Citations
- 446 F. Supp. 2d 297
Full opinion text
FINDINGS OF FACT AND CONCLUSIONS OF LAW
JORDAN, District Judge.
TABLE OF CONTENTS
I. INTRODUCTION.........................................................303
II. FINDINGS OF FACT......................................................303
A. The Parties ...........................................................303
B. Technological Background ..............................................303
1. Alpha-Amylases....................................................303
2. Protein Engineering................................................304
3. Claims of the ’031 Patent............................................305
C. Prosecution History of the ’031 Patent....................................306
1. The Original Claims ................................................306
2. The First Office Action..............................................307
a. Written Description and Enablement..............................307
b. Obviousness....................................................308
3. Novozymes’s Response..............................................308
4. The Second Office Action............................................309
5. Evidence of Unexpected Results......................................310
6. Allowance.........................................................311
D. The Machius Reference.................................................312
E. Marketing of Spezyme Ethyl............................................313
F. Amino Acid Sequences of Spezyme Ethyl and G997.........................313
III. CONCLUSIONS OF LAW..................................................315
A. Claim Construction.....................................................315
1. “Bacillus stearothermophilus Alpha-Amylase”.........................317
a. The Parties’ Proposed Constructions..............................317
b. The Court’s Construction........................................317
i. The Term is Not Limited to SEQ ID NO:3.....................317
ii. The Term is Not Limited to Proteins of a Particular Length.....318
2. “% Homology”.....................................................319
a. The Parties’ Proposed Constructions..............................319
b. The Court’s Construction........................................319
B. Infringement..........................................................321
1. Claim 1 ...........................................................321
2. Claim 3 ...........................................................322
3. Claim 5 ...........................................................322
C. Invalidity.............................................................322
1. Obviousness.......................................................323
a. Suzuki and Bisg°ard-Frantzen ...................................323
b. Unexpected Results.............................................324
i. Suzuki Conditions..........................................324
ii. Experimental Procedures....................................325
iii. Expected Results...........................................327
c. Machius.......................................................328
2. Enablement .......................................................329
D. Unenforceability.......................................................330
1. Inequitable Conduct................................................330
a. The Borchert Declaration........................................331
b. Failure to Disclose the Machius Reference.........................332
2. Prosecution Laches.................................................333
IV. SUMMARY OF CONCLUSIONS..... ......................................333
I. INTRODUCTION
This is a patent infringement case. No-vozymes A/S (“Novozymes”) has sued Genencor International, Inc. (“Genencor”) and Enzyme Development Corporation (“EDC”) (collectively “Defendants”), alleging infringement of U.S. Patent No. 6,867,031 (issued Mar. 15, 2005) (the “ ’031 patent”). Trial of this matter has been bifurcated: a bench trial on patent infringement, invalidity, and unenforceability was held from March 6 to March 9, 2006, and a second bench trial on willfulness and damages is scheduled to begin on October 10, 2006. The following, issued pursuant to Federal Rule of Civil Procedure 52(a), are my findings of fact and conclusions of law as to the liability issues tried last March.
For the reasons that follow, including my decision on claim construction, I conclude that Defendants have infringed claims 1, 3, and 5 of the ’031 patent, that those claims are valid, and that the ’031 patent is enforceable. Accordingly, this case will proceed to the second phase trial to decide the issues of willfulness and damages.
II. FINDINGS OF FACT
A. The Parties
1. Novozymes is a Danish corporation with a place of business in Bagsvaerd, Denmark. (Uncontested Facts, Docket Item [“D.I.”] 101 at ¶ III.A.) Novozymes is the sole assignee of the ’031 patent titled “Amylase Variants.” (’031 patent.)
2. Genencor is a Delaware corporation having a principal place of business in Palo Alto, California. (Uncontested Facts, D.I. 101 at ¶ III.B.) Genencor sells an alpha-amylase product under the brand name Spezyme® Ethyl. (Id. at ¶ III.V.)
3. EDC is a Delaware corporation having a principal place of business in New York, New York. (Id. at ¶ III.C.) EDC is a United States distributor of Genencor’s Spezyme Ethyl. (Id. at ¶ III.W.)
B. Technological Background
1. Alpha-Amylases
4. The ’031 patent relates to alpha-amylase enzymes. (’031 patent, 1:21-22.) Enzymes are catalysts, meaning that they increase the rate of chemical reactions. (Uncontested Facts, D.I. 101 at ¶ III.E; Arnold, D.I. 120, Trial Transcript [“Tr.”] at 143:15-144:1.) The alpha-amylase enzymes described by the ’031 patent are proteins (Arnold, Tr. at 139:4-6) that catalyze the breakdown of alpha-1,4-glucosidic bonds (Uncontested Facts, D.I. 101 at ¶ III.E). Alpha-1,4-glucosidic bonds connect individual glucose molecules together to form starch molecules. (Id.) By breaking those bonds, alpha-amylases “break apart the starch complexes and convert complex starch into smaller, simpler groups of glucose molecules ____” (Id.)
5. “[A]lpha-amylases are useful in a variety of commercial applications that involve the processing of starches [,including] ... the fuel ethanol industry, where ethanol fuel is produced from starch-rich crops such as corn, barley, and wheat.” (Id. at ¶ III.G.) “Alpha-amylases are used in the fuel ethanol industry to liquefy and reduce the viscosity of starch feedstocks so that they are easier to process in the manufacturing plant.” (Id. at ¶ III.H.)
6. In the process of fuel ethanol production, alpha-amylases are typically added to a starch slurry that is subjected to temperatures above 100°C for up to ten minutes, followed by an incubation at 80°C for a few hours. (Borchert, Tr. at 25:8-15.) Hence, the thermostability of the enzyme, its capacity to withstand high temperatures, is important to its effectiveness in industrial applications. (See id. at 25:19-26:4.) By using alpha-amylases with better thermostability, manufacturers can use less of the enzyme and reduce costs. (Id. at 26:5-9, 29:12-20.)
7. One way to improve the thermosta-bility of alpha-amylases is to add high levels of calcium to the starch slurry. (Id. at 26:15-22.) But high calcium levels interfere with later processing, so that the calcium has to be removed, an additional step that is inconvenient and increases costs. (Id. at 26:25-27:14, 29:18-20.)
8. The ’031 patent is directed at alpha-amylases, produced by protein engineering, that are thermostable in industrial applications, without the need for added calcium. (Id. at 26:5-11; see generally ’031 patent, 9:48-11:65.)
2. Protein Engineering
9. Like all proteins, alpha-amylases are polymers composed of amino acids linked together by peptide bonds into a linear chain. (Uncontested Facts, D.I. 101 at ¶ III.I.) Each protein chain includes many amino acids assembled in a particular order, so that a particular protein may be identified by its ordered sequence of amino acids. (Id.) That ordered sequence is referred to as the protein’s “amino acid sequence” or its “primary structure.” (Id.)
10. “One end of a protein chain of amino acids is called the ‘N-terminus,’ and the other end is called the ‘C-terminus.’ ” (Id. at ¶ III.J.) Scientists write an amino acid sequence by listing the amino acids in order from the N-terminus to the C-terminus, using one-letter codes for each of the twenty naturally occurring amino acids. (Id. at ¶ III.K) For example, the amino acid alanine has the one-letter code “A”, and asparagine has the one-letter code “N”. (’031 patent, 6:44-47.) Protein sequences written using the one-letter codes are disclosed in the ’031 patent. (Id. at Fig. 1.)
11. “It can be informative when comparing proteins to compare their respective amino acid sequences.” (Uncontested Facts, D.I. 101 at ¶ III.N.) The amino acid sequences are typically “aligned” with one another to achieve a visual correspondence of individual amino acids or groups of amino acids that are common to the proteins being compared. (Id.) Once sequences are aligned, the percentage of identical amino acid mátches in the aligned proteins can be calculated and reported as a percent of “identity,” also referred to at times as “homology.” (Id. at ¶ III.O.) Generally, the alignments and calculations may be done using computer software. (’031 patent, 4:36-45; Devereux, Tr. at 103:20-104:4, 106:11-107:2 (briefly describing the use of software to align protein sequences).)
12. The function of a protein and the conditions under which it can perform that function are determined at least in part by the protein’s amino acid sequence. (Uncontested Facts, D.I. 101 at ¶ III.M.) That is because a particular linear chain of amino acids will fold into a characteristic three-dimensional structure. (Borchert, Tr. at 21:1-7.) The so-called “tertiary” structure of a protein describes the relative positions in three-dimensional space of the protein’s atoms. (Machius, Tr. at 456:13-19.) That tertiary structure is the specific overall shape of the protein (id. at 456:13-15), which determines the protein’s function (id. at 457:15-22).
13. Because of the relationship between protein sequence, structure, and function, one can alter the function or other properties of a protein by altering its sequence. Protein engineering is “the deliberate modification of the amino acid sequence of [a] protein,” so that the protein’s properties can be studied or improved. (Arnold, Tr. at 135:16-21.) Protein sequences can be modified by making substitutions, insertions, or deletions of amino acids in the sequence. (Borchert, Tr. at 23:12-13.)
14. Protein engineers modify a protein sequence by changing the DNA sequence of the gene that encodes that protein. (Al-ber, Tr. at 202:23-203:11; Arnold, Tr. at 139:21-140:5.) Each amino acid in a protein sequence corresponds to a triplet of nucleotides in the DNA sequence of the corresponding gene. (Alber, Tr. at 202:25-203:3.) The DNA sequence may be modified “very precisely” (id. at 203:5-6), thus allowing the modification of protein sequences.
3. Claims of the ’031 Patent
15. Novozymes is asserting claims 1, 3, and 5 of the ’031 patent. (See, e.g., D.I. 118 at 22-26.) Those claims relate to alpha-amylases originally found in a species of bacteria named Bacillus stearothermo-philus but then engineered for improved thermostability. (’031 patent, 65:11-17, 65:21-66:12, 66:16-19.)
16. Specifically, claims 1, 3, and 5 relate to alpha-amylases that have two particular amino acids deleted, those at positions 179 and 180, using the numbering of a reference Bacillus stearothermophilus alpha-amylase sequence given in the patent, “SEQ ID NO:3.” (’031 patent, 65:11-17, 65:21-66:12, 66:16-19.) Those alpha-amylases have improved thermostability without the need for calcium as an added support at high temperature. (Borchert, Tr. at 26:5-11; ’031 patent, 9:48-59, 9:62-66,10:40-48,11:41-65.)
17. Claim 1 of the ’031 patent reads:
A variant of a parent Bacillus stearoth-ermophilus alpha-amylase, wherein the variant has an amino acid sequence which has at least 95% homology to the parent Bacillus stearothermophilus alpha-amylase and comprises a deletion of amino acids 179 an [sic] 180, using SEQ ID NO:3 for numbering, and wherein the variant has alpha-amylase activity.
(’031 patent, 65:11-17.)
18. Claim 3 reads:
A variant alpha-amylase, wherein the variant has at least 95% homology to SEQ ID NO:3 and comprises a deletion of amino acids 179 and 180, using SEQ ID NO:3 for numbering and wherein the variant has alpha-amylase activity.
(Id. at 65:21-66:12.)
19. Claim 5 reads:
A variant of a Bacillus stearothermophi-lus alpha-amylase, wherein the alpha-amylase variant consists of a deletion of amino acids 179 and 180, using SEQ ID NO:3 for numbering.
(Id. at 66:16-19.)
C. Prosecution History of the ’031 Patent
20. The application that issued as the ’031 patent, Application No. 10/025,648 (the “ ’648 application”), was filed on December 19, 2001. (’031 patent, cover page.) The ’648 application was filed as a division of Application No. 09/902,188, filed July 10, 2001, which was a continuation of Application No. 09/354,191, filed July 15, 1999, which was a continuation of Application No. 08/600,656, filed February 13, 1996, which was a continuation of International Application No. PCT/DK96/00056, filed February 5, 1996. (Id.) The ’648 application claimed priority to a group of four Danish patent applications filed from February 3 to October 6, 1995. (Id.) To support an effective filing date for a United States patent, a foreign priority application must provide a sufficient written description of what is claimed in the United States patent. In re Gosteli, 872 F.2d 1008, 1010-11 (Fed.Cir.1989). The earliest of the four Danish applications that discloses a Bacillus stearothermophilus alpha-amylase is the one that was filed on March 29, 1995, Application No. PA 1995 00336. (Trial Exhibit [“TX”] 101, D.I. 121 at [ A-XXXX-XXXX ].) Thus, according to Defendants (D.I. 115 at 4-5, ¶¶ 22-23), no earlier Danish application will support an effective filing date for the ’031 patent, and the earliest possible effective filing date for that patent is March 29,1995.
1. The Onginal Claims
21. A preliminary amendment to the ’648 application was filed on December 19, 2001. (TX 101, D.I. 121 at A-7045-48.) That amendment canceled claims 1-29 and added 18 new claims, numbered 30-47. (Id.) New claims 30-39 were directed to alpha amylases, and claims 40-47 were directed to DNA, vectors, host cells, and methods of expressing the alpha-amylases. (Id.)
22. Claim 30 was an independent claim that read:
A variant of a parent alpha-amylase enzyme, wherein said parent alpha-amylase has an amino acid sequence which has at least 80% homology to SEQ ID NO:3, and wherein said variant comprises deletions at positions equivalent to positions 179 and 180 in SEQ ID NO:3 (using SEQ ID NO:3 for numbering).
(Id. at A-7045.)
23. Claims 31-34 depended directly from claim 30. (Id. at A-7045-46.) Claims 31-33 specified the homology between the parent alpha-amylase amino acid sequence and SEQ ID NO:3 as at least 85%, 90%, and 95% respectively. (Id. at A-7045.) Claim 34 claimed “[t]he variant of claim 30, wherein the variant further comprises amino acid substitutions of a cysteine at positions equivalent to positions 349 and 428 in SEQ ID NO:3.” (Id. at A-7046.)
24. Claim 35 was an independent claim that read:
An isolated alpha-amylase enzyme comprising an amino acid sequence having an amino acid sequence which has at least 80% homology to SEQ ID NO:3, modified by having deletions at positions equivalent to positions 179 and 180 in SEQ ID NO:3.
(Id.)
25. Claims 36-39 depended directly from claim 35. (Id.) Claim 36 claimed “[t]he alpha-amylase enzyme of claim 35, wherein said alpha-amylase enzyme is further modified by having amino acid substitutions of a cysteine at positions equivalent to 349 and 428 in SEQ ID No:3.” (Id.) Claims 37-39 specified the homology between the alpha-amylase amino acid sequence and SEQ ID NO:3 as at least 85%, 90%, and 95% respectively. (Id.)
2. The First Office Action
26. The examiner issued an office action on July 29, 2003. (Id. at A-7619-29.) After a restriction requirement, the applicants elected to prosecute claims 30-39. (Id. at A-7621, A-7636.)
a. Written Description and Enablement
27. The examiner rejected claims 30-34 for failing to meet the written description requirement of 35 U.S.C. § 112. (Id. at A-7623.) According to the examiner, the specification only described a few representative species of the genus of enzymes that were included in the scope of claims 30-34. (Id.) As written, those claims included variant enzymes “with any number of alterations of the parent enzyme as long as amylase activity is maintained.” (Id.) Given that scope, the specification failed to sufficiently describe the invention so that a skilled artisan would recognize that the applicants were in possession of the invention. (Id.)
28. The examiner also rejected claims 30-34 under 35 U.S.C. § 112, because the specification did not enable one skilled in the art to practice the full scope of the claims. (Id. at A-7624-27.) While claims 30-34 required the parent alpha-amylases to have at least 80% homology to SEQ ID NO:3, the variants were not so limited. (Id. at A-7624.) The claims covered variants “with any number of alterations of the parent enzyme as long as amylase activity is maintained,” and as long as the alterations included the deletions of the two amino acids at positions equivalent to 179 and 180 in SEQ ID NO:3. (Id.) Considering the number of possible variants, the unpredictability of the art of protein engineering, and the lack of any detailed instruction as to which regions of the alpha-amylase enzymes could be modified without destroying the alpha-amylase activity, the examiner concluded that the specification did not enable one to make variants with any number of alterations relative to the parent. (Id. at A-7625-27.)
29. The examiner noted that the written description and enablement rejections for claims 30-34 would be overcome if claim 30 was amended so that the class of claimed variants were required to have “at least 80% sequence identity to SEQ ID NO:3.” (Id. at A-7627.)
b. Obviousness
30. The examiner rejected claims 30-33, 35, and 37-39 under 35 U.S.C. § 103(a) as obvious in light of two references. (Id. at A-7627-28.)
31. The first reference (“the Suzuki reference” or “Suzuki”), titled “Amino Acid Residues Stabilizing a Bacillus a-Amylase against Irreversible Thermoinactivation” and authored by Suzuki et al., was published in the Journal of Biological Chemistry in 1989. (TX 115, D.I. 122 at A-8233-38.) Suzuki disclosed alpha-amylases from Bacillus amyloliquefaciens that were modified by the deletion of two amino acids at positions 176 and 177. (Id. at A-8233, A-8237-38.) Alpha-amylases with those deletions had better thermostability. (Id. at A-8237-38.)
32. The second reference (“the Bisgárd-Frantzen reference” or “Bisgárd-Frantzen”) was a patent application, Publication No. WO 95/10603, published April 20, 1995 and titled “Amylase Variants.” (TX 177, D.I. 122 at A-8403-507.) Bisgárd-Frantzen disclosed that the alpha-amylases of Bacillus amyloliquefaciens, Bacillus stearothermophilus, and Bacillus licheniformis were “highly homologous on the amino acid level.” (Id. at A-8413-14.) A sequence alignment of those alpha-amy-lases showed that positions 176 and 177 of the Bacillus amyloliquefaciens enzyme corresponds to positions 179 and 180 of the Bacillus stearothermophilus enzyme. (Id. at A-8415-16.)
33. According to the examiner, “it would have been obvious to one of ordinary skill in the art to introduce the [deletions] disclosed by Suzuki ... into the corresponding positions [179 and 180] of Bacillus stearothermophilus a-amylase,” in order to increase its thermostability. (TX 101, D.I. 121 at A-7628.) Because of the similarity between the Bacillus amylo-liquefaciens and Bacillus stearothermo-philus alpha-amylases revealed by Bisgárd-Frantzen, one of ordinary skill in the art would, the examiner concluded, reasonably expect that the change in the Bacillus stearothermophilus alpha-amylase would give similar results as those disclosed by Suzuki for the Bacillus amy-loliquefaciens alpha-amylase. (Id.)
3. Novozymes’s Response
34. On January 13, 2004, Jason Gar-bell, an in-house patent attorney for Novo-zymes (Garbell, Tr. at 4:13-17), sent an email message to a group at Novozymes, including the inventors of the ’031 patent, commenting on the examiner’s obviousness rejection. (TX 110, D.I. 122 at A-8169-70.) In that e-mail, Mr. Garbell proposed two options for responding to the rejection: “Option 1” was to show by experiment that the claimed deletion in Bacillus stearothermophilus alpha-amylase yielded unexpected results, and “Option 2” was to add limitations to the rejected claims such as those in claims 34 and 36, which were not rejected for obviousness. (Id. at A-8170.) Mr. Garbell preferred Option 1, because that would not require narrowing the scope of the claims and would limit the design-around opportunities available to competitors. (Id.) Genencor was known to be one of those competitors. (Id. at A-8169 (referring to Genencor as GCI); Borchert, Tr. at 355:8-356:15.)
35. Mr. Garbell needed to respond to the office action by January 29, 2004 (TX 110, D.I. 122 at A-8170), and the scientists at Novozymes informed him that the experimental work required for Option 1 could not be completed by then (id. at A-8169, A-8171). It was suggested that proceeding with Option 2 would “give time” for carrying out the experiments, which might then support broader claims. (Id. at A-8169.)
36. On January 14, 2004, the applicants filed an amendment. (TX 101, D.I. 121 at A-7632-37.) In response to the obviousness rejection, claims 34 and 36 were canceled, and independent claims 30 and 35 were amended to add the limitations of claims 34 and 36, respectively. (Id. at A-7634, A-7637.)
37. In response to the written description and enablement rejections, the applicants amended claim 30 to recite that the variant “has at least 80% identity to said parent alpha-amylase.” (Id. at A-7634, A-7636-37.)
4. The Second Office Action
38. The examiner issued another office action on April 6, 2004. (Id. at A-7717-27.) The obviousness rejection was withdrawn. (Id. atA-7719.)
39. The examiner maintained the rejection of claims 30-33 for failure to meet the written description requirement. (Id. at A-7719-21.) Again, the examiner said that the specification described only a few of the many alpha-amylases covered by those claims, so that one skilled in the art could not conclude that the applicants had possession of the claimed invention. (Id. at A-7720-21.) The examiner suggested that the claims be narrowed by requiring the variants to have alpha-amylase activity. (Id. atA-7721.)
40. The examiner rejected claims 30-33, 35, and 37 for failure to meet the enablement requirement. (Id. at A-7721-26.) While the applicants had stated in their response to the first office action that claim 30 had been amended following the examiner’s suggestion (id. at A-7636-37), the examiner noted that the applicants “did not in fact amend the claim exactly as suggested” (id. at A-7725). Rather than requiring the variant to have at least 80% identity to SEQ ID NO:3, as the examiner had suggested (Finding of Fact [“FF”] ¶ 29), the applicants amended claim 30 to require the variant to have at least 80% identity to the parent alpha-amylase (FF ¶ 37). However, the examiner acknowledged that the amendment was “similar” to her suggestion. (TX 101, D.I. 121 at A-7725.) Still, while the examiner recognized that the scope of claims 30-33 had been narrowed, “upon further reconsideration” she believed that the specification did not enable one of ordinary skill to make variants with at least 80% identity to the parent without undue experimentation. (Id.) As in the first office action, the examiner noted the large number of possible variants, the unpredictability of the art, and the lack of guidance about which regions of the alpha-amylases could be modified without losing enzyme activity. (Id. at A-7721-26.) Claims 35 and 37, which required the variant to have at least 80% and 85% homology to SEQ ID NO:3, respectively, were also not supported by an enabling disclosure, the examiner concluded. (Id.)
41. The examiner noted that the specification was enabling for alpha-amylases “having at least 90% homology to SEQ ID NO:3” and having the claimed modifications. (Id. at A-7721.) Thus, claims 38 and 39, which required at least 90% or 95% homology to SEQ ID NO:3, respectively, would be allowable, the examiner indicated, if they were rewritten in independent form. (Id. at A-7726.)
5. Evidence of Unexpected Results
42. In an interview on September 3, 2004, Mr. Garbell and Dr. Borchert discussed with the examiner the obviousness rejection from the first office action. (Id. at A-7798-99.) The examiner stated that she was shown a draft declaration that “appear[ed] to show results sufficiently unexpected to overcome” the previous obviousness rejection. (Id. at A-7799.) Those unexpected results were later submitted to the examiner in the form of a declaration under 37 C.F.R. § 1.132, dated September 6, 2004 (the “Borchert Declaration”). (Id. at A-7739-56.)
43. The Borchert Declaration described the results of an experiment comparing the thermostability of Bacillus stearothermophilus alpha-amylase (“BSG”), with and without deletion of residues179 and 180, and Bacillus amy-loliquefaciens alpha-amylase (“BAN”), with and without deletion of residues 176 and 177. (Id. at A-7739, ¶ 3.) BAN was the alpha-amylase studied by Suzuki. (FF ¶ 31.)
44. Genes for the variants of BSG and BAN with their respective deletions (“BSGdel” and “BANdel” respectively) were constructed by standard methods and the gene sequences were confirmed by DNA sequencing. (TX 101, D.I. 121 at A-7740, ¶ 4.) Cells producing each of the four enzymes, BSG, BSGdel, BAN, and BAN-del, were grown under identical conditions, and the alpha-amylases were separated from the cells by centrifugation. (Id. at A-7740, ¶ 5.) The alpha-amylase-containing supernatants were diluted in buffer containing 0.1 mM calcium and incubated at 80°C in a PCR machine, and at various times the alpha-amylase activity was measured. (Id.) According to the Borchert Declaration, the incubation temperature of 80°C was “the highest temperature where [all four alpha-amylases] could be reliably compared.” (Id.; see also Borchert, Tr. at 686:8-688:17 (describing the calibration experiments to determine the temperature at which all four enzymes would yield reliable measurements).)
45. The alpha-amylase activity of each sample was measured at various times, and the results were reported in tabular form as a percentage residual activity at each time. (TX 101, D.I. 121 at 7741-42, ¶ 6.) BSGdel maintained its activity for the longest period of time: 61% residual activity was measured at 4200 minutes, the last time point of the experiment. (Id.)
46. Four data points were omitted before the data were analyzed. First, two measurements for BSGdel taken at 2881 minutes were omitted by a Novozymes lab technician, Vibeke Holbo, because she noted that the sample used for those measurements had evaporated during incubation. (Holbo, Tr. at 671:3-14; Borchert, Tr. at 384:22-385:2, 412:17-24.) Two other measurements for BSGdel taken at 2940 minutes were omitted by Dr. Borchert, because he noted that the measurements “were extremely far apart” and one showed activity above 130%. (Borchert, Tr. at 386:9-15, 412:25-413:2, 414:8-17.) Dr. Borchert decided that he could not “with any confidence include such measurements in the data analysis.” (Id. at 414:16-17.)
47. A regression analysis was conducted for each data series, and the half-life for each alpha-amylase, i.e. the time at which the alpha-amylase had half of its original activity (Klibanov, Tr. at 515:18-25), was calculated. (TX 101, D.I. 121 at A-7742, ¶ 7.) The four half-lives were: BAN, 0.9 minutes; BANdel, 9.5 minutes; BSG, 92 minutes; BSGdel, 5775 minutes. (Id.) Based on those numbers, Dr. Borchert reported that the deletion of residues 176 and 177 in BAN improved thermostability 11-fold, and the corresponding deletion of residues 179 and 180 in BSG improved thermostability 63-fold. (Id.) Thus, the thermostability was improved 5.7 times as much in BSG as in BAN (63/11=5.7). (Id.)
48. According to Dr. Borchert, the deletion in BSG “has a pronounced and very surprising effect on the thermal stability.” (Id. at A-7743, ¶ 9.) “[The] results are statistically significant and very surprising as the effect of the double deletion in BSG is significantly greater than what would have been expected based on the combined teachings of Suzuki ... in view of Bis-gaard-Frantzen .... ” (Id. at A-7743-44, ¶ 9.)
6. Allowance
49. After the interview on September 3, the applicants submitted an amendment dated September 6, 2004 (id. at A-7733-56) that cancelled all the pending claims and added five new claims, numbered 48-52 (id. at A-7734). For claims 48, 50, and 52, the applicants removed the requirement for cysteine substitutions at positions 349 and 428, which had been added in response to the obviousness rejection from the first office action. (Id.) According to the applicants, an obviousness rejection based on Suzuki and Bisgárd-Frantzen, if it were reasserted by the examiner in response to the broadened claims, would be overcome by the evidence of unexpected results in the Borchert Declaration. (Id. at A-7736-37.)
50. In response to the earlier enablement and written description rejections, the applicants drafted claims 48-49 and 50-51 to require the variants to have at least 95% homology to the parent Bacillus stearothermophilus alpha-amylase and to SEQ ID NO:3, respectively, and to have alpha-amylase activity. (Id. at A-7734-36.) As to the enablement rejection, the applicants noted the examiner’s suggestion that the claims would be enabled if they required the variants to have at least 90% homology to SEQ ID NO:3. (Id. at A-7735-36; see FF ¶41.) The applicants argued that the enablement rejection was “rendered moot by the new claims as the new claims recite a homology of 95%.” (TX 101, D.I. 121 at A-7736.)
51. On September 21, 2004, the examiner issued a notice of allowance. (Id. at A-7791-97.) In her remarks, the examiner stated that the Borchert Declaration “establishes that the claimed variants exhibit unexpectedly large increases in ther-mostability when compared to the increases in thermostability obtained for the corresponding mutations taught by Suzuki et al. As such the claimed variants are non-obvious over the prior art.” (Id. at A-7796.) The examiner made no remarks concerning the written description and enablement rejections. (Id.)
52. Claims 48-52, submitted by the applicants on September 6, 2004, correspond to claims 1-5, issued without further amendment as the claims of the ’031 patent. (Compare id. at A-7734 with ’031 patent, 65:10-66:19.)
D. The Machius Reference
53. Another reference relating to alpha-amylases that was the subject of repeated emphasis during trial is entitled “Crystal Structure of Calcium-depleted Bacillus lichenifomiis a-amylase at 2.2