DETAILED ACTION
In application filed on 01/02/2024, Claims 1-10 are pending. The claim set submitted on 07/07/2026 is considered because this is the most recent claim set with amendments. Claims 1-10 are considered in the current 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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 01/02/2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Specification
The disclosure is objected to because of the following informalities:
Specification recite “GS1” and “GS2” on page 8.
Are the recitations of “GS1” and “GS2” are abbreviations of a technical term?
Examiner suggests to Applicant to provide the non-abbreviated form of “GS1” and “GS2”.
Appropriate correction is required.
Claim Objections
Claim 10 is objected to because of the following informalities:
Claim 10 recites “GS1” and “GS2” which outlet is configured…” in lines 4-5 of the Claim.
Are the recitations of “GS1” and “GS2” are abbreviations of a technical term?
Examiner suggests to Applicant to provide the non-abbreviated form of “GS1” and “GS2”.
Also, Applicant may provide evidence that the claimed “GS1 and GS2” are terms of the art if applicable.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-10 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more. The claims have been analyzed for eligibility in accordance with their broadest reasonable interpretation. All claims are directed to statutory categories, i.e., a method (Claims 1-10) (Step 1: YES).
Analysis:
Claim 1: Ineligible.
Step 1:
The claim recites a series of steps or acts, including “method for determining a 37°C equilibrium-dialysis- equivalent concentration of free testosterone in a serum test sample”. Thus, the claim is directed to a method, which is one of the statutory categories of invention (Step 1: YES).
Step 2A Prong 1:
Claim 1 recites “obtaining a predetermined linear conversion equation for free testosterone…(math step); “(e) establishing, based on the respective first concentrations y and the respective second concentrations x, the predetermined linear conversion equation:y = 0.8719x - 0.2116,wherein y represents a concentration of free testosterone obtained by ultrafiltration at 25°C, and x represents a concentration of free testosterone obtained by equilibrium dialysis at 37°C;” (mathematical step; step provides a mathematical equation); and “calculating the 37°C equilibrium-dialysis-equivalent concentration xtest of free testosterone in the serum test sample according to: xtest = (ytest + 0.2116)/0.8719) (mental or mathematical step)”.
Therefore, the claim is directed towards an abstract idea, and more specifically to the abstract idea group of a math/mental processes since claim 1 relates to using a math process to “determine a 37°C equilibrium-dialysis- equivalent concentration of free testosterone in a serum test sample” (Step 2A, Prong 1: Patent Ineligible).
Step 2A, Prong 2:
This judicial exception is not integrated into a practical application.
Once the establishment of the predetermined linear equation is done, No further action takes place.
Also the steps of “ adding 900 µL; measuring, by liquid…; placing 200 µL; “measuring , by liquid…” ; “processing… and “measuring , by liquid…” are recited at a high level of generality that it amounts to mere data gathering (insignificant extra-solution activity). See MPEP 2106.05(g).
Step 2B:
Furthermore, the courts have found that limitations adding insignificant extrasolution activity to the judicial exception, such as mere data gathering in conjunction with a law of nature or abstract idea, are limitations found not to be enough to qualify as ‘significantly more’ when recited in a claim with a judicial exception (see the 2014 Interim Guidance on Patent Subject Matter Eligibility of the Federal Register dated December 16, 2014; and MPEP 2106.05(I)(A)). Note that mere data gathering is not significantly more than the abstract idea. See MPEP 2106.05(g).
Here, there are no additional elements which are significantly more than the abstract idea. The steps of “adding 900 µL; measuring, by liquid…; placing 200 µL; “measuring , by liquid…” ; “processing… and “measuring , by liquid…” appears to be well-understood, routine, and conventional (WURC) in the field of clinical diagnostics, as evidenced by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496.). (Step 2B: NO).
Therefore, Claim 1 is ineligible.
Moreover, Claims 2-10 are rejected by virtue of their dependency on Claim 1.
Also, each of the dependent claims 2-10 do not solve the issues of claim 1.
Claim 2: Ineligible.
Step 2A, Prong One and Prong Two: Claim 2 further presents an abstract idea “predetermined linear conversion equation has a coefficient of determination R2 of 0.9897” and further define the data gathering steps which appear to be generic and WURC.
Step 2B: The claims do not recite any elements which are significantly more.
Therefore, Claim 2 is ineligible.
Claims 3-10: Ineligible.
Step 2A, Prong One and Prong Two: Claims 3-10 further define the data gathering steps, which appear to be generic and WURC.
Step 2B: The claims do not recite any elements which are significantly more.
Therefore, Claims 3-10 are ineligible.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 1-3 and 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496).
Regarding Claim 1, Chen teaches a method for determining a 37°C equilibrium-dialysis- equivalent concentration of free testosterone in a serum test sample, comprising:
obtaining a predetermined linear conversion equation for free testosterone (See Page 491…calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run, thereby teaching “obtaining a predetermined linear conversion equation for free testosterone”; Examiner submits, under BRI, that the concentration of unknowns is determined from a “predetermined linear conversion equation” obtained from the standard curve) by:
adding a volume 4-hydroxyethyl piperazine ethanesulfonic acid solution (See Page 491…0.5 mL of serum was diluted with 0.5 mL of HEPES buffer) to a volume of each of a plurality of serum specimens (See Page 492, Method Comparison… Serum specimens from 60 male adult subjects with free testosterone concentrations ranging from 49- 439 pmol/L were included in the comparison study)) transferring a resulting mixture to an activated ultrafiltration tube (referred to as The Centrifree® ultrafiltration device is designed [Page 495]) , and centrifuging (See Page 493…Recovery was virtually the same whether the FT was centrifuged through one or through two unused ultrafiltration devices.) the resulting mixture at 25°C and centrifuged for 1 hour to obtain respective ultrafiltrates (See Page 495…Given this, along with the convenience of running the centrifuge at room temperature, we adopted 25 °C as the temperature to conduct the ultrafiltration step; See Page 491…the mixture was transferred into a Centrifree® UF device and immediately centrifuged at 1800 g, 25 °C in a fixed angle rotor for 1 h.);
measuring, by liquid chromatography-tandem mass spectrometry (See Para 492, Method comparison… The UF-LC-MS/MS method…), respective first concentrations y of free testosterone in the respective ultrafiltrates (See Para 491, LC-MS/MS… calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run. …thereby teaching “respective first concentrations y of free testosterone in the respective ultrafiltrates”)
placing a volume of the 4-hydroxyethyl piperazine ethanesulfonic acid solution (See Page 491… 0.1 mL of serum was added into a Micro DispoDialyzer and dialysed against 0.5 mL HEPES buffer in a borosilicate culture tube) on a buffer side of an equilibrium dialysis plate (referred to as borosilicate culture tube; Under BRI, the borosilicate culture tube side where HEPES buffer is located [Page 491, Equilibrium dialysis), placing a volume of serum of each of the plurality of serum specimens (See Page 492, Method Comparison… Serum specimens from 60 male adult subjects with free testosterone concentrations ranging from 49- 439 pmol/L were included in the comparison study)) on a sample side of the equilibrium dialysis plate ( See 491… 0.1 mL of serum was added into a Micro DispoDialyzer; Under BRI, the sample side where the serum is located), and dialyzing at 37°C for some hours (See Page 491… at 37 °C overnight (16 h)) to obtain respective dialysates (See Page 491… Testosterone-2,2,4,6,6-d5 (40 fmol) was added to 0.4 mL of dialysate);
(d) measuring, by liquid chromatography-tandem mass spectrometry, respective second concentrations x of free testosterone in the respective dialysates (See Page 492, Method comparison… and direct ED-LC-MS/MS), thereby teaching “second concentrations x of free testosterone”); and
(e) establishing, based on the respective first concentrations y (See Para 491, LC-MS/MS… calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run. …thereby teaching “respective first concentrations y of free testosterone in the respective ultrafiltrates”) and the respective second concentrations x (See Page 492, Method comparison… and results from direct ED-LC-MS/MS), thereby teaching “second concentrations x of free testosterone”), the predetermined linear conversion equation (See Page 491…calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run, thereby teaching “obtaining a predetermined linear conversion equation for free testosterone”; Examiner submits, under BRI, that the concentration of unknowns is determined from a “predetermined linear conversion equation” obtained from the standard curve):
y = 0.8719x - 0.2116, wherein y represents a concentration of free testosterone obtained by ultrafiltration at 25°C, and x represents a concentration of free testosterone obtained by equilibrium dialysis at 37°C (See Page 492, Method comparison… and results from direct ED-LC-MS/MS), thereby teaching “second concentrations x of free testosterone”); processing the serum test sample by ultrafiltration at 25°C and 2000 g for 1 hour (See Page 491…0.5 mL of serum was diluted with 0.5 mL of HEPES buffer. After equilibrating at room temperature (RT) for 5 min, the mixture was transferred into a Centrifree® UF device and immediately centrifuged at 1800 g, 25 °C in a fixed angle rotor for 1 h.) to obtain a test ultrafiltrate (See Page 491…Testosterone-2,2,4,6,6-d5 (40 fmol) was added to 0.5 mL of ultrafiltrate and the mixture vortexmixed for 5 s, then incubated for 5 min at RT.);
measuring, by liquid chromatography-tandem mass spectrometry, a test ultrafiltration concentration ytest of free testosterone in the test ultrafiltrate (See Page 491…The residue was dissolved in 70 μL of 50% methanol and a 50 μL aliquot was analyzed by LC-MS/MS.; See Page 492, Method comparison… The UF-LC-MS/MS method, thereby teaching “a test ultrafiltration concentration ytest of free testosterone”); and
calculating the 37°C (See Page 491… Equilibrium dialysis … at 37 °C overnight (16 h).) equilibrium-dialysis-equivalent concentration xtest of free testosterone in the serum test sample (See Page 491…Analyst software (version 1.4.2) was used to control the system, mediate data acquisition, integrate peak-area and calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run, thereby teaching “calculating the 37°C equilibrium-dialysis-equivalent concentration xtest of free testosterone in the serum test sample”) according to: xtest = (ytest + 0.2116)/0.8719).
Chen does not teach:
Volumes of 900 µL and 200 µL of a 4-hydroxyethyl piperazine ethanesulfonic acid solution;
Volumes of 300 µL and 200 µL of each of a plurality of serum specimens;
dialyzing for 22 hours;
processing the serum test sample by ultrafiltration at 2000 g
the predetermined linear conversion equation: y = 0.8719x - 0.2116; and
equation of xtest = (ytest + 0.2116)/0.8719).
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process. In the process of sample preparation for ultrafiltration and sample extraction and equilibrium dialysis, the selection of optimal experimental conditions including buffer volumes, sample volumes, centrifugation speed and dialysis time affects processing time and final concentration limits and the efficiency of contaminant removal and buffer exchange.
Thus, “volumes of 900 µL and 200 µL of a 4-hydroxyethyl piperazine ethanesulfonic acid solution; volumes of 300 µL and 200 µL of each of a plurality of serum specimens; the process of dialyzing for 22 hours; and processing the serum test sample by ultrafiltration at 2000 g” are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide sample preparation conditions including the volumes of 900 µL and 200 µL of a 4-hydroxyethyl piperazine ethanesulfonic acid solution; volumes of 300 µL and 200 µL of each of a plurality of serum specimens; the process of dialyzing for 22 hours; and processing the serum test sample by ultrafiltration at 2000 g, for the benefit of providing a comparison of serum free testosterone between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS (Chen, Abstract), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
In addition, in the computation of linear calibration curves in bioanalysis, the selection of optimal experimental conditions including standard purity & preparation, concentration range & standards, curve weighting, intercept treatment and intercept treatment defines the analyte linear equation. Thus, “the predetermined linear conversion equation: y = 0.8719x - 0.2116; and equation of xtest = (ytest + 0.2116)/0.8719)” are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide sample preparation conditions including the “predetermined linear conversion equation: y = 0.8719x - 0.2116; and equation of xtest = (ytest + 0.2116)/0.8719)”, for the benefit of providing a comparison of serum free testosterone between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS (Chen, Abstract), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Regarding Claim 2, the method of claim 1 is obvious over Chen.
Chen further teaches wherein the plurality of serum specimens comprises 60 serum specimens (See Page 492, Method Comparison… Serum specimens from 60 male adult subjects with free testosterone concentrations ranging from 49- 439 pmol/L were included in the comparison study)), and the predetermined linear conversion equation (See Page 491…calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run, thereby teaching “obtaining a predetermined linear conversion equation for free testosterone”; Examiner submits, under BRI, that the concentration of unknowns is determined from a “predetermined linear conversion equation” obtained from the standard curve) has a coefficient of determination R2 of 0.9897.
Chen does not teach:
the predetermined linear conversion equation has a coefficient of determination R2 of 0.9897.
In addition, in the computation of linear calibration curves in bioanalysis, the selection of optimal experimental conditions including the coefficient of determination (R²) evaluates how well an analytical model or calibration curve fits experimental data. It represents the proportion of variance in the analytical response (e.g., peak area, optical density) that is explained by the analyte concentration. Thus, “the predetermined linear conversion equation has a coefficient of determination R2 of 0.9897” is a result effective variable.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide sample preparation conditions including the “the predetermined linear conversion equation has a coefficient of determination R2 of 0.9897”, for the benefit of calculating the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run (Chen, Page 491, LC-MS/MS), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Regarding Claim 3, the method of claim 1 is obvious over Chen.
Chen teaches that the 60 serum specimens (See Page 492, Method Comparison… Serum specimens from 60 male adult subjects with free testosterone concentrations ranging from 49- 439 pmol/L were included in the comparison study)).
Chen further teaches comprise19 female serum specimens (See Page 491…found in the majority of the sera submitted by nineteen adult women).
Chen does not expressly teach that the 60 serum specimens comprise 30 female serum specimens and 30 male serum specimens.
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process.
In clinical bioanalysis, the selection of optimal experimental conditions such as the use of male and female sera accounts for sex-specific differences in human serum significantly which impacts clinical testing, biomarker baselines, and diagnostic interpretation because roughly 68% of circulating serum molecules show varying concentrations between males and females. Further, the number of serum samples in clinical testing directly affect testing accuracy, turnaround time, and the ability to run repeat or add-on assays. Thus, the use of 60 serum specimens comprising 30 female serum specimens and 30 male serum specimens are a result effective variable.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use 60 serum specimens comprising 30 female serum specimens and 30 male serum specimens for the benefit of providing a comparison of serum free testosterone between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS (Chen, Abstract), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Regarding Claim 8, the method of Claim 1 is obvious over Chen.
Chen teaches wherein the liquid chromatography-tandem mass spectrometry (See Abstract… a method involving liquid chromatography tandem mass spectrometry (LC-IDMS/MS)) uses a column having a dimension (See Page 491… The Eclipse C8 HPLC column (50×3.0 mm, 1.8 µm)) and a particle size of 1.8 µm (See Page 491… The Eclipse C8 HPLC column (50×3.0 mm, 1.8 μm)).
Chen does not explicitly teach the column conditions in chromatography wherein the liquid chromatography-tandem mass spectrometry uses a column having a dimension of 2.1 x50 mm and a particle size of 1.7 um, a column oven temperature of 40°C, mobile phase A comprising 0.1% formic acid and 2 mM ammonium acetate aqueous solution, and mobile phase B comprising methanol.
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process.
In liquid chromatography, the selection of optimal experimental conditions such as column temperature, Column Dimensions (Length and Diameter), Buffer type, column packing quality & uniformity directly impacts retention time, resolution, and separation efficiency. Thus, column conditions are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the column conditions in chromatography wherein the liquid chromatography-tandem mass spectrometry uses a column having a dimension of 2.1 x50 mm and a particle size of 1.7 um, a column oven temperature of 40°C, mobile phase A comprising 0.1% formic acid and 2 mM ammonium acetate aqueous solution, and mobile phase B comprising methanol, for the benefit of performing LC-MS/MS measurement to calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run (Chen, LC-MS/MS, Page 491).which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Regarding Claim 9, the method of Claim 8 is obvious over Chen.
Chen teaches wherein gradient elution is performed according to the following program (See Page 491, LC-MS/MS…Briefly, HPLC was conducted using an Agilent Technologies 1200 series system in linear gradient mode at a flow rate of 0.85 mL/min through an Eclipse C8 column employing a mobile phase consisting of methanol-water (20:80) increasing to 100% methanol over 4 min and maintained at 100% methanol for 1 min).
Chen does not explicitly teach that gradient elution is performed according to the following program: at an initial time, a flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%; at 1.20 min, the flow rate is 0.600 mL/min, mobile phase A is 5%, and mobile phase B is 95%; at 1.40 min, the flow rate is 0.600 mL/min, mobile phase A is 5%, and mobile phase B is 95%; at 1.41 min, the flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%; and at 1.60 min, the flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%.
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process.
In liquid chromatography, the selection of optimal experimental conditions such as a predetermined gradient elution program systematically increases mobile phase strength over time (such as raising organic solvent or salt concentration). This dynamic shift reduces run times, sharpens late-eluting peaks, prevents band broadening, and clears strongly bound matrix components from the column. Thus, gradient elution being performed according to the following program: at an initial time, a flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%; at 1.20 min, the flow rate is 0.600 mL/min, mobile phase A is 5%, and mobile phase B is 95%; at 1.40 min, the flow rate is 0.600 mL/min, mobile phase A is 5%, and mobile phase B is 95%; at 1.41 min, the flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%; and at 1.60 min, the flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%” is a result effective variable.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use gradient elution that is performed according to the following program: at an initial time, a flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%; at 1.20 min, the flow rate is 0.600 mL/min, mobile phase A is 5%, and mobile phase B is 95%; at 1.40 min, the flow rate is 0.600 mL/min, mobile phase A is 5%, and mobile phase B is 95%; at 1.41 min, the flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%; and at 1.60 min, the flow rate is 0.600 mL/min, mobile phase A is 95%, and mobile phase B is 5%, for the benefit of performing LC-MS/MS measurement to calculate the concentration of unknowns against a standard curve derived from calibrators analyzed within the same analytical run (Chen, LC-MS/MS, Page 491), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496) as applied to claim 1 above, and further in view of Grau et al. (US20040248770A1).
Regarding Claim 4, the method of Claim 1 is obvious over Chen.
Chen teaches that the activated ultrafiltration tube (referred to as The Centrifree® ultrafiltration device is designed [Page 495]) is obtained by adding a volume of a concentration of NaOH activating solution (See Materials and Methods…0.9 g/L NaOH) into an ultrafiltration tube (referred to as The Centrifree® ultrafiltration device is designed [Page 495]), centrifuging at 25°C and for some minutes (See Page 495…Given this, along with the convenience of running the centrifuge at room temperature, we adopted 25 °C as the temperature to conduct the ultrafiltration step; (See Page 491…0.5 mL of serum was diluted with 0.5 mL of HEPES buffer. After equilibrating at room temperature (RT) for 5 min, the mixture was transferred into a Centrifree® UF device and immediately centrifuged at 1800 g, 25 °C in a fixed angle rotor for 1 h.), adding a volume of pure water (See Materials and Methods…0.9 g/L NaOH; Examiner submits that 0.9 g/L NaOH has water ), and centrifuging under the same conditions (See Page 495…Given this, along with the convenience of running the centrifuge at room temperature, we adopted 25 °C as the temperature to conduct the ultrafiltration step.).
Chen does not explicitly teach the use of NaOH and pure water.
In the analogous art of method for the ultrafiltration of an antifungal agent using a zirconium-layered ceramic ultrafilter to reduce pyrogens, Grau teaches that the filter system is depyorgenated by soaking with 0.5N NaOH solution over 30 minutes, followed by a dilute NaOCl solution at a concentration of 4 mg/ml. After draining off the NaOCl solution, the ultrafiltered depyrogenated water is injected from the retentate side until the pH of permeate becomes 7 to 8 in order to flush out the remaining NaOCl in the system. The filter system is then placed in an oven at 250° C. for 16 hrs (Para 0029).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Chen, to include “the use of NaOH and pure water”, as taught Grau for the benefit of having the filter system depyorgenated (Grau, Para 0029), allowing for the provision of a method for ultrafiltration on manufacturing scale of an antifungal agent using a zirconium-layered ceramic membrane (Membralox, U.S. Filter) for pyrogen reduction via ultrafiltration in the last step synthesis of bulk drug (Grau, Para 0005).
The combination of Chen and Grau does not explicitly teach:
Volumes of 100 µL of water, 100 µL of 0.1 M NaOH; centrifuging at 2000 g for 10 minutes.
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process. In the process of sample preparation for ultrafiltration and sample extraction and equilibrium dialysis, the selection of optimal experimental conditions including buffer volumes, sample volumes, centrifugation speed and dialysis time affects processing time and final concentration limits and the efficiency of contaminant removal and buffer exchange.
Thus, “volumes of 100 µL of water, 100 µL of 0.1 M NaOH; centrifuging at 2000 g for 10 minutes” are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide sample preparation conditions including the “volumes of 100 µL of water, 100 µL of 0.1 M NaOH; centrifuging at 2000 g for 10 minutes”, for the benefit of providing a comparison of serum free testosterone between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS (Chen, Abstract), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496) as applied to claim 1 above, and further in view of Stephenson, Jr. et al. (US20180233340A1).
Regarding Claim 5, the method of Claim 1 is obvious over Chen.
Chen teaches that the ultrafiltrates and the dialysates are being measured by liquid chromatography-tandem mass spectrometry (See Page 491, Sample preparation; Ultrafiltration and sample extraction…. The residue was dissolved in 70 μL of 50% methanol and a 50 μL aliquot was analyzed by LC-MS/MS; See Page 491, Sample preparation; Equilibrium dialysis… Subsequent organic solvent extraction was the same as described above; See Para 0491, LC-MS/MS… LC-MS/MS measurement was the same as reported recently).
Chen does not expressly teach “that the ultrafiltrates and the dialysates are subjected to solid-phase extraction before being measured by liquid chromatography-tandem mass spectrometry”
In the analogous art of Methods and systems for identification of microorganisms either after isolation from a culture or directly from a sample.
Stephenson, Jr. teaches that “the ultrafiltrates and the dialysates are subjected to solid-phase extraction before being measured by liquid chromatography-tandem mass spectrometry” (See Para 0053…Additional methods of purifying the samples may include, without limitation, liquid chromatography, HPLC, UHPLC, precipitation, solid-phase extraction, liquid-liquid extraction, dialysis, affinity capture, electrophoresis, filtration, ultrafiltration or other suitable methods known in the art, are used for the purification; See Para 0054…various methods have been described involving the use of HPLC for sample clean-up prior to mass spectrometry analysis).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Chen to include that the ultrafiltrates and the dialysates are subjected to solid-phase extraction before being measured by liquid chromatography-tandem mass spectrometry”, as taught by Stephenson, Jr., for the benefit of purifying the samples prior for sample clean-up prior to mass spectrometry analysis (Stephenson, Jr., Para 0054), allowing for the provision of methods and systems are configured to identify microorganisms based on the characterization of proteins of the microorganisms via high-resolution/mass accuracy single-stage (MS) or multi-stage (MSn) mass spectrometry. Included herein are also discussion of targeted detection and evaluation of virulence factors, antibiotic resistance markers, antibiotic susceptibility markers, typing, or other characteristics using a method applicable to substantially all microorganisms and high-resolution/mass accuracy single-stage (MS) or multi-stage (MSn) mass spectrometry (Stephenson, Jr., Abstract).
Claims 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496) in view of Stephenson, Jr. et al. (US20180233340A1) as applied to claim 5 above, and further in view of Zou et al. (US20210163525A1).
Regarding Claim 6, the method of Claim 5 is obvious over Chen in view of Stephenson, Jr.,
The combination of Chen and Stephenson, Jr. does not teach:
“wherein the solid- phase extraction for each ultrafiltrate comprises adding 20 µL of an internal standard solution to 600 µL of the ultrafiltrate, vortexing to obtain a mixture, adding the mixture to an activated HLB solid-phase extraction material, conditioning twice with 300 µL of 10% methanol aqueous solution, eluting twice with 300 µL of 90% methanol aqueous solution, merging eluents, drying the merged eluents by nitrogen, and redissolving a dried product with 100 µL of 50% methanol aqueous solution”.
In ultrasonic-assisted pretreatment method for extraction of multiple steroid hormones in a sediment, belonging to the field of analysis of trace level emerging contaminants in sediments, Zou teaches “wherein the solid- phase extraction (See Para 059…After the supernatant obtained after the three times of extraction was concentrated to obtain 1 mL of sample, 250 mL of ultrapure water was added and completely mixed; See Para 0059…an Oasis HLB solid-phase extraction column (Waters, 6 cc, 500 mg)) for each ultrafiltrate comprises adding a volume of an internal standard solution to a volume of the ultrafiltrate, vortexing to obtain a mixture, adding the mixture to an activated HLB solid-phase extraction material (See Para 0059…hen, the mixture was passed at a flow rate of 10 mL/min through an Oasis HLB solid-phase extraction column (Waters, 6 cc, 500 mg) sequentially activated with 5 mL of methanol and 5 mL of ultrapure water. ), conditioning twice with a volume of a percentage of methanol aqueous solution, eluting twice with a volume of a percentage of methanol aqueous solution, merging eluents, drying the merged eluents by nitrogen, and redissolving a dried product with a volume of percentage of methanol aqueous solution (See Para 0059… After the completion of the sample loading, impurities in the column were washed off with a 5% methanol solution, and the HLB column was blow-dried with nitrogen for 20 min.)”
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Chen and Stephenson, Jr. to include “wherein the solid- phase extraction for each ultrafiltrate comprises adding 20 µL of an internal standard solution to a volume of the ultrafiltrate, vortexing to obtain a mixture, adding the mixture to an activated HLB solid-phase extraction material, conditioning twice with a volume of a percentage of methanol aqueous solution, eluting twice with a volume of a percentage of methanol aqueous solution, merging eluents, drying the merged eluents by nitrogen, and redissolving a dried product with a volume of a percentage of methanol aqueous solution”, as taught by Zou for the benefit of providing SPE cleaning step (Zou, Para 0059), allowing for the provision of an an ultrasonic-assisted extraction method as pretreatment for quantification of multiple steroid hormones in a sediment. By selecting extractants from the combinations of two of methanol, acetonitrile and acetone, the method has the advantages of simple procedures, small amount of organic impurities in the extract, little interference and wide application, and can effectively enhance the recovery and accuracy of steroid hormone detection (Zou, Para 0004).
The combination of Chen, Stephenson, Jr. and Zou does not explicitly teach the claimed liquid/fluid volumes and the claimed percentages concentrations of methanol.
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process. In the process of sample preparation for ultrafiltration and sample extraction and equilibrium dialysis, the selection of optimal experimental conditions including buffer volumes, sample volumes, buffer concentrations affects processing time and final concentration limits and the efficiency of contaminant removal and buffer exchange.
Thus, “the claimed liquid/fluid volumes and the claimed percentages concentrations of methanol” are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide sample preparation conditions including “the claimed liquid/fluid volumes and the claimed percentages concentrations of methanol”, for the benefit of providing SPE cleaning step (Zou, Para 0059), allowing for the provision of an an ultrasonic-assisted extraction method as pretreatment for quantification of multiple steroid hormones in a sediment. By selecting extractants from the combinations of two of methanol, acetonitrile and acetone, the method has the advantages of simple procedures, small amount of organic impurities in the extract, little interference and wide application, and can effectively enhance the recovery and accuracy of steroid hormone detection (Zou, Para 0004).
Regarding Claim 7, the method of Claim 5 is obvious over Chen in view of Stephenson, Jr.,
The combination of Chen and Stephenson, Jr. does not teach:
“wherein the solid- phase extraction for each dialysate comprises adding 20 µL of an internal standard solution to 200 µL of the dialysate, vortexing to obtain a mixture, adding the mixture to an activated HLB solid-phase extraction material, conditioning twice with 300 µL of 10% methanol aqueous solution, eluting twice with 300 µL of 90% methanol aqueous solution, merging eluents, drying the merged eluents by nitrogen, and redissolving a dried product with 100 µL of 50% methanol aqueous solution”.
In ultrasonic-assisted pretreatment method for extraction of multiple steroid hormones in a sediment, belonging to the field of analysis of trace level emerging contaminants in sediments, Zou teaches “wherein the solid- phase extraction (See Para 059…After the supernatant obtained after the three times of extraction was concentrated to obtain 1 mL of sample, 250 mL of ultrapure water was added and completely mixed; See Para 0059…an Oasis HLB solid-phase extraction column (Waters, 6 cc, 500 mg)) for each dialysate comprises adding a volume of an internal standard solution to a volume of the dialysate,, vortexing to obtain a mixture, adding the mixture to an activated HLB solid-phase extraction material (See Para 0059…hen, the mixture was passed at a flow rate of 10 mL/min through an Oasis HLB solid-phase extraction column (Waters, 6 cc, 500 mg) sequentially activated with 5 mL of methanol and 5 mL of ultrapure water. ), conditioning twice with a volume of a percentage of methanol aqueous solution, eluting twice with a volume of a percentage methanol aqueous solution, merging eluents, drying the merged eluents by nitrogen, and redissolving a dried product with a volume of a percentage methanol aqueous solution (See Para 0059… After the completion of the sample loading, impurities in the column were washed off with a 5% methanol solution, and the HLB column was blow-dried with nitrogen for 20 min.)”
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Chen and Stephenson, Jr. to include “wherein the solid- phase extraction for each dialysate comprises adding a volume of an internal standard solution to a volume of the dialysate, vortexing to obtain a mixture, adding the mixture to an activated HLB solid-phase extraction material, conditioning twice with a volume of a percentage of methanol aqueous solution, eluting twice with a volume of a percentage of methanol aqueous solution, merging eluents, drying the merged eluents by nitrogen, and redissolving a dried product with a volume of a percentage of methanol aqueous solution”, as taught by Zou for the benefit of providing SPE cleaning step (Zou, Para 0059), allowing for the provision of an an ultrasonic-assisted extraction method as pretreatment for quantification of multiple steroid hormones in a sediment. By selecting extractants from the combinations of two of methanol, acetonitrile and acetone, the method has the advantages of simple procedures, small amount of organic impurities in the extract, little interference and wide application, and can effectively enhance the recovery and accuracy of steroid hormone detection (Zou, Para 0004).
The combination of Chen, Stephenson, Jr. and Zou does not explicitly teach the claimed liquid/fluid volumes and the claimed percentages concentrations of methanol.
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process. In the process of sample preparation for ultrafiltration and sample extraction and equilibrium dialysis, the selection of optimal experimental conditions including buffer volumes, sample volumes, buffer concentrations affects processing time and final concentration limits and the efficiency of contaminant removal and buffer exchange.
Thus, “the claimed liquid/fluid volumes and the claimed percentages concentrations of methanol” are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide sample preparation conditions including “the claimed liquid/fluid volumes and the claimed percentages concentrations of methanol”, for the benefit of providing SPE cleaning step (Zou, Para 0059), allowing for the provision of an an ultrasonic-assisted extraction method as pretreatment for quantification of multiple steroid hormones in a sediment. By selecting extractants from the combinations of two of methanol, acetonitrile and acetone, the method has the advantages of simple procedures, small amount of organic impurities in the extract, little interference and wide application, and can effectively enhance the recovery and accuracy of steroid hormone detection (Zou, Para 0004).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496) in view of Kobald et al. (US20190049460A1) as applied to claim 5 above, and further in view of Ghoshal et al. (US20100155595A1).
Regarding Claim 10, the method of Claim 1 is obvious over Chen.
Chen does not teach “wherein the liquid chromatography-tandem mass spectrometry is performed using an electrospray ion source in a positive ion mode, a spray voltage of 5500 V, an ion source temperature of 550°C, GS1 of 50 psi, GS2 of 50 psi, curtain gas of 30 psi, and multiple reaction monitoring for testosterone with a parent ion of m/z 289.3 and a daughter ion of m/z 97.1”
In the analogous art of a method and an apparatus for determining a concentration of a target analyte in a sample of bodily fluid, Kobold teaches “wherein the liquid chromatography-tandem mass spectrometry is performed using an electrospray ion source in a positive ion mode, a spray voltage, an ion source temperature , GS1, GS2 and curtain gas i (See Para 0101…The mass spectrometer was an AB Sciex QTrap 6500 System (BL27421409) with Selexion FAIMS module. The ion source was a turbo spray ESI (ESI—electrospray ionization) with equal source parameters for positive and negative polarity: Temperature 300° C., curtain gas 30 psi, ion spray voltage ±4300 V, ion source gas 1 60 psi, ion source gas 2 30 psi and collision gas medium. The Selexion was operated under the following conditions: temperature low, no modifier, separation voltage 3500 V, and resolution enhancement open) and multiple reaction monitoring for testosterone with a parent ion of m/z 289.3 (See Para 0103, Table 1…Testosterone 289.2)”.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Chen to include ““wherein the liquid chromatography-tandem mass spectrometry is performed using an electrospray ion source in a positive ion mode, a spray voltage, an ion source temperature of, GS1, GS2 and curtain gas and multiple reaction monitoring for testosterone with a parent ion of m/z 289.3”, as taught by Kobold for the benefit of determining the concentration of testosterone (Kobold, Para 0093), allowing for the provision of a sample of bodily fluid including the target analyte, providing an internal standard solution including a mixture of components having a plurality of isotopes of the target analyte, wherein a concentration of each isotope is unknown, adding the internal standard solution to the sample, analyzing the sample including the internal standard solution using a mass spectrometer, creating a sample function curve based on signal intensities, wherein the signal intensities define arbitrary units, transferring an analyte signal into a corresponding arbitrary analyte unit by means of the sample function curve, and transferring the arbitrary analyte unit into the concentration of a target analyte by means of a standardization function representing a curve of concentrations depending on the arbitrary units (Kobold, Abstract).
The combination of Chen and Kobold does not teach that testosterone has a parent ion of m/z 289.3 and a daughter ion of m/z 97.1”
In the analogous art of methods for measurement of certain analytes that may indicate congenital adrenal hyperplasia, in particular by tandem mass spectrometry, Ghoshal teaches that testosterone has a parent ion of m/z 289.3 and a daughter ion of m/z 97.1” (See Para 0027…one of the CAH panel analytes is testosterone. In related embodiments, one or more ions from testosterone comprise ions selected from the group consisting of ions with a mass to charge ratio (m/z) of 289.1±0.5, 109.0±0.5, and 97.0±0.5. In particularly preferred embodiments, one or more ions from testosterone comprise a precursor ion with m/z of 289.1±0.5, and one or more fragment ions selected from the group of ions with m/z of 109.0±0.5 and 97.0±0.5).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Chen and Kobold tom include “that testosterone has a parent ion of m/z 289.3 and a daughter ion of m/z 97.1”, as taught by Ghoshal for the benefit of detecting the amount of one or more CAR panel analytes (i.e., pregnenolone, 17-OH pregnenolone, progesterone, 17-OH progesterone, dehydroepiandrosterone (DHEA), androstenedione, testosterone, deoxycorticosterone, 11-deoxycortisol (also known as 11-desoxycortisol), cortisone, corticosterone, dihydrotestosterone, and cortisol) in a sample by mass spectrometry, including tandem mass spectrometry (Ghoshal, Para 0009), allowing for the provision of methods for measurement of certain analytes that may indicate congenital adrenal hyperplasia, in particular by tandem mass spectrometry (Ghoshal, Para 0002).
The combination of Chen, Kobold and Ghoshal does not explicitly teach the claimed values of “an electrospray ion source in a positive ion mode, a spray voltage of 5500 V, an ion source temperature of 550°C, GS1 of 50 psi, GS2 of 50 psi, curtain gas of 30 psi,”
However, MPEP § 2144.05, Part II, Subpart B holds that a particular parameter that is recognized as a result effective variable (“a variable that achieves a recognized result”) would be one, but not the only motivation for a person of ordinary skill in the art to experiment to reach another workable product or process. In the process of using a mass spectrometer for analyte detection/analysis, voltage controls ion movement and detection, pressure dictates how far ions travel without hitting gas molecules, and temperature helps vaporize samples and prevents condensation. These factors directly affect your signal strength, resolution, and ability to analyze fragile molecules.
Thus, “an electrospray ion source in a positive ion mode, a spray voltage of 5500 V, an ion source temperature of 550°C, GS1 of 50 psi, GS2 of 50 psi, curtain gas of 30 psi,” are a result effective variables.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to provide mass spectrometry analysis conditions including ““an electrospray ion source in a positive ion mode, a spray voltage of 5500 V, an ion source temperature of 550°C, GS1 of 50 psi, GS2 of 50 psi, curtain gas of 30 psi,”, for the benefit of providing a comparison of serum free testosterone between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS (Chen, Abstract), which allows for the provision of a simple, rapid, highly selective and sensitive method that accurately determines serum-free testosterone. The minimal sample preparation, reasonable throughput and superior specificity and sensitivity may allow this method to serve both as a reference procedure and a routine method in the routine clinical laboratory practice. (Chen, Page 495).
Response to Arguments
Applicant's arguments filed on 07/07/2026, with respect to the 35 U.S.C. §112(b) rejections on claims 2 and 6-7 have been fully considered and are persuasive.
Applicant submits that the claims are substantially amended by the present response. Amended claim 2 now recites "the plurality of serum specimens comprises 60 serum specimens". Amended claims 6 and 7 no longer recite the previously objected-to phrase "the specimen". Accordingly, the antecedent-basis issues identified in the Office Action have been obviated.
Examiner respectfully agrees and the 35 U.S.C. §112(b) rejections on claims 2 and 6-7 are withdrawn.
Applicant’s arguments, see Page 7, filed 07/07/2026, with respect to the 35 U.S.C. §101 rejections on claims 1-10 have been fully considered and are not persuasive.
Applicant submits that claim 1 is directed to a specific practical laboratory method for determining a 37°C equilibrium-dialysis-equivalent concentration of free testosterone in a serum test sample. The claim is not directed merely to a mathematical relationship or to an abstract idea of establishing a linear equation.
The Office Action stated that claim 1 recited a mathematical step of establishing a linear equation and that, once the establishment of the linear equation was done, no further action took place. The present amendments directly address that concern. Amended claim 1 expressly recites further physical and analytical steps after the conversion equation is established, including processing a serum test sample by ultrafiltration at 25°C and 2000 g for 1 hour, measuring a test ultrafiltration concentration ytest of free testosterone in the test ultrafiltrate by liquid chromatography-tandem mass spectrometry, and calculating a 37°C equilibrium-dialysis- equivalent concentration xtest according to a specific conversion formula.
The recited equation is integrated into a practical application. The equation is not claimed in isolation. It is generated from specific physical sample-processing steps and particular analytical measurements, namely ultrafiltration at 25°C, equilibrium dialysis at 37°C, and liquid
chromatography-tandem mass spectrometry measurement of free testosterone. The equation is then used in a specific laboratory workflow to transform an ultrafiltration measurement of a serum test sample into a 37°C equilibrium-dialysis-equivalent concentration of free testosterone.
Amended claim 1 also recites substantially more than generic data gathering. It requires specific laboratory operations, including preparation of serum specimens, ultrafiltration at a specified temperature, centrifugal force and duration, equilibrium dialysis at a specified temperature and duration, and liquid chromatography-tandem mass spectrometry measurement of free testosterone. The dependent claims further recite specific sample preparation, solid-phase extraction, chromatographic, and mass spectrometric conditions.
Applicant’s arguments with respect to amended claim 1-10 has been considered and Examiner respectfully disagrees.
Examiner assertively identifies the judicial exception (abstract idea) in amended Claim 1 as ““obtaining a predetermined linear conversion equation for free testosterone…(math step); “(e) establishing, based on the respective first concentrations y and the respective second concentrations x, the predetermined linear conversion equation:y = 0.8719x - 0.2116,wherein y represents a concentration of free testosterone obtained by ultrafiltration at 25°C, and x represents a concentration of free testosterone obtained by equilibrium dialysis at 37°C;” (mathematical step; step provides a mathematical equation); and “calculating the 37°C equilibrium-dialysis-equivalent concentration xtest of free testosterone in the serum test sample according to: xtest = (ytest + 0.2116)/0.8719) (mental or mathematical step)” (Step 2A Prong One).
Examiner further found that the additional elements ““ adding 900 µL; measuring, by liquid…; placing 200 µL; “measuring , by liquid…” ; “processing… and “measuring , by liquid…”” do not integrate the exception into a practical application (Step 2A Prong Two), as these additional elements appear to be insignificant post solution activities as they amount to just “data gathering” or “applying it” – both held as not practical applications (MPEP 2106.05f, MPEP 2106.05g).
Further these additional elements do not amount to significantly more than the exception (Step 2B) because they are well-understood, routine and conventional activities in the field of clinical diagnostics (See 101 rejection Supra). These elements are claimed in a merely generic manner (e.g., at a high level of generality) or as insignificant extra-solution activity: See MPEP 2106.05(d), previously known in the field of clinical diagnostics, as evidenced by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496.). (Step 2B: NO).
As a result, Examiner submits that the claims do not amount to significantly more that the judicial exception. The 101 rejection is maintained.
Applicant’s arguments, see Page 8, filed 07/07/2026, with respect to the rejection(s) of claims 1 and 3-9 under 35 U.S.C. §102 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made for amended Claim 1 by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496).
Applicant submits that as amended, independent claim 1 requires, among other limitations, determining a 37°C equilibrium-dialysis-equivalent concentration of free testosterone in a serum test sample by using a specific predetermined linear conversion equation:
y=0.8719x-0.2116,
and calculating:
xtest = (ytest + 0.2116)/0.8719.
Soldin does not disclose or suggest these features.
Soldin is directed to analysis of free thyroxine and free triiodothyronine by mass spectrometry. Soldin does not disclose the presently claimed free testosterone method. In particular, Soldin does not disclose obtaining a free-testosterone-specific conversion equation by measuring free testosterone in ultrafiltrates obtained at 25°C and in dialysates obtained at 37°C, wherein both sets of measurements are performed by liquid chromatography-tandem mass spectrometry…
Further, Soldin's comparison between ultrafiltration and equilibrium dialysis is not the same as the method of amended claim 1. Soldin compares results obtained under different sample-processing and analytical conditions. In particular, the equilibrium dialysis comparison in Soldin is followed by immunoassay, whereas amended claim 1 requires that the first concentrations and the second concentrations are both measured by liquid chromatography- tandem mass spectrometry. Therefore, Soldin's comparison includes multiple changed variables, including sample preparation, temperature, and analytical methodology. Such a comparison does not disclose the claimed free-testosterone-specific LC-MS/MS conversion equation and does not disclose applying that equation to determine a 37°C equilibrium-dialysis-equivalent concentration of free testosterone in a serum test sample.
In addition, Soldin plainly lacks the specific process parameters of amended claim 1.
Because Soldin does not disclose each and every element of amended claim 1 arranged as claimed, amended claim 1 is not anticipated by Soldin. Claims 3-9 depend directly or indirectly from amended claim 1 and are patentable for at least the same reasons.
Applicant’s arguments with respect to amended claim 1 has been considered and Examiner respectfully disagrees.
Examiner submits that the limitations of amended Claim 1 is taught as disclosed in the rejection of amended Claim 1 (Supra) by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496).
In addition, Claims 2-10 are rejected by virtue of dependency on Claim 1.
Applicant’s arguments, see Page 8, filed 07/07/2026, with respect to the rejection(s) of claim 2 under 35 U.S.C. §103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made for amended Claim 2 in view of Claim 1 by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496).
Applicant submits that Amended claim 2 depends from amended claim 1 and further recites that the plurality of serum specimens comprises 60 serum specimens and that the predetermined linear conversion equation has a coefficient of determination R2 of 0.9897.
Neither Soldin nor Jonklaas discloses or suggests the features of amended claim 2. Soldin is directed to thyroid hormone analysis and does not disclose the presently claimed free testosterone conversion method. Jonklaas compares total and free thyroxine and triiodothyronine measurements and discusses discrepancies between assay methodologies, particularly in inpatients. Jonklaas does not disclose or suggest establishing a free-testosterone-specific conversion equation from 25°C ultrafiltration results and 37°C equilibrium dialysis results, nor does it disclose or suggest the specific equation y = 0.8719x - 0.2116, R2 = 0.9897, or the calculation xtest = (ytest + 0.2116)/0.8719…
Moreover, the claimed method is not a routine substitution of one analytical method for another. The specification demonstrates that the conversion equation is obtained under defined sample-processing and analytical conditions. Soldin's thyroid hormone comparison does not provide a reasonable expectation that the specific free testosterone equation recited in the claims would be obtained, or that it could be used to convert a 25°C ultrafiltration measurement into a 37°C equilibrium-dialysis-equivalent concentration.
Accordingly, amended claim 2 is patentable over Soldin in view of Jonklaas.
Applicant’s arguments with respect to amended claim 2 has been considered and Examiner respectfully disagrees.
Examiner submits that the limitations of amended Claim 2 is taught as disclosed in the rejection of amended Claim 2 (Supra) by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496).
In addition, Claim 3 is rejected by virtue of dependency on Claim 2.
Applicant’s arguments, see Page 10, filed 07/07/2026, with respect to the rejection(s) of claim 10 under 35 U.S.C. §103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made for amended Claim 10 in view of Claim 1 by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496) in view of Kobald et al. (US20190049460A1) as applied to claim 5 above, and further in view of Ghoshal et al. (US20100155595A1).
Applicant submits that Amended claim 10 now depends from amended claim 1 and further recites specific liquid chromatography-tandem mass spectrometry conditions for measuring testosterone, including an
electrospray ion source in a positive ion mode, a spray voltage of 5500 V, an ion source temperature of 550°C, GS 1 of 50 psi, GS2 of 50 psi, curtain gas of 30 psi, and multiple reaction monitoring for testosterone with a parent ion of m/z 289.3 and a daughter ion of m/z 97.1.
Soldin does not disclose the amended free testosterone conversion method of claim 1, nor does it disclose the specific testosterone measurement conditions recited in amended claim 10 in combination with the claimed 25°C ultrafiltration-to-37°C equilibrium dialysis conversion. The rejection therefore fails for at least the same reasons discussed above with respect to amended claim 1.
Applicant’s arguments with respect to amended claim 10 has been considered and Examiner respectfully disagrees.
Examiner submits that the limitations of amended Claim 10 is taught as disclosed in the rejection of amended Claim 10 (Supra) by Chen et al. ("Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry." Clinical biochemistry 43.4-5 (2010): 490-496) in view of Kobald et al. (US20190049460A1) as applied to claim 5 above, and further in view of Ghoshal et al. (US20100155595A1).
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 OYELEYE ALEXANDER ALABI whose telephone number is (571)272-1678. The examiner can normally be reached on M-F 7:30am-5:30pm.
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/OYELEYE ALEXANDER ALABI/ Examiner, Art Unit 1797
