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 01/30/2026 has been entered. Claims 1-5, 9-12, 15, 17, 19, 22-24, 27-28, 31, 97, 98, 101, and 102 are pending in the application. Claim 29 has been cancelled without prejudice or disclaimer. Claims 1,2,4 and 23 have been amended. Claims 6-8, 13, 14, 16, 18, 20, 21, 25, 26, 30, 32-96, 99, 100, and 103-116 have previously been canceled without prejudice or disclaimer.
Status of Objections and Rejections
The objection to the claims has been withdrawn in view of Applicant's amendment.
All rejections under 35 U.S.C. 112(b) are withdrawn in view of Applicant's amendment.
The rejection of claim 29 under 35 U.S.C. 103 as being unpatentable over Kraemer in view of Feige and Gjerde is obviated by Applicant's cancellation.
All rejections from the previous office action under 35 U.S.C. 103 are maintained.
New grounds of rejection under 35 U.S.C. 103 are necessitated by the amendments.
Response to Arguments
Applicant's arguments, see pages 6-13, filed 01/30/2026, with respect to the rejections of claims 1-5, 9-12, 15, 17, 19, 22-24, 27-29, 31, 97, 98, 101, and 102 under 35 U.S.C. 103, have been fully considered but they are not persuasive.
Applicant argues (pp. 7-8) that amended claims 1,2, and 4 now require that the biological samples are urine samples, and therefore the cited combination of Kraemer, Feige, and Gjerde fail to teach or suggest the claimed method. Applicant contends that Kraemer only describes SOMamer assays using serum or plasma samples and contains no disclosure suggesting that its methods could be applied to urine. Accordingly, Applicant asserts that the cited references render the amended claims obvious.
The Examiner respectfully disagrees.
The argument is not persuasive because Kraemer’s disclosure of performing the assay on serum or plasma does not limit the method to those specific fluids nor discourage its application to other biological sample types. Kraemer explains that the SOMAmer assay was developed as a general multiplex platform, where target analytes were chosen arbitrarily from a complete SOMAmer menu to be representative of proteins that are most abundant within plasma or serum samples (p. 3, col. 1, Results). These listed target proteins are also well-known urine biomarkers. The assay further included control SOMAmers to monitor plasma and serum-dependent effects as a test for whether the platform was designed to handle matrix variability. This shows that the assay was designed to work across different biological sample types, with controls specifically included to account for differences in sample fluids rather than being limited to plasma or serum. Therefore, a person of ordinary skill in the art would have reasonably expected that SOMAmer reagents used to detect proteins in plasma or serum could also be used to detect the same proteins in urine samples and yield predictable results (See MPEP 2143(I)(B)).
Applicant argues (p. 8) that the Examiner’s reliance on Feige is improper because Feige operates in a different experimental context than Kraemer. Specifically, Feige analyzes mechanically lysed muscle tissue, where solid tissue is pulverized and intracellular proteins are extracted. Protein concentration is then measured using a BCA assay and diluted to 250 ug/mL. This workflow involves tissue lysis and lysate dilution, which the applicant asserts is unrelated to Kraemer’s plasma/serum SOMAmer assays, or claimed methods, which involve biological fluids (e.g., urine) and exclude concentrating protein prior to buffer exchange. Therefore, the Applicant contends that a person of ordinary skill in the art would not be motivated to combine Kraemer with Feige’s tissue-lysis workflow.
The Examiner respectfully disagrees.
The Examiner relies on Feige only for the well-known step of measuring and normalizing protein concentration using a BCA assay, not for its specific tissue lysis procedure. Feige demonstrates that, once a biological sample containing proteins is obtained, it was routine in the art to determine total protein concentration and adjust samples to a consistent working concentration (e.g., by dilution) prior to downstream analysis. This teaching is methodologically general and does not depend on the particular biological source (muscle lysate, plasma, serum, or urine) as BCA assays are broadly used for quantifying protein in diverse biological matrices. A person of ordinary skill in the art would therefore have recognized that the same normalization principle could be applied to Kraemer’s samples in order to ensure comparable protein levels across assays, which improves analytical reproducibility. Accordingly, the Examiner’s combination does not require adopting Feige’s tissue lysis protocol, but merely its routine practice of quantifying protein and adjusting samples to a consistent concentration, rendering the claimed adjustment step obvious.
Applicant argues (pp. 8-9) that the Examiner’s result-effective variable rationale for the claimed 70-100 ug/mL protein concentration range (claims 2 and 4) is improper for two reasons, different sample context and unexpected results. Applicant argues that Feige discloses lysed muscle tissue samples diluted to 250 ug/mL after BCA measurement. The Applicant asserts this context differs from Kraemer, which involves plasma/serum assays, and from the amended claims now directed to urine samples. Therefore, Feige allegedly does not provide a meaningful starting point for determining the claimed 70-100 ug/mL and that the 70-100 ug/mL range yields the best balance of signal strength and linear response for the largest number of analytes (Example 7; Figs. 12A-12B). The Applicant argues this demonstrates the range is not routine optimization but rather a non-obvious range that maximizes multiplex assay performance, and that the cited references do not suggest this range.
The Examiner respectfully disagrees.
Applicant’s argument regarding Example 7 is not persuasive. The cited portion of the specification indicates that linear assay performance occurs across a broad concentration range of approximately 2 ug/mL to 200 ug/mL ([0200]). Disclosing that the range of 70-100 ug/mL provides relatively strong RFU signals and a greater number of analytes within the linear range merely identifies a preferred operating region within this already recognized workable range. Selecting a narrower working range within a known operable interval to balance competing assay parameters (e.g., signal intensity versus linearity) constitutes routine optimization of a result effective variable, which would have been well known by a person of ordinary skill in the art (See MPEP 2144.04).
In particular, once the art recognizes that total protein concentration influences assay signal intensity and linear dynamic range (Median normalized…(RFU) were log 2+transformed before applying [PCA] and linear models; Feige, [0231]), a person of ordinary skill in the art would reasonably expect that different concentrations within the workable interval will produce predictable tradeoffs between sensitivity and linearity. It therefore would have been routine to evaluate concentration within the disclosed 2-200 ug/mL range and select a concentration or sub-range that provides suitable assay performance. Identifying a subrange that yields relatively stronger RFU signals while maintaining acceptable linearity does not demonstrate that the claimed range is critical or unexpected, but rather reflects the expected outcome of routine assay optimization.
Moreover, the instant specification itself characterizes the broader interval as producing a linear range for many analytes, indicating that the assay remains functional throughout the wide spectrum of concentrations ([0200]). The identification of a narrower preferred range that improves performance for a larger number of analytes therefore reflects a degree-of-performance optimization rather than the discovery of a new or unexpected phenomenon occurring uniquely within the claimed 70-100 ug/mL interval. Evidence of improved results within a subset of an otherwise functional range does not establish non-obviousness absent a showing that the claimed range produces unexpected properties or results that differ in kind, rather than merely in degree, from the surrounding concentrations.
Finally, the Applicant’s assertion that the prior art does not disclose the precise 70-100 ug/mL range is not persuasive. The relevant inquiry is not whether the exact range is taught, but whether a person of ordinary skill in the art would have found it obvious to adjust the protein concentration within a known workable range to achieve optimal assay performance. Feige teaches measuring protein concentration of serum samples that were analyzed via the same SOMAscan platform as the instant application, using the same BCA assay, and adjusting samples to a desired working concentration prior to analysis. The selection of an intermediate concentration range that balances signal strength and linearity would have been a predictable and routine matter of optimization. Accordingly, the data cited in Example 7 do not demonstrate that the claimed 70-100 ug/mL range is critical or yields truly unexpected results sufficient to overcome the prima facie case of obviousness.
Applicant argues (pp. 9-10) that there is no motivation to combine the teachings of Kraemer, Feige, and Gjerde, and that the references allegedly teach away from the Examiner’s proposed combination. Kramer prepares samples by serially diluting serum in SB17 buffer (e.g., 6%, 3%. 0.6%, 0.3%). Because all samples are diluted into the same buffer, the Applicant argues the samples would already have uniform buffer composition, eliminating the need for buffer exchange. Kraemer also reports reproducible assay performs (CV 11-12%), which the Applicant asserts shows buffer composition variability is not a problem and therefore Kraemer teaches away from buffer exchange. Feige involves mechanical lysis of muscle tissue, detergent extraction of intracellular proteins, and normalization of lysate concentration. The Applicant contends this context is fundamentally different from biofluid assays such as serum, plasma, or urine, and therefore would not motivate modification of Kraemer’s SOMAmer assays. Gjerde is directed to cell purification and enrichment, particularly separation of intact cells such as circulating tumor cells using size-based methods. The Applicant argues that because Gjerde focuses on cell viability and purification, its gel filtration methods would not be applied to diluted serum samples. Based on these points, the Applicant concludes that the cited references do not provide a motivation to combine and therefore the claims are not obvious.
The Examiner respectfully disagrees.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
The arguments is not persuasive because it improperly requires that the references address identical experimental contexts or explicitly suggest the precise combination proposed by the Examiner. Obviousness does not require that each reference be direct to the same biological sample type or assay, but rather the references collectively teach known techniques that person of ordinary skill in the art would reasonably apply in combination.
Firstly, Kraemer’s dilution of serum samples into SB17 buffer does not teach away from buffer exchange. Kraemer merely describes one sample preparation approach and reports acceptable assay reproducibility under those conditions. However, Kraemer does not criticize, discourage, or discredit the use of additional sample preparation techniques such as buffer exchange or protein normalization. The fact that Kraemer’s method functions without buffer exchange does not mean that such steps would have been considered undesirable or inapplicable. A reference teaches away only when it suggests that the proposed modification would be unsuitable or detrimental, which Kraemer does not do.
Secondly, the examiner does not rely on Feige for its tissue-lysis methodology, but rather for the general and well-established practice of measuring total protein concentration (e.g., via BCA assay) and normalizing samples to a defined concentration prior to proteomic analysis. This principle is broadly applicable to protein assays regardless of the biological source of the proteins. A person of ordinary skill in the art would have recognized that normalizing protein concentration is a common technique used to improve comparability between samples, and this teaching is not limited to muscle lysates.
Thirdly, although Gjerde discusses separation of intact cells, it nevertheless teaches size based separation techniques such as gel filtration that can be sued to separate biological components and perform buffer exchange. The relevance of Gjerde lies in the underlying separation technique, not in the particular biological material being processed. Techniques developed for one biological system are frequently applied to others when they address the same technical problem, such as separating macromolecules from smaller components or exchanging buffers. Finally, the Applicant’s argument improperly analyzes the references in isolation rather than considering what they collectively would have suggested to a person of ordinary skill in the art. Kraemer demonstrates multiplex protein detection in biological samples, Feige teaches protein quantification and normalization, and Gjerde teaches gel filtration-based separation methods. Taken together, these teachings would have suggested applying known sample preparation techniques, including buffer exchange and protein normalization to prepare biological samples for proteomic analysis. Accordingly, the cited references do not teach away from the proposed combination, and the Examiner’s rationale for combining their teachings remains sound. The rejection under 35 U.S.C. 103 is therefore maintained.
Applicant argues (pp. 11-12) that claims 23 and 102 are patentable because they ultimately depend from claim 2 and the combination of Kraemer, Feige, and Gjerde allegedly fails to teach adjusting protein concentration to about 70-100 ug/ml in urine samples. Applicant contends that the additional references, Webber for claim 23 and Neeser for claim 102 do not remedy this alleged deficiency because they also do not disclose the claimed concentration range or urine sample context. Accordingly, Applicant maintains that the rejections of claims 23 and 102 should be withdrawn.
The Examiner respectfully disagrees.
This argument is not persuasive because it does not address the specific limitations for which Webber and Neeser are cited. Webber is relied upon solely to teach the use of a micro-BCA assay and its associated reagents, and Neeser is relied upon solely to teach fluorescence-based total protein assays using reagents such as epicocconone, both of which represent routine protein quantification techniques that would have been obvious to apply in the sample preparation workflow taught by Kraemer, Feige, and Gjerde. Applicant’s arguments merely repeat the contention regarding the 70-100 ug/mL concentration range and urine samples form claim 2, which has already been addressed, and therefore do not rebut the Examiner’s rationale for combining the cited references to teach the additional assay limitations of claims 23 and 102.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1,2,4-5,9-12,15,17,19,22,24,27-28,31,97-98, and 101 are rejected under 35 U.S.C. 103 as being unpatentable over Kraemer et al. (“From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay”; 2011) in view Feige et al. (US 20190255149 A1, EFD 2017-06-01) and in further view of Gjerde (US 20180178142 A1).
Regarding claim 1, Kraemer teaches a method for preparing a plurality of biological samples (Sample preparation…serum…samples; page 10, column 2, paragraph 1, lines 1-3) for detecting a protein (“to detect high and low-abundance analytes,” wherein these analytes are the proteins of Table 2; page 10, column 2, paragraph 1, last 2 lines) comprising:
generating a plurality of test samples (“A portion of the 6% serum stock solution was diluted 10-fold in SB17 to create a 0.6% serum stock,” wherein the test samples are the 6% and 0.6% stock solutions; page 10, column 2, paragraph 1, line 5) using a formulation (“SB17,” wherein the SB17 is SB18 with additional supplements; page 10, column 2, paragraph 1, line 5; page 10, column, paragraph 3, line 3) comprising a buffering agent (HEPES; page 10, column 1, paragraph 3, line 1), one or more salts (NaCl; page 10, column 1, paragraph 3, line 1), a chelating agent (EDTA; page 10, column 1, paragraph 3, line 4) and a nonionic surfactant (Tween 20; page 10, column 1, paragraph 3, line 2), and
generating a plurality of adjusted test samples by adjusting the total protein concentrations of the plurality of test samples (Heat-cooled 2 X SOMAmer mixes (55 mL) were combined with an equal volume of 6% or 0.6% serum dilutions, producing equilibration mixes containing 3% and 0.3% serum; page 10, column 2, paragraph 3, lines 1-7)(These equilibrium mixes were later combined into the wells of a 96-well streptavidin plate (each well now containing both the 3% and 0.3% serum samples), thereby creating a plurality of adjusted test samples that now have a new total protein concentration compared to the 6% and 0.6% test samples; page 11, column 1, paragraph 1, lines 13-16)(The 3% and the 0.3% equilibrium mixes are an intermediate total protein adjustment, and the mixture of the two are the adjusted test samples),
wherein the method does not comprise concentrating the total protein of the plurality of biological samples prior to performing the buffer exchange (Kraemer is free of any teaching of concentrating the total protein of the plurality of biological samples prior to performing the buffer exchange and therefore meets this negative limitation).
Kraemer fails to teach performing a buffer exchange, the total protein concentration of each adjusted test sample is about the same, and the plurality of biological samples are urine samples.
Kraemer instead teaches performing a dilution using a buffer formulation (resulting in a 6% serum sample solution) (page 10, column 2, paragraph 1, line 5) and only states that both equilibrium mixes are combined into each well without stating the ratios of the 3% and 0.3% dilutions (page 11, column 1, paragraph 1, lines 13-16). Kraemer also instead mentions the sample matrix to be serum (p. 10, col. 2, Sample Preparation).
Feige teaches the total protein concentration of each adjusted test sample is about the same (“Protein concentration was determined by a BCA assay and samples were diluted at 250 μg/mL,” wherein the BCA naturally measures the total protein concentration; [0231]]).
Feige is considered to be analogous to the claimed invention because it is in the same field of endeavor for biological fluid analysis for protein biomarker discovery using sample normalization and assay reproducibility strategies. 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 adjusted test samples taught by Kraemer to have about the same total protein concentration as taught by Feige. Kraemer aims “to normalize SOMAmer conformer distributions and thus ensure reproducible SOMAmer activity in spite of variable histories”. Standardizing the total protein concentration in the test samples prior to analysis is merely the application of a known technique to the known problem, inter-sample variability, to achieve the predictable improvement of inter-sample comparability (See MPEP 2143(I)(A)).
Kraemer fails to teach performing a buffer exchange and that the plurality of biological samples are urine samples. Kraemer instead mentions the sample matrix to be serum (p. 10, col. 2, Sample Preparation).
Gjerde teaches performing a buffer exchange (gel filtration can be used for buffer exchange; [0299]) and that the plurality of biological samples are urine samples (The sample can be from any biological source…including…urine).
Gjerde is considered to be analogous to the claimed invention because it is in the same field of endeavor for purifying and detecting biological cells from a complex biological sample (Abstract). 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 substituted the direct buffer dilution step applied to serum samples as taught by Kraemer in view of Feige with Gjerde’s gel-filtration-based buffer exchange as applied to urine samples. Gjerde states that buffer exchange can be used “to enrich a particular cell type by separating cells away from non-cell components or by separating cells from each other based on their size” ([0299]) which would be useful in detecting proteins at a lower concentration without altering the functional purpose (Kraemer, page 10, paragraph 1, last 2 lines). Both techniques were well-known and interchangeable methods and would have yielded the predictable result of reduced sample interference (See MPEP 2143(I)B)). Additionally, Kraemer explains that the SOMAmer assay was developed as a general multiplex platform, where target analytes were chosen arbitrarily from a complete SOMAmer menu to be representative of proteins that are most abundant within plasma or serum samples (p. 3, col. 1, Results). These listed target proteins are also well-known urine biomarkers. The assay further included control SOMAmers to monitor plasma and serum-dependent effects as a test for whether the platform was designed to handle matrix variability. This shows that the assay was designed to work across different biological sample types, with controls specifically included to account for differences in sample fluids rather than being limited to plasma or serum. Therefore, a person of ordinary skill in the art would have reasonably expected that SOMAmer reagents used to detect proteins in plasma or serum could also be used to detect the same proteins in urine samples and yield predictable results (See MPEP 2143(I)(B)).
Regarding claim 2, Kraemer teaches a method for preparing a biological sample (Sample preparation…serum…samples; page 10, column 2, paragraph 1, lines 1-3) for detecting a protein (“to detect high and low-abundance analytes,” wherein these analytes are the proteins of Table 2; page 10, column 2, paragraph 1, last 2 lines) comprising:
generating a test sample (“A portion of the 6% serum stock solution was diluted 10-fold in SB17 to create a 0.6% serum stock,” wherein the test samples are the 6% and 0.6% stock solutions; page 10, column 2, paragraph 1, line 5) using a formulation (“SB17,” wherein the SB17 is SB18 with additional supplements; page 10, column 2, paragraph 1, line 5; page 10, column, paragraph 3, line 3) comprising a buffering agent (HEPES; page 10, column 1, paragraph 3, line 1), one or more salts (NaCl; page 10, column 1, paragraph 3, line 1), a chelating agent (EDTA; page 10, column 1, paragraph 3, line 4) and a nonionic surfactant (Tween 20; page 10, column 1, paragraph 3, line 2);
generating an adjusted test sample by adjusting the total protein concentration of the test sample (Heat-cooled 2 X SOMAmer mixes (55 mL) were combined with an equal volume of 6% or 0.6% serum dilutions, producing equilibration mixes containing 3% and 0.3% serum; page 10, column 2, paragraph 3, lines 1-7)(These equilibrium mixes were later combined into the wells of a 96-well streptavidin plate (each well now containing both the 3% and 0.3% serum samples), thereby creating a plurality of adjusted test samples that now have a new total protein concentration compared to the 6% and 0.6% test samples; page 11, column 1, paragraph 1, lines 13-16)(The 3% and the 0.3% equilibrium mixes are an intermediate total protein adjustment, and the mixture of the two are the adjusted test samples) to a final total protein concentration (the combined equilibrium mixes naturally create a final total protein concentration).
Kraemer fails to teach performing a buffer exchange, that the final total protein concentration in the adjusted test sample is from about 70 µg/mL to about 100 µg/mL, and that the plurality of biological samples are urine samples.
Kraemer instead teaches performing a dilution using a buffer formulation (resulting in a 6% serum sample solution) and only states that both equilibrium mixes are combined into each well without stating the ratios of the 3% and 0.3% dilutions nor total protein concentration. Kraemer also instead mentions the sample matrix to be serum (p. 10, col. 2, Sample Preparation).
Gjerde teaches performing a buffer exchange (gel filtration can be used for buffer exchange; [0299]) and that the plurality of biological samples are urine samples (The sample can be from any biological source…including…urine).
Gjerde is considered to be analogous to the claimed invention because it is in the same field of endeavor for purifying and detecting biological cells from a complex biological sample (Abstract). 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 substituted the direct buffer dilution step taught by Kraemer with Gjerde’s gel-filtration-based buffer exchange. Gjerde states that buffer exchange can be used “to enrich a particular cell type by separating cells away from non-cell components or by separating cells from each other based on their size” ([0299]) which would be useful in detecting proteins at a lower concentration without altering the functional purpose (Kraemer, page 10, paragraph 1, last 2 lines). Both techniques were well known and interchangeable methods and would have yielded the predictable result of reduced sample interference (See MPEP 2143(I)B)). Additionally, Kraemer explains that the SOMAmer assay was developed as a general multiplex platform, where target analytes were chosen arbitrarily from a complete SOMAmer menu to be representative of proteins that are most abundant within plasma or serum samples (p. 3, col. 1, Results). These listed target proteins are also well-known urine biomarkers. The assay further included control SOMAmers to monitor plasma and serum-dependent effects as a test for whether the platform was designed to handle matrix variability. This shows that the assay was designed to work across different biological sample types, with controls specifically included to account for differences in sample fluids rather than being limited to plasma or serum. Therefore, a person of ordinary skill in the art would have reasonably expected that SOMAmer reagents used to detect proteins in plasma or serum could also be used to detect the same proteins in urine samples and yield predictable results (See MPEP 2143(I)(B)).
Kraemer fails to teach the final total protein concentration in the adjusted test sample is from about 70 µg/mL to about 100 µg/mL.
Feige teaches the total protein concentration in the adjusted test sample is 250 µg/mL (“Protein concentration was determined by a BCA assay and samples were diluted at 250 μg/mL,” wherein the BCA naturally measures the total protein concentration; [0231]])
Feige is considered to be analogous to the claimed invention because it is in the same field of endeavor for analyzing proteins in biological samples using the SOMAscan platform by normalizing the total protein concentration (Abstract; [0231]). 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 preparation method taught by Kraemer in view of Gierde by incorporating the teachings of Feige to adjust the test samples to a specific total protein concentration in order to detect both high and low abundance analytes (Kraemer, page 10, paragraph 1, last 2 lines). Although the 250 ug/mL total protein concentration taught by Feige is not within the claimed ranged of 70-100 ug/mL, this is a result-effective variable that would have been modified to achieve the desired protein recovery and quantitation. It is standard practice to dilute or concentrate samples so that their total analyte concentration falls within the middle of a validated linear calibration range with a CV<20% (Kraemer, page 6, column 1, paragraph 1). Kraemer aims to “detect high and low-abundance analytes” (page 10, paragraph 2, last 2 lines), so optimizing the sample concentration to fit in between the upper and lower limits of quantification would have been obvious in order to produce measurable, proportional signals without saturating the detection system (Fig. 3; Table 2; Abstract)(See MPEP 2144.05(II)).
Regarding claim 3, Modified Kramer teaches the method of claim 2.
Modified Kraemer fails to teach determining the total protein concentration of the test sample and/or the biological sample.
Feige teaches determining the total protein concentration of the test sample (“Protein concentration was determined by a BCA assay,” wherein the BCA naturally measures the total protein concentration; [0231]]).
Feige is considered to be analogous to the claimed invention because it is in the same field of endeavor for analyzing proteins in biological samples using the SOMAscan platform by normalizing the total protein concentration (Abstract; [0231]). 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 preparation method taught by Kraemer in view of Gierde and Feige by further incorporating the teachings of Feige to determine the total protein concentration of the test sample. Doing so would verify the initial and new serum sample % after the buffer exchange, accounting for any loss within the column before further dilutions are made for the SOMAscan analysis. Determining the total protein concentration of the test sample is merely an application of a known analytical technique to a known problem, inter-sample variability, and would yield the predictable result of uniform assay input without altering the functional purpose of the method (See MPEP 2143 (I)(A)).
Regarding claim 4, Kraemer teaches a method for preparing a biological sample (Sample preparation…serum…samples; page 10, column 2, paragraph 1, lines 1-3) for detecting a protein (“to detect high and low-abundance analytes,” wherein these analytes are the proteins of Table 2; page 10, column 2, paragraph 1, last 2 lines) comprising:
generating a test sample (“A portion of the 6% serum stock solution was diluted 10-fold in SB17 to create a 0.6% serum stock,” wherein the test samples are the 6% and 0.6% stock solutions; page 10, column 2, paragraph 1, line 5) using a formulation (“SB17,” wherein the SB17 is SB18 with additional supplements; page 10, column 2, paragraph 1, line 5; page 10, column, paragraph 3, line 3) comprising a buffering agent (HEPES; page 10, column 1, paragraph 3, line 1), one or more salts (NaCl; page 10, column 1, paragraph 3, line 1), a chelating agent (EDTA; page 10, column 1, paragraph 3, line 4) and a nonionic surfactant (Tween 20; page 10, column 1, paragraph 3, line 2);
generating an adjusted test sample by adjusting the total protein concentration of the test sample (Heat-cooled 2 X SOMAmer mixes (55 mL) were combined with an equal volume of 6% or 0.6% serum dilutions, producing equilibration mixes containing 3% and 0.3% serum; page 10, column 2, paragraph 3, lines 1-7)(These equilibrium mixes were later combined into the wells of a 96-well streptavidin plate (each well now containing both the 3% and 0.3% serum samples), thereby creating a plurality of adjusted test samples that now have a new total protein concentration compared to the 6% and 0.6% test samples; page 11, column 1, paragraph 1, lines 13-16)(The 3% and the 0.3% equilibrium mixes are an intermediate total protein adjustment, and the mixture of the two are the adjusted test samples) to a final total protein concentration (the combined equilibrium mixes naturally create a final total protein concentration).
Kraemer fails to teach performing a buffer exchange, determining the total protein concentration of the test sample, that the final total protein concentration in the adjusted test sample is from about 70 µg/mL to about 100 µg/mL, and that the plurality of biological samples are urine samples.
Kraemer instead teaches performing a dilution using a buffer formulation (resulting in a 6% serum sample solution) and only states that both equilibrium mixes are combined into each well without stating the ratios of the 3% and 0.3% dilutions nor total protein concentration. Kraemer also instead mentions the sample matrix to be serum (p. 10, col. 2, Sample Preparation).
Gjerde teaches performing a buffer exchange (gel filtration can be used for buffer exchange; [0299]).
Gjerde is considered to be analogous to the claimed invention because it is in the same field of endeavor for purifying and detecting biological cells from a complex biological sample (Abstract). 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 substituted the direct buffer dilution step taught by Kraemer with Gjerde’s gel-filtration-based buffer exchange. Gjerde states that buffer exchange can be used “to enrich a particular cell type by separating cells away from non-cell components or by separating cells from each other based on their size” ([0299]) which would be useful in detecting proteins at a lower concentration without altering the functional purpose (Kraemer, page 10, paragraph 1, last 2 lines). Both techniques were well known and interchangeable methods and would have yielded the predictable result of reduced sample interference (See MPEP 2143(I)B)). Additionally, Kraemer explains that the SOMAmer assay was developed as a general multiplex platform, where target analytes were chosen arbitrarily from a complete SOMAmer menu to be representative of proteins that are most abundant within plasma or serum samples (p. 3, col. 1, Results). These listed target proteins are also well-known urine biomarkers. The assay further included control SOMAmers to monitor plasma and serum-dependent effects as a test for whether the platform was designed to handle matrix variability. This shows that the assay was designed to work across different biological sample types, with controls specifically included to account for differences in sample fluids rather than being limited to plasma or serum. Therefore, a person of ordinary skill in the art would have reasonably expected that SOMAmer reagents used to detect proteins in plasma or serum could also be used to detect the same proteins in urine samples and yield predictable results (See MPEP 2143(I)(B)).
Kraemer fails to teach determining the total protein concentration of the test sample, that the total protein concentration in the adjusted test sample is from about 70 µg/mL to about 100 µg/mL.
Feige teaches determining the total protein concentration of the test and that the total protein concentration in the adjusted test sample is 250 µg/mL (“Protein concentration was determined by a BCA assay and samples were diluted at 250 μg/mL,” wherein the BCA naturally measures the total protein concentration; [0231]])
Feige is considered to be analogous to the claimed invention because it is in the same field of endeavor for analyzing proteins in biological samples using the SOMAscan platform by normalizing the total protein concentration (Abstract; [0231]). 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 preparation method taught by Kraemer in view of Gierde by incorporating the teachings of Feige to determine the total protein concentration of the test sample after the buffer exchange before sample adjustment, as well as adjusting the test samples to a specific total protein concentration in order to detect low abundance analytes. Measuring the sample concentration can be used to verify the initial and new serum sample % after the buffer exchange, accounting for any loss within the column before further dilutions are made for the SOMAscan analysis. Both determining the total protein concentration of the test sample and adjusting the sample to a specific concentration are merely applications of known analytical techniques to a known problem, inter-sample variability, and would yield the predictable result of uniform assay input without altering the functional purpose of the method (See MPEP 2143 (I)(A)). Although the 250 ug/mL total protein concentration taught by Feige is not within the claimed ranged of 70-100 ug/mL, this is a result-effective variable that would have been modified to achieve the desired protein recovery and quantitation. It is standard practice to dilute or concentrate samples so that their total analyte concentration falls within the middle of a validated linear calibration range with a CV<20% (page 6, column 1, paragraph 1). Kraemer aims to “detect high and low-abundance analytes” (page 10, paragraph 2, last 2 lines), so optimizing the sample concentration to fit in between the upper and lower limits of quantification would have been obvious in order to produce measurable, proportional signals without saturating the detection system (Fig. 3; Table 2; Abstract)(See MPEP 2144.05(II)).
Regarding claim 5, Modified Kramer teaches the method of claim 2, comprising generating a plurality of test samples by performing a buffer exchange on a plurality of biological samples (the “100 mL aliquots” taught by Kraemer would undergo the buffer exchange taught by Gjerde, creating a plurality of test samples; page 10, column 2, paragraph 1, line 1), wherein the total protein concentration of each adjusted test sample is about the same (Feige, “Protein concentration was determined by a BCA assay and samples were diluted at 250 μg/mL,” wherein the BCA naturally measures the total protein concentration; [0231]]).
Regarding claim 9, Modified Kramer teaches the method of claim 2, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt and a magnesium salt (Kraemer, MgCl2; page 10, column 2, paragraph 1, line 5).
Regarding claim 10, Modified Kramer teaches the method of claim 2, wherein the one or more salts comprise a sodium salt (Kraemer, “NaCl,” wherein “buffer SB17 is SB18”; page 10, column 1, paragraph 3, lines 1,3), a potassium salt (Kraemer, KCl; page 10, column 1, paragraph 3, line 2) and a magnesium salt (Kraemer, MgCl2; page 10, column 2, paragraph 1, line 5).
Regarding claim 11, Modified Kramer teaches the method of claim 9, wherein the sodium salt is NaCl (Kraemer, “NaCl,” wherein “buffer SB17 is SB18”; page 10, column 1, paragraph 3, lines 1,3), the potassium salt is KCl (Kraemer, KCl; page 10, column 1, paragraph 3, line 2) and the magnesium salt is MgCl2 (Kraemer, MgCl2; page 10, column 2, paragraph 1, line 5).
Regarding claim 12, Modified Kramer teaches the method of claim 11, wherein the NaCl in the formulation is at a concentration of from about 10 mM to about 500 mM, or from about 50 mM to about 250 mM, or from about 100 mM to about 200 mM, or from about 75-125 mM, or about 102 mM, and/or wherein the KCl in the formulation is at a concentration of from about 0.5 mM to about 30 mM, or from about 1 mM to about 20 mM, or from about 2 mM to about 15 mM, or from about 4 mM to about 10 mM, or about 5 mM (Kraemer, 5 mM KCl; page 10, column 1, paragraph 3, line 2), and/or wherein the MgCl2 in the formulation is at a concentration of from about 0.5 mM to about 30 mM, or from about 1 mM to about 20 mM, or from about 2 mM to about 15 mM, or from about 4 mM to about 10 mM, or about 5 mM.
Regarding claim 15, Modified Kramer teaches the method of claim 2, wherein the buffering agent is selected from HEPES (Kraemer, HEPES; page 10, column 1, paragraph 3, line 1), MES, Bis-tris methane, ADA, ACES, Bis-tris propane, PIPES, MOPSO, Cholamine chloride, MOPS, BES, TES, DIPSO, MOB, Acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, Glycinamide, Glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS and CABS.
Regarding claim 17, Modified Kramer teaches the method of claim 2, wherein the chelating agent is selected from EDTA (Kraemer, EDTA; page 10, column 1, paragraph 3, line 4), EGTA, DTPA, BAPTA, DMPS and ALA.
Regarding claim 19, Modified Kramer teaches the method of claim 2, wherein the nonionic surfactant is selected from Polyoxyethylene (20) sorbitan monolaurate (Tween-20) (Kraemer, Tween 20; page 10, column 1, paragraph 3, line 2), Polyoxyethylene (40) sorbitan monolaurate (Tween-40) and Polyoxyethylene (80) sorbitan monolaurate (Tween-80).
Regarding claim 22, Modified Kramer teaches the method of claim 3, wherein the total protein concentration of the test sample or the adjusted test sample is determined with a bicinchoninic acid (BCA) assay (Feige, Protein concentration was determined by a BCA assay and samples; [0231]), wherein the BCA assay is optionally a micro BCA assay.
Regarding claim 24, Modified Kramer teaches the method of claim 2, wherein the formulation comprises 40 mM HEPES (Kraemer, 40 mM HEPES; page 10, column 1, paragraph 3, line 1), 102 mM NaCl (Kraemer, 101 mM NaCl; page 10, column 1, paragraph 3, lines 1,3), 5 mM KCl (Kraemer, 5 mM KCl; page 10, column 1, paragraph 3, line 2), 5 mM MgCl2 (Kraemer, 5 mM MgCl2; page 10, column 2, paragraph 1, line 5), 1 mM EDTA (Kraemer, 1 mM EDTA; page 10, column 1, paragraph 3, line 4), and 0.05% Tween-20 (Kraemer, 0.05% (v/v) Tween 20; page 10, column 1, paragraph 3, line 2).
Regarding claim 27, Modified Kramer teaches the method of claim 2, wherein the buffer exchange is not performed with ultrafiltration (Gjerde is free of any teaching of ultrafiltration and therefore meets this negative limitation).
Regarding claim 28, Modified Kramer teaches the method of claim 2, wherein the buffer exchange is performed with gel filtration chromatography (Gjerde, gel filtration can be used for buffer exchange; [0299]).
Regarding claim 31, Modified Kramer teaches the method of claim 2, wherein the method does not comprise concentrating the biological sample (Kraemer is free of any teaching of concentrating the biological sample and therefore meets this negative limitation).
Regarding claim 97, Modified Kramer teaches the method of claim 2
Modified Kraemer is silent to teaching measuring the total protein concentration of the biological sample prior to the performing of the buffer exchange. However, this is simply an arrangement of method steps that would not have modified the operation of the sample preparation and would have been obvious to implement (See MPEP 2144.04(IV)(C)).
Regarding claim 98, Modified Kramer teaches the method of claim 97, wherein the total protein concentration of the biological sample is measured with an assay selected from the group consisting of a fluorescence readout assay, a bicinchoninic acid (BCA) assay (Feige, Protein concentration was determined by a BCA assay and samples; [0231]), a micro BCA assay, a Lowry assay, and an ELISA assay.
Regarding claim 101, Modified Kramer teaches the method of claim 98, wherein the assay is a fluorescence readout assay (Kraemer, Eight individual measurements of fluorescent signal as a function of analyte concentration in buffer were made for each of nine analytes in multiplexed format; page 12, paragraph 2, lines 1-6).
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Kraemer et al. (“From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay”; 2011) in view Feige et al. (US 20190255149 A1, EFD 2017-06-01) and Gjerde (US 20180178142 A1), as applied to claim 3 above, and in further view of Webber (“Proteomics Analysis of Cancer Exosomes Using a Novel Modified Aptamer-based Array (SOMAscanTM) Platform”; 2014).
Regarding claim 23, Modified Kramer teaches the method of claim 22.
Modified Kraemer is silent to teaching the assay comprises a bicinchoninic acid reagent, and optionally an alkaline tartrate- carbonate buffer and/or a copper sulfate solution
Webber teaches a micro-BCA assay comprises a bicinchoninic acid reagent (“Protein concentrations were determined using a micro-BCA assay (Pierce/Thermo),” wherein the Pierce/Thermo micro-BCA assay naturally includes Reagent A, a carbonate buffer containing BCA reagent, and Reagent B, a cupric sulfate solution; page 1051, column 2, Experimental procedures).
Webber is considered to be analogous to the claimed invention because it is in the same field of endeavor for analyzing proteins in biological samples using the SOMAscan platform (Abstract; [0231]). 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 BCA assay taught by Kraemer in view of Feige and Gierde by incorporating the teachings of Webber by including a bicinchoninic acid reagent since there are only a finite number of identified, predictable means to successfully carry out a BCA assay (See MPEP 2143(I)(E)).
Claim 102 is rejected under 35 U.S.C. 103 as being unpatentable over Kraemer et al. (“From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay”; 2011) in view Feige et al. (US 20190255149 A1, EFD 2017-06-01) and Gjerde (US 20180178142 A1), as applied to claim 101 above, and in further view of Neeser (US 20100254581 A1).
Regarding claim 102, Modified Kramer teaches the method of claim 101.
Modified Kraemer is silent to teaching the fluorescence readout assay comprises a reagent selected from a merocyanine dye and epicocconone.
Neeser teaches the fluorescence readout assay comprises a reagent selected from a merocyanine dye and epicocconone (The total protein fluorescent assay of choice FluoroProfile, supplied by Sigma-Aldrich, results from the reversible reaction between virtually non-fluorescent epicocconone with nucleophilic amines on the protein to yield a red-orange emitting fluorophore; [0091]).
Neeser is considered to be analogous to the claimed invention because it is in the same field of endeavor for reducing measuring total protein content in complex biological matrices. 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 substituted the BCA assay taught by Kraemer in view of Feige and Gjerde with the fluorescence readout assay using a epicocconone reagent taught by Neeser in order to analyze proteins at a lower concentration. Neeser states that “The FluoroProfile assay exhibits a linear range of 40 ng to 200 ug per milliliter of protein and a coefficient of variance among different proteins of 16%, as compared to 11% for the less sensitive, smaller linear range colorimetric BCA assay” ([0091]). Therefore, it would have been obvious to utilize an assay with epicocconone to exhibit a greater sensitivity and wider linear range since epicocconone is a well-known reagent in the art and the substitution would have yielded the predictable benefit of detecting analytes at a lower range for the common purpose of protein characterization (See MPEP 2143(I)(B)).
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