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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 2/20/2026 has been entered.
Status of Claims
Receipt of Arguments/Remarks filed on 2/20/2026 is acknowledged. Claims 9, 22, 31, and 40 were amended. New claims 48-50 were added. Claims 1-9, 15-22, 26-31, 33-36, 38, 40, 42-45, and 48-50 are pending.
Information Disclosure Statement
The information disclosure statement (IDS) filed on 4/14/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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 1-3, 5-9, 15-22, 26-31, 33-35, 38, 40, 42-45, 48, and 50 are rejected under 35 U.S.C. 103 as being unpatentable over Samain et al., US 7,521,212 B1, as evidenced by Kaback et al., The Journal of membrane biology. 2011 Jan;239(1):85-93, and De Mey et al., Journal of Industrial Microbiology and Biotechnology. 2007 Nov 1;34(11):689-700.
Regarding claim 1, Samain teaches a method for producing a mixture of oligosaccharides by a cell (Samain col. 2 lines 34-53; col. 19 Ex. 8). Samain teaches providing a cell that expresses a glycosyltransferase (Samain col. 4 lines 40-67). Samain teaches that the cell is capable of producing sugar-nucleotides which are donors for the glycosyltransferase (Samain col. 2 lines 58-60; col. 8 lines 50-60).
Samain teaches culturing the cell under conditions permissive to express the glycosyltransferase and nucleotide sugar (Samain col. 2 lines 47-63).
Samain teaches adding at least one exogenous precursor to enable the cell to produce oligosaccharides (Samain col. 2 lines 33-52). As set forth in the instant specification, the term "acceptor" as used herein refers to a di- or an oligosaccharide that is taken up by the cell and modified by one or more glycosyltransferases with the addition of one or more monosaccharide units for oligosaccharide assembly (see instant specification pp. 40-41 para. 144). The exogenous precursors taught by Samain may be lactose (a disaccharide) or other di- and oligosaccharides such as lactulose, melibiose, or raffinose (Samain col. 6 lines 54-61). Samain teaches the use of permeases modified by recombinant DNA techniques to allow the internalization of different types of precursor (Samain col. 7 lines 1-5). Samain teaches that the method makes it possible to obtain a large number of different oligosaccharides obtained by glycosylation of exogenous precursors other than lactose and transported by lactose permease and other permeases (Samain col. 8 lines 38-45). Thus, Samain teaches that the cell is capable of taking up and glycosylating multiple exogenous precursors.
Samain teaches adding lactose as an acceptor in a fermentation of a modified E. coli strain (Samain col. 16 lines 60-67). Samain teaches that several oligosaccharides are produced, including lacto-N-neotetraose, lacto-N-neo-hexaose, and lacto-N-neo-octaose, and that the amounts of each oligosaccharide depend on the amount of acceptor, lactose, that was added, because LgtA is capable of using lacto-N-neo-tetraose to form an intermediate pentasaccharide that is glycosylated by LgtB to give lacto-N-neo-hexaose, which is itself the precursor for a
new glycosylation cycle resulting in the formation of lacto-N-neo-octaose, and so on up to lacto-N-neo-decaose (Samain col. 17 lines 18-40). Further, Samain teaches a fermentation wherein the bacteria comprises two plasmids, one of which uses N-acetyllactosamine and lacto-N-neo-tetraose as acceptors, but not lactose (Samain col. 19 lines 49-59). Samain teaches fermentation of this strain with lactose added as an acceptor, wherein four main oligosaccharides are produced (Samain col. 20 lines 7-36).
It would have been obvious for a skilled artisan based on the teachings of Samain to add at least two acceptors that are modified by glycosyltransferases. Samain teaches that at least one precursor, which includes di- and oligosaccharide acceptors, is added to the cultivation, indicating that more than one oligosaccharide acceptor can be added, i.e. two or more (Samain col. 2 lines 33-52). Samain teaches that the method allows for obtaining a large number of different oligosaccharides by glycosylation of exogenous precursors other than lactose (Samain col. 8 lines 38-43). Samain also teaches that some glycosyltransferases, such the fucT gene for α-1,3-fucosyl-transferase, utilize acceptors other than lactose (Samain col. 19 lines 49-59).
Given these teachings, as well as the teaching that the amount of acceptor in the cultivation influences the amount of each oligosaccharide produced, a skilled artisan would have been motivated to add two or more acceptors to the cultivation as a way of controlling and optimizing product outcomes. A skilled artisan would have had a reasonable expectation of success in doing so because Samain teaches that acceptors other than lactose can be transported into the cell and that multiple oligosaccharides can be obtained through glycosylation of different exogenous precursors (Samain col. 8 lines 38-43). Thus, a person having ordinary skill in the art could expect that multiple exogenously added acceptors could be taken up by the cell and glycosylated to form different oligosaccharides.
Regarding claim 2, Samain teaches adding lactose, a disaccharide, as an acceptor (Samain col. 16-17 Ex. 3).
Regarding claim 3, Samain teaches that the cell is metabolically engineered for production of the oligosaccharides (Samain col. 2 lines 33-36; col. 8 lines 42-59).
Regarding claim 5, the limitation “wherein the cell produces” is a functional limitation of the cell with a structure as set forth in claim 1. Absent any unclaimed essential features, any cell having the structure set forth in claim 1 must be capable of performing the claimed function. Samain teaches the active method steps of claim 1, as set forth above, and teaches the structure of the cell used in the method. Thus, the method of Samain reads on claim 5. Further, Samain teaches that the cell produces a mixture of three or more oligosaccharides, lacto-N-neotetraose; lacto-N-neo-hexaose; lacto-N-neo-octaose; lacto-N-neo-
Decaose (Samain col. 17 lines 18-33; Fig. 8).
Regarding claim 6, Samain teaches that the oligosaccharides differ in degree of polymerization, i.e. tetraose, octaose, hexose, decaose (Samain col. 17 lines 18-33).
Regarding claim 7, Samain teaches that the glycosyltransferase is an N-acetyl-glucosaminyl-transferase, galactosyltransferase, fucosyltransferase, sialyltransferase, or N-acetyl-galactosaminyltransferase (Samain col. 4 lines 45-55).
Regarding claim 8, Samain teaches that the cell is modified in the expression and activity of the glycosyltransferase, i.e. introduced to the cell in the form of an expression vector (Samain col. 4 lines 17-34).
Regarding claims 9 and 15, Samain teaches that the glycosyltransferases are capable of stereospecific conjugation of specific activated saccharide units on a specific acceptor molecule (nucleotide sugar), which generally consist of a UDP-saccharide, a GDP-saccharide or a CMP-saccharide (Samain col. 4 lines 60-67). In one embodiment, Samain teaches that the glycosyltransferase is a fucosyltransferase and the nucleotide sugar is GDP-fucose (Samain col. 19 lines 40-60).
Regarding claim 16, Samain teaches that an exogenous precursor, in addition to lactose, can be raffinose, which is a trisaccharide with a degree of polymerization of 3 (Samain col. 6 lines 55-61).
Regarding claim 17, Samain teaches that various exogenous precursors can be added to the cultivation (Samain col. 6 lines 55-61). It would have been obvious to use two acceptors with different degrees of polymerization, for example, lactose and raffinose, in a single cultivation, given the teachings of Samain that one or more precursors may be added with precursors having different degrees of polymerization being exemplified, as discussed above.
Regarding claim 18, Samain teaches adding at least one exogenous precursor to enable the cell to produce oligosaccharides (Samain col. 2 lines 33-52). Samain teaches that the method allows for obtaining a large number of different oligosaccharides by glycosylation of exogenous precursors other than lactose (Samain col. 8 lines 38-43). As discussed above, it would have been obvious to add more than one acceptor, i.e. 3 acceptors, to the cultivation, as Samain teaches adding multiple acceptors to produce multiple oligosaccharides.
Regarding claims 19-22 and 26-27, the limitations “wherein the oligosaccharide mixture comprises” are functional limitations of the cell with a structure as set forth in claim 1, regarding the oligosaccharides that the cell produces when used in the method as claimed. Absent any unclaimed essential features, any cell having the structure set forth in claim 1 must be capable of performing the claimed function. Samain teaches the active method steps of claim 1, as set forth above, and teaches the structure of the cell used in the method. Therefore, the cell of Samain, which has the same structure as set forth in claim 1, is capable of producing the oligosaccharides as set forth in claims 19-22 and 26-27 in a process of claim 1.
Further, Samain teaches that the oligosaccharides produced may be sialylated (charged) or fucosylated (Samain col. 9 lines 55-65). Samain teaches that the cell produces three fucosylated oligosaccharides, lacto-N-fucopentaose, lacto-N-difucohexaose, and lacto-N-difucooctaose (Samain col. 20 lines 12-30).
Regarding claim 28, the limitation “wherein the acceptors are produced by methods comprising” is a product-by-process limitation of the acceptors used in claimed method, and as such patentability is assessed based on the structure implied by the steps, not the manipulations of the recited steps. “[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985)” (see MPEP § 2113 subsection I). Samain teaches acceptors as set forth in claim 1, for example lactose, as discussed above. The acceptors are capable of being glycosylated to form various oligosaccharides (Samain col. 8 lines 38-45). Thus, the acceptors used in the method of Samain read on claim 28.
Regarding claim 29, while Samain does not expressly teach that the acceptors are completely converted into the oligosaccharides, it would have been obvious to a person having ordinary skill in the art that the method as taught by Samain could be carried out in order to completely convert the acceptors to the oligosaccharides, i.e. by reacting for sufficient time or providing the necessary cultivation conditions for all of the acceptor taken up by the cell to be converted to the oligosaccharide of interest, as these are aspects of a method that would be routinely optimized by a person having ordinary skill in the art.
Additionally, the limitation, “wherein any one of the acceptors is completely converted into any one of the oligosaccharides” is a result of the claimed method. Samain teaches a method comprising the active method steps set forth in claim 1, as discussed above. Therefore, it would be expected that the method of Samain would achieve the same result as the claimed method.
Regarding claim 30, the limitations “wherein the cell produces the mixture of at least two oligosaccharides” is a functional limitation of the cell with a structure as set forth in claim 1. Absent any unclaimed essential features, any cell having the structure set forth in claim 1 must be capable of performing the claimed function. Samain teaches the active method steps of claim 1, as set forth above, and teaches the structure of the cell used in the method. Therefore, the cell of Samain, which has the same structure as set forth in claim 1, is capable of producing the mixture of oligosaccharides as set forth in claim 30. Further, Samain teaches that some of the oligosaccharides produced by the cell are intracellular and some are extracellular, i.e. excreted outside the cell (Samain col. 13-14 “Assay of the Oligosaccharides”).
Regarding claim 31, Samain teaches that the cell comprises a membrane protein, a permease, which transports precursors into the bacterium (Samain col. 6 lines 29-47). Samain teaches that the permeases can be modified by mutation to allow transport of additional precursor compounds (Samain col. 6 lines 61-67, col. 7 lines 1-5). Thus, Samain teaches that the cell has modified activity/expression of an endogenous membrane protein that is involved in uptake of a precursor for the synthesis of oligosaccharides.
Regarding claim 33, Samain teaches that the E. coli lactose permease is LacY, which is an MFS transporter (Samain col. 18 lines 60-65; see Kaback Introduction).
Regarding claim 34, Samain teaches a cell with modified expression of a membrane protein as set forth in claim 31. Further, Samain teaches that the modified membrane protein allows for different types of precursors to be taken up by the cell (Samain col. 6 lines 61-67, col. 7 lines 1-5). Samain teaches that the production of oligosaccharides by the cell requires internalization the exogenous precursor which is then modified by glycosyltransferases to produce the oligosaccharides (Samain col. 2 lines 46-54). The precursors are transported by permeases (membrane proteins) and glycosylated (Samain col. 8 lines 38-42). Thus, the expression of membrane proteins, such as permeases as discussed regarding claim 31, would allow for glycosylation of precursors to form oligosaccharides, which would thereby improve the production of the oligosaccharides.
Regarding claim 35, the limitation “wherein the cell resists lactose killing when grown in an environment in which lactose is combined with one or more other carbon source(s)” is a functional limitation of the cell with a structure as set forth in claim 1. Absent any unclaimed essential features, any cell having the structure set forth in claim 1 must be capable of performing the claimed function. Samain teaches the active method steps of claim 1, as set forth above, and teaches the structure of the cell used in the method. Thus, the cell used in the method of Samain reads on claim 35.
Regarding claim 38, the limitation, “wherein the cell is capable of producing phosphoenolpyruvate (PEP)” is a functional limitation of the cell with a structure as set forth in claim 1. Absent any unclaimed essential features, any cell having the structure set forth in claim 1 must be capable of performing the claimed function. Further, the cell taught by Samain, E. coli, is known to be capable of producing phosphoenolpyruvate, PEP, which is an intermediate in glycolysis (see De Mey Fig. 1).
Regarding claim 40, the Samain teaches that the oligosaccharides produced by the cell are mammalian milk oligosaccharides, i.e. lacto-N-neotetraose (Samain col. 17 lines 25-30, see instant specification p. 15 for list of mammalian milk oligosaccharides).
Regarding claim 42, Samain teaches the cell is a bacterium, E. coli (Samain Ex. 3).
Regarding claim 43, Samain teaches that the cell has abolished production of colanic acid (Samain col. 19 lines 43-60).
Regarding claims 44-45, Samain teaches that the separation and purification involve the use of active charcoal (Samain col. 14 lines 25-30).
Regarding claim 48, Samain teaches that the cell expresses two glycosyltransferases, LgtA and LgtB (Samain Example 3). As discussed above regarding claim 18, it would have been obvious to add more than one acceptor, i.e. 3 acceptors, to the cultivation, as Samain teaches adding multiple acceptors to produce multiple oligosaccharides.
Regarding claim 50, Samain teaches that the cell comprises a membrane protein which transports precursors, i.e. acceptors which are glycosylated to form oligosaccharides, into the bacterium (Samain col. 6 lines 29-47; col. 8 lines 39-43). Samain teaches that the permeases can be modified by mutation to allow transport of additional precursor/acceptor compounds (Samain col. 6 lines 61-67, col. 7 lines 1-5).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Samain et al. as applied to claims 1-3, 5-9, 15-22, 26-31, 33-35, 38, 40, 42-45, 48, and 50 above, in view of Peracha et al., WO 2019/025485 A1.
Samain teaches the method of claim 1 as set forth above. Samain teaches strains which overexpresses rcsA (Samain col. 9 lines 30-46; col. 19 Ex. 8).
Samain does not teach a cell with multiple copies of a DNA sequence encoding one protein (claim 4).
Regarding claim 4, Peracha teaches genetically modified microorganisms (E. coli) for the improved production of human milk oligosaccharides (HMOs) and methods of producing HMOs (Peracha p. 1 lines 4-9; p. 3 line 20). Peracha teaches that genes may be overexpressed in the microorganism to improve the production of HMOs such as 2-FL or 3-FL, including overexpression of the gene rcsA (Peracha p. 18 lines 20-27; p. 19 lines 8-12). Peracha teaches that genes can be overexpressed by using common techniques in the art, including additional exogenous gene copies being added to the cell (Peracha p. 9 lines 31-34, p. 10 lines 1-3).
It would have been obvious for a skilled artisan to modify the cell as taught by Samain and incorporate multiple copies of a DNA sequence encoding one protein. Samain teaches overexpression of genes such as rcsA in cells for HMO production. Peracha is also directed to HMO production using modified cells and teaches overexpression of rcsA. Peracha teaches that overexpression can be accomplished using known techniques in the art, which includes adding additional copies of a gene to the cell. It would have been obvious for a skilled artisan to utilize a known method of gene overexpression, inserting multiple copies of a gene, in order to achieve overexpression as taught by Samain. As there are a finite number of known techniques in the art for achieving gene overexpression in bacteria such as E. coli, it would have been obvious for a skilled artisan to choose one of these identified, predictable methods to achieve gene overexpression in an E. coli strain, with a reasonable expectation of success, as both Samain and Peracha are directed to modified E. coli for HMO production, and Peracha teaches inserting multiple copies of a single gene for overexpression in E. coli.
Claim 36 is rejected under 35 U.S.C. 103 as being unpatentable over Samain et al. as applied to claims 1-3, 5-9, 15-22, 26-31, 33-35, 38, 40, 42-45, 48, and 50 above, in view of De Mey M, Journal of Industrial Microbiology and Biotechnology. 2007 Nov 1;34(11):689-700.
Samain teaches the method of claim 1 as set forth above. Samain does not teach that the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor (claim 36).
Regarding claim 36, De Mey teaches that E. coli is one of the most commonly used production organisms in industrial biotechnology, and that aerobic high density cell cultures are used to arrive at high biomass yields (De Mey p. 689 para. 1). De Mey teaches that during aerobic fermentation of E. coli, large amounts of acetate are accumulated, which results in a loss of carbon and is detrimental to E. coli growth (De Mey p. 689 para. 1). De Mey teaches that the amount of acetate can be reduced in E. coli at the bioprocess level and the genetic level (De Mey p. 690 para. 4). Genetic approaches include modifying the central metabolism pathways of the E. coli cell (De Mey p. 691 “Genetic approaches to minimize acetate formation”).
It would have been obvious for a skilled artisan to modify the microorganism used in the method of Samain to have reduced acetate production given the teachings of De May. De May teaches that E. coli produces acetate and teaches various methods of reducing acetate production in E. coli strains, including genetic modification of the cell. Therefore, a skilled artisan would have found it obvious to modify the E. coli cell of Samain to have reduced acetate production as taught by De May.
A person of ordinary skill in the art would have been motivated to reduce acetate production because De May teaches that acetate production by E. coli during fermentation causes many problems when growing E. coli in industrial settings. Specifically, De May teaches that acetate inhibits growth and biomass production, inhibits recombinant protein production, causes reduction in proton motive force, and acidification of the medium (De May p. 690 para. 2). Therefore, a skilled artisan would have found it advantageous to modify the E. coli strain for production of oligosaccharides as taught by Samain to have reduced acetate production, in order to avoid the detrimental effects of acetate accumulation during E. coli fermentation.
A skilled artisan would have had a reasonable expectation of success in reducing acetate production in the cell of Samain because De May teaches multiple techniques for reducing the acetate production of E. coli cells. A person having ordinary skill in the art could therefore expect success in implementing a known technique to solve a known issue in E. coli industrial fermentation in the method of Samain, which is directed to using modified E. coli for production of oligosaccharides.
Claim 49 is rejected under 35 U.S.C. 103 as being unpatentable over Samain et al. as applied to claims 1-3, 5-9, 15-22, 26-31, 33-35, 38, 40, 42-45, 48, and 50 above, in view of Pedersen et al., US 2020/0354761 A1.
Samain teaches the method of claim 1 as set forth above. Samain does not teach that the cell expresses a membrane protein belonging to the family of MFS transporters or sugar efflux transporters that provides enhanced efflux of the mixture of oligosaccharides (claim 49).
Regarding claim 49, Pedersen teaches a genetically modified microorganism (E. coli) for making HMOs and methods of producing HMOs (Pedersen Abstract). Pedersen teaches that at the end of fermentation, the oligosaccharide has accumulated intracellularly and extracellularly, and the transport of the oligosaccharide out of the cell can be facilitated by sugar efflux transporters in the cell (Pedersen p. 7 para. 71). Pedersen teaches that sugar efflux transporters can be heterologously expressed to enhance export of the human milk oligosaccharides (Pedersen p. 7 para. 71).
It would have been obvious for a skilled artisan to modify the cell of Samain and express a sugar efflux transporter to enhance efflux of the oligosaccharide mixture. Both Samain and Pedersen are directed to production of HMOs using modified E. coli. Pedersen teaches that the cells for HMO production can be modified with heterologous sugar efflux transporters to enhance export of the oligosaccharides. Thus, it would have been obvious for a skilled artisan to make this modification in the cell as taught by Samain, used in a method of producing oligosaccharides.
A person of ordinary skill in the art would have been motivated to make this modification to the cell of Samain because heterologous efflux transporters increase the export of oligosaccharides that are produced by the cell, as taught by Pedersen. It would therefore be considered advantageous to express sugar efflux transporters in order to increase oligosaccharide export and recovery of oligosaccharides in a method as taught by Samain for this purpose.
A skilled artisan would have had a reasonable expectation of success in making this modification because Pedersen teaches that sugar efflux transporters can be expressed in modified E. coli to enhance HMO production. As both Samain and Pedersen are directed to modified E. coli for HMO production, a person having ordinary skill in the art could expect success in modifying the cell taught by Samain in the same way as taught by Pedersen to achieve the same purpose, oligosaccharide export, in the same cell type, E. coli.
Response to Arguments
Applicant’s arguments with respect to claims 1-9, 15-22, 26-31, 33-36, 38, 40, and 42-45 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
Claims 1-9, 15-22, 26-31, 33-36, 38, 40, 42-45 and 48-50 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653
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