T 0518/17 - Solubilising subtilisin

 Key points

• The method of claim 1 specifies (after claim construction) as step a) diluting a fermentation broth that comprises subtilisin crystals and/or subtilisin precipitate. The opponent submits that the patent provides no enabling disclosure of providing such a fermentation broth.
• The Board: “the provision of a fermentation broth comprising subtilisin crystals and/or subtilisin precipitate is a feature of the claim that has to be taken into consideration when assessing whether or not the claimed invention is sufficiently disclosed. The board considers it irrelevant in this context that the claim does not explicitly require the provision of such a fermentation broth, since such requirement is a necessary consequence of the express language of the claim and thus implied.”
• Accordingly, the patentee's argument “that it was enough for sufficiency of disclosure that the skilled person could determine whether or not crystals were present in the fermentation broth and that the claimed invention started at the point where the skilled person was faced with the recovery of subtilisin from a fermentation broth in which it was present in crystalline or precipitated form must fail.”
• “ The board concludes from the above that it is a verifiable fact that neither the patent nor the state of the art cited therein provide any guidance for the skilled person with respect to the technical measures to be applied in the fermentation process of subtilisin proteases to achieve such high yields that subtilisin crystallises spontaneously in the fermentation broth during fermentation.”
• “The board concurs with the appellant that it can be inferred from paragraph [0002] of the patent that the claimed method is to be applied in particular in circumstances in which the presence of subtilisin crystals in the fermentation broth is the result of a "dramatic" increase in the fermentation yield.”
• “Notably, the [patentee] had themselves submitted that the "dramatic" increases referred to in paragraph [0002] of the patent "had arisen as a result of years of optimisation and improvement in the production process and production microorganism". However, evidence that the skilled person was aware of the concrete measures of optimisation and improvement to be taken to reliably reach such high yields in fermentation resulting in subtilisin crystals had not been provided by the respondent.”
• As a comment, we are talking about fermentation, so quite likely the patentee, as well as the opponent, has a few closely guarded trade secrets. Quite possibly every industrial company in the field of fermentation production of the compound at issue is able to achieve highly concentrated fermentation broths, but the various secret tricks (and microorganisms) used by them are nowhere to be found in the prior art.

T 0518/17 - 

https://www.epo.org/law-practice/case-law-appeals/recent/t170518eu1.html

Main request (patent as granted) - claim 1

Claimed invention - claim construction

11. The claim is directed to a method of solubilising subtilisin crystals and/or subtilisin precipitate in a fermentation broth. The method comprises three explicit steps, denoted a), b) and c). In step a), the fermentation broth is diluted 100-2000% (w/w).

12. The fermentation broth is not characterised further in the claim. However, the parties concurred that, as a consequence of the purpose indicated in the claim ("solubilizing protease crystals and/or protease precipitate ... wherein the protease is a subtilisin") the skilled person will understand that the fermentation broth referred to in method step a) has to comprise subtilisin crystals and/or subtilisin precipitate. The board concurs with this claim construction. Accordingly, the claimed method comprises as step a) diluting a fermentation broth that comprises subtilisin crystals and/or subtilisin precipitate.

Sufficiency of disclosure (Article 100(b) EPC)

13. In the decision under appeal, the opposition division had held that "the formation of crystals and/or precipitates cannot be considered as a technical step of the method claimed, and thus the points raised by the O's [appellant] in respect of the alleged inability to obtain at least such crystals are of no relevance to sufficiency of disclosure, since these steps do not have to be carried out as part of the claimed invention."

14. The board points out that the underlying purpose of the requirement of sufficient disclosure of an invention is to ensure that the exclusive right conferred by a patent should be justified by the actual technical contribution to the art (see decision T 409/91,

OJ EPO 1994, 653: point 3.5 of the Reasons). A technical contribution to the art requires that the claimed invention can be realised by the skilled person at the effective date of the patent. In the present case, realisation of the method requires that a fermentation broth comprising subtilisin crystals or subtilisin precipitate be available to the skilled person (see point 12. above).

15. Therefore the provision of a fermentation broth comprising subtilisin crystals and/or subtilisin precipitate is a feature of the claim that has to be taken into consideration when assessing whether or not the claimed invention is sufficiently disclosed. The board considers it irrelevant in this context that the claim does not explicitly require the provision of such a fermentation broth, since such requirement is a necessary consequence of the express language of the claim and thus implied.

16. Accordingly, the opposition division's reasoning (see point 13. above) cannot hold, and the respondent's arguments that it was enough for sufficiency of disclosure that the skilled person could determine whether or not crystals were present in the fermentation broth and that the claimed invention started at the point where the skilled person was faced with the recovery of subtilisin from a fermentation broth in which it was present in crystalline or precipitated form must fail.

17. The appellant maintained on appeal that the patent, taking into account the common general knowledge of the skilled person, provided no guidance on how to obtain a fermentation broth comprising subtilisin crystals without undue burden. For this reason, they submitted that the requirement of sufficiency of disclosure was not met.

18. According to established case law of the boards of appeal, a successful objection of sufficiency of disclosure presupposes that there are serious doubts, substantiated by verifiable facts. In inter partes proceedings the burden of proof is initially upon an opponent to establish, on the balance of probabilities, that a skilled person reading the patent, using common general knowledge, would be unable to carry out the invention (see Case Law of the Boards of Appeal, 9th edition 2019 ["CLBA"], section III.G.5.1.2c)). If the opponent has discharged their burden of proof, the patent proprietor seeking to refute the conclusively established facts by way of counter-arguments bears the burden of proving the alleged facts (CLBA, section III.G.5.2.1).

19. In the following, the board considers in turn the teaching of the patent, the common general knowledge and the post-published experimental evidence relied on by the parties.

Teaching of the patent

20. The present invention lies in the field of industrial production of the enzyme subtilisin, a serine protease, through fermentation. Microorganisms producing the enzyme are fermented under conditions leading to the secretion of the subtilisin into the fermentation broth (see paragraphs [0020] to [0025] of the patent). Subsequently, the biomass and further insolubles, commonly referred to as sludge, are separated from the subtilisin in the fermentation broth by filtration or centrifugation (see paragraphs [0039] to [0041]). The patent states that "[t]he fermentation yield of industrial proteases has increased dramatically over the passed [sic] years. The yield is now so high that more than 60% of the protease in the fermentation broth may be present as crystals and/or precipitate" (see paragraph [0002]) and that it provides "an improved method of solubilizing protease crystals and/or protease precipitate in a fermentation broth" (see paragraph [0001]). The method aims at ensuring that all the subtilisin is in solution in the fermentation broth and can be separated from the sludge by known separation methods, such as centrifugation and filtration (see paragraph [0044]).

21. The board concurs with the appellant that it can be inferred from paragraph [0002] of the patent that the claimed method is to be applied in particular in circumstances in which the presence of subtilisin crystals in the fermentation broth is the result of a "dramatic" increase in the fermentation yield.

22. Accordingly, the claimed invention is understood to relate, in one embodiment, to diluting a fermentation broth that comprises subtilisin crystals as a consequence of high fermentation yields.

23. The first issue addressed is whether or not the patent provides the skilled person with guidance for carrying out this embodiment.

24. Although the patent provides information on proteases, microorganisms and fermentation broths that can be used according to the invention in the section of the description entitled "Detailed Description" (see paragraphs [0005] to [0047]), no information is provided on how to obtain a fermentation broth comprising subtilisin crystals as a consequence of high fermentation yields.

25. As for the examples, Example 1 of the patent, entitled "pH and calcium effect on solubilizing protease (subtilisin 309) crystals", discloses that the "subtilisin was subtilisin 309 as described in

EP 0396 608 B1 [document D43 in the appeal proceedings]" and that the "Bacillus species was Bacillus licheniformis with copies of the subtilisin inserted as described in WO 02/00907 [document D45]" (see paragraph [0049]). Next, the solubilisation process is described as starting from a "fermentation broth".

26. Example 2 of the patent, entitled "pH and calcium effect on solubilizing protease (subtilisin 309 variant) crystals", discloses that the "subtilisin was a variant of subtilisin 309 as described in WO 00/37599 [document D44]" and that the "Bacillus species was Bacillus licheniformis with copies of the subtilisin inserted as described in WO 02/00907 [document D45]" (see paragraph [0056]). Again, the solubilisation process is described as starting from a "fermentation broth". Neither example discloses how the fermentation broth was obtained.

27. As for the publications cited in the patent (see paragraphs [0049] and [0056] and points 25. and 26. above), the board notes that these documents report that subtilisin enzyme activity was determined in the supernatant of the untreated fermentation broth and the enzyme was recovered from the supernatant after centrifugation or filtration of the fermentation broth (see document D43, page 12, lines 46 to 56; document D44, page 32, lines 27 to 32; page 45, line 25 to page 46, line 8; document D45, page 50, line 1).

28. The reference in these documents to the recovery of subtilisin from the liquid phase after centrifugation or filtration indicates to the skilled person that the enzyme is present in soluble form. Accordingly, the board concurs with the appellant that the publications cited in the patent do not disclose fermentation conditions that lead to the formation of subtilisin crystals in a fermentation broth. Document D42, referred to in the patent as disclosing a complex fermentation medium (see paragraph [0021]), likewise does not disclose such fermentation conditions.

29. The board notes in this context that according to the respondent's own submissions "whether or not crystals or precipitate are formed depends not only on the [bacterial] strain but also on the components of the fermentation medium and conditions such as temperature". No information on any of these parameters that would lead to subtilisin crystals in the fermentation broth appears to be disclosed in the patent or in the state of the art cited in the patent.

30. The board concludes from the above that it is a verifiable fact that neither the patent nor the state of the art cited therein provide any guidance for the skilled person with respect to the technical measures to be applied in the fermentation process of subtilisin proteases to achieve such high yields that subtilisin crystallises spontaneously in the fermentation broth during fermentation.

31. Accordingly, the respondent's argument that the patent contains repeatable examples and extensive disclosure in paragraphs [0027] to [0046] about how to put the invention into effect also fails.

Common general knowledge of the skilled person

32. The respondent during the oral proceedings before the board conceded that the conditions for achieving a fermentation broth comprising subtilisin crystals were not disclosed in the patent in suit. They however maintained that this was not required in view of the common general knowledge of the skilled person.

33. With respect to documents D2, D4 and D7, relied on by the respondent as evidence that fermentation broths comprising subtilisin crystals were well known in the state of the art, the board notes that document D2 discloses that addition of a flocculant or a precipitation agent, such as a salt or a low-molecular-weight organic solvent, to the fermentation broth containing subtilisin causes the protein to precipitate (see page 6, line 34 to page 7, line 3 and Example 3). Document D4 discloses the formation of alpha-amylase crystals in a fermentation broth (see Example 1), but does not contain any disclosure as regards the formation of subtilisin crystals in a fermentation broth (see column 1, lines 22 to 26 and column 2,

lines 4 to 11 and 17 to 19). In document D7, the fermentation broth containing subtilisin is treated with coagulants or flocculants to produce a crystalline suspension (see abstract and Example 1).

34. The board concurs with the appellant that none of documents D2, D4 and D7 discloses a fermentation broth containing subtilisin crystals formed due to high yield. Instead, they provide evidence that additional measures were employed in the state of the art to actively convert the soluble form of subtilisin to crystals and/or precipitate.

35. The respondent further argued, relying on documents D49 and D53, that improvements in fermentation yields were the prevailing situation in the protease fermentation industry at the priority date of the patent.

36. Document D49 was published after the priority date of the patent (see sections I. and III.). Thus the question arises whether or not document D49 can be accepted at all as being an account of the prevailing situation in the art at the effective date of the patent. In the board's judgement, this question can however be left unanswered since the document in fact does not support the respondent's case. Indeed, document D49 discloses that "[a]dvances in expression technology have resulted in the ability to obtain relatively high enzyme concentrations (e.g., 10-100 g/l) in fermentation broth. In some cases, the expression level exceeds the solubility limit of an enzyme of interest, and the enzyme is present in a precipitated or crystalline form at the end of fermentation" (see page 3, paragraph [11]). The document however does not provide any information on these "advances in expression technology", i.e. is silent on how they were achieved. Further, with respect to subtilisin, document D49 provides no information on a fermentation broth comprising crystals. On the contrary, it reports a fermentation broth containing soluble subtilisin which requires addition of flocculant to form a precipitate (see Example 7).

37. Document D53 discloses, in the context of manufacturing proteases for detergents, that "[y]ields could be improved to high levels and, thanks to the high purity of the secreted enzyme, it could even be crystallized by addition of salts" (emphasis added, see page 253, 4th paragraph).

38. Thus the board considers that neither document D49 nor document D53 provides evidence that improvements in fermentation yield leading to subtilisin concentrations exceeding the solubility limit and resulting in spontaneous crystallisation were widespread in the state of the art.

39. With respect to the respondent's assertion that the fermentation yield could be raised above the solubility limit by optimising the production microorganism using routine techniques that belonged to the common general knowledge of the skilled person, the board notes that no corroborating evidence was provided by the respondent in this respect.

40. To the contrary, it can be inferred from document D53, the sole document relied on by the respondent as representing the common general knowledge of the skilled person, that years of optimisation are required to improve the fermentation yield (see Figure 4). Moreover, yield improvement by the measures studied in document D53, i.e. chemical mutagenesis, medium optimisation and genetic engineering, reach a plateau after seven years of research (see Figure 4), without reportedly leading to the formation of crystals (see page 253, 3rd paragraph).

41. Notably, the respondent had themselves submitted that the "dramatic" increases referred to in

paragraph [0002] of the patent "had arisen as a result of years of optimisation and improvement in the production process and production microorganism". However, evidence that the skilled person was aware of the concrete measures of optimisation and improvement to be taken to reliably reach such high yields in fermentation resulting in subtilisin crystals had not been provided by the respondent.

42. In the board's opinion, a reasonable amount of trial-and-error experimentation may be acceptable to acknowledge that the claimed invention can be carried out without undue burden. This however presupposes that sufficient information is available that leads the skilled person directly towards success through the evaluation of initial failures. In the present case, and based on the evidence on file, the board considers that information on critical process variables (see points 40. and 41. above) is lacking. The skilled person would therefore need to repeat the lengthy optimisation and improvement process, without any guarantee that fermentation conditions resulting in the formation of subtilisin crystals during fermentation due to high yield would be achieved. Such a situation is often described in the case law as an invitation to perform a "research program" and considered to amount to an undue burden (see CLBA, section II.C.6.7).

43. The respondent's proposition that the skilled person only needed to run the fermentation long enough to reach supersaturation is not found to be persuasive either. The skilled person is well aware that during a fermentation process microorganisms eventually stop producing. The skilled person would thus not expect supersaturation to be able to be reached by merely extending the duration of the fermentation process, and in particular not in the absence of any evidence in the state of the art that supersaturation with respect to subtilisin has ever been reached in a fermentation broth and has led to crystal formation (see point 33. above).

44. The respondent also argued that the skilled person could artificially induce crystal formation by changing the factors known to affect the solubility of enzymes in general, e.g. by modifying the salt concentration and/or pH of the fermentation broth or by spiking the fermentation broth by adding crystals and/or amorphous solid subtilisin.

45. However, the embodiment under consideration (see point 22. above) relates to the presence of subtilisin crystals in a fermentation broth due to high yield, and the purpose of the claimed method is to dissolve these subtilisin crystals so that soluble subtilisin can be separated from the sludge. If the fermentation yield is so low that all of the subtilisin is still in the soluble phase, the skilled person cannot be assumed to even consider artificially inducing the formation of crystals only to dissolve them again in the next step. Instead, the skilled person would remove soluble subtilisin from the sludge immediately by centrifugation or filtration. Furthermore, artificial induction of subtilisin crystal formation is not disclosed or even hinted at in the patent.

46. In view of the above considerations, the board concurs with the appellant that there exists no common general knowledge on how to obtain the high yield of subtilisin needed to achieve a fermentation broth with crystals.

Post-published experimental evidence

47. The opposition division in the decision under appeal accepted that the post-published document D29 demonstrated "that crystals can be formed and subsequently solubilized in accordance with the examples of the patent", and the respondent maintained on appeal that document D29 confirmed that subtilisin crystals could be formed in a fermentation broth.

48. Sufficiency of disclosure must, in principle, exist at the effective date of a patent, while post-published documents may be used as evidence that the disclosure is reproducible without undue burden only under certain circumstances (see also CLBA, section II.C.6.8).

49. The board notes that whether or not in the case at hand, where the patent provides no guidance for achieving a fermentation broth comprising subtilisin crystals, post-published evidence can remedy a lack of sufficiency of disclosure can in fact be left unanswered because, in the board's opinion, the disclosure in document D29 is manifestly unsuitable as evidence that "crystals can be formed (...) in accordance with the examples of the patent".

50. In fact, the examples of the patent in suit do not disclose any details as to how a fermentation broth comprising subtilisin crystals is prepared (see

points 25. and 26. above), and this was conceded by the respondent (see point 32. above). Document D29 merely states that "[a] fermentation broth containing subtilisin was prepared according to Example 1 of European patent no 2125865 [the patent]". No experimental protocol and no information on the bacterial strain used or any of the fermentation parameters such as medium components, temperature, pH, duration are disclosed in document D29.

51. Furthermore, the appellant has provided experimental data showing that fed-batch fermentation using complex fermentation broth of a Bacillus licheniformis host cell producing a Bacillus lentus subtilisin (Savinase) did not lead to subtilisin crystals (see document D48, Experiment 1). Adding dialysed Bacillus lentus subtilisin (Savinase), stirring and storing overnight (see document D48, Experiment 1) and increasing the subtilisin concentration to 90 g/kg by adding lyophilised Bacillus lentus subtilisin (Savinase) did not lead to subtilisin crystals either (see

document D48, Experiment 2).

52. Firstly, the board considers that in the circumstances of the present case (see point 30. above) the provision of evidence in the form of experimental data was not even required to discharge the appellant's burden of proof.

53. Secondly, the board finds the respondent's criticisms with respect to these experimental data not to be persuasive. In fact, the very host cell and subtilisin used in Experiment 1 of document D48 are those mentioned in the examples of the patent, the disclosed fermentation process is the preferred one according to paragraph [0025] of the patent, the medium is one of the media disclosed in paragraph [0021] of the patent and the patent states that the "microorganism producing the protease of interest may be fermented by any method known in the art" (see paragraph [0021]. Furthermore, the board points out that providing high yield is the sole measure disclosed in the patent for generating subtilisin crystals, although the fermentation conditions leading to high yields and to subtilisin crystals are not disclosed (see point 30. above). The board notes further that, in the experiments reported in document D48, very high yields of subtilisin were simulated by supplementing the fermentation broth with subtilisin, but no crystallisation was observed.

54. The board is thus satisfied that document D48 provides evidence that the appellant, despite making a reasonable effort, was unable to put the claimed invention into practice.

Conclusion on sufficiency of disclosure (Article 100(b) EPC) - main request

55. The board concludes from the above considerations that the appellant has discharged their burden of proof and that the respondent has not established that the embodiment under consideration was indeed workable for the skilled person without undue burden using common general knowledge at the effective date of the patent.

56. The ground for opposition under Article 100(b) EPC thus prejudices the maintenance of the patent as granted.