Ex Parte Jacobi et al

Patent Trial and Appeal Board

Jun 13, 2018

13212486 (P.T.A.B. Jun. 13, 2018)

UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR
13/212,486 08/18/2011 Alexander Jacobi
28519 7590 06/15/2018
C/0 VP, IP, LEGAL
BOEHRINGER INGELHEIM USA CORPORATION
900 RIDGEBURY RD
PO BOX 368
RIDGEFIELD, CT 06877-0368
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
ATTORNEY DOCKET NO. CONFIRMATION NO.
01-2660-US-l 6336
EXAMINER
SWOPE, SHERIDAN
ART UNIT PAPER NUMBER
1652
NOTIFICATION DATE DELIVERY MODE
06/15/2018 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):
USPTO.e-Office.rdg@boehringer-ingelheim.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte ALEXANDER JACOBI, DOROTHEE AMBROSIUS,
PHILINE DOBBERTHIEN, CHRISTIAN ECKERMANN, and
FRANZ NOTHELFER
Appeal2016-005606
Application 13/212,486
Technology Center 1600
Before DEBORAH KATZ, JOHN E. SCHNEIDER, and
TIMOTHY G. MAJORS, Administrative Patent Judges.
MAJORS, Administrative Patent Judge.
DECISION ON APPEAL
Appellants 1 submit this appeal under 35 U.S.C. § 134(a) involving
claims to processes for irreversibly inactivating mammalian proteases in a
liquid obtained from a mammalian cell culture. The Examiner rejected the
claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b).
We AFFIRM.
1 Appellants identify the Real Party in Interest as BOEHRINGER
INGELHEIM INTERNATIONAL GMBH. App. Br. 1.
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Application 13/212,486
STATEMENT OF THE CASE
Appellants' invention relates to a process for preparing
biopharmaceutical products by the inactivation of proteolytically active
enzymes in the cell-free culture supernatant. Spec. 1:5-7.
The Specification explains that, in a typical process for preparing
proteins, "cells or the culture supernatant is or are harvested and the protein
contained therein is worked up and purified." Id. at 1: 17-22. According to
the Specification, "[i]t is known that proteases are present in the harvested
liquid," and that a protein/biopharmaceutical in the liquid "can be very
rapidly degraded or structurally damaged by proteases." Id. at 1 :24--27. The
Specification, thus, states that "[ t ]he invention relates to a method of
inactivating proteases by repeatedly changing the pH in the cell culture
supernatant at the start of the process for the purification of
biopharmaceuticals." Id. at 2:22-24.
Claims 1-3, 6-8, 10, 11, 14, and 16-30 are on appeal. Claims 1 and
2, the independent claims on appeal, are reproduced below:
1. A process for irreversibly inactivating mammalian
proteases in a liquid that is obtained from a mammalian cell
culture, comprising the steps of:
(a) adjusting the pH of the liquid to 3 to 5, and then
(b) adjusting the pH of the liquid to 7 to 9,
wherein step (a) is carried out at a temperature of20 to 30°C, and
the pH in step (a) is maintained for a period of 5 minutes to 30
minutes,
whereby neutral proteases are irreversibly inactivated in step (a),
acid proteases are irreversibly inactivated in step (b ), and
whereby protein degradation by said neutral and acid proteases
is reduced.
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2. A process for reducing protein degradation in a liquid that
is obtained from a mammalian cell culture, comprising the steps
of
(a) adjusting the pH of the liquid to 3 to 5, and then
(b) adjusting the pH of the liquid to 7 to 9,
wherein the pH in step (a) is maintained for a period of 5 minutes
to 30 minutes, and step (a) is carried out at a temperature of 20
to 30°C,
whereby mammalian neutral proteases are irreversibly
inactivated in step (a), mammalian acid proteases are irreversibly
inactivated in step (b ), and
whereby protein degradation is reduced.
App. Br. 23 (Claims App.).
The claims stand rejected as obvious under 35 U.S.C. § 103(a) over
Laustsen2 and Zapata. 3 Ans. 2-6.
Appellants requested oral hearing in this appeal, but later waived
appearance at the hearing that was scheduled for June 13, 2018. See Waiver
(April 9, 2018).
DISCUSSION
Issue
The issue on appeal is whether a preponderance of the evidence
supports the Examiner's conclusion that claims 1-3, 6-8, 10, 11, 14, and 16-
30 would have been obvious over Laustsen and Zapata.
2 Laustsen et al., WO 97/20921, published June 12, 1997.
3 Zapata, WO 2004/035608 A2, published Apr. 29, 2004.
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Findings of Fact (FF)
The Examiner's findings and statement of the rejection are provided at
pages 2-6 of the Examiner's Answer. See also Non-Final Rejection (Dec. 5,
2015) ("Non-Final Act.") 2-7. We identify the following teachings in
Laustsen and Zapata for emphasis and convenient reference.
FF 1. Laustsen teaches "[a] method for inactivation [ofJ at least one
undesirable enzyme in a mixture of enzymes containing a desirable enzyme,
obtained by varying pH, temperature and holding time." Laustsen Abstract;
see also id. at 21: 1-9 ( claim 1, reciting a method for inactivation of at least
one undesirable enzyme in a mixture by the steps of "a) holding the mixture
at a temperature of from 2°C to 75°C at a pH below 5.0 for at least 20 sec.;
and/orb) holding the mixture at a temperature of from 2°C to 75°C at a pH
above 9.0 for at least 20 sec."); see also id. at 21 :21-22 ( claim 4, reciting
that the mixture "is a culture broth") and 21 :27-28 ( claim 6, reciting that
"the enzymes are produced by a fungus").
FF 2. Laustsen teaches:
The principle we use in the present invention is that an
enzyme normally either is able to tolerate acidic (pH below 5.0)
or alkaline conditions (pH above 9.0). We combine this fact with
the temperature tolerance of the enzyme in question (2°C to
75°C) and the time we hold the enzyme mixture at said
conditions in order to get the optimum conditions where most of
the desirable enzyme is maintained and most of the undesirable
enzyme is inactivated. So the method operates in a system where
three parameters can be varied: the pH, the temperature and the
holding time.
Id. at 3:18-27.
FF 3. In describing undesirable enzymes, Laustsen teaches:
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A very common undesirable class of enzymes is the
proteolytic enzymes (proteases, peptidases) as they are able to
degrade proteins, and as enzymes are proteins, proteolytic
enzymes are able to degrade the desired enzyme.
However, really all enzymes obtained from e.g. animals,
plants or microorganisms, may be undesirable as the industry is
asking for more and more "pure" enzymes, or "mono-
compartment" enzymes, i.e., any enzyme activity but the desired,
is unwanted.
Id. at 4:9--17. Laustsen further teaches that "[a]ny enzyme obtainable from,
e.g., a microorganism, an animal or a plant may be desirable," and that
"[t]he method of the invention is especially useful when the desirable
enzyme is obtainable from a microorganism, an animal or a plant." Id. at
4:24--30.
FF 4. Laustsen teaches:
When having a mixture of enzymes containing a desirable
enzyme and one or more undesirable enzymes, it is advisable
first to look at the pH-curve for the desirable enzyme and see
whether it has optimum to the alkaline or the acidic side. If the
desirable enzyme has optimum to the acidic side, it means that it
is normally not destroyed at a pH below 5.0. It is then
recommended to run a test series combining various pH-values
(e.g. pH 4.5, pH 4.0, pH 3.5) with various temperatures-values
( e.g. 40°C, 50°C, 60°C, 70°C) and various holding times ( e.g. 1
minute, 5 minutes, 60 minutes, 240 minutes). By doing this
optimum conditions can be found where most of the desirable
enzyme is stored and most of the undesirable enzyme(s) are
inactivated.
Id. at 6:23-35. Laustsen teaches ranges for pH, temperature, and holding
times that may be applied and optimized. See id. at 6:36-7: 18 (e.g., pH
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between 3.5-10.0, temperature "in particular in the range of from 10-70°C,"
and holding time "preferably in the range of from 1 minute to 4 hours").
FF 5. Laustsen teaches, in one working example, inactivation of
undesired proteases in a concentrate at low pH leaving a desired lipase
enzyme. Id. at 10: 21-25 (Example I -fungal system/enzyme). This
example describes cloning of Candida antarctica lipase B in an Aspergillus
oryzae host, and obtaining a cell culture broth including the lipase enzyme
that was further subjected to pH and temperature variations according to the
invention in order to inactivate undesired proteases. Id. at 10:23-11: 16.
Laustsen describes that the pH was adjusted to 3.5 at various temperatures
(from 45°-70°C) and "[a]fter various minutes within the interval of 0-60
minutes samples were adjusted to pH=8.0 ... and analyzed for lipase and
protease activity." Id. at 11: 1-3. Laustsen teaches that "treatment at 45°C
for 60 minutes at pH 3 .5 offers a residual yield of 92 % of lipase activity ...
and less than 2 % undesired protease activity." Id. at 11 :7-9; see also id.
Figs. 1-2; see also id. at 11: 10-16 ( describing a treated sample as being
100% stable whereas the untreated sample lost 40% of its initial lipase
activity after 4 days).
FF 6. In another working example, Laustsen teaches "inactivation of
undesired proteases in a concentrate at low pH at low and moderate
temperature leaving the desired cellulase complex from Humicola insolens."
Id. at 16:3-9 (Example 7). In this example, a culture broth containing the
cellulase is obtained, and following a separation and filtration step, the
concentrate was subjected to low pH conditions (e.g., pH 3.0, pH 3.5) for a
holding time of 60 minutes and at various temperatures (3°C and 25°C). Id.
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at 16: 16-17:5 Laustsen teaches "[a]fter said holding time pH in all cases
[was] adjusted to pH 8.0." Id. at 16:27-28; see also id. at 17:1-5 (Table 4,
showing yields of desired cellulase and undesired proteases).
FF 7. Zapata teaches a method for producing antibody fragments
(F(ab')2 fragments), and that the method "comprises concentration of cell
culture media and activation of endogenous enzymes present in the cell
culture media by adjusting the temperature and pH." Zapata Abstract. More
specifically, Zapata describes "enzymatic digestion of the secreted
antibodies by aspartyl proteases, cysteinyl proteases, or a combination of
both types of proteases is initiated by lowering the pH of the cell media to
about pH 3.5 and adjusting the temperature to about 37°C." Id. ,r 12.
FF 8. Zapata teaches "[t]he particular endogenous enzyme that is
activated in the media can be selected by varying the culture conditions." Id.
Zapata teaches that "cysteinyl proteases can be specifically and irreversibly
inhibited ... by increasing the pH of the media to 8.5." Id.
FF 9. In a working example, Zapata teaches the use of CHO cell
cultures, in which the CHO cells were transfected with vectors coding for
the light and heavy chains of a fully human antibody (IgG immunoglobulin).
Id. ,r,r 44--70 (Example 1). 4 Zapata teaches that "[c]ell culture supernatant
was harvested by filtration or centrifugation and passed through a sterile
filter to remove particulate matter and cells prior to being subjected to pH
treatments and activation of enzymatic cleavage." Id. ,r 47. Zapata teaches
that enzymatic digestion of the immunoglobulin was initiated in the cell
4 CHO (Chinese hamster ovary cells) are a mammalian cell line. Spec.
2:27-30.
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culture fluid by adjusting the temperature to 37°C and lowering the pH of
the fluid to approximately 3.5. Id. ,r 49. Zapata teaches that "[a]liquots were
taken at specific intervals [every 0.5 to 1.0 hours] for a time period of 22
hours and adjusted to a pH of 7.0 to stop the digestion." Id. ,r,r 51-52.
FF 10. Further to this working example (FF 9/Example 1), Zapata
teaches enzymatic activity may be controlled to produce a desired antibody
fragment. Id. ,r 63. Zapata teaches that a desired product of the activated
cell culture medium is "the 100 kD F(ab')2 product which is the result of the
aspartyl enzyme activity described above, [whereas] the cysteinyl enzyme
activity which results in the formation of the F(ab')2* fragment can be
prevented by several methods." Id. In one such method, Zapata describes
cysteinyl enzyme in the cell culture media was initially activated
by bringing the media to a low pH for a short period of time,
followed by increasing the pH to 8.5 and incubating for two
hours. After the media was incubated at pH 8.5, irreversible
inactivation of the cysteinyl enzymatic activity was achieved.
The pH of the cell culture media was then lowered to pH 3.5 to
allow for the aspartyl enzyme activity which was not inhibited
by the incubation at pH 8.5. F(ab')2 fragments, without F(ab')2*
fragments, resulted from this incubation.
Id. ,I 64.
Analysis
Claim 1
The Examiner rejected claim 1 as obvious over the combination of
Laustsen and Zapata. Ans. 2-3. The Examiner finds that Laustsen teaches
selective inactivation of enzymes in a culture broth. Id. at 3. The Examiner
finds that Laustsen, in one working example, "teaches inactivation of
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undesired proteases in fungal culture broth, wherein the method includes the
steps of incubating the culture broth at pH 3.5 for 60 minutes at 25°C
followed by adjusting the culture broth to pH 8." Id. According to the
Examiner, Laustsen suggests that the method can be applied to other cell
culture systems ( e.g., animals), and Laustsen teaches that the conditions of
pH, temperature, and holding time are optimizable, results-effective
variables. Id. at 3--4. Because the Examiner finds that "Laustsen does not
reduce to practice inactivation of proteases in a mammalian culture broth,"
the Examiner turns to Zapata. Id. at 4.
The Examiner finds that "Zapata teaches that the activity of proteases
in mammalian culture broth can be activated and inhibited by changes in
pH." Id. (noting, e.g., irreversible inactivation of cysteinyl protease by
adjustment of a mammalian cell culture broth to pH 8.5).
The Examiner concludes it would have been obvious based on
Laustsen and Zapata, "to irreversibly inactivate undesirable proteases in
mammalian culture broth, while retaining desirable enzymes and [ other
protein] products." Id. The Examiner reasons that this method "would
include steps of inactivating undesirable neutral and alkaline protease(s) that
are denatured by acid pH (e.g., pH 3.5, Laustsen) followed by inactivating
undesirable acid protease(s) in the mammalian culture broth that are
denatured by elevated pH (e.g. pH 8, Laustsen; pH 8.5, Zapata)." Id. at 4--5.
The Examiner reasons it would have been obvious "to determine the optimal
conditions for said inactivation, while retaining the activity of the desired
enzymes and products." Id. And, the Examiner reasons, Laustsen provides
a motivation to perform this method because it "teaches the advantage of
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inactivating culture broth proteases in order to reduce proteolysis of the
desired protein(s)," and by further suggesting the application of this method
to animal enzymes. Id.; see also id. at 6 (identifying several supporting
rationales (e.g., "simple substitution of the fungal culture medium of
Laustsen with the mammalian culture medium of Zapata to obtain
predictable result of inactivating low pH- and high pH -sensitive proteases in
mammalian culture medium.").
The Examiner finds that the expectation of success is high because
Laustsen teaches methods for inactivation of proteases in culture broth at
acidic and alkaline pH, and the means to adjust pH, temperature, and time,
thus evidencing that the skilled artisan would be able to readily optimize the
method for the enzyme/protein and cell culture system. Id. at 5---6.
On this record, we agree with the Examiner's reasoning and
conclusion that claim 1 would have been obvious over Laustsen and Zapata.
Laustsen teaches a method with the same two active steps recited in method
claim 1 - a first step where a cell culture liquid is adjusted to a pH of
between 3 to 5, making the liquid more acidic; and a second step where the
cell culture liquid is adjusted to a pH of 7 to 9, making it neutral to alkaline.
FF 1-2. Laustsen further teaches that proteolytic enzymes (proteases) can
be controlled and inactivated by applying these steps based on the
"principle" that acidic enzymes cannot normally tolerate alkaline conditions
and, vice versa, that alkaline enzymes do not tolerate acidic conditions.
FF 2, 4. By inactivating undesired proteases, degradation of protein by those
proteases would be predictably reduced. FF 5---6. Laustsen also discloses
ranges for pH values, temperatures, and holding times for the adjustment
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steps that overlap with the values of those variables recited in claim 1, and
states expressly that these variables are optimizable. FF 2, 4. Laustsen does
not disclose a working example with mammalian cells or proteases, but
Laustsen suggests that its method is likewise applicable to proteins obtained
from animals. FF 3. In any event, Zapata teaches mammalian cell cultures
(CHO) that include mammalian proteins (e.g., fully-human IgG) and
proteases in the extracted cell culture supernatant. FF 9. And, similar to the
principle observed in Laustsen, Zapata teaches that mammalian proteases
can also be activated and inactivated by controlling the conditions of the
culture media (e.g., pH, temperature, etc.). FF 7-9.
Below, we address Appellants' arguments.
Appellants contend that teachings related to a fungal host cell system
and proteases are not applicable to mammalian cells and mammalian
proteases as recited in claim 1. App. Br. 10-12, 14, 17-18; Reply Br. 4--8,
10, 12-13.5 Appellants assert that "proteases produced by fungal cells differ
in significant ways from those made by mammalian cells" and that
"Aspergillus proteases are not irreversibly inactivated at neutral pH, but
rather, [are] simply reversibly inactivated." App. Br. 10. Appellants
contend "the asserted combination of Laustsen and Zapata is silent on the
5 This argument (like others) is spread throughout Appellants' briefing. We
note that Appellants' continuing use of untitled sections labelled "A"
through "I" to repeat portions of earlier remarks and replies and set forth
new contentions did not help define discrete arguments, and instead made it
quite challenging to follow Appellants' contentions on appeal. See App. Br.
passim; Reply Br. passim. (We recognize, however, that the Examiner used
a similar labeling at times in prosecution of this application, and Appellants
may have adopted it with the aim of consistency).
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properties of mammalian cells that are useful in the claimed processes." Id.
at 11. Appellants contend a suggestion to optimize related to fungal systems
does not render obvious the conditions relevant to a mammalian expression
system. Id. at 14. Appellants contend fungal systems can produce inhibitors
for mammalian proteases, revealing a difference in the systems, so this
"forbids conclusions from the fungal to the mammalian protease behavior."
Id. at 17. According to Appellants, fungal proteases and mammalian
proteases "are essentially non-analogous art." Id. at 18. And, Appellants
state, "there is simply no teaching of mammalian cell culture-specific
information in Laustsen, and Zapata - which is directed solely to fungal cell
cultures as well - adds nothing to lead the skilled worker to use any
variation of these methods ... for mammalian cell cultures to irreversibly
inactivate mammalian proteases." Reply Br. 4.
Appellants' argument is unpersuasive. Contrary to Appellants'
contentions, Laustsen suggests that its methods, which are exemplified with
fungal systems/proteases, can be extended to other cell systems and
proteases including those from plants and animals. FF 1, 3; see also
Laustsen 21: 1-9 ( claim 1, reciting a method of inactivating undesirable
enzymes by lowering and raising pH, which is not limited to enzymes
produced by a fungus). Hence, Appellants' assertion that fungal proteases
and mammalian proteases are "non-analogous" is rebutted by Laustsen,
which evidences the opposite. Appellants contend there are differences
between fungal and mammalian proteases. See, e.g., App. Br. 10, 17. But
lacking is evidence sufficient to back up this contention and show how any
purported differences are relevant to the rejection on appeal. Appellants, for
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example, cite no persuasive evidence that fungal proteases are "simply
reversibly inactivated." App. Br. 10. The Examiner, on the other hand,
provides evidence that the proteases in Laustsen are irreversibly inactivated.
See, e.g., Ans. 3, 12; FF 5---6; see also Ans. 25 ( citing Whitaker6 to support
the finding that the pH at which irreversible denaturation of a protease
occurs depends on enzyme structure, not its source). And, even if there are
some differences between proteases, Appellants provide no persuasive
evidence that the "principle" - explained in Laustsen that enzymes tolerate
either acidic or alkaline conditions - does not apply broadly to mammalian
proteases, and that the skilled person would not readily optimize those
conditions consistent with Laustsen's teachings. FF 2--4. 7 Finally,
6 Whitaker et al., Effect of pH on rates of enzyme catalysis, PRINCIPLES OF
ENZYMOLOGY FOR THE FOOD SCIENCES 271-300 (1994) ("Whitaker").
Appellants take issue with the Examiner citing new references (e.g.,
Whitaker) in the Answer. Reply Br. 1. According to Appellants, the use of
these references means that prosecution should be reopened and that
Appellants may assert the Examiner has introduced new grounds of
rejection. Id. at 1-2. Appellants could have sought to reopen prosecution by
following the procedure set forth in 37 C.F.R. § 4I.39(b)(l) if they thought
the Examiner had improperly included new grounds. Appellants did not
and, instead, maintained the appeal by filing a reply brief under§ 41.41. See
37 C.F.R. § 4I.39(b)(2).
7 Appellants' Specification, in suggesting that the described processes also
extend to production organisms other than mammalian cells, provides
further evidentiary support that the principle ( explained in Laustsen) is
broadly applicable. The Specification states that the process for inactivating
acid and neutral proteases by changing the pH twice was carried out with
mammalian cells, and reports ( with no details) that "[ t ]he results can also be
transferred to culture supematants of other production organisms and can be
used within the scope of the requirements of the product protein, particularly
its pH stability." Spec. 6:20-24. Moreover, as the Examiner points out,
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Appellants' assertion that Zapata, like Laustsen, is directed "solely to fungal
cell cultures" is incorrect. Reply Br. 4 (Appellants make similar assertions
throughout the Reply Brief; see, e.g., id. 7, 12). As explained above, Zapata
specifically exemplifies a mammalian cell culture (CHO cells) expressing
human protein. FF 9; see Spec. 8: 16-27 (using CHO production cell lines
and cell culture supernatant from those lines).
Appellants argue that Laustsen and Zapata teach away from the
invention. App. Br. 11-12, 15-17, 19-21. Appellants contend Zapata
"teaches activation and not irreversible inactivation of enzymes." App. Br.
15. According to Appellants, "following the teachings and method of
Zapata would inevitably and necessarily have resulted in activation of
proteases in the mammalian culture fluid, and thus ENHANCED
degradation of proteins in the fluid," which is "CLEARLY a teaching
away." Id. at 16. Appellants assert that Laustsen teaches inactivation of
undesired proteases at low pH, yet "Zapata teaches selectively activating
protease activity to cleave protein product," and thus "[ c ]learly Zapata
claim 1 does not recite any specific mammalian cell, nor does it recite
inactivation of any specific proteases. Ans. 9-1 O; see also id. at 18 ("[T]he
claims do not recite preventing degradation of any specific cell culture
product by inactivating, to any specific extent, any specific mammalian
proteases in a liquid from any specific mammalian cell culture."). The
Specification is also lacking in many details (e.g., little to no identification
of specific proteases that are irreversibly inactivated). Cf In re Epstein, 32
F.3d 1559, 1568 (Fed. Cir. 1994) (noting that a lack of detail in the
Specification may "support[] the Board's finding that one skilled in the art
would have known how to implement the features of the references.").
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suggests the opposite principle of operation, and outcome, from what
Laustsen teaches." Id. at 21.
We disagree. Inasmuch as Appellants contend that Laustsen teaches
away, the basis of such a contention is unclear. If Appellants are suggesting
that Laustsen teaches away because it is purportedly directed to fungal
cells/proteases and does not teach irreversible inactivation of enzymes, we
reject the argument as explained above. 8
Turning to Zapata, contrary to Appellants' assertions, Zapata teaches
both activation and inactivation of proteases. FF 8. Indeed, Zapata teaches
expressly the "irreversible inactivation" of the cysteinyl protease in the
incubated mammalian cell culture media at pH 8.5. FF 8-9. True, Zapata
teaches a first activation step at low pH, but this is not a teaching away for
two reasons. First, claim 1 does not exclude protease activation and, in fact,
Appellants' Specification indicates that the initial step of reducing the pH
according to the method claimed would activate acidic proteases and cleave
protein. See, e.g., Spec. 4:13-14 ("Activated proteases are active at pH< 5
and cleave the substrate"), 5:6-7 ("After acidification of the cell culture
supernatant, activation of the proteases present obviously takes place"), and
10:23-24 ("activation of the acidically active proteases in CCF [ cell culture
fluid] does not take place until the reaction conditions are acidified"). The
claimed method simply addresses acidic activation by, later, making the pH
8 Based on the Specification, it is not required that all protease activity be
eliminated. For example, the Specification indicates that "[a]ctivated
proteases which had been inactivated by a brief incubation at pH 2: 7 exhibit
a residual activity reduced to 35% at pH 3.7." Spec. 4:13-14 and Fig. 6.
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of the liquid neutral to alkaline "whereby" acid proteases are irreversibly
inactivated. App. Br. 23. Second, the Examiner's proposed modification
involves the use of a mammalian cell culture system ( as evidenced in
Zapata) in Laustsen's method of adjusting the pH, temperature, etc. to
inactivate undesired proteases and, thus, preserve and isolate a desired
enzyme/protein. The Examiner is not proposing a wholesale use of Zapata's
method of activating and maintaining a particular type of protein digestion
to produce a particular antibody fragment. FF 7-9. 9 So, the fact that
Zapata's method aims to induce and maintain enzyme activation (by aspartyl
protease, but not cysteinyl protease) to produce a particular type of desired
antibody fragment (F(ab')2, but not F(ab')2*) cleaved from the human
9 Although not decisive in this appeal, we note the following: Zapata
teaches, inter alia, an initial acidification step (pH 3.5, temperature 37°C) of
the CHO cell culture supernatant followed by the removal of an aliquot at 30
minutes that is brought to pH 7 to stop protein (IgG) digestion. FF 9. The
only apparent difference between this process and the actual steps recited in
claim 1 is the temperature (37°C vs. 30°C), but temperature was a known
results-effective variable (FF 4) and the Specification states that the
invention may be carried out at "preferably 20-37 °C." Spec. 6: 17-18.
Zapata does not disclose that neutral and acid proteases are "irreversibly
inactivated" in the removed aliquots, but given that the actual steps are
substantially identical to what is claimed and described in the Specification,
the Office may require Appellants to prove otherwise. In re Best, 562 F .2d
1252, 1255 (CCPA 1977); cf In re Woodruff, 919 F.2d 1575, 1578 (Fed.
Cir. 1990) ("It is a general rule that merely discovering and claiming a new
benefit of an old process cannot render the process again patentable.").
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antibody does not teach away from using a mammalian cell culture in
Laustsen' s method. 10
Appellants contend that the Examiner has not given patentable weight
to the preamble of claim 1 and/or it appears the Examiner is improperly
relying on inherency. App. Br. 11-12; Reply Br. 3. Appellants "assert[]
that their invention irreversibly inactivates mammalian proteases." Reply
Br. 3.
Even if the preamble should be given weight in reciting a "process for
irreversibly inactivating mammalian proteases," we are unpersuaded the
Examiner has ignored it. App. Br. 23. Quite the opposite, the Examiner
reasons that a process for irreversibly inactivating mammalian enzymes
would have been obvious based on the combined teachings in Laustsen and
Zapata. Ans. 9-10. As explained above, we are persuaded on this record
that Laustsen teaches irreversible inactivation of proteases in a fungal cell
culture system, and that the skilled artisan could have, and would have,
extended Laustsen's teachings to mammalian cell culture systems and
proteases - optimizing to the extent necessary to irreversibly inactivate
those proteases to avoid proteolysis of a desired protein. FF 2--4.
As to inherency, the Examiner points out that the claims do not recite
inactivation of any specific protease(s), and the Examiner finds that at least
one or more neutral and acid proteases would be irreversibly inactivated by
subjecting a mammalian cell culture medium to acidic and then alkaline
1° For similar reasons, we are not persuaded that the proposed modification
of the prior art changes the principle of operation of Laustsen as suggested
by Appellants. App. Br. 20-21.
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conditions consistent with Laustsen's and Zapata's teachings. Ans. 10-11.
As already explained above, at least Zapata teaches that cysteinyl protease is
irreversibly inactivated at an alkaline pH. FF 8. And, in further support, the
Examiner cites several references as evidencing irreversible inactivation of
other ubiquitous mammalian proteases (e.g., Turk, 11 as showing irreversible
inactivation of cathepsin Lat acidic pH). 12 See, e.g., Non-Final Act. 5; App.
Br. 7 ( describing Turk as "teach[ing] irreversible inactivation of cathepsin L
at acid pH."). Appellants assert that "many if not most proteases can be
irreversibly inactivated at destructive pHs" (App. Br. 18), yet provide no
persuasive argument or evidence showing why the Examiner's findings
(insofar as they do invoke inherency) are incorrect.
Appellants argue "the claimed invention achieves the surprising
results of irreversibly inactivating proteases rather than merely reversibly
inactivating them." App. Br. 12, 14.
We remain unpersuaded. As already explained, and as pointed out by
the Examiner, the prior art teaches irreversible inactivation of undesired
proteases in a culture broth (both mammalian and fungal). Ans. 12. And at
least Laustsen teaches the variables (pH, temperature, and time) that should
be optimized to inactivate undesired proteolytic enzymes. FF 1--4. If
11 Boris Turk et al., Acidic pH as a physiological regulator of human
cathepsin L activity, 259 EUR. J. BIOCHEM. 926-32 (1999).
12 The evidence indicates that cathepsin L is a protease that is ubiquitous in
mammalian cells, and is also found secreted outside the cells. Spec. 5:6-14
( describing as "ubiquitously expressed in all tissues"); Turk 926 ("Cathepsin
L is ubiquitous in mammalian cells, and all species variants studied have
similar properties").
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anything, on this record, the results - including reducing protein
degradation by irreversibly inactivating proteolytic enzymes - would have
reasonably been expected. In re Skoner, 517 F.2d 947,950 (CCPA 1975)
("Expected beneficial results are evidence of obviousness of a claimed
invention."). Appellants fail to provide persuasive argument, evidence, or
data commensurate in scope with the claims to show why the method was
"surprising" in any way. App. Br. 12.
Finally, Appellants contend the Examiner does not give adequate
reasons for combining Laustsen and Zapata, and that the skilled person
would have had no expectation of success in doing so to produce the claimed
invention. App. Br. 17-20. According to Appellants, the Examiner "does
not provide reasons or rationale for the unsupported assertion that the
claimed methods, comprising two subsequent inactivation steps at different
conditions, would have been obvious in view of the combination of the two
references." Id. at 18. And, Appellants urge, the rejection is based on an
improper "hindsight reconstruction." Id. at 19 ("there are no blazemarks for
the specific ranges taught by either Laustsen or Zapata").
These contentions are unpersuasive largely for reasons already
explained above. A reason for extending Laustsen's teachings to animal
proteases comes from Laustsen itself. FF 3. Moreover, Laustsen teaches -
indeed claims - a process with two inactivation steps at pH values and
temperature ranges and times that encompass or abut the values of those
variables as recited in claim 1. FF 1, 5---6. Also, Laustsen teaches it was
known and within the skill of the ordinary artisan to optimize these variables
for the undesired protease and desired protein. FF 4. Laustsen supplies a
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reasonable expectation of success, particularly when paired with Zapata,
which teaches the activation and inactivation of proteases in mammalian cell
culture (CHO) by adjusting temperature and pH consistent with the
"principle" spelled out in Laustsen. FF 2, 7-9. Pfizer, Inc. v. Apotex, Inc.,
480 F.3d 1348, 1364 (Fed. Cir. 2007) ("[T]he expectation of success need
only be reasonable, not absolute.").
For all the above reasons, we conclude that the preponderance of the
evidence supports the Examiner's rejection of claim 1 as obvious over
Laustsen and Zapata.
Claim 2
Although Appellants state, in a section of their Brief summarizing the
claimed subject matter, that "claims 1 and 2 stand and fall independently of
one another" (App. Br. 5), Appellants provide no specific and separate
argument related to claim 2. We agree with the Examiner on this record that
claim 2 also would have been obvious over Laustsen and Zapata, and find
Appellants' contentions unpersuasive for the reasons explained above.
Conclusion of Law
The preponderance of the evidence on this record supports the
Examiner's conclusion that claims 1 and 2 would have been obvious.
Claims 3, 6-8, 10, 11, 14, and 16-30 have not been argued separately and
therefore fall with claim 1. 37 C.F.R. § 4I.37(c)(l)(iv).
SUMMARY
We affirm the rejection of the claims on appeal for obviousness.
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TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 3 7 C.F .R. § 1.13 6( a )(1 )(iv).
AFFIRMED
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