DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Amendment
Applicant’s remarks and amendments filed 2/03/2026, in response to the communication mailed 9/3/2025 and the non-final rejection mailed 4/30/2025, are acknowledged and have been fully considered. Any previous rejection or objection not mentioned herein is withdrawn.
Applicant’s remarks and supplemental amendment to the claims filed 3/9/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
In the claims submitted 3/9/2026, claims 1-2, 8-14, 21, 26-36 are present. It is noted that claim 14 is marked “Currently Amended” although the Applicant’s remarks and arguments state that claim 14 was cancelled. For the sake of compact prosecution, the discussion in the remarks is assumed to be an inadvertent error and claim 14 will be examined to the extent that it reads on the elected species.
Claims 1, 14, 29, and 36 read upon the elected species (a method for the obtention of proteins and peptides in native state by fractionation of mammalian intestine mucosa, comprising the steps (i), (ii), (iiia), (iva), and (v.b), as recited in the original claim 1.
Claims 1, 14, 29, and 36 have been examined herein on the merits.
Claims 2, 8-13, 21, 26-28, and 30-35 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 3/28/2025.
Claim Interpretation
As previously set forth, the phrase “in native state” as used in reference to the obtained proteins and peptides, is being interpreted according to the broadest reasonable interpretation (B.R.I.), in light of the specification, to refer to proteins or peptides that are obtained “in their properly folded and assembled form with operative structure and function” (see page 3, lines 25-27 of the specification). The specification also states that proteins having intact structure and function that is not altered by heat, chemicals, enzyme reaction, or other denaturants, are referred to as "native proteins" (see specification on page 1, line 36 – page 2, line 3). Thus, in light of the specification, and the level of knowledge in the art, the term in native state is herein being interpreted as encompassing a protein or peptide, obtained in such a way and in accordance with the positively defined method steps of the instant claims, to have intact normal or native functions and structures. In most cases, a protein’s function is defined by its structure, and thus evidence of a protein’s function, when said protein is obtained by a prior art purification method, will be considered evidence attesting to the native structure of said protein being intact.
Response to Arguments
Applicant’s arguments, on pages 15-17 of the remarks filed 3/9/2026, with respect to the previous rejections of claims 1 and 14 under 35 U.S.C. § 112(b) and 35 U.S.C. § 103 as being obvious over the teachings of CN101182495A, “Zhengzhou”, in view of CN107345207A, “Wang”, have been fully considered and are persuasive in light of the amendments. Therefore, the rejections have been withdrawn.
However, upon further consideration, new grounds of rejection are made in view of Applicant’s amendments to the claims, prompting further search of the art, and consideration of the amended subject matter.
New Claim Objections/Rejections, Necessitated By Applicant’s Amendments
Claim Objections
Claim 36 is objected to because of the following informalities:
In claim 36, in step (v.b.’1), the phrase “through an up to µm cut-off filter” is recited. This appears to be an inadvertent error, and should instead say “through an up to 100 µm cut-off filter”, as recited in each of claims 1, 14, and 29.
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, 14, 29, and 36 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.
The phrase “most of the mucosa proteins and peptides”, as recited in newly added step v.b.1’ of claims 1, 14, 29, and 36, is a relative term which renders the claim indefinite. The term “most” 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. One cannot determine whether what amount of the mucosa proteins and peptides is required to fulfill this method step. There is no clear standard to compare the collected “most of the mucosa proteins and peptides” against, and thus how is one performing the method to know whether “most” of the proteins/peptides have been obtained? The resulting claim language is indefinite as the limitation does not set forth proper boundaries of claim protection which is sought, i.e. the metes and bounds are indefinite.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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, 14, 29, and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Zhengzhou Tongyuan Biology Tec (CN101182495A, herein after “Zhengzhou”, a machine generated translation is cited on the accompanying PTO-892, of record) in view of Wang et al. (CN107345207A, a machine generated translation is cited on the accompanying PTO-892, of record), Fahrner et al. (US Pat. No. 7,323,553), and Rivera, E., et al. (2008. Improving Tangential Flow Filtration Yield. BioPharm International, 21(7)).
Zhengzhou discloses a co-production process for producing alkaline phosphatase and heparin sodium using pig small intestine as raw material ([0002]; [0012]).
In regards to obtaining proteins in a native state, Zhengzhou teaches using a Tris-HCl buffer containing a certain concentration of magnesium chloride, sodium chloride and zinc chloride to extract the enzyme, which has the advantage of being able to more effectively protect the activity of the enzyme [0039], thus teaching an enzyme preservative. The method also comprises a method of low-temperature reduced pressure concentration and vacuum freeze-drying to prepare the enzyme preparation, and teaches that the advantages of this method are that the activity of the enzyme is not lost, and it improves the production efficiency ([0041]).
Zhengzhou teaches a method of obtaining a pig intestine mucosa, adding 4-10℃ pre-cooled 0.05mol/L Tris-HCl buffer (pH7.5, containing 0.01mol/L magnesium chloride; 0.01mol/L sodium chloride; 0.01mol/L zinc chloride) at a solid-liquid ratio of 1:3, i.e. thus preparing an aqueous extract of a mammalian intestinal mucosa, and then fully homogenizing the mucosa sample in a beater ([0048]), and that the slurry is kept at a low temperature of 4-10℃. Thus Zhengzhou teaches preserving a mammalian intestinal mucosa extract at a temperature allowing preserving one or more active enzymes (i.e. alkaline phosphatase) and heparin (i.e. by maintaining a temperature lower than 10°C), as in the instant step (i).
Zhengzhou teaches homogenizing the preserved mucosa at a temperature of less than 40°C to lyse the mucosa cells to obtain a homogenate ([0048]) of the mucosa having heparin and proteins as in the instant step (iiia).
Zhengzhou teaches that cooled n-butanol is added to the homogenate and the solution is kept it in a water bath at no higher than 37°C ([0049]). Zhengzhou teaches that after filtering and centrifuging (3000-5000r/m), a filtrate, i.e. a permeate fraction, comprising the crude enzyme (i.e. the native protein solution, having been maintained as low temperatures in non-harsh conditions) is collected and that the precipitate, i.e. the pellet or retentate fraction, comprises heparin ([0050]-[0052]) and additional proteins ([0068]-[0069]), as recited in step (iva) of the instant invention.
Zhengzhou teaches that the enzyme solution was added to a DEAE Sepharose 4B column and eluted into fractions ([0053]), thus teaching performing at least one step of ion exchange chromatography (see also [0014]) in order to separate the proteins and peptides according to at least their isoelectric point, as recited in step (v.b’).
Zhengzhou also teaches that the enzyme protein concentrate was added to a Sephadex G-150 gel filtration column to perform gel chromatography separation ([0055]; [0014]). Zhengzhou teaches assaying the fractions for alkaline phosphatase enzyme activity, thus providing evidence that the obtained enzyme is active and has a native or normal structure and function, according to the B.R.I. of the claims, as discussed above.
However, Zhengzhou does not explicitly teach obtaining both mucosa proteins and peptides, as recited in the elected species of the invention, nor does Zhengzhou explicitly teach
submitting supernatants containing proteins and/or peptides to both a cationic exchange chromatography step and subsequentially to an anionic exchange chromatography step, followed by steps of dialysis/filtering and concentrating the products by means of a tangential flow system through a filter having a cut-off from 1 to 5 kDa, as required in the instant claims.
Wang (CN107345207, of record) is drawn to methods including the co-production of small molecule peptides, heparin sodium and alkaline phosphatase using animal intestinal mucosa (Abstract; [0002]). Wang teaches a method that comprises steps of obtaining and pulverizing a sample of animal intestinal mucosa, performing centrifugation and filtration steps ([0050-0055]), and then performing a step of ultrafiltration (with a molecular weight cutoff of 10 kDa) to collect a permeate and the retentate respectively, wherein the permeate is a small molecule peptide solution ([0063]-[0065]). Wang teaches that the retentate contains the larger proteins, i.e. those greater than 10 kDa, including enzymes such as alkaline phosphatase, which may be further purified ([0066]-[0070]). Wang thus teaches that a protein containing fraction obtained from an animal intestinal mucosa contains both larger proteins and small peptides, which may be separated by means of fractionating, such as by ultrafiltration.
Wang also teaches that the small peptides have numerous useful functions, including enhancing human immune function ([0065]), and other various cellular effects (see [0039]: “Small molecule peptides participate in the synthesis of enzymes, stimulate enzyme activity, enhance enzyme function, and maintain enzyme stability”).
Fahrner ’553 pertains to protein purification and teaches a method for purifying proteins from a composition comprising polypeptides and at least one impurity without the use of affinity chromatography (Abstract, Col 1, lines 15-20). Fahrner teaches lysing cells which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments, and that such disruption releases the entire contents of the cell into the homogenate, and produces subcellular fragments that are difficult to remove due to their small size (Col 1, lines 40-50). Fahrner states that such impurities and cell debris are generally removed by centrifugation or by filtration, e.g., as in the instantly claims steps iva, v.b’, and v.b.1’ (Col 1, lines 45-50). Regarding protein purification in general, Fahrner teaches (in Col 1, lines 52-66):
“Once a solution containing the protein of interest is obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the “flow-through.”
From such teachings in Fahrner, it is evident that means for selectively enriching protein/peptides of desired characteristics are known to the art. Fahrner teaches one such known method is ion-exchange chromatography, named for the exchangeable counterion, a procedure applicable to purification of ionizable molecules wherein ionized molecules are separated on the basis of the non-specific electrostatic interaction of their charged groups with oppositely charged molecules attached to the solid phase support matrix, and differences in molecule charges result in resolution of various molecule types by ion-exchange chromatography. (Col 2, lines 3-24).
Fahrner further teaches that both anion (negative) and cation (positive) exchange chromatography techniques are known to the art (Col 2, lines 25-42).
Fahrner expressly teaches that both forms of ion exchange chromatography may be used sequentially, in either order (Col 4, lines 20-26: “In a preferred embodiment, the first and second non-affinity purification steps are cation exchange chromatography and anion exchange chromatography, in either order. In another preferred embodiment, the first non-affinity purification step is cation exchange chromatography and said second non-affinity purification step is anion exchange chromatography”, see also claims 4 and 5 of Fahrner).
Fahrner also teaches that the cation exchange chromatography step removes part of the unwanted host cell proteins, degradation products, and protein aggregates, and that the anion exchange chromatography step further purifies a desired protein from remaining host cell proteins, endotoxins, and DNA impurities (Col 18, line 65 - Col 19, line 6).
Fahrner thus teaches steps of obtaining bound and unbound (i.e. flow-through) fractions using both anion and cation exchange chromatography, and obtaining acidic peptide fractions and basic peptide fractions would naturally flow from following the methods taught therein.
Fahrner also teaches that the process may further comprise high-performance tangential-flow filtration (HPTFF) (Col 4, lines 26-30, Claim 1). Fahrner teaches that “Tangential flow filtration” or “TFF or “crossflow filtration” refers to a filtration process in which the sample mixture circulates across the top of the membrane, while applied pressure causes certain solutes and Small molecules to pass through the membrane. Typically, the solution flows parallel to the filter membrane. A pressure differential across the membrane causes fluid and filterable solutes to flow through the filter. This can be conducted as a continuous flow process, since the Solution is passed repeatedly over the membrane while that fluid that passes through the filter is continually drawn off into a separate circuit” (Col 14, lines 4-18).
Fahrner teaches that protein separations in HPTFF is accomplished using diafiltration in which the impurities is washed out of the retentate by simultaneously adding fresh buffer to the feed reservoir as filtrate is removed through the membrane and teaches that diafiltration makes it possible to obtain purification factors for products collected in the retentate that are greater than the membrane selectivity (Col 19, lines 31-42).
Rivera et al. discloses optimal methods for maximizing product yield during tangential flow filtration processes (Title, Abstract).
Rivera teaches that the molecular weight cut-off (MWCO) of the membrane used is critical to the retention capability of the target molecule(s), and suggests that it is recommended to use a membrane with an MWCO rating of less than 1/3 the size of desired molecule to recover (see pg. 3, last para, under “Laboratory-Scale Process Development”).
Rivera tests the performance of 8 kD and 5 kD MWCO membranes (Figure 1, pg 4). From the data and discussions, Rivera concludes that a reused cellulose 5kDa membrane resulted in improvements of increased product yield and reduced financial costs (pg. 16, Conclusions).
Therefore, to one of ordinary skill in the art, before the effective filing date of the claimed invention, it would have been obvious to modify the method taught in Zhengzhou for simultaneously obtaining heparin and proteins, namely alkaline phosphatase, from pig intestinal mucosa, according to the teachings in Wang, such that proteins and small peptides would have been obtained in the permeate comprising crude proteins in the native state (having been maintained as low temperatures in non-harsh conditions as taught in Wang) and it would have been further obvious in view of the level of ordinary skill in the art and the teachings of , to further optimize the process according to the teachings of Fahrner and Rivera, including steps for cation and anion exchange chromatography, and steps of diafiltration with a tangential flow system having a 5 kDa cutoff filter, because these are known steps for protein purification which result in improved preparations with less impurities and improved yield.
One would have been motivated to do so because Wang teaches that small peptides found in animal intestinal mucosa have useful biological and medicinal properties, and are known to be useful for improving human immune function. Further, one of ordinary skill in the art would recognize that when obtaining a mixture of proteins and peptides as taught in Zhengzhou, a number of various proteins and peptides would be present, not just the alkaline phosphatase of interest. Zhengzhou teaches obtaining a number of eluted fractions during the ion-exchange chromatography and gel filtration steps and then testing the fractions for the alkaline phosphatase enzyme activity. Yet, one having ordinary skill in the art would be aware that the other fractions obtained therein would also have additional peptides of interest, as suggested in Wang. Thus, the combined teachings of Zhengzhou in view of Wang would have made it obvious to obtain proteins and peptides in their native state, including performing filtration, fractionation, and/or centrifugation steps of extracts of mammalian intestinal mucosa.
Further, Zhengzhou teaches performing ion-exchange chromatography and Fahrner describes well-known methods to improve a protein purification process to remove impurities using cation and anion exchange chromatography columns.
Both Zhengzhou and Fahrner discusses steps of centrifugation and filtration for clearing cell lysates and removing debris, and one having ordinary skill in the art would have been able to arrive at the instantly claimed cut-off filter of up-to-100µm (in step v.b.1’) which is a very permissive filter, removing very large aggregates and cellular debris. Steps for centrifuging and filtering to clear cell lysates are known in the art, as taught in both Zhengzhou and Fahrner.
One would have been motivated to apply additional techniques known in the art for further improving on the protein purification process taught by Zhengzhou and Wang. The teachings of Fahrner and Rivera describe known means for removing impurities and contaminants, and one having ordinary skill in the art would be well-versed in such biochemical purification techniques. The selection of sequential anion and cation exchange chromatography columns followed by tangential flow filtration (or diafiltration), known in the art for obstinately the same purpose of purifying protein, would have thus been a matter of judicious selection and routine optimization to one of ordinary skill in the art. See also MPEP § 2143.I.G, which states:
“The courts have made clear that the teaching, suggestion, or motivation test is flexible and an explicit suggestion to combine the prior art is not necessary. The motivation to combine may be implicit and may be found in the knowledge of one of ordinary skill in the art, or, in some cases, from the nature of the problem to be solved. Id. at 1366, 80 USPQ2d at 1649. "[A]n implicit motivation to combine exists not only when a suggestion may be gleaned from the prior art as a whole, but when the ‘improvement’ is technology-independent and the combination of references results in a product or process that is more desirable, for example because it is stronger, cheaper, cleaner, faster, lighter, smaller, more durable, or more efficient. Because the desire to enhance commercial opportunities by improving a product or process is universal—and even common-sensical—we have held that there exists in these situations a motivation to combine prior art references even absent any hint of suggestion in the references themselves. In such situations, the proper question is whether the ordinary artisan possesses knowledge and skills rendering him capable of combining the prior art references." Id. at 1368, 80 USPQ2d at 1651.”
Here, all of the steps recited in the claimed method are known to the art, and one of ordinary skill in the art would be able to arrive at the particular combination claimed herein. Further, it is known to the art that combining multiple purification steps results in improved process, having less impurities. See e.g. the teachings of Fahrner cited above.
Further, the instant claims do not recite that any particular protein or peptide is isolated, and there is no indication that the selected cut-off filters of the instant claims are critical.
Regarding the particular selection of MWCO filters, the 5kDa filters for the diafiltration are expressly taught in Rivera, and the general filtration step of v.b.1’ is essentially the cell lysate clearing steps discussed in both Fahrner and Zhengzhou. Such high level filtration to remove impurities would be well-known in the art, and arriving at a 100µm filter does not amount to an inventive step. MPEP § 2144.05 describes that the determination of suitable or effective concentration of a known composition (or performing a known method) can be determined by one of ordinary skill in the art through the use of routine or manipulative experimentation to obtain optimal results, as these are variable parameters attainable within the art. Where 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).
Regarding claims 29 and 36, which appear to only differ from claims 1 and 14 by the order of the anionic/cationic exchange steps, Fahrner explicitly teaches that the steps can be performed in either order, and one of skill in the art would know that the anionic exchange chromatography would result in an acidic fraction and the cationic would yield a basic fraction. Further, MPEP § 2144.04. IV.C. indicates that the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946); and that the selection of any order of mixing ingredients is prima facie obvious, In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930). Therefore, it would have been obvious to perform the steps of anion exchange chromatography and cation exchange chromatography, as taught in Fahrner, in either order when purifying proteins.
The cited references are relied upon for the reasons discussed above. Based upon the overall beneficial teaching provided by the references with respect to obtaining heparin and performing protein purification, including steps for filtration, centrifugation, ion exchange chromatography, and diafiltration, the adjustments of particular conventional working conditions (e.g. determining filtration cutoffs, incubation times, or temperature treatments during a purification process) is found to be a matter of judicious selection and routine optimization that is well within the level of ordinary skill in the art.
From the teachings of the cited references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention, because Zhengzhou teaches purifying proteins from animal intestinal samples, thereby obtaining a number of eluted fractions during ion-exchange chromatography and Fahrner teaches sequential steps of anion and cation exchange chromatography for removing impurities. Further, the tangential flow filtration steps taught in Fahrner would be improved by the findings of Rivera, teaching that a 5 kDa cut-off results in improved yields for protein samples.
Therefore, claims 1, 14, 29, and 36, would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made, as evidenced by the references, especially in the absence of evidence to the contrary.
Conclusion
Claims 1, 14, 29, and 36 are rejected.
Claims 2, 8-13, 21, 26-28, and 30-35 remain withdrawn
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655