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 .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1, 3, 7, 11, 13-15, 19-22, 24, 28, 32, 34, 36-37, and 40, as well as the species a lysosomal acid lipase (claim 14), formula I (claim 20), polysorbate 80 (claim 34), and mono- or di-acylglycerol (claim 36) in the reply filed on 12/11/2025 is acknowledged.
Priority
The instant application filed on 02/06/2023 if a 371 of PCT/EP2021/072039 filed on 08/06/2021 and claims priority to EP20190136.0 filed on 08/07/2020. EP20190136.0 finds support for the instantly claimed invention; therefore, the effective filing date of the instant application is 08/07/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 02/06/2023 and 10/10/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Abstract Objections
The abstract of the disclosure is objected to because the abstract is over 150 words. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
Claim Objections
Claim 19 is objected to because of the following informalities: “Hydroxysuccinimid” should be “Hydroxysuccinimide”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b), Indefiniteness
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.
Claims 1, 3, 7, 11, 13-15, 19-22, 24, 28, 32, 34, 36-37, and 40 are rejected under 35 U.S.C. 112(b) 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.
Claim 1 recites steps (i) and (ii); however, it is unclear if steps (i) and (ii) are both required, or if step (i) or step (ii) is required. This is unclear because there is no and/or linking word between the two steps. For the purposes of applying prior art, the Examiner has interpreted both step (i) and step (ii) to be required.
Regarding claim 3, the phrase “for example” " renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d). For the purposes of applying prior art, the Examiner has interpreted the limitations recited after “for example” to be preferred embodiments and not part of the claimed invention.
Regarding claims 20, 28, and 37, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d). For the purposes of applying prior art, the Examiner has interpreted the limitations recited after “preferably” to be preferred embodiments and not part of the claimed invention.
Claim 3 recites “(lipase enzymatic assay for polysorbates (LEAP) assay)” in parentheses; however, it is unclear is what is in parentheses is part of the claimed invention, or a preferred embodiment. For the purposes of applying prior art, the Examiner has interpreted the phrase recited in parentheses to be a preferred embodiment and not part of the claimed invention.
Claims 7, 11, and 13-14 recites the limitation "the hydrolase”. There is insufficient antecedent basis for this limitation in the claim. No hydrolase was previously recited.
Claims 15, 19-22, 24, 32, 34, and 40 are included in this rejection for depending on either rejected independent claim 1 or a rejected dependent claim.
Claim Rejections - 35 USC § 102, Anticipation
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 3, 7, 11, 21-22, and 24 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Sprinzl (US 2009/0258348; Date of Publication: October 15, 2009).
Sprinzl’s general disclosure relates to “the use of an esterase for monitoring and/or tracking the synthesis of a protein, polypeptide or peptide in a cell-free translation system or in an in vivo expression system in which the synthesis of a protein, polypeptide or peptide can occur, wherein said monitoring and/or tracking comprises the detection of the function of said esterase” (see, e.g., Sprinzl, abstract). Additionally, Sprinzl discloses “a method for immobilising a protein, polypeptide or peptide comprising the steps of (a) tagging said protein, polypeptide or peptide with an esterase and (b) binding said esterase to an esterase inhibitor, wherein said esterase inhibitor is immobilized on a solid substrate” (see, e.g., Sprinzl, abstract).
Regarding claim 1 pertaining to steps (i) and (ii), Sprinzl teaches “a method for immobilising a protein, polypeptide or peptide comprising the steps of (a) tagging said protein, polypeptide or peptide with an esterase and (b) binding said esterase to an esterase inhibitor, wherein said esterase inhibitor is immobilized on a solid substrate” (see, e.g., Sprinzl, abstract). Additionally, Sprinzl teaches “The method for immobilising a protein, polypeptide or peptide may comprise further steps, like (c) cleaving said protein, polypeptide or peptide from said esterase and (d) recovering a purified fraction of said protein, polypeptide or peptide. By steps (c) and (d) the immobilized a protein, polypeptide or peptide may be obtained and/or purified from, inter alia, cell-free translation systems” (see, e.g., Sprinzl, [0181]). Furthermore, Sprinzl teaches in example 7 that “it was also surprisingly found that immobilization of inhibitors of esterases allow affinity purification of the synthesized esterase either alone or of a protein, polypeptide or peptide fused to an esterase (see, e.g. example 7). This purification can occur in particular in the format of fusion constructs in the herein described form at "X-esterase" or "esterase-X", wherein said esterase interacts with an immobilized binding partner of the esterase (for example an inhibitor, like trifluoromethyl-alkylketones; FIG. 8(1), example 7, FIG. 9). After binding of the esterase part of the fusion protein with its binding partner (FIG. 8(2)), as described herein (example 7, FIG. 9), the esterase fusion protein can be either released from its binding partner as a whole (FIG. 8(3a)) or the desired protein, polypeptide or peptide may be cleaved of from the esterase part. This may be carried out for example, and as described herein, by cleaving an introduced linker between the esterase and the desired protein (FIG. 8(3b), example 7, FIG. 9). Therefore, present invention provides for an vector comprising the coding sequence of an esterase, preferably esterase 2 from Alicyclobacillus acidocaldarius, and a part X, wherein said part X is a protein, polypeptide or peptide to be purified. Preferably, the esterase and the part X that are comprised in the vector of the invention (as described below), are connected (in frame) via a linker, preferably cleavable by proteases, more preferably cleavable by a factor XA protease. As shown in the appended examples, the vector of the present invention may be used as a template for in vitro synthesis of the encoded esterase fusion protein. The resulting translation mixture, containing the synthesized esterase fusion protein may be incubated in presence of a matrix (i.e, Sepharose CL-6B) carrying an immobilized esterase-binding partner, preferably an esterase inhibitor, (i.e, trifluoromethyl-alkylketones (TFK)). After binding of the esterase fusion protein to the binding partner, the components of the esterase fusion protein may be released as described above and exemplified herein (example 7, FIG. 9). The matrix, the binding partner of the esterase part is immobilised on, may be in form of beads, columns, flat surfaces (i.e. glass slides), coated vials and the like. A skilled person can easily substitute the matrix as exemplified herein (example 7, FIG. 9) with any desired matrix to be used for immobilising the esterase binding partner. Thus, the present invention also provides for the use of an esterase as a reporter enzyme to monitor and/or track the synthesis of proteins, polypeptides or peptides fused to said esterase, preferably to the N-terminus of said esterase, and also as a cleavable tag for the purification of said proteins, polypeptides or peptides. Further illustrative details are given in the appended examples” (see, e.g., Sprinzl, [0054]).
Regarding claim 3 pertaining to the hydrolytic activity of the composition, this hydrolytic activity exhibited by the composition would be inherent. Sprinzl teaches the same method of contacting a composition comprising a protein with a hydrolase inhibitor, wherein the hydrolase inhibitor is immobilized on a solid carrier, and recovering the protein from the composition (see, e.g., Sprinzl, abstract & [0181]-[0186]). Therefore, the prior art teaches the same methods with the same inhibitors and proteins, which would inherently lead to the same effect of reduced hydrolytic activity compared to the stating composition (see, e.g., MPEP 2112.02).
Regarding claim 7 pertaining to adsorbing the hydrolase to the hydrolase inhibitor, Sprinzl teaches “binding said esterase to an esterase inhibitor, wherein said esterase inhibitor is immobilized on a solid substrate” (see, e.g., Sprinzl, abstract).
Regarding claim 11 pertaining to the hydrolases, Sprinzl teaches that the hydrolase is an esterase (see, e.g., Sprinzl, abstract).
Regarding claim 21 pertaining to the order of the steps, Sprinzl teaches “a method for immobilising a protein, polypeptide or peptide comprising the steps of (a) tagging said protein, polypeptide or peptide with an esterase and (b) binding said esterase to an esterase inhibitor, wherein said esterase inhibitor is immobilized on a solid substrate” (see, e.g., Sprinzl, abstract). Additionally, Sprinzl teaches “The method for immobilising a protein, polypeptide or peptide may comprise further steps, like (c) cleaving said protein, polypeptide or peptide from said esterase and (d) recovering a purified fraction of said protein, polypeptide or peptide. By steps (c) and (d) the immobilized a protein, polypeptide or peptide may be obtained and/or purified from, inter alia, cell-free translation systems” (see, e.g., Sprinzl, [0181]). Therefore, step (i) of contacting the protein composition with a hydrolase inhibitor is performed before step (ii), which pertains to recovery of the protein from the composition.
Regarding claim 22 pertaining to protein purification, Sprinzl teaches “The invention also provides for a method for the purification of a protein, polypeptide or peptide, said basically combining the steps provided herein above, namely, comprising the steps of: (a) expressing in vitro said protein, polypeptide or peptide in a format of an esterase fusion construct or tagging said protein, polypeptide or peptide with an esterase; (b) immobilizing said esterase fusion construct or said esterase tag protein, polypeptide or peptide according method provided above, e.g. via an specific esterase binding molecule, for example a (specific) anti-esterase antibody or an esterase inhibitor specifically interacting with esterase; (c) cleaving said protein, polypeptide or peptide from said esterase; and (d) recovering a purified fraction of said protein, polypeptide or peptide (see, e.g., Sprinzl, [0182]-[0186]).
Regarding claim 24 pertaining to the protein, Sprinzl teaches that the protein to can be an antibody (see, e.g., Sprinzl, [0104]).
Claim Rejections - 35 USC § 103, Obviousness
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 13, 28, 32, 34, 36-37, and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Sprinzl in view of Patel (WO 2019/060062; Date of Publication: March 28, 2019 – cited in the IDS filed on 02/06/2023).
The teachings of Sprinzl are discussed above as it pertains to a method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier, and recovering the protein from the composition.
Regarding claim 40 pertaining to pharmaceutical compositions, Sprinzl teaches “proteins produced by this systems are widely employed in biochemical, biostructural and pharmaceutical uses, not only in fundamental research but also in the biochemical, chemical and pharmaceutical industry” (see, e.g., Sprinzl, [0002]).
However, Sprinzl does not teach: wherein the hydrolase is a lipase (claim 13); or wherein the method further comprises adding a surfactant (claim 28); or wherein the fatty acid ester is polyoxyethylene sorbitan or iso-sorbide fatty acid mono-, di- or tri-ester (claim 32); or wherein the fatty acid ester is polysorbate 80 (claim 34); or wherein the surfactant is Mono- or Di-acylglycerol (claim 36); or wherein the degradation of the fatty acid ester is less than 20% (claim 37).
Patel’s general disclosure relates to “biopharmaceutical formulations and drug products exhibiting reduced amounts of subvisible particle formation upon storage, and to methods of their preparation and storage. Specifically, this disclosure is directed to formulations and drug products comprising a protein and a surfactant or stabilizer including high percentage amounts of a long-chain, mono-unsaturated fatty acid ester, and methods of their preparation and storage” (see, e.g., Patel, [0002]). Moreover, Patel discloses “The free fatty acids formed as a result of hydrolysis may aggregate and form particulates in drug products containing such formulations over time. Particulates (both visible and subvisible) can impact product stability, reduce a drug product's shelf life because of its failure to meet compendial particulate matter specifications (e.g., U.S. FDA specifications), and may have clinical effects, such as an immunogenic reaction upon administration” (see, e.g., Patel, [0003]).
Regarding claim 13 pertaining to the hydrolase, Patel teaches that the esterase can be a lipase (see, e.g., Patel, [00039]).
Regarding claim 28 pertaining to addition of a surfactant, Patel teaches “Compositions and drug products disclosed herein comprise a protein and a surfactant or stabilizer including high percentage amounts (e.g., at least 97%) of a long-chain fatty acid ester” (see, e.g., Patel, abstract).
Regarding claims 32, 34, and 36 pertaining to the fatty acid ester, Patel teaches “The term "fatty acid ester" means any organic compound that contains a fatty acid chain linked to a head group via an ester bond. An ester bond is formed when a hydroxyl group (e.g., an alcohol or carboxylic acid) is replaced by an alkoxy group. As used herein, the hydroxyl group can be part of a carboxylic acid, more specifically a fatty acid, and/or an alcohol, such as glycerol, sorbitol, sorbitan, isosorbide, or the like. The alcohol group is generally referred to herein as the head group”. Examples of fatty acid esters generally include phospholipids, lipids (e.g., the head group is glycerol, including monoglycerides, diglycerides, and triglycerides), and surfactants and emulsifiers, including for example polysorbates like polysorbate 20, polysorbate 60, and polysorbate 80, which are non-ionic detergents. Surfactants and emulsifiers are useful as cosolvents and stabilizers. They function by associating with both a hydrophilic surface and a lipophilic surface to maintain dispersion and structural stability of ingredients, like proteins. Surfactants are added to protein formulations primarily to enhance protein stability against mechanical stress, such as air/liquid interface-induced and solid/liquid interface-induced partial unfolding and self-association. Without a surfactant, proteins may in some cases become structurally unstable in solution, and form multimeric aggregates that eventually become subvisible particles” (see, e.g., Patel, [00026]-[0027]).
Regarding claim 37 pertaining to the degradation of fatty acid ester, this degradation would be considered inherent. The combined prior art of Sprinzl and Patel teach the instantly claimed invention, which is a method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier, and recovering the protein from the composition (see, e.g., Sprinzl, abstract). Patel teaches that the esterase can be a lipase (see, e.g., Patel, [00039]), as well addition of a fatty acid ester to the method (see, e.g., Patel, [00026]-[00027]). Therefore, since the combined prior art of Sprinzl and Patel teach the instantly claimed invention, it would be inherent that this would lead to degradation of the fatty acid ester less than 20% (see, e.g., MPEP 2112.02).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform Sprinzl’s method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier and recovering the protein from the composition, wherein the hydrolase inhibitor is further contacted with hydrolase, which can be an esterase, as taught by Patel. One would have been motivated to do so because Patel teaches “The term "esterase" means an enzyme that catalyzes the hydrolysis of an ester bond to create an acid and an alcohol. Esterases are a diverse category of enzymes, including acetyl esterases {e.g., acetylcholinesterase), phosphatases, nucleases, thiolesterases, lipases and other carboxyl ester hydrolases (EC 3.1. As its name implies a carboxyl ester hydrolase (a.k.a. carboxylesterase, carboxylic-ester hydrolase, and EC 3.1.1.1) uses water to hydrolyze a carboxylic ester into an alcohol and a carboxylate” (see, e.g., Patel, [00039]). Additionally, Patel teaches that esterases can contribute to polysorbate instability (see, e.g., Patel, [00041]). Furthermore, Patel teaches “The inclusion of an esterase inhibitor during purification of a protein of interest or in the final formulation may prevent or slow the hydrolysis of non-ionic detergents like polysorbate 80, which in turn are expected to prevent or reduce subvisible particle formation” (see, e.g., Patel, [00044]). Moreover, Sprinzl teaches “synthesis of a protein, polypeptide or peptide in a cell-free translation system in an in vivo expression system can be easily and efficiently be detected when said protein, polypeptide or peptide is synthesized with, preferably, a covalently attached/bound esterase (or an esterase activity)” (see, e.g., Sprinzl, [0014]). Therefore, based on the teachings of Sprinzl and Patel, it would have been obvious to employ an esterase as the hydrolase within the composition comprising the protein and the immobilized hydrolase inhibitor in order to prevent or slow the hydrolysis of non-ionic detergents like polysorbate 80, which in turn are expected to prevent or reduce subvisible particle formation, and detect protein expression.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform Sprinzl’s method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier and recovering the protein from the composition, wherein the composition further comprises a surfactant, as taught by Patel. One would have been motivated to do so because Patel teaches “Surfactants are added to protein formulations primarily to enhance protein stability against mechanical stress, such as air/liquid interface-induced and solid/liquid interface-induced partial unfolding and self-association. Without a surfactant, proteins may in some cases become structurally unstable in solution, and form multimeric aggregates that eventually become subvisible particles” (see, e.g., Patel, [00027]). Furthermore, Patel teaches “Polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 are widely employed in the pharmaceutical, cosmetic, and food industries as stabilizers and emulsifiers” (see, e.g., Patel, [00030]). Moreover, Sprinzl teaches cell-free protein translation systems for the in vitro synthesis of proteins (see, e.g., Sprinzl, [0001]). Therefore, based on the teachings of Sprinzl and Patel, it would have been obvious to add a surfactant to the protein composition in order to enhance stability of the protein composition. One would have expected success because Sprinzl and Patel both teach protein compositions comprising hydrolase inhibitors.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Sprinzl as applied to claims 1, 3, 7, 11, 21-22, and 24 above, and further in view of Rosenbaum (Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick Type C Disease Therapeutics; 2010).
The teachings of Sprinzl are discussed above as it pertains to a method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier, and recovering the protein from the composition.
However, Sprinzl does not teach: wherein the hydrolase is lysosomal acid lipase (claim 14).
Rosenbaum’s general disclosure relates to testing various thiadiazole carbamates, amides, esters, and ketones for inhibition of lysosomal acid lipase (LAL) (see, e.g., Rosenbaum, abstract). Moreover, Rosenbaum discloses that “only thiadiazole carbamates are effective inhibitors of LAL” and that “LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat” (see, e.g., Rosenbaum, abstract).
Regarding claim 14 pertaining to lysosomal acid lipase, Rosenbaum teaches the enzyme lysosomal acid lipase (see, e.g., Rosenbaum), and further teaches that orlistat, as well as thiadiazole carbamates are potent inhibitors of lysosomal acid lipase (see, e.g., Rosenbaum, Introduction pg. 5282 & Figure 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform Sprinzl’s method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier and recovering the protein from the composition, wherein the hydrolase inhibitor is further contacted with hydrolase, which can be a lysosomal acid lipase, as taught by Rosenbaum. One would have been motivated to do so because Rosenbaum teaches that orlistat, which is a lipase inhibitor, and thiadiazole carbamates are potent inhibitors of lysosomal acid lipase (see, e.g., Rosenbaum, Introduction pg. 5282 & Figure 3). Moreover, Sprinzl teaches the use of inhibitors in the cell-free protein expression system, wherein the inhibitor is immobilized on a solid substrate (see, e.g., Sprinzl, abstract). Therefore, based on the teachings of Sprinzl and Rosenbaum, it would have been obvious to employ lysosomal acid lipase as the hydrolase because this would allow for lysosomal acid lipase to bind to the inhibitor immobilized on the solid carrier. One would have expected success because Sprinzl and Rosenbaum both teach enzyme inhibitors.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Sprinzl and Rosenbaum as applied to claims 1, 3, 7, 11, 14, 21-22, and 24 above, and further in view of Patel (WO 2019/060062; Date of Publication: March 28, 2019 – cited in the IDS filed on 02/06/2023).
The teachings of Sprinzl and Rosenbaum, herein referred to as modified-Sprinzl-Rosenbaum, are discussed above as it pertains to a protein with a hydrolase inhibitor immobilized on a solid carrier, and recovering the protein from the composition.
However, modified-Sprinzl-Rosenbaum does not teach: wherein the immobilized inhibitor is selected from the group consisting of orlistat or bis-enol-ester (claim 15).
The teachings of Patel are discussed above.
Regarding claim 15 pertaining to the immobilized inhibitor, Patel teaches “the applicants envision that the inclusion of an esterase inhibitor in a protein formulation containing a fatty acid ester surfactant may help maintain protein stability and help reduce SVP formation. Common esterase inhibitors known in the art include orlistat (tetrahydrolipistatin; an inhibitor of carboxylesterase 2 and lipoprotein lipase), diethylumbelliferyl phosphate (a cholesterol esterase [lipsase A] inhibitor), URB602 ([l- -biphenyl]-3-tl-carbamicacid cyclohexyl ester; a monoacylglycerol lipase inhibitor), and 2-butoxyphenylboronic acid (an inhibitor of hormone-sensitive lipase). The inclusion of an esterase inhibitor during purification of a protein of interest or in the final formulation may prevent or slow the hydrolysis of non-ionic detergents like polysorbate 80, which in turn are expected to prevent or reduce subvisible particle formation” (see, e.g., Patel, [00044]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform Sprinzl’s method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier and recovering the protein from the composition, wherein the hydrolase inhibitor is orlistat, as taught by Patel. One would have been motivated to do so because Patel teaches “inclusion of an esterase inhibitor during purification of a protein of interest or in the final formulation may prevent or slow the hydrolysis of non-ionic detergents like polysorbate 80, which in turn are expected to prevent or reduce subvisible particle formation” (see, e.g., Patel, [00044]), wherein the esterase inhibitor can be orlistat (see, e.g., Patel, [00044]). Moreover, modified-Sprinzl-Rosenbaum teaches that orlistat is a “well-documented inhibitor of several lipases” (see, e.g., Rosenbaum, Introduction, pg. 5282). Therefore, based on the teachings of modified-Sprinzl-Rosenbaum and Patel, it would have been obvious to employ orlistat as the immobilized inhibitor since orlistat is a potent inhibitor of several lipases. One would have expected success because modified-Sprinzl-Rosenbaum and Patel teach compositions comprising enzyme inhibitors.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Sprinzl as applied to claims 1, 3, 7, 11, 21-22, and 24 above, and further in view of Kihara (US 2019/0247827; Date of Publication: August 15, 2019).
The teachings of Sprinzl are discussed above as it pertains to a method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier, and recovering the protein from the composition.
However, Sprinzl does not teach: wherein the inhibitor is immobilized on a solid carrier via the reaction of an amino group and an N-hydroxysuccinimide group (claim 19).
Kihara’s general disclosure relates to “A mixed mode affinity chromatography carrier includes a substrate, a hydrophilic polymer, an antibody-binding cyclic peptide, and a cation exchange group” (see, e.g., Kihara, abstract). Moreover, Kihara discloses “An object of the present invention is to provide a mixed mode affinity chromatography carrier excellent in all of antibody adsorption capacity, impurities removal function, and drug resistance. As a result of intensive examination in order to achieve the object, the present inventors have found that a mixed mode affinity chromatography carrier including a substrate, a hydrophilic polymer, a cyclic peptide, and a cation exchange group is excellent in all of antibody adsorption capacity, impurities removal function, and drug resistance, thereby completing the present invention” (see, e.g., Kihara, [0011]-[0012]).
Regarding claim 19 pertaining to immobilization of the inhibitor, Kihara teaches “The method of introducing (immobilizing) an affinity ligand into the cation exchange group-introduced carrier is not particularly limited as long as it is capable of binding the affinity ligand to the cation exchange group-introduced carrier by a covalent bond. Examples of the method of introducing (immobilizing) an affinity ligand into the cation exchange group-introduced carrier include a method in which a part of cation exchange groups is converted into N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) and reacted with an affinity ligand, and the unreacted EDC/NHS-converted cation exchange group is regenerated after the reaction; and a method in which, in the case of an affinity ligand having an amino group, a cation exchange group is protected, a formyl group is introduced, and then the affinity ligand is introduced into the formyl group by reductive amination through an amino group” (see, e.g., Kihara, [0495]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform Sprinzl’s method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier and recovering the protein from the composition, wherein the hydrolase inhibitor is immobilized on the carrier via the reaction of an amino group and an N-hydroxysuccinimide group, as taught by Kihara. One would have been motivated to do so because Kihara teaches that N-hydroxysuccinimide can be employed to immobilize affinity ligand to a cation exchange group via a covalent bond (see, e.g., Kihara, [0495]). Furthermore, Kihara teaches that mixed mode affinity chromatography carrier including a substrate, a hydrophilic polymer, a cyclic peptide, and a cation exchange group is excellent in all of antibody adsorption capacity, impurities removal function, and drug resistance, thereby completing the present invention” (see, e.g., Kihara, [0011]-[0012]). Moreover, Sprinzl teaches “a method for immobilising a protein, polypeptide or peptide comprising the steps of (a) tagging said protein, polypeptide or peptide with an esterase and (b) binding said esterase to an esterase inhibitor, wherein said esterase inhibitor is immobilized on a solid substrate” (see, e.g., Sprinzl, abstract). Therefore, based on the teachings of Sprinzl and Kihara, it would have been obvious to immobilize a hydrolase inhibitor to a solid carrier via N-hydroxysuccinimide. One would have expected success because Sprinzl and Kihara both teach methods of protein immobilization onto carriers.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Sprinzl, Rosenbaum, and Patel as applied to claims 1, 3, 7, 11, 14-15, 21-22, and 24 above, and further in view of Yang (Activity-Based Proteome Profiling of Potential Cellular Targets of Orlistat – An FDA-Approved Drug with Anti-Tumor Activities; 2009 – cited in the IDS filed on 10/10/2025).
The teachings of Sprinzl, Rosenbaum, and Patel, herein referred to as modified-Sprinzl-Rosenbaum-Patel, are discussed above as it pertains to a hydrolase inhibitor immobilized on a solid carrier, and recovering the protein from the composition, wherein the hydrolase inhibitor is orlistat.
However, modified-Sprinzl-Rosenbaum-Patel does not teach: wherein the inhibitor is consistent with formula (1) (claim 20).
Yang’s general disclosure relates to identification of previously unknown targets of orlistat (see, e.g., Yang, abstract). Moreover, Yang discloses proteins involved in cellular proliferation/division and protein degradation that orlistat targets (see, e.g., Yang, Table 1). Furthermore, Yang discloses the use of tetrahydrolipstatin (THL)-like protein reactive probes (i.e., THL-R, THL-L, and THL-T), wherein “(1) THL (being derived from a natural product) is cell-permeable, making our probes applicable for direct in situ cell-based screening; (2) THL reacts with its known cellular targets via a covalent reaction through its reactive β-lactone moiety and the nucleophilic active-site residue (typically Ser/Cys residues, e.g. Ser2308 in FAS (3b)) of the target protein, resulting in the formation of an isolatable protein/THL complex; (3) previous minor structural modifications at either the 16-carbon or 6-carbon aliphatic chains of THL did not significantly alter its native biological activities” (see, e.g., Yang, “Design and Synthesis of Orlistat-like Probes”, pgs. 657-659).
Regarding claim 20 pertaining to formula 1, Yang teaches the same chemical structure as instant formula 1 within the boxed section of Yang’s figure 1, wherein the THL-R molecule, without the probe, is performing a nucleophilic attack of the β -lactone ring to the target probe (see, e.g., Yang, Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform Sprinzl’s method of contacting a composition comprising a protein with a hydrolase inhibitor immobilized on a solid carrier and recovering the protein from the composition, wherein the hydrolase inhibitor is further contacted with hydrolase, which can be formula (1), as taught by Yang. One would have been motivated to do so because Yang teaches instantly claimed formula (1) (see, e.g., Yang, Figure 1) which is designated as THL-R, in order to produce THL-like protein-reactive probes (see, e.g., Yang, “Design and Synthesis of Orlistat-like Probes”, pg. 657). Furthermore, Yang teaches “We took advantage of several key properties known to THL in the design of our activity-based probes: (3, 5) (1) THL (being derived from a natural product) is cell-permeable, making our probes applicable for direct in situ cell-based screening; (2) THL reacts with its known cellular targets via a covalent reaction through its reactive β-lactone moiety and the nucleophilic active-site residue (typically Ser/Cys residues, e.g. Ser2308 in FAS (3b)) of the target protein, resulting in the formation of an isolatable protein/THL complex; (3) previous minor structural modifications at either the 16-carbon or 6-carbon aliphatic chains of THL did not significantly alter its native biological activities. (5b) Accordingly, probes 1 and 2, in which a C−C triple bond was introduced into THL to replace the terminal C−C single bond of the aliphatic chains, were synthesized. We also synthesized probe 3 by substituting the CHO group in the N-formyl-L-leucine moiety of THL with a propiolic acyl group (Figure 1, shaded in purple). These extremely conservative modifications of introducing an alkyne handle in the parental THL structure were aimed at maintaining the native biological properties of Orlistat, while providing the necessary functionality for identification and characterization (i.e., imaging) of previously unknown cellular targets by downstream conjugation of the protein/probe complex to reporter tags via the bio-orthogonal click chemistry” (see, e.g., Yang, “Design and Synthesis of Orlistat-like Probes”, pgs. 657-658). Therefore, based on the teachings of Yang, the C-C triple bond introduced to the THL molecule, which would give the instantly claimed structure, were provided in order to allow for conjugation of the protein/probe complex to the reporter tags via the bio-orthogonal click chemistry. Moreover, modified-Sprinzl-Rosenbaum-Patel teaches “inclusion of an esterase inhibitor during purification of a protein of interest or in the final formulation may prevent or slow the hydrolysis of non-ionic detergents like polysorbate 80, which in turn are expected to prevent or reduce subvisible particle formation” (see, e.g., Patel, [00044]), wherein the esterase inhibitor can be orlistat (see, e.g., Patel, [00044]). Moreover, modified-Sprinzl-Rosenbaum-Patel teaches that orlistat is a “well-documented inhibitor of several lipases” (see, e.g., Rosenbaum, Introduction, pg. 5282). Therefore, based on the teachings of modified-Sprinzl-Rosenbaum-Patel and Yang, it would have been obvious to employ the inhibitor corresponding to instantly claimed formula (1) because inhibitor has the same functional properties as the native orlistat, while allowing for the addition of a probe to observe downstream cellular processes. One would have expected success because modified-Sprinzl-Rosenbaum-Patel and Yang teach immobilization of inhibitors, wherein the inhibitor can be orlistat.
Conclusion
Claims 1, 3, 7, 11, 13-15, 19-22, 24, 28, 32, 34, 36-37, and 40 are rejected.
No claims are allowed.
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653