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 .
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-20 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.
Regarding claims 2-5, 7-9, 10-15 and 17-20, the term “about” is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. See MPEP 2173.05(b)(III)(A). Claim 16-20 is rejected by virtue of its dependency on claim 15. Claims 5-8 rejected by virtue of its dependency on claim 4. Claims 10-14 rejected by virtue of its dependency on claim 9.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-3 are rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47; citation made with respect to the copy provided with IDS dated 06/07/2023) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Part C, Volume 28, Pages 85-103) in further view of Snyder et al. (Snyder et al., “Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328).
With respect to claim 1, Seyama et al. teaches a method of isolating desmosine and isodesmosine in elastin, (Page 41, Rat aortic elastin preparation) comprising:
hydrolyzing a sample of elastin (Page 41, paragraph starting with "Fischer strain" teaches the elastin fraction was hydrolyzed in 6 N HCL for 48 hours at 110 degrees) and
separating the desmosine and isodesmosine in the hydrolyzed sample of elastin within a high-performance liquid chromatography (HPLC) column (Page 41, paragraph starting with "The HPLC” teaches an aliquot of aortic elastin fraction hydrolysate was used for determination of D and ID; Page 41, paragraph starting with "Fischer strain” teaches D and ID and other amino acids were separated on the reverse phase HPLC column) using a mobile phase gradient comprising a first solvent solution of MSA and HSA (Page 41, paragraph starting with "The HPLC” teaches 0.1 M methane sulfonic acid at pH 2.0 and the retention time of D and ID were controlled with the addition of sodium heptane sulfonate).
However, Seyama et al. fails to teach the mobile phase gradient comprising a second solvent solution of formic acid in Acetonitrile.
Xu et al. teaches the quantification of collagen extracted from tissues (Page 102, paragraph starting with “ In this article”). Xu et al. teaches analyzing the collagen using HPLC (Page 95, paragraph starting with “ The accurate quantitative”) and liquid chromatography mass spectrometry (Page 97, paragraph starting with “ The marker peptide”). Xu et al. analyzed marker peptides to identify collagen in tissue (Page 97, paragraph starting with “ The sample was”). Xu et al. teaches that the marker peptide method was useful for amino acid determination in collagen (Page 96, paragraph starting with “The marker peptide”) by hydrolyzing the collagen sample (Page 97, paragraph starting with “The marker peptides”). Xu et al. further teaches the detection of total collagen in tissue by hydrolyzing the sample with hydrochloric acid which allowed for the detection of hydroxyproline and collagen using HPLC (Page 98, starting with the paragraph “The contents of”).
Xu et al. further teaches a second solvent solution of formic acid in Acetonitrile (Page 97, paragraph starting with “The marker peptides” teaches identifying the collagen V marker peptides ; Page 97, paragraph starting with “The marker peptides” teaches where the LC, Zorbax SB C18 was used, and the column temperature was 25°C ; Page 97, paragraph starting with “The marker peptides” teaches Formic acid water solution, 0.1%, was used as the mobile phase A, and 0.1% formic acid acetonitrile solution was used as the mobile phase B; Page 97, paragraph starting with “The marker peptides” teaches the gradient elution procedure was performed as given below: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B, Page 96, paragraph starting with “The molecular-weight,” Xu et al. teaches acetonitrile denoted as ACN and Page 97, paragraph starting with “For the LC,” Xu et al. teaches a 60% ACN with 0.1% FA solution as mobile phase B).
Snyder et al. teaches methods for method development of HPLC instruments (Page 327, Section 7.3.3). Snyder et al. teaches separation conditions can be varied in order to improve relative retention and maximize resolution (Page 328, Section 7.3.3.2). Snyder further teaches the simultaneous variation of two different separation conditions will generally prove more effective (Page 328, Section 7.3.3.2).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the mobile phase of Seyama et al. to incorporate the teachings of 0.1% formic acid in acetonitrile as mobile phase B for the mobile phase gradient as taught by Xu et al. (Page 97, paragraph starting with “The marker peptides) and Snyder’s motivation of simultaneous variations of two different separation conditions proving more effective (Page 328, paragraph starting with “Any of”) to provide: the mobile phase gradient comprising a second solvent solution of formic acid in Acetonitrile. Doing so would have a reasonable expectation of successfully optimizing selectivity of analytes for liquid chromatography analysis as discussed by Snyder et al. (Page 328, paragraph starting with “Any of” and Page 379, paragraph starting with “Once separations”). See MPEP 2143 (I)(D). The person of ordinarily skill in the art would further have predicted that the modification would allow for specific detection of the extracellular matrix components because Xu et al. teaches that amino acids were detected as the marker peptides of collagen V (Page 97, paragraph starting with “The marker peptides”).
With respect to claim 2, modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 1. Modified Seyama et al. further teaches wherein the HPLC column comprises a C18 HPLC column operating at about 30 to about 40 degrees Centigrade : (Page 41, paragraph starting with "The HPLC,” Seyama et al. teaches the HPLC resin used was a reversed phase type, i. e., RP-18, 7 or Nucleosil C-18; and page 41, paragraph starting with "The HPLC “ teaches the column temperature was maintained at 25°C). Note: The term “about” is not defined by the claim (see above). Therefore, the term “about” was interpreted as a value 5 digits away from the claimed range of “about 30 to about 40 degrees Centigrade “.
In the alternative, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." MPEP § 2144.05(II)(A). Therefore, it would have been obvious to one skilled in the art to use a temperature of about 30 to about 40 degrees C because Snyder et al. teaches HPLC separation is usually carried out at temperatures between ambient and 50◦C; and the selection of a specific temperature within this range is often made on the basis of optimum selectivity (Page 64, Section “High Temperature Operation”).
With respect to claim 3, modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 1.
Modified Seyama et al. further teaches wherein the first solvent solution comprises about 0.098 M to about 0.102 M MSA and about 5.88 mM to about 6.12 mM HSA at about pH 1.90 to about pH 2.1 (the separation of desmosine and Isodesomosine by HPLC was 0.1 MSA (pH 2.0)/acetonitrile=90/10 containing 6mM HSA) (Seyama et al., Page 41, paragraph starting with “The HPLC”).
However, Modified Seyama et al. does not teach and the second solvent solution comprises about 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes.
Xu et al. teaches the quantification of collagen extracted from tissues (Page 102, paragraph starting with “ In this article). Xu et al. teaches analyzing the collagen using HPLC (Page 95, paragraph starting with “ The accurate quantitative”) and liquid chromatography mass spectrometry (Page 97, paragraph starting with “ The marker peptide”). Xu et al. analyzed marker peptides to identify collagen in tissue (Page 97, paragraph starting with “ The sample was”) Xu et al. teaches that the marker peptide method was useful for amino acid determination in collagen (Page 96, paragraph starting with “The marker peptide”) by hydrolyzing the collagen sample (Page 97, paragraph starting with “The marker peptides”). Xu et al. further teaches the detection of total collagen in tissue by hydrolyzing the sample with hydrochloric acid which allowed for the detection of hydroxyproline and collagen using HPLC (Page 98, starting with the paragraph “The contents of”).
Xu et al. further teaches a second solvent solution comprising about 0.196% to about 0.204% formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28: (Page 97, paragraph starting with “The marker peptides,” Xu et al. teaches identifying the collagen V marker peptides; Page 97, paragraph starting with “The marker peptides,” Xu et al. teaches where the LC, Zorbax SB C18 was used, and the column temperature was 25°C; Page 97, paragraph starting with “The marker peptides” Xu et al. teaches Formic acid water solution, 0.1%, was used as the mobile phase A, and 0.1% formic acid acetonitrile solution was used as the mobile phase B; Page 97, paragraph starting with “The marker peptides” teaches the gradient elution procedure was performed as given below: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B, Page 96, paragraph starting with “The molecular-weight,” Xu et al. teaches acetonitrile denoted as ACN and Page 97, paragraph starting with “For the LC,” Xu et al. teaches a 60% ACN with 0.1% FA solution as mobile phase B).
Snyder et al. teaches methods for method development of HPLC instruments (Page 327, Section 7.3.3). Snyder et al. teaches separation conditions can be varied in order to improve relative retention and maximize resolution (Page 328, Section 7.3.3.2). Snyder et al. further teaches optimized separation can be found by trial-and-error changes in %B or temperature (Page 323, paragraph starting with “A maximum”) and once %B is selected, the next step is adjustment of separation selectivity for optimal relative retention and maximum resolution (Page 320, paragraph starting with “Following”).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the mobile phase conditions of Seyama et al. to incorporate the teachings of 0.1% formic acid in acetonitrile as mobile phase B for the mobile phase gradient and a gradient elution procedure of: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B as taught by Xu et al. (Page 97, paragraph starting with “The marker peptides” and Page 97, paragraph starting with “The marker peptides”) and Snyder’s motivation of simultaneous variations of two different separation conditions proving more effective (Page 328, paragraph starting with “Any of”) to provide: the second solvent solution comprises about 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes through routine experimentation (see MPEP 2144.05 (II)). Doing so would utilize known variation of mobile phase gradients as discussed by Snyder et al. by optimizing selectivity of analytes for liquid chromatography analysis (Page 328, paragraph starting with a “Any of” and Page 379, paragraph starting with “Once separations”). Furthermore, starting with a 0.1% formic acid in acetonitrile as mobile phase B and a gradient elution of 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B, one of ordinary skill would have been motivated to modify Xu et al. to have an elution gradient of 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes through routine experimentation to optimize and control factors of selectivity such as improving the selectivity of the HPLC method and to ensure that the separation is complete by the time the liquid reaches the end of the column, i.e. reducing the gradient elution to about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes would allow for optimized separation of the analytes.
Claims 4 is rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47; citation made with respect to the copy provided with IDS dated 06/07/2023) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Part C, Volume 28, Pages 85-103) in further view of Snyder et al. (Snyder et al., “Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328) as applied to claim 1, and further in view of Duan et al. (CN113698472A; see machine translation).
With respect to claim 4, modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 1.
Modified Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with HCl (Page 41, paragraph starting with “The HPLC).
However, modified Seyama et al. does not teach wherein the hydrolyzing comprises washing the sample of elastin in chilled alcohol at less than about 10 degrees Centigrade.
Duan et al. teaches the processing of fish byproducts and preparation method of
high-purity small-molecule fish skin collagen peptides (Page 1, paragraph starting with "As can be"). Duan et al. teaches a pretreatment of the fish skin to extract collagen (Page 3, paragraph starting with "The fish skin"). Duan et al. also teaches the invention that relates to a fish skin collagen peptide spray, which is prepared from the small molecular fish skin collagen peptide, is prepared into an external spray for beauty repair, is uniformly dispersed and easily absorbed by skin, and is suitable for various beauty repair and health care products (Page 8, paragraph starting with "The fish collagen peptide").
Duan et al. further teaches washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade (Page 7, paragraph starting with “Precooling” teaches precooling the absolute ethyl alcohol to 4-6 ℃, and removing residual fat at 4-6 ℃ ; Page 6, paragraph starting with “step 2” teaches mixing and stirring the fish skin and absolute ethyl alcohol according to a material-liquid ratio of 1: 10-1: 20(g/mL or kg/L) to remove residual fat).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Seyama et al. of washing the sample of elastin with cold saline and drying with 2:1 chloroform-methanol then acetone with the method of Duan et al. of washing the sample of elastin with precooling absolute ethyl alcohol to 4-6 ℃ and stirring to remove fat to provide: wherein the hydrolyzing comprises washing the sample of elastin in chilled alcohol at less than about 10 degrees Centigrade. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade would result in removing residual fat (Page 6, paragraph starting with “step 2”). The skilled artisan would have had a reasonable expectation of success in hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade because Seyama et al. teaches the purification of elastin and Duan et al. teaches a preparation method of high-purity fish skin collagen peptide (Page 6, paragraph starting with “The purpose of”). See MPEP 2143 (I)(B).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47; citation made with respect to the copy provided with IDS dated 06/07/2023) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Pa) in further view of Snyder et al. (Snyder, Lloyd; Kirkland Joseph and Dolan John, Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328, Page 322, 327-328) in further in view of Duan et al. (CN113698472A; see machine translation) as applied to claim 4, and further in view of Nilsuwan et al. (Nilsuwan et al., Development of Hydrolysis and Defatting Processes for Production of Lowered Fishy Odor Hydrolyzed Collagen from Fatty Skin of Sockeye Salmon (Oncorhynchus nerka), September 23 2021, Foods, Volume 10, Issue 10, 2257).
With respect to claim 5, Modified Seyama et al. and Duan et al. teaches all of elements of the current invention as stated above with respect to claim 4.
Modified Seyama et al. teaches wherein the washing comprises mixing the sample of precooling the absolute ethyl alcohol to 4-6 ℃, and removing residual fat at 4-6 ℃ (On Page 6, starting at “step 2”, Duan et al. teaches mixing and stirring the fish skin and absolute ethyl alcohol for 6 hours to remove residual fat).
However, Duan et al. fails to teach for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade.
Nilsuwan et al. teaches the hydrolysis of collagen removed from salmon (Page 3, Section 2.3.1). Nilsuwan et al. teaches solvent extraction was also conducted to investigate fat removal processes using DSCS and subsequent solvent extraction to yield HC powder (Page 2, paragraph starting at “To reduce”). Nilsuwan et al. teaches removing fat by mixing the HC powder with alcohol, specifically isopropanol (Page 3, paragraph starting with “HC defatted”). Nilsuwan et al. teaches amino acid after defatting (Page 11, Section 3.4.2).
Nilsuwan et al. further teaches the mixture was further mixed with isopropanol and the aid of ultrasonic processor with pulse mode (on/off at 10 s) for 10 min (Page 3, Section 2.5). The mixture was then stirred at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuged at 10,000× g for 20 min at 4 ◦C (Page 3, Section 2.5). Nilsuwan et al. further teaches fat content of HC from salmon skin hydrolysate after fat removal at different stirring cycles of 0, 3, 6, and 9 (Table 2).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the defatting stirring conditions of Seyama et al. to incorporate the teachings of pulse mode (on/off at 10 s) for 10 min where the mixture was stirred at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuged at 10,000× g for 20 min at 4 ◦C as taught by Nilsuwan et al. (Page 3, Section 2.5) to provide: for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade through routine experimentation (see MPEP 2144.05 (II)). Doing so would utilize known variation of defatting conditions as discussed by Nilsuwan et al. by optimizing the amount of fat removed for amino acid determination (Page 11, Section 3.4.2). Furthermore, starting with a mixing with isopropanol and the aid of ultrasonic processor with pulse mode (on/off at 10 s) for 10 min (Page 3, Section 2.5) then stirring at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuging at 10,000× g for 20 min at 4 ◦C, one of ordinary skill would have been motivated to modify Nilsuwan et al. to have mixed with chilled alcohol for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade through routine experimentation to optimize and control factors of defatting such as improving the recovery of amino acids from collagen hydrolysate, i.e. changing the defatting conditions to mixing with chilled alcohol for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade would allow for optimized recovery of amino acids and defat the hydrolysate.
Claims 6 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Pa), in further view of Snyder et al. (Snyder et al., Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328), in further in view of Duan et al. (CN113698472A; see machine translation) as applied to claim 4 above, and further in view of Daamen et al. (Daamen et al., Isolation of intact elastin fibers devoid of microfibrils. Tissue Eng. 2005 Jul-Aug;11(7-8):1168-76).
With respect to claim 6, modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 4 (see above).
Modified Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Seyama et al., Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with HCl (Seyama et al., Page 41, paragraph starting with “The HPLC).
However, Modified Seyama et al. does not teach hydrolyzing the washed sample of elastin by vacuum hydrolysis with HCl.
Daamen et al. teaches a novel protocol for the isolation of elastin (Page 1169, paragraph starting with "Equine ligamentum"). Daamen et al. teaches that previous methods fail at removing additional extracellular matrix components such as collagen (Page 1168, paragraph starting with " Evidence exists"). Daamen et al. teaches three protocols for extracting elastin for amino acid determination (Page 1171, paragraph starting with "Elastin samples").
Daamen et al. further teaches followed by vacuum hydrolyzing the washed sample of elastin within HCl (On page 1169, Procedure B and Figure 1 teaches washing elastin samples with water in procedure B , followed by defatting samples for procedure A; On page 1171, paragraph starting with “Elastin samples,” Daamen et al. further teaches amino acid determination by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. of hydrolyzing a sample of elastin with the method of Daamen et al. of hydrolyzing elastin samples under vacuum with 6M HCl to provide: hydrolyzing the washed sample of elastin by vacuum hydrolysis with HCl. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by hydrolyzing the washed sample of elastin by vacuum hydrolysis with HCl would result in highly purified elastin preparation (Daamen et al., Page 1168-1169, paragraph starting with “We have previously”). The skilled artisan would have had a reasonable expectation of success in hydrolyzing the washed sample of elastin by vacuum hydrolysis with HCl because Seyama et al. teaches the purification of elastin and Daamen et al. teaches purifying elastin for amino acid determination (Page 1173, Table 1). See MPEP 2143 (I)(B).
With respect to claim 7, modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 4 (see above).
Modified Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Seyama et al., Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with 6 N HCl for 48 hours at 110°C (Page 41, paragraph starting with “The HPLC).
However, Modified Seyama et al. does not teach comprising vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours.
Daamen et al. teaches a novel protocol for the isolation of elastin (Page 1169, paragraph starting with "Equine ligamentum"). Daamen et al. teaches that previous methods fail at removing additional extracellular matrix components such as collagen (Page 1168, paragraph starting with " Evidence exists"). Daamen et al. teaches three protocols for extracting elastin for amino acid determination (Page 1171, paragraph starting with "Elastin samples").
Daamen et al. further teaches vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hour (Page 1169, Procedure B teaches washing elastin samples with water in procedure B; Page 1169, Figure 1 teaches followed by defatting samples with acetone for procedure A; Page 1171, paragraph starting with “Elastin samples” teaches amino acid determination by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. of hydrolyzing a sample of elastin with the method of Daamen et al. of hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C to provide: vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hour. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours would result in highly purified elastin preparation (Page 1168-1169, paragraph starting with “We have previously”). The skilled artisan would have had a reasonable expectation of success in vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours because Seyama et al. teaches the purification of elastin and Daamen et al. teaches purifying elastin for amino acid determination (Page 1173, Table 1). See MPEP 2143 (I)(B).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47; citation made with respect to the copy provided with IDS dated 06/07/2023) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Pa), in further view of Snyder et al. (Snyder et al., “Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328,), in further in view of Duan et al. (CN113698472A; see machine translation) in further view of Daamen et al. (Daamen et al., Isolation of intact elastin fibers devoid of microfibrils. Tissue Eng. 2005 Jul-Aug;11(7-8):1168-76)as applied to claim 4, and further in view of Shiraishi et al. (Shiraishi et al., "Development of a Robust LC-MS/MS Method for Determination of Desmosine and Isodesmosine in Human Urine," Journal of Oleo Science; Vol. 59, No. 8; February 10, 2010; pp. 431-439; cited in the IDS dated 06/07/2023).
With respect to claim 8, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 4. Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with 6 N HCl for 48 hours at 110°C (Page 41, paragraph starting with “The HPLC). Seyama et al. teaches evaporating the hydrolysate and drying over P2O5 and solid NaOH in a desiccator.
However, Seyama et al. fails to teach drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade.
Shiraishi et al. is used to remedy this, as Shiraishi et al. teaches desomosine and isodesmosine as markers of elastin degradation found in urine (Page 432, paragraph starting with "In this study"). Elastin was added to urine samples and hydrolyzed using hydrochloric acid and analyzed using UPLC MS/MS. (Page 432, paragraph starting with "A volume" and Page 433, paragraph starting with "Ultra-performance").
Shiraishi et al. further teaches comprising drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade (These eluates were evaporated to dryness under nitrogen gas flow at 60°C) (Page 432, paragraph starting with “A volume”).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. evaporating the hydrolysate and drying over P2O5 and solid NaOH in a desiccator with the method of Shiraishi et al. of eluates being evaporated to dryness under nitrogen gas flow at 60°C to provide: drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees; would result in an shorter separation time for isodesomosine and desmosine (Page 434-435, paragraph starting with “A general”). The skilled artisan would have had a reasonable expectation of success in eluates were evaporated to dryness under nitrogen gas flow at 60°C; because Seyama et al. teaches the purification of elastin and Shiraishi et al. teaches a robust analytical method for measuring desmosine and isodesmosine (Page 438, paragraph starting with “This study”). See MPEP 2143 (I)(B).
Claims 9-11, 13 and 15-17 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Part C Methods), in further view of Snyder et al. (Snyder et al., Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328), in further in view of Duan et al. (CN113698472A; see machine translation) in further view of Daamen et al. (Daamen et al., Isolation of intact elastin fibers devoid of microfibrils. Tissue Eng. 2005 Jul-Aug;11(7-8):1168-76).
With respect to claim 9, Seyama et al. teaches a method of measuring desmosine and isodesmosine in elastin, (Page 43, Measurement of desmosine and isodesmosine in hydrolysates of commercial elastin by HPLC and AAA method) comprising:
hydrolyzing a sample of elastin (Page 41, paragraph starting with "Fischer strain");
separating the desmosine and isodesmosine in the hydrolyzed sample of elastin within a high-performance liquid chromatography column (Page 41, paragraph starting with "The HPLC” teaches an aliquot of aortic elastin fraction hydrolysate was used for determination of D and ID; Page 41, paragraph starting with "Fischer strain” teaches D and ID and other amino acids were separated on the reverse phase HPLC column) using a mobile phase gradient comprising a first solvent solution of MSA and HSA (Page 41, paragraph starting with "The HPLC” teaches 0.1 M methane sulfonic acid at pH 2.0 and the retention time of D and ID were controlled with the addition of sodium heptane sulfonate); and
measuring the amount of desmosine and isodesmosine as eluents from the HPLC column at about 275 nanometers based upon a HPLC chromatogram (Fig. 5 shows the retention times and amounts of isodesmosine and desmosine after elution from the HPLC column using a UV detector at 275 nm .
Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches hydrolyzing the washed sample of elastin with HCl (Page 41, paragraph starting with “The HPLC); but Seyama et al. fails to teach: hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade followed by vacuum hydrolyzing the washed sample of elastin within HCl; and the mobile phase gradient comprising a second solvent solution of formic acid in Acetonitrile.
Duan et al. teaches the processing of fish byproducts and preparation method of
high-purity fish skin collagen peptides (Page 1, paragraph starting with "As can be"). Duan et al. teaches a pretreatment of the fish skin to extract collagen (Page 3, paragraph starting with "The fish skin"). Duan et al. also teaches the invention that relates to a fish skin collagen peptide spray, which is prepared from the small molecular fish skin collagen peptide, is prepared into an external spray for beauty repair, is uniformly dispersed and easily absorbed by skin, and is suitable for various beauty repair and health care products (Page 8, paragraph starting with "The fish collagen peptide").
Duan et al. further teaches washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade (Page 7, paragraph starting with “Precooling” teaches precooling the absolute ethyl alcohol to 4-6 ℃, and removing residual fat at 4-6 ℃ Page 6, paragraph starting with “step 2” teaches mixing and stirring the fish skin and absolute ethyl alcohol according to a material-liquid ratio of 1: 10-1: 20(g/mL or kg/L) to remove residual fat.
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Seyama et al. of washing the sample of elastin with cold saline and drying with 2:1 chloroform-methanol then acetone with the method of Duan et al. of with precooling absolute ethyl alcohol to 4-6 ℃ and stirring to remove fat to provide: hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade would result in removing residual fat (Page 6, paragraph starting with “step 2”). The skilled artisan would have had a reasonable expectation of success in hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade because Seyama et al. teaches the purification of elastin and Duan et al. teaches a preparation method of high-purity fish skin collagen peptide MPEP 2143 (I)(B) (Page 6, paragraph starting with “The purpose of”).
Modified Seyama et al. fails to teach followed by vacuum hydrolyzing the washed sample of elastin within HCl; and the mobile phase gradient comprising a second solvent solution of formic acid in Acetonitrile.
Daamen et al. teaches a novel protocol for the isolation of elastin (Page 1169, paragraph starting with "Equine ligamentum"). Daamen et al. teaches that previous methods fail at removing additional extracellular matrix components such as collagen (Page 1168, paragraph starting with " Evidence exists"). Daamen et al. teaches three protocols for extracting elastin for amino acid determination (Page 1171, paragraph starting with "Elastin samples").
Daamen et al. further teaches followed by vacuum hydrolyzing the washed sample of elastin within HCl (On page 1169, Figure 1, washing elastin samples with water in procedure B, followed by defatting samples for procedure A; On page 1171, paragraph starting with “Elastin samples,” Daamen et al. further teaches amino acid determination by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. hydrolyzing a sample of elastin with the method of Daamen et al. hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C to provide: followed by vacuum hydrolyzing the washed sample of elastin within HCl. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by vacuum hydrolyzing the washed sample of elastin within HCl; would result in highly purified elastin preparation (Page 1168-1169, paragraph starting with “We have previously”). The skilled artisan would have had a reasonable expectation of success in vacuum hydrolyzing the washed sample of elastin within HCl because Seyama et al. teaches the purification of elastin and Daamen et al. teaches purifying elastin for amino acid determination. (Page 1173, Table 1) MPEP 2143 (I)(B).
Modified Seyama et al. fails to teach the mobile phase gradient comprising a second solvent solution of formic acid in Acetonitrile.
Xu et al. teaches the quantification of collagen extracted from tissues (Page 102, paragraph starting with “ In this article”). Xu et al. teaches analyzing the collagen using HPLC (Page 95, paragraph starting with “ The accurate quantitative”) and liquid chromatography (LCQ) mass spectrometry (Page 97, paragraph starting with “ The marker peptide”). Xu et al. analyzed marker peptides to identify collagen in tissue (Page 97, paragraph starting with “ The sample was”). Xu et al. teaches that the marker peptide method was useful for amino acid determination in collagen (Page 96, paragraph starting with “The marker peptide”) by hydrolyzing the collagen sample (Page 97, paragraph starting with “The marker peptides”). Xu et al. further teaches the detection of total collagen in tissue by hydrolyzing the sample with hydrochloric acid which allowed for the detection of hydroxyproline and collagen using HPLC (Page 98, starting with the paragraph “The contents of”).
Xu et al. further teaches and a second solvent solution of formic acid in Acetonitrile (Page 97, paragraph starting with “The marker peptides” teaches identifying the collagen V marker peptides ; Page 97, paragraph starting with “The marker peptides teaches where the LC, Zorbax SB C18 was used, and the column temperature was 25°C ; Page 97, paragraph starting with “The marker peptides” teaches Formic acid water solution, 0.1%, was used as the mobile phase A, and 0.1% FA-can solution was used as the mobile phase B; Page 97, paragraph starting with “The marker peptides” teaches the gradient elution procedure was performed as given below: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B).
Snyder et al. teaches methods for method development of HPLC instruments (Page 327, Section 7.3.3). Snyder et al. teaches separation conditions can be varied in order to improve relative retention and maximize resolution (Page 328, Section 7.3.3.2). Snyder further teaches The simultaneous variation of two different separation conditions will generally prove more effective (Page 328, Section 7.3.3.2).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the mobile phase of Seyama et al.to incorporate the teachings of 0.1% formic acid in acetonitrile as mobile phase B for the mobile phase gradient as taught by Xu et al. (Page 97, paragraph starting with “The marker peptides) and Snyder et al. motivation of simultaneous variations of two different separation conditions proving more effective (Page 328, paragraph starting with “Any of”) to provide: the mobile phase gradient comprising a second solvent solution of formic acid in Acetonitrile. Doing so would have a reasonable expectation of successfully optimizing selectivity of analytes for liquid chromatography analysis as discussed by Snyder et al. (Page 328, paragraph starting with “Any of” and Page 379, paragraph starting with “Once separations”). See MPEP 2143 (I)(D). The person of ordinarily skill in the art would further have predicted that the modification would allow for specific detection of the extracellular matrix components because Xu et al. teaches that amino acids were detected as the marker peptides of collagen V (Page 97, paragraph starting with “The marker peptides”).
With respect to claim 10, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 9. Modified Seyama et al. further teaches wherein the HPLC column comprises a C18 HPLC column operating at about 30 to about 40 degrees Centigrade (On Page 41, paragraph starting with "The HPLC” states that the HPLC resin used was a reversed phase type, i.e., RP-18, 7 or Nucleosil C-18 The column temperature was maintained at 25°C (Seyama et al., Page 42, paragraph starting with "The HPLC). Note: The term “about” is not defined by the claim (see above). Therefore, the term “about” was interpreted as a value 5 digits away from the claimed range of “about 30 to about 40 degrees Centigrade.”
In the alternative, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." MPEP § 2144.05(II)(A). Therefore, it would have been obvious to one skilled in the art to use a temperature of about 30 to about 40 degrees C because Snyder et al. teaches HPLC separation is usually carried out at temperatures between ambient and 50◦C; and the selection of a specific temperature within this range is often made on the basis of optimum selectivity (Page 64, Section “High Temperature Operation”).
With respect to claim 11, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 9. Modified Seyama et al. further teaches wherein the first solvent solution comprises about 0.098 M to about 0.102 M MSA and about 5.88 mM to about 6.12 mM HSA at about pH 1.90 to about pH 2.1 (Seyama, page 41, paragraph starting with “The HPLC”, the separation of desmosine and Isodesomosine by HPLC was 0.1 MSA (pH 2.0)/acetonitrile=90/10 containing 6mM HSA) (Seyama et al., Page 41, paragraph starting with “The HPLC”).
However, Modified Seyama et al. does not teach and the second solvent solution comprises about 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes.
Xu et al. teaches the quantification of collagen extracted from tissues (Page 102, paragraph starting with “ In this article). Xu et al. teaches analyzing the collagen using HPLC (Page 95, paragraph starting with “ The accurate quantitative”) and liquid chromatography mass spectrometry (Page 97, paragraph starting with “The marker peptide”). Xu et al. analyzed marker peptides to identify collagen in tissue (Page 97, paragraph starting with “The sample was”). Xu et al. teaches that the marker peptide method was useful for amino acid determination in collagen (Page 96, paragraph starting with “The marker peptide”) by hydrolyzing the collagen sample (Page 97, paragraph starting with “The marker peptides”). Xu et al. further teaches the detection of total collagen in tissue by hydrolyzing the sample with hydrochloric acid which allowed for the detection of hydroxyproline and collagen using HPLC (Page 98, starting with the paragraph “The contents of”).
Xu et al. further teaches a second solvent solution comprising about 0.196% to about 0.204% formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 (Page 97, paragraph starting with “The marker peptides” teaches identifying the collagen V marker peptides ; Page 97, paragraph starting with “The marker peptides” teaches where the LC, Zorbax SB C18 was used, and the column temperature was 25°C ; Page 97, paragraph starting with “The marker peptides” teaches Formic acid water solution, 0.1%, was used as the mobile phase A, and 0.1% formic acid acetonitrile solution was used as the mobile phase B; Page 97, paragraph starting with “The marker peptides” teaches the gradient elution procedure was performed as given below: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B, Page 96, paragraph starting with “The molecular-weight,” Xu et al. teaches acetonitrile denoted as ACN and Page 97, paragraph starting with “For the LC,” Xu et al. teaches a 60% ACN with 0.1% FA solution as mobile phase B.
Snyder et al. teaches methods for method development of HPLC instruments (Page 327, Section 7.3.3). Snyder et al. teaches separation conditions can be varied in order to improve relative retention and maximize resolution (Page 328, Section 7.3.3.2). Snyder et al. further teaches optimized separation can be found by trial-and-error changes in %B or temperature (Page 323, paragraph starting with “A maximum”) and once %B is selected, the next step is adjustment of separation selectivity for optimal relative retention and maximum resolution (Page 320, paragraph starting with “Following”).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the mobile phase conditions of Seyama et al. to incorporate the teachings of 0.1% formic acid in acetonitrile as mobile phase B for the mobile phase gradient and a gradient elution procedure of: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B as taught by Xu et al. (Page 97, paragraph starting with “The marker peptides” and Page 97, paragraph starting with “The marker peptides”) and Snyder et al. motivation of simultaneous variations of two different separation conditions proving more effective (Page 328, paragraph starting with “Any of”) to provide: the second solvent solution comprises about 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes through routine experimentation (see MPEP 2144.05 (II)). Doing so would utilize known variation of mobile phase gradients as discussed by Snyder et al. by optimizing selectivity of analytes for liquid chromatography analysis (Page 328, paragraph starting with a “Any of” and Page 379, paragraph starting with “Once separations”). Furthermore, starting with a 0.1% formic acid in acetonitrile as mobile phase B and a gradient elution of 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B, one of ordinary skill would have been motivated to modify Xu et al. to have an elution gradient of 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes through routine experimentation to optimize and control factors of selectivity such as improving the selectivity of the HPLC method and to ensure that the separation is complete by the time the liquid reaches the end of the column, i.e. reducing the gradient elution to about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes would allow for optimized separation of the analytes.
With respect to claim 13, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 9.
Modified Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Seyama et al., Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with 6 N HCl for 48 hours at 110°C (Page 41, paragraph starting with “The HPLC).
However, Modified Seyama et al. does not teach comprising vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours.
Daamen et al. teaches a novel protocol for the isolation of elastin (Page 1169, paragraph starting with "Equine ligamentum"). Daamen et al. teaches that previous methods fail at removing additional extracellular matrix components such as collagen (Page 1168, paragraph starting with " Evidence exists"). Daamen et al. teaches three protocols for extracting elastin for amino acid determination (Page 1171, paragraph starting with "Elastin samples").
Daamen et al. further teaches vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hour (Page 1169, Procedure B teaches washing elastin samples with water in procedure B; Page 1169, Figure 1 teaches followed by defatting samples with acetone for procedure A ; Page 1171, paragraph starting with “Elastin samples” teaches amino acid determination by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C ).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. of hydrolyzing a sample of elastin with the method of Daamen et al. of hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C to provide: vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hour. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours; would result in highly purified elastin preparation (Page 1168-1169, paragraph starting with “We have previously”). The skilled artisan would have had a reasonable expectation of success in vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours because Seyama et al. teaches the purification of elastin and Daamen et al. teaches purifying elastin for amino acid determination (Page 1173, Table 1). See MPEP 2143 (I)(B).
With respect to claim 15, Seyama et al. teaches a method of measuring desmosine and isodesmosine in elastin, (Page 43, Measurement of desmosine and isodesmosine in hydrolysates of commercial elastin by HPLC and AAA method) comprising:
hydrolyzing a sample of elastin (Page 41, paragraph starting with "Fischer strain");
separating the desmosine and isodesmosine in the hydrolyzed sample of elastin within a high-performance liquid chromatography column (Page 41, paragraph starting with "The HPLC” teaches an aliquot of aortic elastin fraction hydrolysate was used for determination of D and ID; Page 41, paragraph starting with "Fischer strain” teaches D and ID and other amino acids were separated on the reverse phase HPLC column) using a mobile phase gradient comprising a first solvent solution of MSA and HSA (Page 41, paragraph starting with "The HPLC” teaches 0.1 M methane sulfonic acid at pH 2.0 and the retention time of D and ID were controlled with the addition of sodium heptane sulfonate); and
measuring the amount of desmosine and isodesmosine as eluents from the HPLC column at about 275 nanometers based upon a HPLC chromatogram (Fig. 5 shows the retention times and amounts of isodesmosine and desmosine after elution from the HPLC column using a UV detector at 275 nm.
Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with HCl (Page 41, paragraph starting with “The HPLC); but Seyama et al. fails to teach hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade followed by vacuum hydrolyzing the washed sample of elastin within HCl and a second solvent solution comprising about 0.196% to about 0.204% formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes.
Duan et al. teaches the processing of fish byproducts and preparation method of high-purity fish skin collagen peptides (Page 1, paragraph starting with "As can be").Duan et al. teaches a pretreatment of the fish skin to extract collagen (Page 3, paragraph starting with "The fish skin"). Duan et al. also teaches the invention that relates to a fish skin collagen peptide spray, which is prepared from fish skin collagen peptide, is prepared into an external spray for beauty repair, is uniformly dispersed and easily absorbed by skin, and is suitable for various beauty repair and health care products (Page 8, paragraph starting with "The fish collagen peptide").
Duan et al. further teaches hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade (On page 7, paragraph starting with “Precooling,” precooling the absolute ethyl alcohol to 4-6 ℃, and removing residual fat at 4-6 ℃ ; and mixing and stirring the fish skin and absolute ethyl alcohol according to a material-liquid ratio of 1: 10-1: 20 (g/mL or kg/L) to remove residual fat ).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Seyama et al. of washing the sample of elastin with cold saline and drying with 2:1 chloroform-methanol then acetone with the method of Duan et al. of with precooling absolute ethyl alcohol to 4-6 ℃ and stirring to remove fat to provide: teach hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade would result in removing residual fat (Page 6, paragraph starting with “step 2”). The skilled artisan would have had a reasonable expectation of success in hydrolyzing a sample of elastin by washing the sample of elastin in chilled alcohol at less than 10 degrees Centigrade because Seyama et al. teaches the purification of elastin and Duan et al. teaches a preparation method of high-purity fish skin collagen peptide (Page 6, paragraph starting with “The purpose of”). See MPEP 2143 (I)(B).
Modified Seyama et al. does not teach followed by vacuum hydrolyzing the washed sample of elastin within HCl and a second solvent solution comprising about 0.196% to about 0.204% formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes.
Daamen et al. is used to remedy this, as Daamen et al. teaches a novel protocol for the isolation of elastin (Page 1169, paragraph starting with "Equine ligamentum"). Daamen et al. teaches that previous methods fail at removing additional extracellular matrix components such as collagen (Page 1168, paragraph starting with " Evidence exists"). Daamen et al. teaches three protocols for extracting elastin for amino acid determination (Page 1171, paragraph starting with "Elastin samples").
Daamen et al. further teaches followed by vacuum hydrolyzing the washed sample of elastin within HCl (On page 1169, Procedure B and Figure 1 teaches washing elastin samples with water in procedure B , followed by defatting samples for procedure A; On page 1171, paragraph starting with “Elastin samples,” teaches amino acid determination by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. acid hydrolyzing a sample of elastin with the method of Daamen et al. hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C to provide: followed by vacuum hydrolyzing the washed sample of elastin within HCl. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C; would result in highly purified elastin preparation (Page 1168-1169, paragraph starting with “We have previously”). The skilled artisan would have had a reasonable expectation of success in vacuum hydrolyzing the washed sample of elastin within HCl because Seyama et al. teaches the purification of elastin and Daamen et al. teaches purifying elastin for amino acid determination. (Page 1173, Table 1) MPEP 2143 (I)(B).
Modified Seyama et al. does not teach and a second solvent solution comprising about 0.196% to about 0.204% formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes.
Xu et al. teaches the quantification of collagen extracted from tissues (Page 102, paragraph starting with “ In this article”). Xu et al. teaches analyzing the collagen using HPLC (Page 95, paragraph starting with “ The accurate quantitative”) and liquid chromatography mass spectrometry (Page 97, paragraph starting with “ The marker peptide”). Xu et al. analyzed marker peptides to identify collagen in tissue (Page 97, paragraph starting with “ The sample was”). Xu et al. teaches that the marker peptide method was useful for amino acid determination in collagen (Page 96, paragraph starting with “The marker peptide”) by hydrolyzing the collagen sample (Page 97, paragraph starting with “The marker peptides”). Xu et al. further teaches the detection of total collagen in tissue by hydrolyzing the sample with hydrochloric acid which allowed for the detection of hydroxyproline and collagen using HPLC (Page 98, starting with the paragraph “The contents of”).
Xu et al. further teaches and a second solvent solution comprising about 0.196% to about 0.204% formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 (Page 97, paragraph starting with “The marker peptides” teaches identifying the collagen V marker peptides ; Page 97, paragraph starting with “The marker peptides teaches the LC, Zorbax SB C18 was used, and the column temperature was 25°C ; Page 97, paragraph starting with “The marker peptides” teaches Formic acid water solution, 0.1%, was used as the mobile phase A, and 0.1% FA-can solution was used as the mobile phase B; Page 97, paragraph starting with “The marker peptides” teaches the gradient elution procedure was performed as given below: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B).
Snyder et al. teaches methods for method development of HPLC instruments (Page 327, Section 7.3.3). Snyder et al. teaches separation conditions can be varied in order to improve relative retention and maximize resolution (Page 328, Section 7.3.3.2). Snyder et al. further teaches optimized separation can be found by trial-and-error changes in %B or temperature (Page 323, paragraph starting with “A maximum”) and once %B is selected, the next step is adjustment of separation selectivity for optimal relative retention and maximum resolution (Page 320, paragraph starting with “Following”).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the mobile phase of Modified Seyama et al. to incorporate the teachings of 0.1% formic acid in acetonitrile as mobile phase B for the mobile phase gradient and the gradient elution procedure was performed as given below: 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B as taught by Xu et al. (Page 97, paragraph starting with “The marker peptides” and Page 97, paragraph starting with “The marker peptides”) and Snyder‘s motivation of simultaneous variations of two different separation conditions proving more effective (Page 328, paragraph starting with “Any of”) to provide: the second solvent solution comprises about 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes through routine experimentation (see MPEP 2144.05 (II)). Doing so would utilize known variation of mobile phase gradients as discussed by Snyder et al. by optimizing selectivity of analytes for liquid chromatography analysis (Page 328, paragraph starting with a “Any of” and Page 379, paragraph starting with “Once separations”). Furthermore, starting with a 0.1% formic acid in acetonitrile as mobile phase B and a gradient elution of 0–5min, 5% B; 5–40min, 5–40% B; and 40–80min, 40–75% B, one of ordinary skill would have been motivated to modify Xu et al. to have an elution gradient of 0.196% to about formic acid in Acetonitrile at about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes through routine experimentation to optimize and control factors of selectivity such as improving the selectivity of the HPLC method and to ensure that the separation is complete by the time the liquid reaches the end of the column, i.e. reducing the gradient elution to about 0-5% in about 4 to about 6 minutes, 5-12% in about 12-18 minutes, and 12-5% in about 12 to about 28 minutes would allow for optimized separation of the analytes.
With respect to claim 16, modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 15. Modified Seyama et al. further teaches wherein desomosine and isodesmosine peaks are identified in the chromatogram and compared with a reference standard (On page 43, paragraph starting with “We found,” Seyama et al teaches that the eluted peaks of D and ID were symmetrical when Nucleosil C-18 was used for separation isodesmosine and desomosine by HPLC Fig 4 shows the elution of isodesmosine and desomosine samples and Fig. 5 shows the elution pattern of standard isodesmosine and desomosine).
With respect to claim 17, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 15. Modified Seyama et al. further teaches wherein the HPLC column comprises a C18 HPLC column operating at about 30 to about 40 degrees Centigrade : (Page 41, paragraph starting with "The HPLC,” Seyama et al. teaches the HPLC resin used was a reversed phase type, i. e., RP-18, 7 or Nucleosil C-18; and page 41, paragraph starting with "The HPLC “ teaches the column temperature was maintained at 25°C). Note: The term “about” is not defined by the claim (see above). Therefore, the term “about” was interpreted as a value 5 digits away from the claimed range of “about 30 to about 40 degrees Centigrade “.
In the alternative, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." MPEP § 2144.05(II)(A). Therefore, it would have been obvious to one skilled in the art to use a temperature of about 30 to about 40 degrees C because Snyder et al. teaches HPLC separation is usually carried out at temperatures between ambient and 50◦C; and the selection of a specific temperature within this range is often made on the basis of optimum selectivity (Page 64, Section “High Temperature Operation”).
With respect to claim 19, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 15.
Modified Seyama et al. teaches washing the elastin sample by removing the aorta of rat and removed the tissue by washing with cold saline and drying with 2:1 chloroform-methanol then acetone (Seyama et al., Page 41, paragraph starting with “Fischer strain”). Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with 6 N HCl for 48 hours at 110°C (Page 41, paragraph starting with “The HPLC).
However, Modified Seyama et al. does not teach vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours.
Daamen et al. teaches a novel protocol for the isolation of elastin (Page 1169, paragraph starting with "Equine ligamentum"). Daamen et al. teaches that previous methods fail at removing additional extracellular matrix components such as collagen (Page 1168, paragraph starting with " Evidence exists"). Daamen et al. teaches three protocols for extracting elastin for amino acid determination (Page 1171, paragraph starting with "Elastin samples").
Daamen et al. further teaches vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hour (Page 1169, Procedure B teaches washing elastin samples with water in procedure B; Page 1169, Figure 1 teaches followed by defatting samples with acetone for procedure A ;Page 1171, paragraph starting with “Elastin samples” teaches amino acid determination by hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C ).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. of hydrolyzing a sample of elastin with the method of Daamen et al. of hydrolyzing elastin samples under vacuum with 6M HCl for 22 hours at 110°C to provide: vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hour. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours; would result in highly purified elastin preparation (Page 1168-1169, paragraph starting with “We have previously”). The skilled artisan would have had a reasonable expectation of success in vacuum hydrolyzing the washed sample of elastin at about 100 to 120 degrees Centigrade for about 20 to 28 hours; because Seyama et al. teaches the purification of elastin and Daamen et al. teaches purifying elastin for amino acid determination (Page 1173, Table 1). See MPEP 2143 (I)(B).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47; citation made with respect to the copy provided with IDS dated 06/07/2023) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Pa), in further view of Snyder et al. (Snyder et al., “Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328), in further in view of Duan et al. (CN113698472A; see machine translation) in further view of Daamen et al. (Daamen et al., Isolation of intact elastin fibers devoid of microfibrils. Tissue Eng. 2005 Jul-Aug;11(7-8):1168-76) as applied to claim 9, and further in view of Shiraishi et al. (Shiraishi et al., "Development of a Robust LC-MS/MS Method for Determination of Desmosine and Isodesmosine in Human Urine," Journal of Oleo Science; Vol. 59, No. 8; February 10, 2010; pp. 431-439; cited in the IDS dated 06/07/2023).
With respect to claim 14, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 9.
Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with 6 N HCl for 48 hours at 110°C (Page 41, paragraph starting with “The HPLC). Seyama et al. teaches evaporating the hydrolysate and drying over P2O5 and solid NaOH in a desiccator.
However, Seyama et al. fails to teach comprising drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade.
Shiraishi et al. is used to remedy this, as Shiraishi et al. teaches desomosine and isodesmosine as markers of elastin degradation , found in urine (Page 432, paragraph starting with "In this study"). Elastin was added to urine samples and hydrolyzed using hydrochloric acid and analyzed using UPLC MS/MS. (Page 432, paragraph starting with "A volume" and Page 433, paragraph starting with "Ultra-performance").
Shiraishi et al. further teaches drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade (Page 432, paragraph starting with “A volume” teaches these eluates were evaporated to dryness under nitrogen gas flow at 60°C) .
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. evaporating the hydrolysate and drying over P2O5 and solid NaOH in a desiccator with the method of Shiraishi et al. of eluates being evaporated to dryness under nitrogen gas flow at 60°C to provide: drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees; would result in an shorter separation time for isodesomosine and desmosine (Page 434-435, paragraph starting with “A general”). The skilled artisan would have had a reasonable expectation of success in eluates were evaporated to dryness under nitrogen gas flow at 60°C because Seyama et al. teaches the purification of elastin and Shiraishi et al. teaches a robust analytical method for measuring desmosine and isodesmosine (Page 438, paragraph starting with “This study”). See MPEP 2143 (I)(B).
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47; citation made with respect to the copy provided with IDS dated 06/07/2023) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Pa), in further view of Snyder et al. (Snyder, Lloyd; Kirkland Joseph and Dolan John, Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328, Page 322, 327-328), in further in view of Duan et al. (CN113698472A; see machine translation) in further view of Daamen et al. (Daamen et al., Isolation of intact elastin fibers devoid of microfibrils. Tissue Eng. 2005 Jul-Aug;11(7-8):1168-76)as applied to claim 15, and further in view of Shiraishi et al. (Shiraishi et al., "Development of a Robust LC-MS/MS Method for Determination of Desmosine and Isodesmosine in Human Urine," Journal of Oleo Science; Vol. 59, No. 8; February 10, 2010; pp. 431-439; cited in the IDS dated 06/07/2023).
With respect to claim 20, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 15.
Seyama et al. also teaches acid hydrolyzing the washed sample of elastin with 6 N HCl for 48 hours at 110°C (Page 41, paragraph starting with “The HPLC). Seyama et al. teaches evaporating the hydrolysate and drying over P2O5 and solid NaOH in a desiccator.
However, Seyama et al. fails to teach comprising drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade.
Shiraishi et al. is used to remedy this, as Shiraishi et al. teaches desomosine and isodesmosine as markers of elastin degradation found in urine (Page 432, paragraph starting with "In this study"). Elastin was added to urine samples and hydrolyzed using hydrochloric acid and analyzed using UPLC MS/MS. (Page 432, paragraph starting with "A volume" and Page 433, paragraph starting with "Ultra-performance").
Shiraishi et al. further teaches drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade (Page 432, paragraph starting with “A volume” teaches these eluates were evaporated to dryness under nitrogen gas flow at 60°C) .
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to substitute the methods of Modified Seyama et al. evaporating the hydrolysate and drying over P2O5 and solid NaOH in a desiccator with the method of Shiraishi et al. of eluates being evaporated to dryness under nitrogen gas flow at 60°C to provide: drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees Centigrade. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that by drying the hydrolyzed sample of elastin using nitrogen gas at about 50 to about 70 degrees; would result in an shorter separation time for isodesomosine and desmosine (Page 434-435, paragraph starting with “A general”). The skilled artisan would have had a reasonable expectation of success in eluates were evaporated to dryness under nitrogen gas flow at 60°C because Seyama et al. teaches the purification of elastin and Shiraishi et al. teaches a robust quantification method of DES and IDES from urine samples with added elastin (Page 432, Section 2.3)
Claims 12 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Seyama et al. (Seyama et al., Improved Rapid and Simple Determination of Desmosine and Isodesmosine by High-Performance Liquid Chromatography, 1987, J-STAGE, Volume 16 Issue 1, Pages 38-47) in further view of Xu et al. (Xu et al., Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry, March 28 2022, Tissue Engineering: Part C Methods), in further view of Snyder et al. (Snyder et al., Introduction to Modern Liquid Chromatography, 2009, Wiley, 3rd Edition, Page 322, 327-328), in further in view of Duan et al. (CN113698472A; see machine translation) in further view of Daamen et al. (Daamen et al., Isolation of intact elastin fibers devoid of microfibrils. Tissue Eng. 2005 Jul-Aug;11(7-8):1168-76) and further in view of Nilsuwan et al. (Nilsuwan et al., Development of Hydrolysis and Defatting Processes for Production of Lowered Fishy Odor Hydrolyzed Collagen from Fatty Skin of Sockeye Salmon (Oncorhynchus nerka), September 23 2021, Foods, Volume 10, Issue 10, 2257).
With respect to claim 12, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 9. Modified Seyama et al. teaches wherein the washing comprises mixing the sample of precooling the absolute ethyl alcohol to 4-6 ℃, and removing residual fat at 4-6 ℃. (On Page 6, starting at “step 2”, Duan et al. teaches mixing and stirring the fish skin and absolute ethyl alcohol for 6 hours to remove residual fat).
However, Modified Seyama et al. fails to teach for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade.
Nilsuwan et al. teaches the hydrolysis of collagen removed from salmon (Page 3, Section 2.3.1). Nilsuwan et al. teaches solvent extraction was also conducted to investigate fat removal processes using DSCS and subsequent solvent extraction to yield HC powder (Page 2, paragraph starting at “To reduce”). Nilsuwan et al. teaches removing fat by mixing the HC powder with alcohol, specifically isopropanol (Page 3, paragraph starting with “HC defatted”). Nilsuwan et al. teaches amino acid after defatting (Page 11, Section 3.4.2).
Nilsuwan et al. further teaches the mixture was further mixed with isopropanol and the aid of ultrasonic processor with pulse mode (on/off at 10 s) for 10 min (Page 3, Section 2.5). The mixture was then stirred at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuged at 10,000× g for 20 min at 4 ◦C (Page 3, Section 2.5). Nilsuwan et al. further teaches fat content of HC from salmon skin hydrolysate after fat removal at different stirring cycles of 0, 3, 6, and 9 (Table 2).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the defatting stirring conditions of Seyama et al. to incorporate the teachings of pulse mode (on/off at 10 s) for 10 min where the mixture was stirred at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuged at 10,000× g for 20 min at 4 ◦C. as taught by Nilsuwan et al. (Page 3, Section 2.5) to provide: for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade through routine experimentation (see MPEP 2144.05 (II)). Doing so would utilize known variation of defatting conditions as discussed by Nilsuwan et al. by optimizing the amount of fat removed for amino acid determination (Page 11, Section 3.4.2). Furthermore, starting with a mixing with isopropanol and the aid of ultrasonic processor with pulse mode (on/off at 10 s) for 10 min (Page 3, Section 2.5) then stirring at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuging at 10,000× g for 20 min at 4 ◦C, one of ordinary skill would have been motivated to modify Nilsuwan et al. to have mixed with chilled alcohol for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade through routine experimentation to optimize and control factors of defatting such as improving the recovery of amino acids from collagen hydrolysate, i.e. changing the defatting conditions to mixing with chilled alcohol for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade would allow for optimized recovery of amino acids and defat the hydrolysate.
With respect to claim 18, Modified Seyama et al. teaches all of elements of the current invention as stated above with respect to claim 15. Modified Seyama et al. teaches wherein the washing comprises mixing the sample of precooling the absolute ethyl alcohol to 4-6 ℃, and removing residual fat at 4-6 ℃ (On Page 6, starting at “step 2”, Duan et al. teaches mixing and stirring the fish skin and absolute ethyl alcohol for 6 hours to remove residual fat).
However, Duan et al. fails to teach for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade.
Nilsuwan et al. teaches the hydrolysis of collagen removed from salmon (Page 3, Section 2.3.1). Nilsuwan et al. teaches solvent extraction was also conducted to investigate fat removal processes using DSCS and subsequent solvent extraction to yield HC powder (Page 2, paragraph starting at “To reduce”). Nilsuwan et al. teaches removing fat by mixing the HC powder with alcohol, specifically isopropanol (Page 3, paragraph starting with “HC defatted”). Nilsuwan et al. teaches amino acid after defatting (Page 11, Section 3.4.2).
Nilsuwan et al. further teaches the mixture was further mixed with isopropanol and the aid of ultrasonic processor with pulse mode (on/off at 10 s) for 10 min (Page 3, Section 2.5). The mixture was then stirred at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuged at 10,000× g for 20 min at 4 ◦C (Page 3, Section 2.5). Nilsuwan et al. further teaches fat content of HC from salmon skin hydrolysate after fat removal at different stirring cycles of 0, 3, 6, and 9 (Table 2).
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the defatting stirring conditions of Seyama et al. to incorporate the teachings of pulse mode (on/off at 10 s) for 10 min where the mixture was stirred at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuged at 10,000× g for 20 min at 4 ◦C. as taught by Nilsuwan et al. (Page 3, Section 2.5) to provide: for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade through routine experimentation (see MPEP 2144.05 (II)). Doing so would utilize known variation of defatting conditions as discussed by Nilsuwan et al. by optimizing the amount of fat removed for amino acid determination (Page 11, Section 3.4.2). Furthermore, starting with a mixing with isopropanol and the aid of ultrasonic processor with pulse mode (on/off at 10 s) for 10 min (Page 3, Section 2.5) then stirring at 150 rpm for 20 min with a magnetic stirrer at 4–8 ◦C and subsequently centrifuging at 10,000× g for 20 min at 4 ◦C, one of ordinary skill would have been motivated to modify Nilsuwan et al. to have mixed with chilled alcohol for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade through routine experimentation to optimize and control factors of defatting such as improving the recovery of amino acids from collagen hydrolysate, i.e. changing the defatting conditions to mixing with chilled alcohol for about 20 to 40 seconds and letting stand at about −30 to about −10 degrees Centigrade for about 5 to 15 minutes, followed by mixing for about 5 to 15 minutes at about 0 to 10 degrees Centigrade would allow for optimized recovery of amino acids and defat the hydrolysate.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure of claims 3, 11 and 15, Shiraishi et al. (Shiraishi et al., "Development of a Robust LC-MS/MS Method for Determination of Desmosine and Isodesmosine in Human Urine," Journal of Oleo Science; Vol. 59, No. 8; February 10, 2010; pp. 431-439; cited in the IDS dated 06/07/2023) teaches desomosine and isodesmosine as markers of elastin degradation found in urine (Page 432, paragraph starting with "In this study"). Shiraishi et al. teaches elastin was added to urine samples and hydrolyzed using hydrochloric acid and analyzed using UPLC MS/MS (Page 432, paragraph starting with "A volume" and Page 433, paragraph starting with "Ultra-performance"). Shiraishi et al. further teaches the mobile phase A was 5mM heptafluorobutyric acid in water and B was acetonitrile; the gradient was as follows: 2% of B for the run time from 0 to 1 min, followed by a linear gradient from 2 to 10% of B for 1 to 6 min, a linear gradient from 10 to 20% of B for 6 to 8 min, a linear gradient from 20 to 30% of B for 8 to 8:01 min, 30% of B for 8:01 to 10 min, a linear gradient from 30 to 2% of B for 10 to 10:01 min, and then 2% of B for 10:01 to 15 min (Page 433, paragraph starting with "Ultra-performance").
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/S.J.B./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
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