Ex Parte Glaser et al

Board of Patent Appeals and Interferences

Jan 17, 2012

11003149 (B.P.A.I. Jan. 17, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte STEPHAN GLASER, FRANK GEIPEL,
THOMAS KIRSCHBAUM, BERNHARD REXER,
JOHANN-PETER THALHOFER, RAINER MUELLER,
CLAUDIA GIESSEL, HELLMUT ECKSTEIN, and ELVIRA WOLF
__________

Appeal 2011-009105
Application 11/003,149
Technology Center 1600
__________

Before DONALD E. ADAMS, FRANCISCO C. PRATS, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a method
of producing purified protein with carboxypeptidase B activity. The
Examiner rejected the claims as obvious. We have jurisdiction under 35
U.S.C. § 6(b). We affirm.

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Statement of the Case
Background
The Specification teaches that “the invention pertains to the
production and further processing of an enzymatically inactive precursor of
a protein with carboxypeptidase B activity. An aspect of the invention
pertains to the activation and concomitant purification of the protein with
carboxypeptidase B activity” (Spec. 1 ¶ 0001).
The Claims
Claim 1-15 are on appeal. Claim 1 is representative and reads as
follows:
1. A method for producing a purified protein with
carboxypeptidase B activity, the method comprising the steps
of:
(a) providing a histidine-tagged pro-carboxypeptidase B
wherein the histidine tag binds to a particulate metal chelate
affinity matrix,
(b) contacting the histidine-tagged pro-carboxypeptidase
B with the affinity matrix whereby the histidine-tagged pro-
carboxypeptidase B binds to the affinity matrix,
(c) washing the affinity matrix and the bound histidine-
tagged pro-carboxypeptidase B,
(d) incubating the affinity matrix and the bound histidine-
tagged procarboxypeptidase B in a buffer containing trypsin,
wherein the procarboxypeptidase B is cleaved to release a
protein with carboxypeptidase B activity from a
carboxypeptidase B propeptide, and wherein the propeptide
remains bound to the affinity matrix, and
(e) separating the protein with carboxypeptidase B
activity from the propeptide bound to the affinity matrix,
thereby purifying the protein with carboxypeptidase B activity.

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The issues

The claims stand rejected as follows:
 claims 1-12 under 35 U.S.C. § 103(a) as obvious over Walker,1
Habermann,
2
and Clauser
3
(Ans. 3-6);
 claims 13-15 under 35 U.S.C. § 103(a) as obvious over Walker,
Habermann, Clauser, Smolyar,
4
and Simard
5
(Ans. 7-9).
Appellants acknowledge that the claims stand or fall together based on
claim 1 (see App. Br. 5), so we will consider the rejections together. 37
C.F.R. § 41.37(c)(1)(vii).
The Examiner finds that Walker teaches “a single step protein
purification system from utilizing a His-tagged fusion protein and His-
tagged proteolytic enzyme. Upon the immobilization of both the tagged
proteins on nickel columns, the desired protein is released by the action of
the protease while the His-tag and the tagged protease remain on the
column” (Ans. 4).

1
Walker et al., Efficient and Rapid Affinity Purification of Proteins
Using Recombinant Fusion Proteases, 12 BIO/TECHNOLOGY 601-
605 (1994).
2
Habermann, Paul, US 6,531,294 B1, issued Mar. 11, 2003.
3
Clauser et al., Structural Characterization of the Rat
Carboxypeptidase Al and B Genes, 263 J. BIOLOGICAL CHEMISTRY
17837-17845 (1988).
4
Smolyar, Alex, WO 02/095018 A1, published Nov. 28, 2002.
5
Simard et al., Limited proteolysis of Escherichia coli cytidine 5’-
triphosphate synthase. Identification of residues required for CTP
formation and GTP-dependent activation of glutamine hydrolysis,
270 EUROPEAN J. BIOCHEMISTRY 2195-2206 (2003).
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The Examiner finds that Habermann teaches
purifying carboxypeptidase B by (a) expressing a natural or
unnatural enzymatically inactive precursor form of
carboxypeptidase B in a fusion construct, which contains
signal sequence in methylotrophic yeast strain Pichia
pastoris, (b) purifying the precursor form expressed with
histidine tag sequence using nickel chelating column, and (c)
converting the purified precursor form into the active form
by an enzymatic cleavage by trypsin treatment

(Ans. 4). The Examiner finds that Clauser teaches “a procarboxypeptidase
protein, which is 100% identical to SEQ ID NO: 2” (Ans. 5).
The Examiner finds it obvious to “release the active carboxypeptidase
B protein of Clauser et al. by efficient proteolytic cleavage, while the protein
bound to the column as taught by Walker et al., the affinity tail would
remain attached to the column” (Ans. 6). The Examiner finds it “would
have been further obvious to use trypsin to cleave the fusion protein and
activate CPB in one step as taught by Habermann” (Ans. 6).
Appellants “submit that the Examiner is improperly ignoring the
context of Walker and applying it nebulously and nonspecifically for
teaching „the concept‟ of single-step activation by cleavage from a column”
(App. Br. 13). Appellants contend that the “activation method of
Habermann results in the undesirable, non-covalent bonding of the
propeptide to the CPB and the additional processing/separation steps sought
to be avoided by the instant methods” (App. Br. 14). Appellants contend
that “the combination of references fails to teach or fairly suggest on-column
activation of CPB from proCPB, as required by instant claim 1, and fails to
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teach or fairly suggest effective separation of CPB from the propeptide
fragment as required by instant claim 1” (App. Br. 14).
Appellants contend that
the Examiner has improperly adopted and used motivation
provided by the instant specification with respect to the
desirability of a single-step cleavage/activation and
separation method for the production of activated CPB free
of propeptide remnants in order to avoid the non-covalent
association of the fragments that causes industrial
production yield problems, in particular with production of
insulin, in order to improperly avoid acknowledging and
addressing deficiencies in the applied combination of
references.

(App. Br. 15-16).
Appellants contend that Walker‟s “aligned on-column binding is not
enabled with respect to trypsin and proCPB by any of the references or by
knowledge common to the art. Second, if one assumes it is, it is still not
Appellants‟ invention, which does not require binding of trypsin to the
column.” (App. Br. 17).
The issue with respect to this rejection is: Does the evidence of record
support the Examiner‟s conclusion that the combination of Walker,
Habermann, and Clauser render claim 1 obvious?
Findings of Fact
1. Walker teaches “schemes for the application of fusion proteases
in the removal of affinity tails from recombinant fusion proteins” (Walker
601, col. 2). Walker teaches that the “recombinant protein is purified in a
single chromatographic step which removes both the affinity tail and the
fusion protease” (Walker 601, abstract). Walker teaches that the
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“advantages over existing methods include much improved specificity of
proteolytic cleavage, complete removal of the protease and the affinity tail in
one step, and the option of adding any desired amount of fusion protease to
ensure efficient cleavage” (Walker 601, abstract).
2. Walker teaches that in
Figure 1A, the fusion protease . . . is bound to an affinity
matrix to which the target protein is already bound.
Cleavage of the fusion protein into affinity tail and the
desired protein occurs efficiently while the two species of
fusion protein are simultaneously bound to the matrix. As
the fusion protease and the released affinity tail both contain
GST, they remain firmly bound to the column and the
recombinant protein is recovered by draining or centrifuging
the column. Thus, a single column can be used to rapidly
and efficiently produce pure protein from crude bacterial
extracts

(Walker 601, col. 2).
3. Walker teaches that a “single fusion protease can be used to
cleave recombinant proteins with a variety of fusion tails . . . This allows
flexibility in the choice of affinity tail . . . For example. GST-3C
pro
may be
used to cleave a fusion protein containing from the aminoterminus, a six-
histidine tail” (Walker 601, col. 2).
4. Walker teaches that to “illustrate the general applicability of on-
column cleavage of fusion proteins with GST-3C
pro
, two other GST proteins
were purified on glutathione-Sepharose . . . The on-column cleavage was
performed . . . and each protein was recovered rapidly . . . and in good yield .
. . Each protein had the expected aminoterminal sequence” (Walker 602,
col. 2).
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5. Walker teaches “[e]xpression and purification of the substrate
for 6His-3C
pro
(6His·PKC1) was performed exactly as for 6His-3C
pro
. These
two proteins were mixed, incubated overnight and then added to a 2 ml Ni
2+
-
NTA column . . . The flow-through contained purified PKC1 . . . and its
aminoterminus was found to contain the expected sequence” (Walker 603,
col. 1).
6. Walker teaches a method where
instead of eluting GST·2A
pro
from the column, the
glutathione-Sepharose was extensively washed and purified
GST-3C
pro
was applied . . . After further washing to remove
traces of unbound GST-3C
pro
, the column was sealed, the
desired volume of buffer was added, and the column was
incubated for 16 hours at 4°C. Finally, purified 2A
pro
was
washed off the column . . . and its aminoterminal sequence .
. . was confirmed to be the same as previously published.

(Walker 602, col. 2).
7. Habermann teaches
a process for the preparation of pancreatic carboxypeptidase
B . . . where (a) a natural or unnatural precursor form of
carboxypeptidase B or an isoform . . . is expressed in a
microorganism by secretion, (b) the precursor form
expressed by secretion is purified and (c) the purified
precursor form is converted into the active carboxypeptidase
B . . . by means of an enzymatic treatment

(Habermann, col. 2, ll. 40-51).
8. Habermann teaches that a
preferred process is one of the type in which the unnatural
precursor form has the following structure:
S-(As)x-E-CB, (1)
where
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S is a signal peptide which brings about the secretion from
the respective microorganism, of the fusion protein
formed during expression;
As is any desired genetically encodable amino acid;
E is a peptide linker consisting of an endopeptidase
recognition sequence;
CB is the amino acid sequence of carboxypeptidase B or an
isoform or a mutein of carboxypeptidase B; and
x is an integer from 0-100.

(Habermann, col. 2, l. 57 to col. 3, l. 5).
9. Habermann teaches “purification of the precursor form by
affinity chromatography, particularly preferably a process of the type in
which the sequence attached for the purification by affinity chromatography
is 1 to 6, preferably 4, histidine residues” (Habermann, col. 3, ll. 8-12).
10. Habermann teaches “expression and the HIS-
procarboxypeptidase B protein is directly purified by means of a nickel
affinity chromatography step after precipitation with ammonium sulfate in
solution” (Habermann, col. 7, ll. 48-51).
11. Habermann teaches “the activation of procarboxypeptidase B
by reaction with trypsin . . . the propeptide fragments removed from the
carboxypeptidase B and other constituents are separated off from the
carboxypeptidase B by microfiltration by means of a Centriprep filter unit”
(Habermann, col. 7, l. 64 to col. 8, l. 7).
12. The Examiner finds that “Clauser et al. teach a
procarboxypeptidase protein, which is 100% identical to SEQ ID NO: 2”
(Ans. 5).

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Principles of Law
The Supreme Court has emphasized that “the [obviousness] analysis
need not seek out precise teachings directed to the specific subject matter of
the challenged claim, for a court can take account of the inferences and
creative steps that a person of ordinary skill in the art would employ.” KSR
Int’l v. Teleflex Inc., 550 U.S. 398, 418 (2007). As noted by the Court in
KSR, “[a] person of ordinary skill is also a person of ordinary creativity, not
an automaton.” Id. at 421. “The combination of familiar elements according
to known methods is likely to be obvious when it does no more than yield
predictable results.” Id. at 416.
Analysis
Walker teaches a method for producing a purified protein which
comprises steps including providing a histidine tagged protein, binding that
protein to an affinity matrix, washing the affinity matrix and bound histidine
tagged protein, incubating the protein with an endopeptidase to release the
bound histidine tagged protein, and eluting the cleaved protein from the
affinity column (FF 1-6). The Examiner acknowledges that Walker does not
“teach purifying active carboxypeptidase B protein, or using trypsin for
cleaving inactive pro-carboxypeptidase B to active carboxypeptidase B
protein” (Ans. 4).
Habermann teaches expression of a histidine tagged
procarboxypeptidase B protein, purifying the protein on an affinity column,
and releasing the active carboxypeptidase B from the column using trypsin
(FF 7-11).
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Applying the KSR standard of obviousness to the findings of fact, we
agree with the Examiner that it would have been obvious to substitute
Habermann‟s carboxypeptidase B as the protein of interest in Walker and to
substitute Habermann‟s trypsin for the protease in Walker (Ans. 5-6). Such
a combination is merely a “predictable use of prior art elements according to
their established functions.” KSR, 550 U.S. at 417.
Appellants “submit that the Examiner is improperly ignoring the
context of Walker and applying it nebulously and nonspecifically for
teaching „the concept‟ of single-step activation by cleavage from a column”
(App. Br. 13).
We are not persuaded. Walker teaches that “a single column can be
used to rapidly and efficiently produce pure protein from crude bacterial
extracts” (Walker 601, col. 2; FF 2). Walker “illustrate[s] the general
applicability of on-column cleavage of fusion proteins” using different
affinity tags including Histidine tags and different proteases (Walker 602,
col. 2; FF 4-6). These are not “nebulous concepts” but rather specific and
concrete examples of single step purification of proteins using a single
affinity column (FF 1-6).
Appellants contend that the “activation method of Habermann results
in the undesirable, non-covalent bonding of the propeptide to the CPB and
the additional processing/separation steps sought to be avoided by the instant
methods” (App. Br. 14). Appellants contend that “the combination of
references fails to teach or fairly suggest on-column activation of CPB from
proCPB, as required by instant claim 1, and fails to teach or fairly suggest
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effective separation of CPB from the propeptide fragment as required by
instant claim 1” (App. Br. 14).
We are not persuaded. “[W]hen the question is whether a patent
claiming the combination of elements of prior art is obvious,” KSR directs
that “a court must ask whether the improvement is more than the predictable
use of prior art elements according to their established functions.” KSR, 550
U.S. at 417. Here, the prior art elements are used in a predictable manner,
entirely following the established methods of Walker and Habermann.
Walker teaches a single step process in which an endoprotease releases the
protein of interest from the column while avoiding further processing steps,
including the on column cleavage of the target protein by a protease and
separation of the target protein from the tag which remains bound to the
affinity column (FF 1-6). Habermann teaches that procarboxypeptidase B
can be linked to a histidine affinity tag and that cleavage with trypsin will
separate the affinity tag and propeptide fragment from the desired protein
(FF 7-11).
In combination, Walker and Habermann teach each element of the
claimed invention and the ordinary artisan would have recognized that the
single step method of Walker would simplify the purification procedure of
Habermann (FF 1-11). See In re Merck & Co., 800 F.2d 1091, 1097 (Fed.
Cir. 1986) (“Non-obviousness cannot be established by attacking references
individually where the rejection is based upon the teachings of a
combination of references.”)
Appellants contend that
the Examiner has improperly adopted and used motivation
provided by the instant specification with respect to the
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desirability of a single-step cleavage/activation and
separation method for the production of activated CPB free
of propeptide remnants in order to avoid the non-covalent
association of the fragments that causes industrial
production yield problems, in particular with production of
insulin, in order to improperly avoid acknowledging and
addressing deficiencies in the applied combination of
references.

(App. Br. 15-16).
We are not persuaded. While we are fully aware that hindsight bias
often plagues determinations of obviousness, Graham v. John Deere Co.,
383 U.S. 1, 36 (1966), we are also mindful that the Supreme Court has
clearly stated that “if a technique has been used to improve one device, and a
person of ordinary skill in the art would recognize that it would improve
similar devices in the same way, using the technique is obvious unless its
actual application is beyond his or her skill.” KSR, 550 U.S. at 417.
In the instant situation, the person of ordinary skill and creativity
would have recognized that the carboxypeptidase B purification method of
Habermann would have been improved using the single step purification
method of Walker (FF 1-11). Walker teaches that the “recombinant protein
is purified in a single chromatographic step which removes both the affinity
tail and the fusion protease” (Walker 601, abstract; FF 1). Walker teaches
that the “advantages over existing methods include much improved
specificity of proteolytic cleavage, complete removal of the protease and the
affinity tail in one step, and the option of adding any desired amount of
fusion protease to ensure efficient cleavage” (Walker 601, abstract; FF 1).
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The ordinary artisan would have modified Habermann‟s method to
adopt the single chromatographic step method of Walker for the express
benefits of removal of protease and affinity tag in one step (FF 1).
Appellants contend that Walker‟s “aligned on-column binding is not
enabled with respect to trypsin and proCPB by any of the references or by
knowledge common to the art. Second, if one assumes it is, it is still not
Appellants‟ invention, which does not require binding of trypsin to the
column.” (App. Br. 17).
We are not persuaded. Appellants have provided no evidence that
trypsin, whether simply added to the column directly or added linked to an
affinity tag as suggested by Walker, would not function to cleave the
procarboxypeptidase B protein. See In re Pearson, 494 F.2d 1399, 1405
(CCPA 1974) (“Attorney‟s argument in a brief cannot take the place of
evidence.”). Further, Walker never teaches or suggests that trypsin or other
larger proteases will not function. Instead, Walker is concerned with
specificity of cleavage, which Habermann demonstrates is not an issue for
the cleavage of procarboxypeptidase B with trypsin (FF 11). This supports a
reasonable expectation of success, consistent with Kubin, which stated that
“[r]esponding to concerns about uncertainty in the prior art influencing the
purported success of the claimed combination, this court [in O’Farrell]
stated: „[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) (citing In re O’Farrell, 853 F.2d 894, 903-
904 (Fed. Cir. 1988)).
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We also are not persuaded by Appellants‟ argument that the invention
“does not require binding of trypsin to the column.” (App. Br. 17.) Claim 1,
step (d) simply requires incubation of the affinity matrix and bound
carboxypeptidase B in a buffer containing trypsin. There is no limitation in
the claim that excludes the use of affinity tagged trypsin which would bind
to the column. “[L]imitations are not to be read into the claims from the
specification.” In re Van Geuns, 988 F.2d 1181, 1184 (Fed. Cir. 1993).
Conclusion of Law
The evidence of record supports the Examiner‟s conclusion that the
combination of Walker, Habermann, and Clauser render claim 1 obvious.
SUMMARY
In summary, we affirm the rejection of claims 1-12 under 35 U.S.C. §
103(a) as obvious over Walker, Habermann, and Clauser.
We affirm the rejection of claims 13-15 under 35 U.S.C. § 103(a) as
obvious over Walker, Habermann, Clauser, Smolyar, and Simard.

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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