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 June 4, 2026 is acknowledged. Claims 3, 10-13, 15, 19-31, 34-35, 38, and 42 are cancelled, and claims 1-2 and 17-18 are amended. Claims 1-2, 4-9, 14, 16-18, 32-33, 36-37, 39-41, and 43-45 are pending, wherein claims 39-41 and 45 are withdrawn.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.
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 Alakomi (Academic Dissertation, 2007, VTT Technical Research Centre of Finland, pgs. 1-95) and 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). Friedrich teaches after an induction period of 2.5 h, cells were harvested by centrifugation and resuspended during 10 min in ice-cold periplasmic fractionation buffer (500 mM sucrose, 1 mM EDTA, 200 mM boric acid/NaOH pH 8.0; 2 ml per L and OD55o). After adding 15 mM EDTA and 250 pg/mL lysozyme, the cell suspension was incubated for 20 min on ice, centrifuged several times, and the cleared supernatant containing the recombinant protein was recovered [000175]. 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). Although Friedrich teaches the periplasmic releasate was obtained with EDTA, Alakomi discloses polyethylenimine (PEI) is a potent permeabilizer of the outer membrane (OM) of pathogenic Gram-negative bacteria, as it sensitized E. coli, P. aeruginosa, and S. Typhimurium to hydrophobic antibiotics and detergents, as well as can intercalate the OM and increase the membrane surface area without liberation of LPS-associated cell material from Salmonella (pg. 57, para 3). Therefore, it would have been an obvious, routine, and well-understood practice to substitute the periplasmic extraction buffer containing EDTA taught by Friedrich with another known periplasmic extraction chemical PEI, that would liberate the fusion protein from the periplasmic space for further purification.
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, as evidence by Alakomi, 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 June 4, 2026 have been fully considered but they are not persuasive.
Regarding Remarks directed to the 35 USC § 103 rejections, Applicant argues Friedrich does NOT teach "applying a load solution comprising the cell lysate or periplasmic releasate to a hydrophobic interaction chromatography (HIC) as a first chromatography step," wherein the "periplasmic releasate" is obtained as recited in the pending claims, Thus, Friedrich's method involves applying to AEX (rather than HIC) a protein solution containing a fraction of the periplasmic releasate that has been ammonium sulfate precipitated, resuspended in a buffer, and centrifuged to remove insoluble matter. Friedrich's protein solution is different from "a load solution comprising the cell lysate or periplasmic releasate" recited in instant claims. Indeed, as described in the instant application, when a load solution comprising periplasmic releasate was applied to AEX, sufficient purification of the PASylated protein was NOT obtained (see para [0182], Fig. 1, Lane 14), while "pure protein" was obtained with Friedrich's method (cf. Fig. 6A and 6B of Friedrich). 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 even assuming one were to replace the AEX of Friedrich with the HIC of Koehnlein, one would still not have arrived at the presently claimed invention, at least because the first chromatography step of Friedrich was NOT performed with the presently claimed a cell lysate or periplasmic releasate for reasons discussed above, and given the large number of chromatography options and the unpredictability of the biochemistry art, only through innovative experimentation were the inventors able to arrive at the presently claimed invention, especially since the inventors showed that HIC can be reliably used as a first step of chromatography for the efficient purification of PASylated fusion proteins from a cell lysate or periplasmic releasate to at least 45% pure after the first chromatography step (e.g., Examples 4-6).
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 purifying the PASylated asparaginase from periplasmic extract except utilizing HIC as a first chromatography step, and Koehnlein teaches PEGylated proteins are purified from a mixture of various sized fusion proteins (non-PEGylated, mono-PEGylated, and oligo-PEGylated) using HIC as a first chromatography step. Furthermore, regarding Applicant’s arguments about Friedrich precipitating the releasate by ammonium sulfate then subjecting to the AEX, therefore has fewer 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). Similarly, the amended claims now recite the periplasmic releasate was obtained by selectively disrupting the bacterial OM by treatment with PEI, which as evidenced by Alakomi, is a well-understood and routine laboratory chemical for OM disruption, the same as EDTA used in the Friedrich’s method. Furthermore, the argument that the periplasmic releasate in the method taught by Friedrich is a ‘fraction’ of the releasate due to the ammonium sulfate precipitation contrary to the claimed method is not persuasive, as the newly amended claims now recite ‘optionally the periplasmic releasate is filtered and/or dialyzed prior to the HIC’, which is the same treatment in the Friedrich method in [000176]. Therefore, one of ordinary skill in the art would have reasonably arrived at a method for purifying PASylated fusion proteins utilizing HIC as a first chromatography step from periplasmic releasate based on the teachings of Friedrich, Koehnlein, Alakomi, and Brieback, especially in view of the well-understood and routine practice in the art of purifying fusion proteins using conventional laboratory methods.
Conclusion
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.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657
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