T 2605/22 (Disulfide bonds in polypeptides/GENETECH) 12-02-2025
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Prevention of disulfide bond reduction during recombinant production of polypeptides
Grünecker Patent- und Rechtsanwälte
PartG mbB
Michalski Hüttermann & Partner
Patentanwälte mbB
Bayer Intellectual Property GmbH
Oetke, Cornelia
Mathys & Squire LLP
Maiwald GmbH
df-mp Dörries Frank-Molnia & Pohlman
Summary of Facts and Submissions
I. The appeals by opponents 1, 2, 4, 7, 8, 9 and 11 (appellants I, II, III, IV, V, VI and VII, respectively) lie from the decision of the opposition division that European patent No. 2 188 302, entitled "Prevention of disulfide bond reduction during recombinant production of polypeptides", in amended form based on the set of claims of the main request filed with letter of 7 April 2021, met the requirements of the EPC. The decision was issued in corrected form on 26 January 2023.
II. The patent was granted on European patent application No. 08 781 481.0 filed as an international patent application under the PCT which had been published as WO 2009/009523 (application as filed).
III. The patent had been opposed on the grounds of Article 100(a) EPC, in relation to novelty (Article 54 EPC) and inventive step (Article 56 EPC), and of Article 100(b) and (c) EPC.
IV. The appellants submitted various documents with their statements of grounds of appeal and with subsequent letters.
Opponents 3, 5, 6 and 10 did not make submissions during the appeal proceedings. They are parties as of right to the appeal proceedings.
With its reply to the appeals, the respondent (patent proprietor) filed sets of claims for a main request and for auxiliary requests 1 to 41 and submitted various documents. The main request is identical to the main request allowed in opposition proceedings.
By letter dated 15 May 2024, the respondent filed a set of claims for auxiliary request 42.
V. The board summoned the parties to oral proceedings and informed them of its preliminary opinion in a communication under Article 15(1) RPBA dated 12 September 2024.
VI. By letter dated 9 January 2025, the patent proprietor informed the board that it was withdrawing its request for oral proceedings and also withdrawing all auxiliary requests. The patent proprietor further stated "We do not intend to make any further submissions or to file any further claim requests on the understanding that the patent will be revoked based on the state of the file without any oral proceedings being held."
By letter dated 9 January 2025, opponent 3 indicated that it would not attend the oral proceedings.
By letter dated 10 January 2025, opponent 5 indicated that it would not attend the oral proceedings.
VII. The board cancelled the oral proceedings and continued the proceedings in writing.
VIII. Claim 1 of the main request reads as follows:
"1. A method for the prevention of the reduction of a disulfide bond in a polypeptide expressed in a recombinant host cell during processing following fermentation, comprising, following fermentation, supplementing the pre-harvest cell culture fluid (CCF) or harvested cell culture fluid (HCCF) of said recombinant host cell with a thioredoxin inhibitor, wherein said polypeptide is an antibody, or a biologically functional fragment of an antibody, wherein said recombinant host cell is a eukaryotic host cell, and wherein said thioredoxin inhibitor is:
(i) a direct inhibitor of thioredoxin;
(ii) a specific inhibitor of thioredoxin reductase;
(iii) is cupric sulfate
(a) added in a concentration between about 5 myM and about 100 myM;
(b) added in a concentration between about 10 myM to about 80 myM; or
(c) added in a concentration between about 15 myM and about 50 myM;
(iv) an inhibitor of G6PD, selected from the group consisting of pyridoxal 5'-phosphate, 1 fluoro-2,4 dinitrobenzene, dehydroepiandrosterone (DHEA) and epiandrosterone (EA);
(v) an inhibitor of hexokinase activity, which inhibitor is
(a) EDTA added in a concentration of between about 10 mM and about 50 mM; or
(b) selected from the group consisting of sorbose-1-phosphate, polyphosphates, 6-deoxy-6- fluoroglucose, 2-C-hydroxy-methylglucose, xylose, and lyxose ;
(vi) an antibody specifically binding to a thioredoxin reductase; or
(vii) a measure indirectly resulting in the inhibition of thioredoxin activity, which measure is air sparging the harvested culture fluid of said recombinant host cell."
IX. The appellants' submissions are summarised as follows:
Main request - claim 1
Claim interpretation
The feature "following fermentation" was ambiguous. It described multiple technically logical scenarios, for example: following some fermentation (i.e. once fermentation has started), following production of a recombinant antibody molecule by fermentation (i.e. once at least one antibody molecule has been produced by a recombinant host cell), following the termination of all fermentation (i.e. when all the CCF was harvested or the cells were lysed), or (as the respondent argued) following the end of a stage in an industrial antibody production method that was associated with the bulk of the antibody.
Amendments (Article 123(2) EPC)
The amendment "following fermentation, supplementing the pre-harvest cell culture fluid (CCF) [...] with a thioredoxin inhibitor" ignored a fundamental difference in the application's teaching, between the timing of the effect (when reduction was prevented) and the timing of the intervention (when the method step was performed).
The application did not directly and unambiguously disclose that the method step (supplementing the culture fluid with a Trx inhibitor) used to achieve the purpose took place following fermentation. In other words, the application taught that the effect was achieved following fermentation, but it did not disclose that the intervention taken to achieve this effect occurred following fermentation.
The expression "following completion of the cell culture process" (see page 31, lines 20 to 24 of the application as filed) was not the same as "following fermentation". At best, there was similarity between "following completion of the cell culture process" and one specific interpretation of "following fermentation" (i.e. following termination of all fermentation). Moreover, the terms "following completion of the cell culture process" and "following fermentation" were clearly not synonyms in the context of continuous cell culture processes which were explicitly mentioned in the application as filed (see page 34, lines 26 to 27). The section on page 31 could not, therefore, provide direct and unambiguous disclosure for an ambiguous amendment that could define other timings for the intervention (e.g. following some fermentation, i.e. after fermentation has started).
The section on page 30 (line 31) through to the section on page 31 (lines 20 to 24) discussed non-directed methods (air sparging and lowering of pH), both of which it explicitly taught were applied to the harvested CCF. Accordingly, when the skilled person read "following completion of the cell culture process" in lines 20 to 24 on page 31, they understood it to mean "after harvest". This, however, was inconsistent with the claim's reference to "pre-harvest CCF".
The only interpretation of "following fermentation" that fitted consistently with the original application was "after harvest". The application repeatedly drew a conceptual line for the timing of the intervention at the point of harvesting the CCF. It disclosed that the intervention (supplementing with a Trx inhibitor) could be done before and/or after harvest. The claim, however, defined a new, specific period during which the intervention should be applied: after fermentation but before harvesting.
The respondent's (patent proprietor's) submissions are summarised as follows:
Main request - claim 1
Claim interpretation
"following fermentation, supplementing the pre-harvest cell culture fluid (CCF)"
The skilled person, avoiding artificial and semantic constructions and with a mind willing to understand, would not interpret "following fermentation" as "during fermentation", "following some fermentation", or "following the start of fermentation", as suggested by the appellants. The normal implicit meaning was "after the end of fermentation".
The skilled person would arrive at this interpretation based on their common general knowledge that "fermentation" referred to the growth and production process by which recombinant eukaryotic cells express recombinant proteins. This process was controlled through the culture conditions of the cells and formed part of conventional antibody manufacture. A "fermentation process" also encompassed the times when the culture received feeds, which would be understood as occurring during the growth and production phases of cell culture.
The skilled person would not interpret fermentation as referring to an individual molecular event, such as the secretion of a single antibody from a single cell, and there was no evidence cited for such interpretation.
The "pre-harvest CCF" in claim 1 was self-defining and meant the CCF before harvest. It referred to the fluid containing the antibody, as well as host cells and impurities, before harvest.
In summary, the term "following fermentation" in claim 1 referred to a time in an antibody production process, which time was after the end of the growth and production phases.
Amendments (Article 123(2) EPC)
The way in which the application as filed discussed the methods of the invention with respect to timing and the nature of the thioredoxin inhibitor informed the way in which the skilled reader would understand the passages on pages 26, 29 and 31.
The summary of the invention did not merely state that an inhibitor was added to a "culture fluid". Instead, it specified that it was added to a culture fluid at a time that was defined relative to the harvest: "pre-harvest" or "harvested" (see page 2, lines 21 to 28, claims 2 and 3 as filed). Using the harvest of cell culture fluid as the anchor point for the timing of the supplementing implied that it took place relatively close to harvest, relatively, that is, as compared with the other standard events and stages of a recombinant antibody production process, e.g. inoculation, feeds, growth and production phases.
When read by a skilled person, the three relevant passages of the application as filed, on page 26, lines 20 to 24, page 29, lines 1 to 12, and page 31, lines 20 to 25, meant that a method in which the supplementing of the pre-harvest CCF was further specified as "following fermentation" did not constitute new technical teaching.
When these passages were read together, from a practical and reasonable standpoint, and in the context of the application as a whole, it was directly and unambiguously derivable that the invention included a method in which the pre-harvest CCF was supplemented with thioredoxin inhibitor following fermentation.
X. The requests relevant to this decision are as follows.
The appellants request that the decision under appeal be set aside and the patent revoked.
The respondent (patent proprietor) requests that the appeals be dismissed and that the decision to maintain the patent based on the set of claims of the main request filed with the reply to the appeals be upheld.
Reasons for the Decision
Main request
1. The claim interpretation and assessment of added subject-matter in this decision is limited to the first alternative in claim 1 "following fermentation, supplementing the pre-harvest cell culture fluid (CCF)[...] of said recombinant host cell with a thioredoxin inhibitor" (underlining by the board). The second alternative "supplementing the [...] harvested cell culture fluid (HCCF)" has not been assessed by the board.
Claim interpretation - claim 1
2. The method involves a starting material:
"a polypeptide expressed in a recombinant host cell [..., which] is an antibody"
to which a single process step is applied:
"supplementing the pre-harvest cell culture fluid (CCF) [...] of said recombinant host cell with a thioredoxin [Trx] inhibitor"
(wherein, for the sake of ease of discussion, "supplementing" is understood as "adding")
resulting in an antibody in which the reduction of a disulfide bond has been prevented.
"following fermentation, supplementing the pre-harvest cell culture fluid (CCF) [...] of said recombinant host cell with a thioredoxin inhibitor"
3. The expression "following fermentation" in the context of the claim does not provide the skilled person with a clear point in time or time period at which a Trx inhibitor is to be supplemented to the CCF. As outlined by appellant VI (see point 64. of its statement of grounds of appeal), multiple technically sensible interpretations exist. For example, the expression could mean "following the start of fermentation", "following some fermentation", "following production of an antibody molecule by fermentation", "following a discrete round of fermentation", "following the end of a production phase in industrial antibody production", or "following termination of all antibody synthesis". The board finds none of these interpretations illogical or not technically sensible (see Case Law of the Boards of Appeal of the EPO, 10th edition 2022, I.C.4.1).
4. The claim itself does not contain a definition of the expression "following fermentation". Even if the description of the application as filed or the patent is taken into account as a further means of interpretation, it does not provide an explicit definition of "following fermentation" either, but merely states that "[f]ollowing fermentation proteins are purified" (see page 35, line 14, of the application as filed) and mentions that "the invention concerns methods for preventing the reduction of disulfide bonds of recombinant proteins during processing following fermentation" (see page 26, lines 23 to 24, of the application as filed). The skilled person therefore only learns that proteins can be processed and purified "following fermentation", but not when this time period starts. In any case, a definition in the description is not to be used to introduce limiting features into a claim (see Case Law of the Boards of Appeal of the EPO, 10th edition 2022, II.A.6.3.4).
5. The respondent argued that the gist of the invention was the recognition that reduction of disulfide bonds in the harvested antibodies occurred during processing of the cell culture fluid. This was likely to be due to the action of the enzymes of the Trx system released from lysed cells (see example 1, paragraph [0209] of the patent). The skilled person would therefore interpret the claim to mean that the supplementing had to occur at the end of fermentation, shortly before the CCF was separated from the whole cells or even after separation in the HCCF, i.e. "following fermentation in claim 1, refers to a time in an antibody production process, which time is after the end of the growth and production phases" (see reply to the appeals, point 82.).
6. The board does not agree. While recognising the effect which the invention aims to achieve, namely, as outlined in claim 1, "prevention of the reduction of a disulfide bond in a polypeptide expressed in a recombinant host cell during processing following fermentation", the board cannot see how achieving this effect at a certain process stage ("during processing following fermentation") would exclude any of the technically sensible interpretations for the time period "following fermentation, supplementing" at which a Trx inhibitor is added to the CCF.
7. The board, in view of the diverging, but technically sensible and not illogical interpretations outlined by the appellants, finds the expression "following fermentation" to be ambiguous (see point 3. above) and not limited to a "time in an antibody production process, which time is after the end of the growth and production phases", as argued by the respondent.
Added subject-matter (Article 123(2) EPC)
8. It is undisputed that the expression "following fermentation, supplementing the pre-harvest cell culture fluid (CCF) or harvested cell culture fluid (HCCF) of said recombinant host cell with a thioredoxin inhibitor" in claim 1 does not appear verbatim in the application as filed.
9. The expression "following fermentation, supplementing the pre-harvest cell culture fluid (CCF)" is ambiguous in the context of claim 1 (see points 6. and 7. above). Therefore only an explicit disclosure in combination with the other features of the claim would allow all possible interpretations to be directly and unambiguously derived from the application as filed (see Case Law of the Boards of Appeal of the EPO, 10th edition 2022, II.E.1.3.9 (e)). This is obviously not the case. For this reason alone, the subject-matter of claim 1 extends beyond the content of the application as filed (see also statement of grounds of appeal of appellant VI, point 47.).
10. Nevertheless, further arguments by the respondent are addressed below.
11. The passages in the application as filed provided by the respondent as basis for the feature "following fermentation, supplementing the pre-harvest cell culture fluid (CCF)" can be separated into two sets of disclosures, the first dealing with the effect to be achieved and the second dealing with the intervention or process step to achieve this effect.
12. The first set of disclosures concerns the effect to be achieved by the claimed method.
The start of the summary on page 2, lines 22 to 25, describes the invention as generally relating to "a method for preventing reduction of a disulfide bond in a polypeptide expressed in a recombinant host cell". This is defined more precisely on page 26, lines 22 to 24: "preventing the reduction of disulfide bonds of recombinant proteins during processing following fermentation" and on page 29, lines 9 to 11: "Alternatively or in addition other non-specific methods can also be used to prevent the reduction of disulfide bond reduction [sic] following fermentation during the recombinant production of recombinant proteins, such as air sparging or pH adjustment" (underlining by the board).
13. The respondent argued in this regard that "methods for preventing the reduction of disulfide bonds of recombinant proteins during processing following fermentation" (page 26, lines 22 to 24) would have been understood by the skilled person according to "its ordinary practical meaning. Namely that the method is carried out (and prevents antibody reduction) at a time during manufacturing that is 'following fermentation'" (see point 147. in the reply to the appeals).
14. The board does not agree with this interpretation. It is not implicit in the disclosure on page 26, lines 22 to 24, that carrying out the intervention, i.e. supplementing with a Trx inhibitor, and achieving the effect, i.e. preventing the reduction of a disulfide bond in an antibody, have to occur at the same point in time/time period during the antibody production process (see also the corresponding claim interpretation in point 6. above). Rather, it was common general knowledge at the filing date that ingredients added at the beginning of or during cell culture, if sufficiently stable, can remain in the cell culture fluid during the entire growth and production phase, including a period of time after cell growth and recombinant protein production have been stopped, e.g. by harvesting the cells. They can therefore also act at a later processing stage. This is also apparent from the application as filed, which states: "Since some cell cultures already contain copper (e.g. about 0.04 myM CuSO4 for the CHO cell cultures used in the Examples herein), this amount is in addition to the copper, if any, already present in the cell culture" (sentence bridging pages 31 and 32). In other words, a disclosure of a time period when the effect of reduction prevention is achieved does not disclose the time period when the Trx inhibitor is supplemented.
15. The respondent further argued that "to prevent the reduction of disulfide bond reduction [sic] following fermentation" with non-directed approaches such as air sparging or pH adjustment (see page 29, lines 9 to 11 of the application as filed) meant that the intervention had to be applied at the time when the effect was to be achieved, i.e. following fermentation. The implication, or necessary consequence, was that the skilled person would have interpreted the passage in the preceding paragraph: "Trx inhibitors may be added before harvest and/or during harvest, preferably before harvest" (discussed in the preceding paragraph as preferably before harvest) as equally relating to a stage following fermentation.
16. The board does not agree. Firstly, the expression "added before harvest" does not imply a timing close to harvest (see also points 20. and 21. below). Secondly, the skilled person knew that air sparging and pH adjustment are transient measures which have to be applied at the time when the effect is to be achieved, while Trx inhibitors can be present in the culture fluid for an extended period of time and can achieve their effect also at a later stage. The skilled person would therefore not have concluded that the timing "following fermentation" disclosed for air sparging and pH adjustment also implicitly applied to the addition of Trx inhibitors.
17. The passages on page 26, lines 22 to 24, and page 29, lines 7 to 11 thus disclose that the prevention of disulfide bond reduction has to occur "following fermentation" or "during processing following fermentation", but not at which stage or point in time/time period of the process the "supplementing [...] with a thioredoxin inhibitor" takes place (see also corresponding point 4. above with regard to claim interpretation). In any case, these passages do not resolve the ambiguity of the term "following fermentation".
18. The second set of disclosures concerns the intervention to achieve the effect:
page 2, lines 23 to 24, and claim 1 as filed:
"supplementing the pre-harvest or harvested culture fluid";
page 2, line 26, and claim 2 as filed:
"added to the pre-harvest culture fluid";
page 29, lines 2 to 4:
"The Trx inhibitors are typically added to the cell culture fluid (CCF), which contains the recombinant host cells and the culture media, and/or to the harvested cell culture fluid (HCCF), which is obtained after harvesting by centrifugation, filtration, or similar separation methods."
19. The respondent argued that the addition of the Trx inhibitor was defined relative to the harvest by the terms "pre-harvest" or "harvested". This meant that the harvest of cell culture fluid was the "anchor point for the timing of the supplementing" and implied that it had to take place "relatively close to harvest" (see reply to the appeals, points 162. and 163.).
20. The board does not agree because the application as filed does not disclose a point in time or a time period "relatively close to harvest" at which the addition of Trx inhibitors had to occur.
21. None of the expressions "pre-harvest" (page 2, lines 24 and 26; claims 1 and 2), "before harvest and/or during harvest" (page 9, line 7) or "before harvest" (page 9, line 8) implies a time period "following fermentation". Firstly, these expressions define open periods of time for which no starting point is provided, i.e. they include the whole of the cell culture period before harvest. Secondly, they have the endpoint "harvest", which, however, is not the only possible endpoint for fermentation. Other technical possibilities to end fermentation are commonly known, such as freezing, chemical inhibition, lysis etc. (see also statement of grounds of appeal by appellant VI, point 67.).
22. A further disclosure, cited by the respondent, is to be found on page 31, lines 20 to 23 as filed:
"Disulfide bond reduction can be inhibited (i.e., partially or fully blocked) by using one or more Trx inhibitors and/or applying non-directed approaches following completion of the cell culture process, preferably to CCF prior to harvest or in the HCCF immediately after harvest."
23. Due to the presence of the "and/or" conjunction, it is not clear from this passage whether the Trx inhibitors are to be used "following completion of the cell culture process" or if this timing applies only to the "non-directed approaches". Table 1 on pages 29 and 30, which shows "Reduction Inhibition Methods" including Trx inhibitor addition, states in footnote 1 below the table that the methods are "[a]pplied to CCF prior to harvest or in HCCF immediately after harvest", i.e. it does not mention "following completion of the cell culture".
24. The quoted passage on page 31 is thus not an unambiguous disclosure that Trx inhibitors are to be used following completion of the cell culture process. Even if the board, following the interpretation of the respondent (see reply to the appeals, point 195.), were to start from the assumption that the passage discloses "using one or more Trx inhibitors [...] following completion of the cell culture process [...], preferably to CCF prior to harvest" (page 31, line 21 to 22), this would not equate to "following fermentation, supplementing the CCF with a Trx inhibitor" in claim 1.
25. Firstly, "following fermentation" is not the same as "following completion of the cell culture process". In view of the claim interpretation by the board, the ambiguous term "following fermentation" in the context of supplementing the CCF does not necessarily mean that the culture process has been completed (see point 7. above). In contrast, the cell culture process, in the context of antibody production, is generally completed with the harvest or lysis of the cells. Moreover, "following completion of the cell culture process" and "following fermentation" are not synonyms in the context of fed-batch or continuous cell culture processes where culture medium (with or without the cells) is continuously or intermittently introduced into and removed from the culturing vessel (see application as filed, page 34). Such processes are explicitly mentioned in the application as filed (see page 34, lines 26 to 27), and the interpretation of a term or an expression in the application as filed therefore needs to be technically sensible for continuous cell culture processes as well.
26. Secondly, the term "using" is substantially broader then "supplementing" and includes, for example, processes in which the Trx inhibitor is added at an earlier stage, remains present in the culture fluid and is used "following completion of the cell culture process".
27. The board therefore concludes that the application as filed does not disclose a process in which, following fermentation, the CCF is supplemented with a Trx inhibitor.
28. Claim 1 extends the subject-matter beyond the content of the application as filed (Article 123(2) EPC).
Order
For these reasons it is decided that:
1. The decision under appeal is set aside.
2. The patent is revoked.