Ex Parte Dave et al

Patent Trial and Appeal Board

Aug 28, 2018

13390094 (P.T.A.B. Aug. 28, 2018)

UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR
13/390,094 02/10/2012
21186 7590 08/30/2018
SCHWEGMAN LUNDBERG & WOESSNER, P.A.
P.O. BOX 2938
MINNEAPOLIS, MN 55402
UNITED ST A TES OF AMERICA
Nitesh Dave
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.
4129.019US1 1195
EXAMINER
BRADLEY, CHRISTINA
ART UNIT PAPER NUMBER
1675
NOTIFICATION DATE DELIVERY MODE
08/30/2018 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@slwip.com
SLW@blackhillsip.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte NITESH DA VE,
KRISHANA CHAIT ANY A GULLA,
SUND ARE SH SHANKAR, and HARISH IYER 1
Appeal2016-007333
Application 13/390,094
Technology Center 1600
Before JOHN G. NEW, TA WEN CHANG, and DAVID COTTA,
Administrative Patent Judges.
CHANG, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134(a) involving claims to a
chromatographic process for purification of a polypeptide, which have been
rejected as obvious. We have jurisdiction under 35 U.S.C. § 6(b ).
We AFFIRM.
STATEMENT OF THE CASE
Reversed-phase high-performance liquid chromatography
(RP-HPLC) involves the separation of molecules on the
basis of hydrophobicity. The separation depends on the
1 Appellants identify Biocon Limited as the real party in interest. (Appeal
Br. 2.)
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Application 13/390,094
hydrophobic binding of the solute molecule from the
mobile phase to the immobilized hydrophobic ligands
attached to the stationary phase, i.e., the sorbent. ... The
solute mixture is initially applied to the sorbent in the
presence of aqueous buffers, and the solutes are eluted by
the addition of organic solvent to the mobile phase.
Elution can proceed ... by gradient elution whereby the
amount of organic solvent is increased over a period of
time. The solutes are, therefore, eluted in order of
increasing molecular hydrophobicity.
( Aguilar 9. 2)
The Specification states that "usage of ion pairing agents ... in ...
analytical method development and its effect on protein peak resolution is
well known." (Spec. 1.) In particular, the Specification explains that, when
used in RP-HPLC ion pairing agents such as trifluoroacetic acid (TFA)
adsorb to the stationary phase via a hydrocarbon chain and also paired with
the surface charges of a protein solute to increase the hydrophobicity of the
protein. (Id. at 1-2.) The Specification further explains that, because the
interaction of the ion pairing agent and protein depends on the surface
charges of the protein, which differ among different proteins in a mixture,
usage of the ion pairing agents results in differential binding of proteins in a
mixture to the stationary phase, which in tum improves the resolution
between the protein peaks as the proteins are eluted. (Id. at 2.)
According to the Specification, "[h ]igher loading on the column
beyond the protein injected on the column for analytical detection[] is
crucial for process development and dictates the cost and yield of a given
2 Marie-Isabel Aguilar, Reversed-Phase High-Performance Liquid
Chromatography, 251 METHODS IN MOLECULAR BIOLOGY, HPLC OF
PEPTIDES AND PROTEINS: METHODS AND PROTOCOLS 9 (M.-I. Aguilar ed.
2004).
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process." (Id. at 2.) Further according to the Specification, the disclosures
in the Specification show that "ion pairing agents have dramatic effect in the
purity of proteins even after the protein loading was increased" and
"demonstrates the use of ion pairing in improving yield of the
chromatographic step." (Id.)
Claims 1-7 and 15-19 are on Appeal. 3 Claim 1 is illustrative and
reproduced below:
1. A chromatographic process for purification of a
polypeptide from a mixture having at least one related
impurity, at a pH ranging from about 2.5 to about 8.5,
said process comprising steps of:
packing RP-HPLC column with silica (C4-C18)
based resin, equilibrated with about 5% to about 85 %
organic modifier;
loading the polypeptide mixture on the column at a
flow rate of about 180 cm/hr to about 360 cm/hr;
washing the column with an ion pairing agent
having a concentration ranging from about 0.05% to
about 1 % in combination with the organic modifier
having a concentration ranging from about 5% to about
85%, at pH ranging from about 2.5 to about 8.5 and;
performing a linear gradient of about 10% to about
70% of the organic modifier for eluting a purified
polypeptide from the column, wherein the polypeptide is
an insulin analogue of a purity ranging from about 90%
to about 100% after a single chromatographic process.
(Appeal Br. 15 (Claims App.).)
3 Claims 13 and 14 have been withdrawn. (Appeal Br. 4.)
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The Examiner rejects claims 1-7 and 15-19 under pre-AIA 35 U.S.C.
§ 103 (a) as being unpatentable over Bhopale, 4 Mills, 5 Chen, 6 Chance,7
Klyushnichenko, 8 Romanchikov, 9 Aguilar, and Grace Vydac. 10 (Ans. 2.)
DISCUSSION
Issue
The Examiner finds that Bhopale teaches purification of recombinant
insulin analogues "involv[ing] 2--4 chromatographic steps selected from
reverse phase, size exclusion chromatography, hydrophobic interaction,
4 G. M. Bhopale et al., Recombinant DNA expression products for human
therapeutic use, 89 CURRENT SCIENCE 614 (2005) (hereinafter "Bhopale").
5 Janine B. Mills et al., One-step purification of a recombinant protein from
a whole cell extracy by reversed-phase high-peiformance liquid
chromatography, 1133 J. CHROMATOGRAPHY A 248 (2006). Page citations
herein are to author manuscript version available in PMC 2009 August 6
(hereinafter "Mills").
6 Yuxin Chen et al., Preparative reversed-phase high-peiformance liquid
chromatography collection efficiency for an antimicrobial peptide on
columns of varying diameters (1mm to 9.4 mm I.D.), 1140 J.
CHROMATOGRAPHY A 112 (2007). Page citations herein are to author
manuscript version available in PMC 2009 October 8 (hereinafter "Yuxin").
7 Chance et al., US 5,700,662, issued Dec. 23, 1997 (hereinafter "Chance").
8 V.E. Klyushnichenko et al., Recombinant human insulin V optimization of
the reversed-phase high-performance liquid chromatographic separation,
662 J. CHROMATOGRAPHY B. 363 (1994) (hereinafter "Klyushnichenko").
9 A.B. Romanchikov et al., Human recombinant insulin. VII. Improvement
of chromatographic separation efficiency using the principle of
bifunctionality, 23 BIO-ORGANIC CHEMISTRY 98 (1997) (hereinafter
"Romachikov"). Page citations herein are to version in the record,
apparently obtained via Infotrieve.
lO Grace Vydac, THE HANDBOOK OF ANALYSIS AND PURIFICATION OF
PEPTIDES AND PROTEINS BY REVERSED-PHASE HPLC (3d ed. 2002)
(hereinafter "Grace Vydac").
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charge transfer, ion exchange and affinity chromatography." (Ans. 2.) The
Examiner finds that Chance, Klyushnichenko, and Romanchikov teach "the
use ofRP-HPLC [reverse phase high-performance liquid chromatography]
to purify insulin and its analogues, albeit in multi-step procedures." (Id. at
6.)
The Examiner finds that Bhopale, Chance, Klyushnichenko, and
Romanchikov do not teach a purification protocol for insulin analogues
comprising RP-HPLC as the single chromatographic process. (Id. at 3, 6.)
However, the Examiner finds that "Mills teaches 'a general one-step facile,
flexible and readily scalable purification method for ... recombinant
proteins[] based on RP-HPLC."' (Id. at 3.) The Examiner also finds that
Chen teaches that "the slow acetonitrile gradients (0.1---0.2% CH3CN/min)
developed by Mills for single step purification of recombinant proteins can
also be used for single step purification of peptides from mixtures of close-
related impurities." (Id. at 4.)
The Examiner further cites Aguilar and Grace Vydac as evidence of
the "high level of knowledge regarding the use of RP-HPLC for protein
purification." (Id. at 7.)
The Examiner concludes that a skilled artisan would have found it
obvious to combine the cited prior art to arrive at the claimed invention. (Id.
at 8.) In particular, the Examiner finds that a skilled artisan would have
been motivated to modify Bhopale's method with the single-step RP-HPLC
purification protocol taught by Mills and Chen, with a reasonable
expectation of success, because Mills teaches that such single-step protocols
are superior to the multi-step protocols taught by Bhopale in, e.g.,
purification time and yield, because Mills teaches that the one-step RP-
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HPLC protocol is a "facile, flexible and readily scalable purification method
for proteins, specifically recombinant proteins," and because Mills teaches
purifying a recombinant protein to 94% purity in a single step from a crude
cell lysate. (Id. at 8.)
As to the specific RP-HPLC conditions recited in the claims, the
Examiner finds that such conditions are either disclosed by the prior art
and/or are obvious to optimize through routine experimentation. (Id. at 9--
10.) For instance, the Examiner finds that Mills, Chen, Chance,
Klyushnichenko, Aguilar, Grace Vydac, and Romanchikov all teach silica
based C8 resin; that Chance, Klyushnichenko, and Romanchikov all disclose
purifying insulin analogues by RP-HPLC using an organic modifier
(acetonitrile) in 0.1-2% ion pairing agent (trifluoroacetic acid (TFA)) at a
pH within the claimed range of about 2.5 to about 8.5; that Mills and Chen
teach a method of determining concentrations of acetonitrile required to
elute a protein of interest, which may then be used to determine the
concentration of acetonitrile in "the column equilibration mobile phase, ...
the wash step, and ... the elution gradient;" and that Aguilar and Grace
Vydac show a skilled artisan would have been able to "optimize both the
concentration of organic modifier and ion pairing agent to achieve optimal
purification" as well as the sample flow rate. (Id. at 9--10.)
Appellants contend that a skilled artisan at the time of the invention
"would not have expected to succeed in purifying insulin or its analogs in
one step, to achieve the claimed level of purity." (Appeal Br. 8, 10-11.)
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Appellants do not separately argue the claims. 11 Accordingly, we
limit our analysis to claim 1 as representative. 3 7 C.F .R. § 41.3 7 ( c )( 1 )(iv).
The issue with respect to this rejection is whether a skilled artisan would
have had a reasonable expectation of successfully combining the cited prior
art to arrive at the invention of claim 1.
Findings of Fact
1. Aguilar teaches that RP-HPLC allows for "excellent resolution
... under a wide range of chromatographic conditions for very closely
related molecules." (Aguilar 9.)
2. Aguilar teaches that
[ t ]he RP-HPLC experimental system for the analysis of
peptides and proteins usually consists of an n-alkylsilica-
based sorbent from which the solutes are eluted with
gradients of increasing concentrations of organic solvent
such as acetonitrile containing an ionic modifier such as
trifluoroacetic acid (TF A) (1,2). Complex mixtures of
peptides and proteins can be routinely separated and low
picomolar-femtomolar amounts of material can be
collected for further characterization. Separations can be
easily manipulated by changing the gradient slope, the
11 Appellants purport to separately argue Claim 2, which depends from
claim 1 and further requires that the insulin analogue be "selected from the
group consisting of Aspart, Lispro, Glargine and any combination thereof."
(Appeal Br. 4, 13, and 15 (Claims App.).) However, Appellants' argument
with respect to claim 2 in the Appeal Brief consists solely of a statement that
"the references do not render [obvious] a process geared to purify the
specific insulin analogs Aspart, Lispro, Glargine, and combinations thereof,
in a single chromatographic process." (Id. at 13.) Proper separate argument
of a claim requires "more ... than a mere recitation of the claim elements
and a naked assertion that the corresponding elements were not found in the
prior art." In re Lovin, 652 F.3d 1349, 1357 (Fed. Cir. 2011). Furthermore,
as the Examiner points out, Chance discloses purifying Lispro using a
chromatographic process. (Ans. 6.)
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operating temperature, the ionic modifier, or the organic
solvent composition.
(Id. at 10; see also id. at 13 (describing the "wide range of operating
parameters" that may be changed to manipulate the resolution of protein
mixtures in RP-HPLC), 17 (teaching that solute retention and resolution may
be manipulated through changes in the composition of the mobile phase and
that proteins are routinely eluted by a gradient of increasing organic solvent
concentration in RP-HPLC), 20 (choice of gradient conditions depend on
nature of molecule of interest).)
3. Aguilar teaches that
RP-HPLC is extremely versatile for the isolation of
peptides and proteins from a wide variety of synthetic or
biological sources and is used for both analytical and
preparative applications .... Preparative RP-HPLC is ...
used for . . . large-scale purification of synthetic peptides
and recombinant proteins .... The isolation of proteins
from a biological cocktail derived from a tissue extract or
biological fluid for example, often requires a combination
of techniques to produce a homogenous sample. HPLC
techniques are then introduced at the later stages following
initial precipitation, clarification, and preliminary
separations using soft gels.
(Id. at 11 ( citations omitted).)
4. Aguilar teaches C 18-, C4-, and CS-based sorbents are
commonly employed in RP-HPLC analysis of peptides and proteins. (Id. at
13-14.)
5. Aguilar teaches that RP-HPLC is "generally carried out with an
acidic mobile phase." (Id. at 17 .)
6. Agular teaches that different flow rates may be used for
columns of different sizes in RP-HPLC. (Id.)
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7. Grace Vydac teaches that RP-HPLC "has become a widely
used, well-established tool for the analysis and purification of biomolecules"
because it is able to "separate polypeptides of nearly identical sequences, not
only for small peptides ... but even for much larger proteins." (Grace
Vydac 2.)
8. Grace Vydac teaches that "[p ]olypeptides which differ by a
single amino acid residue can often be separated by RP-HPLC" and that
"most variants [ of insulin] can be separated by RP-HPLC" even though
"[i]nsulin variants have molecular weights of around 5,300 with only
slightly different amino acid sequences." (Id.)
9. Grace Vydac teaches using RP-HPLC to separate rabbit and
human insulin, which differs only by a single amino acid, using a silica C4
column and an eluent of 27-30% acetonitrile in 0.1 % TF A over 25 minutes
at 1.5 mL/minute. (Id.)
10. Grace Vydac teaches that, in RP-HPLC, polypeptides adsorb to
the surface of the hydrophobic stationary phase after entering the HPLC
column and remain adsorbed until the concentration of organic modifier
reaches the critical concentration necessary to cause desorption. (Id. at 4.)
More particularly, Grace Vydac teaches that
[ t ]he desorption and elution of polypeptides from RP
HPLC columns is accomplished with aqueous solvents
containing an organic modifier and an ion-pair reagent or
buffer. The organic modifier solubilizes and desorbs the
polypeptide from the hydrophobic surface while the ion-
pair agent or buffer sets the eluent pH and interacts with
the polypeptide to enhance the separation. Elution is
accomplished by gradually raising the concentration of
organic solvent during the chromatographic nm (solvent
gradient). When the solvent reaches the precise
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concentration necessary to cause desorption, the
polypeptide is desorbed and elutes from the column.
(Id. at 17.)
11. Grace Vydac teaches that "[ t ]he sensitivity of polypeptide
desorption to precise concentrations of organic modifier accounts for the
selectivity of RP-HPLC in the separation of polypeptides." (Id. at 5; see
also id. at 6 (teaching that "polypeptides are very sensitive to organic
modifier concentration).)
12. Grace Vydac teaches that gradient elution is usually preferred
over isocratic elution in RP-HPLC and that "[s]lowly raising the
concentration of organic solvent results in the sharpest peaks and best
resolution." (Id. at 6, 18 ( emphasis omitted).) Grace Vydac recommends
"beginning gradients at no less than 3 to 5% organic modifier concentration"
and "ending gradients at no more than 95% organic modifier." (Id. at 18.)
13. Grace Vydac teaches that TFA is widely used as an ion-pairing
agent and that it is normally used at concentrations of about 0.1 % (w/v). (Id.
at 19. Grace Vydac further teaches that,
[ a ]lthough TF A is typically present in the mobile phase at
concentrations of 0.05% to 0.1 %, varying the
concentration of TF A has a subtle [ e ]ffect on peptide
selectivity .... This means that, for good reproducibility,
it is important to control the TF A concentration very
carefully in peptide separation methods. This provides
another tool for optimizing peptide resolution. After the
column and gradient conditions have been selected, it is
possible to vary the TF A concentration slightly to further
optimize resolution between peptide pairs.
(Id. at 20.)
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14. Grace Vydac teaches that "[p ]eptide separations are often
sensitive to the eluent pH" and that pH "can be a useful tool in optimizing
peptide separations." (Id. at 22.)
15. Grace Vydac teaches that, while protein resolution is relatively
independent of mobile phase flow rate, flow rate may influence aspects of a
separation such as detection sensitivity, sample solubility, column back-
pressure, and gradient slope and shape. (Id. at 24--25.)
16. Grace Vydac teaches that "[p]reparative RP-HPLC is frequently
used to purify synthetic peptides in milligram and gram quantities." (Id. at
3; see also id. at 40 (explaining that RP-HPLC is used to purify microgram
to milligram quantities of polypeptides for research purposes as well as to
purify up to gram quantities of recombinant proteins for use in clinical trials
or for marketed products).)
1 7. Grace Vydac teaches that scaling up laboratory separations to
process scale may involve increasing the size of the column and elution flow
rate, a change in elution solvents, use of different ion-pairing agents or
buffers, and/or a change in gradient conditions. (Id. at 40; see also id. at 42.)
18. Bhopale teaches that "[ r ]ecombinant DNA (rDNA) technology
has made a revolutionary impact in the area of human healthcare by enabling
mass production of safe, pure and effective rDNA expression products" and
that, "[ c ]urrently, several categories of rDNA products, viz. hormones of
therapeutic interest ... are being produced using rDNA technology for
human use." (Bhopale Abstract (emphasis omitted).)
19. Bhopale teaches a general process for purification ofrDNA
products. (See, e.g., id. at Fig. 2.) Bhopale teaches that, while "[t]here are
several different methods that can be used for purification of rDNA
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products,[] only chromatographic purification methods are generally used."
(Id. at 616.) Bhopale teaches that "[a] combination of two to four different
chromatographic techniques is generally employed in a typical downstream
processing procedure." (Id.) Bhopale teaches reversed phase (RP)
chromatography as one of the chromatographic purification methods used
for rDNA products. (Id. at 616 (Table 1).)
20. Bhopale teaches human insulin, insulin aspart, insulin glargine,
insulin lispro, and insulin glulisine as some of the "important rDNA
products ... approved by FDA for human therapeutic use." (Id. at 617
(Table 2); see also id. at 618---619 ( describing use of human insulin produced
using S. cerevisae or E. coli to treat diabetes mellitus).)
21. Romanchikov studies the retention mechanisms of insulin and
an analog in RP-HPLC. (Romanchikov Abstract.)
22. Romanchikov teaches that separation selectivity and resolution
of these proteins were dependent on the composition and properties (e.g.,
salt buffer type, pH, ionic strength) of the mobile phase and also teaches that
separation selectivity may be "regulated by altering the contribution of each
of the two separation mechanisms," (i.e., interaction with the hydrophobic
and the ion-exchange groups on the silica gel). (Id.; see also id. at 3, 7, 9--
10, and Fig. 3.)
23. Romanchikov teaches that the concentration and size of
hydrophobic section of the ion pairing agent also influence insulin retention.
(Id. at 7.)
24. Romanchikov illustrates the dependence of insulin retention
coefficient on salt concentration using a column with silica (C8) based resin
(Armsfer-Si-100 Cs (PR)) and mobile phases comprising an organic
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modifier and ion pairing agent (20% acetonitrile (CH3CN) and 0.3% TFA, or
16.2% acetonitrile and 0.1 % TFA). (Id. at Fig. 4.)
25. Romanchikov illustrates the dependence of the insulin retention
coefficient on ion pairing agent concentration in mobile phase using a
column with silica (C8) based resin (Armsfer-Si-100 Cs (PR)) and mobile
phases comprising an organic modifier (16.6% or 22.4% acetonitrile) and an
ion pairing agent (0.1 o/o--0.5% TFA or heptafluorobutyric acid (HFBA)).
(Id. at Fig. 5.)
26. Romanchikov illustrates the dependence of the insulin retention
coefficient on organic modifier concentration using a column with silica
(C8) based resin (Armsfer-Si-100 Cs (PR)) and mobile phases comprising
acetonitrile as the organic modifier and 0.1, 0.3, or 0.5% TFA or HFBA as
ion pairing agent. (Id. at Fig. 6.)
27. Romanchikov teaches separation of insulin and its analog
(deamido insulin) using a column with silica (C8) based resin (Armsfer- Si
- 100 Cs (PR)) in gradient mode. (Id. at Fig. 7.)
28. Romanchikov teaches that"[ o Jptimal parameters of reverse-
phase system were found for effective separation of insulin and deamido
insulin on bifunctional sorbent both for analysis as well as for preparative
obtaining of highly purified insulin" and further teaches that "[k ]nowing the
interaction mechanisms of proteins with mobile and stationary phases has
given new possibilities to control the main characteristics of
chromatographic process - separation selectivity coefficient and resolution
capability of system[-] by changing the parameters of mobile and stationary
phases." (Id. at 10.)
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29. Klyushnichenko relates to the applicability of RP-HPLC to
analysis of the products of recombinant insulin and investigates the
"influence of several mobile phases in reversed-phase ... HPLC on
selectivity, resolution and sensitivity." (Klyushnichenko Abstract; see also
id. at 364, left column.)
30. Klyushnichenko teaches establishing optimum conditions for
separation of insulin-related proteins on commercial and laboratory-made
supports via optimization of selectivity and resolution as a function of pH
and ionic strength. (Id.; see also id. at 366-367, and 368-369 (optimization
for scaling up).)
31. Klyushnichenko teaches that, "[i]n spite of small differences in
the structure and sequence of animal and human insulin, RP-HPLC ensures
the separation of these proteins and their derivatives and precursors. The
flexibility of this method is demonstrated by the quality of the separation of
insulin, proinsulin and desamidinsulin peaks." (Id. at 363, right column
( citations omitted).)
32. Klyushnichenko teaches that "HPLC is an effective method not
only for analytical separations, but also for the preparative isolation of
insulin with high purity." (Id. at 364, left column.)
33. Klyushnichenko illustrates separation of recombinant insulin by
HPLC using various columns with silica based resin (C4-C18). (Id. at 364,
left column.)
34. Klyushnichenko illustrates separating insulin and its analog
using mobile phases comprising 10% acetonitrile-0.1 % TF A and 100%
acetonitrile-0.1 % TFA. (Id. at Fig. 2, Fig. 5.)
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35. Klyushnichenko concludes that its proposed techniques
"ensures a high quality of active insulin production." (Id. at Abstract.)
3 6. Chance teaches "[ r ]ecombinant processes for preparing insulin
analogs modified at position 29 of the B-chain and, optionally, at other
positions." (Chance Abstract.)
3 7. Chance teaches that a particularly preferred insulin analog of its
invention is "one wherein B28 is lysine and B29 is proline, i.e., an inversion
of the native human insulin amino acid sequence at positions 28 and 29 of
the B-chain." (Id. at 3:49-52; see also id. at Example 1 (Lys(B28),
Pro(B29) Human Insulin).)
38. Chance teaches purifying a sample of an insulin analog
(Lys(B28), Pro(B29) human insulin) as follows:
Th[ e] sample was further purified by reverse-phase HPLC
(using a 2.12x25 cm DuPont C8 column eluted at room
temperature, at 2.6 ml/min, using a linear gradient of
increasing acetonitrile in O. IM NaH2P04, pH 2.2). The
effluent was monitored at 2 7 6 nm. Selected fractions were
assayed by analytical HPLC. The desired fractions were
pooled and further purified using pH 7 HPLC as follows.
The pool from the low pH HPLC preparation run
was diluted approximately 2 times in an ice bath with
O. IM (NH4)2HP04. The pH was adjusted to 7 with cold
2NNaOH in an ice bath. The sample was loaded onto and
eluted from the same HPLC column using the same
conditions as the low pH preparation run except the eluting
buffer was O. IM, pH 7 (NH4)2HP04/acetonitrile.
The pool from the pH 7 HPLC preparative run was
chilled in an ice bath and diluted two times with 0.1 %
aqueous trifiuoroacetic acid (T'FA). IN HCl was added
(cold, sample in ice bath) to lower the pH to 3. The sample
was loaded onto a Vydac C4 or, alternatively a DuPont C8
HPLC column (2.12x25 cm) and eluted with a linear
gradient of increasing acetonitrile in 0.1 % aqueous TFA.
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The effluent was monitored at 214 nm or 276 nm. The
desired fractions were pooled and lyophilized giving a
sample yield of 41 mg of the desired analog of greater than
97 percent purity by reverse-phase HPLC.
(Id. at 9: 1-25.)
39. Chance also teaches eluting an insulin analog using linear
gradient of increasing acetonitrile in 0.1 % or 0.5% TF A using a C-8
Ultrasphere HPLC column or a C-18 Vydac column. (See e.g., id. at 9:55-
59, 10:24--28, 10:50-54, 11:8-14, 11:34--38, 11:61---63, 13:12-21, 13:51-55,
14:10-14, 14:36-40, 14:62---66, 15:20-24, 15:46-50, 16:4--8, 16:29-33,
16:54--58, 17: 11-15, 17:37--41, 17:63---67, 18:22-26, 18:48-52, 19:6-10,
19:29-33, 19:54--58, 20: 11-15, 42:51-56, 43:61---65, 44:40--45, 46: 1---6; see
also id. at 43:29--41 (purifying insulin analog using C-18 Vydac HPLC
column, washing with a buffer consisting of 10 parts acetonitrile and 90
parts 0.5% TFA solution (buffer A), applying a linear gradient from 0-20%
of a buffer consisting of 50 parts acetonitrile and 50 parts 0.5% TFA (buffer
B) at 2.4 ml/min, and applying a linear gradient of 20-70% of buffer Bat 8
ml/ min).)
40. Mills teaches that
both affinity and immunoaffinity chromatography
techniques are often critical in developing purification
procedures for recombinant proteins, often as the first step
in a multicolumn purification approach. . . . Although
multicolumn protocols (generally employing
combinations of affinity, ion-exchange and size-exclusion
chromatography) are effective[], complications remain
... leading to an increase in purification time as well as a
concomitant loss of purified sample yield. Further, even
if a one-step affinity approach to purification of a protein
on a small scale was favoured, scale-up to larger sample
amounts may become prohibitively expensive.
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(Mills 1 ( citations omitted).)
41. Mills teaches "a one-step facile, flexible and readily scalable
purification method for a recombinant protein, TM 1-99 ( 113 amino acid
residues; 12,837 Da) based on reversed-phase high-performance liquid
chromatography (RP-HPLC) from an E. coli cell lysate." (Mills Abstract.)
42. Mills describes carrying out its method on a Zorbaz 300SB-C8
column. (Id. at 2.)
43. Mills teaches performing analytical and preparative RP-HPLC
under the following conditions:
2.4.1. Analytical RP-HPLC-Analytical runs and fraction analyses
were carried out by a linear AB gradient (1 % B/min for analytical
profiles and 2% B/min for fraction analysis) at a flow-rate of 0.3
ml/min where Eluent A is 0.05% aq. TFA and Eluent B is 0.05% TFA
in acetonitrile; temperature, 25 °C. TF A concentrations of 0.05---0.1 %
are characteristic of most separations of peptides and proteins by RP-
HPLC (for both analytical and scale-up purposes[)]. ....
2.4.2. Preparative RP-HPLC-Preparative purification was carried
out by a linear AB gradient (Eluent A is 0.05% aq. TFA, pH 2.0, and
Eluent Bis 0.05% TFA in acetonitrile) of 2% B/min up to 24% B,
followed by a slow (0.1 % B/min) gradient up to 40% B. A rapid rise
(4% B/min) up to 60% B was then followed by an isocratic wash with
60% B .... Flow-rate, 0.3 ml/min; temperature, 25 °C
(Id. at 3.)
44. Mills teaches that "[a] key advantage of [its] slow gradient
approach is the high sample loading concentrating the desired material on
the column, thus aiding its separation from closely adjacent impurities." (Id.
at 4.) Mills further teaches that another advantage of its method is that "the
slow gradient spreads the desired product over a large number of fractions,
the bulk of which would contain purified product only, with just the first and
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last product fractions containing hydrophilic and hydrophobic impurities,
respectively." (Id.)
45. Mills teaches a "'rule of thumb' approach whereby the shallow
gradient is started 15% below the acetonitrile concentration required to elute
the protein of interest in an analytical run can be applied as a general rule of
thumb for polypeptides of IO-residue length and longer." (Id.) Mills
teaches that this rule is based on the fact that "peptides and proteins exhibit
only narrow partitioning windows," has the advantage of being able to
separate a large range of sample loads, and "is a good compromise between
the time taken for efficient product purification and obtaining maximum
sample load, i.e., a lower or greater starting acetonitrile concentration would
extend the run time or decrease maximum sample load, respectively." (Id. at
4--5.)
46. Mills teaches that, using its method, "[l]oads of 23 and 48 mg
of lyophilized crude cell extract produced 2.4 and 4.2 mg of purified product
(>94% pure), respectively." (Id. at Abstract.) Mills states that its results
"show the excellent potential of one-step RP-HPLC for purification of
recombinant proteins from cell lysates, where high yields of purified product
and greater purity are achieved compared to affinity chromatography." (Id.)
47. Mills concludes that "a one-step RP-HPLC slow gradient
approach shows excellent potential for purification of recombinant proteins
from cell lysates, where high yields of purified product are achieved
compared to affinity chromatography." (Id. at 6.)
48. Chen teaches that "[t]o obtain antimicrobial peptides, peptide
synthesis is the favored method especially for de nova antimicrobial peptide
design," but "[t]he impurities created during peptide synthesis are usually
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closely related structurally to the peptide of interest and hence often pose
difficult purification problems." ( Chen 1; see also id. at 4 ( describing
antimicrobial peptide used in the study and stating that "both hydrophilic
and hydrophobic impurities are present in the crude peptide mixture, a
number of which are eluted close to the peptide of interest").)
49. Chen teaches that
[t]he excellent resolving power of reversed-phase high-
performance liquid chromatography (RP-HPLC) has
made it the predominant high-performance liquid
chromatography (HPLC) technique for preparative
peptide separations. Indeed, not only is RP-HPLC usually
superior to other modes of HPLC with respect to both
speed and efficiency, but it also offers the widest scope for
manipulation of mobile phase conditions and choices of
different columns to optimize separations.
(Id. at 1 ( citations omitted).)
50. Chen describes "the application of a one-step preparative RP-
HPLC protocol employing slow acetonitrile gradients to the purification of
an amphipathic, a-helical antimicrobial peptide on reversed-phase columns
of varying column diameters (1 mm to 9.4 mm I.D.)." (Id. at 2.) Chen
teaches that one-step RP-HPLC purification procedures may avoid loss of
product yield, "which is frequently a feature of multi-step protocols." (Id.)
51. Chen's study was conducted using columns with silica (C8)
based resin. (Id. at 3, 5.)
52. Chen teaches the following HPLC conditions for its study:
All preparative runs were carried out with a linear AB
gradient (1 % acetonitrile/min for 30 min up to 30%
acetonitrile, followed by 0.1 % acetonitrile/min for 250
min up to 55% acetonitrile) at room temperature and a
flow-rate of 2 ml/min, 1 ml/min, 0.25 ml/min or 0.08
ml/min for the semi-preparative column, analytical
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column, narrowbore column and micro bore column,
respectively, where eluent A was 0.2% aq. trifluoroacetic
acid (TFA) pH 2 and B was 0.2% TFA in acetonitrile.
Chen et al. had previously demonstrated that 0.2---0.25%
TF A in the mobile phase achieved optimum resolution of
peptide mixtures compared to the 0.05---0.1 % TFA
concentrations traditionally employed. Following the slow
gradient step, the column was washed with 70%
acetonitrile for 3 0 min.
All samples were loaded at the same flow-rate as the flow-
rate used for column elution.
Analytical RP-HPLC was carried out on the Zorbax 300
SB-Cs narrowbore column with a linear AB gradient (1 %
acetonitrile/min) at a flow-rate of 0.25 ml/min, where
eluent A was 0.2% aq. TFA, pH 2, and eluent B was 0.2%
TF A in acetonitrile.
(Id. (citation omitted).)
53. Chen teaches that, "[i]n order to design a slow gradient
preparative protocol, it is first necessary to determine the concentration of
acetonitrile required to elute the peptide of interest during an initial
analytical run" and that, to establish the percentage acetonitrile at which the
0.1 % acetonitrile/min slow gradient should begin, the "rule of thumb" for
polypeptides of 10 residues in length and longer is to "start at an acetonitrile
concentration 12% below that required to elute the peptide in the 1 %
acetonitrile/min analytical run." (Id. at 5.)
54. Chen teaches that
[a] key advantage of such a slow gradient approach is that,
at high sample loads, sample displacement can occur
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(Id.)
where the hydrophobic impurities displace the product of
interest and the product of interest displaces the
hydrophilic impurities during partitioning of product and
impurities, thus, aiding in the separation of product from
closely adjacent impurities. [] In addition, the desired
product may be eluted over a number of fractions, the bulk
of which would contain purified product only, with just the
first and last product fractions containing hydrophilic and
hydrophobic impurities, respectively, leading to improved
resolution and yield of the product of interest.
5 5. Chen teaches that "excellent product purity (>99%) and yield"
was obtained by its slow gradient approach to purification of the 200 mg
sample load using the semi-preparative column. (Id. at 6.)
56. Chen teaches that a recombinant protein (114 amino acid
residues) of >94% purity was obtained using its one-step slow gradient
protocol. (Id. at 8.)
57. Chen states that one of its objectives is "to examine the
potential of [a] slow gradient protocol as a general method for purifying
peptides with closely-related impurities from a wide range of sample loads."
(Id. at 2; see also id. at 5 (stating that "it is our intent to make [a] purification
protocol generally applicable to polypeptides in general") and 6 ( explaining
that the slow gradient approach is effective for resolution of desired peptide
product from even closely structurally related synthetic purities because
"only one fraction for each sample load contained overlapping product and
hydrophilic impurities of any significance").) Chen teaches that its
"straightforward one-step slow gradient protocol shows excellent potential
as a universal method for purification of synthetic peptides with closely
structurally related impurities." (Id. at 9; see also id. at Abstract.)
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Analysis
Except as otherwise noted, we adopt the Examiner's findings,
analysis, and conclusions with respect to claim 1, including with regard to
the scope and content of, and motivation to modify or combine, the prior art,
as set forth in the Final Action and Answer. (Final Act. 2-11, 12-15; Ans.
2-10, 11-27, FF1-FF57.) We address Appellants' arguments below.
Appellants concede that RP-HPLC "and other separation techniques
have been used to purify proteins in general, even at [the] preparative scale,"
and that "insulin or its analogs could be purified to a high level of purity
using RP-HPLC." (Appeal Br. 12.) However, Appellants contend that a
skilled artisan at the time of the invention "would not have expected to
succeed in purifying insulin or its analogs in one step, to achieve the claimed
level of purity." (Id. at 8, 10-11.) More particularly, Appellants contend
that: (1) "one cannot ... generalize a one-step purification of prior art
proteins that are in no way related to insulin or its analogs, to the purification
of the claimed proteins" (id. at 8, 11-12), and (2) the Examiner "appears to
ignore the stark differences between separation methods developed for
analytical scale and those developed for preparative scale." (Id. at 8, 12-
13.)
We are not persuaded. With respect to Appellants' argument that the
one-step RP-HPLC method references such as Mills and Chen cannot be
generalized to the purification of insulin analogs, we agree with the
Examiner that a skilled artisan would have a reason for, as well as a
reasonable expectation of success in, adapting the one-step method taught by
Mills and Chen to purify insulin, given that: (1) references such as Bhopale
teach the desirability of purifying insulin analogs from related impurities; (2)
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"Mills and Chen successfully appl[ied] the single[-step] RP-HPLC method
to proteins that are different from insulin but are also very different from
each other, underscoring the versatility of the approach"; and (3) references
such as Grace Vydac show that there is high level of skill and knowledge in
the art such that HPLC conditions are routinely optimized for protein
purification. (Ans. 12, 21-22.) We further note that Chen explicitly states
that its one-step purification protocol "shows excellent potential as a
universal method for purification of synthetic peptides with closely
structurally related impurities." (FF57 ( emphasis added).)
Appellants contend in the Reply Brief that "Bhopale published very
close in time to Chen and Mills" and that "Bhopale strongly suggests that at
about the same time that Mills and Chen [were] conducting their purification
on proteins wholly unrelated to the claimed proteins, those of ordinary skill
in the art would have at best expected a multi-step purification process to
purify the claimed protein." (Reply Br. 2-3.) Appellants similarly cite
Chance as suggesting that a skilled artisan would have expected multi-step
purification process to be required for purifying insulin analogs. (Id. at 3.)
We are not persuaded. Appellants base their argument on Bhopale
and Chance but do not sufficiently take Chen, Mills, and other cited
references into account. 12 However, "[ n ]on-obviousness cannot be
12 Appellants contend that 'just because Chen and Mills were lucky in
purifying proteins wholly unrelated to the claimed proteins does not
necessarily mean that those of ordinary skill in the art would have had the
reasonable expectation of success in purifying the claimed proteins at the
claimed level of purity." (Reply Br. 3.) We are not persuaded for the
reasons already discussed above. Chen and Mills do not suggest that the
results of their purification methods are a matter of luck. Likewise, we
emphasize that, for purposes of an obviousness analysis, "expectation of
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established by attacking references individually where the rejection is based
upon the teachings of a combination of references." In re Merck & Co., 800
F .2d 1091, 1097 (Fed. Cir. 1986). Instead, the references "must be read, not
in isolation, but for what [they] fairly teach[] in combination with the prior
art as a whole." Id. We agree with the Examiner that Bhopale and Chance,
when read in combination with Chen and Mills, provide a skilled artisan
with a reasonable expectation of success in arriving at a one-step
chromatographic process for purifying insulin analogs.
As to Appellants' argument that the prior art relates to purification
methods on the analytical scale, we note as does the Examiner that the claim
is not limited to purification on a preparative scale. (Ans. 20.) Furthermore,
even assuming the claim requires purification on a preparative scale, a
skilled artisan would have a reasonable expectation of success in combining
the cited references to arrive at the claimed purification method on such a
scale. As the Examiner points out, Mills teaches that its one-step
purification process is readily scalable, Chen provides examples wherein
100 mg and 200 mg of peptides are purified and further describes its method
as a "one-step slow gradient preparative protocol," and Grace Vydac
suggests that a skilled artisan would be able to scale up laboratory
separations to process scale separations by adjusting particular HPLC
conditions. (Ans. 20, 24--25; FFl 7; FF41; Chen Fig. 4 and Abstract.)
Finally, to the extent Appellants argue that the subject matter of claim
1 exhibits unexpected results, we are not persuaded. In particular,
Appellants argue that the results achieved by the method of claim 1 is
success need only be reasonable, not absolute." Pfizer, Inc. v. Apotex, Inc.,
480 F.3d 1348, 1364 (Fed. Cir. 2007).
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"unexpected based on Bhopale' s teachings" and that "[ t ]he claimed process
is ab initio 'superior to the procedures in the art' because it achieves a result
that has never been described in the art." (Reply Br. 3; see also id. at Appeal
Br. 8.) Once again, however, Appellants attack a reference (Bhopale)
individually without taking into account the combination of cited references
including Chen and Mills. Likewise, the fact that the result of a claimed
method is superior or has not previously been described in the art does not
automatically render the result unexpected, particularly in the context of an
obviousness rejection. Pfizer, Inc. v. Apotex, Inc., 480 F.3d 1348, 1371
(Fed. Cir. 2007) (explaining that a superior property must still be unexpected
to be considered evidence of non-obviousness).
Appellants argue in the Reply Brief that - contrary to the Examiner's
contention that Appellants have not established unexpected results because
the examples in the Specification do not sufficiently describe the contents of
their starting materials (Ans. 16-18)-the examples in the Specification
identify at least some impurities in Appellants' starting materials. (Reply 3-
4.) While we agree with Appellants that the examples appear to identify at
least some of the impurities present in their starting materials, we also agree
with the Examiner's broader point that Appellants have not persuasively
shown that the presence of the particular impurities in the starting samples
render the result of the purification process unexpected.
Moreover, the Examiner finds that the data supplied by the examples
in the Specification are not commensurate with the broad scope of the claim,
as required by a showing of unexpected results. (Ans. 16-18.) In the Reply
Brief, Appellants contend that the Specification describes purification results
for insulin analogs Aspart, Lispro, and Glargine and that they thus "have
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shown that a sufficient number of species encompassed by the claimed
genus demonstrate the unexpected results." (Appeal Br. 4.)
We are not persuaded by Appellants' argument. Appellants fail to
provide persuasive evidence or explanation why Aspart, Lispro, and
Glargine are sufficiently representative of insulin analogs to render the
examples in the Specification commensurate with the scope of claim 1.
"Attorneys' argument is no substitute for evidence." Johnston v. IVAC
Corp., 885 F.2d 1574, 1581 (Fed. Cir. 1989). In addition, claim 1
encompasses a wide variety of types of impurities, organic modifiers, ion
pairing agents, and HPLC conditions. Thus, assuming that Aspart, Lispro,
and Glargine are representative of insulin analogs, Appellants still fail to
explain how the examples in the Specification provides evidence of
unexpected results commensurate with the scope of the claim. For this
additional reason, we are not persuaded that the alleged unexpected results
proffered by Appellants demonstrate the process of claim 1 to be non-
obvious.13
Finally, Appellants contend that the rejection is based on
impermissible hindsight. (Reply Br. 5.) We are not persuaded. "Any
judgment on obviousness is in a sense necessarily a reconstruction based
13 Appellants further argue that, even if the data in the Specification is not
commensurate with the scope of claim 1, the same would not be true of
claim 2. (Reply Br. 4--5.) As we noted earlier, however, Appellants did not
properly separately argue claim 2 in the Appeal Brief. See supra note 11. In
any event, as discussed above, while claim 2 limits the insulin analog to the
group consisting of Aspart, Lispro, Glargine, and combinations thereof,
Appellants have neither shown that the results achieved by the claimed
method is unexpected in the first place, or that the data provided in the
Specification shows unexpected results commensurate with the scope of
other limitations in claim 2.
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upon hindsight reasoning, but so long as it takes into account only
knowledge which was within the level of ordinary skill at the time the
claimed invention was made and does not include knowledge gleaned only
from applicant's disclosure, such a reconstruction is proper." In re
McLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209,212 (CCPA 1971). For
the reasons already discussed, we find that the Examiner's rejection of claim
1 is properly based on the knowledge within the level of ordinary skill at the
time of the claimed invention, as evidenced by the cited prior art, rather than
any knowledge gleaned only from Appellants' disclosure.
Accordingly, we affirm the Examiner's rejection of claim 1. Claims
2-7 and 15-19, which are not separately argued, fall with claim 1. 37
C.F .R. § 41.3 7 ( c )(1 )(iv).
SUMMARY
For the reasons above, we affirm the Examiner's decision rejecting
claims 1-7 and 15-19.
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).
AFFIRMED
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