T 1252/11 (Bead arrays/ILLUMINA) 19-01-2016

Admission of the set of claims according to the main request into the proceedings (Article 13 RPBA)

1. The set of claims according to the respondent's main request is identical to the claims filed as an auxiliary request in response to the board's communication under Article 15(1) RPBA. Since these claims must be regarded as an amendment to the respondent's case after it had filed its reply to the grounds of appeal, it is at the board's discretion to admit and consider them (see Article 13(1) RPBA).

2. The board, exercising its discretion taking into account the criteria specified in Article 13(1) and (3) RPBA, decides to admit and consider the amended claims of the main request. While the appellant is right when saying that these claims could have been filed at an earlier stage of the proceedings, the board holds that the sole amendment introduced into the feature "... said secondary enzymes being attached to said sites on said array" in step c) of the method of claim 1, neither increases the complexity of the case nor raises any issues which the board or the other parties cannot reasonably be expected to deal with without adjournment of the oral proceedings. Thus, the admission of the amended claims into the appeal proceedings does not run contrary to the need for procedural efficiency.

Rule 80 EPC and Article 84 EPC

3. The findings in the decision under appeal concerning amendments introduced into the claims during opposition proceedings (see section 3.1 of the decision) have not been contested by the appellant.

4. As regards the objection under Rule 80 EPC to the amendment introduced into claim 1 of the present main request, the board does not share appellant's view that the insertion of the wording "said [sites]" in step c) of the claimed method has not been occasioned by a ground for opposition.

5. In its statement of grounds of appeal (see page 5, section (c) of the statement), the appellant raised an objection under Article 123(2) EPC arguing that, while the passage on page 87, lines 24 to 34 of the application on file read "... said secondary enzymes being attached to the sites on said array" (emphasis added by the board), the article "the" had been omitted in step c) of the method claimed in claim 1 of the main request then on file.

6. Moreover, in its communication under Article 15(1) RPBA the board indicated that the feature in step c) lacked a clear reference to the sites to which the hybridization complexes are attached. In the board's provisional view, this issue could be relevant to the assessment of inventive step (Article 56 EPC), in particular with regard to the question whether or not the technical effect purported to be achieved ("... increased concentration of the required enzymes is obtained in the immediate vicinity of the reaction ..."; see column 76, lines 43 and 44 of the patent in suit) would be in fact achieved if the secondary enzymes were not attached to the substrate at sites in the immediate vicinity of the hybridization complexes (see section 17 of the communication).

7. In view of the above, the board holds that the amendment introduced into claim 1 of the present main request has been occasioned by either, or both, of the objection(s). These objections are grounds for opposition under Article 100 EPC.

8. As regards the appellant's further objection that, as a result of the introduction of the wording "said" in step c), claim 1 does not meet the clarity requirement of Article 84 EPC, the board disagrees. The wording "... attached to said sites on said array" in step c) of the method of present claim 1 clearly refers to the wording "... said hybridization complexes are attached to sites on an array" in step a). Contrary to appellant's view, the board cannot see any ambiguity in this respect. Hence, the objection under Article 84 EPC is not justified.

Article 123(2)(3) EPC

9. In the decision under appeal, the opposition division found that the subject-matter of the amended claims according to the main request did not extend beyond the content of the application as filed, and that the scope of the claims as granted had not been extended (see section 3.2.4 of the decision).

10. In appeal proceedings, the appellant contested the findings of the opposition division concerning the alleged omission of the feature "capture probe" in step a) of claim 1.

11. The board shares the opposition division's view that a person skilled in the art would not derive from the application as filed that the use of capture probes in the sequencing method disclosed therein was compulsory. For instance, claims 9 and 10 of the application as filed do not require a capture probe for the attachment of the hybridization complexes to a surface of a substrate. Moreover, in the description of the application as filed various possibilities for the attachment of the target sequences to the surface of an array are disclosed. In particular, in the passage starting on page 93, line 19 it is disclosed that the target sequences (and, consequently, the hybridization complexes) are attached directly to the sites of the array. It is apparent from the passage bridging pages 93 and 94 that the attachment can be effected by, e.g., incorporating biotinylated nucleotides into the nucleic acids and coating the microspheres with streptavidin. Chemical crosslinking is also contemplated (see page 94, line 15). In view of this disclosure, the board cannot accept the appellant's argument that the use of the term "directly" must be restricted to the attachment via a capture probe.

12. Nor can the board accept the appellant's argument that the attachment of the hybridization complexes to sites on an array as specified in claim 1 has no basis in the application as filed. Attachment of the target sequences to the sites of an array is - undisputedly - disclosed in the application as filed (see section "Attachment of Target Sequences to Arrays" starting on page 91). Since the hybridization complexes are formed by hybridization of the sequencing primer to the target sequence, this implies that also the hybridization complexes must be attached to those sites.

13. In view of the above, the board concludes that appellant's objections under Article 123(2) EPC are not justified.

14. The opposition division's findings on Article 123(3) EPC were not contested in appeal proceedings.

Article 83 EPC

15. In its statement of grounds of appeal, the appellant contested the opposition division's findings on Article 83 EPC (see section 3.3 of the decision under appeal) relying on essentially the same arguments as in opposition proceedings.

16. As regards the appellant's argument concerning the allegedly insufficient disclosure of an attachment of the hybridization complexes and the secondary enzymes to one and the same bead, the opposition division referred to the passage on page 88, lines 2 to 15 of the application as filed describing the attachment of enzymes to array sites either directly or using flexible linkers. Attachment of the target sequences to the array is described in the passage from page 91, line 1 to page 94, line 17. The appellant has not brought forward any verifiable facts that would preclude attachment of hybridization complexes and secondary enzymes to the same bead using the methods disclosed in the application as filed.

17. It is, in the board's view, doubtful whether the appellant's further objection based on the absence in the claims of an - allegedly essential - feature requiring an encoding/decoding process may be regarded as an objection of lack of sufficient disclosure in the application as filed. But even if so, the board observes that encoding/decoding systems which can be used for random arrays are disclosed in the passage starting on page 101, line 14 of the application as filed.

18. The board therefore concludes that appellant's objections under Article 83 EPC are not justified.

Article 54 EPC

19. The findings in the decision under appeal on the issue of novelty were not contested by the appellant, and the board has no reason to doubt that these findings are correct.

Article 56 EPC

Document (8) as the closest state of the art

20. Document (8) describes a "real time" method of sequencing DNA, based on the detection of base incorporation by the release of pyrophosphate (PPi) which is detected enzymically, e.g. by the generation of light in the luciferase-luciferin reaction. The method allows rapid detection and provision of sequence information. This is achieved by using, in place of dATP, a dATP analogue which does not interfere with the luciferase reaction, and by performing the chain extension and detection, or signal-generation, reactions substantially simultaneously, by including the PPi-detection enzymes in the polymerase reaction step (see page 3, first full paragraph, and page 4, second and third full paragraphs). The DNA may be attached, directly or indirectly, to a solid support which may take the form of microtitre wells or comprise particles, fibres or capillaries (see paragraph bridging pages 8 and 9).

21. In a particular embodiment of the sequencing method, an array format is used. Samples are distributed over a surface, for example a microfabricated chip, allowing analysis of many samples in parallel. The solution containing the enzymes and one nucleotide flows over the surface and the signal produced by each sample is detected (see paragraph bridging pages 14 and 15).

22. In the decision under appeal, the opposition division found that the method of claim 1 as then on file differed from that described in document (8) in that (i) the secondary enzymes, i.e. the enzymes required for detecting PPi, are attached to sites on the array and (ii) the hybridization complex is attached to microspheres associated with discrete individual sites on said array (see section 3.5.4.4 of the decision). According to present claim 1, the secondary enzymes are attached to the sites on the array where the hybridization complexes are attached.

23. As indicated in the passage on page 87, lines 28 to 30 of the application as filed, the technical effect associated with this feature is a faster reaction rate for detection, and the use of less enzyme. This is to be regarded as an improvement over the method of document (8). Hence, the objective problem to be solved is not to provide an alternative sequencing method - as the appellant argued -, but rather to provide an improved sequencing method for a plurality of nucleic acid sequences, as the opposition division stated in the decision under appeal (see page 20, second paragraph).

24. Even though the application as filed does not include experimental data showing that this technical problem is in fact solved by the method of claim 1, it is, in the board's view, credible that co-immobilization of the hybridization complexes and the secondary enzymes at the same sites results in a faster reaction rate for detection of PPi, and evidence to the contrary has not been brought forward, either in opposition or appeal proceedings.

25. In the appellant's view, the solution proposed in claim 1 was obvious in view of document (18). This document describes a luminescent immobilized enzyme test system for the detection of PPi using a column filled with Sepharose beads on which a pyrophosphatase (e.g. ATP sulfurylase) and firefly luciferase are co-immobilised, and a continuous flow of saturating concentrations of substrate through the column. The sample containing PPi is injected in the system and luminescence is detected using a luminometer (see Figure 2). The system is said to allow for automation (see lines 10 to 12 of the abstract). It is stated on page 271, right-hand column:

"The catalytic responsiveness of a system of enzymes that carry out consecutive reactions is significantly greater when they are coimmobilized than when they are free in solution, as reflected in the rapidity with which the steady-state rate is attained (21) and in the overall reaction rate when the first reaction is reversible (22). This is assumed to be due to the spatial proximity of the coimmobilized enzymes and the high local concentration of the intermediate that arises because of the diffusional resistances within the unstirred solvent layers (23)."

26. It should be noted that the statements in this passage of document (8) are of a general nature and do not specifically relate to the enzymes involved in PPi detection. A person skilled in the art could possibly derive from this passage that co-immobilisation of the two enzymes involved in the PPi detection reaction onto Sepharose beads may result in a greater catalytic responsiveness and a faster reaction rate. However, neither in this passage nor anywhere in document (8) is it suggested to immobilise the enzymes on an array at the sites where the hybridization complexes are attached, i.e. where PPi is released during the sequencing reaction. Thus, even if the skilled person had combined the teachings of documents (8) and (18), he/she would have not arrived at the method of present claim 1 without applying inventive skills. Hence, in view of these two documents the subject-matter of claim 1 is considered to involve an inventive step within the meaning of Article 56 EPC.

Document (5) or (6) as the closest state of the art

27. Both documents (5) and (6) describe the same method of sequencing DNA which is based on measuring of the PPi generated by a polymerisation reaction and makes use of a series of precisely ordered capillary columns each containing an enzyme covalently attached to Sepharose 4B. Document (6) is a scientific publication published in 1988 and document (5) the corresponding US patent granted and published two years later. A schematic diagram of the DNA sequencer used in the method is shown in Figure 1 of either document (5) or (6):

FORMULA/TABLE/GRAPHIC

28. In the decision under appeal (see section 3.5.4.2), the opposition division held that the method described in documents (5) and (6) differs from the method of claim 1 as then on file in that (i) the hybridization complexes are not attached to microspheres associated with individual sites on a solid support, in particular an array and (ii) the secondary enzymes (e.g. ATP sulfurylase and luciferase) are not attached to sites on the same array, but to different solid supports, specifically columns. The opposition division observed that the technical effect underlying the method of the present invention, i.e. an increased reaction rate resulting from the secondary enzymes being attached in immediate vicinity of the polymerisation reaction, could not be achieved by applying the method of documents (5) and (6). Nor could this method be applied to the sequencing of a plurality of target nucleic acids in parallel. This applies, mutatis mutandis, also to claim 1 of the present main request.

29. In the view of the opposition division, starting from document (5) and/or (6) the problem to be solved was to provide a method of sequencing a plurality of target nucleic acids using the PPi-based sequencing-by-synthesis with an increased reaction rate. The board agrees. As stated in paragraph 24 above, from a technical point of view - and in the absence of any evidence to the contrary - it is credible that the method proposed in claim 1 solves this technical problem.

30. The sole issue that remains to be considered is whether or not the proposed solution was obvious to a person skilled in the art. The board notices that in the passage bridging pages 434 and 435 of document (6) various suggestions are made as to how to improve the method described therein. This passage reads:

"Other areas of research can improve this method. Solid support matrixes such as silica gel or glass beads may allow faster flow rates that will decrease the sequencing time. An analog of dATP that is normally incorporated into the DNA by the polymerase but is unable to bind to luciferase or development of a luciferase that shows greater substrate specificity would clearly be useful. Another possible area of investigation is development of a solid support, coupled to a mixture of luciferase and ATP sulfurylase, which is also able to bind the DNA sample. The entire sequencing could be carried out in one column. The use of chain-terminating nucleotides to keep sequencing in phase for more complicated templates should be investigated."

31. This passage merely hints at a possible development of a single column to which a mixture of secondary enzymes as well as the DNA sample is attached. However, there is no suggestion whatsoever as regards the use of an array having individual sites to which the DNA samples and the secondary enzymes are attached. Even if, as the appellant contended, a person skilled in the art could have resorted to other pieces of prior art, such as documents (8) and/or (18), for the reasons given in paragraph 26 above he/she would not have arrived at the claimed invention without applying inventive skills. In the board's view, the appellant's line of argument on lack of inventive step relying on a combination of up to four different documents (documents (5)/(6), (8), (18) and (7) (see section X above) is clearly tainted with hindsight.

32. Summarising the above, the board concludes that, in view of the documents relied upon by the appellant in appeal proceedings, its objection of lack of inventive step is not justified.

Adaptation of the description - Remittal (Article 111 EPC)

33. Since the claims according to the main request on file have been found to meet the requirements of the EPC, the board decides to remit the case to the opposition division for adaptation of the description to the amended claims.

34. In accordance with the jurisprudence of the Boards of Appeal (see e.g. decision T 1808/06 of 14 February 2008; see section 2 of the Reasons), Article 84 EPC is to be taken into account for the adaptation of the description (see Article 84 EPC, second sentence: "[The claims] shall ... be supported by the description"). Inconsistencies between the claims and the description must be avoided, and references to embodiments which are not encompassed by the amended claims should normally be deleted.

Conclusion

35. In view of the findings above, the appellant's request to revoke the patent must fail.