T 0366/16 (Method and kit for nucleic acid isolation/CAMBRIDGE) 03-09-2021

Reasons for the Decision

Admission of documents D13 to D16 into the appeal proceedings

1. The board sees no reason to overturn the opposition division's decision to admit documents D13 to D16 into the proceedings. These and other documents were submitted by both parties only a few days prior to the oral proceedings before the opposition division. The opposition division "decided to admit all of these documents, since they were considered important to establish common practise in the field regarding inclusion of hazardous agents in nucleic acid isolation kits", and based its decision in part on these documents, after hearing the parties on this issue (see decision under appeal, points 2.3.3.2.1 and 2.3.3.2.2, Minutes, points 14 to 16). In the board's opinion the opposition division may establish, based on documentary evidence, what was common practise for the skilled person at a particular date. It has the discretion to take into account this evidence according to Article 114(2) EPC, even if the evidence is late filed. In this context, prima facie relevance of late filed documents is one of the admission criteria to be considered.

2. The admission of documents D13 to D16 was thus not incorrect. The EPC does not provide an express legal basis for excluding in appeal proceedings documents which were admitted into the first-instance proceedings. At least if the opposition division correctly used its discretion to admit the documents and based the decision under appeal on them (see e.g. T 1852/11, Reasons 1.3), as was done in the present case, the Board sees no reason to exclude them from the appeal proceedings.

Auxiliary request 6

Claim interpretation - claim 1

3. Claim 1 is directed to a method for isolating a nucleic acid comprising at least a binding, a washing and an elution step as defined in steps (i) to (iii). Claim 1 further specifies that "the wash solution comprises a buffer with a buffering range that encompasses a pH that is higher than the first pH [i.e. "an acidic pH" applied for binding the nucleic acid to a solid phase, comment added by the board], and the wash solution is at a pH that is within a buffering range of the binding buffer but lower than the buffering range of the buffer of the wash solution, and wherein the pH of the wash solution is pH 6.0 or less".

4. Thus, claim 1 defines for the wash buffer various pH-related requirements, which affect the binding buffer cited in step (i) of claim 1 too:

4.1 The wash buffer's buffering range as defined in step (ii) of claim 1 has to encompass at least one pH value that is higher than the "acidic" pH used for the binding buffer in step (i) of claim 1. A definition of "acidic" is not given in the patent. According to the term's common meaning in the art, a pH below 7.0 is generally considered as acidic, a pH above 7.0 as alkaline, while pH 7.0 is neutral. Accordingly, claim 1 encompasses wash buffers with "acidic" and "alkaline" buffering ranges (i.e. their buffering range is either below or above pH 7.0) that include either the "acidic pH" of the binding buffer, or not. It has not been contested that the term "buffering range" in claim 1 describes the buffer's pH range wherein added acids and bases are effectively neutralised, while a relatively constant pH is maintained. In other words, the term relates to the buffer's effective buffering capacity.

4.2 The wash buffer must further be used at a pH which lies within the "buffering range" of the binding buffer, but below its own buffering range, wherein the highest pH is 6.0 or lower.

4.3 The buffering range of the binding buffer is not defined in claim 1, except that it must comprise the pH used for the wash buffer. The binding buffer is used at an "acidic" pH, which in the absence of any definition, lies within or outside of the buffer's own buffering range. Moreover, according to step (iii) of claim 1, the binding buffer must be used at a pH that is lower than pH 6.5.

4.4 The elution of the bound nucleic acids as defined in step (iii) of claim 1 is performed in the pH range "6.5-10", which has to be higher than the pH used for the binding buffer in step (i). The type of solvent used for eluting the nucleic acids is not defined in step (iii). Hence, water or buffers may be used. Furthermore, since the buffering range of the wash buffer is not defined in claim 1 in relation to the solvent used for elution, the claim encompasses wash buffers with buffering ranges that overlap with the pH of the solvent used for elution, or not.

4.5 Accordingly, claim 1 comprises various combinations of binding buffers, wash buffers, and solvents for eluting the bound nucleic acids, comprising buffers or water. For illustration purposes only, two examples of such buffer combinations falling within claim 1 are given in the following to demonstrate the interrelationship of the various parameters defined in steps (i) to (iii) of claim 1:

An acetate binding buffer (buffering range: pH 3.6 to 5.6) can be used between pH 1.0 < 5.9 (i.e. all pHs are "acidic", below pH 6.5, and lower than the upper limit of pyridine's buffering range), if combined with a pyridine wash buffer (buffering range: pH 4.9 to 5.9) used between pH 3.6 and 4.8 (i.e. all pHs are <= 6.0, lie within the buffering range of the acetate binding buffer, but below pyridine's buffering range);

a citrate binding buffer (pK2, buffering range: 3.0 to 6.2) can be used at pH 1 < 5.6 (i.e. all pHs are "acidic", below pH 6.5, and lower than the upper limit of acetate's buffering range), if combined with an acetate wash buffer (buffering range: pH 3.6 to 5.6) when used between pH 3.0 and 3.5 (i.e. all pHs are <= 6.0, lie within citrate's buffering range, but below acetate's buffering range). The buffering ranges of the exemplary buffer combinations indicated above are disclosed in Table 1 on pages 3 to 5 of the patent.

The elution step for both examples can be performed by any buffer or water at a pH between 6.5 to 10.

4.6 Claim 1 further excludes from the method the presence of chaotropic agents and organic solvents.

Novelty

5. At the oral proceedings, the board concluded that auxiliary request 6 was novel over the disclosure of document D2. In view of the finding that the method of claim 1 of auxiliary request 6 lacks an inventive step (see below), the board sees in the present case no merit in providing a detailed reasoning as to why the method of claim 1 is considered novel.

Inventive step

Closest prior art and technical problem

6. Document D2 is one of the documents cited by both parties as closest prior art for the method of claim 1.

7. Document D2 discloses a method for isolating nucleic acids from liquid samples based on solid carriers (see column 1, lines 4 to 6). The binding of the nucleic acids to the solid material is performed under acidic conditions in the range of pH 1 to 6. The method uses further a wash step, likewise performed under acidic conditions (<= pH6.5), followed by an elution step of the nucleic acids from the carrier under alkaline conditions in the range of pH 7.5 to 9 (see column 2, lines 5 to 10, 31 to 35).

7.1 Document D2 discloses in the context of binding that the binding is "carried out by means of simple lowering of the pH to below pH 6. For this, a binding buffer is used which can maintain a pH range from 1 to 6, preferably from 3 to 5. As is known, the purine bases of the nucleic acids have improved stability in the preferred pH range. Suitable buffers are, for example, formate, acetate, citrate buffers or other buffer systems which have adequate buffer capacity in the pH range mentioned" (see column 3, lines 11 to 18).

7.2 Regarding the washing, document D2 mentions that the "carrier material with the nucleic acids bound thereto can be washed with the described binding buffer or with other phosphate-free buffers, where the pH of this buffer should be between 4 and 7, preferably between 4.5 and 6.5", and that the "wash buffers disclosed according to the invention do not elute the bound nucleic acids even if these have been adsorbed on the carrier material with addition of chaotropic substances. In a wash buffer according to the present invention, additions of organic solvents and/or chaotropic substances are not necessary in order to avoid the premature elution of nucleic acids" (see column 3, lines 36 to 51).

7.3 As regards the elution of the bound nucleic acids, document D2 reports that "it has been found that the nucleic acid purified in this way can be dissolved from the solid phase by a simple increase in the pH to above 7.5. The elution buffer used for this should maintain a pH range from 7.5 to 9, preferably 8 to 8.5. Suitable buffers are, for example, tris HCl buffer, tricine, bicine and other buffers which buffer in this pH range, preferably tris/HCl" (see column 3, lines 60 to 65).

7.4 In summary, the generic concept underlying the nucleic acid isolation method of document D2 is that the binding and washing steps are performed under acidic conditions (preferably in the pH ranges of 3 to 5, and 4.5 to 6.5, respectively), which cause a stable binding of the nucleic acids to the solid material, while the nucleic acids are eluted under alkaline conditions (preferably in the pH range of 8 to 8.5). The method does not use any organic solvents and chaotropic agents, since a simple pH switch between acidic and alkaline conditions is sufficient for obtaining high yields of isolated nucleic acids. While document D2 reports that for achieving this purpose the binding and elution buffers should "maintain" the pH in the respective ranges indicated above, i.e. show an "adequate buffer capacity" or buffering range, no such maintenance is mentioned in the context of washing which is performed at a pH within the ranges indicated above.

7.5 Examples 1 to 3 of document D2 disclose the exclusive use of Tris-HCl as buffer for washing and eluting the bound nucleic acids at pHs 6.5 and 9, respectively (see Table in Example 1 of column 4). It is uncontested that the Tris-HCl buffer was intentionally selected in document D2 for these purposes. The use of Tris-HCl allows, after washing, the direct elution of the bound nucleic acids by switching the pH from 6.5 to 9 without changing the buffer, since pH 9 lies within Tris-HCl's buffering range of pH 7.1 to 9. Therefore, Tris-HCl is indicated in document D2 as the preferred elution buffer (see point 7.3 above).

7.6 Example 3 of document D2 further discloses that the yield of the isolated nucleic acids strongly depends on the pH used for the binding reaction (see the Table in column 6). A maximum yield of 90% is obtained at pH 4.5, which decreases stepwise by 10% after increasing the binding by 0.5 pH steps until pH 5.5 (70% yield) is reached. A yield drop to 50% is observed if the binding is performed at pH 6.0, falling to a 20% yield at pH 6.5, and a 2% yield at pH 7.0. Example 3 thus demonstrates, in line with the generic concept indicated above, that nucleic acids bind to a solid carrier material at an acidic pH (pH 4.5 to 5.5), while significantly less nucleic acids bind under weak acidic conditions (pHs 6.0, or 6.5), or almost none under neutral pH binding conditions (pH 7.0).

7.7 Example 3 discloses different binding buffers: Acetate/KOH and Acetate/NaOH (buffering range: pH 3.6 to 5.6), MES (buffering range: pH 5.5 to 6.7), and Tris-HCl (buffering range: pH 7.1 to 9.0). Tris-HCl at pH 7.0 as binding buffer is used as a control.

7.8 The acetate binding buffer embodiments of Example 3 of document D2 differ from the method of claim 1 in two features. Firstly, in that the Tris-HCl wash buffer's pH 6.5 lies not within the buffering range of the acetate binding buffer (see point 7.7), and secondly in that the pH of the wash buffer is not pH 6.0 or lower. Despite these two differences the yield of nucleic acids by the acetate binding buffer embodiments of Example 3 is 90% and 80%, depending on the pH used for binding (pHs 4.5 and 5.0, respectively).

7.9 The MES binding buffer embodiment of Example 3 in document D2 differs from the method of claim 1 in one feature only, i.e. the use of the Tris-HCl wash buffer at pH 6.5 instead of pH 6.0 or lower. The overall yield of nucleic acids obtained by the MES binding buffer embodiment is 70% or 50%, depending on the binding pH used (pHs 5.5, and 6.0, respectively). The yield is thus lower than that for the acetate binding buffer embodiments. This demonstrates the importance of a low acidic pH for the binding reaction, because the overall conditions between the acetate and MES binding buffer embodiments of Example 3 differ in a single feature only: the binding pH (pH 4.5 or 5.0, instead of pH 5.5 or 6.0, respectively).

7.10 The MES binding buffer embodiment of Example 3 in document D2 represents, in line with the established case law, the closest prior art for the claimed method since it differs in one feature only (see Case Law, I.D.3.1.). This embodiment will be considered in the following.

8. In order to formulate the objective technical problem underlying the subject-matter of claim 1, the effect of the distinguishing feature (here: a 0.5 pH difference of the wash buffers) with respect to the MES binding buffer embodiment of document D2 must be determined.

9. It is established case law that the technical problem has to be determined on the basis of the effect actually achieved by the distinguishing feature vis-a-vis the closest prior art. Moreover, this effect has to be achieved over the entire area claimed. If this is not the case, the problem must be reformulated. The submission of comparative tests is not absolutely necessary in support that the claimed effect has indeed been achieved over the entire range covered by a claim, since it suffices that this is credible on the basis of facts derived from the patent taking the skilled person's common general knowledge into account (see Case Law, I.D.4.1, I.D.4.3.).

10. Comparative data of the claimed method with the MES binding buffer embodiment of document D2 under consideration have not been submitted by appellant I. Accordingly, the question arises whether based on facts derivable from the patent it is credible that the use of a wash buffer at pH 6.0, i.e. at a pH 0.5 lower, results in the provision of a method wherein all of the different binding buffers, different wash buffers and different combinations of binding and wash buffers falling within claim 1 still obtain a high yield, as stated by appellant I.

11. As mentioned above, the MES binding buffer embodiment of document D2 achieves an absolute yield of 70% or 50% of isolated nucleic acids at a binding pH of 5.5 or 6.0, respectively.

12. The term "high yield" used by appellant I is relative in the absence of a reference value, since depending on the circumstances absolute yields, for example, in between 10% and 100% may be regarded as high.

13. The patent, including Examples 1 to 4, is silent on a reference value for the yield obtained by the various embodiments falling within the claimed method. The patent discloses merely a relative yield of the isolated RNA molecules in the different working examples (see e.g. Figures 1 and 3). This means that the absolute yield obtained by the various binding/wash buffer combinations, for example, in Examples 1 and 3 of the patent cannot be determined individually. Nor can the yield be compared between the combinations of binding/wash buffers in the different working examples of the patent either. This was not contested by appellant I. In other words, based on the evidence derivable from the patent, it cannot be established whether or not the different combinations of binding/wash buffers in Examples 1 and 3 of the patent achieve a comparable "high yield" under all of the conditions tested, since the information about the absolute yield is lacking in all working examples.

14. In this situation the yield can neither be compared between the claimed method and the MES binding buffer embodiment of document D2, nor can it be assessed whether the claimed method achieves a "high yield" over all of the binding/wash buffer combinations that fall within claim 1. Based on the experimental evidence provided in the patent, it can be established only that the claimed method isolates nucleic acids, albeit at an unknown efficiency, i.e. it remains unknown whether the yield is high or low.

15. Appellant I submitted that the experimental data in Examples 1, 3 and 4 supported an overall improvement of the claimed method across the whole range claimed over the disclosure of document D2.

16. For the reasons outlined above, the board does not agree, since based on the evidence disclosed in the patent and in the absence of an absolute yield, it cannot be credibly established that the claimed method achieves a high yield of isolated nucleic acids across the whole breadth of claim 1.

17. Appellant I further submitted that Example 4 provided comparative data that the method of the invention provided comparable high yields without using hazardous ingredients, such as chaotropic agents and organic solvents. The board is not convinced by this argument either, since the closest prior art method in document D2 does likewise not use chaotropic agents and organic solvents. Furthermore, Example 4 provides a comparative test with an unrelated commercial product of unknown composition only. Accordingly, the results of Example 4 are irrelevant for deciding whether or not a technical effect can be ascribed to the distinguishing feature outlined above.

18. Appellant I further submitted that document D2 disclosed solely a single combination of a binding buffer and wash buffer that obtained a high yield, i.e. the acetate binding buffer embodiments. The board does not agree, since, as set out above, based on the evidence derivable from the patent, it cannot be credibly established that the claimed method achieves a high yield, either for a single combination of binding/wash buffers, let alone for all of the combinations falling within the whole breadth of the claim.

19. In view of the considerations above, the board is not convinced that the distinguishing feature is associated with an advantageous technical effect, at least not across the whole range covered by claim 1.

20. Accordingly, a less ambitious technical problem has to be defined, which is the provision of an alternative method for the isolation of nucleic acids.

21. In view of the experimental data in Examples 1, 3 and 4 of the patent, the board is satisfied that the method of claim 1 solves this problem.

Obviousness

22. It remains to be assessed whether or not the skilled person starting from the MES binding buffer embodiment in Example 3 of document D2, and faced with the technical problem identified above, would have arrived at the claimed method in an obvious manner.

23. Appellant I submitted that the skilled person had no motivation to further lower the pH of the Tris-HCl wash buffer to pH 6.0 or lower, since pH 6.0 instead of 6.5 was even further away from Tris-HCl's buffering range.

24. The board does not agree. Document D2 reports in column 3, lines 37 to 40 that the washing can be done either by the "described binding buffer or with other phosphate-free buffers, where the pH of this buffer should be between 4 and 7, preferably between 4.5 and 6.5". The skilled person knows that Tris-HCl is a phosphate-free buffer, and as set out above, document D2 mentions that this buffer is preferably used for washing and eluting purposes. Thus starting from the Tris-HCl wash buffer at pH 6.5 in Example 3 and looking for alternative wash conditions, the skilled person would have taken into consideration all of document D2's teaching for this purpose, in particular parameters that have been explicitly mentioned as preferred, such as the use of a washing pH across the whole range of "4.5 and 6.5" (see column 3, line 40).

25. The skilled person would have only refrained from applying this teaching if reasons for doing so were apparent. Appellant I submitted that the lowering of the pH from 6.5 to 6.0 would have removed Tris-HCl even further away from its buffering range of pH 7.1 to 9 which harbored the risk that the pH for washing became unstable. This is, however, not convincing. Tris-HCl's pH of 6.5 as disclosed for washing in Example 3 of document D2 lies already outside of it's buffering range. Since Tris-HCl cannot therefore buffer at pH 6.5, it can neither buffer at pH 6.0 too. This means that pH stability cannot be an issue in the present case, as long as the pH remains acidic during the washing to prevent a premature elution of the bound nucleic acids from the solid material (see document D2, column 3, lines 45 to 47). A condition certainly achieved by lowering the pH of Tris-HCl to 6.0.

26. The board therefore concludes that the method of claim 1 is obvious for the skilled person based on the teaching of document D2 alone. Auxiliary request 6 therefore lacks an inventive step (Article 56 EPC).

Auxiliary requests 7 and 8

27. Claims 1 of auxiliary requests 7 and 8 differ from claim 1 of auxiliary request 6 in that the feature "wherein the second pH is within the buffering range of the wash buffer" has been added. Claim 1 of auxiliary request 8 is further amended compared to claim 1 of auxiliary request 7, in that the feature "pH 6.5-10" has been replaced by "pH 7-9" in step (iii).

Inventive step

28. The additional/amended features in claims 1 of auxiliary requests 7 and 8 do not further distinguish the claimed methods from the disclosure of the MES binding buffer embodiment in Example 3 of document D2. The Tris-HCl buffer in Example 3 is used at pH 9 for eluting the bound nucleic acid from the solid carrier. This pH value for elution (i.e. the "second pH" according to claim 1) lies within Tris-HCl's buffering range of pHs 7.1 to 9, i.e. of the buffer that has been likewise used for washing. Furthermore, an elution at pH 9 as disclosed in Example 3 of document D2 falls within the pH range cited in step (iii) of the claimed methods.

29. Accordingly, the reasons set out above under lack of inventive step for the method of claim 1 of auxiliary request 6 apply mutatis mutandis to the methods of claims 1 of auxiliary requests 7 and 8 (Article 56 EPC).

Admission of auxiliary requests 9 to 11 filed at the oral proceedings into the appeal proceedings

30. Since the summons to the oral proceedings were notified on 15 April 2020, Article 13 RPBA 2020 is to be applied for questions regarding any amendment to a party's appeal case (Article 24(1) RPBA 2020).

31. Appellant I filed auxiliary requests 9 to 11 during the oral proceedings in reply to an allegedly new argument raised by the board during the oral proceedings.

32. Auxiliary requests 9 to 11 represent an amendment to appellant I's case under Article 13(2) RPBA 2020 according to which any amendment to a party's appeal case made after notification of a summons to oral proceedings shall, in principle, not be taken into account unless there are exceptional circumstances, which have been justified with cogent reasons by the party concerned.

33. Auxiliary requests 9 to 11 are identical to auxiliary request 6, except that the method of claim 1 of auxiliary request 9 was limited to an elution in the presence of an elution buffer at a pH ("second pH") that was within the buffering range of the elution buffer. Moreover, all claims relating to a kit have been deleted.

Claim 1 of auxiliary request 10 compared to claim 1 of auxiliary request 9 was further limited in that "the second pH" (i.e. the elution pH) was within the buffering range of the wash buffer too. In other words, the elution pH was within the buffering ranges of the buffers used for washing and eluting.

Lastly, claim 1 of auxiliary request 11 compared to claim 1 of auxiliary request 10 was further limited in that "the second pH" (i.e. the elution pH) was "in the range of pH 7-9", instead of "pH 6.5-10".

34. Auxiliary requests 9 to 11 constitute an attempt to overcome an allegedly new objection under Article 56 EPC raised by the board during the oral proceedings. It concerned the board's question whether or not it was credible based on the evidence in the patent and in light of the closest prior art method that the technical problem formulated by appellant I (see above) was solved across the whole breadth of claim 1 for the embodiment of the claimed method using strong acidic binding and wash buffers, in combination with water for eluting the bound nucleic acids from the solid material.

35. The objection that no advantageous technical effect is achieved by the distinguishing feature of claim 1 compared to the closest prior art method of document D2 over the whole range claimed was raised by appellant II already during the written phase of the opposition proceedings, and was maintained in their statement of grounds of appeal (see submission dated 12 October 2015, on page 7, paragraphs 1 to 4; statement of grounds of appeal, page 28, last paragraph to page 29, second paragraph). Although the opposition division did not find this objection convincing and acknowledged inventive step in the decision under appeal, the board's provisional opinion deviated from this finding, at least in so far as the board was not convinced that the technical effect of a high yield was obtainable across the whole scope of claim 1 (see point 39 of the board's communication). Furthermore, point 38.4 of the board's communication mentions that claim 1 encompasses the combination of acidic wash buffers and water for elution purposes, and that in view of the evidence provided in Example 1 of the patent the achievement of a high yield across the whole scope of claim 1 appeared doubtful. Moreover, the fact that the method of claim 1 encompassed the combination of acidic binding and wash buffers was likewise mentioned in the board's communication (see point 21.3).

36. In light of these considerations, the board is not convinced by appellant I's submission that auxiliary requests 9 to 11 were filed to address a new argument of the board raised for the first time during the oral proceedings.

37. Irrespective of the above, the amendments in claims 1 of auxiliary requests 9 to 11 do not have the effect that the technical problem defined by appellant I for the method of claim 1 (see above) is solved across the whole range claimed. This is so because the fundamental issue remains as set out above, namely that based on the data in the patent it cannot be established that a high yield is obtained by all combinations of binding/wash and elution buffers encompassed by claim 1. In the absence of an absolute yield or a reference yield, the combinations of various buffers and their effect on the yield can neither be determined nor compared. Accordingly, an advantageous effect cannot be ascribed to the distinguishing feature of the claimed methods vis-a-vis the MES binding buffer embodiment of document D2, and hence the technical problem underlying the claimed method remains, as for the higher ranking auxiliary requests above, the provision of an alternative method for isolating nucleic acids.

38. The amendments in claims 1 of auxiliary requests 9 to 11 do also not further distinguish the claimed method over the MES binding buffer embodiment of document D2. This embodiment uses a Tris-HCl buffer at pH 9.0 for elution, i.e. "the second pH", which lies within the buffering range (pH 7.1 - 9.0) of the elution buffer. Moreover, pH 9.0 of the Tris-HCl buffer used in the MES binding buffer embodiment for elution purposes lies within the elution pH range cited in step(iii) of the claimed methods.

39. Consequently, exceptional circumstance according to Article 13(2) RPA 2020 do not apply in the present case. Moreover, it is not apparent that the amended claimed methods overcome prima facie the objections raised above under Article 56 EPC against the method of claim 1 of auxiliary requests 6 to 8. The board therefore decided not to admit auxiliary requests 9 to 11 into the appeal proceedings.