DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Amendment
The Amendment filed 10/23/2025 has been entered. Claims 1-3, 5-14 remain pending in the application. Claims 1, 2, 3, 5, and 8 have been amended, and claim 4 has been cancelled. Claims 10-14 are newly added.
Status of Objections and Rejections
The rejection of claims 1-3, 5-9 under 35 USC 112(b) are withdrawn in view of Applicant's amendment.
The rejection of claim 4 under 35 USC 112(b) and 35 U.S.C. 103 is obviated by Applicant's cancellation.
All rejections from the previous office action under 35 U.S.C. 103 are maintained.
New grounds of objection are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 112(b) are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 103 are necessitated by the amendments.
Response to Arguments
Applicant's arguments, see pages 5-8, filed 10/23/2025, with respect to the rejection of claims 1-9, under 35 U.S.C. 103, have been fully considered but they are not persuasive.
Applicant argues (pp. 5-6, #1) that amended claim 1, which recites sonication of the sample using a low power range, is not taught or suggested by Bao in view of Weng. Applicant argues that Bao merely discloses dissolving relatively simple hydrogels by shaking and does not recognize difficulties associated with dispersing more complex, crosslinked HA hydrogels. According to Applicant, neither Bao nor Weng identifies the technical problem of achieving homogenous dispersion without degrading the analyte, nor do they suggest using controlled, low-power ultrasound. Applicant therefore asserts that the prior art lacks both motivation to combine and any teaching of the claimed specific power range.
The Examiner respectfully disagrees.
Although Bao does not disclose sonication, Bao does provide sample preparation to cross-linked HA hydrogels (Title) contrary to Applicant’s assertion that Bao only dissolves simple hydrogels. Weng remedies this deficiency by applying ultrasound to hyaluronic acid gel systems, demonstrating that ultrasound power is an adjustable operational parameter (See wider range of ultrasonic power between 1000-3000W in claim 9). This establishes that HA hydrogels are known to be structurally affected by controlled ultrasound treatment. A supporting reference, Oueslati et al. (“CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form”; 2014) confirms that cross-linked HA forms aggregates in solution and “has to be partially degraded by sonication to obtain a homogenous solution,” and demonstrates that sonicated cross-linked HA can be accurately quantified by the carbazole method (also claimed in amended claim 1) without hindering analysis (p. 106, col. 2, last para.-p.107, col. 1, first par.). Thus it is well-known in the art both that sonication modifies HA gel structure and that such treatment can be applied prior to carbazole quantification yielding the predictable result of homogenous solutions. 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 have modified Bao to incorporate the teachings of Weng by treating the test solution with ultrasounds, so as to prepare fully mixed cross-linked hydrogel samples to more uniformly absorb the reagents A and B as well as temperature changes. (See MPEP 2143(I)(A)).
Applicant argues (p. 6, #2) that Weng is unrelated to analytical quantification because it discloses ultrasound only in the context of manufacturing injectable HA formulations. Weng allegedly applies very high power ultrasound (1000-3000 W) during gel formation and does not address spectrophotometric analysis, analyte integrity, or reproducibility of absorbance readings. Applicant contends that a person of ordinary skill in the art seeking to improve analytical quantification, as in Bao, would not consult a manufacturing reference like Weng because the contexts, product synthesis versus analytical measurement, are fundamentally different.
The Examiner respectfully disagrees.
While Weng discloses ultrasound in a manufacturing context, it nevertheless establishes that hyaluronic acid gel systems are structurally affected by ultrasound and that ultrasound power is an adjustable parameter. The relevant inquiry under 35 U.S.C. 103 is not whether Weng is directed to analytical quantification per se, but whether it teaches that HA hydrogels respond to controlled ultrasound treatment. That principle is directly applicable to the dissolution and homogenization of cross-linked HA prior to analysis. Moreover, Oueslati demonstrates that cross-linked HA forms aggregates and must be partially degraded by sonication to obtain a homogenous solution suitable for carbazole quantification, confirming that ultrasound treatment in analytical preparation was known in the art. Although Bao does not mention crosslinking aggregation, a person of ordinary skill in the art seeking to improve homogeneity and reproducibility in Bao’s carbazole assay would have reasonably applied known ultrasound treatment of HA gels and adjusted power as needed. The distinction between manufacturing and testing does not negate the predictability that ultrasound modifies HA gel structure, nor does it preclude routine optimization of ultrasound intensity for analytical purposes (See MPEP 2144.05). Accordingly, the rejection remains proper under 35 U.S.C. 103.
Applicant argues (p. 6, #3) that the amended 0.5-50 W/kg ultrasound power range is critical and non-obvious because it uniquely solves an unrecognized problem of achieving homogenous dispersion of complex HA hydrogels without impairing quantification accuracy. Applicant asserts that the instant application power below 0.5 W/kg results in inadequate dispersion and irreproducible absorbance readings, while power above 50 W/kg allegedly strengthens crosslinking and hinders dispersion. Applicant further argues that neither Bao nor Weng teaches or suggests this specific range, noting that Bao does not disclose ultrasound at all and that Weng uses much higher power levels (1000-3000 W) for manufacturing rather than analytical purposes.
The Examiner respectfully disagrees.
Weng discloses sonication at power levels between 1000-3000W (claim 9), and Oueslati discloses sonication at approximately 22 W/g (i.e., ~22,000 W/kg)(p. 103, col. 2, para. 2), establishing that very high specific power densities were known and operable in polymer systems. Oueslati further demonstrates that sonicated cross-linked HA can be accurately quantified by the carbazole method under these high sonication power ranges without hindering analysis (p. 107, col. 1, para. 1, last 5 ll.). This directly undermines Applicant’ assertion that power above 50 W/kg would necessarily impair measurement. Even if Weng applies higher absolute power during manufacturing, such power would likewise disrupt gel structure and achieve dispersion when applied for analytical preparation. Because ultrasound intensity directly influences polymer disruption and degradation, power constitutes a known, result-effective variable. The instant specification does not provide comparative data demonstrating inoperability outside the claimed 0.5-50 W/kg range contrary to Applicant’s arguments, nor does it establish a critical threshold at the claimed limits; rather, it merely shows operability within selected examples. In view of Oueslati’s operability at vastly higher specific power and Weng’s teaching of adjustable ultrasound power in HA systems, selecting a lower power window to balance dispersion and analyte preservation represents routine optimization (See MPEP 2144.04). Accordingly, the rejection under 35 U.S.C. 103 remains proper.
Applicant argues (p. 7, #4) that there is no motivation to combine Bao and Weng because Bao’s carbazole method is self-contained and does not identify dispersion deficiencies that would prompt the use of ultrasound. Applicant further contends that Weng is directed to gel manufacturing and does not address analytical quantification or measurement accuracy, and therefore lies in a different technical field. Applicant asserts that combining Bao and Weng would impair the carbazole reaction and that the claimed low-power sonication range addresses an unrecognized problem and yields unexpected improvements in homogeneity and analyte preservation.
The Examiner respectfully disagrees.
In response to applicant's argument that Weng is nonanalogous art, it has been held that a prior art reference must either be in the field of the inventor’s endeavor or, if not, then be reasonably pertinent to the particular problem with which the inventor was concerned, in order to be relied upon as a basis for rejection of the claimed invention. See In re Oetiker, 977 F.2d 1443, 24 USPQ2d 1443 (Fed. Cir. 1992). Weng is analogous art because it concerns ultrasound treatment of hyaluronic acid hydrogel systems and teaches that ultrasound power affects HA gel structure. This teaching is directly relevant to applying controlled sonication to HA gels in analytical preparation.
In this case, the motivation to combine need not arise from an express deficiency identified in Bao; rather, it is sufficient that the references relate to the same material and that the proposed modification would have been predictable to a person of ordinary skill in the art. Bao discloses carbazole-based quantification of cross-linked hyaluronic acid, while Weng teaches that hyaluronic acid gel systems are structurally affected by adjustable ultrasound treatment “to obtain the fully mixing,” (page 4, step (3)). Further, Oueslati recognizes that cross-linked HA forms aggregates in solution and must be partially degraded by sonication to obtain a homogenous solution suitable for carbazole quantification (p. 106, col. 2, last para.-p.107, col. 1, first par.). Thus, the art demonstrates both that HA gels can require mechanical disruption prior to analysis and that sonication does not prevent accurate carbazole measurement. The manufacturing context of Weng does not negate its teaching that ultrasound modifies HA gel structure; a person of ordinary skill in the art would reasonably apply known ultrasound treatment to improve homogeneity in analytical preparation. Moreover, Oueslati’s disclosure of operability at very high specific power densities (e.g. 22 W/g)(p. 103, col. 2, para. 2), undermines the assertion that ultrasound conditions would naturally impair the carbazole reaction. The instant specification does not establish criticality or unexpected results at the claimed 0.5-5 W/kg limits, but merely demonstrates operability under selected examples. Accordingly, selecting an appropriate ultrasound power to achieve dispersion without undue degradation represents routine optimization of a known result-effective variable, and that rejection under 35 U.S.C. 103 is maintained.
Claim Objections
Claims 1-3, 5-14 are objected to because of the following informalities:
Claim 1, step h., l. 1, recites “comprised between”. The phrase “comprised between” is objected to as informal and non-standard claim terminology. Applicant may correct the claim by omitting “comprised” to read “between”.
Claims 2-3, 5-14 are objected to based upon dependency from objected claim 1.
Claim 1, step h., l. 2, recites “500”. It is unclear as to whether this is 500 nm, mm, cm, etc. Applicant may correct this to read “500 nm”.
Claims 2-3, 5-14 are objected to based upon dependency from objected claim 1.
Claims 2-3, 5-9, l.1 are objected to for absence of punctuation after the recitation of the independent claim of dependence. Applicant may correct this by including punctuation of a comma after the recitation of “claim 1” to read “claim 1,”.
Claim 1, step g., l. 3 and step h., l. 2 is objected to for absence of punctuation after the term “optionally”. Applicant may correct this by including punctuation of a comma after the recitation of “optionally” to read “optionally,”.
Claims 2-3, 5-14 are objected to based upon dependency from objected claim 1.
Claim 1, step g., l. 3 states “optionally solution”. Applicant may correct to read “optionally, a solution”.
Claims 2-3, 5-14 are objected to based upon dependency from objected claim 1.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the second paragraph of 35 U.S.C. 112(b):
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.
Claim 13 is 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.
Regarding claim 13, l. 1 recites “75”. It is unclear as to whether this is 75 °C, °F, or K. Applicant may correct this to read “75 °C”.
Appropriate correction is required.
Claim Interpretation
The claims contain limitations which are directed to intended uses or capabilities of the claimed invention. These limitations are only given patentable weight to the extent which effects the structure of the claimed invention. Please see MPEP 2114. Note that functional limitations are emphasized in italics herein.
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.
Claims 1-3, 5-7, 9-14 are rejected under 35 U.S.C. 103 as being unpatentable over Bao (CN 108801952 A, citations herein made with respect to English Machine Translation attached; 2018) , in view of Weng (CN 110327366 A, citations herein made with respect to English Machine Translation attached; 2019-10-15).
Regarding claim 1, Bao teaches a method for the determination of the hyaluronic acid content of a hydrogel (a medical cross-linked sodium hyaluronate gel free hyaluronic acid sodium content detection method; Abstract), the method comprising the following steps:
a. preparing, as reagent A, a solution of sodium tetraborate in sulfuric acid (sodium tetraborate sulfuric acid solution; Abstract) ;
b. preparing reagent B by dissolving carbazole in ethanol (carbazole ethanol solution; Abstract);
c. preparing test solutions by dissolving the hydrogel in an aqueous solution (weighing the product two proper parts, placing the volumetric flask, adding water to dissolve; Abstract);
e. preparing a reference stock solution by dissolving glucuronic acid (preparing…glucuronic acid standard solution; Abstract; See Table 1 below that shows that glucuronic acid was dissolved with water), or a glucuronic acid-containing substance in an aqueous solution;
f. preparing at least 3 reference solutions by dilution of the reference stock solution in aqueous solution (Table 1, below showing 5 reference solutions by water dilution and page 2, lines 5-6; test tube 0 is the blank);
g. preparing the test tubes by admixing (Abstract explains that the reference solutions (standard solutions), blank, and the samples are each placed in separate test tubes and the reagents are added while fully shaking) reagent A (sodium tetraborate sulfuric acid solution is slowly added to each tube; Abstract), reagent B (adding carbazole-ethanol solution…into each test tube; Abstract) and one of the following: reference solution (standard solution; Abstract), test solution (the sample; Abstract), aqueous solution (blank solution; Abstract), and optionally solution for interference.
place each test tube in a water bath for at least 5 min, then cool them to room temperature (water bath heating for 15 min and cool to room temperature; Abstract);
h. reading an absorbance for the test tubes at a wavelength comprised between 500 and 580 nm, (each pipe-absorbance measured at a wavelength of 530nm; Abstract), against the blank (0 # tube as reference; Abstract)(test tube 0 of the table on page 3 and also depicted below is the blank solution with 0ml of glucuronic acid and 1 ml of water)(Blank solution: 1 ml aqueous solution; page 2, step (2), line 2) and optionally the sample for interference.
Bao does not teach d. treating the test solutions with ultrasounds for a period of time sufficient to obtain a macroscopically homogeneous solution. Bao does teach fully vibrating mixing them uniformly (Abstract).
Weng teaches treating a solution with ultrasounds for a period of time sufficient to obtain a macroscopically homogeneous solution wherein the ultrasound is applied at a power (“the hyaluronic acid gel crosslinked gel obtained in the step (1) and step (2) to obtain the fully mixing, ultrasonic (ultrasonic power is 1000W at room temperature) for 30 minutes,” wherein fully mixed would naturally be macroscopic; page 4, step (3)).
Weng is considered to be analogous to the claimed invention because it is in the same field of endeavor for hyaluronic acid content in a hydrogel. Even if Weng applies sonication during manufacturing, such power would likewise disrupt gel structure and achieve dispersion when applied in analytical preparation for the benefit of fully mixing the hydrogel (see citation above). 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 have modified Bao to incorporate the teachings of Weng by treating the test solution with ultrasounds, so as to prepare fully mixed cross-linked hydrogel samples to more uniformly absorb the reagents A and B as well as temperature changes. A person of ordinary skill in the art would have recognized that applying ultrasonic power to the diluted hydrogel sample is a technique already known in the art and would have yielded the predictable solution of a more well mixed sample of Bao’s cross-linked hydrogel (See MPEP 2143(I)(A))(See also support from a reference Oueslati et al. (“CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form”; 2014)(“The following study focuses on the quantification of cross linked HA. This last HA form leads to aggregates in solution. And then, it has to be partially degraded by sonication to obtain a homogeneous solution,” wherein Oueslati explains that such sonication did not hinder the quantification of HA using the carbazole method, the same method used in Bao; (p. 106, col. 2, last para.-p.107, col. 1, first par.).
Modified Bao is silent to teaching sonication is performed at a specific power between 0.5 W/kg and 50 W/kg.
Weng instead teaches sonication is at 1000 W at room temperature; page 4, Example 1, step (3), line 2)(1000-3000W in claim 9;
Although the range of power taught by Weng is not a specific power within the claimed range, the instant claims state that the purpose of sonication is “to obtain a macroscopically homogenous solution,” while Weng discloses applying sonication “ to obtain the fully mixing”. Weng therefore meets the same goal as the instant claims. Reference Oueslati recognizes that cross-linked HA forms aggregates in solution and must be partially degraded by sonication to obtain a homogenous solution prior to carbazole quantification (p. 106, col. 2, last para.-p.107, col. 1, first par.) and discloses sample sonication at approximately 22 W/g (i.e., ~22,000 W/kg)(p. 103, col. 2, para. 2). Oueslati further demonstrates that sonicated cross-linked HA at this power can be accurately quantified by the carbazole method without hindering analysis, thereby confirming that ultrasound treatment prior to carbazole assay was known in the art. Because ultrasound intensity directly influences polymer disruption and degradation, power constitutes a known, result-effective variable. The instant specification does not provide comparative data demonstrating inoperability outside the claimed 0.5-50 W/kg range, nor does it establish a critical threshold at the claimed limits; rather, it merely shows operability within selected examples. In view of Oueslati’s operability at vastly higher specific power and Weng’s teaching of adjustable ultrasound power in HA systems (between 1000-3000W stated in claim 9), selecting a lower power window to balance dispersion and analyte preservation represents routine optimization (See MPEP 2144.04). 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 have modified the sample preparation method taught by Bao in view of Weng by selecting an appropriate sonication power within the claimed range between 0.5 W/kg and 50 W/kg, depending on the sample type, volume, viscosity, or desired degree of mixing or breakdown, in order to achieve the full mixing taught by Weng and partial degradation taught by Oueslati.
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Table 1 reference solutions of Bao, page 2, step 3, machine-translated
Regarding claim 2, Modified Bao teaches the method according to claim 1.
Modified Bao fails to teach after step c, the following step: c2. heating the test solutions to a temperature higher than room temperature.
Although Bao in view of Weng does not explicitly disclose a pre-reaction heating step, heating dissolved polymer gels to facilitate dissolution is a routine and predictable laboratory technique. Bao already discloses boiling conditions (~100°C) during the carbazole reaction (Abstract), demonstrating that HA solutions tolerate elevated temperatures. 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 have modified the hydrogel dissolution step taught by Bao in view of Weng by heating the test solution to improve the dissolution efficiency, representing routine optimization of a known variable (temperature) particularly in the absence of criticality (See MPEP 2144.04).
Regarding claim 3, Modified Bao teaches the method of claim 1 wherein in step g, the water bath is boiling water and the time is about 15 minutes (boiling water bath heating for 15min; Abstract).
Regarding claim 5, Modified Bao teaches the method of claim 1.
Modified Bao is silent to teaching the ultrasound is performed at a specific energy comprised between 500 J/kg and 30 kJ/kg.
Weng teaches the ultrasound is performed at an energy (ultrasonic power is 1000W at room temperature) for 30 minutes; page 4, Example 1, step (3), line 2);
Modified Bao is silent to teaching the ultrasound is performed at a specific energy comprised between 500 J/kg and 30 kJ/kg. However, specific energy is a direct function of power, treatment time, and mass. Selection of appropriate time and mass conditions to achieve a desired specific energy would have been within the level of ordinary skill in order to achieve the full mixing taught by Weng. 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 have adjusted the sonication parameters based on routine optimization to achieve this range since specific energy is a result effective variable. (MPEP 2144.05).
Regarding claim 6, Modified Bao teaches the method according to claim 1, wherein reagent A is a solution 0.95 % w/v of disodium tetraborate in sulfuric acid (0.025mol/L sodium tetraborate in sulfuric acid solution; Bao, Abstract), and reagent B is a solution 0.125 % w/v of carbazole in ethanol (carbazole ethanol solution of 0.125%; Bao, Abstract).
Regarding claim 7, Modified Bao teaches the method according to claim 1, wherein in step c. the test solution is prepared at least in double (weighing the product two proper parts; Bao, Abstract).
Regarding claim 9, The method of claim 1, wherein, after step h., the percentage content of sodium hyaluronate is calculated by interpolating the experimental absorbance values with the reference standard curve (using the standard tube drawing concentration-absorbance linear regression curve and obtaining linear regression equation so as to calculate the glucuronic acid content using the absorbance of the sample solution, thereby calculating the dissociative hyaluronic acid sodium content; Bao, page 2, step (4), last 4 lines); See % sodium hyaluronate in translated table 4 of Bao below).
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Table 4, Bao, machine-translated
Regarding claim 10, The method of claim 1, wherein a concentration of the at least 3 reference solutions is between 0.0005% w/v and 0.0100% w/v (10-50 ug/mL corresponds to 0.0010%-0.0050% w/v as shown in Table 3 of Bao below).
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Table 3 of Bao
Regarding claim 11, The method of claim 1, wherein a concentration of the at least 3 reference solutions is between 0.0010 % w/v and 0.0050 % w/v (10-50 ug/mL corresponds to 0.0010%-0.0050% w/v as shown in Table 3 of Bao above).
Regarding claim 12, The method of claim 1, wherein the wavelength is at about 530 nm (each pipe-absorbance measured at a wavelength of 530nm; Bao, Abstract).
Regarding claim 13, The method of claim 2.
Bao fails to teach the temperature is between 75 and 85°C (The Examiner interprets 75 to be 75°C).
Bao instead teaches only dissolving the test solution in water and then vibrating to mix. Although Bao in view of Weng does not explicitly disclose a pre-reaction heating step, heating dissolved polymer gels to facilitate dissolution is a routine and predictable laboratory technique. Bao already discloses boiling conditions (~100°C) during the carbazole reaction (Abstract), demonstrating that HA solutions tolerate elevated temperatures. 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 have modified the hydrogel dissolution step taught by Bao in view of Weng by heating the test solution to a temperature of 75°C-85°C for a defined period to improve the dissolution efficiency representing routine optimization of a known variable (temperature) particularly in the absence of criticality (See MPEP 2144.04).
Regarding claim 14, The method of claim 2.
Modified Bao fails to teach test solutions are heated for at least 2 hours.
Although Bao in view of Weng does not explicitly disclose a pre-reaction heating step for a defined amount of time, heating dissolved polymer gels to facilitate dissolution for a period of time is a routine and predictable laboratory technique. Bao already discloses boiling conditions (~100°C) during the carbazole reaction (Abstract), demonstrating that HA solutions tolerate elevated temperatures. 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 have modified the hydrogel dissolution step taught by Bao in view of Weng by heating the test solution to a temperature of 75°C-85°C for 2hrs to improve the dissolution efficiency, representing routine optimization of known variables (temperature and time) particularly in the absence of criticality (See MPEP 2144.04).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Bao (CN 108801952 A, citations herein made with respect to English Machine Translation attached; 2018) in view of Weng (CN 110327366 A, citations herein made with respect to English Machine Translation attached; 2019-10-15), as applied to claim 1 above, and in further view of Bitter (“A Modified Uronic Acid Carbazole Reaction”; 1962) and Ding (CN 107653280 A, citations herein made with respect to English Machine Translation attached; 2018).
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Table 1 reference solutions of Bao, page 2, step 3, machine-translated
Regarding claim 8, Modified Bao teaches the method according to claim 1, wherein step g. is performed as follows:
adding 5 parts of reagent A to each test tube (sodium tetraborate in sulfuric acid solution 5ml to each tube; Bao, Abstract);
Layer each of the following solutions, each in a separate test tube (slowly adding 0.025mol/L sodium tetraborate in sulfuric acid solution 5ml to each tube; Abstract);
Blank: 1 part of water solution (preparing blank solution; Abstract)(See test tube 0 with water in Table 1 above);
Reference solutions: 1 part of each dilution of the Reference stock solution (See 5 test tubes of GA Standard solution/ml at different concentrations in row 2 of Table 1 above);
Test solutions: 1 part of each Test solution (absorbing 1ml detectings tube; Bao, Abstract)
shake the tubes (while shaking; Bao, Abstract) to dissolve the double-phase created by the solution (shaking the test tubes is capable of producing the results of dissolving the double-phase created by the solution);
put each test tube containing the layered solution in a water bath for at least 5 minutes, then cool each test tube containing the layered solution down (after finishing adding mixing the boiling water bath to boil for 10min and then taking out, cooling in ice water bath; Bao, Abstract);
add 0.2 parts of reagent B to each test tube containing the layered the solution (adding carbazole ethanol solution of 0.125% 0.2ml into each test tube; Abstract), shake each test tube containing the layered solution (fully shaking; Bao, Abstract) to dissolve the double-phase and put each test tube containing the layered solution in a water bath for at least 5 minutes (water bath heating for 15min; Abstract)(shaking the test tubes is capable of producing the results of dissolving the double-phase created by the sample).
Modified Bao fails to teach the steps in the order claimed, specifically adding reagent A to each test tube first, then adding the blank, reference solutions, and test solutions to the test tubes as well as cooling them down to room temperature before adding reagent B. Bao teaches adding the blank, reference solutions (standard solution series of Bao) and the test solutions (sample test tubes of Bao) to the test tubes first then adding reagent A (sodium tetraborate in sulfuric acid) to each test tube. Bao also teaches cooling the test tubes in an ice bath after reagent A and the solutions are added and only specifies cooling down to room temperature after reagent B is added (cooling to room temperature; Abstract).
Bitter teaches the steps in the order as claimed:
adding 5 parts of reagent A to each test tube (“5 ml of sulfuric acid reagent is placed in tubes fixed in a rack and cooled to 4°C,” wherein the sulfuric acid reagent is “sodium tetraborate · 10H2O (analytical grade) in sulfuric acid”; page 331, Procedure, lines 1-2; page 330, Reagents (a));
layer each of the following solutions onto the reagent A, each in a separate test tube (sample or standard is carefully layered on to the acid; page 331, Procedure, lines 2-3):
Reference solutions: 1 part of each dilution of the Reference stock solution (“standard is
carefully layered on to the acid,” wherein “Glucuronolactone standards of 440 ,ug/ml were prepared by dilution; page 330, Reagents, step (c));
Test solutions: 1 part of each test solution (sample…is carefully layered on to the acid; page 331,
Procedure, lines 2-3;
Bitter is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining hyaluronic acid content in a sample. Bitter teaches the following advantages of layering using this method: (a) further increase of sensitivity beyond that recorded by Gregory (11)) (b) immediate appearance of color, (c) marked increase of stability of the color, (d) greater reproducibility, and (e) reduction of interference by chlorides and oxidants. A preliminary report of some of these results has been given (13). Even so, Bitter also admits that “No difference was observed if the acid reagent was added to the sample or vice versa” (page 331, last 4 lines). 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 have modified Bao in view of Weng to incorporate the teachings of Bitter by rearranging the order of steps for the benefits listed above (See MPEP 2144.04(IV)(C)).
Modified Bao does not explicitly teach that the blank solution is added between the first and second reagent and also undergoes the same procedure as the reference and test solutions in the claim. Bitter does however teach “The OD of the blank against sulfuric acid should be below 0.025” (page 331, Procedure, paragraph 3, line 4).
Ding teaches the blank solution is added between reagent A and B and also undergoes the same procedure as the reference and test solutions in the claim (Page 6, paragraph 2 explains adding reagent A (borax and 5 mL sulfuric acid solution) to 6 test tubes then adding a blank solution and standard solutions, followed by shaking them until uniformly mixed, placing them in a water bath and cooling down, then adding reagent B (carbazole reagent) to the test tubes, and placing the test tubes in a warm water bath for 15 mins).
Ding is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining hyaluronic acid content in a sample. Ding teaches the blank to be a control group. It is commonly known in the art to create a method blank to help identify contamination that might be introduced during the entire analytical process (sample preparation, reagents, test tubes, instrumentation, etc.) From here a limit of detection can be created that ensures that reported results are above the noise (or contamination) of the system, which is taught by Ding to be below 0.025. Page 330, paragraph 1 of Bitter states that when “estimating uranic acids in chromatographic fractions…the color is unstable and sensitive to… impurities in the reagents or the sample interfere with it”. Additionally page 2, paragraph 2 of Bitter explains there to be an unwanted color change due to “contamination of chromatographic samples by dust or chlorinated tap water”. 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 have modified Bao in view of Weng and Bitter to incorporate the teachings of Ding by subjecting the blank to the same sample processing steps of the reagent and test solutions so as to more accurately quantify the content of sodium hyaluronate in the hydrogels (See MPEP 2143(I)(A)).
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Oueslati et al., 2017 (instant PTO-892) teaches sonication of HA hydrogel for quantification using a carbazole assay.
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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/V.S./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758