T 2836/19 (A method of culturing a cell/CHUGAI SEIYAKU KABUSHIKI KAISHA) 03-06-2022

Main request (claims 1-5 claims)

Article 123(2) EPC

1. The main request is identical to auxiliary request 13 of the decision under appeal.

1.1 In the decision under appeal, the method of claim 1 was held to comply with Article 123(2) and (3) EPC but to offend against Article 56 EPC.

1.2 Appellant agreed with the findings of the opposition division under Article 123(2) EPC.

1.3 Respondents contended that the method according to claim 1 contravened the requirements of Article 123(2) EPC. There was no disclosure of the features

(a) "adding a fed-batch medium comprising serine, cysteine and tyrosine",

(b) "wherein the concentration of cysteine in the culture solution is maintained at 0.4 mM or higher" and

(c) "maintaining the concentration of serine, tyrosine and cysteine during a period from a fourth day to a tenth day of the culture by addition of the fed-batch medium".

2. A central question to be answered when assessing whether the amendment complies with Article 123(2) EPC is whether the combination of features of claim 1 of the main request, can be seen by the skilled person as directly and unambiguously derivable from the content as a whole of the parent application as filed. In this respect the notion of selection from two lists provides valuable guidance in the application of the so-called gold standard (see the landmark decision T 12/81 and the Case Law of the Boards of Appeal, 9th edition, 2019, II.E.16.2).

2.1 It is undisputed that the patent application does not explicitly disclose the method of claim 1 combining the features (a), (b) and (c) cited above. Thus, it remains to be assessed whether the claimed subject matter is implicitly, yet directly and unambiguously, disclosed.

2.2 An implicit disclosure means a disclosure which any person skilled in the art would objectively consider as necessarily implied in the explicit content of the patent application as a whole. It means nothing more than the clear and unambiguous consequence of what is explicitly mentioned (see e.g. T 947/05 of 27.11.2007, reasons 2.1 and T 860/00 of 28.09.2004, reasons 1.1).

Feature (a): "adding a fed-batch medium comprising serine, cysteine and tyrosine"

2.3 The decision under appeal considered that claim 16 referring back to claim 15, reading "wherein the serine and tyrosine and/or cysteine are fed into the culture solution in multiple batches sequentially or continuously", formed a basis for the feature (a) in claim 1 because "fed ... in multiple batches" implied that a fed-batch medium containing the listed amino acids had to be added.

The only difference was that the conjunction "and/or" used in claim 16 was replaced by an "and" in the present claim 1. From the fed-batch medium defined in claim 24, comprising serine as well as cysteine and tyrosine, referring back to claims 21 or 22, the skilled person could directly and unambiguously derive from the conjunction "and/or" that the embodiment including all three amino acids was preferred.

2.4 The board agrees that in the patent application claim 16 depends on claim 15 and that the "and/or" in claim 16 covers also one embodiment comprising all three amino acids.

2.4.1 However, the board cannot concur with the reasoning in point 2.1.2 of the decision under appeal that "wherein the serine and tyrosine and/or cysteine are fed into the culture solution in multiple batches sequentially or continuously" (claim 16, emphasis added) necessarily implies that the fed-batch culture comprises a fed-batch medium comprising all of serine, tyrosine and cysteine.

Claims 15 or 16 do not specify that the fed-batch culture comprised the addition of a specific set of amino acids consisting of serine, tyrosine and cysteine into the culture solution by means of a fed-batch medium. The addition of amino acids via a fed-batch medium is not the only option, as also "only specific components may be added" (see paragraph [0036] of the patent application). The wording of claims 15 and 16 does not exclude the feeding of the amino acids serine, tyrosine and/or cysteine as components, even separately from each other, and not in a fed-batch medium. Finally, the fed-batch medium comprising serine, tyrosine and cysteine according to claim 24 required the concentration of serine in the fed-batch medium to range between 10 mM to 1000 mM or 20 mM to 500 mM (see claims 21 and 22), which is absent in the present claim 1.

2.4.2 Thus, even if the method according to claim 25 of the patent application is taken into account, there is no direct and unambiguous disclosure of a method as defined in present claim 1, comprising the step of adding a fed-batch medium comprising serine, tyrosine and cysteine, wherein serine is present at any concentration, but nonetheless sufficient to maintain the serine concentration in the culture solution above a specific threshold concentration level.

"[M]aintaining the concentration of serine, cysteine and tyrosine in the culture medium by addition of the fed-batch medium"

2.5 The decision under appeal considered that paragraphs [0038] and [0039] of the patent application provide a basis for the maintenance of the concentration of all three amino acids in the culture solution.

2.6 The board agrees that in case of employing the fed-batch culture, the medium that is to be added during the culture may contain a high concentration of serine (and tyrosine and/or cysteine). It is important "to maintain the concentration of serine, or respective concentrations of serine and tyrosine, or respective concentrations of serine and cysteine, or respective concentrations of serine, tyrosine, and cysteine, at a predetermined concentration or higher at least during a predetermined stage of the culture, as described above." (see paragraph [0038] of the patent application).

The maintenance of the concentration of serine (and tyrosine and/or cysteine) in the culture solution at a predetermined concentration can be achieved either by addition of a high concentration of serine (and tyrosine and/or cysteine) to the medium at an early stage of the cell culture, or alternatively by addition of a medium comprising a high concentration of serine (and tyrosine and/or cysteine) during the culture to supplement the medium with amino acids (see paragraph [0031] of the patent application).

It is only the maintenance of the concentration of serine by adding fed-batch medium in the culture solution that finds a basis in paragraph [0015] of the patent application.

2.6.1 Although the addition of "a" fed-batch medium containing a high concentration of serine (and tyrosine and/or cysteine) for maintaining the concentration of serine, tyrosine and/or cysteine in the culture solution is directly and unambiguously derivable from paragraphs [0031], [0038] and [0042] of the patent application, the board cannot derive therefrom that the different serine, tyrosine and cysteine concentrations in the culture solution should be maintained by the addition of a single fed-batch medium comprising serine, tyrosine and cysteine.

2.7 Even if the language of claims 15 and 16 is read in the light of the patent application as a whole, the board cannot share appellant's conclusion that the reference in the patent application to "a" -indefinite article- or "the" -definite article- medium would be read by the skilled person as one single medium, i.e. a single fed-batch medium comprising serine, tyrosine and cysteine.

2.7.1 The burden of demonstrating that the method according to claim 1 has a basis in the patent application rests with the appellant (i.e. proprietor). The indication that a method, which uses several different fed-batch media, has no basis in the patent application is insufficient to establish that the method of claim 1 using a single fed-batch medium complies with Article 123(2) EPC.

2.7.2 Firstly, [i]n the fed-batch culture, a (indefinite article) medium is fed continuously or sequentially during cultivation [...] (see paragraph [0036] of the patent application). The medium to be added in the fed-batch culture is referred to as "fed-batch medium" and does not necessarily have to be the same medium as that used in the culture (hereinafter "initial medium"); namely a different medium may be added, or only specific components may be added.

2.7.3 Secondly, the medium that is to be added during the culture may contain a high concentration of serine (and tyrosine and/or cysteine) (see paragraph [0038] of the patent application). In the fed-batch culture, it is important "to maintain the concentration of serine, or respective concentrations of serine and tyrosine, or respective concentrations of serine and cysteine, or respective concentrations of serine, tyrosine, and cysteine, at a predetermined concentration or higher at least during a predetermined stage of the culture". " ... the concentration of serine (and tyrosine and/or cysteine) in the culture solution may be monitored to adjust the concentrations of these amino acids in the medium to be added (i.e. fed-batch medium) so that the concentrations of these amino acids in the culture solution can be controlled".

2.7.4 From paragraphs [0036] and [0038], it cannot be directly and unambiguously derived that the medium to be added during the fed-batch culture must be one single fed-batch medium, just because the medium to be added must be different from the initial medium or because only specific components may be added. "[T]he" fed-batch medium is a medium which is fed during fed-batch culture. "The" fed-batch medium refers to the type of medium fed during culture, not to the composition of the medium itself. The fact that the concentration of serine (and tyrosine and/or cysteine) in the medium to be added (i.e. fed-batch medium) may be adjusted so that the concentrations of these amino acids in the culture solution are maintained at or above the desired concentration threshold, confirms this view and does not directly and unambiguously establish that one single fed-batch medium is used in the fed-batch culture of the invention.

2.8 In addition, paragraph [0042] of the patent application mentions that in case of a fed-batch culture, serine and tyrosine and/or cysteine are dissolved at high concentrations to be enriched in a fed-batch medium and a fed-batch medium is added either continuously or sequentially during cultivation so that the concentrations of these amino acids are maintained at predetermined or higher concentrations.

2.8.1 The use of the conjunction "and/or" to define the presence of a high concentration of serine (and tyrosine and/or cysteine) in the feed batch medium or that these high concentrations of amino acids may be contained in the culture medium from an initial stage of the cell culture in paragraphs [0017], [0038] and [0042] do not directly and unambiguously disclose that the fed-batch culture uses "a" single fed-batch medium containing all of the amino acids that are to be maintained in the culture solution.

2.8.2 Thus, neither the preparation of "a" fed-batch medium nor the addition of "a" medium to maintain the concentration of desired amino acids at or above a concentration threshold in the culture solution teaches directly and unambiguously the use of one single fed-batch medium, even less of one single fed-batch medium containing all the amino acids to be maintained.

2.8.3 The skilled person would have understood that the method according to claim 1 is at most reproduced in Example 3 among all the examples. In this example the concentration of serine and tyrosine in the culture solution was maintained at or above the claimed concentration threshold while no indication is given as to whether the concentration of cysteine was maintained or not. Even if a fed-batch medium comprising serine, cysteine and tyrosine at high specific concentrations was used in example 3, it neither directly nor unambiguously discloses the use of one single fed batch medium comprising any concentration of serine, tyrosine and cysteine in a fed batch culture for maintaining the concentration of the serine and tyrosine and cysteine at or above the claimed concentration threshold in the culture solution.

Feature (b):"[W]herein the concentration of cysteine in the culture solution is maintained at 0.4 mM or higher".

2.9 The decision under appeal considered that paragraphs [0016] and [0024] of the patent application provide explicit basis for "concentration of cysteine in the culture solution may be 0.4 mM or higher". Paragraph [0027] also states that "the concentration of cysteine in the culture solution may be maintained at 0.4 mM or higher".

2.9.1 Appellant indicated that the "concentration of cysteine is maintained at 0.4 mM or higher" was based on claim 12 and paragraphs [0017], [0024] and [0027] of the patent application.

2.10 The board agrees that claim 12 referring back to claim 11 relates to a method of culturing a cell, further characterized in that a concentration of tyrosine in the culture solution is maintained at 1 mM or higher, and/or a concentration of cysteine in the culture solution is maintained at a concentration of the cysteine in an initial medium or higher or at 0.4 mM or higher at least during a part of or an entire period from the third day to the tenth day of the culture.

2.10.1 The board agrees that an animal cell culture medium comprising 1 mM or higher of serine or a salt thereof, and at least 1 mM or higher of tyrosine and/or 0.4 mM or higher of cysteine is disclosed (see paragraph [0017] of the patent application). This medium refers not only to a medium comprising cysteine at a concentration of 0.4 mM or higher in an initial medium but also a medium adjusted such that a concentration of cysteine in the culture solution is maintained at 0.4 mM or higher by addition of a fed-batch medium or the like at least during a certain period from or after the onset of the cell growth phase. Since a typical concentration of cysteine in the initial medium is about 0.4 mM, the concentration of cysteine in the culture solution may be 0.4 mM or higher, preferably 1 mM or higher, during a part of or an entire culture period from the third day of the culture, regardless of the concentration of cysteine in the initial medium (see paragraphs [0024] and [0027] of the patent application). Thus, feature (b) has a basis in the patent application.

Feature (c): "[M]aintaining the concentration of serine, cysteine and tyrosine during a period from a fourth day to a tenth day of the culture by addition of the fed-batch medium."

2.11 Respondents argued that the selection of the sub-period of maintenance of the concentration of serine, cysteine and tyrosine in the culture solution from day 4 to 10 was a threefold selection -for each amino acid separately- which was nowhere disclosed in combination with the lower concentration threshold defined in claim 1 (see paragraphs [0022], [0028], [0038]).

Moreover, Example 3 could not really support the method of claim 1.

2.12 Appellant asserted that feature (c) of claim 1 was based on claims 12, 13 and paragraphs [0027] to [0029] of the patent application. This concept was further elaborated in paragraphs [0019], [0022] and [0026]).

2.13 The board concurs with appellant's view. Claims 12 and 13 of the patent application disclose a method of culturing a cell to produce a desired protein, characterized in that a concentration of serine in a culture solution is maintained at 2 mM or higher at least during a part of or an entire period from the third day to the tenth day of the culture. Further, a concentration of tyrosine in the culture solution is maintained at 1 mM or higher, and/or a concentration of cysteine in the culture solution is maintained at a concentration of the cysteine in an initial medium or higher or at 0.4 mM or higher at least during a part of or an entire period from the third day to the tenth day of the culture (emphasis added).

The highlighted expression stands for a culture period from the fourth, fifth, sixth, or seventh day of the culture, or a culture period from the start point of the culture or from the first or second day of the culture including a part of or an entire culture period from the third day to the tenth day of the culture (see paragraph [0028] of the patent application). Hence, the period of time combining a starting point of this period "from a fourth day" with the end-point "to a tenth day" has a direct and unambiguous basis in this paragraph. The now claimed shortened time span necessitates only one selection from a single list of equal possibilities.

Combination of features recited in claim 1

2.14 The board, in applying the so-called gold standard (see point 2. of the reasons above), notes that there is neither an explicit nor an implicit yet direct and unambiguous disclosure in the patent application of a method combining the features (a) to (c) as in claim 1: wherein all of serine, tyrosine and cysteine are selected and maintained at different concentrations: 2 mM, 1 mM and 0.4 mM, respectively, each of them from the/a fourth to the/a tenth day of the culture, by means of an addition of the fed-batch medium comprising all the three amino acids.

2.14.1 Since a fed-batch medium for use in a fed-batch culture method is defined in the patent application as a medium comprising serine and tyrosine and/or cysteine, i.e. comprising either serine and tyrosine; serine and cysteine; or serine, tyrosine and cysteine (see claim 16; paragraphs [0042] and [0038]), the skilled person has to first select a fed-batch medium comprising the combination of serine, tyrosine and cysteine from the three alternatives described above.

2.14.2 There is no pointer to a fed-batch medium having serine, tyrosine and cysteine, neither in the claims or paragraphs [0038] and [0042], nor in example 3 of the patent application. Each of the fed-batch media is equally preferred and suitable for fed-batch culture.

2.14.3 The board notes that Example 3 does not exactly reproduce the method of claim 1. Although the addition of fed-batch medium comprising serine, tyrosine and cysteine is more effective than fed-batch media containing only some of them, in fed-batch culture they all achieve a better protein yield than the control fed-batch culture. Again, based on example 3, there is neither a pointer nor a direct and unambiguous disclosure that the use of the claimed fed-batch medium comprising serine, tyrosine and cysteine would be preferable to any other disclosed fed-batch media.

2.15 Claims 11 to 13 disclose methods of culturing cells comprising maintaining the concentrations of serine, tyrosine and/or cysteine in the culture solution. Claim 13 refers back to claims 11 or 12. Claim 13 relates to an embodiment where serine is maintained at 2 mM in the culture solution. Claim 12 specifies that tyrosine is maintained at 1 mM and/or that cysteine is maintained at a concentration of an initial medium or higher or at 0.4 mM or higher in the culture solution respectively. Claim 11 requires maintenance of the serine concentration at 1 mM or higher.

Thus, claims 11 to 13 disclose the following combinations of amino acid concentrations (or higher) to be maintained: 1) Ser 1 mM (claim 11); 2) Ser 2 mM (claim 13 referring to claim 11); 3) Ser 1 mM, Tyr 1mM (claim 12); 4) Ser 1 mM, Cys initial concentration (claim 12); 5) Ser 1 mM, Cys 0.4 mM (claim 12); 6) Ser 1mM, Tyr 1mM, Cys initial concentration (claim 12); 7) Ser 1mM, Tyr 1mM, Cys 0.4 mM (claim 12); 8) - 12) combinations 3) to 7) but with 2 mM Ser (claim 13 referring to claim 12).

All these combinations of features are equally preferred without any pointer to a particular combination.

2.15.1 The skilled person has to select a particular combination from this list to specifically maintain serine, tyrosine and cysteine at a concentration at or above 2 mM, 1 mM and 0.4 mM, respectively in the culture solution. This constitutes a second selection, for which there is neither a pointer nor a direct and unambiguous disclosure thereof. Indeed, the adjusted medium comprising 1 mM or higher of serine or a salt thereof, and at least 1 mM or higher of tyrosine and/or 0.4 mM or higher of cysteine in paragraphs [0017], [0042] or examples 1 to 3 of the patent application fail to disclose the maintenance of serine, tyrosine and cysteine in the culture solution at or above 2 mM, 1 mM and 0.4 mM respectively in combination.

2.16 Finally, the method of culturing cells according to claims 11 to 13 of the patent application specifies that a concentration of serine, tyrosine and/or cysteine is maintained at least during a part of or an entire period from a third day to a tenth day of the culture. The expression "at least during a part of or an entire period from a third day to a tenth day of the culture" is further defined in paragraphs [0022] and [0028] and refers to a culture period from the fourth, fifth, sixth, or seventh day of the culture or a culture period from the start point of the culture or from the first or second day of the culture including a part of or an entire culture period from the third day to the tenth day of the culture.

The skilled person has to select from the definition provided in paragraph [0028] that the culture period is shortened from the fourth day to a tenth day of culture among all the equal possibilities to arrive at the method of claim 1. This constitutes a third selection from a list for which there is neither a pointer nor a direct and unambiguous disclosure thereof.

2.16.1 In view of the above considerations, the skilled person has to select the claimed features from three alternatives and two different lists of a certain length and equally weighted possibilities in the application as filed to arrive at the combination of features of the method according to claim 1. A method with such a combination of features for which there is no clear pointer extends beyond the content of the application as filed (see e.g T 686/99 of 22 January 2003, reasons 4.3.3).

2.17 The board therefore concludes that the method of claim 1 contravenes Article 123(2) EPC.

Admission of auxiliary requests 2 and 3 filed with appellant's statement of grounds of appeal into the proceedings.

3. Auxiliary requests 2 and 3 were first filed with the statement of grounds of appeal.

4. The respondents requested not to admit them into the proceedings.

5. Pursuant to Article 12(4) RPBA 2007, which applies to the present case under Article 25(2) RPBA 2020, the board has a discretion to hold inadmissible facts, evidence or requests, which could have been presented or were not admitted into the first instance proceedings.

6. Auxiliary requests 2 and 3 derive from the main request and auxiliary request 1, respectively, by combination of claims 1 to 4. Formally, the amendment corresponds to the deletion of claims 1 to 3 while retaining claim 4. The deletion of the subject-matter defined in claims 1 to 3 from the main and auxiliary request 1 cannot have taken the respondents by surprise as the proposed amendments do not change the legal and factual scope of the proceedings. Claim 4 was already present in auxiliary requests 13 and 14 in the opposition proceedings.

6.1 Since the above deletion neither creates a fresh case nor affects procedural economy, the board, availing itself of its discretionary power under Article 12(4) RPBA 2007, decided to admit the new auxiliary requests 2 and 3 into the appeal proceedings.

Auxiliary requests 1 to 3

Article 123(2) EPC

7. The findings on Article 123(2) EPC in connection with claim 1 of the main request (see paragraphs 2.4 to 2.17 above) equally apply to claim 1 of each of the auxiliary requests 1 to 3 in that they combine the same technical features as claim 1 of the main request.

8. Since no allowable request is on file, the appeal must be dismissed.