The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 May 19, 2026 has been entered.
The following in an analysis of claim 1. The preamble state that claim 1 is a method for determining the amount of an insulin analog in a sample by tandem mass spectrometry. Step (a) requires immunocapturing an insulin analog with detemir being the claimed analog. Instant paragraph [0028] list different analogs including detemir (LEVEMIR®) so that both the claimed name and the tradename will be treated as meeting the required analog when considering the teachings of a reference. It is clear from this step that as long as the detemir (LEVEMIR®) analog is captured, it doesn’t matter how many insulin analogs are immunocaptured. Since there are no specifics relative to the immunocapturing step and because the claim is not limited to capturing a single analog, examiner is treating the limitation as covering any type of immunocapturing step. Immunocapturing a specific analog or a more general procedure that captures a plurality of analogs will be treated as meeting the requirement of step (a). Step (b) requires subjecting the immunocaptured insulin analog to an ionization source under conditions suitable to generate one or more insulin analog ions detectable by mass spectrometry, wherein the one or more insulin analog ions comprise one or more detemir (LEVEMIR®) precursor ions with a mass-to-charge ratio (m/z) of 987.2 ± 0.5. This step requires the captured insulin analog to be ionized in a manner that one or more detemir (LEVEMIR®) ions having a mass-to-charge ratio (m/z) of 987.2 ± 0.5 to be produced. Examiner notes that paragraph [00327] of the instant specification appears to contain Table 18 which gives the parent ions for 6 analogs. From that table, it appears that the Levemir insulin analog is the only analog capable of producing the required ions. For examination purposes, step (b) will be treated as requiring the immunocaptured insulin analog(s) to be subjected to an ionization source that produces one or more insulin analog ions that can include multiply charged ions from the 4+, 5+ and/or 6+ states as long as the required ions are produced. In other words step (b) requires at least the Levemir insulin analog to be immunocaptured in step (a). Step (c) requires fragmenting the one or more detemir precursor ions to produce one or more detemir fragment ions. Step (d) requires determining an amount of the one or more detemir fragment ions by tandem mass spectrometry (examiner notes that ionizing to produce precursor ions, producing fragment ions from the precursor ions and determining the amount of one or more fragment ions by mass spectrometry constitutes tandem mass spectrometry). Examiner notes that this step does not require the amount of any specific detemir fragment ion to be determined. As a result it covers determining an amount of any of the detemir fragment ion produced in step (b). This would include multiply charged detemir precursor ions from the 4+ and/or 5+ states as well as the detemir precursor ions with a mass-to-charge ratio (m/z) of 987.2 ± 0.5. Thus, as long as there is evidence that a detemir precursor ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 would have been produced when immunocaptured insulin analogs are ionized, determining the amount of any single and/or combination of the detemir fragment ions produced in the ionization source meets the requirement of step (d).
With respect to claim 24, the above analysis of claim 1 applies. The differences are that the required analog is glulisine, the glulisine precursor fragment ion has a mass-to-charge ratio (m/z) of 971.5 ± 0.5 and the claim no longer requires the immunocapturing step. The lack of an immunocapturing step requirement means that any purification method such as liquid-liquid extraction and/or solid-phase extraction are also covered by the claim. Paragraph [0028] mentioned above lists the required analog as glulisine (APIDRA®) so that the tradename will be treated as equivalent to the required glulisine when considering art. Table 18 mentioned above teaches that both Novalog and Apidra can be ionized to produce the required precursor ion. However the fragment ions are different for the two insulin analogs so that the two analogs can be distinguished based on the fragment ions The requirement that the amount of the one or more glulisine fragment ions is correlated with the amount of the glulisine in the sample is being treated as inherently met if the amount of the glulisine is determined from the one or more glulisine fragment ions. This would include glulisine fragment ions from multiply charged glulisine precursor ions from the 4+ and/or 5+ states as well as the glulisine precursor ions with a mass-to-charge ratio (m/z) of 971.5 ± 0.5, as long as there is evidence that a glulisine precursor ion with a mass-to-charge ratio (m/z) of 971.5 ± 0.5 would have been produced when the glulisine insulin analog was ionized.
The disclosure is objected to because of the following informalities: the instant specification has a table 14 (see example 9, instant paragraph [00316]), two example 10s (see the headings prior to instant paragraphs [00317] and [00320]), a table 16 (see the second example 10, instant paragraph [00320]) and two table 18s (see paragraphs [00324] and [00327]).
Appropriate correction is required. Examiner notes that correction of the above issues may require renumbering of additional examples, tables and/or changing references to examples and/or tables in other parts of the instant description besides the specific paragraphs noted above. Thus a careful review of the example numbers, table numbers and their references in the instant disclosure is suggested to prevent the correction of the above noted issues from creating further issues.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 9-10 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for treating the sample to enhance stability of the insulin analog prior to mass spectrometry, does not reasonably provide enablement for subjecting the sample to basic conditions prior to mass spectrometry. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the invention commensurate in scope with these claims. With respect to the breadth of the claim(s) Wands factor, claim 9 covers any treatment that would cause the sample to become basic. However, instant paragraph [00270] is the only paragraph of the instant specification in which the treatment of the sample with a base (the base trizma) in described. In that paragraph, there is no disclosure that the addition of trizma base causes the sample to become basic. Rather it is clear that trizma base was added to enhance the stability of the peptides in the sample. With respect to the direction provided by the inventor and the presence of working examples Wands factors trizma base is the only example given. Thus there is no support that any treatment of the sample under basic conditions would perform the same function associated with the addition of trizma base taught by applicant in this paragraph. Additionally, claim 1 requires subjecting the insulin analog to an ionization source under conditions to generate detemir precursor ions having a mass-to-charge ratio (m/z) of 987.2 ± 0.5. Instant paragraph [00245] teaches that ionization of insulin may result in multiply charged precursor ions (such as precursor ions of 4+, 5+, 6+, etc.). In particular, ionization conditions, specifically the pH of the buffer utilized during electrospray, greatly influences the identity and quantity of generated insulin precursor ions. For example, under acidic conditions, positive electrospray ionization may predominately generate 5+ and 6+ charged insulin precursor ions with m/z of 1162.5±0.5 and 968.5±0.5, respectively. However, under basic conditions, positive electrospray ionization may predominately generate 4+ and 5+ charged insulin precursor ions with m/z of 1453.75±0.5 and 1162.94±0.5, respectively. The methods may utilize either acidic or basic conditions; preferably acidic conditions. Based on the proximity to the different m/z of the multiply charged precursor ions of insulin, the detemir precursor ion required by claim 1 is a 6+ charged ion so that based on the above description the pH of the sample should be acidic during ionization. That creates some inconsistencies and/or questions with respect to being able to generate the required detemir precursor ion under the requirements of claims 9-10. Specifically with respect to claim 10, the subjecting the sample to basic conditions is further defined to comprise to subjecting the sample to “trizma and/or ethanol” (emphasis added). The “and/or” language means that the can be provided through trizma base by itself, ethanol by itself or a combination of the two. The instant disclosure fails to describe how basic conditions are established with ethanol by itself.
For these reasons, claims 9 and 10 are only enabled for a portion of their scope, but not enabled for their full scope.
Claims 9-10 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claim 1 an immunocapturing step prior to ionization, fragmenting and determining steps that are potentially part of mass spectrometry. Claim 9 is not clear whether subjecting the sample to basic conditions prior to mass spectrometry occurs before or after at least the immunocapturing step. Additionally, claim 1 requires subjecting the insulin analog to an ionization source under conditions to generate detemir precursor ions having a mass-to-charge ratio (m/z) of 987.2 ± 0.5. Instant paragraph [00245] teaches that ionization of insulin may result in multiply charged precursor ions (such as precursor ions of 4+, 5+, 6+, etc.). In particular, ionization conditions, specifically the pH of the buffer utilized during electrospray, greatly influences the identity and quantity of generated insulin precursor ions. For example, under acidic conditions, positive electrospray ionization may predominately generate 5+ and 6+ charged insulin precursor ions with m/z of 1162.5±0.5 and 968.5±0.5, respectively. However, under basic conditions, positive electrospray ionization may predominately generate 4+ and 5+ charged insulin precursor ions with m/z of 1453.75±0.5 and 1162.94±0.5, respectively. The methods may utilize either acidic or basic conditions; preferably acidic conditions. Based on the proximity to the different m/z of the multiply charged precursor ions of insulin, the detemir precursor ion required by claim 1 is a 6+ charged ion so that the buffer used to produce it during ionization must be acidic. Claims 9-10 require a basic treatment prior to mass spectrometry. Since instant paragraph [00245] clearly teaches that the ionization can be done under both acidic and basic conditions and that the 6+ charged ions are mainly produced when the ionization is under acidic conditions, it is not clear how the requirement for subjecting the sample to basic conditions prior to mass spectrometry leads to ionization under conditions suitable to generate the required 6+ charged precursor ion. Is it possible to generate 6+ charged precursor ions under basic conditions in the presence of the trizma and/or ethanol so that the specific basic conditions are required rather than the general scope of claim 9? Alternatively, is the “under basic conditions” language actually not representative of the reason for the addition of trizma to the sample? In this respect, instant paragraph [00270] clearly teaches that the trizma base is added to the sample to enhance stability of the peptides in the elution plate. Since there is no specific description that points to the sample becoming basic because of this addition of trizma base. While that may be the case, the instant disclosure does not teach that it happens. Thus, it appears that the subjecting the sample to basic conditions is not clear with respect to what is actually happening when trizma base is added to the sample. With respect to claim 10, as noted above the subjecting the sample to basic conditions includes subjecting the sample to ethanol by itself. Thus it is not clear how subjecting the sample to ethanol which is not a base constitutes subjecting the sample to basic conditions.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 5-8, 12-14, 16, 18-21 and 24 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Peterman (Proteomics 2014). In the paper Peterman teaches that the detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays. With specific reference to claim 1, Peterman teaches a method for determining the amount of an insulin analog in a sample by mass spectrometry, by a) immunocapturing an insulin analog wherein the insulin analog is detemir (immunoaffinity retrieval described in section 2.3); b) subjecting the immunocaptured insulin analog to an ionization source under conditions suitable to generate one or more insulin analog ions detectable by mass spectrometry, wherein the one or more insulin analog ions comprise a precursor ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 (see section 2.1, Levemir® is the tradename for detemir; section 2.4: LC-MS/MS, section 2.5: Data Analysis, to “provide additional levels of qualitative analysis, the three most abundant precursor charge states per insulin variant were used as well as the six most abundant isotopes per charge state.", section 3.1: LC-MS Qual/Quan data extraction strategies using HR/AM MS, the +5 and +6 charge states are relatively close in measured abundance and the +4 precursor is ca. 13% in relative abundance, the analysis software co-added 6 peaks [area under curve (AUC) values] from each of the "three primary precursor charge states, the +4, +5 and +6 charge states, for detection and quantitation to increase signal without significantly increasing noise). This means that positive electrospray ionization produced measurable ions from each of the +4, +5 and +6 charge states for each of the insulins measured. Figure 2 shows the detection and verification scheme for 60 pM Humulin S (R) extracted from plasma. The Pinpoint software utilizes theoretical isotopic m/z values for the top six isotopes per precursor charge state for data extraction, verification, and quantification. Figure 2A shows the overlaid extracted ion chromatogram (XIC) profile for the 18 different m/z values representing three different precursor charge states. Relevant to the instant claims are the values around m/z of 968 and 969 shown in figure 2B for the +6 state. Since this was done for each of the analogs, as shown by figure 3, and the materials included the Levemir® (detemir) and Apidra® (glulisine) analogs, at least the +6 state of the Levemir® analog would have produced one or more ions meeting the requirement of one or more detemir precursor ions of the required m/z as well as precursor ions of the +5 and +4 states.); and c) fragmenting the one or more detemir precursor ions to produce one or more detemir fragment ions (section 2.4 points to the formation of detemir fragment ions from the one or more detemir precursor ions); d) determining the amount of one or more detemir ions by mass spectrometry (see at least the abstract, section 2.5 and section 3.2); e) determining the amount of one or more detemir fragment ions to determine the amount of detemir in the sample (see at least the abstract, section 2.5, section 3.1 and section 3.2). Examiner notes that since the claims do not require the fragmenting of any specific detemir precursor ion to produce the detemir fragment ions used in step e), the claim covers using detemir fragment produced from any of the detemir precursor ions including those from the +4 and +5 states. For that reason claim 1 is anticipated by Peterman even though Peterman may not have used the +6 detemir precursor ion to quantify the analog. With respect to claim 5 see the article title. With respect to claims 6 and 17-18 see the first paragraph of section 3.1 (intact human insulin ionizes under positive electrospray conditions). With respect to claims 7-8 see the first paragraph of section 2.4 (each sample was separated using a linear gradient (10–50% in ten minutes) composed of (A) 0.1% formic acid in water and (B) 0.1% formic acid in MeCN). With respect to claims 12-14 and 16 see the abstract and section 2.4 (quantification by LC-MS with high resolution and accurate mass (HR/AM) spectrometric detection). With respect to claims 19-21 see section 2.1 (Immunoaffinity pipette tips (MSIA D.A.R.Ts -- Disposable Automated Research Tips) derivatized with mouse antihuman insulin antibody). With respect to claim 24, since the claim is generally broader than claim 1, except in the particular insulin analog (glulisine/apidra) and the required m/z of the glulisine precursor ion. Since Peterman teaches and presents data for the Apidra® (glulisine) analog, the analysis above for claim 1 applied to the Apidra® (glulisine) analog shows the anticipation of claim 24.
Claims 1, 5-7, 12-14, 16, 18-21 and 24 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Hess (Analytical and Bioanalytical Chemistry 2012) in view of the teachings of Peterman as described above. In the paper Hess teaches simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or six-fold protonated molecule ions and diagnostic product ions. As described above, Peterman clearly teaches that electrospray ionization would have produced one or more insulin analog ions from each of the +4, +5 and +6 states for each of the analogs. With specific reference to claim 1, Hess teaches a method for determining the amount of an insulin analog in a sample by mass spectrometry, by a) immunocapturing an insulin analog wherein the insulin analog is detemir (see table 1, insulin detemir and the sample preparation section on page 1814); b) subjecting the immunocaptured insulin analog to an ionization source under conditions suitable to generate one or more detemir precursor ions detectable by tandem mass spectrometry, wherein the one or more insulin analog ions comprise a precursor ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 (see the paragraph bridging pages 1814 and 1815, the fact that Hess used at least one insulin analog ion from the 6-fold protonated molecular ions and Peterman teaches that electrospray ionization produced one or more insulin analog ions from each of the +4, +5 and +6 states for each of the analogs combined with the presence of the Levemir/detemir the Apidra/glulisine analogs means that the specifically claimed insulin detemir precursor ions would have been produced along with other Detemir precursor ions from the +4 ad +5 states); and c) fragmenting the one or more detemir precursor ions to produce detemir fragment ions (see table 1 for the particular detemir precursor ion and its fragment ions), d) determining the amount of one or more detemir fragment ions by mass spectrometry (see at least table 1 and the paragraph bridging pages 1814 and 1815); and e) using the amount of the one or more detemir fragment ions to determine the amount of detemir in the sample spectrometry (see at least table 1 and the paragraph bridging pages 1814 and 1815). Examiner notes that since the claims do not require the fragmenting of any specific detemir precursor ion to produce the detemir fragment ions used in step e), the claim covers using detemir fragment produced from any of the detemir precursor ions including those from the +4 and +5 states. For that reason claim 1 is anticipated by Hess even though Hess may not have used the +6 detemir precursor ion to quantify the analog. With respect to claim 5 see the article title. With respect to claims 6 and 17-18 see the paragraph bridging pages 1814-1815 (molecules were ionized by electrospray ionization in positive-ion mode). With respect to claim 7 see the paragraph bridging pages 1814-1815 (separation was carried out with mobile phases consisting of 0.2 % acetic acid with 0.01 % trifluoroacetic acid (TFA) (phase A) and 0.04 % acetic acid and 0.002 % TFA in acetonitrile (phase B)). With respect to claims 12-14 and 16 see the title and the paragraph bridging pages 1814-1815. With respect to claims 19-21 see the sample preparation section on page 1814. With respect to claim 24, since the claim is generally broader than claim 1, except in the particular insulin analog (glulisine/apidra) and the required m/z of the glulisine precursor ion. Since Hess teaches and presents data for the Apidra® (glulisine) analog, the analysis above for claim 1 applied to the Apidra® (glulisine) analog shows the anticipation of claim 24.
Claim 24 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Thomas (Methods 2012, hereinafter called Thomas ‘12) in view of the teachings of Peterman as described above. In the paper Thomas ‘12 teaches hyphenated purification and enrichment steps prior to mass spectrometric detection of insulin. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra (glulisine), Lantus DesB30–32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1–29)) and CJC-1295 (GH-RH analogue), LongR3-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. As described above, Peterman clearly teaches that electrospray ionization would have produced one or more insulin analog ions from each of the +4, +5 and +6 states for each of the analogs with an acidic pH during ionization producing precursor ions from at least the +5 and +6 states. With respect to claim 24 Thomas ‘12 teaches a method for determining the amount of an insulin analog in a sample by mass spectrometry, by a) immunocapturing an insulin analog comprising glulisine (purifying the insulin analog, see the abstract, table 2 and section 2.6); b) subjecting the immunocaptured insulin analog to an ionization source under conditions suitable to generate one or more insulin analog ions detectable by mass spectrometry, wherein the one or more insulin analog ions comprise a precursor ion with a mass-to-charge ratio (m/z) of 971.5 ± 0.5 (see section 2.7 in combination the Peterman teaching noted above and the fact that the Apidra analog is present points to the presence of the required ions in the ionized sample); c) fragmenting the one or more glulisine precursor ions to produce glulisine fragment ions (see section 2.8 and table 2), d) determining the amount of one or more glulisine fragment ions and e) using the amount of glulisine fragment ions to determine the amount of glulisine in the sample (see section determining the amount of one or more insulin analog ions by mass spectrometry and see at least paragraph bridging the columns of page 234 -- for quantitative data interpretation). Examiner notes that since the claims do not require the fragmenting of any specific glulisine precursor ion to produce the glulisine fragment ions used in step e), the claim covers using glulisine fragment produced from any of the glulisine precursor ions including those from the +4 and +5 states. For that reason claim 1 is anticipated by Thomas ‘12 even though Thomas ‘12 may not have used the +6 glulisine precursor ion to quantify the analog.
Claims 24 is rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Chambers (Analytical Chemistry 2014) in view of peterman as described above. In the paper Chambers teaches a multidimensional method for the simultaneous, direct quantification of intact human insulin and five insulin analogs in human plasma. Figure 1 shows the structures of the different insulins including the following analogs: Lantus (insulin glargine), Apidra (insulin glulisine), Levemir (insulin detemir), NovoLog (insulin aspart) and Humalog (insulin lispro). The method uses a mixed-mode SPE and a multidimensional LC method including a solid-core particle column containing an anion exchange stationary phase. Matrix factors for all analogs were calculated in 6 sources of human plasma and CVs of the matrix factors were <15% in all cases supporting the selectivity of the method, while achieving LLOQs of 50−200 pg/mL (1.4−5.6 μIU/mL) for each insulin from 250 μL of human plasma. With respect to claims 24 and 25, Chambers teaches a method for determining the amount of an insulin analog in a sample by mass spectrometry (see at least the title), the method comprising: (a) purifying the insulin analog comprising glulisine from the sample (see the "Protein Precipitation (PPT) Pretreatment" and "Solid-Phase Extraction (SPE)" paragraphs on pages 696-697); subjecting the purified insulin analog to an ionization source under conditions suitable to generate one or more glulisine precursor ions detectable by mass spectrometry, wherein the one or more insulin analog ions comprise a glulisine precursor ion with a mass-to-charge ratio (m/z) of 971.5 ± 0.5 (see the "Mass Spectrometry and Software" paragraph on page 697 and the MRM transitions of Table 1 teaching a precursor ion at an m/z of 971.8 and fragment ions at an m/z of 1179, 454.4, 660.8 and 346.2 for aspart and Peterman teaching that electrospray ionization produced one or more insulin analog ions from each of the +4, +5 and +6 states for each of the analogs combined with the presence of the Apidra/glulisine analogs means that the specifically claimed insulin glulisine precursor ions would have been produced along with other glulisine precursor ions from the +4 ad +5 states); and (c) fragmenting the glulisine precursor ions to produce glulisine fragment ions (see at least table 1), determining an amount of the one or more glulisine fragment ions by mass spectrometry, wherein the amount of the one or more glulisine fragment ions is correlated with the amount of the glulisine in the sample (see at least the title, the "Mass Spectrometry and Software" paragraph on page 697 and Table 1). Examiner notes that since the claims do not require the fragmenting of any specific glulisine precursor ion to produce the glulisine fragment ions used in step e), the claim covers using glulisine fragment produced from any of the glulisine precursor ions including those from the +4 and +5 states. For that reason claim 1 is anticipated by Chambers even though Chambers may not have used the +6 glulisine precursor ion to quantify the analog.
Claims 1, 5-8, 12-14, 16 and 18-24 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Thomas (Drug Testing and Analysis 2014, hereinafter called Thomas ’14) in view of Peterman as described above. In the paper Thomas ’14 teaches the determination of human insulin and its analogues in human blood using liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS). The qualitative and quantitative determination of insulin from human blood samples is an emerging topic in doping controls as well as in other related disciplines (e.g. forensics). Beside the therapeutic use, insulin represents a prohibited, performance enhancing substance in sports drug testing. In both cases accurate, sensitive, specific, and unambiguous determination of the target peptide is of the utmost importance. The challenges concerning identifying insulins in blood by liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS) are detecting the basal concentrations of approximately 0.2 ng/mL and covering the hyperinsulinaemic clamps at >3 ng/mL simultaneously using up to 200 μL of plasma or serum. This is achieved by immunoaffinity purification of the insulins with magnetic beads and subsequent separation by micro-scale liquid chromatography coupled to ion mobility/high resolution mass spectrometry. The method includes human insulin as well as the synthetic or animal analogues insulin aspart, glulisine, glargine, detemir, lispro, bovine, and porcine insulin. The method validation shows reliable results considering specificity, limit of detection (0.2ng/mL except for detemir: 0.8 ng/mL), limit of quantification (0.5ng/mL for human insulin), precision (CV<20%), linearity (r>0.99), recovery, accuracy (>90%), robustness (plasma/serum), and ion suppression. For quantification of human insulin a labelled internal standard ([[2H10]-LeuB6,B11,B15,B17] human Insulin) is introduced. By means of the additional ion mobility separation of the different analogues, the chromatographic run time is shortened to 8 min without losing specificity. As proof-of-concept, the procedure was successfully applied to different blood specimens from diabetic patients receiving recombinant synthetic analogues. As described above, Peterman clearly teaches that electrospray ionization would have produced one or more insulin analog ions from each of the +4, +5 and +6 states for each of the analogs. With respect to claims 1 and 24, Thomas '14 teaches a method for determining the amount of an insulin analog in a sample by mass spectrometry (see at least the title, abstract, table 1 ), the method comprising: (a) immuno extracting (purifying) the insulin analog from the sample (see at least the abstract and the "sample preparation" paragraph on page 1127 and the mass spectrometry); subjecting the purified insulin analog (detemir or glulisine) to an ionization source under conditions suitable to generate one or more detemir or glulisine precursor ions detectable by mass spectrometry, wherein the one or more insulin analog ions comprise a detemir or glulisine precursor ion with a respective mass-to-charge ratio (m/z) of 987.2 ± 0.5 or 971.5 ± 0.5 (see at least the "Ion mobility - mass spectrometry" paragraph on page 1127 and the "Mass spectrometry" paragraph on pages 1129-1130 teaching fragment ions (diagnostic product ions) at an m/z of 315.15, 454.36 and 346.16 in combination the Peterman teaching noted above relative to the presence of +6 multiply charge ions in combination with +5 multiply charged precursor ions and the fact that the glulisine (Apidra) and detemir (Levemir) analogs are present points to the presence of the required precursor ions in the ionized sample); (c) fragmenting the detemir or glulisine precursor ions to produce detemir or glulisine fragment ions, d) determining an amount of the one or more detemir or glulisine fragment ions by mass spectrometry, wherein the amount of the one or more detemir or glulisine fragment ions is correlated with the amount of the detemir or glulisine insulin analog in the sample (see at least the title, the "Ion mobility - mass spectrometry" paragraph on page 1127 and Tables 1 and 2). Examiner notes that since the claims do not require the fragmenting of any specific detemir or glulisine precursor ion to produce the detemir or glulisine fragment ions used in step e), the claims cover using detemir or glulisine fragment produced from any of the detemir or glulisine precursor ions including those from the +4 and +5 states. For that reason claims 1and 24 are anticipated by Thomas ‘14 even though Thomas ‘14 may not have used the +6 detemir or glulisine precursor ion to quantify the analog. With respect to claim 5 see the “Blood specimens” paragraph on page 1126. With respect to claims 6 and 18 see the “Ion mobility – mass spectrometry” paragraph on page 1127 in combination with the ”Liquid chromatography (LC)” paragraph on page 1127 ((AQUITY nanoflow UPLC coupled to nano-electrospray ionization, positive ion mobility mode). With respect to claims 7-8 see the ”Liquid chromatography (LC)” paragraph on page 1127 (The aqueous solvent (A) consisted of a mixture of 0.1% of formic acid in water, and the organic phase (B) was acetonitrile). With respect to claims 12-14 and 16 see at least the "Ion mobility - mass spectrometry" paragraph on page 1127 and the "Mass spectrometry" paragraph on pages 1129-1130 (ultra-high performance liquid chromatography (UHPLC) and high resolution/high accuracy mass spectrometry (HRMS)). With respect to claims 19-23 see the “Chemicals and reagents” paragraph on page 1126 (paramagnetic secondary antibody-coated (anti-mouse IgG) beads)).
Claim 24 is rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Thevis (Analytical Chemistry 2006) in view of the Peterman teachings as described above. In the paper Thevis teaches doping control analysis of intact rapid-acting insulin analogues in human urine by liquid chromatography-tandem mass spectrometry. Insulin and related synthetic therapeutics have been prohibited by the World Anti-Doping Agency for athletes demonstrably not suffering from diabetes mellitus. The primary specimen for doping controls has been urine, but the renal excretion of intact human insulin as well as synthetic analogues such as the rapid-acting products Humalog LisPro, Novolog Aspart, and Apidra Glulisine has been reported negligible owing to metabolic degradation. Nevertheless, employing solid-phase extraction in combination with immunoaffinity purification followed by a top-down sequencing-based mass spectrometric approach, an assay was established allowing the identification of three intact rapid-acting synthetic insulins in doping control urine samples. A volume of 25 mL of urine was concentrated, insulin analogues were isolated from the concentrate by immunoaffinity chromatography, and the eluate was analyzed using microbore liquid chromatography/tandem mass spectrometry. Characteristic product ion spectra obtained from 5-fold protonated intact analytes as well as isolated insulin B-chains allowed the unambiguous identification of target analytes with detection limits of 0.05 ng/mL (9 fmol/mL). Moreover, assay validation demonstrated recoveries between 72 and 80% for Humalog LisPro, Novolog Aspart, and Apidra Glulisine, and assay precisions ranged from 9 to 16%. A reliable tool is provided that allows the qualitative determination of rapid-acting insulins in urine specimens collected for sports drug testing. As described above, Peterman clearly teaches that electrospray ionization would have produced one or more insulin analog ions from each of the +4, +5 and +6 states for each of the analogs. With respect to claim 24, Thevis teaches a method for determining the amount of an insulin analog in a sample by mass spectrometry (see at least the title and abstract), the method comprising: (a) purifying the insulin analog from the sample using a solid-phase extraction step in combination with an immunocapture step (see the abstract, the "Materials and Chemicals" paragraph on pages 1898-1899 and the "Sample Preparation" paragraph on page 1899) wherein the insulin analog comprises glulisine (Apidra); subjecting the purified insulin analog to an ionization source under conditions suitable to generate one or more glulisine precursor ions detectable by mass spectrometry, wherein the one or more insulin analog ions comprise a precursor ion with a mass-to-charge ratio (m/z) of 971.5 ± 0.5 (see the "Liquid Chromatography-Tandem Mass Spectrometry" paragraph on page 1899, the paragraph bridging pages 1900-1901 teaching that the Apidra yielded product ions at m/z 346, 328, and 227 in combination the Peterman teaching noted above relative to the presence of +6 multiply charge ions in combination with +5 multiply charged precursor ions and the fact that the Apidra Glulisine analog is present points to the presence of the required precursor ions in the ionized sample); (c) fragmenting the detemir or glulisine precursor ions to produce detemir or glulisine fragment ions, d) determining an amount of the one or more insulin analog ions by mass spectrometry, wherein the amount of the glulisine fragment ions is correlated with the amount of the glulisine insulin analog in the sample (see at least the title and abstract). Examiner notes that since the claims do not require the fragmenting of any specific glulisine precursor ion to produce the glulisine fragment ions used in step e), the claims cover using glulisine fragment produced from any of the detemir or glulisine precursor ions including those from the +4 and +5 states. For that reason claim 24 is anticipated by Thevis even though Thevis may not have used the +6 glulisine precursor ion to quantify the analog.
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 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Peterman or Hess or Thomas ’14 in view of the Peterman teachings as applied to claim 1 above, and further in view of Chen (Clinical Chemistry 2013). Hess, Peterman, 12 or Thomas ‘14 do not teach that the sample is subjected to basic conditions prior to mass spectrometry.
In the paper Chen teaches quantitative analysis of insulin using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The standards and reagents section on page 1350 teaches that a 1.5 mol/L Tris (Trizma) base solution was from Sigma. The sample preparation section which bridges pages 1350-1351 teaches that patient serum was thawed and vortex mixed, and 150 µL was vigorously mixed with 350 µL of basic ethanol (85% ethanol, 15% Tris base) and allowed to incubate for 60 min at -20 °C. The resulting precipitate was pelleted by centrifugation for 10 min at 5200g to produce clarified supernatant which was used to determine insulin. the following two paragraphs on page 1351 describe the solid-phase sample cleanup, liquid chromatography and mass spectral analysis.
It would have been obvious to one of ordinary skill in the art to use the basic ethanol (Trizma) reagent to treat the Hess, Peterman, Thevis, Thomas ‘12 or Thomas ’14 samples as taught by Chen because of its ability to selectively remove undesirable components of the sample for insulin measurement prior to chromatography and mass spectral detection of the insulin as taught by Chen.
Applicant's arguments filed May 19, 2026 have been fully considered but they are not persuasive. In response to the amendments examiner has modified the claim interpretation above to show applicant how the current independent claim language is being interpreted. In this respect, examiner notes that the independent claims require the generation of precursor ions with a specific mass-to-charge ratio (m/z), but fail to require that those precursor ions are the ions fragmented to produce the fragment ions for which an amount is determined and used to determine the concentration of an insulin analog in the sample. Thus any precursor ion and/or fragment ion from the two specific analogs being claimed can be used to meet that requirement of the claims as long as there is evidence that the required precursor ion would be formed in the ionization step. Based on the interpretation, the previous anticipation rejections have been modified to cover the appropriate claims and show that the required ions would have been produced during the ionization step, The specification has been objected to for minor problems and new rejections based on 35 U.S.C. 112(a) and 35 U.S.C. 112(b) have been added. The arguments are moot with respect to the new objections/rejections.
With respect to the anticipation by Hess, Peterman, Thomas ’14, Chambers, Thevis and Thomas ’12, the language of step (b) in claims 1 and 24 is of a scope that the detemir precursor ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 or the glulisine precursor ion with a mass-to-charge ratio (m/z) of 971.5 ± 0.5 are not the only precursor ions present. Thus as long as the required precursor ions are present, other precursor ions from detemir, glulisine or other possible insulin analogs can also be present. In this respect, Peterman clearly teaches that ions of the +4, +5 and +6 states are produced by the electrospray ionization process, the +5 and +6 charge states are relatively close in measured abundance and the +4 precursor is ca. 13% in relative abundance. From this one of ordinary skill in the art would have expected the presence of a +6 precursor ion in addition to at least the +5 precursor ion when ionization occurred under acidic conditions. From Table 18 of the instant specification the ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 or 971.5 ± 0.5 are derived from ionizing the Levemir (detemir) and Apidra (glulisine) analogs. Each of these references is looking at quantifying at least one of these analogs. Thus, if, as Peterman clearly teaches, +6 and +5 precursor ions are both present when insulin analogs are ionized prior to mass spectral analysis, an ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 or 971.5 ± 0.5 would inherently result from subjecting the respective detemir and glulisine analogs to an electrospray ionization source under acidic conditions. Thus contrary to the argument that none of these references teach an ion with a mass-to-charge ratio (m/z) of 987.2 ± 0.5 or 971.5 ± 0.5, there is evidence in Peterman that points directly to the creation of these ions in an electrospray ionization source under the ionization conditions taught by each anticipatory reference. For that reason step (b) of claims 1 and/or 24 is anticipated by teachings of each of the applied anticipatory references. Step (c) of claims 1 and 24 requires that the one or more detemir or glulisine precursor ions are fragmented to produce one or more detemir or glulisine fragment ions. Since this step does not require the specific detemir or glulisine precursor ions of step (b) to be fragmented, the step covers fragmenting any of the detemir or glulisine precursor ions present after step (b) (i.e. if covers fragmenting the +5 and/or +4 precursor ions in addition to the +6 precursor ions that are specifically required to be present by step (b). Similarly, steps (d) and (e) do not specify which detemir or glulisine fragment ions have their amount determined and are used to determine the amount of detemir or glulisine in the sample. Thus they are of a scope that covers determining the amount of detemir or glulisine fragment ions from +5 or +4 detemir or glulisine precursor ions for steps (d) and (e). Thus Peterman, Hess, Thomas ’12, Chambers, Thomas ’14 and Thevis are still within the scope of steps (d) and (e), contrary to the argument of applicant. Thus, the argument is not persuasive for claim 1 and the claims which depend therefrom or claim 24.
With respect to the arguments directed specifically toward the Peterman references the first paragraph of section 4.2 teaches that immunoextraction with a pan-insulin Ab was sensitive and effective for the six insulin variants analyzed in the study including Humulin S®, Apidra®, Lantus®, NovoRapid®, and porcine insulin. This statement only list five insulin variants. Section 2.1 lists six insulin variants with Levemir® being the additional insulin variant. Thus Levemir® is one of the insulin variants that was studied. The third full paragraph on page 1446 teaches that they presented a multiplexed mass spectrometric immunoassay (MSIA) for intact insulin and its analogues that couple immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range. The assay is sensitive, selective, and automated and can be applied to detect new insulin isoforms allowing their further incorporation into second generation assays. The multiplexed assay means the ability to detect and quantitate each of the insulin variants. Thus contrary to the urging of applicant, Peterman studied the measurement of both the detemir (Levemir®) and glulisine (Apidra®) analogs of insulin. Section 2.4 describes the LC-MS/MS portion. The second paragraph of section 4.2 teaches that in the described experiments, full scan MS data were analyzed due to the low noise and selectivity of the MSIA extraction. This approach enabled quantification of HR/AM MS data using the precursor charge state distribution as well as the isotopic distribution analysis for evaluation of potential background interference for quantification. Also, the described method provides sufficient cycle time to trigger MS/MS data for verification.
With respect to the other applied references, Peterman is a teaching reference showing that under acidic ionization conditions one of ordinary skill in the art would have expected the presence of insulin precursor ions from the +4, +5 and +6 multiply charged states. Peterman does not “modify” what is taught by another anticipatory reference. It is simply evidence that for ionization of the detemir insulin analog under acidic conditions an ion with a mass-to-charge ratio of 987.2 ± 0.5 would have been expected to be generated. Thus showing that the requirement of paragraph (b) of claim 1 was inherently met. Likewise, it is simply evidence that for ionization of the glulisine insulin analog under acidic conditions an ion with a mass-to-charge ratio of 971.5 ± 0.5 would have been expected to be generated. Thus showing that the requirement of paragraph (b) of claim 24 was also inherently met.
If applicant would like further evidence of this, the teachings of Loo (Biomedical and Environmental Mass Spectrometry 1990, previously cited) or Chen (US 2012/0164741, previously cited and hereinafter called Chen ‘741) can be consulted.
For the Loo paper in particular, the sample preparation paragraph on page 287 teaches that for ESI, sample solutions (100 mM) were prepared in distilled water with glacial acetic acid added at 1-5% so that the ionization occurred under acidic conditions. The last full paragraph on page 288 teaches that the ESI spectrum of bovine insulin showed 4+, 5+ and 6+ ions with the [M + 5H]5+ ion as the base peak (see figure 1(a)). This is similar to the teaching of Peterman and further supports the inherent generation/production of 6+ ions when ionizing insulin analogs under acidic conditions.
The Chen ‘741 patent publication appears to be applicant’s own work. In particular paragraph [0133] teaches that ionization of insulin may result in multiply charged precursor ions (such as precursor ions of 4+, 5+, 6+, etc.). Ionization conditions, particularly the pH of the buffer utilized in electrospray techniques, greatly influence the identity and quantity of insulin precursor ions generated. For example, under acidic conditions, positive electrospray ionization may predominately generate 5+ and 6+ charged insulin precursor ions with m/z of 1162.5 ± 0.5 and 968.5 ± 0.5, respectively (see, for example Figure 3A). However, under basic conditions, positive electrospray ionization may predominately generate 4+ and 5+ charged insulin precursor ions with m/z of 1453.75 ± 0.5 and 1162.94 ± 0.5, respectively. The methods may utilize either acidic or basic conditions; preferably acidic conditions. Here again the teachings of Peterman relative to at least the inherent generation of 5+ and 6+ charged insulin precursor ions under acidic conditions is supported.
Applicant’s claims do not distinguish over the applied art based on the generation of the required insulin analog precursor ions. The evidence is clear that the required fragment ions are inherently generated when detemir or glulisine are ionizes in an electrospray under acidic conditions. The failure of the instant claims to move away from anticipation by the applied art is that the subsequent steps do not limit the fragmentation of the precursor ions to fragmentation of the precursor ion required in paragraph (b) of claims 1 and 24 and the determination of the amount of those fragment ions and use of that fragment ion amount to determine the amount of the particular insulin analog in the sample. As noted above, as long as there is evidence that the required precursor ion would be generated during ionization of the particular insulin analog, claims 1 and 24 cover fragmenting any of the generated precursor ions from the particular insulin analog, determining the amount of one or more of those fragment ions and using the determined amount to determine the amount of the particular insulin analog in the sample. That is why Hess, Peterman, Thomas ’14, Chambers, Thevis and Thomas ’12 still anticipate one or more of the instant claims and the arguments are not persuasive.
With respect to the obviousness rejection, as explained above, Hess, Peterman, Thomas ’14 still anticipate claim 1. Thus, the teachings of Chen are not needed for that purpose. However the teachings of Chen are needed for claims 9 and 10 which the arguments fail to address. Thus the argument is not persuasive.
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/ARLEN SODERQUIST/ Primary Examiner, Art Unit 1797
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