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 .
Priority
This application is a 371 of PCT/1B2020/059201 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, 4, 6, 7 and 21 are amended.
Claims 1, 2, 4, 6-11, 13-17 and 19-23 are pending (claim set filed 06/22/2026) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 06/22/2026 are acknowledged. The Declaration under 37 CFR 1.132 of Nikolay Khanzhin filed 06/22/2026 is 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The previous claim 1 objection has been withdrawn necessitated by amendment of claim 1.
The previous claims 1, 2, 4, 6-11, 13-17, 19-23 rejections under 35 U.S.C. 112(b) have been withdrawn necessitated by amendment of claims 1, 4, 6 and 21.
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, 2, 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 (p. 15, lines 12-14). Thus, Chassagne and Bahler teachings render claims 2 and 4 obvious.
Regarding claim 6, Chassagne teaches 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 describes that the nanofiltration membrane preferably comprises a thin film composite membrane (column 6, liners 30-32) and 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 and Declaration under 37 CFR 1.132 of Nikolay Khanzhin filed 06/22/2026 have been fully considered but they are not persuasive.
Applicant and Dr. Khanzhin argue (addressing p. 7 of the Remarks and p. 2-4 of the Declaration of Dr. Khanzhin) that Chassagne and Bahler teachings have numerous differences, i.e. Chassagne teaches isolation of oligosaccharide from the supernatant of genetically engineered cells (E. coli) and Bahler discloses production of a large and viscous polysaccharide released in the growth medium upon Streptococcus pneumoniae lysis and Bahler polysaccharide is structurally and functionally different from HMO of Chassagne. These arguments are not persuasive because:
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, although Chassagne and Bahler teach production of different saccharides and different protocol of production and isolation, the common feature in Chassagne and Bahler teachings are isolation of saccharides from other components, including proteins by filtration. Bahler teaches that higher temperature and lower pH cause protein precipitation and aggregation (column 4, lines 48-52, column 6, lines 40-43) and Chassagne teaches ultrafiltration with heat treatment (p. 27, lines 7-8) that provides motivation to add low pH treatment the fermentation broth from Chassagne teaching heated at 60-65°C prior to ultrafiltration. Thus, heat and pH treatments can be used for the same purpose of removal proteins prior to filtration and that will reduce number of components from which neutral HMOs are isolated and provide better flow during ultrafiltration in Chassagne teaching and facilitate purification of HMOs.
Dr. Khanzhin further argues (addressing p. 5-7 of the Declaration) that Bahler demonstrated much lower filterability of supernatant after centrifugation at pH 3 and even at pH 5 and hence skilled artisan would expect lower permeate flow in the ultrafiltration step of Chassagne. Additionally, since Bahler uses cell lysis, it would provide substantially higher amount of protein than in the Chassagne and detergent used during cell lysis will increase amount of released proteins and also cause additional protein precipitation at pH 3. Dr. Khanzhin mentions complexity of protein solubility and provides prior art of Cao in support. Additionally, Dr. Khanzhin argues that lowering pH and heating in Bahler is followed by centrifugation, increasing pH and then filtering and claimed method do not include centrifugation after precipitation. These arguments are not persuasive because:
It is acknowledged that lysis in Bahler provides higher amount of proteins and that lowering of pH decrease filterability in Bahler teaching (Figure 7). However, (i) since Chassagne does not teach cell lysis, the amount of proteins to remove would be less and the effect of filterability is expected to be less; (ii) Figure 7 in Bahler shows that lowering pH to 3 drastically reduced total protein amount and that recovering of pH to 7.0 restores high filterability and (iii) since instant claim 1 has “comprising” language, additional steps between pH adjustment and ultrafiltration can be included such as centrifugation and increase in pH. It is noted that in the instant Example 2 the fermentation broth was centrifuged after the pH treatment (the specification p. 48, lines 19-24). One of ordinary skill in the art would recognize that the additional steps of centrifugation and increase in pH can be applied depending on the experimental conditions such as amount of protein in the reaction milieu and type of filtration used to avoid reduced filterability.
Dr. Khanzhin argues (addressing p. 7-8 of the Declaration) that Chassagne does not identify protein removal as a problem and describes method to remove impurities including proteins by ion-exchanger. The method Dr. Khanzhin refers to is related to the fermentation process when the enzyme is bound to the surface of the cell or to enzymatic process in cell-free medium (p. 23, lines 6-11) and that method is suggested to be preceded by ultrafiltration and nanofiltration that would remove proteins (p. 25, line 12). As described above, low pH and heat treatments are expected to provide less amount of proteins and hence better flow during ultrafiltration.
Applicant and Dr. Khanzhin argue (addressing p. 9-11 of the Remarks and Declaration of Dr. Khanzhin) that the claimed invention is associated with unexpected results of increased purity and yield and that ”The exemplified conditions cover the claimed pH, temperature, UF and NF cut-off ranges and therefore the claims are commensurate with the obtained results. Also, the method was demonstrated to work equally well for two distinct HMOs, i.e. for exemplified tri-saccharide 2'FL containing fucose and tetra-saccharide LNnT containing N-acetylglucosamine moiety”. 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.
As described in the previous Office action, 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 other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range.” (MPEP 716(d)). 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 over the entire claimed range because: (i) the temperature used in Examples 2 and 3 is 60°C (p. 48 and 49) and the temperature range in the claim 1 is 50-65°C and in claim 21 is 45-75°C which 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 (pH 3-5.5), (ii) although have 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. Thus, the claims are not commensurate in scope with the unexpected results.
Therefore, the 35 U.S.C. 103 rejection is maintained and modified necessitated by amendment of claims.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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
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