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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 December 16, 2025 has been entered.
DETAILED ACTION
Claim 7 is cancelled. Claims 1-6 and 8-20 are under examination.
Priority
This application is a 371 of PCT/US21/23100 filed on March 19, 2021, which claims priority to U.S. Provisional Application 62/992,540 filed on March 20, 2020. A certified copy of this document was filed on September 9, 2022.
Therefore, the effective filing date of the invention is March 20, 2020.
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.
Claims 1-5 and 8-20 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 regards as the invention.
Claims 1 and 20 recite one or more sites within the G-CSF polypeptide and only once between N-terminal methionine at position one “and Lysine at position 16” in claim 1, line 6 and in claim 20, line 5. The specification identifies the sequence of the N-terminal 17 residues of filgrastim as MTPLGPASSLPQSFLLK (p.10, 2nd paragraph), which contains leucine in positions 15 and 16 and lysine in position 17. It is unclear what the limitation “lysine at position 16” is referring to, because position 16 contains a leucine and lysine is in position 17.
Claims 2-5 and 8-19 are rejected for depending from rejected claim 1 but failing to remedy the indefiniteness therein.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-6 and 10-19 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, at the time the application was filed, had possession of the claimed invention.
Applicant is referred to MPEP 2163(II)(A)(3)(a)(i and ii), which states that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure indicates that the patentee has invented species sufficient to constitute the genus. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.
Claims 1-6 and 10-19 are drawn to a method of measuring the amount of unmodified N-terminus of a human granulocyte colony-stimulating factor (G-CSF) peptide, comprising: “(a) incubating a sampling comprising the G-CSF polypeptide with pepsin under conditions that allow cleavage at one or more sites within the G-CSF polypeptide and only once between N-terminal Methionine at position 1 and Lysine at position 16; separating the cleavage products generated in step (a); and (c) measuring the amount of unmodified, free N-terminus of the G-CSF polypeptide by comparing to a control standard comprising a mixture of N-terminally modified G-CSF and polypeptides and unmodified G-CSF polypeptides”. The specification does not disclose a representative number of conditions that allow cleavage “only once between N-terminal methionine at position 1 and Lysine at position 16”.
The current specification identifies that pepsin is a non-specific protease, and it can potentially cut at various sites between the two residues (N-terminal methionine and lys-17) (Specification p.10, 3rd paragraph). The current specification describes a single example of pepsin digestion of filgrastim at pH 2.2, followed by incubation at 50°C for 5 minutes or 37°C for 15 minutes (Specification p.13, B. Pepsin Digestion of Filgrastim).
The specification does not provide any guidance on the effect of longer digestion times, different temperatures, or other pH values which would provide the desired cleavage only once between N-terminal methionine at position 1 and Lysine at position 16. The specification does not suggest any relationship between pH, temperature and incubation time that would produce the desired cleavage, so it is highly unpredictable as to what other combinations of pH, temperature and incubation time would provide the instantly recited peptide cleavage product.
The disclosure of a single condition of pH, temperature and incubation time that produces the desired result of cleaving the G-CSF polypeptide only once between N-terminal methionine at position 1 and lysine at position 16 is not considered to constitute a representative number of species of the genus of conditions of pH, temperature and incubation time to achieve pepsin digestion, in view of the lack of guidance of what other conditions may achieve the desired result of a single cleavage between Met-1 and Lys-16. Thus, one of ordinary skill in the art could not conclude that Applicant was in possession of the conditions required to obtain pepsin digestion. This disclosure does not constitute a representative number of species of the genus in view of the potential breadth and variability in the genus, and so there is a failure to satisfy the written description requirement for the genus.
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, 8-18 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Tsuchida et al. (“Characterization of Stress-Exposed Granulocyte Colony Stimulating Factor Using ELISA and Hydrogen/Deuterium Exchange Mass Spectrometry”, Journal of The American Society for Mass Spectrometry, 2014, Vol. 25, No.10, pp.1747-1754; previously cited) in view of Kinstler et al. (US 5,824,784 issued October 20, 1998).
Regarding claims 1 and 10, Tsuchida teaches granulocyte colony-stimulating factor (G-CSF) (Filgrastim) (p.1748, 1st column last paragraph under Sample Preparation). Tsuchida teaches samples were loaded onto an online pepsin-digestion column (immobilized pepsin column), in which the digestion and peptide trapping were performed, and the digested peptides were then introduced to a UPLC column and separated with a gradient elution of solvent A and solvent B (p.1749, 1st column – H/D Exchange Mass Spectrometry (HDX-MS)). Tsuchida teaches that G-CSF has a leucine residue at position 15 and position 16 (Figure 4). Tsuchida teaches the samples were loaded onto an online pepsin-digestion column, in which the digestion and peptide trapping were performed for 4 min at 20°C at pH 2.5 (p.1749, 1st column – 2nd paragraph.
Tsuchida does not teach conditions that make pepsin cleave G-CSF only once between N-terminal methionine at position 1 and leucine at position 16.
However, the instant specification identifies incubation conditions of pH 2.2, a temperature of 37°C or 50°C, for a time of 5 minutes or 15 minutes (specification p.13, B. Pepsin Digestion of Filgrastim). Thus, Tsuchida’s method of digesting G-CSF with pepsin would necessarily result in the production of cleaving G-CSF only once between N-terminal methionine at position 1 and lysine at position 16.
Tsuchida does not teach measuring the amount of unmodified free N-terminus G-CSF polypeptide by comparing to a control standard comprising a mixture of N-terminally modified G-CSF polypeptides and unmodified G-CSF polypeptides.
However, Kinstler teaches methods and compositions relating to the attachment of water-soluble polymers to proteins, including novel N-terminally chemically modified G-CSF compositions and related methods of preparation (Abstract). Kinstler teaches using SEC-HPLC to identify N-terminally monopegylated rh-G-CSF, as well as unmodified G-CSF to determine the percent composition of N-terminally pegylated G-CSF (Column 15, Table 2; FIG. 2). Kinstler teaches a sample containing a peak eluting as monopegylated G-CSF and a minor peak of unreacted G-CSF (Col.19, lines 5-7, GIF. 7). Kinstler further teaches determining peak area relative to standard reference maps (Col. 16, lines 49-53).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add a step of quantifying the percentage of free N-terminal methionine by comparing the peak area of the N-terminal peptide M1-L15 in the sample to the peak area of the N-terminal peptide M1-L15 in the control standard obtained from Tsuchida, because Kinstler teaches samples containing mono-PEG-GCSF and unmodified GCSF using SEC-HPLC and measuring peak height and determining peak area to obtain percent composition of N-terminally pegylated G-CSF. One of ordinary skill in the art would reasonably expect that using a known method of comparing peak areas for samples and control standards obtained using HPLC would predictably result in quantifying the percentage of free N-terminal methionine in a sample, because Kinstler teaches quantifying the percent of unmodified G-CSF and pegylated G-CSF in a sample.
Regarding claim 8, Tsuchida teaches the samples were loaded onto an online pepsin-digestion column, in which the digestion and peptide trapping were performed for 4 min at 20°C at pH 2.5 (p.1749, 1st column – 2nd paragraph).
Regarding claim 9, since neither the instant claims nor the instant specification provide a limiting definition of “about”, the examiner is interpreting the conditions of 4min, 20°C and pH 2.5 as falling within the scope of “about” 15min; “about” 37°C, and “about” pH 2.2.
Regarding claims 11-13, Tsuchida teaches peptide mapping with HPLC-UV detection, and digested samples were separated with a reversed-phase column on a UPLC system (relevant to claim 11: separation step is selected from chromatography; relevant to claim 12: chromatography is selected from ultra-high performance liquid chromatography (UPLC); relevant to claim 13: the HPLC is reverse-phase HPLC) (p.1748, 2nd column – Peptide mapping).
Regarding claim 14, Tsuchida teaches digested samples were separated with a reversed-phase column by a gradient elution of solvent A [0.1% trifluoroacetic acid] and solvent B [90% acetonitrile/10% water / 0.1% trifluoroacetic acid] on a UPLC system (p.1748, 2nd column – peptide mapping), which falls within the claimed range of trifluoroacetic acid of the invention.
Regarding claims 15-16, since neither the instant claims nor the instant specification provide a limiting definition of “about”, the examiner is interpreting 0.1% v/v as falling within the scope of “about”. Tsuchida teaches digested samples were separated with a reversed-phase column by a gradient elution of solvent A [0.1% trifluoroacetic acid] and solvent B [90% acetonitrile/10% water / 0.1% trifluoroacetic acid] on a UPLC system (p.1748, 2nd column – Peptide mapping).
Regarding claims 17-18, Tsuchida teaches mass information was collected using a quadrupole time-of-flight mass spectrometer (SYNAPT G2 HDMS; Waters) in the MSE mode (p.1748, 2nd column – Peptide mapping).
Regarding claim 20, Tsuchida teaches granulocyte colony-stimulating factor (G-CSF) (Filgrastim) (p.1748, 1st column last paragraph). Tsuchida teaches samples were loaded onto an online pepsin-digestion column (immobilized pepsin column), in which the digestion and peptide trapping were performed, and the digested peptides were then introduced to a UPLC column and separated with a gradient elution of solvent A and solvent B (p.1749, 1st column – H/D Exchange Mass Spectrometry (HDX-MS)). Tsuchida teaches that G-CSF has a leucine residue at position 15 and position 16. Tsuchida teaches the samples were loaded onto an online pepsin-digestion column, in which the digestion and peptide trapping were performed for 4 min at 20°C at pH 2.5 (p.1749, 1st column – 2nd paragraph).
Since neither the instant claims nor the instant specification provide a limiting definition of “about”, the examiner is interpreting the conditions of 4min, 20°C and pH 2.5 as falling within the scope of “about” 15min; “about” 37°C, and “about” pH 2.2.
Tsuchida does not teach conditions that make pepsin cleave G-CSF only once between N-terminal methionine at position 1 and leucine at position 16.
However, the instant specification identifies incubation conditions of pH 2.2, a temperature of 37°C or 50°C, for a time of 5 minutes or 15 minutes (specification p.13, B. Pepsin Digestion of Filgrastim). Thus, Tsuchida’s method of digesting G-CSF with pepsin would necessarily result in the production of cleaving G-CSF only once between N-terminal methionine at position 1 and lysine at position 16.
Tsuchida does not teach measuring the amount of unmodified free N-terminus G-CSF polypeptide by comparing to a control standard comprising a mixture of N-terminally modified G-CSF polypeptides and unmodified G-CSF polypeptides.
However, Kinstler teaches methods and compositions relating to the attachment of water-soluble polymers to proteins, including novel N-terminally chemically modified G-CSF compositions and related methods of preparation (Abstract). Kinstler teaches using SEC-HPLC to identify N-terminally monopegylated rh-G-CSF, as well as unmodified G-CSF to determine the percent composition of N-terminally pegylated G-CSF (Column 15, Table 2; FIG. 2). Kinstler teaches a sample containing a peak eluting as monopegylated G-CSF and a minor peak of unreacted G-CSF (Col.19, lines 5-7, GIF. 7). Kinstler further teaches determining peak area relative to standard reference maps (Col. 16, lines 49-53).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Tsuchida by adding a step to quantify the percentage of free N-terminal methionine by comparing the peak area of the N-terminal peptide M1-L15 in the sample to the peak area of the N-terminal peptide M1-L15 in the control standard obtained from Tsuchida, because Kinstler teaches samples containing mono-PEG-GCSF and unmodified GCSF using SEC-HPLC and measuring peak height and determining peak area to obtain percent composition of N-terminally pegylated G-CSF. One of ordinary skill in the art would reasonably expect that using a known method of comparing peak areas for samples and control standards obtained using HPLC would predictably result in quantifying the percentage of free N-terminal methionine in a sample, because Kinstler teaches quantifying the percent of unmodified G-CSF and pegylated G-CSF in a sample.
Claims 2-5 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Tsuchida et al. (“Characterization of Stress-Exposed Granulocyte Colony Stimulating Factor Using ELISA and Hydrogen/Deuterium Exchange Mass Spectrometry”, Journal of The American Society for Mass Spectrometry, 2014, Vol. 25, No.10, pp.1747-1754; previously cited) in view of Kinstler et al. (US 5,824,784 issued October 20, 1998) as applied to claim 1 above, and further in view of Scaramuzza et al. (“A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization”, Journal of Controlled Release, 2012, Vol. 164, Issue 3, pp.355-363; previously cited).
Regarding claim 2, Tsuchida and Kinstler do not teach a recombinant form of filgrastim.
However, Scaramuzza teaches a recombinant form of filgrastim (methionyl human granulocyte colony stimulating factor (rh-Met-G-CSF), BK0026 (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the filgrastim taught by Tsuchida with the recombinant filgrastim taught by Scaramuzza, because it would amount to a simple substitution of one known filgrastim for another, and it was known in the art at the time of invention that recombinant filgrastim could be digested into fragments using enzymes.
Regarding claim 3, Tsuchida teaches samples containing unmodified G-CSF.
Tsuchida and Kinstler do not teach modified G-CSF samples or PEG-modified G-CSF polypeptide.
However, Scaramuzza teaches samples containing PEGylated filgrastim using PEG20kDa-NH2 (p.356, 2nd column, 2.3. Filgrastim PEGylation).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add modified G-CSF taught by Scaramuzza to the unmodified G-CSF taught by Tsuchida. Each of Tsuchida and Scaramuzza teach using enzyme cleavage of G-CSF to obtain cleavage products. One of ordinary skill in the art would reasonably expect that combining unmodified G-CSF with modified G-CSF would predictably result in a sample mixture that could be incubated with a protease to allow cleavage of the G-CSF polypeptide, because it would amount to a combination of known elements together, and it was known in the art at the time of invention that both modified G-CSF and unmodified G-CSF could be cleaved to produce cleavage peptides detectable by chromatography and mass spectrometry.
Regarding claims 4-5, Tsuchida and Kinstler do not teach pegfilgrastim.
Scaramuzza teaches a monoPEGylated derivative of a recombinant form of filgrastim (also known as methionyl human granulocyte colony stimulating factor (rh-Met-G-CSF) (i.e. pegfilgrastim), BK0026 (abstract). Scaramuzza teaches NH2-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim (abstract). Scaramuzza teaches the new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace unmodified filgrastim taught by Tsuchida with pegfilgrastim taught by Scaramuzza, because Scaramuzza teaches that pegylated filgrastim and non-PEGylated filgrastim demonstrated no differences in primary structure or peptide mapping. One of ordinary skill in the art would have been motivated to use a monoPEGylated filgrastim because Scaramuzza teaches that monoPEGylated filgrastim is a promising candidate for developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.
Regarding claim 19, Tsuchida teaches using non-stressed proteins along with the degraded proteins as samples (p.1748, 1st column Sample Preparation).
Tsuchida and Kinstler do not teach wherein the control standard comprises a known amount of modified G-CSF polypeptide and a known amount of unmodified G-CSF polypeptide.
Scaramuzza teaches modified G-CSF and non-PEGylated filgrastim (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine control samples containing modified G-CSF taught by Scaramuzza with control samples containing unmodified G-CSF taught by Tsuchida to create a mixed control sample. Each of Tsuchida and Scaramuzza teach cleavage of G-CSF to obtain cleavage products. One of ordinary skill in the art would reasonably expect that combining modified G-CSF with unmodified G-CSF would predictably result in a control sample mixture that could be used as a mixed control sample, because it would amount to a combination of known elements together, and it was known in the art at the time of invention that both modified G-CSF and unmodified G-CSF could be used as controls detectable by chromatography and mass spectrometry.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Tsuchida et al. (“Characterization of Stress-Exposed Granulocyte Colony Stimulating Factor Using ELISA and Hydrogen/Deuterium Exchange Mass Spectrometry”, Journal of The American Society for Mass Spectrometry, 2014, Vol. 25, No.10, pp.1747-1754; previously cited) in view of Kinstler et al. (US 5,824,784 issued October 20, 1998) as applied to claim 1 above, and further in view of Monogioudi et al. (“Effect of enzymatic cross-linking of β-casein on proteolysis by pepsin”, Food Hydrocolloids, 2011, Vol. 25, Issue 1, pp.71-81; previously cited).
The teachings of Tsuchida et al. and Kinstler et al. are discussed above.
Regarding claim 6, Tsuchida teaches granulocyte colony-stimulating factor (G-CSF) (Filgrastim) (p.1748, 1st column last paragraph). Tsuchida teaches samples were loaded onto an online pepsin-digestion column (immobilized pepsin column), in which the digestion and peptide trapping were performed, and the digested peptides were then introduced to a UPLC column and separated with a gradient elution of solvent A and solvent B (p.1749, 1st column – H/D Exchange Mass Spectrometry (HDX-MS)). Tsuchida teaches that G-CSF has a leucine residue at position 15 and position 16.
Tsuchida and Kinstler do not disclose whether the pepsin cleaves G-CSF between leucine at position 15 and leucine at position 16.
However, Monogioudi teaches β-casein proteolysis using pepsin (abstract). Monogioudi teaches the optimum pH for pepsins of various origins varies between 2 and 7 (p.72, 1st column 2nd paragraph). Monogioudi further teaches that the cleavage sites include mainly phenylalanine and leucine (p.72, 1st column 2nd paragraph). Monogioudi teaches pepsin from porcine gastric mucosa (product: P6887, activity: 3200 U/mg) was obtained from Sigma-Aldrich (p.72, 2nd column – 2.2. Enzymes), which is identical to the pepsin disclosed in the specification (Specification p.14, Table 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention for pepsin to cleave the G-CSF taught by Tsuchida between leucine at position 15 and leucine at position 16 to produce a fragment of 15 amino acids in length, because Monogioudi teaches that pepsin splits bonds involving leucine most readily. One of ordinary skill in the art would reasonably expect that exposing G-CSF to pepsin would predictably result in the pepsin cleaving between the leucine at position 15 and leucine at position 16 to obtain a peptide of 15 amino acids, because it was known in the art at the time of invention that pepsin readily splits bonds involving leucine, and Monogioudi teaches the same pepsin P6887 product from Sigma used by applicant. Thus, the pepsin taught by Monogioudi would necessarily possess the property of cleaving G-CSF only once between Leucine 15 and Leucine 16, because it is an inherent property of the pepsin, and as per MPEP §2112.01: “Products of identical chemical composition cannot have mutually exclusive properties.” In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Response to Arguments
Applicant summarizes the teachings of Scaramuzza and argues that Scaramuzza’s BK0026 is PEGylated at glutamine 135 and not at the N-terminus, and Scaramuzza does not teach or suggest quantifying the percentage of free N-terminal methionine by comparing peak areas of the N-terminal peptide M1-L15 between a sample and a control standard (See Remarks dated 12/16/2025, p.6 3rd paragraph).
Applicant argues that Tsuchida does not teach or suggest using pepsin digestion to measure the amount of unmodified N-terminus of a G-CSF polypeptide, nor does Tsuchida teach or suggest quantifying the percentage of N-terminal methionine by comparing peak areas of the N-terminal peptide M1-L15 between a sample and a control standard (See Remarks dated 12/16/2025, p.6 last paragraph to top of p.7).
Applicant argues that Monogioudi teaches pepsin digestion of β-casein not G-CSF, and provides no teaching or suggestion regarding measuring unmodified N-terminus of G-CSF polypeptides, let alone quantifying the percentage of free N-terminal methionine by comparing peak areas of the N-terminal peptide M1-L15 (See Remarks dated 12/16/2025, p.7 1st paragraph).
Applicant argues that the prior art of record does not teach or suggest the newly added limitation of “quantifying the percentage of the free N-terminal methionine by comparing the peak area of the N-terminal peptide M1-L15 in the sample to the peak area of the N-terminal peptide M1-L15 in the control standard”; and further that Scaramuzza’s peptide mapping produces a fragment from residue numbers 1-12, not the M1-L15 peptide required by the claimed method (See Remarks dated 12/16/2025, p.7 2nd paragraph).
Regarding claims 8-9, applicant argues that for the reasons set forth above regarding claim 1, the Office has failed to establish a prima facie case of obviousness, and the addition of Hornbuckle does not cure the deficiencies in the rejection of claim 1 (See Remarks dated 12/16/2025, p.8, 1st paragraph). Applicant argues that Hornbuckle does not teach the use of a control standard comprising a mixture of N-terminally modified and unmodified G-CSF polypeptides, nor does it suggest the specific peak area comparison method now recited in claim 1 (See Remarks dated 12/16/2025, p.8, 1st paragraph).
Regarding claim 20, Applicant argues that this claim now also recites a method of measuring the amount of unPEGylated, free N-terminus of Pegfilgrastim, and for the same reasons discussed above for claim 1, none of the cited references teach or suggest the specific quantification method recited in claim 20 (See Remarks dated 12/16/2025, p.8 last 2 paragraphs).
Applicant argues none of the cited references teach or suggest a control standard comprising a mixture of N-terminally modified G-CSF polypeptides and unmodified G-CSF polypeptides as required (See Remarks dated 12/16/2025, p.7 last paragraph).
Applicant's arguments filed December 16, 2025 have been fully considered but they are not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant’s arguments regarding the last of teaching of the newly added limitation is directed to a feature that was not present in the previously rejected claims. The new limitation is addressed in the rejection above through the teachings of Kinstler et al.
Each of Tsuchida, Scaramuzza and Monogioudi teach the digestion of peptides using proteases. As discussed in the rejection above, Tsuchida teaches G-CSF, digestion of G-CSF using pepsin, separating the digested peptides using UPLC and that G-CSF contains Leucine at position 15 and position 16. Scaramuzza teaches a monoPEGylated derivative of filgrastim, which has an unmodified free N-terminus. Scaramuzza teaches comparing the digested samples against controls, including non-digested G-CSF and PEG solutions. Therefore, Scaramuzza teaches measuring the amount of unmodified N-terminus G-CSF. Monogioudi teaches proteolysis using pepsin, and that the cleavage sites include mainly phenylalanine and leucine. As Tsuchida teaches samples were loaded onto an online pepsin-digestion column, in which the digestion and peptide trapping were performed for 4 min at 20°C at pH 2.5, Tsuchida’s method of digesting G-CSF with pepsin would result in the production of cleaving G-CSF only once between N-terminal methionine at position 1 and Leucine at position 16.
Regarding the control standard, one of ordinary skill in the art would be expected to select control samples that would serve as accurate reference samples for the experimental samples being tested. As Tsuchida teaches running control samples comprising undigested peptide, and Kinstler teaches mixed samples containing pegylated and unpegylated G-CSF, one of ordinary skill in the art would know to select control standards comprising unmodified and modified G-CSF polypeptides as references for determining the amount of unmodified N-terminus G-CSF, because it was known in the art at the time of invention that control standards serve as references for quantifying modifications to G-CSF polypeptides.
Tsuchida teaches that G-CSF comprises a leucine at position 15 and position 16. Tsuchida also teaches pepsin digestion at for 4 min at 20°C at pH 2.5. Thus, one of ordinary skill in the art would reasonably expect that a non-specific pepsin would cleave G-CSF between the two leucines at position 15 and position 16 to generate an M1-L15 fragment.
Regarding claims 8-9, applicant’s arguments are not found persuasive for the same reasons discussed for claim 1 above.
Regarding claim 20, applicant’s arguments are not found persuasive for the same reasons discussed for claim 1 above.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DEEPA MISHRA whose telephone number is (571) 272-6464. The examiner can normally be reached Monday - Friday 9:30am - 3:30pm EST.
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/DEEPA MISHRA/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657