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 .
Continued Examination Under 37 CFR 1.114
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on January 30, 2026 has been entered.
Claims 1-3, 12 and 21 are now pending and being examined. Claim 1 is amended.
Claim Objections
2. Claim 1 is objected to because of the following informalities: There appears to be a typo in the acronym “MALDI-TOP” that should be corrected to “MALDI-TOF” Appropriate correction is required.
Maintained Rejections
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.
3. Claim(s) 1, 12, and 21 remain rejected under 35 U.S.C. 103 as being unpatentable over Figuero-Espinosa et al (Journal of Microbiological Methods, April 2019, 159:120-127), in view of Bethel et al (Antimicrobial Agents and Chemotherapy, 2011, 55:3465-3475); Shapiro et al (ACS Infections Diseases, 2017, 3:833-844); Spakman, D (2014. “The use of MALDI-TOF MS for the detection of β-lactamase activity in Gram-negative anerobic bacteria”. Bachelor’s Thesis, Biology. University of Groningen, Groningen, The Netherlands); Olsson et al (1976, Antimicrobial Agents and Chemotherapy, 9:727-735); Yano et al (Chinese Journal of Oceanology and Limnology); Dortet (1) (Journal of Clinical Microbiology, 2014, 52:2359-2364); Dortet (2) (Journal of Medical Microbiology, 2014, 63:772-776); and Literacka et al (Eur. J. Microbiol. Infect. Dis., 2017, 36:2281-2287).
Figuero-Espinosa teach detecting pathogenic strains of bacteria in patient samples to determine an effective treatment in a timely manner is a key factor involving a critical patient’s survival. Delays in effective treatment may reduce the survival chances as much as 10% daily (p. 120, col. 1-2). Figuero-Espinosa teach their method of detecting pathogenic strains by direct detection of mature β-lactamase protein in blood samples is quick and easy (p. 121, col. 1 at the bottom).
Figuero-Espinosa teach a method of direct detection of a pathogenic strain of bacteria having resistance to β-lactam antibiotics in biological samples (Table 1; Materials and Methods, sections 2.1-2.4), the method comprising:
(a) isolating KPC-2 β-lactamase protein expressed by a pathological strain of Gram-negative bacilli in the samples (Materials and Methods, section 2.5);
(b) performing top-down mass spectrometry on the intact protein present in the samples, using MALDI-TOF-MS (Materials and Methods, sections 2.5-2.8);
(c) determining the mass of the protein in a range between 17,000 and 50,000 Da (section 2.6-2.8; Table 1; Results section 3; Figure 1); wherein the pathogenic bacteria express CTX-M resistance markers including CTX-M2, CTX-M9, and CTX-M15 (Table 1); wherein a common KPC-2 protein peak at ~28,477 Da or ~28,544 Da was detected and characterized as an identifying protein mass peak for the KPC-2 pathogenic strain (Table 1; p. 123, section 3; Figure 1).
Figuero-Espinosa suggests using their methods of direct detection of β-lactamase protein to detect other β-lactamase proteins, such as CTX-Ms (p. 126, col. 1 at the bottom).
Detecting mass value of 27,946/ 27,944, or 28,107 Da that has the same value as a β-lactamase protein lacking N-terminal 28 amino acids:
Although Figuero-Espinosa suggests using their methods of direct detection of β-lactamase protein in samples by mass spectrometry to detect CTX-M proteins, Figuero-Espinosa does not exemplify using their method to detect a CTX-M protein, or specific mass 27,946 Da and mass 28,108 Da that has the same mass value of a β-lactamase from which 28 amino acid residues at the N-terminus have been removed.
Bethel exemplifies successfully determining the mass of intact β-lactamase CTX-M9 protein using electrospray ionization mass spectrometry (EIS-MS) (p. 3467, col. 1, “ESI-MS”), wherein Bethel detected a mass at 27,946±3 Da (mature protein lacking the N-terminal signal peptide that encompasses 27,944 Da) (p. 3469, col. 102; Table 2; Figure 2A). Bethel teaches CTX-M15 is another known β-lactamase and it has had significant impact on the treatment of hospital- and community-acquired infections caused by enteric bacilli (p. 3465, col. 1). Mass 27,944 Da is present in instant claim 12 representing CTX-M98 or CTX-M129.
Shapiro demonstrates successfully detecting the mass of intact CTX-M15 protein using LC-MS (p. 842, col. 1), wherein Shapiro determined the mass of intact CTX-M15 to be ~28,108 or 28,107 Da (Figures 4-7). Mass 28,108 Da is present in instant claim 12, corresponding to CTC-M28 or CTX-M142 protein.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to detect β-lactamase CTX-M proteins in the method of Figuero-Espinosa and at mass value 28,107 Da or 27,944 Da. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Figuero-Espinosa expressly suggests conducting the method to detect CTX-M proteins; (2) Figuero-Espinosa demonstrates their method works successfully to directly detect β-lactamase protein in biological samples and to detect protein masses present in the 17,000 to 50,000 Da range; and (3) Bethel and Shapiro demonstrate CTX-M proteins can be successfully detected using mass spectrometry and at the same 28,107 Da or 27,944 Da mass values instantly claimed.
Using NaCl solution to apply osmotic pressure:
Figuero-Espinosa teaches subjecting bacteria to formic acid – isopropanol – water at a 17:33:50 ratio for protein extraction (section 2.5) and does not teach utilizing NaCl for osmotic pressure, or adding a hypotonic solution to lyse the cells for protein extraction.
Spakman also teaches the need for rapid β-lactamase detection, and teaches the known use of MALDI-TOF MS as a tool for the detection of β-lactamase (abstract). Spakman teaches one problem for rapid detection of β-lactamase in Gram-negative bacteria is the low quantity of extracellular β-lactamase present in samples, because most of the β-lactamase is located intracellularly in gram negative bacteria (abstract). Spakman suggests this problem can be remedied by using osmotic shock (citing Olsson below) or by adding surfactant SDS to the bacteria in order to increase the amount of extracellular β-lactamase. Spakman suggests freeing the bacteria’s intracellular portion of β-lactamase for assay by causing lysis of the bacterial cells, wherein lysis can be achieved enzymatically by lytic enzymes or mechanically by several methods including sonication, homogenization, bead beating, and freezing and grinding. Spakman suggests performing osmotic shock and adding SDS, or performing lysis to increase the amount of extracellular β-lactamase in order to increase detection (Discussion section 4).
Olsson (cited by Spakman) teaches and successfully demonstrates a method of releasing β-lactamase from Gram-negative bacteria for detection by osmotic shock of the bacteria using MgCl2, followed by isoelectric focusing and determination of molecular weight of the isolated intact protein (p. 729, col. 1-2). Olsson determined that osmotic shock was superior to polymyxin B for lysing cells and releasing β-lactamase (p. 732, col. 1-2).
Yano demonstrates successfully releasing β-lactamase extracellularly from bacteria culture by subjecting the bacterial cells to osmotic pressure/shock using NaCl solution by contacting the bacterial cells with 0.3 mol/L NaCl, then transferring the cells to hypotonic medium lacking NaCl (Materials and Methods; p. 105-108; Figure 2 and 4).
Dortet (1) also teaches the need to for rapid identification of bacteria expressing carbapenemase and resistant to β-lactam antibiotics (p. 2359; Table 1). Dortet teaches a commercially available test, “CarbAcineto” for rapid detection of carbapenemase-producing bacteria in biological sample that is superior to the known “Carba NP” test because it utilizes a step of osmotic pressure with a hypertonic NaCl solution to lyse the cells, whereas the “Carba NP” test uses a non-ionic detergent (Materials and Methods; p. 2362, col. 1). Dortet teaches that the NaCl solution is superior because it leads to an efficient lysis of the bacteria, providing a higher yield of carbapenemase, resulting in a significant improvement in detection of carbapenemase activity, specificity, and sensitivity (p. 2361, col. 2 to p. 2362, col. 1). Dortet teaches their improved test detects all types of carbapenemases (abstract).
Dortet (2) teaches the “Carba NP” test successfully detects carbapenemase activity, including for CTX-M proteins, and from a variety of pathogenic bacteria (Table 1).
Literacka demonstrates the commercialized “Carba NP” and “CarbAcineto” assay successfully detect a variety of carbapenemases in several different pathogenic gram-negative bacteria (abstract; Tables 1-4).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to subject the bacterial cells from the sample to osmotic pressure, particularly by using NaCl, as well as adding a hypotonic solution to the cells for lysis to isolate β-lactamase in the method of Figuero-Espinosa. One would have been motivated to, and have a reasonable expectation of success to do so: (i) in order to release β-lactamase from the bacterial cells of the sample and increase the extracellular amount of β-lactamase for detection, as taught and/or successfully demonstrated by Spakman, Yano, Dortet (1) and (2), and Literacka; (ii) because Olsson successfully demonstrates that β-lactamase isolated by osmotic pressure is successfully subsequently analyzed for mass; and (iii) Dortet teaches that the NaCl solution is superior for isolation of carbapenemase because it leads to an efficient lysis of the bacteria, providing a higher yield of carbapenemase, resulting in a significant improvement in detection of carbapenemase activity, specificity, and sensitivity
4. Claim(s) 2 and 3 remain rejected under 35 U.S.C. 103 as being unpatentable over Figuero-Espinosa et al (Journal of Microbiological Methods, April 2019, 159:120-127), Bethel et al (Antimicrobial Agents and Chemotherapy, 2011, 55:3465-3475); Shapiro et al (ACS Infections Diseases, 2017, 3:833-844); Spakman, D (2014. “The use of MALDI-TOF MS for the detection of β-lactamase activity in Gram-negative anerobic bacteria”. Bachelor’s Thesis, Biology. University of Groningen, Groningen, The Netherlands); Olsson et al (1976, Antimicrobial Agents and Chemotherapy, 9:727-735); Yano et al (Chinese Journal of Oceanology and Limnology); Dortet (1) (Journal of Clinical Microbiology, 2014, 52:2359-2364; Dortet (2) (Journal of Medical Microbiology, 2014, 63:772-776); and Literacka et al (Eur. J. Microbiol. Infect. Dis., 2017, 36:2281-2287).; as applied to claims 1, 12, and 21 above, and further in view of Ourghanlian et al (Antimicrobial Agents and Chemotherapy, 2017, 61:e02510-16, internet pages 1-10).
Figuero-Espinosa, Bethel, Shapiro, Spakman, Yano; Dortet (1); Dortet (2); and Literacka (the combined references) teach a method of detecting a pathogenic strain having resistance to β-lactam antibiotics in a biological sample comprising: (a) isolating a protein expressed by a pathogenic strain in a biological sample isolated from a subject by applying osmotic pressure to the biological sample using NaCl; and (b) performing top-down mass spectrometry on the isolated protein, as set forth above.
The combined references do not teach isolating the β-lactamase protein for mass spectrometry analysis by performing anion exchange chromatography.
Ourghanlian demonstrates successfully isolating β-lactamase proteins, including CTX-M15 and KCP-2, from bacteria grown in brain heart infusion broths for mass spectrometry analysis by performing anion exchange chromatography (Figure 2; p.8-9, Materials and Methods). Ourghanlian teach they used a commercially available anion-exchange chromatography column (p.8, Materials and Methods, “Production and purification of β-lactamases”).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to isolate β-lactamase proteins using anion exchange chromatography in order to perform mass spectrometry in the method of the combined references. One would have been motivated to, and have a reasonable expectation of success to, because the combined references teach methods for detecting specific of β-lactamase proteins from pathogenic strains of bacteria in biological samples by mass spectrometry, and Ourghanlian teaches a commercially available, successful method of anion-exchange chromatography for isolating β-lactamase proteins from pathogenic strains of bacteria grown in biological samples and subsequently analyzed by mass spectrometry.
Response to Arguments
5. Applicants argue that it is not obvious to combine the NaCl-induced osmotic lysis methods taught by Dortet (1) and Yano with eh MALDI-TOF methods of Spakman. Applicants argue that Spakman discloses using NaCl-induced osmotic lysis in the context of increasing extracellular fraction of β-lactamase for better β-lactamase detection, but not detection by MALD-TOF. Applicants argue Spakman teaches osmotic shock as tool for increasing permeabilization of cells, rather than for lysing cells. Applicants argue that the state of the art is such that salts like NaCl are widely recognized as signal-suppressing contaminants in MALDI-TOF mass spectrometry. Applicants argue that MALDI-TOF is highly sensitive to salts present in a sample. Applicants argue that one would not be motivated to apply NaCl osmotic pressure in combination with MALDI-TOF mass spectrometry.
Applicants argue that enhanced MALDI-TOF signal intensity is an unexpected result of NaCl induced osmotic shock. Applicants argue they demonstrate experimentally that NaCl-osmotic shock produces significantly stronger MALDI-TOF detection of β-lactamases than osmotic treatments using organic osmolytes or alternative salts (MgCl2, CaCl2), despite all conditions being capable of inducing osmotic stress and protein release. Applicants argue that the mass spectrometry signal was not comparable across all methods of protein release. Applicants argue that Cheon demonstrated detergent-based lysis methods, such as OG or SDS treatment, yielded substantially higher total protein amounts as measured by SDS-PAGE band density compared to NaCl-induced osmotic shock. However, Cheon demonstrated that NaCl produced a stronger MALDI-TOF signal for KPC-2 than for OG and SDS. Applicants conclude that despite yielding lower total protein amounts, NaCl-induced osmotic shock produces a markedly stringer MALDI-TOF signal intensity for the target β-lactamase protein than detergent-based methods using OG or SDS. Applicants argue their data demonstrates that protein extraction efficiency, as assessed by SS-PAGE, does not correlate with MALDI-TOF peak intensity. The enhanced MALDI-TOF detection achieved by NaCl-induced osmotic shock cannot be attributed merely to increased protein release. Rather, the data demonstrates that NaCl-induced osmotic shock unexpectedly yields an extract protein composition that is substantially more compatible with MALDI-TOF ionization and detection, despite a lower overall amount of protein released. Applicants argue that the enhanced sensitivity of MALDI-TOF achieved with NaCl-induced osmotic shock is neither taught noir suggested by any of the prior art references, and the state of the art is such that salts are incompatible with MALDI-based analysis, making the enhanced detection unexpected.
Applicants argue that Dortet and Literacka are technically unrelated to the detection of β-lactamase using MALDI-TOF. Applicants argue these references used NaCl-induced osmotic shock to release β-lactamase for detection using colorimetric assays (Cara NP and CarbAcineto). Therefore, these references do not teach the unexpected result of enhanced detection by MALDI-TOF.
6. The arguments have been carefully considered but are not persuasive. Applicants are arguing unexpected results and advantages for limitations that are not recited in the claims nor disclosed in the instant specification. MPEP 716.02(d) states: Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range.
In the instant case, Applicants argue that there is an unexpected enhanced MALDI-TOF MS CTX-M protein signal detection by direct analysis of bacterial cell samples osmotically lysed by NaCl and distilled water. Applicants argue the direct analysis of samples containing the NaCl from lysis unexpectedly yielded improved protein signals by MALDI-TOF MS analysis, where presence of NaCl in samples is normally expected to be inhibitory. Claim 1 recites:
(a) isolating a protein expressed by a pathogenic strain in a biological sample isolated from a subject by applying osmotic pressure to the biological sample using a NaCl solution; and
(b) performing MALDI-TOF mass spectrometry on the isolate protein.
Claim 1 does not recite osmotically lysing bacterial cells with NaCl and directly applying the lysed sample to MALDI-TOF-MS analysis, which is required to achieve the unexpected and improved results argued by Applicants. Instead, claim 1 broadly recites a method “using NaCl” to apply osmotic pressure to the biological sample. There is no limitation reciting how the NaCl is used for osmotic pressure, no limitation requiring osmotic lysis, no limitation reciting what concentration of NaCl is used to achieve the unexpected results argued, and no limitation requiring the NaCl sample to be directly applied to MADLI-TOF MS analysis after osmotic pressure or lysis and without any purification steps between to remove or reduce the salt.
The instant specification discloses osmotic lysis of samples can be achieved using NaCl:
[0076] (3) Cell Lysis by Osmotic Pressure
[0077] The strain culture was added to a washing buffer (pH 8.0 Tris-HCl+500 mM NaCl), and then incubated at room temperature for 10 minutes. Next, the supernatant was removed by centrifugation at 14,000 g for 10 minutes, and triple distilled water was added to the pellet, followed by incubation for 10 minutes. This pretreated sample was further separated into a supernatant (crude enzyme solution) and a precipitate by centrifugation. From the crude enzyme solution, the expression and size of the target protein were analyzed by SDS-PAGE analysis (FIG. 3).
However, there is no disclosure in the specification to directly apply the NaCl-lysed sample to MALDI-TOF MS analysis in order to observe and gain any of the advantages argued by Applicants. Instead, the instant specification discloses purifying the lysed sample to remove the salt prior to MALDI-TOF MS analysis using a column comprising PLRP-S resin and nanoflow liquid chromatography, then the protein sample was spot-dried on a plate and subject to mass spectrometry:
[0103] (1) Separation of Crude Protein Extract
[0104] About 0.1 μg of a protein sample was mixed with a 1% formic acid solution at a ratio of 1:1 and adjusted to approximately pH 3, and then the sample was loaded into a column. The crude protein extract was separated using a column (150 μm×20 cm) packed with PLRP-S resin and nanoflow liquid chromatography. The gradient conditions for sample loading and separation used at this time are as follows.
Buffer A: 0.1% formic acid in water/buffer B: 0.1% formic acid in acetonitrile
sample loading: from 0 to 10 min, 5% (B), 5 μL/min flow rate
Concentration gradient for separation:
[0108] from 10 to 10.01 min, from 5% to 10% (B), 300 nL/min flow rate
[0109] from 10.01 to 40 min, from 10% to 40% (B), 300 nL/min flow rate
[0110] from 40 to 41 min, from 40% to 80% (B), 300 nL/min flow rate
[0111] from 41 to 42 min, 80% (B), 300 nL/min flow rate
[0112] from 42 to 43 min, from 80% to 5% (B), 300 nL/min flow rate
[0113] from 43 to 60 min, 5% (B), 300 nL/min flow rate.
[0120] (3) Mass Spectrometry of CTX-M Protein Using Low-Resolution Mass Spectrometer
[0121] The mass spectrometry spectrum of CTX-M protein was obtained using the Bruker Biotyper MALDI-TOF MS system. First, 1 μL of a sinapinic acid (SA, present at 10 mg/mL in 0.1% TFA/50% acetonitrile) matrix and about 100 ng of CTX-M protein were placed on the plate spot, dried completely, and subjected to mass spectrometry. At this time, the maximum energy used was 30%, random position acquisition was performed, a total of 2,000 laser shots (40 shots per time) were irradiated, and each spectral data was cumulatively obtained. Mass spectrometry spectra were obtained for the range of 10,000 to 40,000 m/z, and CTX-M protein with a charge state of +1 as well as CTX-M protein with a charge state of +2 were simultaneously detected (FIG. 9).
Thus, Applicants are arguing unexpected results and advantages for method steps not recited in the claims or disclosed in the instant specification.
Although claim 21 recites the osmotic pressure is performed by an osmotic lysis, claim 21 still does not recite the specific limitations argued by Applicants that are required to observe and gain the advantages argued.
Although Applicants point to the Cheon 2021 publication (Cheon et al, Proteomics Clinical Applications, 2021, 15:2100044, internet pages 1-9) to argue evidence supporting the direct analysis of NaCl-lysed bacterial samples yields unexpectedly improved MALDI-TOF detection signals of CTX-M proteins, the method taught by Cheon discloses specific steps to achieve the advantages argued by Applicants that are not recited in the instant claims or disclosed in the instant specification. Cheon discloses specific steps of osmotically lysing E. coli bacterial cells with NaCl and distilled water (osmotic shock, OS), collecting the supernatant, performing top-down MALDI-TOF MS analysis directly on the OS supernatant and detecting a KPC-2 peak, wherein MALDI-TOF MS signal for KPC-2 peak was strongest for OS samples using NaCl OS, compared to using sugar OS, or detergents for lysis. Cheon teaches their method comprised the following steps in Supplemental Figure S2:
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The instant specification does not disclose this method, and the claims are not limited to this method.
Additionally, Cheon also teaches that not all concentrations of NaCl yielded the improved signal results in MALDI-TOF that are argued by Applicants. MALDI-TOF signals using a salt gradient from 500 mM NaCl to distilled water (DW) showed outstanding performance in bacterial cell lysis and the highest sensitivity in MALDI-TOF MS analysis, but the KPC-2 protein signals “were dramatically declined at the higher concentration of 500 mM NaCl (1-5 M)” (p. 6, col. 2). Therefore, Cheon demonstrates that the unexpected results argued by Applicants are also limited in range to a specific NaCl concentration for lysis. The instant claims and instant specification do not recite/disclose/recognize any specific concentration of NaCl lysis that yields the beneficial results argued by Applicants, and the instant specification does not disclose any step of directly applying the supernatant from NaCl-lysed samples to MALDI-TOF MS analysis, and instead, discloses purifying the protein samples to remove the salt prior to MALDI-TOF analysis, which is routine.
The claims as currently constituted do not exclude steps of purifying/isolating the protein after osmotic pressure or osmotic lysis of the sample using NaCl and prior to application for MALDI-TOF MS analysis. Examiner maintains the cited prior art renders obvious “using NaCl solution” for osmotic pressure and osmotic lysis of samples to generate sufficient CTM-X proteins for analysis by MALDI-TOF MS, for the reasons of record.
7. Conclusion: No claim is allowed.
8. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642