T 0919/21 (Glycosylation of FIX/WYETH) 23-11-2023
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Production of coagulation factor IX with improved glycosylation pattern
Grounds for opposition - insufficiency of disclosure (yes)
Late-filed request - should have been submitted in first-instance proceedings (yes), circumstances of appeal case justify admittance (no)
Summary of Facts and Submissions
I. The appeal of the opponent (appellant) lies from the opposition division's decision to reject the opposition filed against European patent No. 2 495 307 entitled "Production of coagulation factor IX with improved glycosylation pattern".
II. The opposition proceedings were based on the grounds for opposition in Article 100(a) EPC, in relation to novelty (Article 54 EPC) and inventive step (Article 56 EPC), and those in Article 100(b) and (c) EPC.
III. With the reply to the appeal, the patent proprietor (respondent) submitted sets of claims of auxiliary requests 1 to 15. Auxiliary requests 1 to 13 were later withdrawn.
Claim 1 of the main request (patent as granted) reads as follows:
"1. A method for producing coagulation factor IX with an improved glycosylation pattern in a cell culture comprising:
culturing mammalian cells that contain a gene encoding a coagulation factor IX in a cell culture medium comprising between 10 nM and 600 nM manganese under conditions and for a time sufficient to permit expression of coagulation factor IX, wherein the glycosylation pattern of the expressed coagulation factor IX is more extensive than the glycosylation pattern observed under otherwise identical conditions in otherwise identical medium that lacks the manganese, wherein a more extensive glycosylation pattern is a greater number of covalently linked sugar residues in the oligosaccharide chain."
Claim 1 of each of auxiliary requests 14 and 15 reads as follows:
"1. A method for producing coagulation factor IX with an improved glycosylation pattern in a cell culture comprising:
culturing mammalian cells that contain a gene encoding a coagulation factor IX in a cell culture medium comprising approximately 40 nM manganese under conditions and for a time sufficient to permit expression of coagulation factor IX, wherein the glycosylation pattern at Ser-61 of the expressed coagulation factor IX is more extensive than the glycosylation pattern at Ser-61 observed under otherwise identical conditions in otherwise identical medium that lacks the manganese, wherein a more extensive glycosylation pattern is a greater number of covalently linked sugar residues in the oligosaccharide chain."
IV. The board summoned the parties to oral proceedings in accordance with their requests and, in a communication pursuant to Article 15(1) RPBA, expressed its preliminary opinion on, inter alia, sufficiency of disclosure and admittance and consideration of auxiliary requests 14 and 15 in the appeal proceedings.
V. In a further submission, the appellant provided arguments on, inter alia, admittance and consideration of auxiliary requests 14 and 15 in the appeal proceedings.
VI. Oral proceedings were held on 23 November 2023.
VII. The following document is referred to in this decision:
D35|Declaration by D. R. Lasco dated 25 September 2017|
VIII. The appellant's arguments relevant to the decision are summarised as follows.
Main request - claim 1
Sufficiency of disclosure (Article 100(b) EPC)
The data in the patent did not demonstrate that the addition of manganese in an amount between 10 nM and 600 nM to a cell culture medium resulted in the production of coagulation factor IX (FIX) with a more extensive glycosylation pattern, i.e. a greater number of covalently linked sugar residues in the oligosaccharide chain.
Figures 2 and 4 did not show any effect of manganese on glycosylation of FIX compared to the control sample. Regarding the data in Figures 5 and 6, it was acknowledged in the patent and by the respondent's expert in the declaration D35 that neither the tetra-oligosaccharide (K4-species) nor the tri-oligosaccharide or the di-oligosaccharide peak could be trusted. Three of the four data peaks were hence meaningless and unreliable. Since the values reported for each peak were the ratios of the area of each of the four peaks to their sum (paragraph [00163] of the patent), the remaining data peak was also unreliable.
Additionally, in Figure 6, the positive control had a lower peak for the K4-species and a higher peak for the fucose-only species (K4'-species) than the negative control and, thus, no interpretation of or conclusion from any of the data points in these experiments was possible. The two duplicate assay reference samples displayed a large variation of almost 10% in the K4-species peaks, a fact that further demonstrated that none of these data had any significance (document D35, page 7).
In any case, a decrease in the peak of the K4'-species did not necessarily mean that the glycosylation pattern had to be more extensive since this effect could be negated by a greater reduction in the tetra-oligosaccharide peak.
Auxiliary requests 14 and 15
Admittance (Article 12 RPBA)
Auxiliary requests 14 and 15 had only been submitted in the appeal proceedings. They could have been submitted earlier as the objection that only the glycosylation at Ser-61 in FIX had been investigated in the patent whereas FIX contained multiple other glycosylation sites had been raised in the notice of opposition (page 17). The respondent had not submitted any reasoning why this amendment had been filed so late.
Moreover, the amendment did not consist of the addition of a feature from a dependent claim but of a new feature from the description and therefore added complexity to the case. It was not allowable under Article 123(2) EPC since the amended claim was the result of an intermediate generalisation.
IX. The respondent's arguments relevant to the decision are summarised as follows.
Main request - claim 1
Sufficiency of disclosure (Article 100(b) EPC)
The imperfections in the data shown in Examples 2 and 3 of the patent could not discredit the data, especially as the deficiencies were explained in the patent (paragraphs [0144], [0150], [0153] and [0160]). The addition of a single sugar residue in the oligosaccharide chain was sufficient to fulfil the requirement of a more extensive glycosylation pattern as defined in the claim. If the K4'-species was decreased, this could only mean that one or more of the other three glycosylation species were increased and the requirement was fulfilled. It was therefore sufficient to merely analyse the K4'-species.
Figure 2 concerned a preliminary experiment in which the tetra-oligosaccharide data could be relied on as no degradation issues had been observed for the controls. It showed a trend towards an increase in the K4-species and a decrease in the K4'-species when 10 nM manganese was added to the cell culture medium. Figure 4 showed the same trend since the values for the K4'-species in the two control experiments ("Expt."; 13% and 12%) were higher than those for the K4'-species in the experiment where 10 nM manganese was added ("Expt. + Mn"; 11% and 10%). Figure 5 demonstrated that the addition of 15 nM manganese decreased the K4'-species (10% vs 15%). Figure 6 showed that the addition of 10 nM to 500 nM manganese decreased the K4'-species, as explained in paragraph [0159] of the patent.
As explained in document D35 (second full paragraph on page 7), despite the unusual result for the positive control in the experiment of Figure 6, the skilled person would not have had any doubts about this effect supported by four different experiments.
Auxiliary requests 14 and 15
Admittance (Article 12 RPBA)
It had not been necessary to submit auxiliary requests 14 and 15 in opposition proceedings since the opposition division's preliminary opinion had been positive. They were filed at the first opportunity in appeal with the reply to the appeal. The amendment did not create new issues but overcame one of the objections on the glycosylation pattern on other sites in FIX. This should not come as a surprise to the appellant as glycosylation at position Ser-61 was investigated in Examples 2 and 3 of the patent.
X. The parties' requests relevant for the decision are as follows.
The appellant requests that the decision under appeal be set aside and amended such that the patent be revoked, and that auxiliary requests 14 and 15 not be admitted and considered in the proceedings.
The respondent requests that the appeal be dismissed, or that the patent be maintained in amended form based on the set of claims of one of auxiliary requests 14 and 15, both submitted with the reply to the appeal.
Reasons for the Decision
Main request (patent as granted) - claim 1
Sufficiency of disclosure (Article 100(b) EPC)
1. Claim 1 concerns a method for producing coagulation factor IX (FIX) with an improved, more extensive glycosylation pattern, which is defined as "a greater number of covalently linked sugar residues in the oligosaccharide chain" and which is, according to the claim, achieved by culturing mammalian cells that express FIX in a cell culture medium comprising between 10 nM and 600 nM manganese (see section III. for the full wording of the claim).
2. Reference is made to the corrected patent publication B9. Examples 2 and 3 of the patent disclose the analysis of the effect of different additives, including manganese, on the glycosylation at amino acid serine 61 (Ser-61) of FIX. For this analysis, FIX was digested with a lysyl endopeptidase resulting, inter alia, in a so-called K4 peptide that comprised Ser-61 (paragraph [0143] of the patent). Four different K4 peptide species were found that either comprised a complete tetra-oligosaccharide (K4-species), a desialylated tri-oligosaccharide, a di-oligosaccharide, or only fucose (K4'-species).
3. Results from experiments in which manganese was added to the cell culture medium are shown in Figures 2, 4, 5 and 6 of the patent. According to the respondent, these results demonstrate that the addition of between 10 nM and 600 nM manganese to a cell culture medium results in the production of FIX with a more extensive glycosylation pattern. However, this is not persuasive.
Figure 2
4. Figure 2 shows the results of a small-scale experiment described in Example 2 of the patent in which the effects of different additives on glycosylation at Ser-61 of FIX were assessed. In this experiment, only the "supplemented" cell culture medium ("Supp." in Figure 2) led to a significant increase in the amount of the K4-species; the addition of 1 nM or 10 nM manganese to the cell culture medium did not (see paragraph [0148] and Figure 2). Contrary to the respondent's assertion, no trend towards an effect of 10 nM manganese on the amount of the K4-species is shown in Figure 2 as the amount of the K4-species in one of the duplicate experiments is identical to that observed in the control experiments. The slight increase of about 2% from about 74% in the controls to 76% in the second duplicate experiment is hence not significant and in any case lies within the deviation observed in other duplicate experiments shown in this figure.
5. Consequently, the data in Figure 2 of the patent do not support that the claimed method, which encompasses the addition of 10 nM manganese to the cell culture medium, achieves the effect of a more extensive glycosylation pattern.
Figure 4
6. Figure 4 shows the results of a further experiment in which, inter alia, the FIX glycosylation pattern was analysed under control conditions ("Control"), under conditions designated "Expt." and under conditions designated "Expt. + Mn" (10 nM manganese, MnSO4) to explore "additives that might complement the original medium additives" (see paragraph [0152] of the patent). Hence, to assess a possible effect of 10 nM manganese on FIX glycosylation, the results for the conditions "Expt." and "Expt. + Mn" must be compared. This comparison does not show any difference in the amount of the K4-species.
7. According to the respondent, a trend towards an increase in FIX glycosylation caused by the addition of 10 nM manganese to the cell culture was evident from the amount of the K4'-species, which decreased from 13% and 12% in the "Expt." duplicate experiments to 11% and 10% when 10 nM manganese were added. However, this difference is marginal, and similar variations were observed between the duplicate experiments for the different conditions that were tested, including the assay reference and the control (see Figure 4). It is therefore not statistically significant and does not allow drawing any conclusion on the effect of the addition of 10 nM manganese to a cell culture medium on FIX glycosylation.
8. Additionally, as described in paragraph [0150] of the patent, "for unknown reasons, the de-sialylated form of K4' started showing up in greater abundance in both the test samples and the assay reference" in, inter alia, the experiment shown in Figure 4. Moreover, as described in paragraph [0152] of the patent, also "for unknown reasons, the control and experimental conditions gave very similar K4 species distributions that were more like what had previously been observed for the control conditions". These facts demonstrate further that the data in Figure 4 cannot be reliably interpreted (see also points 9. to 15. below).
Figures 5 and 6
9. In the experiments shown in Figures 5 and 6, glycosylation at Ser-61 of FIX was investigated when 15 nM manganese (Figure 5) or when between 10 nM and 500 nM manganese (Figure 6) was added to the cell culture medium. In each of these experiments, the desialylated form of the K4-species was detected in high and variable amounts, including in the assay control, and the relative amounts of the K4-species demonstrated a large variation between the duplicate experiments, i.e. the same phenomena were observed as described in point 8. above for Figure 4.
10. In addition, only a very small difference between the negative ("Control") and the positive ("Supp.") control was observed in the experiment of Figure 5. In the experiment of Figure 6, lower relative amounts of the K4-species and higher relative amounts of the K4'-species were observed for the positive control compared to the negative control, i.e. the opposite of what would have been expected.
11. A direct consequence of these deficiencies in the data shown in Figures 5 and 6 is that the relative amounts of the different K4 peptide species, in particular the K4-species comprising full tetra-oligosaccharide and species comprising the desialylated tri-oligosaccharide, as shown in these figures, do not reflect the actual amounts of these species in the samples. This fact was also acknowledged in the patent (paragraphs [0152], [0158], [0159] and [0160]) and in the expert declaration D35 submitted by the respondent during the opposition proceedings (sections 5, 6 and 7 of D35).
12. However, according to the respondent, these deficiencies in the experimental data could be ignored as it was sufficient to consider the relative amount of the fucose-only K4'-species in these experiments (paragraphs [0159] and [0160] of the patent; second full paragraph on page 6 and first, second and third full paragraphs on page 7 of D35). A decrease in this species automatically meant that one or more of the other species were increased and that, therefore, the overall FIX glycosylation pattern was extended.
13. The board is not persuaded that the data in Figures 5 and 6 can be analysed in this manner. First of all, a decrease in the relative amount of the K4'-species does not necessarily mean that the overall FIX glycosylation pattern must be more extensive since this effect could be negated by a greater reduction in the amount of the K4-species.
14. Second, the data can only be evaluated relative to appropriate control reactions, which, however, in particular in the data of Figure 6, are also unreliable as in the alleged positive control reaction, lower relative amounts of the K4-species and higher relative amounts of the K4'-species than in the negative control were detected (see point 10. above). These results cast doubt on the validity of any of the data disclosed in this figure since it is not clear which of the control data, if not both, are erroneous. Therefore, none of the data points shown in this figure can reliably be interpreted, and no conclusion can be drawn on the extent of FIX glycosylation under any of the different assay conditions relative to each other.
15. In view of these considerations, the patent does not contain any data that credibly demonstrate that a FIX with a more extended glycosylation pattern can be produced in a cell culture medium comprising between 10 nM and 600 nM manganese than in an otherwise identical medium that lacks the manganese. In fact, the results shown in Figure 2, the only experiment in which the data did not suffer from the deficiencies identified above, demonstrate that a more extended FIX glycosylation pattern was not obtained in a cell culture medium comprising 10 nM manganese (see points 4. and 5. above).
16. In the absence of any data supporting a conclusion that a more extended FIX glycosylation pattern can be obtained in a cell culture medium comprising between 10 nM and 600 nM manganese, and in view of the fact that the patent contains data that even point to the contrary, the patent does not disclose the claimed method in a manner sufficiently clear and complete for it to be carried out by the skilled person (Article 100(b) EPC).
Auxiliary requests 14 and 15
Admittance (Article 12 RPBA)
17. With the reply to the appeal, the respondent submitted auxiliary requests 14 and 15, which are new to the proceedings (see section III.). Under Article 12(6) RPBA, the board must not admit, inter alia, requests which should have been submitted in the proceedings leading to the decision under appeal unless the circumstances of the appeal case justify their admittance, in line with the requirement set out in Article 12(2) RPBA that a party?s appeal case must be directed to the requests on which the decision under appeal was based. Moreover, a party must clearly identify any amendment to their case in appeal and must provide reasons for submitting it at that stage and, in the case of amendments to the claims, why they overcome the objections raised (Article 12(4) RPBA).
18. Claim 1 of each of auxiliary requests 14 and 15 comprises the feature that the glycosylation pattern of the expressed FIX is at Ser-61 (see section III.). When submitting these claim requests, the respondent indicated for auxiliary request 14 that the amendment was made "in response to the objections raised by the opponents under A100 (a) and (b) EPC" and that it "may address, if necessary, any argument of the opponent that increasing the extent of glycosylation is not relevant for factor IX as therapeutic agent" (section 7.8 of the reply to the appeal).
19. No further substantiation was given in the reply to the appeal as to why the amendment overcame the objections raised. Only in a later submission (see section V.) and during the oral proceedings before the board, the respondent claimed that this feature overcame the appellant's objection that glycosylation of FIX was only analysed at this amino acid residue and that it had not been necessary to submit auxiliary requests dealing with this objection during the opposition proceedings because the opposition division's preliminary opinion had been in favour of the respondent in this aspect.
20. Apart from being late filed, contrary to the obligation under Article 12(4) RPBA that a party should provide reasons when filing an amendment, the respondent's reasons are not persuasive. The objection that only the glycosylation at Ser-61 in FIX had been investigated in the patent although FIX contained multiple other glycosylation sites had been raised in the notice of opposition (page 17). Therefore, auxiliary requests responding to this objection could have been submitted in the opposition proceedings.
21. The respondent's further argument (submitted in reply to the board's communication) that the amendments did not create any new issues and did not add any complexity cannot be accepted either, at least because the newly added feature that the glycosylation at Ser-61 of FIX is assessed was added from the description of the application and was not present in any dependent claim of the patent as granted. Prior to the filing of auxiliary requests 14 and 15, the appellant therefore did not have to consider the impact of this feature on any of their objections.
22. Consequently, auxiliary requests 14 and 15 should have been submitted in the proceedings before the opposition division. No particular circumstances of the appeal case are apparent or were invoked by the respondent that could justify the filing of these auxiliary requests only in the appeal proceedings. Therefore, auxiliary requests 14 and 15 cannot be considered in the appeal proceedings, pursuant to Article 12(6) RPBA.
Order
For these reasons it is decided that:
1. The decision under appeal is set aside.
2. The patent is revoked.