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 .
DETAILED ACTION
The instant application claims benefit to provisional application 63/130295, filed on December 23, 2020.
The amendment filed August 11, 2025 is acknowledged. Claims 3, 10-13, 15, 19-31, 34-35, 38, and 42 are canceled, and claims 1-2 and 17-18 are amended. Currently claims 1-2, 4-9, 14, 16-18, 32-33, 36-37, 39-41, and 43-45 are pending, wherein claims 39-41 and 45 are withdrawn.
Claims 1-2, 4-9, 14, 16-18, 32-33, 36-37, and 43-44 are under examination.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 August 11, 2025 has been entered.
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 18 and 44 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.
Claim 18 contains the trademark/trade name POROS Benzyl ultra resin, Hexyl-650C resin, and Phenyl-600M resin. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe types of resins and, accordingly, the identification/description is indefinite.
Claim 44 is likewise rejected as being dependent on claim 18.
Claim Rejections - 35 USC § 103
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 7-9, 14, 16-17, 32-33, 36-37, and 43 are rejected under 35 U.S.C. 103 as being unpatentable over Friedrich et al. (WO2018234492A1, previously cited in PTO-892 mailed 12/11/2024, hereinafter “Friedrich”) in view of Koehnlein (US11518781B2, previously cited in PTO-892 mailed 12/11/2024), as evidenced by Breiback et al. (Biopolymers. 2018;109:e23069, pgs. 1-12, previously cited in PTO-892 mailed 12/11/2024, hereinafter “Breiback”).
Friedrich teaches a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, wherein the modified protein can be produced by expressing in host cells which meets the limitations of claims 1, 2, 16, 17, 32, and 43 (abstract). Friedrich teaches methods for the isolation of the modified protein and/or the polypeptide as defined herein and/or of the asparaginase comprise, without limitation, purification steps such as affinity chromatography (preferably using a fusion tag such as the Strep-tag II or the His6-tag), gel filtration (size exclusion chromatography), anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), high pressure liquid chromatography (HPLC), reversed phase HPLC, ammonium sulfate precipitation or immunoprecipitation (para 122). Friedrich teaches the modified protein comprises a polypeptide that is a random coil polypeptide which meets the limitation of claim 14 (para 76). Friedrich teaches many L-asparaginase proteins have been identified in the art, the most widely used L-asparaginases are derived from E. coli or from Erwinia chrysanthemi, both of which share 50% or less structural homology which meets the limitation of claim 33 (para 58). Friedrich teaches the modified protein of the invention can be a recombinant protein produced in an E. coli strain, preferably a protein from an Erwinia species, which meets the limitation of claims 36 and 37 (para 61).
Friedrich teaches PASylated Cristanspase (asparaginase) chemically conjugated to Pga-P/A peptides of varying lengths and purified the periplasmic extracts from cell cultures transformed with the nucleic acid encoding the fusion protein, via anion exchange chromatography which meets the limitations of claims 1, 2, and 17 (para 171, 176). Friedrich teaches SDS-PGE analysis of the mature PA-Cristanspase fusion protein after periplasmic extraction, ammonium sulfate precipitation (ASP) and anion exchange chromatography (AEX) in Figure 6, which shows the mature fusion protein is substantially purified after AEX (para 152, Figure 6A). Homogeneous protein preparations without signs of aggregation were obtained with a final yield of 128 mg for PA#1(200)-Crisantaspase and 48 mg for PA#1(400)- Crisantaspase from one 8 L fermenter, respectively (para 177).
Friedrich teaches the molecular weight (MW) of Cristanspase is 105 kDa (true mass 140 kDa), the PA200-Crisantaspase is 595 kDa (true mass 205 kDa) and the PA400-Crisantaspase is 1087 kDa (true mass 269 kDa), which calculates the MW of the charge shielding domain as approximately 46%-92% of the molecular weight of the biologically active domain, which meets the limitations for claims 1, 2, 8 (para 153). Friedrich teaches the P/A (200) polypeptide as set forth in SEQ ID NO: 7 has a calculated MW of 16.1 kDa, which meets the limitation of claim 9 (pg. 60). As evidenced by Breiback, PASylated proteins show a trend toward increasing hydrodynamic volume with rising Pro content of the P/A polypeptide, which meets the limitation of increasing the hydrodynamic radius of the protein in claim 7 (pg. 4, col. 2, para 3).
Friedrich does not explicitly teach purifying the fusion protein from a periplasmic releasate using a hydrophobic interaction chromatography as the first chromatography step, nor that the purity of the protein is at least 45% pure after the first chromatography step.
However, Koehnlein teaches a process for producing PEGylated protein compositions, subjecting a mixture comprising the PEGylated proteins to a hydrophobic interaction chromatography (HIC) (abstract). Koehnlein teaches the processes involving HIC are advantageous, because they can be performed at a pH close to the physiological pH, which may improve stability of the protein (col. 4, lines 4-8). Koehnlein teaches the purification of mono-PEGylated proteins is particularly desirable because it provides improved stability without significantly compromising therapeutic efficacy (col. 1, lines 34-36). Koehnlein teaches PEGylation reactions tend to produce mixtures comprising non-PEGylated, mono-PEGylated, and oligo-PEGylated proteins, and efforts to utilize cation exchange chromatography for separating the mixtures in sequential elution have been performed (col. 2, lines 30-44). Koehnlein teaches the use of a two-stage HIC step, which selectively binds oligo and mono PEGylated proteins (col. 2, lines 56-65). Purification of proteins by HIC is based on hydrophobicity of the protein rather than charge which may be advantageous because they are relatively unaffected by acidic forms or by glycosylation variants that affect protein charge (col. 4, lines 33-38). Koehnlein teaches the PEGylated protein is an erythropoietin (EPO) protein, which can be prepared via expression in eukaryotic cells (col. 22, lines 14-23). Koehnlein teaches the PEGylated EPO are prepared for HIC by concentrating 0.87 mg/mL recycled EPO, diluted, then added to the column at 0.5mg per sample (col. 26, lines 30-60). High performance liquid chromatography (HPLC) is used to analyze the HIC fractions for purity (col. 27, lines 45-48). Koehnlein teaches 16 HIC resins were tested for their effectiveness of separating the PEGylated proteins, Toyopearl Phenyl-650M and Phenyl Sepharose HP were the most efficient at the separation, wherein the 650M was the more hydrophobic and eluted 100% of the mono PEGylated proteins in the second fraction which meets the limitation of claim 3 (col. 30, lines 49-55, Table 3). Koehnlein teaches the buffer solutions may contain varying amounts of salt, such as Na2SO4, wherein equilibration buffers and wash buffers contain relatively high salt concentrations (0.4-0.6M) at or around pH 7.0-8.0, which meets this limitation in claims 2 and 17 (col. 5, lines 40-48).
Although neither Friedrich nor Koehnlein explicitly discloses the pH of the load solution comprising Na2SO4 at a pH of 5.5-6.5, the adjustment of specific concentrations clearly would have been a routine matter of optimization using standard laboratory techniques available at the time of filing on the part of the artisan of ordinary skill, said artisan recognizing that the effectiveness of the composition would have been affected by these concentrations.
Neither Friedrich nor Koehnlein explicitly teach purifying PASylated fusion proteins utilizing HIC as a first chromatography step.
However, Breiback discloses the biochemical and biophysical characterization of PAS polypeptides is astonishingly similar to the chemical PEG polymer in several aspects, in particular the high solubility in water as well as polar organic solvents and the disordered, expanded random chain behavior (pg. 11, col. 1, para 4).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of purifying a charge-shielded PAS fusion protein, such as Erwinia asparaginase from a cell lysate utilizing chromatographic methods taught by Friedrich and substitute the AEX chromatography in Friedrich with the HIC as the first chromatography step as taught by Koehnlein with a reasonable expectation of success. It would have been obvious for a person of ordinary skill in the art to try well-known laboratory chromatography methods to efficiently separate PASylated fusion proteins from cell lysate by utilizing HIC as a first chromatography step, due to similar methods of separating PEGylated fusion proteins purified with HIC as a first chromatography as taught by Koehnlein. One of ordinary skill in the art would have been motivated to try the finite predictable potential solutions of known chromatography methods for higher yield isolation and purification of PASylated fusion proteins. “A person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product/method [was] not of innovation but of ordinary skill and common sense.” KSR, 550 U.S. at 421, 82 USPQ2d at 1397. Furthermore, one of ordinary skill in the art would have known, as evidenced by Brieback, PASylated fusion proteins have very similar biophysical and biochemical properties to PEGylated fusion proteins, and one of ordinary skill would have had a reasonable expectation of success by combining the teachings of Koehnlein and Friedrich to advantageously produce and purify PASylated asparaginase with HIC as a first chromatography step.
Claims 4-6, 18, and 44 are rejected under 35 U.S.C. 103 as being unpatentable over Friedrich, Koehnlein, and Brieback as applied to claims 1-2, 7-9, 14, 16-17, 32-33, 36-37, and 43 above, and further in view of Defrees et al. (WO 2008/057683 A2, previously cited in PTO-892, filed 1/4/2024, hereinafter “Defrees”).
As discussed above, Friedrich teaches purification of PAS fused L-asparaginase can be achieved utilizing a number of chromatography methods, such as HIC, AEX, and CEX, wherein Friedrich purifies the modified protein utilizing AEX (para 122, 152). Friedrich teaches the molecular weight (MW) of Cristanspase is 105 kDa (true mass 140 kDa), the PA200-Crisantaspase is 595 kDa (true mass 205 kDa) and the PA400-Crisantaspase is 1087 kDa (true mass 269 kDa), which calculates the MW of the charge shielding domain as approximately 46%-92% of the molecular weight of the biologically active domain, which meets this limitation in claim 18 (para 153). Koehnlein teaches PEGylated fusion proteins have conventionally been purified utilizing CEX, but notes CEX separates protein molecules according to their charge, basic variants of a species will elute later than acidic variants, which can result in poor recovery of the mono-PEGylated acidic EPO forms, thus HIC is advantageous as a first step, because they are relatively unaffected by acidic forms that affect protein charge (col. 4, lines 18-41).
Neither Friedrich or Koehnlein explicitly teach the method further comprises an additional AEX or CEX in purifying the charge-shielded fusion protein, nor the sequential chromatography steps recited in claims 6 and 18.
However, Defrees teaches purification of polypeptide conjugates that include poly(ethylene glycol) (PEG) and the use of hydrophobic interaction chromatography (HIC) to resolve different glycoforms of glycoPEGylated polypeptides (abstract). Defrees teaches that HIC followed by cation exchange can resolve EPO(erythropoietin)-PEG3 species from EPO-PEG2 species (para 6). Defrees teaches AEX used in the method is employed to isolate the polypeptide conjugate from contaminants such as particulates, chemicals and proteins/peptides (e.g., enzymes used in a glycoPEGylation reaction) (para 212). Defrees also teaches the use of various chromatography methods, such as (a) contacting a mixture containing the first polypeptide conjugate with a hydrophobic interaction chromatography (HIC) medium; and (b) eluting the first polypeptide conjugate from the HIC medium (para 89). The method may further include: (c) eluting the first polypeptide conjugate from an anion exchange or mixed-mode chromatography medium (para 89). Defrees teaches the method may further include: (d) eluting the first polypeptide conjugate from a cation exchange chromatography medium (para 89).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try known methods of purifying charge shielded fusion proteins taught by Friedrich, Koehnlein, and Defrees to determine an optimal purification scheme with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to determine a capture and purification scheme based on known sequential chromatography methods that was advantageously tailored for a particular fusion protein, and would have utilized an HIC followed by a cation exchange chromatography to resolve the separation of closely structured species of conjugates as taught by Defrees (para 6). One of ordinary skill in the art would have pursued the known potential solutions of utilizing well-known chromatography methods in sequential orders based on the physiochemical properties of the conjugated compound and the teachings of the prior art.
Response to Arguments
Applicant's arguments filed August 11, 2025 have been fully considered but they are not persuasive.
Regarding remarks directed to the 103 rejections, Applicant argues Koehnlein does NOT teach purifying PEGylated proteins from a cell lysate or periplasmic releasate. As described in Koehnlein, the starting point for a PEGylation reaction is a purified protein, and the ending point of the PEGylation reaction is a mixture of the protein with zero (non-PEGylated), one (mono-PEGylated), or more (oligo-PEGylated) PEG groups added. Koehnlein describes purification of the mono-PEGylated protein from a mixture of non-PEGylated protein, mono-PEGylated protein and oligo-PEGylated protein. Applicant argues purifying a mono-PEGylated protein from a mixture containing a single protein with different levels of PEG modification is very different from the claimed method of purifying a PASylated protein from a cell lysate or periplasmic releasate containing a variety of proteins with varying degrees of hydrophobicity. Thus, results of Koehnlein could not reasonably predict the outcome of using HIC as the first chromatography step to purify a PASylated asparaginase from cell lysate or periplasmic releasate. Applicant argues the reasonable expectation of success is further lacking in view of the known differences between PEGylated proteins and PASylated proteins, e.g., "PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity" (Breiback, Abstract, also page 10). Applicant argues that even assuming one were to replace the subtractive anion exchange chromatography (AEX) of Friedrich with the HIC of Koehnlein, one would not have arrived at the presently claimed invention, at least because the first chromatography step of Friedrich was NOT performed with a cell lysate or periplasmic releasate as the presently claimed. Rather, in Friedrich, periplasmic extracts were precipitated by ammonium sulfate to a saturation of 25% at 25 "C, resuspended in AEX running buffer, dialyzed and centrifuged to remove insoluble matter before subjected to subtractive AEX (paragraph [000176]). As known to those skilled in the art, different proteins may precipitate (salt out) at different concentrations of ammonium sulfate and many proteins might not be precipitated by ammonium sulfate to a saturation of 25% at 25°C. Further, some of the ammonium sulfate precipitated proteins stayed insoluble after resuspension and were removed by centrifugation in Friedrich. Thus, the first chromatography step of Friedrich was conducted with fewer proteins having less variation in hydrophobicity than that of the claimed cell lysate or periplasmic releasate. Applicant argues only through innovative experimentation, the inventors were able to arrive at the claimed invention, by using HIC as a first step to reliably and efficiently purify the PASylated fusion proteins, which the applicant claims are superior and unexpected over the prior art.
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, Friedrich teaches all of the limitations of the PASylated asparaginase except utilizing HIC as a first chromatography step, and Koehnlein teaches PEGylated proteins are purified from periplasmic releasate using HIC as a first chromatography step (NOTE instant claim 1 recites a method of applying a load solution comprising the cell lysate or perisplasmic releasate). Furthermore regarding Applicant’s arguments about Friedrich precipitating the releasate by ammonium sulfate then subjecting to the AEX, therefore has few proteins than the periplasmic releasate used in the claimed invention, it is noted that this feature upon which applicant relies is not recited in the rejected claims, therefore these limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Brieback provides evidential support of the closely related physiochemical properties between PEG and PAS peptides, such as both charge-shielding domains that are hydrophilic in nature and exhibit similar random coil behavior in aqueous solution as PEG. Furthermore, Brieback states PAS polypeptides is a promising PEG mimetic for biopharmaceutical applications, although Brieback does disclose PAS polypeptides in general are more hydrophilic. Similarly, Friedrich teaches PEGylated asparaginase has been commonly used in disease treatment in an attempt to reduce immunogenicity associated with administration of the microbial L-asparaginase, however the PEGylated protein has reduced in vitro activity compared to the unmodified asparaginase, which Friedrich’s invention attempts to solve with an alternative ‘shielding’ fusion protein, the PASylated asparaginase. Thus, one of ordinary skill in the art would have been motivated to follow the teachings of Friedrich and Brieback that PASylated fusion proteins are highly similar to PEGylated fusion proteins, and try different chromatography methods, such as the HIC utilized as a first step in the purification of PEGylated proteins taught by Koehnlein with a reasonable expectation of success. In response to Applicant’s assertion the claimed method has unexpected results over the prior art due to the use of HIC as a first chromatography step for PASylated proteins, Koehnlein’s method (col. 30, Table 3) utilized the same screening process of HIC material for purifying the PEGylated proteins with reliably high purity and yield as the claimed method, thus one of ordinary skill in the art would be motivated to try the same resins with a PASylated protein as taught by the prior art with the expectation of same or similar results.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657