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 instant application is a U.S. national phase of PCT/EP2019/053716, filed February 14, 2019.
Applicant filed a claim amendment on May 12, 2026. Claims 1-20, 25-26, 31-32, and 34-35 are canceled. Claims 21 and 38 are amended.
Claims 21-24, 27-30, 33, and 36-38 are pending. Claims 36-37 are withdrawn.
Claims 21-24, 27-30, 33, and 38 are examined.
The previous objections to claims 21, 33, and 38; and 112(b) rejection of claim 23 are withdrawn.
Claim Objections
Claims 21 and 38 are objected to because of the following informalities: line 6, need to add “recovering a culture supernatant of the C. histolyticum as the mixture of substances;” to maintain clam language consistency. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
(new rejection necessitated by amendment) Claims 21-24, 27-30, 33, and 38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Newly amended claims 21 and 38 recite a method for purification of collagenase type I, collagenase type II, neutral protease and clostripain, from a mixture of substances, comprising the steps of culturing Clostridium histolyticum, recovering the culture supernatant as the mixture of substances, loading the mixture to a hydrophobic interaction chromatography (HIC) having a stationary phase that comprises polypropylene glycol or butyl Sepharose, and an aqueous solution as a mobile phase comprising ammonium sulfate or potassium chloride; separating the aforementioned enzymes from one another by the HIC in a single chromatographic step, so the collagenase I & II are mixed in a common fraction, and the neutral protease and the clostripain are separated into two further fractions. To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described by their complete structure.
In the instant case, the specification merely describes two working examples with specific conditions: In Example 1, the separation of the enzymes using a stationary phase consisting of propylene glycol with an aqueous phase of ammonium sulfate under an isocratic elution in three elution steps, wherein the clostripain is eluted in a first fraction, the collagenases I & II are mixed in a common second fraction, and the neutral protease is in a third fraction. In Example 2, all four enzymes are separated into 4 fractions using a stationary phase consisting of butyl-sepharose with an aqueous phase of potassium chloride carried out as a gradient with linearly decreasing salt concentrations. Both Examples disclose specific elution conditions, including different salt concentrations for elution of the respective fractions, especially Example 1, in which the third fraction containing the neutral protease is eluted without ammonium sulfate. These specified elution conditions along with the specific stationary and mobile phase combinations are the only combinations disclosed.
While the specification demonstrates possession of particular chromatographic conditions capable of providing the claimed three-fraction separation, it does not provide sufficient description demonstrating possession of the broader genus as presently claimed, which encompasses various combinations of the recited stationary phases and mobile phase salts without limitation to the chromatographic conditions and elution conditions disclosed as producing the required function of separation in a single chromatographic step, specifically the chromatographic conditions applicable across the claimed alternatives that result in collagenases type I and II in a common fraction, while neutral protease and clostripain are separately recovered in two additional fractions.
The inventions of claims 22-24, 27-30, and 33 require the use of the inventions of claims 21 and 38, and therefore are likewise rejected under 35 U.S.C. 112, first paragraph, as failing to comply with the written description requirement.
Applicant’s attention is directed to the Guidelines for the Examination of Patent Applications Under the 35 U.S.C. 112(a) or Pre-AIA 35 U.S.C. 112, first paragraph, "Written Description" Requirement (MPEP2163).
In conclusion, Applicant’s disclosure of the species of separating the aforementioned enzymes in the claimed separate fractions in a single HIC chromatographic step using a stationary phase of either propylene glycol or butyl-sepharose, and a mobile phase of either ammonium sulfate or potassium chloride, of the claimed broad genus is not deemed sufficient to reasonably convey to one skilled in the art that Applicant was in possession of the claimed broad genus at the time the application was filed. Thus, it is concluded that the written description requirement is not satisfied for the claimed genus.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 21-24, 27-30, 33, and 38 are rejected under 35 U.S.C. 103 as being unpatentable over JP 2004535197 A (cited in PTO-892 mailed 11/13/2025, hereinafter “JP”) in view of Kula et al. (Biochem and Biophys Research Comm. 1976. Vol. 69, No. 2, pgs. 389- 396, cited in PTO-892 mailed 10/17/2024, hereinafter “Kula”) and Fres et al. (Journal of Lipid Research Volume 51, 2010, pgs. 2454-2459, cited in PTO-892 mailed 11/13/2025, hereinafter “Fres”).
JP teaches a method for partially or completely purifying at least one kind of enzyme contained in an excess fermentation product of Clostridium histolyticum (abstract). JP teaches the microorganism Clostridium histolyticum extracellularly produces a complex enzyme mixture containing collagenase, various proteinaceous enzymes and low molecular weight components when cultured in a peptone-containing nutrient medium, wherein the main components are type I and type II collagenases, clostripain, and a neutral protease [0002]. JP teaches the enzyme of the Clostridium histolyticum overferment is separated by a single-stage or preferably a multi-stage chromatographic method exclusively using chromatographic materials based on styrene / divinylbenzene and / or especially on the hydroxylapatite ceramics, which results in a very rapid and cost-effective enzyme purification [0007]. JP teaches hydroxylapatite is a calcium phosphate represented by the general formula Ca10 (PO4 )6 (OH)2 that allows protein binding for ionic electrostatic exchange and strong ionic complex salt binding [0007]. JP teaches three elution stages, Fraction 1 obtained in the first elution stage contains exclusively low molecular weight components. Fraction 2 of the second elution stage also contains a neutral protease (caseinase) in addition to low molecular weight components. Fraction 3 of the third elution step also contains low molecular weight components, including all clostripains and all type I and type II collagenases [0017]. Fraction 3 is subjected to a second chromatography step to further separate the clostripains and collagenases into further fractions [0018]. JP teaches the method does not have any protein precipitation steps that can cause unwanted structural changes of the protein, such as ammonium sulfate [0011].
JP does not teach the method utilizes hydrophobic interaction chromatography comprising material propylene glycol or butyl sepharose, nor the type of salt used in the aqueous solution in the mobile phase, such as ammonium sulfate or potassium chloride. JP also does not teach the clostripain and collagenases are further separated in the single chromatography step.
However, Kula teaches a purification method of collagenase A and clostripain from Clostridium histolyticum (title). Kula teaches the use of hydrophobic chromatography (HIC) utilizing butyl Sepharose to separate clostripain in a homologous series of hydrocarbon-coated agaroses for the development of fractionators (abstract), which meets the limitation of claim 21. Kula teaches clostripain was extracted by passing the culture filtrate though the butyl agarose, and then collagenase A was extracted by passing the excluded mixture through heptylagarose (abstract). Kula teaches the enzymes were a lyophilized collagenase preparation containing several extracellular proteins from Clostridium histolyticum served as a source for both collagenase and clostripain (pg. 390, para 2). Kula teaches separation in a step elution scheme by passage of the crude protein first on the butyl sepharose to extract the clostripain, and then by applying the excluded proteins on the heptyl agarose in order to purify the collagenase (pg. 392, para 3).
JP and Kula do not teach butyl sepharose that is a cross-linked agarose with 3-n-butoxy-2-hydroxypropyl residues.
However, Fres teaches optimizing separation and purification of a recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase, using Butyl Sepharose High Performance (HP) (abstract, pg. 2455, col. 2, para 1). As disclosed in the specification, the claimed method utilizes butyl Sepharose HP which is a cross-linked agarose with 3-n-butoxy-2-hydroxypropyl residues (pg. 5, para 2). Fres teaches baseline purification of the farnesylated protein was made possible by using the new HIC resin, Butyl Sepharose HP, which separation of the farnesylated and unmodified hGBP1 had not been possible with the previously available butyl Sepharose 4 Fast Flow (pg. 2456, col. 1, para 4-5). Fres also teaches the elution profile of hGBP1 from a preparative HIC using Butyl Sepharose High Performance (GE Healthcare) with an ammonium sulfate gradient from 0.5 M to 0 M, which meets the limitations recited in claims 21-23, 27-29, and 38 (pg. 2456, Fig. 1).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to optimize the JP method of separating collagenase type I & II, clostripain, and neutral protease from a C. histolyticum supernatant into separate fractions using a single chromatography step, as taught by JP, by replacing the chromatography materials with the newly available butyl Sepharose HP, which is a cross-linked agarose with 3-n-butoxy-2-hydroxypropyl residues as the stationary phase in the HIC, as taught by Fres. One of ordinary skill in the art would have been motivated to substitute chromatography materials because Fres discloses the more effective separation of closely related proteins utilizing butyl Sepharose HP. There would have been a reasonable expectation of success because Kula discloses separation of clostripain from collagenase in a step elution scheme using butyl sepharose.
Further, it would have been prima facie obvious to optimize the method of separating the aforementioned enzymes utilizing the HIC by incorporating a gradient or step elution scheme as taught by the prior art. One of ordinary skill in the art would have been motivated to separate the clostripain from collagenase fractions in the method taught by JP, as both are useful due to their narrow specificity, and thus depends on their being free of other proteolytic activities as taught by Kula (pg. 390, para 1). Thus, the method of purifying these four enzymes as described in JP could have been easily conceived by a person skilled in the art based on the technical matter described in Kula and Fres, wherein one of ordinary skill in the art would optimize the appropriate elution scheme based on well-known newer chromatography techniques in the art.
Response to Arguments
Applicant's arguments filed May 12, 2026 have been fully considered but they are not persuasive.
Regarding Remarks directed to the 35 USC § 103 rejection, Applicant argues JP does not disclose the presently claimed invention, specifically separating the mixture of enzymes in a single HIC chromatography step. Rather, JP discloses a three-stage method for the partial or complete purification at least of one enzyme contained in an excess fermentation of Clostridium histolyticum, characterized in that the enzymes of the excess fermentation are separated by a chromatography method exclusively using styrene/divinyl-benzene base and/or hydroxyapatite as the chromatography materials. Applicant argues JP mentions in passing the method can be performed in a single step, however the abstract of JP only refers to multi- step methods and there is no disclosure of any details concerning a single stage method or its results anywhere in JP. Applicant argues the filing date of the present application is February 14, 2019, and an argument that a skilled person "today" would know how to implement a single-step chromatography process based on an older reference constitutes an impermissible hindsight analysis. Without specific evidence that the claimed single-step hydrophobic interaction chromatography using butyl Sepharose and achieving the claimed separation was part of the common general knowledge before the 2019 filing date, such reasoning cannot support an obviousness rejection. Applicant argues Kula does not disclose or suggest the use of butyl Sepharose and explicitly teaches away from the use of Sepharose, by teaching the consecutive use of omega-aminoalkylagaroses, which are agaroses substituted by H2N- (CH2)n-NH- groups, and the “Seph-Cn series” are dismissed as unsuitable for the separation of the enzymes, thus the stationary phases disclosed in Kula are therefore structurally distinct from butyl Sepharose and Kula does not teach or suggest the use of butyl Sepharose as claimed. Applicant argues Fres discloses a process that includes three chromatographic steps and not a single chromatographic step, wherein the second chromatographic step, farnesylated hGBP1 and unmodified hGBP1 are separated on Butyl Sepharose HP with an ammonium sulfate gradient from 0.5 to 0 M. Applicant argues based on the disclosure of Fres, one skilled in the art would see no advantage in using butyl Sepharose instead of styrene/divinyl-benzene based materials and/or hydroxyapatite (from JP), because it does not simplify the method, thus Fres does not disclose a more effective (simpler) separation. Applicant argues JP clearly states that the chromatography materials disclosed therein are instrumental for achieving a high separation performance (see the second sentence of paragraph [0008] of the published US application). One skilled in the art would therefore be discouraged from improving the method by changing the stationary phase. JP discloses that the use of ammonium sulfate is detrimental to a good separation (the published US application, paragraph [0012], last sentence). In contrast, Fres uses ammonium sulfate and one skilled in the art would therefore be discouraged to use the teaching of Fres in combination with the teaching of JP.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, JP teaches separation of the enzymes from a mixture of substances from the culture supernatant of C. histolyticum using chromatography, albeit disclosing in the examples different chromatographic materials in 3 steps, JP does explicitly suggest utilizing one chromatography step, and Kula teaches the use of HIC (which is considered as a single chromatography step) to separate collagenase and clostripain from a mixture of enzymes, wherein the stationary phase was compared between omega-aminobutylagaroses and activated butyl-sepharose(Seph-Cn-NH2 represents butyl Sepharose, Sepharose 4B, activated with CNBr and reacted with an α,ω-diaminoalkane n-carbon-atoms long- pg. 389, Abbrev., Footnotes). Fres further teaches an optimized method utilizing the Butyl Sepharose HP or Butyl Sepharose FF (that is used in the Examples of the present application) for purification, which dramatically improves the separation of enzymes in comparison to the previously available HIC resin (pg. 2457, col. 2, para 3).
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Thus, it is obvious to a person skilled in the art at the time of filing the claimed invention to modify and/or optimize ion-exchange chromatography in general and hydrophobic interaction chromatography in general, which are well-known and commonly used as means for purifying antibodies and enzymes, with newer chromatographic technologies in the art to arrive at the presently claimed invention.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA EDWARDS whose telephone number is (571)270-0938. The examiner can normally be reached M-F 8am-5pm EST.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657
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