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 .
DETAILED ACTION
The preliminary amendment filed 06/16/2023 is acknowledged. Claims 1-6 are pending and under consideration in this action.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
The instant claims are entitled to an effective filing date of 03/11/2021, which is the filing date of PCT/MX2021/000007.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
The instant abstract recites “[t]he invention refers to a process for producing human collagen structures with controlled characteristics”, which includes an implied phrase “[t]he invention refers to” and repeats information given in the title “producing human collagen structures with controlled characteristics”.
The abstract of the disclosure is objected to because it contains a minor informality.
Specifically, the abstract recites “Tissue” and “Pre-treatment”, which should be lowercased to “tissue” and “pre-treatment” because the terms are in the middle of a sentence.
A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 in part b) recites “a mold concentration”, which should be replaced with either “a mole” or “a molar concentration”, because based on the context “mold” is a misspelling of mole or molar.
In part c), claim 1 recites “Extraction”, which should be lowercased to “extraction” because the term is in the middle of a sentence.
In part e), claim 1 recites “ 0.20 -0.28”, which is an improperly spaced numerical range that should be replaced with either “0.20 – 0.28” or “0.20-0.28”, because as currently written the numerical range could be misread as a negative number “-0.28”.
In part f), claim 1 recites “form”, which should be replaced with “from” because based on the context “form” is a misspelling.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 1-6 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 recites the limitation "it" in line 4. There is insufficient antecedent basis for this limitation in the claim. Specifically, claim 1 recites “a) tissue conditioning, where tissues or fluids are not of interest for the method are removed and it is reduced to particle size of 0.5 to 1 mm in a uniform way”. It is unclear which structural element is required to be reduced to a particle size of 0.5 to 1 mm in a uniform way. In other words, it is unclear whether “it” refers to a structural element of interest or the tissue or fluids removed that are not of interested. Consequently, one of ordinary skill in the art cannot ascertain the metes and bounds of the required tissue conditioning step. For compact prosecution, the tissue conditioning step will be interpreted as requiring any unremoved tissue to be reduced in particle size because part b) refers to the previously conditioned tissue.
Claim 1 recites “using 100 mL per gram of dry tissue” in part b), which because it is unclear whether the dry tissue intends to reference the previously conditioned tissue, such that the previously conditioned tissue is required to be dried, or whether the claim intends to reference a separate dried tissue.
Claim 1 recites “efficient” in part b), which renders the claim indefinite. The term “efficient” is not defined by the claims, 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. Specifically, claim 1 requires “efficient magnetic agitation”, however the way in which “efficient” magnetic agitation differs from other forms of magnetic agitation is unclear.
Claim 1 recites “together with 200 g of pepsin per kg of dry tissue”, which is indefinite because it is unclear whether the “dry tissue” intends to reference the neutralized tissue of part b1) or a separate tissue that is dried.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites the broad recitations “ acetic acid at a molar concentration of 0.02- 0.5 M” and “200 g of pepsin per kg of dry tissue” in part c), and the claim also recites “(2.27% pepsin in 0.5 M acetic acid)” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. To obviate this rejection, the entirety of the parenthetical phrase “(2.27% pepsin in 0.5 M acetic acid)” can be deleted, or the claim can be amended to limit the pepsin and acetic acid to the 2.27% pepsin in 0.5 M acetic acid embodiment.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites the broad recitation “the resulting collagen solution” in part d), and the claim also recites “(450 mL of solution per g of dry tissue)” in part d) which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. To obviate this rejection, the parenthetical phrase “(450 mL of solution per g of dry tissue)” can be deleted, or the claim can be amended to limit the resulting solution to 450 mL of solution per g of dry tissue.
Claim 1 recites “high” in part d), which renders the claim indefinite. The term “high” is not defined by the claims, 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. Claim 1 requires “a high salt concentration”, but it is unclear which salt concentrations are considered to be high salt concentrations because the term “high” is not defined in the claim or by the specification.
Claim 1 recites the term “about” in part d), which is a term of approximation. The specification does not define the term and the specification does not provide any values, such that one of ordinary skill in the art could ascertain the values. Therefore, the amount of permissible variation is unclear. For example, it is unclear whether the “about 2-6 hours” range encompasses 1.5-10 hours, 1-7 hour etc. because the upper and lower limits are not clearly defined for the range. Consequently, one of ordinary skill in the art cannot ascertain the metes and bounds of the claimed range.
Claim 1 recites “350 mL of acetic acid per g dry tissue” in part d), which is indefinite because it is unclear whether the dry tissue intends to reference a specific dry tissue recited earlier in the claim or any dry tissue. In other words, claim 1 recites “dry tissue” throughout the claim, and it is unclear whether every recitation refers to the same dry tissue, different dry tissue, or any dry tissue.
Claim 1 recites “the solution” and “the collagen solution” in part e), which render the claim indefinite for two reasons. First, it is unclear whether “the solution” and “the collagen solution” are the same solution or separate solutions. Claim 1 recites “to purify the solution from the excess of salt present in the collagen solution”. Therefore, the collagen solution may be identical to “the solution” or it may be structurally distinct from the “the solution” due to the purification step, such that “the solution” contains excess salt and “the collagen solution” is purified from the excess salt. Second, it is unclear whether “the solution” and/or “the collagen solution” intend to reference the resulting solution of part d), or a separate solution.
Claim 1 recites “the acetic acid solution” in part e) and it is unclear whether “the acetic acid solution” intends to reference the dialysis buffer consisting of a solution with a low concentration of acetic acid or a separate acetic acid solution. To obviate this rejection “the acetic acid” can be replaced with “the dialysis buffer”.
Claim 1 recites “the contact surface” in part e), which is indefinite because it is unclear whether “the contact surface” intends to reference a contact surface of the porous membrane or a separate contact surface. To obviate this rejection “the contact surface” can be replaced with “the porous membrane”.
Claim 1 recites “the buffer” three times in part e). These recitations render the claim indefinite because it is unclear whether “the buffer” intends to reference the dialysis buffer or a separate buffer. To obviate this rejection “the buffer” can be replaced with “the dialysis buffer”.
Claim 1 recites “the method is stopped” in part e), which is indefinite because it is unclear whether “the method” intends to reference just the dialysis step of part e) or the method as a whole. If the entirety of the method is required to be stopped, then it is also unclear whether steps f-h are optional or required.
Claim 1 recites “at the beginning of the stage” in part e), which is indefinite because it is unclear which stage is being referenced. The claim may intend to reference the beginning of the dialysis stage of part e) or any stage of parts a-d recited previously in the claim. Furthermore, “the beginning” is not defined as a timeframe in the claim or the specification. Therefore, it is unclear whether the timeframe of “the beginning” encompasses 10 min from the start of the stage, 2 min from the start of the stage etc.; alternatively, the claim may intend to encompass the entirety of the stage.
Claim 1 requires “the method” to be “stopped when the collagen reaches the same conductivity as the buffer at the beginning of the stage of a value between 0.20 -0.28 mS/cm” in step e), which is indefinite for two reasons. First, it is unclear whether the method as a whole should be stopped or just the dialysis step of part e). If the entire method is required to be stopped, it is also unclear whether parts f-h of claim 1 are required or optional. Second, it is unclear whether the collagen is required to reach a conductivity of 0.20-0.28 mS/cm, because the claim may only require the buffer to be at 0.20-0.28 at the beginning of the stage.
Claim 1 recites “the solution” in part f), which is indefinite because it unclear whether “the solution” refers to “the purified collagen solution” or a separate solution.
Claim 1 recites “where depending on the application and expected function” in part g), which renders the claim indefinite because it is unclear whether part g) is required or optional depending on the application and expective function.
Claim 1 recites “a concentration between 2.5-10 mg/mL is chosen” in part g), which renders the claim indefinite because the way in which this limitation modifies the claim is unclear. It is unclear whether the 2.5-10 mg/mL is required or merely chosen [e.g. mentally]. If such concentration is required, it is further unclear which solution or composition is required to be at such concentration.
Claim 1 recites “the desired structure” in part g). There is insufficient antecedent basis for this limitation in the claim. “The” is a definite article that refers to a specific or particular noun. Therefore, it is unclear which desired structure is being referenced.
Claim 1 recites the parenthetical phrase “(which consists of removing heat by gradually lowering the temperature of the plate with which the mold and collagen solution are in contact, allowing the water and acetic acid crystals to be uniform and varied, accommodating the fibers)” in part g), which renders the claim indefinite for three reasons. First, it is unclear whether the parenthetical phrase should be read into the claim as a limitation or not. In other words, the parentheses indicate that the limitation may not be required. Second, if the parenthetical phrase is required, the term “gradually” renders the claim indefinite because it is not defined in the claims or specification as to allow one of ordinary skill in the art to ascertain the metes and bounds of a ‘gradual’ lowering. Third, it is unclear whether “the water” refers to the water present in the acetic acid with a molarity between 0.02 – 0.5 M or separate water. Fourth, the way in which crystals can be both “uniform and varied” is unclear.
In part g) of claim 1, it is unclear whether both molding and second lyophilization are required. Part g) states “molding or second lyophilization”. However, the part g) goes on to describe steps for molding and lyophilization as if both are required. Specifically, part g) recites “once solubilized, collagen is placed in molds that allow to generate the desired structure; once again, the solution is lyophilized”, which may indicate that both molding and lyophilization are required. Due to this contradiction, one of ordinary skill in the art cannot ascertain the metes and bounds of part g).
In part g) claim 1 recites “where depending on the application and expected function, a concentration of between 2.5-10 mg/mL is chosen and collagen is solubilized again in an acetic acid solution; where between 2.5 – 10 mg of collagen per mL of acetic acid with a molarity between 0.02-0.5 M are used”, and it is unclear how this recitation limits the claim. In other words, it is unclear whether the “concentration of between 2.5-10 mg/mL” limitation is required, or optional depending on the application and expected function. Furthermore, it is unclear whether the 2.5-10 mg/mL recitation references the mg of collagen and mL of acetic acid. Moreover, it is unclear how the “2.5 – 10 mg of collagen per mL of acetic acid with a molarity between 0.02-0.5 M” are required to be used, e.g. for molding, for the second lyophilization or both.
Part h) recites “the exposure time”. There is insufficient antecedent basis for this limitation in the claim. It is unclear whether “the exposure time” intends to reference the exposure time of the collagen pieces to the formaldehyde, or a different exposure time.
Claims 2-6 depend from claim 1 and are rejected for the reasons set forth above.
The terms “fine” and “highly” in claim 3 are relative terms which renders the claim indefinite. The terms “fine” and “highly” are 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. In the instant case, the density of the college structures is unclear because one of ordinary skill in the art cannot determine a density point at which such collagen structures are considered to be “fine” and “highly dense”.
Claim 5 recites “characterized in that collagen has a purity of ≥ 95%”, which renders the claim indefinite because it is unclear whether the claim intends to reference any collagen or the collagen obtained from the method of claim 1.
Claim 6 recites “the collagen obtained allows the cultivation and co-cultivation of primary and/or line human cells; likewise, it functions as a deposit of growth factors, proteins and exosomes, for its possible use in regenerative therapy”, which is indefinite for two reasons. First, the way in which the collagen obtained “allows” for such cultivation and co-cultivation is unclear. The specification does not describe any particular structure associated with the ability to allow for such cultivation and co-cultivation. Second, the way in which the obtained collagen can “function as a deposit of growth factors…and exosomes” is unclear. Collagen alone is not known to function as a growth factor, or exosome. Therefore, it is unclear whether claim 6 intends for the obtained collagen to be combined with growth factors and exosomes.
Claim Interpretation
The intended purpose of the method is to produce human collagen structures with controlled characteristics. This intended purpose does not structurally limit the method, because the method steps in the body of the claim do not require human tissue. As such, the claim encompasses any collagen. Furthermore, the claimed “controlled characteristics” encompass any structural and functional characteristic of a collagen produced using the claimed method. For brevity, intended-use recitations that do not structurally limit the method are mostly omitted from the description below. Claim 1 is a method that requires the following active steps.
A) For tissue conditioning, a tissue of interest is isolated by removing any tissues or fluids not of interest. The tissue of interest is reduced to a particle size of 0.5 – 1 mm, such that the particles are considered uniform due to their similar sizes.
B) The conditioned tissue is exposed to a sodium hydroxide (NaOH) solution that is at a concentration of 0.05 - 2 M. The solution mixture comprising 100 mL sodium hydroxide solution per gram of preconditioned tissue [or any dry tissue] is obtained. The solution mixture is magnetically agitated for 2-6 h.
B1) Series [i.e. any multiplicity] of final washes with distilled water are carried out. The non-limiting intended purpose of the washes is to obtain neutralized tissue with a pH between 7-8.
C) Enzymatic hydrolysis is performed by combining the washed tissue with an acetic acid solution and pepsin. The resulting combination comprises 450 L of acetic acid solution and 200 g pepsin per kg of tissue [washed or preconditioned or any dry tissue]. Furthermore, the claim requires carrying out an extraction by enzymatic hydrolysis over a period of three days by adding an intermediate acid solution comprising 200 mL of acetic acid per gram of the tissue at the start of the three days, and after 48 hours adding an intermediate acid solution comprising 250 mL of acetic acid per g of neutralized tissue are added [to the mixture comprising pepsin]. Filtration is performed (e.g. centrifugation is a form of filtration).
D) The resulting collagen solution is brought to a “high” salt concentration [i.e. any salt concentration] by adding sodium chloride. The claim recites “adding sodium chloride at a rate of 58.44 g of sodium chloride per L”. There are 58.44 g/mol of NaCl and molarity is mole/L, so this limitation is interpreted as adding NaCl to any molarity. The collagen solution comprising sodium chloride is homogenized with magnetic stirring to allow for their precipitation at a time of about 2-6 hours. The resulting solution is sieved by any means. Since sieving doesn’t alter the size of a particle, the non-limiting intended use of the sieving step is to arrive at a particle size between 125-850 µm. Fibers [collagen fibers] recovered from the sieve are solubilized in an acetic acid solution of 350 mL per g of tissue.
E) The resulting collagen solution from part d) is purified from excess salt [any amount of salt removal] using any dialysis system. Collagen [e.g. collagen within the resulting collagen solution from part d] is placed inside a porous membrane with a pore size between 12-14 kD and a dialysis buffer is used for the dialysis process. The dialysis buffer is required to consist of acetic acid from 0.02 -5 M, which is considered a “low concentration”. The intended purpose of this dialysis step is to expel impurities and allow for the exchange of salt molecules between both solutions, i.e. the resulting collagen solution from part d) which includes 1-2 M of salt and the dialysis buffer that does not include salts. The exchange is accelerated [i.e. facilitated] by a contact surface of the porous membrane (e.g. dialysis tubing) and the flow of the dialysis buffer. This dialysis step lasts 3-4 days and the dialysis buffer is changed after 48 hours due to the recirculation of the dialysis buffer. Conductivity of the dialysis buffer and the collagen solution are monitored, but, as discussed above, it is unclear whether or when the dialysis step is required to be stopped and it is unclear whether the 0.2-0.28 mS/cm is required.
F) The purified collagen from step e) is lyophilized by freezing it to -40˚C, and then subjecting it to vacuum pressure for two days at a pressure of 0.04-0.2 mbar, which is equivalent to 4-20 Pa. The resulting lyophilized collagen is weighed and distributed in any way.
G) This stage requires molding or a second lyophilization. For the second lyophilization, the solution is lyophilized at a temperature of -40˚C and a vacuum pressure between 0.04- 0.2 mbar. For molding, the solution is only required to be placed in a mold and any resulting structure is considered a desired structure.
H) The collagen pieces are crosslinked in the presence of formaldehyde vapor atmosphere. The exposure time is not required to be set and any environment in which crosslinking occurs can be considered a crosslinking apparatus. The concentration of the formaldehyde vapor reagent cloud is required to be between 0.2 -1.6 ppm.
Claim 2 requires pressing or compressing the collagen by subjecting it to a determined mechanical force of 400-5000 N to compact its dimensions to a desired value from 0.01 to 10 mm, which will inherently increase its fibrillar density and generate fine and highly dense collagen structures.
Claim 4 requires a “yield of 35% collagen”. A percentage is part of a whole and the whole is not limited in anyway. Therefore, the yield may be determined and calculated in anyway.
For clarity, relevant prior art teachings will be organized according to the a-h stages discussed above. Any limitations not taught by the prior art will be listed numerically and corresponding rationales will follow the same numerical system.
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.
Claims 1 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Wei (Journal of Wuhan University of Technology-Mater. Sci. Ed., 34(4), 950-959) in view of He (Journal of Food Process Engineering, 42(6), e13214), Nalinanon (Journal of the Science of Food and Agriculture, 90(9), 1492-1500), and Smestad(US 2006/0002967).
Regarding claim 1, Wei teaches extracting and characterizing the collagen biomaterials from the skin of hybrid sturgeon. See the last sentence of section 1.
Wei teaches removing skin as a byproduct in fillet processing. Wei teaches slicing hybrid sturgeon skin into small pieces of 0.3 cm x 0.3 cm (i.e. 3 mm x3 mm). See section 2.1. Thus, Wei teaches removing tissues or fluids that are not of interest (i.e. the hybrid sturgeon body besides the skin) and reducing the skin in a uniform way to a particle size of 3x3 mm, which is not within the instantly claimed 0.5-1 mm particle size range (see number 1 below).
Wei teaches immersing prepared skin from the fish in 0.1 M NaOH at a ratio of skin and alkali solution 1: 20 w/v. Wei discloses that all steps are in continuous stirring. See section 2.4. Thus, Wei teaches exposing previously conditioned tissue to a solution of sodium hydroxide at a mole concentration between 0.05 – 2 M, but Wei does not teach using 100 mL per gram of dry tissue (see number 2 below); and Wei suggests performing steps under stirring (e.g. agitation), but Wei does not teach magnetic agitation and for a time of 2-6 hours (see number 3 below).
b1) Wei teaches rinsing the treated skin with cold distilled water to neutral pH. Lipid content is removed and the defatted skins are washed with cold distilled water. The solvent is changed every 6 hours. See section 2.4. Thus, Wei teaches carrying out a series of final washes with distilled water for the intended purpose of neutralizing the pH.
Wei teaches extracting skin with acetic acid at a skin and acid ratio of 1:15 (w/v). The mixtures are filtered through two layers of gauze. The acid-soluble collagen (ASC) is salted-out by adding 2.6 M NaCl. The precipitate is redissolved in acetic acid and dialyzed against 0.1 M acetic acid. The dialysis fluid is changed per 6 hour. The dialysis fluid is freeze-dried and referred to as ASC. The acid extractant is optimized. Undissolved residues of ASC are added to 0.5 M acetic acid at a ratio of residue and acetic acid 1:15 w/v, while porcine pepsin (120 U/g residue) is added to the mixture. The mixtures are stirred continuously for 48 h. The mixture is filtered through two layers of gauze, salted out and dialyzed. See section 2.4. Thus, Wei teaches performing enzymatic hydrolysis by subjecting the tissue to 0.5 M, which is within the instantly claimed 0.02-0.5 M range. However, Wei does not teach using 450 L [acetic acid] per kg of dry tissue together with 200 g of pepsin per kg of dry tissue (see number 4 below). Furthermore, Wei teaches extraction over a period of 2 days, but not the instantly claimed three days (see number 5 below), and Wei does not teach adding intermediate solution starting with 200 mL of acetic acid per g of dry tissue and adding 250 mL of acetic acid per g of dry tissue after 48 hours (see number 6 below).
Wei teaches salting-out by adding sodium chloride until 2.6 M NaCl (e.g. high salt concentration; and 58.44 g/mol NaCl, M=mol/L) solutions are under acidic conditions. The salted-out precipitate is collected by centrifugation at 15,000 r/min for 30 min. The precipitate is redissolved in 0.5 M acetic acid, and the solutions are dialyzed. See section 2.4. Furthermore, Wei teaches a step of filtering mixtures through two layers of gauze (e.g. sieve). See section 2.4. Thus, Wei teaches bringing a resulting collagen solution to a high salt concentration of 2.6 M and the rate is understood to be at 58.44 g NaCl per L as claimed because there are 58.44 g/mol NaCl and M=mol/L; furthermore, Wei suggests that the precipitation time includes the 30 min of centrifugation time and 30 min is considered to be about 2-6 hours as instantly claimed; and Wei teaches a sieving step; and redissolving in acetic acid. However, Wei does not teach homogenization with magnetic stirring (see number 7 below), and Wei does not teach an acetic acid redissolving step that uses 350 mL of acetic acid per g of dry tissue (see number 8 below).
Wei teaches redissolving the salted-out precipitate in 0.5 M acetic acid solution and dialyzing it against 0.1 M acetic acid (e.g. within the instantly claimed 0.02- 0.5 M) and distilled water for 24 h. The dialysis fluid is changed per 6 h (e.g. recirculation). The dialysate is freed-dried. See section 2.4. Thus, Wei teaches a dialysis step in which the collagen solution is introduced to a dialysis buffer consisting of acetic acid 0.1 M, which is within the instantly claimed 0.02- 0.5 M; Wei suggests that the collagen solution may have a salt concentration of 2.6 M, not the instantly claimed 1-2 M salt concentration (see number 9 below); Wei teaches a 24 h stage, and not a stage that lasts three to four days and includes a buffer change after 48 hours as claimed (see number 10 below).
Wei teaches freeze drying the dialyzates. See section 2.4. However, Wei is silent regarding the specific process.
(1) Wei does not teach a particle size of 0.5 to 1 mm (relevant to instant part a).
(2) Wei does not teach 100 mL [sodium hydroxide] per gram of “dry tissue” (relevant to instantly claimed part b).
(3) Wei does not teach efficient magnetic agitation for 2-6 hours (relevant to instantly claimed part b).
(4) Wei does not teach using 450 L [acetic acid] per kg of “dry tissue” together with 200 g of pepsin per kg of dry tissue for enzymatic hydrolysis (relevant to instant part c).
(5) Wei does not teach extraction over a period of three days (relevant to instant part c).
(6) Wei does not teach adding intermediate acid solution starting with 200 mL of acetic acid per g of dry tissue and after 48 hours 250 mL of acetic acid per g of dry tissue are added (relevant to instant part c).
(7) Wei does not teach homogenization by magnetic stirring (relevant to instant part d).
(8) Wei does not teach solubilizing fibers recovered from a sieve again in an acetic acid solution using 350 mL of acetic acid per g of dry tissue (relevant to instant part d).
(9) Wei does not teach the instantly claimed 1-2 M salt concentration (relevant to instant part e).
(10) Wei does not teach a dialysis stage that lasts three to four days and includes a buffer change after 48 hours as claimed (relevant to part e)
(11) Wei does not teach a porous membrane with a pore size between 12-14 kD (relevant to instant part e).
(12) Wei does not teach monitoring the conductivity of both solutions and stopping the method when collagen reaches the same conductivity as the buffer at a value between 0.2-0.28 mS/cm (relevant to instant part e)
(13) Wei is silent regarding the lyophilization method. As such, Wei does not teach freezing to -40˚C, and subsequently subjecting it to vacuum pressure of 0.04-0.2 mbar for a period of two days, the resulting collagen is weighed and distributed (relevant to instant part f).
(14) Wei does not teach molding or a second lyophilization process, wherein the molding requires placing the collagen in molds, or the second lyophilizing requires to be at a temperature of -40˚C, and a vacuum pressure of 0.04-0.2 mbar (relevant to part g).
(15) Wei does not teach subjecting collagen pieces to a formaldehyde vapor atmosphere in a crosslinking apparatus, wherein the formaldehyde vapor is at a 0.2 – 1.6 ppm concentration (relevant to part h).
He teaches extracting collagen from fishbone. See the abstract. He teaches an enzymatic hydrolysis process with pepsin in which fish-bone samples are weighed and prepared into 1% solutions, then mixed uniformly with magnetic stirrer. See section 2.5 (relevant to number 3 and 7 above). Thus, He suggests that magnetic stirring can be used for uniformly mixing solutions comprising collagen.
Wei and He do not teach numbers 1-2, 4-6, 8-15 above.
Nalinanon teaches extracting collagen from prepared the skin of arabesque greenling. Nalinanon teaches combining the supernatant from a centrifuged solution of skin and acetic acid with NaCl to obtain a final concentration of 2.6 mol/L in Tris-HCl. The resultant precipitate is collected by centrifugation. The pellet is dissolved in acetic acid and the solution obtained is dialyzed against 0.1 mol/L acetic acid in a dialysis bag with a molecular weight cut-off of 14 kDa for 12 hours, with a change of dialysis solution every 4 hours. See the last paragraph on page 2 and the paragraph spanning pages 2-3 (relevant to number 11 above). Furthermore, Nalinanon suggests measuring conductivity in mS cm-1. See figure 2A and B (relevant to number 12 above).
Wei, He and Nalinanon do not teach numbers 1-2, 4-6, 8-10, and 13-15.
Smestad teaches fabricating a collagen matrix in example 1. Smestad teaches freezing [collagen] fibers at -20˚C. The fibers are then resuspended in phosphate buffered saline and put back into the mold in the desired orientation and compressed with a piston. The compressed fibers are then refrozen at -20˚C, and then thawed at room temperature. See [0058]. The matrix is ten cross-linked using any alternative procedure selected from a list that includes (d) the matrix is crosslinked with formaldehyde vapor generated from a 2% HCHO solution at 22 ˚C for 5-24 hours. The crosslinked matrix is rinsed repeatedly. See [0059]. The crosslinked and rinsed matrix is then lyophilized. See [0060]. Smestad suggests that the matrix can be lyophilized at -20˚C under vacuum of less than 400 millitorr (i.e. less than 0.533 mbar). See [0052]. Smestad teaches weighing a type I or type II collagen dispersion. See [0065]. Thus, Smestad, with respect to the instant claims, teaches lyophilizing collagen matrix at -20˚C, not the instantly claimed -40˚C, under a vacuum pressure that overlaps with the instantly claimed 0.04 – 0.2 mbar range for a period of 48 hours or two days, and Smestad teaches weighing collagen and distributing collagen into a mold (relevant to instant part f and numbers 13 above); furthermore, Smestad teaches placing collagen in molds to allow to generate a desired structure (relevant to instant part g and number 14 above); and Smestad teaches crosslinking collagen in the presence of formaldehyde vapor, but Smestad is silent regarding the instantly claimed 0.2 – 1.6 ppm vapor concentration (relevant to part h and number 15 above)
Wei, He, Nalinanon and Smestad do not explicitly teach 1-2, 4-6, and 8-10 above.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention: (1) optimize the 3x3mm skin cuts of Wei; (2) to optimize the amount of NaOH per gram of tissue taught by Wei; (3 and 7) to replace the stirring technique of Wei with the magnetic stirring technique of He and to further optimize the stirring time; (4) to optimize the acetic acid and pepsin amounts taught by Wei; (5) to optimize the 2 day extraction period of Wei; (6) to repeat the acetic acid addition step taught by Wei at any time during extraction using any amount of acetic acid; (8) to optimize the acetic acid amount in the redissolving step of Wei; (9) to optimize the 2.6 M salt concentration of Wei; (10) optimize the 24 h dialysis stage and 6h fluid change (e.g. recirculation) of Wei; (11) to apply the 12-14 kDa dialysis tubing of Nalinanon to the dialysis process of Wei; (12) to apply the conductivity measurement technique of Nalinanon to the dialysis solution of Wei; (13) to apply the lyophilization method of Smestad to the collagen of Wei and to further optimize the temperature during lyophilization; and (14-15) to further apply the molding and crosslinking techniques of Smestad to the collagen of Wei.
(1) One would be motivated to optimize the skin cuts of Wei, because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Wei demonstrates cutting the skin to 3x3 mm, which may serve as a starting point from which one could optimize. MPEP 2144.05(II)(A) indicates that “[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
(2) One would be motivated to optimize the amount of NaOH (mL) per g tissue because Wei teaches using a specific weight by volume ratio of the tissue to NaOH. There would be a reasonable expectation of success because Wei demonstrates combining skin tissue and NaOH in a 1:20 weight to volume ratio. MPEP 2144.05(II)(A) states that “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”.
(3 and 7) One would be motivated to replace the stirring of Wei with the magnetic stirring of He because He suggests that magnetic stirring is a way of uniformly stirring. There would be a reasonable expectation of success because He demonstrates stirring collagen containing solution with magnetic stirring; and Wei suggests continuous stirring throughout multiple steps. One would be further motivated to optimize the time of the stirring because a person of ordinary skill in the art has good reason to pursue the known options with in their technical grasp. There would be a reasonable expectation of success because MPEP 2144.05(II)(A) indicates that “[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
(4) One would be motivated to optimize the acetic acid and pepsin amounts of Wei because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Wei demonstrates using a 1:15 w/v residue to acetic acid ratio from which one could use to arrive at any liter acetic acid to kg residue ratio, such as the instantly claimed 450 L per kg of tissue. Furthermore, Wei demonstrates using 120 U/g pepsin per gram tissue residue, which can serve as a starting point from which one could optimize. MPEP 2144.05(II)(A) states that “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”.
(5) One would be motivated to optimize the 48-hour extraction period of Wei, because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Wei demonstrates a 2-day extraction period. MPEP 2144.05(II)(A) indicates that “[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
(6) One would be motivated to repeat the acetic acid addition step of Wei with any amount of acetic acid at any point during extraction because Wei suggests optimizing acid extraction (first passage page 952). There would be a reasonable expectation of success because Wei demonstrates repeating steps (e.g. see figure 1). MPEP 2144.05(II)(A) indicates that “[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
(8) One would be motivated to optimize the amount of acetic acid used in the redissolving step of Wei because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Wei demonstrates redissolving a solution in acetic acid after filtering through two layers of gauze. MPEP 2144.05(II)(A) indicates that “[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
(9) One would be motivated to optimize the salt concentration of the collagen solution used in the dialysis process of Wei because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Wei suggests that the concentration may be 2.6 M, which is merely close to the instantly claimed 1-2 M range. MPEP 2144.05(II)(A) states that “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”.
(10) One would be motivated to optimize the 24-hour dialysis and 6 hour fluid change of Wei because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Wei teaches a 24-hour dialysis stage, from which one could optimize to, for example, three or four days and, consequently, the fluid would be changed for the 8th time at hour 48 because 48/6h is 8.
(11) One would be motivated to use the 14 kDa dialysis tubing of Nalinanon in the dialysis method of Wei because Nalinanon suggests using the dialysis tubing for the dialysis of collagen against acetic acid. There would be a reasonable expectation of success because Wei and Nalinanon demonstrate performing dialysis of collagen solutions against acetic acid; thus, one would reasonably expect the dialysis tubing of Nalinanon to serve the same function in the dialysis method of Wei.
(12) One would be motivated to apply the conductivity measurement technique of Nalinanon to the dialysis solution of Wei because Nalinanon teaches characterizing the collagen from the skin of a species of mackerel fish. Thus, one would be motivated to further characterize the sturgeon fish skin of Wei using the conductivity measurement technique of Nalinanon. There would be a reasonable expectation of success because Wei and Nalinanon are in the same field of endeavor.
(13) One would be motivated to apply the lyophilization method of Smestad to the collagen of Wei because Smestad suggests that the method is suitable for preparing biocompatible matrices comprising collagen ([0023]). There would be a reasonable expectation of success because Wei demonstrates freeze drying dialysate comprising collagen (first passage page 952), and Smestad teaches lyophilization of collagen fibers. One would be further motivated to optimize the -20˚C temperature during the lyophilization method of Smestad because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because MPEP 2144.05(II)(A) states that “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”.
(14-15) One would be motivated to apply the molding and crosslinking technique of Smestad to the collagen of Wei because Wei teaches pepsin-soluble collagen that could be widely used in medical biomaterials (abstract) and Smestad teaches biocompatible implants comprising collagen (e.g. [0011] and [0023]). There would be a reasonable expectation of success because Smestad demonstrates performing the molding and formaldehyde vapor crosslinking techniques on collagen. One would be further motivated to optimize the concentration of the formaldehyde vapor used in the crosslinking technique of Smestad because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Smestad teaches generating formaldehyde vapor generated from a 2% HCHO solution. Furthermore, MPEP 2144.05(II)(A) states that “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”.
Regarding claim 4, Wei discloses that the yield of the pepsin-soluble collagen isolated from the skin of the hybrid sturgeon is 10.26 ±0.39% on a weight basis. See the left column on page 954. Wei suggests that collagen is the most abundant protein in animal origin, approximately accounting for 30% of total protein in connective tissue of animals. See the first paragraph of section 1.
Wei, He, Nalinanon, and Smestad do not teach a collagen yield of 35%.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize the collagen yield obtained from the method of Wei, He, Nalinanon, and Smestad. One would be motivated to do so because Wei suggests that collagen may account for approximately 30% of connective tissue. There would be a reasonable expectation of success because Wei teaches a collagen yield of 10.26 ±0.39% on a weight basis.
Claims 2-3 are rejected under 35 U.S.C. 103 as being unpatentable over Wei (Journal of Wuhan University of Technology-Mater. Sci. Ed., 34(4), 950-959), He (Journal of Food Process Engineering, 42(6), e13214), Nalinanon (Journal of the Science of Food and Agriculture, 90(9), 1492-1500), and Smestad(US 2006/0002967), as applied to claim 1 above, and further in view of Hu (Biomedical microdevices, 12(4), 627-635).
The teachings of Wei, He, Nalinanon and Smestad with respect to instant claim 1 are discussed above.
Regarding claims 2-3, Smestad, in example 1, teaches resuspending collagen fibers in phosphate buffer saline, putting it back into the mold in a desired orientation and compressing it with a piston. See [0058]. Smestad teaches slowly dehydrating fibers until the desired dimension (about 8 mm thickness) is reached. See [0066]. Thus, Smestad teaches subjecting a collagen structure to a piston mechanical force, and a desired dimension of 8mm, which is within the instantly claimed 0.01-10 mm.
Wei, He, Nalinanon and Smestad do not teach a mechanical force of 400-5000 N.
Hu teaches preparing compressed collagen gel. After gel formation, the gel is weighed and then compressed on a bed of absorbent paper bounded by a nylon mesh using a mass of 48 g for 1 min, stress equivalent to approximately 1.1 kN m-2 (i.e. 1,100 N m-2). See section 2.2 and figure 1.
It would have been obvious to a person of ordinary skill in the art prior to the effect filing date of the instantly claimed invention to optimize the piston pressure of Smestad in view of the 1,100 N m-2 pressure taught by Hu within the method of Wei, He, Nalinanon and Smestad discussed above. In the process, one would arrive at a fine and highly dense collagen structure, absent evidence to the contrary. One would be motivated to do so because Hu suggests that the compression of collagen gel can improve its mechanical property. There would be a reasonable expectation of success because MPEP 2144.05(II)(A) indicates that “[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Wei (Journal of Wuhan University of Technology-Mater. Sci. Ed., 34(4), 950-959), He (Journal of Food Process Engineering, 42(6), e13214), Nalinanon (Journal of the Science of Food and Agriculture, 90(9), 1492-1500), and Smestad(US 2006/0002967), as applied to claim 1 above, and further in view of Bai (ACS Sustainable Chemistry & Engineering, 5(8), 7220-7227).
The teachings of Wei, He, Nalinanon and Smestad with respect to instant claim 1 are discussed above. As discussed in the claim interpretation section above, it is unclear which collagen claim 5 intends to limit.
Regarding claim 5, Wei discloses that the yield of the pepsin-soluble collagen isolated from the skin of the hybrid sturgeon is 10.26 ±0.39% on a weight basis. See the left column on page 954. Wei suggests that different kinds of acids have different effects on the internal structure of collagen molecules due to their different chemical properties. See the left column on page 954.
Smestad, in example 1, suggests using highly purified type I collagen fibrils for fabrication. See [0056].
Wei, He, Nalinanon and Smestad do not teach a collagen purity ≥95%.
Bai teaches obtaining higher and lower molecular weight collagen peptides with respective extraction efficiencies up to 91.57% and 96.01% and corresponding purities of up to 93.14% and 100%, respectively. See the abstract. Ba