glutamine-free
medium. Bebbington discloses transforming glutamine-auxotrophic cell
lines with a selectable glutamine synthase gene so that the transformed cell
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lines could be used for production of heterologous proteins such as tPA in
glutamine-free media.
Brandt discloses the use of human cell lines for the production of
pharmaceutical proteins with a glycosylation pattern comparable to that of
the naturally occurring protein. Gawlitzek discloses that glutamine-
associated ammonium inhibits galactosylation and sialylation of proteins by
increasing the pH of the trans-Golgi compartment. In view of these
disclosures, it would have been obvious to practice Bebbington’s method of
transforming glutamine-auxotrophic cells with a glutamine synthetase gene
and culturing them on glutamine-free media with human cells, for the
production of glycosylated human pharmaceutical proteins, in order to avoid
the deleterious effects of glutamine-associated ammonium on protein
sialylation and cell viability.
Appellants argue that “[t]here is no suggestion in the cited art or
reasonable expectation from the cited art that a glutamine auxotrophic cell
line … could grow in a glutamine-free media … in such a manner to
increase sialylation and/or N-glycan charge and extend cell viability”
(Appeal Br. 12). Appellants argue that the Examiner erred in relying on
Gawlitzek to provide a suggestion for increasing sialylation and/or N-glycan
charge of a glycosylated protein by limiting glutamine and thereby
ammonium in the media because Gawlitzek’s Figure 7 “demonstrates that
similar enzyme activities were found when comparing cells cultivated under
low and high ammonium concentrations” (id. at 15). Appellants further
argue that Gawlitzek’s Figure 6 demonstrates that “similar mRNA levels of
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the enzymes were found when comparing … cells cultivated under low or
high ammonium concentrations” (id. at 17).
This argument is not persuasive. The results shown in Gawlitzek’s
Figures 6 and 7 demonstrate similar RNA and enzyme activities,
respectively, with low and high ammonia concentrations (see FF 9).
However, Gawlitzek concludes that its data show that ammonium decreases
the activity of glycosylation enzymes by increasing the pH of the trans-
Golgi compartment (see FF 10). The data shown in Gawlitzek’s Figures 6
and 7 do not contradict this conclusion: Figure 6 shows mRNA levels, not
enzyme activities; Figure 7, which does show enzyme activities, assayed
those activities in cell lysates in order to determine whether ammonium has
a direct effect on enzyme expression (see Gawlitzek 643: “To determine
whether ammonia has a direct effect on enzyme expression, intracellular
mRNA levels and enzyme activities of both glycosyltransferases were
monitored over time in cells cultured under control and high ammonium
conditions.”).
Appellants argue that the claimed methods “provide unexpected
benefits in increasing sialylation and/or N-glycan charge of a glycosylated
protein and in allowing for a greater rate of protein synthesis and increased
maximum product concentration” (Appeal Br. 22-23).
This argument is not persuasive. Unexpected results must be
supported by factual evidence, and attorney argument is not evidence. See
In re Pearson, 494 F.2d 1399, 1405 (CCPA 1974). Appellants have not
provided any evidence that the Specification shows that the claimed method
is unexpectedly superior to the closest prior art.
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Conclusions of Law
The evidence of record supports the Examiner’s conclusion that the
method of claim 32 would have been prima facie obvious in view of the
cited references. Appellants have not provided evidence of unexpected
results that outweighs the evidence supporting the prima facie case of
obviousness.
SUMMARY
We affirm the rejection of claims 32-41 under 35 U.S.C. § 103(a).
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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