95000146 (B.P.A.I. Mar. 9, 2010)

Ex Parte 6838235 et al

Board of Patent Appeals and InterferencesMar 9, 2010

95000146 (B.P.A.I. Mar. 9, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE ____________________ BEFORE THE BOARD OF PATENT APPEALS AND INTERFERENCES ____________________ MICHAEL D. CECCHI, LIFE GLOBAL, LLC, GENX INTERNATIONAL INC., and IVF ONLINE LLC, Requester and Respondent1 v. Patent of VITROLIFE AB, Patent Owner and Appellant ____________________ Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 Technology Center 3900 ____________________ Decided: March 9, 2010 ____________________ Before CAROL A. SPIEGEL, DONALD E. ADAMS, and ROMULO H. DELMENDO, Administrative Patent Judges. SPIEGEL, Administrative Patent Judge. DECISION ON APPEAL 1 According to a Replacement Request for Reexamination filed 19 June 2006, the real parties in interest are Michael D. Cecchi, Life Global, LLC, Genx International, Inc., and IVF Online LLC (see page 20 of 33). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 2 I. Statement of the Case Appellant VITROLIFE, AB appeals under 35 U.S.C. Â§Â§ 134(b) and 315(a) from an Examiner's final rejection of all pending claims, claims 1-12, 14-15, and 19-23 (App. Br.2 2, 4; Resp. Br.3 2-3; Ans.4 3-4; App. Rebuttal Br.5 2). We have jurisdiction under 35 U.S.C. Â§Â§ 134(b) and 315(a). We AFFIRM. A. The subject matter on appeal The subject matter on appeal is directed to a method of performing an in vitro fertilization (hereinafter "IVF") process comprising placing a gamete, zygote or embryo in a first IVF fertilization medium containing alanyl glutamine (hereinafter "ala-gln") in lieu of glutamine and performing a first IVF procedure including embryonic development to the 8-cell stage. Claim 1, the only independent claim on appeal, and claim 14 are illustrative and read (App. Br. Clms. App'x 27 and 14, underlining and bracketing indicate additions and deletions, respectively, to the originally issued claims): 1. A method for performing an in vitro fertilization process, comprising: [a.] (a) placing a gamete, zygote or embryo in a first fertilization medium during a first in vitro fertilization procedure wherein the in vitro fertilization medium comprises: (i) water, (ii) a buffer present in an amount to buffer the in vitro 2 Appeal Brief under 37 C.F.R. Â§ 41.67 filed 15 February 2008 (hereinafter "App. Br."). 3 Third Party Requester's Response to Patent Owner's Appeal Brief filed 7 August 2009, i.e., Respondent Brief (hereinafter "Resp. Br."). 4 Examiner's Answer mailed 10 June 2008 (hereinafter "Ans."). 5 Rebuttal Brief under 37 C.F.R. Â§ 41.71(a) filed 9 July 2008 (hereinafter "App. Rebuttal Br."). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 3 fertilization medium with physiological pH, (iii) ionic constituents present in amounts that support growth and development growth and development of the gamete, zygote or embryo, wherein the ionic constituents include sodium, potassium, calcium and magnesium, and (iv) alanyl glutamine and one or more non-essential amino acids present in amounts to prevent osmotic shock of the gamete, zygote or embryo; and (b) performing a first in vitro fertilization procedure on the gamete, zygote or embryo in the first in vitro fertilization medium, wherein the first in vitro fertilization procedure comprises embryonic development to the eight-cell stage. 14. The method of claim 1 further comprising: [(b)] (c) transferring the gamete, zygote or embryo from the first in vitro fertilization medium into a second in vitro fertilization medium for a second in vitro fertilization procedure, wherein the first and second in vitro fertilization media have different compositions but integrated formulations sharing a core group of ionic and non-essential amino acid constituents thereby minimizing trauma to the gamete, zygote or embryo when they are moved from the first in vitro fertilization medium to the second in vitro fertilization medium. B. The maintained rejections The Examiner has rejected the claims as unpatentable as follows:6 GROUND #8: claims 1-6 and 20-22 under 35 U.S.C. Â§ 103(a) over Nakazawa7 and Roth8 alone or further with Christie9 or Minamoto,10 6 We have retained the numbering system, i.e., grounds 8, 9, 10, and 14, used by the Appellant, the Examiner, and the Respondent for consistency with the record before us. 7 Composition for an In Vitro Fertilization Medium, Canadian Patent CA 2,231,148, (filed September 4, 1996) (issued December 4, 2007), to Nakazawa et al., based on PCT application JP 1996/002503, published 13 March 1997; Appeal Brief Exhibit 10; Respondent Brief Exhibit 2 (hereinafter "Nakazawa"). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 4 GROUND #9: claims 1-6, 8, 10-12, 14, 15, and 19-23 under 35 U.S.C. Â§ 103(a) over Gardner 199711 and Roth alone or further with Christie or Minamoto, GROUND #10: claims 7 and 9 under 35 U.S.C. Â§ 103(a) over Gardner 1997, Roth, Christie, and Minamoto as applied to claims 1-6, 8, 10-12, 14, 15, and 19-23, and further with Conway-Myers,12 Cohen,13 and Huszar,14 and 8 Use of Glutamine in the Form of Its Di- and Tripeptide in a Nutrient Medium for Cell Culture, European Patent 220,379 B1, published May 6, 1987, Erich Roth, Inventor; Appeal Brief Exhibit 11; Respondent Brief Exhibit 1 (hereinafter "Roth"). This decision cites to the English translation of record. 9 A. Christie and M. Butler, "Glutamine-based dipeptides are utilized in mammalian cell culture by extracellular hydrolysis catalyzed by a specific peptidase," 277-90, 37 Journal of Biotechnology (1994); Appeal Brief Exhibit 12; Respondent Brief Exhibit 3 (hereinafter "Christie"). 10 Yoshiki Minamoto et al., "Development of a serum-free and heat- sterilizable medium and continuous high-density cell culture," 35-51 Cytotechnology 5 (1991); Appeal Brief Exhibit 13; Respondent Brief Exhibit 4 (hereinafter "Minamoto"). 11 David K. Gardner and Michelle Lane, "Culture and selection of viable blastocysts: a feasible proposition for human IVF?" 367-82, Vol. 3, 4, Human Reproduction Update, European Society for Human Reproduction and Embryology, (1997); Appeal Brief Exhibit 14; Respondent Brief Exhibit 5 (hereinafter "Gardner 1997"). 12 Human Embryo Co-Culture System and Uses Thereof, U.S. Patent 5,837,543, (filed May 22, 1997) (issued November 17, 1998) to Barbara- Ann Conway-Myers et al.; Appeal Brief Exhibit 15; Respondent Brief Exhibit 6 (hereinafter "Conway-Myers"). 13 Storage System Comprising an Emptied Zone Pellucida and Spermatozoa Placed Therein and a Method of Cryopreservation, U.S. Patent 6,132,952, (filed June 1, 1998) (issued October 17, 2000) to Jacques Cohen et al., based Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 5 GROUND #14: claims 14, 15, and 19 under 35 under 35 U.S.C. Â§ 112, second paragraph (indefinite) (Ans. 13). C. The positions of the parties As to grounds 8 and 9, the Examiner found that both Nakazawa and Gardner 1997 teach all of the limitations of claim 1 except for using the claimed ala-gln instead of glutamine in their first IVF media (Ans. 6-7, 8- 10). The Examiner found that Roth teaches substituting "masked glutamines," preferably the dipeptide ala-gln, for glutamine because ala-gln provides the same "growth factor" as glutamine, but without glutamine's toxicity and instability (Ans. 7, 10). The Examiner also found that Christie and Minamoto teach substituting dipeptide glutamines, such as ala-gln and glycyl-glutamine (hereinafter "gly-gln"), for glutamine in mammalian cell cultures because the dipeptide glutamines are less toxic and more stable than glutamine (Ans. 8, 11). The Examiner concluded that it would have been obvious to modify the IVF medium of Nakazawa or Gardner 1997 by using ala-gln instead of glutamine to provide increased stability and lower toxicity as taught by Roth, Christie, and Minamoto (Ans. 8, 11). As to ground 10, the Examiner concluded that it would have been obvious to modify Gardner 1997 by using HEPES buffer (claims 7 and 9) and PVP (claim 9) in view of the conventional incorporation of HEPES and PVP in IVF medium as taught on application 09/088,392, filed 1 June 1998; Appeal Brief Exhibit 16; Respondent Brief Exhibit 8 (hereinafter "Cohen"). 14 Process and System for Selection of Mature Sperm by Surface Membrane Determinants for Assisted Reproduction, U.S. Patent 5,897,988, (filed January 21, 1998) (issued April 27, 1999) to Gabor B. Huszar, based on application 09/010,247, filed 21 January 1998; Appeal Brief Exhibit 17; Respondent Brief Exhibit 7 (hereinafter "Huszar"). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 6 by Conway-Myers (HEPES buffer), Huszar (PVP to diminish sperm velocity and, thereby, facilitate fertilization), and Cohen (HEPES and PVP) (Ans. 12- 13). As to grounds 8 and 9, Appellant argues that: (i) Roth's culture medium does not enable embryonic development; (ii) absent knowledge that embryos prior to the 8-cell stage could metabolize ala-gln, one of ordinary skill in the art would not have reasonably expected that ala-gln could be successfully used to grow embryonic cell cultures as claimed; and, (iii) increased cell death during the first 24-48 hours of culturing cells as shown by Minamoto and Christie teaches away from using glutamine dipeptides in early stage embryo culture (App. Br. 5; App. Rebuttal Br. 3-7). As to ground 11, Appellant argues that Conway-Myers, Cohen, and Huszar fail to remedy the deficiencies of Gardner 1997, Roth, Christie, and Minamoto (App. Br. 24). Appellant submits Gardner I Declaration,15 Hamberger Declaration,16 Gardner 2000,17 Devreker,18 Katz-Jaffe,19 Gardner 2005,20 Gardner II 15 Declaration of David K. Gardner dated 19 October 2006, Appeal Brief Exhibit 1 (hereinafter "Gardner I Decl."). 16 Declaration of Lars A. Hamberger, M.D., Ph.D., dated 8 April 2007, Appeal Brief Exhibit 2 (hereinafter "Hamberger Decl."). 17 David K. Gardner and Michelle Lane, "EDTA Stimulates Cleavage Stage Bovine Embryo Development in Culture but Inhibits Blastocyst Development and Differentiation," 1-6, 56 Molecular Reproduction and Development (2000), Appeal Brief Exhibit 3 (hereinafter "Gardner 2000"). 18 F. Devreker, M. Van den Bergh, J. Biramane, R.M .L. Winston, Y. Englert and K. Hardy, "Effects of taurine on human embryo development in vitro," 14 Human Reproduction, 2350-56, 14, 9 Human Reproduction (1999), Appeal Brief Exhibit 4 (hereinafter "Devreker"). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 7 Declaration,21 the IVF screen prints,22 and Blake23 to support its position (App. Br. Evidence App'x 31-32). As to grounds 8 and 9, Respondent asserts that Roth recognizes the problem associated with using glutamine in embryonic and somatic cell culture media, i.e., production of cytotoxic ammonia from the breakdown of glutamine, as well as the solution, i.e., using ala-gln instead of glutamine (Resp. Br. 4, 7). Citing the Cohen Declaration,24 Respondent notes that Ham's F-10 medium (a conventional medium) was used for both somatic cell and pre-implantation embryo culturing in the same IVF clinic that employed Dr. Gardner (Resp. Br. 4). Respondent further asserts that Christie and Minamoto also recognize the same glutamine-generated 19 Mandy G. Katz-Jaffe, William B. Schoolcraft, and David K. Gardner, "Analysis of protein expression (secretome) by human and mouse preimplantation embryos," 678-85, 86 Fertility and Sterility, American Society for Reproductive Medice (Elsevier, Inc. 2006), Appeal Brief Exhibit 5 (hereinafter "Katz-Jaffe"). 20 David K. Gardner, Laura Reed, Donald Linck, Courtney Sheehan, Michelle Lane, "Quality Control in Human In Vitro Fertilization," 319-23, Vol. 23, 4 Seminars in Reproductive Medicine (Thieme Medical Publishers, Inc. 2005), Appeal Brief Exhibit 6 (hereinafter "Gardner 2005"). 21 Declaration of David K. Gardner under 37 C.F.R. 1.131, dated 13 August 2007, Appeal Brief Exhibit 7 (hereinafter "Gardner II Decl."). 22 Prints of five selected screens from the websites of Irvine Scientifics (2), MediCult (1), Sage (1), and Cook Medical (1) of poor legibility downloaded 25 July 2007, Appeal Brief Exhibit 8 (hereinafter "IVF screen prints"). 23 D. Blake, M. Proctor, N. Johnson and D. Olive, "Cleavage stage versus blastocyst stage embryo transfer in assisted conception (Review)," 1-80 in The Cochrane Collaboration, (Wiley Publishers 2007), Appeal Brief Exhibit 9 (hereinafter "Blake"). 24 Declaration of Jacques Cohen, dated 15 November 2006; Respondent Brief Exhibit 12 (hereinafter "Cohen Decl."). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 8 ammonia problem in tissue culture media as well as the same solution, i.e., using a glutamine dipeptide, e.g., ala-gln, instead of glutamine (Resp. Br. 5, 7). According to Respondent, there is nothing unpredictable about the problem of glutamine-generated ammonia production in culture media or about the way to address the problem, i.e., using ala-gln instead of glutamine (Resp. Br. 5-7). Respondent also points out that substantially fewer than 50% of the cultured embryos are actually used in an IVF procedure, with the remainder being frozen or discarded (Resp. Br. 6, 7). Thus, increased cell death during the first 24-48 hours of culture would not teach away from using ala-gln instead of glutamine in an embryo culture medium for an IVF procedure (id.). The Examiner also relied on the following evidence submitted by Respondent to establish that early stage embryos are able to metabolize glutamine dipeptides (Ans. 35): the Rieger Declaration;25 Hagemann;26 and, Quinn.27 Respondent submitted the Cohen Declaration28 in response to the Gardner I Declaration (Resp. Br. 4). 25 Declaration of Donald Rieger, Ph.D., dated 15 November 2006; Appeal Brief Exhibit 20; Respondent Brief Exhibit 9 (hereinafter "Rieger Decl."). 26 Lora J. Hagemann, Lydia L. Weilbert, Susan E. Beaumont and H. Robin Tervit, "Development of Bovine Embryos in Single In Vitro Production (sIVP) Systems," 143-47, 51 Molecular Reproduction and Development, AgResearch, Dairy & Beef Division, Ruakura Research Centre (Wiley-Liss, Inc. 1998); Appeal Brief Exhibit 18 (hereinafter "Hagemann"). 27 Patrick Quinn, "Human embryo culture media," message dated 02/13/97 downloaded 5 April 2006 from http://embryomail.anri.barc.usda.gov/embryomail/message.tpl?MsgID=269 ; Appeal Brief Exhibit 19 (hereinafter "Quinn"). 28 Declaration of Jacques Cohen dated 15 November 2006 (hereinafter "Cohen Decl."). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 9 As to ground 14, according to the Examiner, the method of independent claim 1 produces a first IVF medium containing an embryo and, therefore, does not provide antecedent basis for the terms "gamete" and "zygote" in dependent claims 14, 15, and 19 (Ans. 13). As to ground 14, Appellant requests that the Board enter the Amendment filed August 16, 2007 to address this informality which was refused entry by the Examiner (App. Br. 24-25). Alternatively, Appellant argues that one of ordinary skill in the art would immediately recognize that, despite the recitation of three alternatives, the phrases "transferring the gamete, zygote or embryo" in claims 14 and 15 and "cryopreservation of the gamete, zygote or embryo" in claim 19 must be interpreted to read "transferring the embryo" and "cryopreservation of the embryo," respectively (App. Br. 25). As to ground 14, Respondent points out that neither a gamete nor a zygote is an embryo (Resp. Br. 7). Based upon Appellant's separate patentability arguments, we decide this appeal on the basis of claims 1 and 14. 37 C.F.R. Â§ 41.37(c)(1)(vii). D. The issues At issue is (1) whether Roth contains an enabling disclosure of embryonic development in culture medium; (2) whether one of ordinary skill in the art would have had a reasonable expectation of success of growing embryos in culture media containing ala-gln instead of glutamine as claimed; (3) whether increased cell death during the first 24-48 hours of culturing cells as disclosed by Minamoto and Christie teaches away from using glutamine dipeptides in early stage embryonic culture; and, (4) Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 10 whether independent claim 1 provides antecedent basis for the phrase "transferring the gamete, zygote or embryo" as recited in claim 14. II. Findings of Fact The following findings of fact (hereinafter "FF") are supported by a preponderance of the evidence of record. A. The 235 patent 1. "Background of the Invention" [1] According to the 235 patent, IVF tries to duplicate the conditions and processes normally occurring within the female reproductive tract necessary for oocyte development, fertilization, and early embryonic development (235 patent 1:17-21). [2] A culture medium is used as a substitute for the biological fluid that would ordinarily surround the gametes, zygotes, and developing embryo (235 patent 1:33-36). [3] Most IVF laboratories use a single culture medium, such as Ham's F- 10, as a single medium throughout the various IVF procedures (235 patent 1:36-41). [4] However, "to the extent tissue culture media contain components that are generally needed by the gametes and the embryo, the media are not formulated to provide the components at levels appropriate to support healthy gamete and embryonic development" (235 patent 1:45-50). [5] Other culture media, such as Earle's, T-6, and human tubal fluid (HTF) media, containing balanced salt solutions supplemented with energy sources, such as glucose, pyruvate, and lactate, generally lack Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 11 key components of the female reproductive tract, such as non- essential amino acids, and the concentrations of their components are not optimized to meet the needs of gametes and developing embryos (235 patent 1:51-60). [6] The development of dual culture media G1 and G2 in the prior art, such as described in Gardner 1997, has led to significant advancements in adapting culture media to the physiological needs of the cleavage stage embryo and the embryo in the eight-cell through blastocyst stage of development (235 patent 2:12-29). [7] According to the 235 patent, research suggests that the G1 and G2 media contribute to higher pregnancy rates and reduce the need for transfer of multiple embryos (235 patent 2:36-40). [8] However, according to the 235 patent, neither G1 nor G2 medium is optimized for supporting the gametes, oocyte maturation, or fertilization (235 patent 2:40-42). 2. terminology [9] The 235 patent equates 4- and 8-cell stage embryos with days two and three of culture, respectively (235 patent 2:1-2). [10] According to the 235 patent, a cleavage stage embryo is an embryo up to the 8-cell stage (235 patent 3:3-4) and a blastocyst is an embryo of around 100 cells (id. 13:35). B. The Cohen Declaration [11] Jacques Cohen, Ph.D., testifying on behalf of Respondent as an expert in reproductive science and embryology, stated that he is in charge of Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 12 the IVF laboratory at Walter Reed Army Medical Center (Cohen Decl. Â¶Â¶ 1-3). [12] Dr. Cohen stated that in his opinion, "IVF culture and cell tissue culture are essentially the same and interchangeable" (Cohen Decl. Â¶ 6), with the proviso that it "has been known since the 60s and 70s (from hundreds of scientific publications), that lactate and pyruvate are essential for culturing early mammalian embryos" (id. Â¶ 4). [13] Dr. Cohen testified that embryologists would add pyruvate and lactate to the Roth medium for use in IVF "since the essential knowledge of these two energy sources has been known to embryologists for decades" (Cohen Decl. Â¶ 7). C. Nakazawa [14] According to Nakazawa, chemically defined media conventionally used in IVF, such as Ham's F-10, have not been optimized to meet the nutritional needs of early embryos (Nakazawa 2, Â¶ 2). [15] Nakazawa discloses a chemically defined medium designed to meet the nutritional needs of sperm or ovum (i.e., gametes) as well as for growing early embryos (Nakazawa 5, Â¶ 3). [16] Unlike previous chemically defined media, the Nakazawa medium comprises all 21 amino acids contained in ovarian follicular fluid, and at the relatively lower concentrations found in follicular fluid, especially relative to methionine (0.21-4.3 mg/L), leucine (0.79-15.8 mg/L), L-aspartic acid (0.09-1.71 mg/L), and cystine (0.14-2.7 mg/L) (Nakazawa 6, Â¶2; 7-8, Table 1; 8, Â¶ 2; 13, Table 4; 14, Â¶ 2). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 13 [17] In addition to the 21 amino acids, all of the exemplary Nakazawa media include sodium pyruvate (0.036 mg/L), sodium lactate (2.398 mg/L), and glucose (0.500 mg/L) (Nakazawa 20-24, Tables 5-11). [18] According to Nakazawa, the amino acids used to formulate its medium may be used in free form, as a pharmacologically acceptable salt, or in any form which may be hydrolyzed to provide the free amino acid, including as di- or tri-peptides (Nakazawa 14, Â¶ 3). [19] All of the exemplary formulations described in Nakazawa used free form amino acids, including glutamine (id. 20-24, Tables 5-11). [20] Nakazawa concluded that its medium provided improved growth stimulation and qualitative stabilization of early embryos (Nakazawa 32, last Â¶). D. Gardner 1997 [21] According to Gardner 1997, "media for embryo culture need to . . . reflect the environment of the female reproductive tract. . . . [and] the composition of such media should change as embryo development proceeds" (Gardner 1997, 369, Â¶ 3), i.e., "one must alter media composition at key times during the preimplantation period in order to meet the needs of the embryo and minimize cellular trauma in the embryo" (id. 371, Â¶ 2). [22] For example, unlike most somatic cells, a mammalian zygote through 8-cell stage embryo use pyruvate, lactate, and/or amino acids, instead of glucose, as the main energy source (Gardner 1997 371, Â¶ 3; 374, Â¶ 1), i.e., the levels of glucose in the oviduct were lowest at the time when the oocyte/early embryo would be present (0.5 Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 14 mM), whilst pyruvate was at its highest (0.32 mM). Conversely, glucose reached its peak concentration in the uterus (3.15 mM) and pyruvate its lowest (0.1 mM â€¦). (id. 371, Â¶ 3). [23] According to Gardner 1997, there is a growing tendency to remove glucose from human embryonic development media because of its detrimental effect on embryos, e.g., impairment of oxidative capacity, when present is simple culture media (Gardner 1997 371, Â¶Â¶ 3-4). [24] Gardner 1997 teaches that glucose becomes increasingly important once the embryonic genome is activated and biosynthetic levels increase and, therefore, its complete removal from media designed to support embryo development beyond the first few cleavage divisions cannot be considered as physiological (Gardner 1997 371, Â¶ 4). [25] Further according to Gardner 1997, the highest percentage of blastocyst development and the highest implantation rates and fetal development occurred when mouse zygotes were cultured for the first 48 hours in the presence of "non-essential amino acids"29 (i.e., alanine, asparagine, aspartate, glutamate, glycine, proline, and serine) and glutamine, followed by culture for a further 48 hours in the presence of 20 of Eagle's amino acids, i.e., both non-essential and essential groups (Gardner 1997 373, Â¶ 1 and Table III). [26] Gardner 1997, reports that it has been proposed that the non-essential amino acids and glutamine function as (i) regulators of energy 29 Eagle's non-essential amino acids are those amino acids not required for the development of somatic cells in culture (Gardner 1997 372, last full sentence). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 15 metabolism, (ii) osmolytes, and (iii) buffers of pH in the cleavage stage embryo (Gardner 1997 373, last Â¶). [27] However, amino acids are not only metabolized by the embryo, they also spontaneously breakdown to release ammonia into the culture medium (Gardner 1997 375, last Â¶). [28] "Unfortunately, ammonium is embryo toxic, not only impairing development in the culture, but also significantly reducing viability" (Gardner 1997 375, last Â¶, citation omitted). [29] While replacing the culture medium every 48 hours would prevent the toxic buildup of ammonium, it would also remove any produced autocrine factor(s) that stimulate embryo development (Gardner 1997 375, last Â¶). [30] Therefore, glutamate dehydrogenase, Î±-ketoglutarate, and NADH were added to the culture medium after 48 hours to enzymatically convert the ammonium present to glutamate (Gardner 1997 376, Â¶ 1). [31] In view of the changing requirements of the embryo, Gardner 1997 formulated two media for the culture of the human zygote to the blastocyst stage, i.e., G1, formulated to support development of the zygote to the 8-cell stage, and G2 formulated to support development from the 8-cell stage to the blastocyst (Gardner 1997 377, Â¶ 3), the compositions of which are shown in Gardner 1997 Table IV. [32] In particular, while both G1 and G2 media contained glutamine and all the non-essential amino acids, only the G2 medium contained all the essential amino acids (Gardner 1997 376, Table IV). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 16 [33] In addition, G1 medium contains 0.32 mM pyruvate, 10.5 mM lactate, and 0.50 mM glucose, while G2 medium contains 0.10 mM pyruvate, 5.87 mM lactate, and 3.15 mM glucose, consistent with activation of the embryonic genome and increasing biosynthesis levels (Gardner 1997 371, Â¶Â¶ 3-4; 376, Table IV). E. Roth [34] According to Roth, due to the relative instability of glutamine, culture media is usually sold essentially free of glutamine, which is then added by the end user (Roth 1:20-36). [35] Roth teaches that certain di-/tri-peptides of glutamine can be used by cells as a source of glutamine and use of such di-/tri-peptides in cell culture media allow for heat-sterilization and improved stability (Roth 2:60-70). [36] Roth discloses growing three different cell lines, K-562, fibroblasts, and HELA cells, in glutamine-free medium and in medium supplemented with glutamine or the glutamine dipeptides ala-gln or gly-gln (Roth 4:118-125). [37] According to Roth, [t]he cells were cultivated in glutamin[e]-free RPMI 1640 (5% CO2 incubator). 1 l[iter] medium containted [sic] 900 ml RPMI 1640, 100 ml fetal calf serum (dialyzed over night against PBS) and 2 ml gentamycine solution. 10 ml of a solution containing 100 mmol glutamin[e], ALA-GLN, GLY-GLN or acetylglutamine were added to the medium (sterile filtration with a Acro Disc 200 Âµm filter). The K-562 cells were split three times a week to 1/3, the HELA cells were split three times to 1/5 and the fibroblasts were split twice to 1/3 (Roth 4:122-127). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 17 [38] Roth found that the growth rate of individual cell lines in culture media containing glutamine, ala-gln, or gly-gln was almost identical (Roth 5:134-137). [39] Roth also described a sample culture medium on page 4 as follows: Component mg/L Component mg/L L-arginine 200.00 biotin 0.20 L-asparagine H2O 56.82 D-calciumpantothenate 0.25 L-cystine Na2 59.15 folic acid 1.00 L-glutamic acid 20.00 I-inosit 35.00 L-alanyl-glutamine 500.00 nicotinamide 1.00 glutathione 1.00 p-aminobenzoic acid 1.00 glycine 10.00 pyridoxine HCl 1.00 L-histidin 15.00 riboflavine 0.20 L-hydroxyproline 20.00 thiamine HCl 1.00 L-isoleucine 50.00 vitamine B12 0.005 L-leucine 50.00 Ca(NO2)2 69.49 L-lysine HCL 40.00 KCl 400.0 L-methionine 15.00 MgSO4 7 H2O 100.0 L-phenylalanine 15.00 NaCl 6000 L-proline 20.00 NaHCO2 2000 L-serine 30.00 Na2HPO4 800.7 L-threonine 20.00 glucose 2000 L-trytophane 5.00 phenolred 5.00 L-tyrosine 20.00 aqua dest. ad 1 l L-valine 20.00 [40] According to Roth, culture media "using glutamine peptides are also in particular suited as cultivation media for the development of the oocyte to the multi cellular embryo in extracorporeal insemination" (Roth 3:78-80), e.g., where a fertilized egg is cultured for 48 to 60 hours to the embryonic 4- or 8-cell stage before transfer into the uterus (id. 1:14-19). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 18 F. Minamoto [41] According to Minamoto, glutamine is the most heat labile component in culture medium and easily decomposes to toxic ammonia (Minamoto 38, Â¶ 2). [42] Minamoto teaches substituting ala-gln or gly-gln for glutamine to provide a heat-stable, serum-free medium suitable for growing and maintaining mammalian cells, especially at saturation density, e.g., in perfusion and hybridoma cell cultures (Minamoto abstract; 50, Â¶ 1). [43] Minamoto noted a difference in the utilization of the dipeptides, likely due to the substrate specificity of the amino peptidase activity (which hydrolyzes the dipeptide) in each cell line tested, and concluded that ala-gln was the better substitute for glutamine (Minamoto Â¶ bridging 38-39). [44] Minamoto observed that [a]lthough the lag time of cell growth was slightly prolonged, the cell growth and maintenance of viable [mouse hybridoma 10 H] cells were . . . significantly improved in the RITC 62-8 medium [serum-free medium containing ala-gln] after saturation density had been reached, and the accumulation of antibodies in the serum- free medium was four times higher than that in RPMI 1460 + 10% FBS [fetal bovine serum] (Minamoto 42, Â¶ 2). G. Christie [45] Christie characterizes the production of ammonia from glutamine in cell culture as a widely recognized problem (Christie 287, Â¶ 4). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 19 [46] According to Christie, dipeptide-based media produce lower levels of accumulated ammonia than glutamine-based media (Christie 289, last Â¶). [47] Specifically, Christie concluded that (i) high cell yields can be obtained in murine hybridoma cell cultures containing either ala-gln or gly-gln providing the concentrations are optimal for extracellular hydrolysis by a peptidase derived from the cytosol, (ii) optimal concentrations may vary with cell type, and (iii) dipeptide-based media produce lower levels of accumulated ammonia, reduce the requirement for glycolysis, and reduce lactate production (id.). [48] Although Christie observed a decrease in viable CC9C10 hybridoma cell concentration during a lag phase (initial 24 hours) of the gly-gln (20 mM) and ala-gln cultures, Christie also observed that this resulted in the maximum cell yield occurring later in the culture compared to the glutamine-based control culture (Christie 280, last Â¶). [49] According to Christie, this lag period was associated with dipeptide hydrolysis and a concomitant increase in glutamine concentration in the medium (Christie 281, Â¶ 2). H. Appellant's rebuttal evidence 1. Gardner I Declaration [50] David K. Gardner, Ph.D., testifying on behalf of Appellant as an expert in embryology, stated that he is the Scientific Director of the Colorado Center for Reproductive Medicine (Gardner I Decl. Â¶Â¶ 1-2). [51] Dr. Gardner evaluated the ability of the medium described on page 4 of Roth with added 10% fetal calf serum (hereinafter "the sample Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 20 Roth medium") to support embryo development versus the ability of the G1.2 and G2.2 dual IVF media described in the 235 patent (Gardner I Decl. Â¶Â¶ 5-6). [52] According to Dr. Gardner, two comparative experiments were performed with the sample Roth medium -- one evaluating the sample Roth medium's ability to support development of zygotes to the eight-cell stage versus the G1.2 medium, and the other the ability of the sample Roth medium to support development from the eight- cell stage to the blastocyst stage versus the G2.2 medium (Gardner I Decl. Â¶Â¶ 9-10). [53] According to Dr. Gardner's data, none of the 95 zygotes used in the first experiment developed to the 8-cell stage and none of the 20 8-cell embryos used in the second experiment developed to the blastocyst stage using the sample Roth medium versus 84% and 77% of the tested zygotes and eight-cell embryos using the G1.2 and G2.2 media, respectively (Gardner I Decl. Â¶ 10). [54] Dr. Gardner concluded that the [sample] Roth Medium is incapable of supporting the development of any mammalian embryos, including human embryos, for at least the following reasons: 1) the [sample] Roth Medium lacks pyruvate and/or lactate; 2) the glucose concentration is too high; 3) the NaCl concentration is too high; and 4) the nicotinamide level is too high (Gardner I Decl. Â¶ 12, original emphasis). 2. Embryonic gene activation a. Katz-Jaffe [55] According to Katz-Jaffe, Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 21 [t]hree important events must occur for continued embryonic development, namely, the destruction of maternal transcripts, the replacement of essential common maternal transcripts with embryonic transcripts, and the expression of new embryonic transcripts. . . . In mice, transcription occurs in a series of waves, one up to the 2-cell stage (activation of the embryonic genome) and one after the 4-cell stage (differentiation) . . . . (Katz- Jaffe 682, Â¶ 2). [56] As of 2006, Katz-Jaffe reports that relatively little is known about the protein production of mouse or human preimplantation embryos (Katz-Jaffe 681, Â¶ 4). b. Devreker [57] According to Devreker, "[e]mbryo viability is partially compromised by suboptimal culture conditions although chromosomal abnormalities (Kola et al., 1993) or failure to activate the embryonic genome are other possible factors (Tesarik 1994)" (Devreker 2350, Â¶ 1). [58] Further according to Devreker, studies in the mouse show genome activation during the first 48 hours of culture, while embryonic genome activation is believed to occur between the 4- and 8-cell stage in humans (Devreker 2355, penultimate Â¶). c. Gardner 1997 [59] According to Gardner 1997, human gene activation occurs between the 4- and 8-cell stage of embryonic development (Gardner 1997 368, Â¶ 2). [60] Gardner 1997 teaches that analysis of an embryo's ability to regulate pH suggests that after compaction (occurring at 8-cell stage), the Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 22 blastomeres start to acquire a more somatic cell-like physiology with the formation of an epithelium (Gardner 1997 374, penultimate Â¶). 3. Gardner II Declaration [61] Appellant submitted the Gardner II Declaration to remove Hagemann as a prior art reference (App. Br. 15). [62] Dr. Gardner testified that he conceived of the subject matter of at least claims 1-6 of the 235 patent as amended April 9, 2007, prior to October 1998 and exercised reasonable diligence from a date prior to October 1998 to the filing of application 09/201,594 (Gardner II Decl. Â¶Â¶ 2, 5). I. Respondent's evidence of peptidase activity in early embryos 1. Rieger Declaration [63] Donald Rieger, Ph.D., testified that (i) the importance of amino acids, specifically, glutamine, to mammalian cell culture was recognized by 1959, (ii) the specific importance of glutamine to embryo metabolism and development in culture was recognized by 1984, (iii) the efficacy of replacing glutamine in cell culture with ala-gln and gly-gln was established by 1994, and (iv) ala-gln and gly-gln were also found to be effective replacements for glutamine in parenteral feeding solutions (Rieger Decl. Â¶Â¶ 3-6). [64] In particular, Dr. Rieger testified that Hagemann and Quinn "clearly establish" replacing glutamine with glutamine dipeptides, gly-gln and/or ala-gln in embryo culture media (Rieger Decl. Â¶Â¶ 8-10). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 23 2. Hagemann [65] Hagemann relates to the development of bovine embryos in single in vitro production systems and states that their data demonstrates that embryo development is equal in media supplemented with either GlutaMax-2 [i.e., gly- gln] or L-glutamine. Further work may show similar positive results with supplementation of media with GlutaMax-1, an[d] L-alynyl-L-glutamine dipeptide [i.e., ala-gln], or a combination of both GlutaMax-1 and -2. (Hagemann 146, Â¶ 3.) 3. Quinn [66] Quinn describes an embryonic culture medium, D3+HTF, containing no phosphate, low glucose, EDTA, and glutamine in the form of ala- gln, which is intended for culture of embryos beyond the 8-cell stage (Quinn 1). III. Discussion A. Grounds #8, #9, and #10 1. legal principles An invention is obvious if "the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious . . . to a person having ordinary skill in the art to which said subject matter pertains." 35 U.S.C. Â§ 103. The factual inquiries underlying obviousness include (1) the scope and content of the prior art, (2) the differences between the prior art and the claims at issue, (3) the level of ordinary skill in the art at the time the invention was made, and (4) any objective evidence of nonobviousness. Graham v. John Deere Co. of Kansas City, 383 U.S. 1, 17-18 (1966). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 24 In determining whether obviousness is established by combining the teachings of the prior art, â€œthe test is what the combined teachings of the references would have suggested to those of ordinary skill in the art.â€ In re Keller, 642 F.2d 413, 425 (CCPA 1981). In addition, a reference disclosure is not limited only to its preferred embodiments, but is available for all that it discloses and suggests to one of ordinary skill in the art. In re Lamberti, 545 F.2d 747, 750 (CCPA 1976); In re Mills, 470 F.2d 649, 651 (CCPA 1972). Further, one skilled in the art must be presumed to know something about the art apart from what the references disclose. In re Jacoby, 309 F.2d 513, 516 (CCPA 1962). Skill in the art is presumed. In re Sovish, 769 F.2d 738, 743 (Fed. Cir. 1985). A specification need not disclose what is well known in the art. Lindemann Machinenfabrik GMBH v. American Hoist & Derrick Co., 730 F.2d 1452 (Fed. Cir. 1984). Moreover, as explained in KSR Int'l Co. v. Teleflex, Inc., 550 U.S. 398, 406 (2007), "[i]f a person of ordinary skill in the art can implement a predictable variation, and would see the benefit of doing so, Â§ 103 likely bars it patentability." "Obviousness does not require absolute predictability of success . . . all that is required is a reasonable expectation of success." In re O'Farrell, 853 F.2d 894, 903 (Fed. Cir. 1988). The presence of a reasonable expectation of success is measured from the perspective of a person of ordinary skill in the art at the time the invention was made. Life Techs., Inc. v. Clontech Labs., Inc., 224 F.3d 1320, 1326 (Fed. Cir. 2000). However, "[a] reference may be said to teach away when a person of ordinary skill, upon reading the reference, would be discouraged from following the path set out in the reference, or would be led in a direction Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 25 divergent from the path that was taken by the applicant." In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). According to 37 C.F.R. Â§ 1.131, in relevant part, when any claim of a patent under reexamination is rejected, the owner of the patent under reexamination may submit a declaration to establish invention of the subject matter of the rejected claim prior to the effective date of the reference on which the rejection is based. 2. analysis As stated above, based on Appellant's separate patentability arguments, we decide grounds of rejection 8-10 on the basis of claim 1. 37 C.F.R. Â§ 41.37(c)(1)(vii). The Examiner has rejected claim 1 as unpatentable under 35 U.S.C. Â§ 103 over Nakazawa and Roth alone or further with Christie or Minamoto (ground 8) or over Gardner 1997 and Roth alone or further in view of Christie or Minamoto (ground 9) (Ans. 6-11). The subject matter of claim 1 is directed to a method of performing an IVF process comprising placing a gamete, zygote or embryo in a first IVF medium comprising ala-gln and performing a first IVF procedure on the gamete, zygote or embryo which includes embryonic development to the eight-cell stage (App. Br. Clms. App'x 27). Conventional tissue culture media has been used to grow gametes, zygotes, and embryos for use in IVF procedures (FF 3, 5, 12, 14). IVF processes try to duplicate the conditions normally occurring within the female reproductive tract necessary for development of the oocyte (egg), fertilization, and early embryonic by using the culture medium as a Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 26 substitute for the biological fluid that would ordinarily surround the gamete, zygote, or developing embryo (FF 1, 2). Consequently, the prior art, i.e., Nakazawa and Gardner 1997, has altered conventional culture media to meet the needs of sperm, eggs, zygotes, and early embryos (FF 6, 15-17, 21-26) in order to provide improved growth conditions and higher pregnancy rates (FF 7, 20, 25). Specifically, Nakazawa and Gardner have formulated IVF culture media containing all 21 amino acids (including glutamine) found in, and at the concentrations present in, ovarian follicular fluid, as well as lactate, pyruvate, and glucose at concentrations found in the female reproductive tract (FF 16, 22, 25, 31, 32). As explained by Gardner 1997, mammalian zygotes through 8-cell embryos, unlike most somatic cells, use pyruvate and/or lactate, instead of glucose, as their main energy source (FF 22) (see also the testimony of Dr. Cohen that addition of lactate and pyruvate energy sources is essential for culturing early mammalian embryos (FF 12-13)). Moreover, too much glucose in human embryonic culture media has a detrimental effect on embryos (FF 23). Thus, as noted by the Examiner (Ans. 28-29), Nakazawa and Gardner 1997 disclose IVF culture media suitable for growing gametes and zygotes through at least 8-cell embryos which differ from the medium recited in claim 1 by using glutamine instead of ala-gln. Roth teaches that glutamine is relatively unstable, so much so that culture media is usually sold free of glutamine, which is then added by the end user (FF 34). Roth discloses growing three different cell lines, K-562, fibroblasts, and HELA cells, in medium supplemented with glutamine, ala- gln, or gly-gln and finding almost identical growth rates (FF 37-39). In Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 27 4addition, Roth teaches that culture media "using glutamine peptides are also in particular suited as cultivation media for the development of oocyte to the multi cellular embryo in extracorporeal insemination", e.g., to the four- to eight-cell embryo stage (FF 40). Similar to Roth, Minamoto teaches the heat lability and decomposition to ammonia of glutamine in culture media, as well as substituting ala-gln or gly-gln, preferably ala-gln, for glutamine in culture media (FF 41-43). Likewise, Christie characterizes the ammoniagenic property of glutamine in cell culture media as a widely recognized problem and teaches that dipeptide-based media, e.g., media containing ala-gln or gly-gln, produce lower levels of accumulated ammonia (FF 45-47). Therefore, we agree with the Examiner (see e.g., Ans. 8, 11, 27-28, and 43) that it would have been prima facie obvious to modify the IVF culture medium of either Nakazawa or Gardner 1997 by using ala-gln instead of glutamine to address glutamine's art recognized problems of instability and toxic ammonia generation in cell cultures as taught by each of Roth, Minamoto, and Christie. We note that Gardner 1997 expressly acknowledges that amino acids spontaneously breakdown to release embryo toxic ammonia into the culture medium and the ammonia should be removed to avoid impairing embryonic development and reducing viability (FF 27- 28). KSR, 550 U.S. at 406. Appellant argues that (i) Roth's culture medium does not enable embryonic development, (ii) absent known that embryos prior to the 8-cell stage could metabolize ala-gln, one of ordinary skill in the art would not have reasonably expected that ala-gln could be successfully used to grow Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 28 embryonic cell cultures as claimed, and (iii) increased cell death during the first 24-28 hours of culturing cells as shown by Minamoto and Christie teaches away from using glutamine dipeptides in early stage embryo culture (App. Br. 5; App. Rebuttal Br. 3-7). As noted by Appellant (App. Br. 7), the cell culture example provided by Roth involves somatic cells (FF 36-39). As noted by the Examiner, Nakazawa and Gardner 1997 describe the requirements of IVF cell culture for gametes, zygotes, and embryos (Ans. 29, 43), including pyruvate, lactate, and glucose at concentrations found in the female reproductive tract (FF 17, 22). As expressly explained by Gardner 1997, a mammalian zygote through 8-cell stage embryo, unlike most somatic cells, use lactate and/or pyruvate, instead of glucose, as the main energy source and too much glucose in the culture medium is detrimental to embryos (FF 22-24). Therefore, Dr. Gardner's data showing that none of the zygotes or 8-cell embryos tested in the two experiments using the sample Roth medium for somatic cells developed into 8-cell embryos or blastocysts, respectively, is not unexpected. Specifically, the sample Roth medium as described on page 4 of Roth not only lacked lactate or pyruvate, the main energy source of mammalian zygotes through 8-cell stage embryos, but also contained 2000 mg/L glucose, an amount far above the 0.500 mg/L and 0.50 to 3.15 mM amounts taught by Nakazawa and Gardner 1997 (FF 17, 22, 33). Indeed, Dr. Gardner himself concluded that the inability of the sample Roth medium is at least due to its lack of pyruvate and/or lactate and its high glucose concentration (FF 54). Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 29 The fact that Roth does not use a specific IVF/embryo culture medium to illustrate the use of ala-gln instead of glutamine does not mean that Roth's disclosure is not enabled for culture of gametes, zygotes, or embryos. A reference disclosure is not limited to its examples, but is available for all it teaches or suggests to one of ordinary skill in the art. In re Lamberti, 545 F.2d at 750; In re Mills, 470 F.2d at 651. Furthermore, one skilled in the art is presumed to know something about the art apart from what the reference discloses. In re Jacoby, 309 F.2d at 516. In this case, one skilled in the art would know that a medium for culturing gametes, zygotes, or embryos requires pyruvate and/or lactate as a main energy source and that too much glucose in the medium is detrimental to the embryo. Notably, Devreker teaches that embryonic viability is partially compromised by suboptimal culture conditions (FF 57). Here, Roth expressly teaches or suggests the use of ala-gln instead of glutamine in both early embryonic and somatic cell cultures to avoid the instability and toxic ammonia generating problems associated with use of glutamine (see e.g., Ans. 29). Thus, the Gardner I Declaration is insufficient to overcome the Examiner's prima facie conclusion of obviousness based on the totality of the evidence of record, especially where the rejection is based on the combined teachings of either Nakazawa or Gardner 1997 and Roth. We also do not find the embryonic genome activation and teaching away arguments sufficient to overcome the Examiner's prima facie conclusion of obviousness. Initially, we note that claim 1 is not limited to development of any particular embryo, e.g., a mammalian human embryo, to the 8-cell stage or beyond. Nonetheless, taking a human embryo for Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 30 example, the evidence of record suggests that human embryonic genome activation occurs about the 8-cell stage (FF 33, 58, 59). As discussed above, glutamine spontaneously breaks down to release embryo toxic ammonia into the culture medium whether or not the embryonic genome is activated. Moreover, according to Christie, the lag phase in which increased cell death occurred in somatic cell culture was associated with dipeptide hydrolysis and a concomitant increase in glutamine concentration in the medium (FF 49). Thus, an inability of a human embryo up to the 8-cell stage to metabolize ala-gln due to whole or partial inactivation of its embryonic genome would reasonably be expected to reduce, if not preclude, the amount of glutamine released into the culture medium by hydrolysis of ala-gln. Reducing or eliminating the amount of glutamine in the culture medium can hardly be said to teach away from the path set out in Roth, Minamoto or Christie. In re Gurley, 27 F.3d at 553. Thus, one of ordinary skill in the art would have had a reasonable expectation of success of growing embryos up to the 8-cell stage in culture media containing ala-gln instead of glutamine. In re O'Farrell, 853 F.2d at 903. Furthermore, as embryonic genome activation occurs, the physiology of the developing embryo starts to acquire a more somatic cell-like physiology (see e.g., FF 60). In concert with the Examiner (Ans. 35), we credit the testimony of Dr. Rieger supporting the inherent ability of early embryos to utilize glutamine dipeptides, specifically ala-gln. In particular, Dr. Rieger testified that Hagemann and Quinn "clearly establish" replacing glutamine with ala-gln in embryo culture media (FF 64). Assuming arguendo that the Gardner II Declaration is sufficient to antedate Hagemann, Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 31 Hagemann has not been relied upon by the Examiner to reject claim 1 (Ans. 38) and is cumulative with Quinn. Therefore, it is unnecessary to consider either Hagemann or the Gardner II Declaration. Quinn suggests that embryos beyond the 8-cell stage grow in culture containing low glucose and glutamine in the form of ala-gln (FF 66). In particular, as noted by the Examiner (Ans. 31), claim 1 is open to embryonic development beyond the 8-cell stage. Thus, as a human embryo, for example, continues to develop beyond the eight cell stage it gains the capacity to utilize ala-gln and a more somatic-cell like physiology (see e.g., FF 60). Therefore, consistent with Roth, Minamoto, Christie, and Gardner 1997, an ordinarily skilled artisan would have had a reasonable expectation of success of growing embryos beyond the 8-cell stage in a culture medium containing ala-gln in place of glutamine. In re O'Farrell, 853 F.2d at 903. 3. conclusion Based on the foregoing, we conclude that (1) the disclosure of Roth enables embryonic development in culture medium, (2) one of ordinary skill in the art would have had a reasonable expectation of success of growing embryos in culture media containing ala-gln instead of glutamine as claimed, and (3) increased cell death during the first 24-48 hours of culturing cells (lag phase) as disclosed by Minamoto and Christie does not teach away from using glutamine dipeptides in early stage embryonic culture. Therefore, we sustain the rejection of independent claim 1 under Â§ 103 over the combined teachings of either Nakazawa or Gardner and Roth alone or further with Minamoto or Christie. Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 32 B. Ground #14 1. legal principle Claims are definite if they "set out and circumscribe a particular area with a reasonable degree of precision and particularity. It is here where the definiteness of the language employed must be analyzed -- not in a vacuum, but always in light of the teachings of the prior art and of the particular application disclosure as it would be interpreted by one possessing the ordinary level of skill in the pertinent art." In re Moore, 439 F.2d 1232, 1235 (CCPA 1971). 2. analysis Claim 1, the only independent claim on appeal, recites a method comprising performing a first IVF procedure which results in "embryonic development to the eight-cell stage." As noted by the Examiner, an eight- cell embryo differs in scope from a "gamete, zygote or embryo" as recited in dependent claims 14, 15, and 19 (Ans. 47). Appellant requests that the Board enter the Amendment filed August 16, 2007 addressing this informality which was denied entry by the Examiner (App. Br. 24-25) for reasons provided in the Right of Notice of Appeal mailed November 19, 2007 (hereinafter "RAN") (Ans. 46; RAN 4- 7). The Amendment filed August 16, 2007 was denominated "Response to Second Action Closing Prosecution Under 37 C.F.R. Â§ 1.951(a) In Inter Partes Reexamination." When an amendment submitted by the Patent Owner after the Action Closing Prosecution is denied entry by the Examiner, the Patent Owner may file a petition under 37 C.F.R. Â§ 1.181 requesting entry of the amendment. See Manual of Patent Examining Procedure, Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 33 eighth edition, Â§ 2673.02. Denial of entry of an amendment is not an appealable matter within the jurisdiction of the Board. Alternatively, Appellant argues that one skilled in the art would immediately recognize that the phrase "transferring the gamete, zygote or embryo" as recited in claims 14 and 15 and the phrase "cryopreservation of the gamete, zygote or embryo" in claim 19 must be interpreted as "transferring the embryo" and "cryopreservation of the embryo," respectively (App. Br. 25). This argument fails because none of a gamete, a zygote, or an embryo is of the same scope as an eight-cell embryo. For example, Appellant has not directed our attention to where the specification of the 235 patent defines an embryo as "development to the eight-cell stage," as opposed to development to the four-cell or sixteen-cell stage. 3. conclusion Based on the foregoing, we conclude that independent claim 1 does not provide antecedent basis for the phrase "transferring the gamete, zygote or embryo," as recited in claim 14. Therefore, we sustain the rejection of claims 14, 15, and 19 under 35 U.S.C. Â§ 112, second paragraph, as indefinite in scope. IV. Order Upon consideration of the record, and for the reasons given, it is ORDERED that the decision of the Examiner to reject claims 1-6 and 20-22 as unpatentable under 35 U.S.C. Â§ 103(a) over Nakazawa and Roth alone or further with Christie or Minamoto is AFFIRMED; FURTHER ORDERED that the decision of the Examiner to reject claims 1-6, 8, 10-12, 14, 15, and 19-23 as unpatentable under 35 U.S.C. Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 34 Â§ 103(a) over Gardner 1997 and Roth alone or further with Christie or Minamoto is AFFIRMED; FURTHER ORDERED that the decision of the Examiner to reject claims 7 and 9 as unpatentable under 35 U.S.C. Â§ 103(a) over Gardner 1997 and Roth alone or further with Christie or Minamoto as applied to claims 1- 6, 8, 10-12, 14, 15, and 19-23 and further with Conway-Myers, Cohen, and Huszar is AFFIRMED; FURTHER ORDERED that the decision of the Examiner to reject claims 14, 15, and 19 as unpatentable under 35 U.S.C. Â§ 112, second paragraph, as indefinite is AFFIRMED; and, FURTHER ORDERED that requests for extensions of time in this inter partes reexamination are governed by 37 C.F.R. Â§ 1.956. See 37 C.F.R. Â§ 41.79. AFFIRMED rvb Patent Owner FOLEY & LARDNER LLP 150 East Gilman Street P.O. Box 1497 Madison, WI 53701-1497 Appeal 2009-015409 Reexamination Control 95/000,146 Patent 6,838,235 B2 35 Third Party Requester WILLIAM W. JONES Patent Counsel 6 Juniper Lane Madison, CT 06433