DETAILED ACTION
The amendment filed on 06/11/2026 has been entered and fully considered. Claim 2 is canceled. Claims 1 and 3-18 are pending, of which claim 1 is amended, and Claims 17-18 are newly added.
Response to Amendment
In response to amendment, the examiner modifies rejection over the prior art established in the previous Office 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 .
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1, 3-15 and 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ma et al. (Journal of Liquid Chromatography & Related Technologies, 2014) (Ma) in view of Agilent (Control pH During Method Development for Better Chromatography, 2015).
Regarding claim 1, Ma teaches a method for detecting contents and fingerprints of active ingredients in Compound Kushen Injection. Ma teaches that qualitative and quantitative analyses by HPLC and LC-DAD-MS/MS were conducted for quality consistency evaluation of Compound Kushen Injection, and that 27 batches of CKI samples were analyzed by HPLC to evaluate batch-to-batch quality consistency (page 209). Ma further teaches that chromatographic fingerprints were generated for CKI samples (Fig. 2, page 212), and that the chromatographic peaks were identified by comparing retention time, on-line UV spectra, and mass spectra with authentic substances and reported data (Table 1).
Ma teaches performing detection by using high-performance liquid chromatography. Ma teaches that the analyses were performed on an HPLC system equipped with a quaternary pump, diode array detector, autosampler, and column compartment, and that the samples were separated on a C18 column (page 209).
Ma teaches that the conditions for the high-performance liquid chromatography comprise a C18 chromatographic column having a dimension of 5 µm and 4.6 mm × 250 mm, because Ma teaches separating CKI samples on a Phenomenex Luna C18 column (5 µm, 4.6 × 250 mm) (page 209). Although Ma does not expressly teach the particular commercial Waters XSelect CSH™ C18 column, Ma teaches the same C18 stationary-phase type and the same particle size and column dimensions. Waters XSelect CSH™ C18 was a known commercial reversed-phase C18 HPLC column. It would have been obvious to one of ordinary skill in the art to select the recited Waters XSelect CSH™ C18 column as the C18 chromatographic column for Ma’s HPLC method because the claimed column is a known C18 column suitable for the same reversed-phase HPLC purpose, and the selection of a known commercial C18 column of the same dimensions for the same type of HPLC separation would have been a predictable substitution of one known C18 column for another.
Ma teaches the active ingredients comprise matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, sophoridine, and 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid, also referred to as piscidic acid (Fig. 1, Table 1). Ma identifies or characterizes CKI compounds including piscidic acid, macrozamin, oxymatrine, oxysophocarpine, sophoridine, matrine, and sophocarpine (Fig. 1, Table 1).
Ma teaches a mobile phase comprising an organic phase and an aqueous phase and teaches gradient elution (page 210). Ma’s qualitative HPLC method uses water containing 0.01 mol/L ammonium acetate adjusted to pH 8.0 as mobile phase A and acetonitrile-water containing 0.01 mol/L ammonium acetate as mobile phase B, with a linear gradient program (page 210). Ma’s quantitative HPLC method similarly uses an aqueous ammonium acetate phase and an acetonitrile-water ammonium acetate phase with a gradient program (page 210).
Ma does not expressly teach that the mobile phase comprises methanol in an organic phase and 0.1%-0.34% potassium dihydrogen phosphate-methanol gradient elution, with the pH value of potassium dihydrogen phosphate adjusted to 2.9-3.1 with phosphoric acid.
However, Agilent teaches that in reversed-phase liquid chromatography, pH and ionic strength of the aqueous portion of mobile phases are important in developing rugged methods not sensitive to small variations in conditions (page 1). Agilent teaches that, with ionic compounds, retention shows significant changes with pH, and that it is important to control pH in reversed-phase systems to stabilize retention and selectivity (page 1). Agilent further teaches that a pH between 2 and 4 generally provides stable retention conditions and is recommended for starting method development with most samples, including basic compounds and typical weak acids (peg 1). Agilent also teaches that the pH of the mobile phase can affect selectivity, peak shape, and retention, and that ionizable compounds such as acids and bases cause significant changes in retention factor and selectivity with changes in pH (page 1).
Agilent further teaches choosing mobile phase pH to optimize retention and selectivity during method development, that buffering capacity is related to prepared molarity and closeness to the pK of the buffering ion, and that pH adjustments should be made to the aqueous solution before addition of organic modifier (Table 1, page 3). Agilent identifies phosphate as a mobile phase modifier and identifies phosphate pK1 as having an effective pH range including the claimed pH range of 2.9-3.1 (Table 1, page 3).
Therefore, it would have been obvious to one of ordinary skill in the art, when developing or optimizing Ma’s reversed-phase HPLC method for Compound Kushen Injection, to modify Ma’s mobile phase and pH conditions to use an acidic phosphate-buffered aqueous phase adjusted to about pH 3, including potassium dihydrogen phosphate adjusted with phosphoric acid, in order to stabilize retention and selectivity and improve peak shape for the ionizable acidic and basic compounds in CKI. The motivation comes from Agilent’s express teaching that pH and buffer selection are important result-effective variables in reversed-phase HPLC method development.
It further would have been obvious to use methanol as the organic phase in place of, or as an alternative to, acetonitrile because methanol and acetonitrile are both conventional organic modifiers used in reversed-phase HPLC method development. Selection between known organic modifiers would have been a routine optimization of the mobile phase to obtain workable separation of CKI analytes.
Regarding the claimed gradient elution table, Ma teaches gradient elution for CKI HPLC analysis (page 210). Agilent teaches that method development involves modifying chromatographic conditions to improve separation and resolution, including modification of gradients depending on where analytes elute (page 7). It would have been obvious to one of ordinary skill in the art to optimize Ma’s gradient program when changing the mobile phase to methanol/phosphate buffer at acidic pH, because gradient profile is a known result-effective variable affecting retention time, peak separation, and resolution in reversed-phase HPLC. The claimed gradient, which varies methanol from 3% to 85% and then returns to 3%, represents an optimized gradient within ordinary reversed-phase HPLC method development for separating multiple CKI constituents.
Ma teaches a column temperature of 35°C (page 210). The claimed column temperature is 28-32°C. It would have been obvious to optimize Ma’s column temperature from 35°C to about 30°C because column temperature is a known HPLC parameter affecting retention, selectivity, and peak shape, and 30°C is a conventional operating temperature close to Ma’s 35°C condition.
Ma teaches a flow rate of 0.8 mL/min (page 210). The claimed flow rate is 0.58-0.62 mL/min. It would have been obvious to optimize Ma’s flow rate to about 0.6 mL/min because flow rate is a known HPLC parameter affecting retention time, resolution, and column pressure. When modifying the mobile phase, pH, and gradient conditions, one of ordinary skill in the art would have had reason to adjust the flow rate to obtain desired separation and acceptable run conditions.
Ma teaches that the DAD detector recorded UV spectra in the range from 190 nm to 400 nm and that the HPLC chromatogram was monitored at 225 nm (page 210). The claimed detection wavelength is 209-213 nm. It would have been obvious to select a detection wavelength within Ma’s taught DAD scanning range of 190-400 nm, including about 211 nm, because UV detection wavelength is routinely selected based on the absorption characteristics of the analytes being detected, and Ma teaches DAD detection over a range that includes the claimed wavelength.
Ma teaches an injection volume of 10 µL (page 211). The claimed injection amount is 3-20 µL. Therefore, Ma teaches an injection amount within the claimed range.
Accordingly, Ma teaches HPLC detection of contents and fingerprints of active ingredients in Compound Kushen Injection using a C18 column, gradient elution, DAD detection, sample injection, chromatographic fingerprinting, and identification/determination of CKI constituents. Agilent teaches that pH, buffer selection, ionic strength, mobile phase conditions, and related chromatographic parameters are result-effective variables in reversed-phase HPLC method development, and specifically teaches low-pH conditions useful for stabilizing retention and selectivity of ionizable analytes. It would have been obvious to one of ordinary skill in the art to modify and optimize Ma’s CKI HPLC method to use the claimed acidic methanol/potassium dihydrogen phosphate gradient conditions and associated HPLC parameters with a reasonable expectation of success in obtaining a workable CKI separation method.
Regarding claim 3, Ma teaches that the method further comprises a mobile phase comprising an organic phase and a gradient elution in the aqueous phase (page 209, par 3). Methanol is commonly used in organic phase and phosphate is commonly used in aqueous phase for HPLC C18 column elution.
Regarding claim 4, Ma does not explicitly teach that pH value of potassium dihydrogen phosphate is adjusted to 2.9-3.1, more preferably to 3.0, with phosphoric acid.
However, pH of the mobile phase affects the elution of the compounds in C18 HPLC. Agilent teaches that “In reversed-phase liquid chromatography, pH and ionic strength of the aqueous portion of mobile phases are important in developing rugged methods not sensitive to small variations in conditions. With ionic compounds, retention of typical species shows significant changes with pH. It is very important to control pH in reversed phase systems to stabilize retention and selectivity. A pH between 2 and 4 generally provides the most stable conditions for retention versus small changes in pH, and this pH range is recommended for starting method development with most samples, including basic compounds and typical weak acids” (Introduction). The active ingredients as recited in claim 1 are weak basic compounds and weak acids.
Thus, it would have been obvious to one of ordinary skill in the art to adjust the pH value of the mobile phase to 3.0, in order to provides the most stable conditions for retention versus small changes in pH.
Regarding claim 5, as has been discussed regarding claim 4, it would have been obvious to one of ordinary skill in the art to adjust the pH value of the mobile phase to 3.0, in order to provides the most stable conditions for retention versus small changes in pH.
it would also have been obvious to one of ordinary skill in the art to optimize the gradient of the elution by routine experimentation.
Regarding claim 6, it would have been obvious to one of ordinary skill in the art to optimize the column temperature (from 35 to 30 °C) by routine experimentation.
Regarding claim 7, it would have been obvious to one of ordinary skill in the art to optimize the flow rate (from 0.8 to 0.6 ml/min) routine experimentation.
Regarding claim 8, it would have been obvious to one of ordinary skill in the art to optimize the detection wavelength (from 225 to 211 nm) by routine experimentation.
Regarding claim 9, Ma teaches wherein conditions for the high performance liquid chromatography in the method comprise an injection amount of 3-20 μl, preferred 5-15 μl, more preferred 8-12 μl, and most preferably 10 μl (page 211, par 2).
Regarding claim 10, it is a common practice in the art to prepare blank solution similar to the elution condition.
Regarding claim 11, it is a common practice in the art to prepare reference substance solutions separately or semi separately. Ma also teaches similar way of preparing reference substance solutions separately or semi separately (page 210-211)
Regarding claim 12, it is a routine to prepare the test substance solution by accurately weighing the test substance solution, adding a blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution. Ma teaches similar way of preparing test substance solution (page 211).
Regarding claim 13 and 15, as has been discussed regarding claims 3-12 above, it would have been obvious to one of ordinary skill in the art to adjust the pH value of the mobile phase to 3.0, in order to provides the most stable conditions for retention versus small changes in pH.
It would also have been obvious to one of ordinary skill in the art to optimize the HPLC conditions, such as column, mobile phase, flow rate, column temperature, gradient, reference substance solution, etc. by routine experimentation.
It is a common practice the art:
(4) Injecting samples in the order of the blank solution, reference substance solution, and the test substance solution, and
(5) Detection: injecting samples in the order of the blank solution, the reference substance solution, and the test substance solution to perform detection, and calculating the content using an external standard method.
Ma teaches construct a fingerprint of the Compound Kushen Injection containing matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, and sophoridine (page 213, Fig. 2).
Regarding claim 14, Ma teaches that wherein the method comprises constructing a fingerprint of the Compound Kushen Injection containing matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, and sophoridine (page 213, Fig. 2).
Regarding claim 17, Ma teaches that wherein conditions for the high performance liquid chromatography in the method comprise an injection amount of 8-12 μl (page 211).
Regarding claim 18, Ma teaches that wherein conditions for the high performance liquid chromatography in the method comprise an injection amount of 10 μl (page 211).
Allowable Subject Matter
Claim 16 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter: The prior art of record does not teach or fairly suggest that wherein the fingerprint in step (4) has 10 common characteristic peaks, wherein, based on peak 7-oxymatrine as a reference, the relative retention time of peak 1-sophoramine is 0.442; the relative retention time of peak 2-macrozamin is 0.603; the relative retention time of peak 3-matrine is 0.693; the relative retention time of peak 4-sophocarpine is 0.816; the relative retention time of peak 5-sophoridine is 0.845; the relative retention time of peak 6-oxysophocarpine is 0.941; the relative retention time of peak 7-oxymatrine is 1.0; the relative retention time of peak 8-2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl ]butanedioic acid is 1.149; the relative retention time of peak 9 is 1. 639; and the relative retention time of peak 10-trifolirhizin is 1.888.
Response to Arguments
Applicant's arguments filed 06/11/2026 have been fully considered and are persuasive in part.
Applicant argues that Ma does not anticipate amended claim 1 because Ma does not disclose each and every element of amended claim 1, including the Waters XSelect CSH™ C18 column, methanol/potassium dihydrogen phosphate mobile phase, pH of 2.9-3.1, the specific gradient program, column temperature, flow rate, detection wavelength, and injection amount. The Examiner acknowledges that, in view of the amendments to claim 1, Ma no longer discloses every limitation of claim 1 as required for anticipation. Accordingly, the rejection of claim 1 under 35 U.S.C. 102(a)(1) over Ma is withdrawn.
However, Applicant's arguments are not persuasive with respect to the rejection of claims 1, 3-15, 17, and 18 under 35 U.S.C. 103 over Ma in view of Agilent. Ma remains directed to the same subject matter as the claimed invention, namely HPLC analysis of Compound Kushen Injection for quality consistency evaluation, including chromatographic fingerprint analysis and identification/determination of CKI constituents. Ma teaches analyzing 27 batches of CKI samples by HPLC, generating chromatographic fingerprints, identifying characteristic peaks, and identifying or characterizing CKI constituents including piscidic acid, macrozamin, oxymatrine, oxysophocarpine, sophoridine, matrine, sophocarpine, and trifolirhizin. Ma also teaches a C18 chromatographic column having the same 5 µm, 4.6 mm × 250 mm dimensions, gradient elution, DAD detection, sample preparation, reference solution preparation, filtration, injection, and chromatogram recording.
Applicant argues that Ma uses a Phenomenex Luna C18 column rather than the claimed Waters XSelect CSH™ C18 column. This argument is not persuasive because Ma teaches use of a C18 column of the same particle size and dimensions for the same purpose, namely reversed-phase HPLC analysis of CKI. The claimed Waters XSelect CSH™ C18 column is a known commercial C18 column used for reversed-phase HPLC. Substitution of one known C18 column for another known C18 column for the same purpose would have been an obvious selection of a known equivalent column absent evidence of unexpected results attributable specifically to that commercial column.
Applicant further argues that Ma uses acetonitrile/ammonium acetate at pH 8.0 rather than methanol/potassium dihydrogen phosphate adjusted to pH 2.9-3.1 with phosphoric acid. This argument is not persuasive because Agilent teaches that, in reversed-phase liquid chromatography, pH and ionic strength of the aqueous portion of the mobile phase are important for developing rugged methods, and that pH affects retention, selectivity, and peak shape. Agilent further teaches that ionizable compounds such as acids and bases can significantly change retention and selectivity with changes in pH, and that a pH between 2 and 4 generally provides stable retention conditions and is recommended for starting method development with most samples, including basic compounds and weak acids. Agilent also identifies phosphate as a common mobile phase modifier with an effective pH range that includes the claimed pH range.
Accordingly, one of ordinary skill in the art seeking to develop or improve Ma's reversed-phase HPLC method for CKI would have had reason to adjust the mobile phase pH and buffer system, including use of acidic phosphate-buffered conditions, in order to improve or stabilize retention, selectivity, and peak shape of the ionizable CKI constituents. The reason to modify Ma is not based on hindsight, but on Agilent's express teaching that pH and buffer selection are result-effective variables in reversed-phase HPLC method development.
Applicant also argues that Ma is already a complete working method and therefore provides no reason to modify its conditions. This argument is not persuasive. A prior art method need not be defective before one of ordinary skill in the art would have reason to improve or optimize it. Ma is concerned with quality consistency evaluation of CKI, and one of ordinary skill in the art would have been motivated to optimize Ma's HPLC conditions, including column selection, mobile phase, pH, gradient, column temperature, flow rate, detection wavelength, and injection volume, to improve separation and detection of known CKI constituents.
Applicant further argues that the claimed parameters are not the result of routine optimization and cites In re Stepan. The Examiner respectfully disagrees. The present rejection identifies why the variables would have been optimized: Agilent expressly teaches that pH, ionic strength, buffer selection, and mobile phase conditions affect retention, selectivity, and peak shape in reversed-phase HPLC. These are result-effective variables. Ma already provides the CKI sample context and a working reversed-phase C18 HPLC method. Therefore, optimizing the HPLC conditions to arrive at workable or improved separation conditions would have been within the level of ordinary skill in the art and would have had a reasonable expectation of success.
Applicant's assertion of unexpected results is also not persuasive. Applicant has not provided persuasive comparative evidence showing unexpected results over the closest prior art under comparable testing conditions. The specification's discussion of testing different columns, mobile phases, pH values, phosphate concentrations, and gradient programs is consistent with ordinary HPLC method development and optimization. In addition, the claims do not recite a particular resolution value, peak symmetry value, limit of detection, accuracy, precision, or other performance threshold. Therefore, the alleged superior separation efficiency, higher peak resolution, or optimal elution program is not commensurate in scope with the claims.
With respect to claim 16, Applicant's arguments are persuasive. Claim 16 recites a fingerprint having 10 common characteristic peaks with specific relative retention times based on peak 7-oxymatrine as a reference. The Examiner acknowledges that Ma does not expressly teach the claimed 10-peak fingerprint with the specific relative retention times recited in claim 16. Accordingly, the prior rejection of claim 16 is withdrawn. Claim 16 is objected to as being dependent upon rejected claim 15, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to XIAOYUN R XU, Ph. D. whose telephone number is (571)270-5560. The examiner can normally be reached M-F 8am-5pm.
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797
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