DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/20/2025 has been entered.
Priority
This application is a 371 of PCT/IB2020/059201 filed 10/01/2020. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Acknowledgment is made of applicant’s claim for foreign priority based on application in DENMARK PA 2019 01154 filed 10/01/2019. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Status of the Claims
Claims 1, 2, 4, 6, 21, 22 and 23 are amended. Claim 3 is cancelled.
Claims 1, 2, 4, 6-11, 13-17 and 19-23 are pending (claim set filed 11/20/2025) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 11/20/2025 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
The previous claims 1-4, 6-11, 14-17, 19, 20 and 23 rejections under 35 U.S.C. 112(b) have been withdrawn necessitated by amendment of claims 1, 6 and 23 and cancellation of claim 3.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 recites: “contacting the reaction milieu in step i) to an ultrafiltration (UF) membrane”. Applicant is suggested to replace recitation with: “contacting the reaction milieu in step i) with an ultrafiltration (UF) membrane”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 4, 6-11, 13-17 and 19-23 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Independent claims 1 and 21 recite: “the reaction milieu”. There is insufficient antecedent basis for this limitation in claims 1 and 21. It is not clear what reaction milieu Applicant is referring to since it was not mentioned before that recitation. The scope and boundaries of claims 1 and 21 are not certain making claims indefinite. The dependent claims 2, 4, 6-11, 13-17, 19, 20, 22 and 23 do not resolve the issue mentioned above and are rejected.
Claim 4 has two antecedent issues. Claim 4 recites: “the non-polymeric membrane”. There is insufficient antecedent basis for this limitation in the claim since claim 1, from which claim 4 depends, does not recite “a non-polymeric membrane”.
Claim 4 recites: “step iii)”. There is insufficient antecedent basis for this limitation in the claim since claim 1 does not recite “step iii)”. It is not clear to what membrane and in what step claim 4 refers to. The scope and boundaries of claim 4 are not certain making claim 4 indefinite. For examination claim 4 is interpreted as referring to UF membrane of step ii) of claim 1 being ceramic membrane.
Claim 6 recites in line 3: “the permeate obtained in step iii)”. There is insufficient antecedent basis for this limitation in the claim since claim 1, from which claim 6 depends, does not recite “step iii)”. It is not clear what permeate needs to contact with a nanofiltration membrane. The scope and boundaries of claim 6 are not certain making claim 6 indefinite. For examination claim 6 is interpreted as referring to permeate obtained in step ii) of claim 1.
Claims 7-11, 19 and 20, dependent on claim 6, do not resolve the issue mentioned above and are rejected.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-4, 6, 8, 13-17 and 21-23 are rejected under 35 U.S.C. 103 as being unpatentable over Chassagne (WO 2017182965 A1 on record in IDS) in view of Bahler (US 7659090 B2).
Regarding claims 1 and 21, Chassagne teaches a method for obtaining an N-acetylglucosamine neutral oligosaccharide from a fermentation broth (p. 3, lines 15-16), wherein the containing N-acetylglucosamine neutral oligosaccharide is an N-acetylglucosamine containing neutral human milk oligosaccharide (HMO) (p. 3, lines 27-28). Chassagne describes method of obtaining LNT and LNnT HMOs which are tetra-saccharides (p. 8, lines 23-25) that reads on claims 1 and 21 limitations. The method comprises purification/separations steps of ultrafiltration, nanofiltration and treatment with an ion exchange resin performed in any order (p. 10, lines 7-11). Chassagne describes embodiment with the first step of ultrafiltration of the fermentation broth and collection of the permeate (p. 10, lines 16-17). Chassagne provides example of fermentation broth ultrafiltration which is performed at 60-65°C (p. 27, lines 7-8). Chassagne discloses that the ultrafiltration membrane can have a molecular weight cut-off between about 1 and about 500 kDa (p. 15, lines 12-14) that reads on claims 1 and 21 limitations.
Chassagne does not teach setting the pH to 3-5.5 (claim 1 limitation) or to at least 3 and less than 4.0 (claim 21 limitation) prior to ultrafiltration.
Bahler teaches method for the reduction or removal of protein impurities from a complex cellular lysate or centrate of Streptococcus pneumoniae comprising polysaccharides (Abstract). The methods includes heating and pH adjustment. Bahler discloses that exposure to heat disrupts the native structure of proteins and denatures them without affecting the polysaccharide and the denatured proteins aggregate and precipitate enabling their easy removal by centrifugation or filtration (column 4, lines 48-52). Bahler mentions that this method can be applied to biological mixtures containing significant levels of soluble protein impurities (column 4, lines 54-55). Bahler describes heating the lysate to at least 60°C for at least 30 minutes to cause protein aggregation and precipitation (column 2, lines 6-8). Bahler discloses that lowering pH to about 3.0 to about 5.0 also causes protein aggregation and precipitation (column 6, lines 40-43). Bahler mentions that treatments can be combined and such pH adjustment can comprise heating to at least 50-60°C for at least 15-30 minutes to cause protein aggregation and precipitation (column 6, lines 60-64).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add adjustment of pH to about 3-5 for the fermentation broth from Chassagne teaching heated at 60-65°C prior to ultrafiltration during isolation of neutral HMO as taught by Chassagne. One would have been motivated to add pH adjustment since Bahler teaches that both heating and lowering pH cause protein aggregation and precipitation, that these treatments can be combined and can be applied to biological mixtures containing soluble protein impurities which the fermentation broth of Chassagne teaching is. Besides, Bahler mentions that precipitated proteins can be easily removed by filtration or centrifugation and the removal of protein impurities will facilitate purification of HMO due to reducing number of components from which neutral HMOs are purified. A skilled artisan would have reasonably expected success in the combination because Chassagne and Bahler teach isolation of saccharides from the fermentation broth. Thus, Chassagne and Bahler teachings render claims 1 and 21 obvious.
Regarding claims 2 and 4, Chassagne teaches the ultrafiltration membrane with the molecular weight cut-off between about 1 and about 500 kDa and describes that the membrane material can be ceramic which is non-polymeric (p. 15, lines 12-14). Thus, Chassagne and Bahler teachings render claims 2 and 4 obvious.
Regarding claim 6, Chassagne teaches ceramic membrane for ultrafiltration as described above (p. 15, line 14). Chassagne discloses method comprising collecting permeate after ultrafiltration and subjecting it to nanofiltration and collecting the retentate (p. 10, lines 16-19). Thus, Chassagne and Bahler teachings render claim 6 obvious.
Regarding claim 8, Chassagne teaches treatment of nanofiltration retentate with ion-exchange resin: “treatment of the NFR (nanofiltration retentate) with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form, and collecting the resin eluate (RE)” (p. 12, lines 6- 9). Thus, Chassagne and Bahler teachings render claim 8 obvious.
Regarding claims 13-17 and 23, Chassagne teaches production of neural oligosaccharide by a genetically modified microorganism (p. 6, lines 6-7). Chassagne describes method of obtaining LNT and LNnT HMOs from the fermentation broth wherein the fermentation is performed by genetically modified E. coli of LacY+ phenotype or LacZ-, LacY+ phenotype (p. 8, lines 8-10). Thus, Chassagne and Bahler teachings render claims 13-17 and 23 obvious.
Regarding claim 22, Chassagne describes ultrafiltration of the fermentation broth performed at 60-65°C (p. 27, lines 7-8). Thus, Chassagne and Bahler teachings render claim 22 obvious.
Claims 9-11, 19 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Chassagne (WO 2017182965 A1 or record in IDS) in view of Bahler (US 7659090 B2) as applied to claims 1, 6 and 8 above, and further in view of Montane (Montane et al. Ind. Eng. Chem. Res., 2006, 45, 2294-2302).
The teachings of Chassagne and Bahler have been set forth above.
Regarding claims 9-11, Chassagne teaches the active charcoal treatment after nanofiltration, collecting the charcoal eluate and treatment of charcoal eluate with a strong cation exchange resin in H+ form and a weak anion exchange resin in free base form (p. 11, lines 13-15). In another embodiment Chassagne teaches the eluate from the ion exchange treatment to undergo activated charcoal treatment (p. 12, lines 7-9). Chassagne does not teach the parameters of activated charcoal treatment, i.e. temperature and amount of charcoal.
Montane teaches purification of xylo-oligosaccharides by activated carbon treatment which is performed in batch and column modes (Abstract). Montane discloses performance of three commercial activated carbons for purification of xylo-oligosaccharides by absorption of lignin and impurities. Montane describes that 10 ml of oligosaccharides dissolved at 20 g/l are treated with 15-500 mg of activated carbon at 30°C (p. 2295, right column, 2nd paragraph) that reads on claimed limitations. 10 ml of 20 g/l provides 200 mg of oligosaccharides and activated carbon is added at as low as 15 mg which is 7.5 weight%.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow guidance of Montane on activated charcoal treatment at 30°C and at as low as 7.5 weight % of activated charcoal relative to oligosaccharides and use it for charcoal treatment in method of obtaining HMO based on Chassagne and Bahler teachings. One would have been motivated to do so since Montane described purification of oligosaccharides and compared performance of commercial activated carbons. A skilled artisan would have reasonably expected success in the combination because Chassagne and Montane teach purification of oligosaccharides involving activated charcoal treatment. Thus, Chassagne, Bahler and Montane teachings render claims 9-11 obvious.
Regarding claims 19 and 20, Chassagne teaches that the activated charcoal treatment can be performed by adding charcoal powder to the solution containing neutral oligosaccharides or by chromatography on activated charcoal: “ The charcoal treatment can be conducted by adding charcoal powder to the aqueous solution of the N-acetylglucosamine containing neutral oligosaccharide under stirring, filtering off the charcoal, re-suspending in aqueous ethanol under stirring and separating the charcoal by filtration. In higher scale purification, the aqueous solution of N-acetylglucosamine containing neutral oligosaccharide after step i), step ii) or step iii) is preferably loaded to a column packed with charcoal, which may be a granulated charcoal or may optionally be mixed with celite, then the column is washed with the required eluent. The fractions containing the N-acetylglucosamine containing neutral oligosaccharide are collected” (p. 22, lines 6-12). Thus, Chassagne, Bahler and Montane teachings render claims 19 and 20 obvious.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Chassagne (WO 2017182965 A1 on record in IDS) in view of Bahler (US 7659090 B2) as applied to claims 1 and 6 above, and further in view of Tang (US 6068705 A).
The teachings of Chassagne and Bahler have been set forth above. Chassagne and Bahler do not teach claimed parameters for nanofiltration.
Tang teaches fractionation of starch hydrolysate using nanofiltration (column 4, lines 5-8). Tang describes that nanofiltration allows to separate lower oligosaccharides and retain oligosaccharides of larger molecular weight and reduce the DE factor after several rounds of nanofiltration (column 7, lines 59-67). Tang discloses that the nanofiltration membrane can be polyamide membrane and have molecular weight cut-off of 400-4000 Da: “ … nanofiltration membrane is selected from the group consisting of polyamide membranes and polysulfonated polysulfone membranes having a molecular weight cut-off within a range of about 400 daltons to about 4,000 daltons.” (claim 2). Tang provides example of nanofiltration of hydrolyzed corn syrup on a thin film composite polyamide membrane with the rejection factor for MgSO4 of 50% (column 17, lines 12-32) that reads on claim 7 limitations. Tang describes that the nanofiltration resulted in oligosaccharides fractionation and reduction of the DE factor from 42 to 15 and higher amount of larger oligosaccharides in the retentate (column 17, lines 12 and 57-59 and column 18, Table).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow guidance of Tang and use polyamide nanofiltration membrane with cut-off of 400-4000 Da and 50% rejection factor for MgSO4 described in Tang teaching in the nanofiltration step of the method of obtaining HMO based on Chassagne and Bahler teachings. One would have been motivated to do so since Tang showed separation of oligosaccharides with reduction in DE factor by described nanofiltration. A skilled artisan would have reasonably expected success in the combination because Chassagne describes method of isolation of oligosaccharides including nanofiltration and Tang provides parameters for nanofiltration. Thus, Chassagne, Bahler and Tang teachings render claim 7 obvious.
Response to Arguments
Applicant's arguments filed 11/20/2025 have been fully considered but they are not persuasive.
Applicant’s arguments with respect to prior art of Dekany and Zhong are moot because the current rejection is based on combination of the prior art of Chassagne and Bahler as described above.
In response to Applicant’s arguments (addressing pages 8-9 of the Remarks) that the claimed invention is associated with unexpected properties: “Not only is there a notable reduction in protein in the supernatant (Specification, pp. 48-49) as discussed in Applicant's previous remarks, the specification also describes an increase in yield by performing the claimed method. E.g., Specification, p. 49.”. Applicant disagrees that the unexpected results are not commensurate with the scope of the claims as was discussed in the previous Office action and states that: “The inventors have provided data outside the claimed range ( 40 °C) and inside the claimed range (50°C and 60°C).”. These arguments are not persuasive because:
It is acknowledged that the results of Examples 2 and 3 demonstrate significant reduction in protein content with lowering pH and results of Example 4 show increase in the yield of LNnT with temperature increase. However, as taught by Bahler, each of heat treatment at 50-60°C and pH treatment at pH 3.0-5.0 causes protein aggregation and precipitation and hence addition of low pH treatment from Bahler teaching (Abstract) to heat treatment at 60-65°C of the fermentation broth during isolation of neutral HMO in Chassagne teaching (p. 27, lines 7-8) is expected to further reduce the protein impurities. Regarding increase in the yield of neutral HMO demonstrated in Example 4, the yield was shown to increase at constant pH of 5.0 and temperature increase from 40°C to 50°C and 60°C. The reduction of soluble protein due to heat treatment and prior to ultrafiltration is expected to prevent unspecific binding of HMO with the proteins and thus increase production yield. Besides, the prior art of Ren (Ren et al. J. Biotechnol., 2007, 129, 668-673) teaches that heat treatment at 60°C disrupts E. coli cells and results in the release of the recombinant protein (Abstract and p. 670, Figure 1). The results of the increase of the amount of protein at 50°C and 60°C compared to 40°C in Example 4 confirms that. Therefore, it can be predicted that in addition to secreted HMO, heat treatment at 60°C can release HMO synthesized intracellularly and present in the cells and thus the increase in HMO yield in UF permeate with the increase in temperature is expected.
As described in the previous Office action, the advantage of the combination of pH and temperature recognized by the Applicant does not make the combination of prior art non-obvious because the prior art does not need to point out all advantages if there is a motivation to combine the prior art. MPEP 2145: “The fact that appellant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). In instant case, low pH and higher temperature can be used for the same purpose, i.e. to increase protein aggregation and precipitation and removal of proteins will facilitate purification of HMOs by reducing number of components from which neutral HMOs are purified providing motivation for combination of prior art of Chassagne and Buhler.
Assuming arguendo applicant has shown unexpected data, claims are not commensurate in scope with the unexpected results. MPEP 716.02: “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In instant case, as was discussed in the previous Office action, claims are not commensurate in scope with the unexpected results of reduction in the protein content. Briefly, (i) the temperature used in Examples 2 and 3 is 60°C (p. 48 and 49) and the temperature range in the claims is much broader, (ii) Examples 2 and 3 are directed to isolation of one of the neutral HMOs, i.e. 2’-FL, while 6 neutral HMOs are recited in claim 1 and even broader limitation is recited in claim 21 and (iii) the results of Example 4 show increase of the protein in UF permeate with increasing temperature and not the decrease and therefore contradict the unexpected results of protein removal. Regarding unexpected results of increased yield demonstrated in Example 4, the results of Example 4 are: (i) performed at pH 5.0 and do not include the whole range of pH recited in the claims, (ii) although show temperature within the claimed range, do not include the whole range of temperature recited in the claims and (iii) Example 4 is directed to isolation of one of the neutral HMO, i.e. LNnT, while multiple HMO are within the scope of claims 1 and 21. Therefore, the claims are not commensurate in scope with the unexpected results and the 35 U.S.C. 103 rejection is maintained and modified as described above.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to LIOUBOV G KOROTCHKINA whose telephone number is (571)270-0911. The examiner can normally be reached Monday-Friday: 8:00-5:30.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653