VIB VZW et al.

Patent Trials and Appeals Board

Feb 25, 2021

2019005598 (P.T.A.B. Feb. 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.
14/322,827 07/02/2014 Nico L.M. CALLEWAERT 10488-07362 US 1088
93219 7590 02/25/2021
Patent Law Works, LLP
310 East 4500 South, Suite 400
Salt Lake City, UT 84107
EXAMINER
LIU, SAMUEL W
ART UNIT PAPER NUMBER
1656
NOTIFICATION DATE DELIVERY MODE
02/25/2021 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):
docketing@patentlawworks.net
patents@patentlawworks.net
PTOL-90A (Rev. 04/07)

UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte NICO L.M. CALLEWAERT, KAREN DE POURCQ,
STEVEN GUYSENS, and LEANDER MEURIS
Appeal 2019-005598
Application 14/322,827
Technology Center 1600
Before DONALD E. ADAMS, RACHEL H. TOWNSEND, and
TAWEN CHANG, Administrative Patent Judges.
CHANG, Administrative Patent Judge.
DECISION ON APPEAL
Pursuant to 35 U.S.C. § 134(a), Appellant1 appeals from the
Examiner’s decision to reject claims 1–5, 7–9, 11–13, 25, and 26. We have
jurisdiction under 35 U.S.C. § 6(b).
We AFFIRM IN PART.

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 VIB VZW
and Universiteit Gent. Appeal Br. 2.
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STATEMENT OF THE CASE
“Glycosylation is one of the most common post-translational
modifications of proteins in eukaryotes.” Spec. ¶ 4. “Glycoproteins are an
important class of biomolecules that play crucial roles in many biological
events such as cell adhesion, tumor metastasis, pathogen infection, and
immune response.” Id. ¶ 3.
The Specification states:
Both natural and recombinant glycoproteins are typically produced as
a mixture of glycoforms that differ only in the structure of the pendent
oligosaccharides. This heterogeneity in glycosylation is a major
problem in structural and functional studies of glycoproteins (e.g.,
crystallization studies), as well as in development of glycoprotein
drugs. The attached sugar chains may, for instance, have profound
effects on protein folding, stability, action, pharmacokinetics, and
serum half-life of the glycoprotein, and some sugar chains are very
immunogenic.
Id.
According to the Specification, “there is a need to have a cell system
or synthesis method providing homogeneous (uniform) glycosylation on a
population of glycoproteins, either already with a correct glycoprofile or as a
starting point for subsequent transglycosylation.” Spec. ¶ 13. Further
according to the Specification, the invention relates to “systems and methods
for obtaining desired glycosylation profiles of a glycoprotein that are
economical in both cost and time,” wherein “[c]orrect glycosylation of the
glycoprotein (or an essentially homogeneous glycosylated population of an
intermediate glycoform of the glycoprotein) is achieved by producing the
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glycoprotein and an endoglucosaminidase enzyme in the same cellular
system.”2 Id. ¶ 14.
CLAIMED SUBJECT MATTER
The claims are directed to a eukaryotic cell comprising certain
exogenous proteins or nucleic acid molecules. Independent claims 1, 25,
and 26 are reproduced below:
1. An eukaryotic cell comprising:
an exogenous endoglucosaminidase enzyme which
endohydrolyzes a N,N’-diacetylchitobiosyl unit in an N-
glycan linked to an exogenous glycoprotein; and
the exogenous glycoprotein;
wherein the endoglucosaminidase enzyme is retained in or
passes through a secretory pathway and deglycosylates
the exogenous glycoprotein;
wherein the exogenous glycoprotein comprises, attached to an
Asn glycosylation site, an N-glycan containing a single
N-acetylglucosamine residue of a N,N’-
diacetylchitobiosyl unit;
wherein the N-glycan does not comprise a N,N’-
diacetylchitobiosyl unit; and

2 The Specification and the cited prior art refer variously to
endoglucosaminidase, endoglycosidase, and endoglycanase.
Glucosaminidases are enzymes that catalyze the hydrolysis of a glucosamine
residue. Kobayashi et al., US 6,815,191 B1, issued Nov. 9, 2004
(“Kobayashi”) 1:25–33. The terms glycanase and glycosidase are
sometimes used interchangeably and refer more broadly to an enzyme that
normally catalyzes the hydrolysis of a glycosidic bond. Defrees et al., US
2005/0064540 A1, published Mar. 24, 2005 (“Defrees”) ¶¶ 25, 85. The
prefix “endo,” in contrast to “exo,” refers to the fact that the bond acted
upon by the enzyme is a nonterminal bond. Id. ¶¶ 25, 27. Endo-ß-N acetyl
glucosaminidase H (Endo H), which is referred to throughout this opinion as
well as in the Specification and cited prior art, is a glycanase/glycosidase as
well as an endoglucosaminidase. See, e.g., Spec. ¶ 12, Defrees ¶¶ 33, 34.
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wherein the exogenous glycoprotein is produced by the cell
with a uniform glycoprofile.

25. An eukaryotic cell comprising:
a first exogenous nucleic acid molecule encoding Endo H
operably linked to an endoplasmic reticulum or Golgi
localization signal; and
a second exogenous nucleic acid molecule encoding a
glycoprotein.

26. An eukaryotic cell comprising:
a first exogenous nucleic acid molecule encoding Endo M
operably linked to an endoplasmic reticulum or Golgi
localization signal; and
a second exogenous nucleic acid molecule encoding a
glycoprotein.

Appeal Br. 19, 21 (Claims App.).

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REJECTION(S)
A. Claims 1–5, 8, 9, 11, 12, and 25 are rejected under 35 U.S.C. § 103(a)
as being unpatentable over Defrees, Rao,3 Uchiyama,4 Kobayashi,
Chiba,5 Zhu,6 and Palling.7 Ans. 4.
B. Claim 7 is rejected under 35 U.S.C. § 103(a) as being unpatentable
over Defrees, Rao, Uchiyama, Kobayashi, Chiba, and Palling. Ans.
12.
C. Claim 13 is rejected under 35 U.S.C. § 103(a) as being unpatentable
over Defrees, Rao, Uchiyama, Kobayashi, Chiba, Palling, and
Crispin.8 Ans. 11–12.

3 Vibha Rao et al., Crystal Structure of Endo-ß-N-Acetylglucosaminidase H
at 1.9 Å Resolution: Active-Site Geometry and Substrate Recognition,
3 STRUCTURE 449 (1995) (“Rao”).
4 Taku Uchiyama et al., Uptake of N, N’-Diacetylchitobiose [(GlcNAc)2] via
the Phosphotransferase System Is Essential for Chitinase Production by
Serratia marcescens 2170, 185 J. BACTERIOLOGY 1776 (2003)
(“Uchiyama”).
5 Yasunori Chiba et al., Production of Human Compatible High Mannose-
type (Man5GlcNAc2) Sugar Chains in Saccharomyces cerevisiae, 273 J.
BIOLOGICAL CHEMISTRY 26298 (1998) (“Chiba”).
6 Guofen Zhu et al., ß1,4 N-Acetylgalactosaminyltransferase
(GM2/GD2/GA2 Synthase) Forms Homodimers in the Endoplasmic
Reticulum: A Strategy to Test for Dimerization of Golgi Membrane Proteins,
7 GLYCOBIOLOGY 987 (1997) (“Zhu”).
7 Palling, US 2010/0113517 A1, published May 6, 2010 (“Palling”).
8 Max Crispin et al., Inhibition of Hybrid- and Complex-type Glycosylation
Reveals the Presence of the GlcNAc Transferase I-Independent Fucosylation
Pathway, 16 GLYCOBIOLOGY 748 (2006) (“Crispin”).
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D. Claim 26 is rejected under 35 U.S.C. § 103(a) as being unpatentable
over Defrees, Rao, Uchiyama, Kobayashi, Chiba, Palling,9 and
DeFrees ’521.10 Ans. 13.
OPINION11
A. Obviousness rejections over independent claim 1 and dependent
claims 2–5, 7–9, and 11–13
1. Issue
The Examiner rejects independent claim 1 and dependent claims 2–5,
8, 9, 11, and 12 as obvious over Defrees, Rao, Uchiyama, Kobayashi, Chiba,
Zhu, and Palling.12 The Examiner rejects dependent claim 7 as obvious over
Defrees, Rao, Uchiyama, Kobayashi, Chiba, and Palling. The Examiner
rejects dependent claim 13 as obvious over Defrees, Rao, Uchiyama,
Kobayashi, Chiba, Palling, and Crispin. The same issue is dispositive for
these rejections; we therefore discuss them together.
The Examiner finds that Defrees teaches a method of modifying the
glycosylation patterns of glycoproteins to achieve a homogeneous
glycosylation pattern. Ans. 4. The Examiner finds that Defrees’ method

9 The Examiner does not list Palling as part of the cited prior art combination
in the heading regarding the rejection of claim 26. However, the Examiner
discusses the “’571” in the body of the rejection. We understand the
reference to ’571 as a typographical error that is intended to refer to Palling,
or the ’517 patent.
10 DeFrees et al., US 2006/0030521 A1, published Feb. 9, 2006 (“DeFrees
’521”).
11 We limit our consideration of the merits of the appealed rejections to the
elected species. See Ex parte Ohsaka, 2 USPQ2d 1460, 1461 (BPAI 1987).
See Response to Election/Restriction filed on March 11, 2016.
12 The Examiner also rejects independent claim 25 as obvious over the same
set of references. Claim 25 is separately discussed in the next section.
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comprises a first step of treating the glycoprotein with Endo H, an
endoglycanase / endoglucosaminidase, “to remove undesired carbohydrate
from said protein,” before catalyzing the ligation of the desired
oligosaccharide moiety to the treated glycoprotein using a mutant
endoglycanase. Id. at 4–6, 8.
More particularly, the Examiner finds that Defrees teaches a first step
of using Endo H to “endohydrolyze[] a N,N’-diacetylchitobiosyl unit in an
N-glycan linked to an exogenous glycoprotein,” as recited in independent
claim 1. Ans. 5–6, 8. The Examiner further finds that Defrees teaches that
this reaction produces a glycoprotein having the structure recited in claim 1,
i.e., comprising, “attached to an Asn glycosylation site, an N-glycan
containing a single N-acetylglucosamine residue of a N,N’-
diacetylchitobiosyl unit, wherein the N-glycan does not comprise a N,N’-
diacetylchitobiosyl unit.” Id.
With respect to the limitation in claim 1 that “the
endoglucosaminidase enzyme is retained in or passes through a secretory
pathway,” the Examiner finds “[i]t has been known in the prior art that Endo
H enzyme only has activity to cleave glycolpolypeptides which have
acquired [endoplasmic reticulum (ER)] glycosylation (high mannose N-
linked) when this enzyme has been localized to the ER.” Ans. 6; see also id.
at 8. The Examiner further finds that Chiba teaches that, “in order to
successfully localize a deglycosylation enzyme to yeast ER, an ER retention
signal [should be] attached to said enzyme so as to allow the enzyme to pass
through the host yeast secretion pathway.” Id. The Examiner concludes
that, “[t]hus, it is obvious to target the Endo H enzyme to endoplasmic
reticulum (ER) . . . by linking said enzyme with ER-retention signal peptide
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. . . which leads the enzyme passing through yeast secretion pathway.” Id. at
6–7. The Examiner further notes that Golgi localization/retention signals are
also known in the art. Id. at 7.
Finally, the Examiner finds that Defrees teaches that the glycoproteins
used in its method may be recombinantly produced in a eukaryotic cell such
as a yeast cell or a mammalian cell. Ans. 4, 6. The Examiner finds that
Defrees similarly teaches that the mutated endoglycanase used in its method
may be recombinantly produced. Id. at 6. The Examiner concludes that,
because Defrees teaches that its method “involves the construction of
recombinant nucleic acids and the expression of genes in transfected host
cells, e.g., eukaryotic yeast cells,” and further teaches that “the ‘modified
glycopeptide’ (final product) . . . can be produced intracellularly (i.e., in a
cell),” Defrees suggests that its method may be carried out in a recombinant
eukaryotic cell that “co-express exogenous endoglucosaminidase ‘Endo H’ .
. . and the glycoprotein,” which would read on claim 1. Id.
The Examiner notes that, even if Defrees does not expressly teach a
eukaryotic host cell co-expressing an exogenous endoglucosaminidase (e.g.,
Endo H) and its exogenous glycoprotein substrate, a skilled artisan would
have had motivation to engineer such a cell because, among other things:
• Both Defrees and Chiba teach that glycopeptides of pharmaceutical or
biological interest can be produced in commercially feasible quantities
in a cell host system;
• Defrees teaches producing its modified glycopeptide “intracellularly
(i.e., in a cell)” or through “a large scale fermentation,” and it would
have been obvious to “recombinantly (exogenously) co-express the
glycoprotein and endoglycanase enzyme . . . in a host cell in order to
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obtain the final product” in the cell and “to obtain desired glycol-
pattern”;
• Defrees teaches that techniques for expressing exogenous genes are
well known in the art and specifically teaches recombinantly
producing glycoproteins, and Kobayashi teaches recombinant
expression of an endo-ß-acetylglucosaminidase in yeast.
Ans. 8–10; see also id. at 10–11 (providing “[a]dditional reason/motivation
to combine the references Defrees with Kobayashi” and “[f]urther
motivations of using an eukaryotic cell for co-expressing the glycoprotein
and endoglycanase ‘Endo H’”).
The Examiner notes that, since recombinant expression of (1)
glycoproteins and (2) endoglucosaminidase / endoglycanase enzymes that
catalyze the reaction recited in claim 1 are both known in the prior art, a
skilled artisan “would have co-expressed recombinant genes encoding said
glycoprotein and said endoglycanase in order to modify glycosylation
pattern of the glycoprotein . . . which is guided by the mechanistic scheme
shown in Figure 3 of Defrees . . . with reasonable expectation of success.”
Ans. 10.
Appellant contends that a skilled artisan would not have had
motivation to combine the references to arrive at the claimed invention.
Appeal Br. 9. In particular, Appellant contends that “there is no motivation
or suggestion to combine the expression of an endoglycanase with
endohydrolyzing activity AND expression of a glycoprotein in the same
eukaryotic cell.” Id. at 10. Appellant contends that, in fact, “there are
several teachings away (or statements which would demotivate a skilled
person from making this combination) in Defrees.” Id. Finally, Appellant
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contends that the subject matter of the invention exhibits unexpected results.
Id. at 12–13.
The issues with respect to this rejection are (1) whether a skilled
artisan would have had a reason to combine the cited prior art references to
arrive at the claimed invention, with a reasonable expectation of success,
and, if so, (2) whether Appellant has provided evidence of unexpected
results that, when considered together with the evidence of obviousness,
shows the claims to be non-obvious.

2. Analysis
Claim Construction
We begin our analysis by construing the limitation “glycoprotein . . .
with a uniform glycoprofile.” During examination of a patent application,
pending claims are given their broadest reasonable construction consistent
with the claim language itself, as well as the Specification, which includes
taking into account any definitions present in the Specification and the use
of the words in the context of the written description. See, In re ICON
Health and Fitness, Inc., 496 F.3d 1374, 1379 (Fed. Cir. 2007). The
Specification uses the terms homogeneous and uniform interchangeably and
as a contrast to heterogeneous. See Spec. ¶¶ 8–9, 13. There is, however, no
specific definition of these terms in the Specification, but we find the terms
are used in accordance with their ordinary meaning, for the following
reasons.
The plain and ordinary meanings of the term “uniform” include
“having always the same form, manner, or degree : not varying or variable,”
“of the same form with others : conforming to one rule or mode,” and
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“presenting an unvaried appearance of surface, pattern, or color.”13
Likewise, the plain and ordinary meaning of “homogeneous” include “of the
same or a similar kind or nature” and “of uniform structure or composition
throughout.”14
The use of the term “uniform” in the claim is consistent with the
foregoing meaning. Moreover, the uses of these terms in the Specification
are consistent with their plain and ordinary meaning. For example, the
Specification describes an embodiment wherein “[t]he glycoproteins with a
single GlcNAc residue may be the only glycoform of the glycoprotein
produced by the cell, i.e., a uniform glycopopulation is produced.” Spec. ¶
32 (emphasis added); see also, e.g., id. ¶ 25 (stating that a mixed population
comprising two populations of glycoproteins “requires a separation step
before a uniformly glycosylated population is obtained”), ¶ 85 (stating that
“[t]he eukaryotic . . . cells as described herein may produce uniformly . . .
glycosylated glycoproteins that are ready to use” or, alternatively, “produce
two populations of easily separable, differentially glycosylated
glycoproteins”), ¶ 14 (describing embodiment where “the yeast cells only
secrete glycoproteins with the desired . . . glycosylation pattern”).
Accordingly, we construe the term “wherein the exogenous
glycoprotein is produced by the cell with a uniform glycoprofile” to mean
that only a single glycoform of the exogenous glycoprotein is produced by
the cell.

13 “uniform.” MERRIAM-WEBSTER, https://www.merriam-
webster.com/dictionary/uniform (last visited Feb. 11, 2021).
14 “homogeneous.” MERRIAM-WEBSTER, https://www.merriam-
webster.com/dictionary/homogeneous (last visited Feb. 11, 2021).
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The Examiner’s prima facie case
On balance, we agree with Appellant that the Examiner has not
established a prima facie case that independent claim 1 is obvious over the
cited prior art. In particular, in light of our construction of the phrase
“wherein the exogenous glycoprotein is produced by a cell with a uniform
glycoprofile,” the Examiner has not established that a skilled artisan would
have combined the cited prior art with a reasonable expectation of success of
arriving at the claimed invention.
The Examiner asserts that Defrees suggests eukaryotic cells that co-
expresses an exogenous endoglucosaminidase (e.g., Endo H) and an
exogenous glycoprotein or, alternatively, that the cited prior art combination
suggests such eukaryotic cells and that a skilled artisan would have been
motivated to make such cells. Ans. 4, 6, 8–11.
As discussed below with respect to claim 25, we agree with the
Examiner that a skilled artisan would have had reason to make a eukaryotic
cell co-expressing an exogenous protein and an exogenous
endoglucosaminidase enzyme (e.g., endo H). However, the Examiner has
not pointed to persuasive evidence that a skilled artisan would have
reasonably expected such a cell to produce “the exogenous glycoprotein . . .
with a uniform glycoprofile,” i.e., to produce only a single glycoform of the
exogenous glycoprotein.
As an initial matter, there is no dispute that Defrees teaches
recombinantly produced glycopeptides. Defrees ¶ 37 (stating that,
“[t]ypically, the glycoprotein [used in its invention] will be recombinantly
expressed in a prokaryotic . . . or eukaryotic cell”). Likewise, it does not
appear disputed that endoglucosaminidase enzymes such as Endo H may be
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produced recombinantly. Ans. 6, 9–10; Kobayashi Example 8 (describing
expression of novel endo-ß-N-acetylglucosaminidase in yeast cell); cf.
Defrees ¶ 63 (discussing recombinant production of mutant endoglycanases
such as Endo H). In short, as Defrees teaches, “[t]he practice of [its]
invention can involve the construction of recombinant nucleic acids and the
expression of genes in transfected host cells.” Defrees ¶ 80.
As the Appellant points out, however, Defrees also teaches that, while
“[i]n principle, mammalian, insect, yeast, fungal, plant or prokaryotic cell
culture systems can be used for production of most therapeutic and other
glycopeptides,” “[i]n practice . . . a desired glycosylation pattern on a
recombinantly produced protein is difficult to achieve.” Defrees ¶ 17.
Similarly, Defrees teaches that “[h]eterogeneity in the glycosylation of a
recombinantly produced glycopeptides arises because the cellular machinery
(e.g., glycosyltransferases and glycosidases) may vary from species to
species, cell to cell, or even from individual to individual.” Id. ¶ 18.
Defrees does teach that “[e]fforts to remedy the deficiencies of the
glycosylation of a particular host cell have focused on engineering the cell to
express one or more missing enzymes integral to the [desired (e.g., human)]
glycosylation pathway,” and teaches that there has been some success in this
regard. Defrees ¶ 20. For instance, Defrees reports the production in the
prior art of Chinese hamster ovary (CHO) cell transfected with the gene
encoding expression of a specific enzyme, which has been shown to produce
glycopeptides equipped with residues not normally found in recombinant
glycopeptides produced by regular CHO cells and having improved
pharmacokinetics as compared to such recombinant glycopeptides. Id.
However, the Examiner has not pointed to persuasive evidence that the
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glycopeptides expressed from such “universal host” cells have a uniform
glycoform. Indeed, Defrees teaches that its invention fulfills, among other
needs, the need “for an in vitro procedure to enzymatically modify
glycosylation patterns” on “commercially important recombinantly and
transgenically produced glycopeptides.” Defrees ¶ 32 (emphasis added).
Thus, we are not persuaded that a skilled artisan would, based on the
teachings that glycoproteins and endoglucosaminidase enzymes may both be
recombinantly produced in Defrees’ method, have had a reasonable
expectation of successfully producing an eukaryotic cell co-expressing an
exogenous glycoprotein and an exogenous endoglucosaminidase enzyme,
wherein the exogenous glycoprotein is produced by the cell with a uniform
glycoprofile, as that limitation is construed above.
We acknowledge the Examiner’s citation to paragraph 186 of Defrees,
which discusses protein (glycoprotein) purification where “modified
glycopeptide is produced intracellularly,” as well as paragraph 191 of
Defrees, which discusses “modified glycopeptide of the invention resulting
from a large-scale fermentation.” Spec. ¶¶ 186, 191 (emphasis added). The
Examiner asserts that “modified glycopeptide” in these contexts necessarily
refers to the “final product” of Defrees’ method and appears to assert that, as
such, the glycopeptide are produced with a homogeneous glycosylation
pattern. Ans. 6, 8–9.
We are not persuaded that a skilled artisan would reasonably expect,
from the above paragraphs of Defrees, that the “modified glycopeptide”
produced intracellularly or resulting from a large-scale fermentation would
have a “uniform glycoprofile” as recited in claim 1 and construed above.
First, Defrees teaches a method for modifying glycosylation patterns of
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glycoproteins, including recombinantly produced glycoproteins, and,
separately, a glycoprotein composition in which the glycoproteins have a
homogeneous glycosylation pattern. Defrees Abstract. Thus, a “modified
glycopeptide” according to Defrees’ invention may not necessarily be part of
a composition of glycopeptides having a homogeneous glycosylation
pattern. See, e.g., Defrees ¶ 91 (describing “resulting polypeptide ha[ving] a
substantially uniform glycosylation pattern” as a “preferred embodiment”);
id. ¶ 88 (regarding obtaining “glycoprotein species that have a substantially
uniform glycosylation pattern”: “The methods of the invention are useful for
remodeling or altering the glycosylation pattern present on a glycoprotein
upon its initial expression”). In other words, although Defrees does teach
that its methods generally provide glycopeptides characterized by
substantially uniform glycosylation pattern, Defrees ¶¶ 84, 93, the Examiner
has not cited to persuasive evidence that such homogeneity could be
achieved in a cellular rather than an in vitro context.
For example, Defrees teaches using its method to “modif[y] . . .
glycopeptides that are incompletely glycosylated during production in cell
culture cells,” id. ¶ 84, suggesting that, even if the glycopeptides are
modified intracellularly as a first step, homogeneity may not be achieved
until after production of the glycopeptides by the cell. See also, e.g., id. ¶ 95
(explaining that “method of the invention also provides for modification of
incompletely glycosylated peptides that are produced recombinantly,” since
“[m]any recombinantly produced glycopeptides are incompletely
glycosylated”), ¶¶ 180–181 (teaching that its method for “remodeling” a
glycopeptide may be practiced “in any useful order on peptides and
glycopeptides that are in crude form, e.g., as expressed, are partially purified
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or are fully purified”), ¶ 38 (explaining that its method would “allow
switching from a production cell line . . . limited in expression level, to a
production cell line that has the capability of producing significantly greater
amounts of product, but yielding an inferior glycosylation pattern,” by
“modif[ying] [the glycosylation pattern] in vitro to match that of the desired
product”) (emphasis added). Likewise, in discussing the scenario where
“the modified glycopeptide is produced intracellularly,” Defrees goes on to
describe various purification steps and further states that, “[s]ome or all of
the foregoing purification steps, in various combinations, can also be
employed to provide a homogeneous modified glycopeptide.” Defrees ¶¶
185–192 (emphasis added). This again suggests the peptide of interest, as
produced by a cell, may not have a uniform glycoform without further
processing.
Finally, we note that Defrees teaches that “‘[s]ubstantially uniform
glycoform’ or a ‘substantially uniform glycosylation pattern,’ when referring
to a glycopeptide species, refers to the percentage of acceptor moieties that
are glycosylated by the glycosyl donor of interest.” Defrees ¶ 78. However,
in contrast to our construction of the phrase “glycoprotein . . . with a uniform
glycoprofile,” Defrees teaches that “‘substantially’ in the above definitions
of ‘substantially uniform’” encompasses a scenario where only “at least
about 40% . . . of the acceptor moieties for a particular mutant
endoglycanase or glycosyltransferase are glycosylated.” Id. ¶ 79; see also
id. ¶ 194 (describing an exemplary embodiment where “the invention
provides a glycopeptide in which at least about 80% of a population of a
selected acceptor moiety on the glycopeptide is glycosylated with the
glycosyl residue added by the mutant endoglycanase”).
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Accordingly, for the reasons above, we reverse the Examiner’s
rejection of claim 1 as obvious over Defrees, Rao, Uchiyama, Kobayashi,
Chiba, Zhu, and Palling. We reverse the rejections of claims 2–5, 7–9, and
11–13, which depend directly or indirectly from claim 1, for the same
reasons. In re Fritch, 972 F.2d 1260, 1266 (Fed. Cir. 1992) (“[D]ependent
claims are nonobvious if the independent claims from which they depend are
nonobvious.”).

B. Obviousness rejection over independent claim 25
1. Issue
Independent claim 25 recites “[a]n eukaryotic cell comprising: a first
exogenous nucleic acid molecule encoding Endo H operably linked to an
endoplasmic reticulum or Golgi localization signal; and a second exogenous
nucleic acid molecule encoding a glycoprotein.” The Examiner rejects the
claim as obvious over Defrees, Rao, Uchiyama, Kobayashi, Chiba, Zhu, and
Palling. Ans. 4.
In support of the conclusion that claim 25 is prima facie obvious over
the cited prior art, the Examiner cites to the same passages of Defrees as
discussed above with respect to claim 1. In particular, the Examiner finds
that Defrees teaches a method of modifying glycosylation patterns of
glycoproteins, including glycoproteins recombinantly produced in a
eukaryotic cell, comprising a first step of treating the glycoprotein with
Endo H “to remove undesired carbohydrate from said protein.” Ans. 4–6.
The Examiner finds that it would have been obvious to link Endo H to an ER
localization signal because “[i]t has been known in the prior art that Endo H
enzyme . . . must be localized to the ER to have enzymatic activity” and that,
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“in order to successfully localize a deglycosylation enzyme to yeast ER, an
ER-retention signal should be attached to said enzyme.” Id. at 6–7. Finally,
the Examiner finds that a skilled artisan would have found it obvious to co-
express the exogenous Endo H enzyme and exogenous glycoprotein for the
reasons discussed above with respect to claim 1. Id. at 6, 8–11.
Appellant does not separately argue claim 25. Thus, we understand
that Appellant’s arguments with respect to claim 25 to be identical to its
arguments with respect to claim 1, i.e., that a skilled artisan would not have
had reason to combine the cited prior art to arrive at the claimed invention
and that Defrees teaches away. Appeal Br. 9–12, 13–14. As discussed
above, Appellant also contends that the subject matter of the claims exhibits
unexpected results. Id. at 12–13, 15. The issues with respect to this
rejection are (1) whether a skilled artisan would have had reason to combine
the cited prior art to arrive at the invention of claim 25, with a reasonable
expectation of success, and, if so, (2) whether Appellant has provided
evidence of unexpected results that, when considered together with evidence
of obviousness, shows the claim to be non-obvious.
2. Analysis
We agree with the Examiner that a skilled artisan would have had
reason to combine the cited prior art to arrive at the invention of claim 25,
with reasonable expectation of success.
As an initial matter, techniques for construction of recombinant
nucleic acids and expression of genes in transfected host cells are well
known in the art. Defrees ¶ 80. With respect to the specific exogenous
nucleic acid molecules recited in claim 25, Defrees explicitly teaches
recombinantly expressing a glycoprotein in a eukaryotic cell. Defrees ¶¶ 33,
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37. As for Endo H, an endoglycanase and endo-ß-N-acetylglucosaminidase,
see supra n. 2, Defrees teaches recombinantly expressing an endoglycanase
such as Endo H, albeit a mutant version, in a eukaryotic cell. Defrees ¶ 33,
34, 63. Similarly, Kobayashi teaches recombinantly expressing a novel,
enzymatically active endo-ß-acetylglucosaminidase in a yeast (i.e.,
eukaryotic) cell. Id. at 1:6–13, 8:44–57, 10:56–63, 15:62–19:44.
Furthermore, Defrees and Kobayashi both teach that enzymes such as
Endo H are useful in remodeling and/or analyzing glycoproteins. For
example, Defrees teaches that “[c]loned endo- and exo-glycosidases are
standardly used to release monosaccharides and N-glycans from
glycopeptides,” and that glycoaminidases and endoglycosidases are used in
the synthesis of carbohydrates. Defrees ¶¶ 14, 23, 33. Kobayashi similarly
teaches that various glycosidases are known to be useful in “cleaving a sugar
chain from a glycoprotein or when identifying the structure of a sugar
chain,” for purposes of “research[ing] . . . the correlation between sugar
chain structure and its function” in order to clarify the role of “the sugar
chains of glycoproteins . . . in mechanisms such as cell differentiation,
carcinogenesis and intercellular recognition.” Kobayashi 1:20–28. In
particular, Kobayashi teaches that endo-ß-N-acetylglucosaminidase “acts on
the asparagine-linked sugar chain (N-linked sugar chain, N-sugar chain) and
has the action of cleaving the diacetyl-chitobiose portion that exists within
the sugar chain thereby liberating the sugar chain,” and further teaches that,
“[s]ince endo-ß-N-acetylglucosaminidase can liberate the sugar portion of a
glycoprotein from the protein portion, it is thought to be important in the
analysis of the function and structure of sugar chains in glycoproteins.” Id.
at 1:28–37. Finally, as discussed above, Defrees teaches that it is known in
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the prior art to remedy the deficiencies of the glycosylation of a particular
host cell by engineering the host cell to express one or more recombinant
enzymes needed for a particular glycosylation pathway. Defrees ¶ 20.
We therefore conclude that, to the extent Defrees does not explicitly
teach a eukaryotic cell comprising both an exogenous nucleic acid molecule
encoding Endo H and a second exogenous nucleic acid molecule encoding a
glycoprotein, such a cell would have been obvious to a skilled artisan based
on the combined teachings of the cited prior art. First, such combination is
no more than “[t]he combination of familiar elements according to known
methods,” i.e., recombinantly expressing two molecules known to be useful
in the prior art, which the Supreme Court has explained is “likely to be
obvious when it does no more than yield predictable results.” KSR Int’l Co.
v. Teleflex Inc., 550 U.S. 398, 416 (2007).15 A skilled artisan would have
had further reason to co-express exogenous nucleic acids encoding Endo H
and a glycoprotein, because, as discussed above, the prior art teaches that
Endo H acts on glycoproteins and further teaches engineering host cells to
express recombinant enzymes in order to remodel glycoproteins.
Finally, we find that a skilled artisan would have had reason to
operably link the nucleic acid encoding Endo H to an endoplasmic reticulum
(ER) or Golgi localization signal, as recited in claim 25. In particular, the
prior art teaches an ER retention/retrieval tag that successfully localize a
functional enzyme involved in glycoprotein processing (i.e., α-1,2-
mannosidase) to the ER, Chiba Abstract, and, indeed, the Specification notes

15 As we discuss below, Appellant has not demonstrated persuasively that
the claimed subject matter exhibits unexpected results.
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that ER and Golgi localization signals are “known in the art and may be
derived from proteins that are normally localized in the ER or Golgi for their
function.” Spec. ¶ 22. Furthermore, the prior art teaches that proteins are
glycosylated in the ER before being further processed in the Golgi, Defrees
¶ 6, and further teaches that “Endo H only cleaves proteins which have
acquired ER glycosylation (high mannose N-liked), i.e., which are localized
to the ER,” Palling ¶ 102. Accordingly, it would have been obvious to a
skilled artisan to operably link the nucleic acid encoding Endo H to an
endoplasmic reticulum (ER) or Golgi localization signal: First, such a
combination is no more than “[t]he combination of familiar elements
according to known methods” that “does no more than yield predictable
results.” KSR, 550 U.S. at 416. Second, Endo H is known to be useful in
remodeling certain glycoproteins and the prior art teaches that such
glycoproteins are localized to the ER.
We turn next to Appellant’s arguments. Only those arguments
timely made by Appellant in the Appeal Brief (no Reply Brief was
submitted) have been considered; arguments not so presented in
the Brief are waived. See 37 C.F.R. § 41.37(c)(1)(iv) (2015); see also Ex
parte Borden, 93 USPQ2d 1473, 1474 (BPAI 2010) (informative) (“Any
bases for asserting error, whether factual or legal, that are not raised in the
principal brief are waived.”).
Appellant contends that there is no motivation to combine the
references. More particularly, Appellant contends that Defrees teaches an
“in vitro synthetic approach to enzymatically modify glycosylation on
glycoproteins,” namely “a method which uses mutant endoglucosaminidases
to synthesize desired complex glycan structures on glycoproteins.” Appeal
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Br. 9. Appellant contends that, “[a]lthough the glycoprotein that is used for
treatment with the mutant endoglycanases may be produced recombinantly,
as well as the mutant endoglycanase itself, the working examples only
provide in vitro conditions . . . to glycosylate the glycoproteins.” Id.
Appellant contends that,
[e]ven if the second synthesis step [of Defrees] could possibly occur
in the cell, one could implicitly or indirectly derive from Defrees that
it is not expected that expression of a wild-type endoglycanase
(needed for the first step of Defrees) within a cell with the aim to
produce a glycoprotein co-expressed with the endoglycosidase would
lead to the desired substrate suitable for the second step of Defrees
where a mutant endoglycanase is used.
Id. at 11. Similarly, although Appellant concedes that Kobayashi teaches the
recombinant production of a wild type endoglycanase, Appellant contends
that “there is no motivation or suggestion to combine the expression of an
endoglycanase with endohydrolyzing activity AND expression of a
glycoprotein in the same eukaryotic cell.” Id. at 10; see also id. at 12
(stating that “Kobayashi has not characterized the N-glycans of the
endogenous yeast proteins” and also “does not hint at a co-expression of the
EndoM endoglycosidase and an exogenous glycoprotein in the hope of
obtaining a uniform glycosylation pattern on an exogenous glycoprotein”).
We are not persuaded. As discussed above, the combination of
Defrees and Kobayashi teaches that glycoproteins and enzymes such as
Endo H may be recombinantly expressed in a eukaryotic cell. It also teaches
that enzymes such as Endo H act on glycoproteins and are useful in
remodeling and analyzing glycoproteins. Finally, Defrees teaches that it is
known in the prior art “to remedy the deficiencies of the glycosylation of a
particular host cell” by “engineering the cell to express one or more . . .
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enzymes.” Defrees ¶ 20; see also id. ¶¶ 186, 191 (discussing “modified
proteins” of its invention being produced “intracellularly” or in “large-scale
fermentation”). Thus, as also discussed above, the co-expression of
exogenous glycoproteins and Endo H in a eukaryotic cell is obvious both
because it is no more than “[t]he combination of familiar elements according
to known methods,” KSR, 550 U.S. at 416, and because a skilled artisan
would be motivated to engineer a cell to express Endo H in order to analyze
and/or modify glycosylation in the host cell. Neither has Appellant shown
that such co-expression yielded anything other than predictable results.
With respect to Appellant’s argument that Defrees teaches an in vitro
“method which uses mutant endoglucosaminidases to synthesize desired
complex glycan structures on glycoproteins,” Appeal Br. 9, we note that “a
reference . . . is prior art for all that it teaches” to a skilled artisan, not merely
its preferred embodiment or even its particular invention. Beckman
Instruments, Inc. v. LKB Produkter AB, 892 F.2d 1547, 1551 (Fed. Cir.
1989).
With respect to Appellant’s argument that “it is not expected that
expression of a wild-type endoglycanase . . . with the aim to produce a
glycoprotein co-expressed with the endoglycosidase would lead to the
desired substrate suitable for the second step of Defrees,” Appeal Br. 9, we
note that, unlike in claim 1, claim 25 does not require any particular
interaction between the exogenous enzyme and glycoprotein, or any
particular resulting substrate suitable for further processing. Similarly, with
respect to Appellant’s argument that Kobayashi does not suggest co-
expression of an endoglycosidase and exogenous glycoprotein “in the hope
of obtaining a uniform glycosylation pattern on an exogenous glycoprotein,”
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id. at 12, we note that unlike in claim 1, claim 25 does not require any
particular degree of uniformity in the glycosylation patterns of the
glycoproteins produced by the eukaryotic cell.
Appellant contends Defrees teaches away from the claimed invention
because “use of a cell would not be the most optimal system” for producing
glycoproteins with a desired glycopattern. Appeal Br. 10. Appellant
contends that Defrees suggests that “a cellular system results in incomplete
glycosylation and does not result in uniform desired proteins (or for making
a eukaryotic cell expressing a glycoprotein AND an endoglycanase).” Id. at
11. We are not persuaded for the same reasons already discussed above,
namely that a reference is prior art for all that it teaches and not merely for
its preferred embodiment or particular invention, and that claim 25 does not
require production of glycoproteins with any particular desired glycopattern.
Appellant further contends “Kobayashi clearly uses intracellular
expression of EndoM.” Appeal Br. 10 (emphasis added). Thus, Appellant
contends, “the cytoplasmic endoglucosaminidase as disclosed by Kobayashi
would not be retained in or pass through the secretory pathway” as recited in
claim 1, and “glycosylated proteins (which pass through the secretory
pathway) would not encounter” such an endoglucosaminidase. Id.
We are not persuaded. “Non-obviousness cannot be established by
attacking references individually where the rejection is based upon the
teachings of a combination of references. . . . [The reference] must be read,
not in isolation, but for what it fairly teaches in combination with the prior
art as a whole.” In re Merck & Co., 800 F.2d 1091, 1097 (Fed. Cir. 1986).
In this case, although Kobayashi does not explicitly teach operably linking
the Endo H to an endoplasmic reticulum (ER) or Golgi localization signal,
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the prior art teaches that Endo H acts on glycoproteins in the endoplasmic
reticulum. Thus, a skilled artisan motivated to produce an Endo H enzyme
that would act on glycoproteins in the cell would have reason to operably
link Endo H to an ER-targeting signal.
Neither has Appellant provided evidence of unexpected results that,
when considered together with evidence of obviousness, shows the claim to
be non-obvious. Appellant cites to Example 4 of the Specification, as well
as Meuris16 and Piron,17 as showing that “in 3 eukaryotic expression
systems (yeast, mammalian cells and plants) the simultaneous expression of
an exogenous endoglucosaminidase and an exogenous glycoprotein leads to
the production of homogeneous and unexpected unnatural glycan
structures present on the exogenous glycoprotein.” Appeal Br. 13.
Appellant contends that “the presence of such homogenous glycan profile
produced within an eukaryotic cell and the nature of these specific glycans
could not be predicted from the prior art, in particular not from Defrees and
Kobayashi.” Id.
We are not persuaded. As an initial matter, “[i]t is well settled
that unexpected results must be established by factual evidence. Mere
argument or conclusory statements in the specification does not suffice.” In
re De Blauwe, 736 F.2d 699, 705 (Fed. Cir. 1984). Here, we note that the
discussion of Example 4, with the exception of the statement in paragraph

16 Leander Meuris et al., GlycoDelete Engineering of Mammalian Cells
Simplifies N-Glycosylation of Recombinant Proteins, 32 NATURE
BIOTECHNOLOGY 485 (2014).
17 Robin Piron et al., Using GlycoDelete to Produce Proteins Lacking Plant-
Specific N-Glycan Modification in Seeds, 33 NATURE BIOTECHNOLOGY 1135
(2015).
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170 regarding the relative location of proteins fused to targeting signals
Mnn2p versus Kre2p, are written in the present tense and therefore are
presumed to be prophetic. See Atlas Powder Co. v. E.I. du Pont De
Nemours & Co., 750 F.2d 1569, 1578 (Fed. Cir. 1984) (explaining that “the
examples were written in the present tense to conform with the PTO
requirements on prophetic example”). “To be particularly probative,
evidence of unexpected results must establish that there is a difference
between the results obtained and those of the closest prior art, and that the
difference would not have been expected by one of ordinary skill in the art at
the time of the invention.” Bristol-Myers Squibb Co. v. Teva Pharms. USA,
Inc., 752 F.3d 967, 977 (Fed. Cir. 2014) (emphasis added). Thus, Example 4,
which is prophetic and not evidence of “results obtained,” does not provide
the factual evidence needed to support unexpected results.
Likewise, Appellant does not cite to evidence, for instance statements
in the Specification or declarations by persons of skill in the art, that the
results described in Meuris and Piron are unexpected.18 While Appellant
contends in its Appeal Brief that the results described in Meuris and Piron
(and Example 4) are “totally unexpected,” “surprising,” and/or “could not be
predicted from the prior art,” “[a]ttorneys’ argument is no substitute for
evidence.” Johnston v. IVAC Corp., 885 F.2d 1574, 1581 (Fed. Cir. 1989).

18 Appellant cites Meuris for mammalian cells producing a “homogeneous
population of glycan structures” that “consists of only three specific
structures.” Appeal Br. 12 (emphasis added). However, we note that
production of such glycoproteins would not meet our construction of the
phrase “wherein the exogenous glycoprotein is produced by the cell with a
uniform glycoprofile,” as discussed above.
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We further note that Appellant’s proffered evidence of alleged
unexpected results are not commensurate with the scope of the claims. First,
none of the examples cited by Appellant falls within the invention of claim
25, because each of them uses Endo T rather than Endo H as recited by
claim 25. Appeal Br. 12–13. Furthermore, although claim 25 encompasses
any eukaryotic cell comprising (1) a nucleic acid molecule encoding Endo H
operably linked to any endoplasmic reticulum or Golgi localization signal
and (2) any exogenous nucleic acid molecule encoding a glycoprotein,
Meuris and Piron describe only a limited number of combinations of a
glycoprotein and a fusion protein comprising Endo T and a Golgi-
localization signal.19 Neither did Appellant provide any explanation why the
proffered evidence provides a reasonable basis for concluding that the
embodiments encompassed by claim 25 would behave in the same manner
as the eukaryotic cells described in those two references. See In re Lindner,
457 F.2d 506, 508 (CCPA 1972).20
Accordingly, we affirm the Examiner’s rejection of claim 25 as
obvious over Defrees, Rao, Uchiyama, Kobayashi, Chiba, Zhu, and Palling

19 Meuris describes cells comprising (1) “ST-endoT fusion protein” wherein
catalytic domain of endoT, an endo-β-N-acetylglucosaminidase, is fused to
the Golgi targeting domain of the human ß-galactoside-α-2,6-
sialyltransferase 1 (ST6GAL1), Meuris 485, right column, and (2) either
granulocyte-macrophage colony-stimulating factor (GM-CSF) or
monoclonal anti-CD20 antibody, id. at 486, left column and 487, right
column. Piron describes cells comprising (1) fusion protein comprising
EndoT and targeting signal of the Arabidopsis Golgi ß-1,2-xylosyltranserase
and (2) activation associated secretory protein 1 (ASP1). Piron 1136.
20 As already explained, Example 4 of the Specification does not add to the
evidence for unexpected results because it appears to be prophetic.
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C. Obviousness rejection over independent claim 26
1. Issue
The Examiner rejects the claim as obvious over Defrees, Rao,
Uchiyama, Kobayashi, Chiba, Palling, and DeFrees ’521. Ans. 13. Claims
25 and 26 are identical, except that claim 26 recites an eukaryotic cell
comprising an exogenous nucleic acid molecule encoding Endo M rather
than Endo H as in claim 25. Appeal Br. 21 (Claim App.). The Examiner
relies on the same reasoning as in claim 25 to establish the prima facie
obviousness of claim 26, and additional relies on DeFrees ’521 as follows:
’521 teaches Endo-M . . . has been successfully used to produce
glycoform of protein with advantage that use of Endo-M allows
to transfer oligosaccharide other than a monosaccharide to
glycosyl acceptor (that is a substrate) so as to produce desired
recombinant modified glycoproteins in host cell such as
mammalian cells, wherein said recombinant glycoproteins so
produced have N-linked glycan structures different from the
naturally occurring glycans, particularly when the polypeptides
are to be used as therapeutic agents.
Ans. 13 (citations omitted).
The Examiner concludes that, based on the above, a skilled artisan
would have been motivated to make a recombinant eukaryotic cell
expressing Endo M, with a reasonable expectation of success, in order to
produce proteins with desired glycoforms for use as, e.g., therapeutic agents,
especially because Endo M allows for transfer of oligosaccharide to a
substrate so as to achieve more efficient glycosylation. Ans. 13–14.
Although Appellant “agree[s] that ’521 application mentions the
advantages of EndoM,” Appellant contends that “there is no teaching or
suggestion in any of the references regarding an eukaryotic cell co-
expressing EndoH/M and a target glycoprotein.” Appeal Br. 17.
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The issue with respect to this rejection is whether a preponderance of
evidence supports the Examiner’s conclusion that claim 26 is obvious over
the cited combination of prior art.
2. Analysis
We agree with the Examiner that claim 26 is obvious over the
combination of cited prior art for the same reasons discussed above with
respect to claim 25, and further because, as Appellant concedes, Kobayashi
teaches recombinantly producing Endo M and DeFrees ’521 teaches that
endoglycosidases such as Endo M are useful for adding oligosaccharide
fragments to substrates and in synthesizing glycopeptides. Appeal Br. 10,
17; DeFrees ’521 ¶¶ 20–21. We are not persuade by Appellant’s argument
for the same reasons discussed above with respect to claim 25.

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CONCLUSION
In summary:
Claims
Rejected
35 U.S.C.
§
Reference(s)/Basis Affirmed Reversed
1–5, 8, 9,
11, 12, 25
103(a) Defrees, Rao,
Uchiyama, Kobayashi,
Chiba, Zhu, Palling
25 1–5, 8, 9,
11, 12
7 103(a) Defrees, Rao,
Uchiyama, Kobayashi,
Chiba, Palling
7
13 103(a) Defrees, Rao,
Uchiyama, Kobayashi,
Chiba, Palling, Crispin
13
26 103(a) Defrees, Rao,
Uchiyama, Kobayashi,
Chiba, Palling,
Defrees ’521
26
Overall
Outcome
25, 26 1–5, 7–9,
11–13

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 IN PART