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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12 November 2025 has been entered.
Priority
Applicant’s claim for the benefit of a prior-filed application (371 of PCT/EP2022/054912, filed 28 February 2022) under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Acknowledgment is made of applicant’s claim for foreign priority (BE2021/5138, filed 26 February 2021) under 35 U.S.C. 119 (a)-(d). Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Claim Interpretation
The cited “polyoxyethylene isooctylcyclohexyl ether” in independent Claim 3 is more commonly known in the art and is synonymous with “reduced Triton X-100” (or “Triton X-100 reduced”), CAS Number 92046-34-9.
The protein of interest of SEQ ID NO: 1 cited in Claim 9 is interpreted to be the protein CRM 197 as identified on pg. 5, bottom paragraph of the disclosure.
Claim 3 recites in step (c) “filtering a composition obtained at the elution step through an anion exchange membrane” (emphasis added). As noted in the Specification, this purification step involves “a step of passing through a Mustang® Q, Mustang® E or Sartobind® Q or Sartobind® STIC anion exchange membrane and/or a sterile filtration step” (pg. 4, par. 9). Thus, Applicant has acknowledged that Mustang® Q, Mustang® E or Sartobind® Q or Sartobind® STIC commercial products are known anion exchange membranes usable for a further purification step.
Response to Amendment
The amendments filed 12 November 2025 have been entered.
Claims 1, 2, 22, and 25-28 have been canceled; new Claims 34-36 have been added. Claims 3, 5-17, 29-36 are pending.
Regarding the rejections of Claims 11, 16, 28, 31, and 33 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite, Applicant’s amendments to Claims 3, 11, 16, and 31 and cancellation of Claim 28 are sufficient; the rejections of Claims 11, 16, 28, and 31 under 35 U.S.C. 112(b) have been withdrawn. However, amendments to Claim 33 are not persuasive; the as-amended claim remains indefinite.
Response to Arguments
Applicant's arguments filed 12 November 2025 have been fully considered.
Regarding the rejections of Claim(s) 3, 7, 8, 11, 17, 28, and 31 under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021) and ZHANG et al. (US 2021/0370199 A1), Applicant argues that Claim 3 would not have been obvious over the prior art because the combination of prior art is untenable due to their unrelated technologies and lack of motivation to combine absent impermissible hindsight (pg. 11, par. 1), e.g., STANEK is directed to protein purification, ANON is directed to nucleic acid purification, ZHANG is directed to rAAV particles (pg. 11, par. 1). Further, regarding ZHANG, Applicant argues ZHANG fails to teach filtration using an anion exchange membrane (pg. 12, par. 2-3), ZHANG teaches away from filtration due to use of a Sartobind Q column for binding target AAV particles (pg. 12, par. 4), and ZHANG does not teach isolation/purification from endotoxins (par. spanning pg. 12-13).
Regarding the rejection of Claim 9 under 35 U.S.C. 103 as being unpatentable over STANEK, ANON, ZHANG, and further in view of BLATTNER et al. (US 2017/0073379 A1), Applicant argues Claim 9 is allowable because “Claim 9 depends from claim 3, and, therefore, is patentable for at least the same reasons” (pg. 13, par. 3).
Regarding the rejection of Claim 33 under 35 U.S.C. 103 as being unpatentable over STANEK, ANON, ZHANG, BLATTNER, and further in view of KREMER et al. (WO 2011/098526 A1), Applicant argues Claim 33 is allowable because “Claim 33 depends from claim 9, which depends on claim 3, [and] is allowable for at least the same reasons” (pg. 13, par. 6).
Regarding the rejections of Claim(s) 5, 6, 12-15, and 30 under 35 U.S.C. 103 as being unpatentable over STANEK in view of ANON and KREMER et al. (WO 2011/098526 A1), Applicant argues the prior art fail to teach or suggest the recited method of Claim 5 because “at least Kremer, fails to teach the step of binding the protein of interest on the hydrophobic interaction matrix and actually teaches the exact opposite”, “Kremer is using the chromatographies in "flow-through" mode”, and “Kremer explicitly teaches away from using the first mode of chromatographic separation” (par. spanning pg. 14-15). Applicant further argues “a person of ordinary skill in the art would not be motivated to multiply the steps in a purification because it would increase the cost to run said purification” with respect to the Office’s rejection based on a duplication of parts because “Kremer teaches away from increasing purification costs, for example, criticizing previous chromatography methods for their "high variable costs (for example due to the necessity of large column capacity, which is inherently required for a binding plus elution step, and hence large amounts of costly resins needed) and high fixed costs (due to labor costs)”” (par. spanning pg. 15-16).
Regarding Claim 14, Applicant has amended Claim 14 to be an independent claim and to incorporate limitations supposedly suggested by the Examiner in the previous Office action to be considered allowable (pg. 17, par. 1). With respect to an elution step using a solution having conductivity between 40 and 105 mS/cm, Applicant argues “the claimed value is unexpected at least in part due to the fact that the claimed conductivity is substantially higher than the value disclosed in Kremer (5 mS)” (pg. 17, par. 1).
Regarding the rejection of Claims 10 and 32 under 35 U.S.C. 103 as being unpatentable over STANEK in view of ANON, KREMER, and ISLAM et al. (Micromachines, 2017, 8, 83), Applicant argues ISLAM is merely a review publication that relies on Fonseca and Chen for disclosing the use of osmotic shock with gram-negative bacteria but only teaching low extraction yield which is “unacceptable for an industrial point of view, since this implies to markedly increase (almost a factor of 2) all the costs for the fermentation of the cells and of the protein of interest” (par. spanning pg. 18-19). Further, regarding Fonseca, Applicant argues “one of ordinary skill in the art would find no motivation to practice such heavy, time-consuming, and expensive tasks, just to test if, by coincidence, there would be an added value to rely on an osmotic shock” (pg. 19, par. 1). Applicant provides more arguments, regarding Chen, i.e., “Chen clearly teaches that the creatinase recovery corresponds to the amount of lipopolysaccharide (LPS) released. (See Chen at page 213, left-hand column, lines 14-17). As LPS are endotoxins, Chen teaches away from relying on osmotic shock to separate endotoxins from a protein of interest because Chen optimizes LPS release” (par. spanning pg. 19-20) and that “Chen's teaching would result in less protein of interest and more endotoxins; therefore, absent the present disclosure, a person of skill in the art would not be motivated to combine the alleged prior art to arrive at the claimed invention” (pg. 20, par. 1).
Regarding new Claims 34-36, Applicants assert the cited limitations are neither “shown nor suggested by any of the prior art or any combination thereof” (par. spanning pg. 20-21) and Claims 34-36 are allowable because Claim 36 “recites more than a fivefold excess of salts, which is logical since Kremer does not want to bind the protein of interest (antibodies), which is opposite to the present invention” (par. spanning pg. 20-21). Applicants also state that “Similarly, claims 37-38 recite conditions for the loading step of the chromatography on hydrophobic support” (par. spanning pg. 20-21).
The Examiner respectfully disagrees.
Regarding the arguments pertaining to the rejections of Claim(s) 3, 7, 8, 11, 17, 28, and 31 under 35 U.S.C. 103, Applicant is arguing that due to the disparity in applications of each listed prior art, one of ordinary skill would not be motivated to combine their teachings absent 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). The claimed invention is directed toward the removal of contaminants from a sample, i.e., the inventive aspect is not found in protein purification. While ANON may have disclosed the use of reduced TX-100 in purifying/maintaining the yield of DNA from a sample of bacteria, ANON nevertheless discloses the use of reduced TX-100 in reducing endotoxin levels in the sample of bacteria. Such a reduction will inherently result in increased purification of the target analyte, whether protein or DNA. Similarly, although ZHANG teaches purification of protein vectors, ZHANG nevertheless discloses further downstream processing utilizing AEX media (p0286), including Sartobind® Q AEX media (p0309), which Applicant has identified to be applicable for a membrane filtration step as required in step (c) of Claim 3 (see Specification, pg. 4, par. 9). Such a use of an AEX membrane will inherently result in any remaining endotoxins in the composition to be fixed to the membrane whether purifying proteins or rAAV particles. Regarding Applicant’s argument that ZHANG fails to teach filtration using an anion exchange membrane (pg. 12, par. 2-3), as mentioned earlier, ZHANG explicitly discloses the use of Sartobind® Q, which Applicant themselves has identified as a suitable AEX membrane. Regarding Applicant’s argument that ZHANG teaches away from filtration due to use of a Sartobind Q column for binding target AAV particles (pg. 12, par. 4), it is again noted that Applicant themselves has specifically identified Sartobind® Q matrix as suitable for use as a filtration membrane. Thus, Claims 3, 7, 8, 11, 17, and 31 are not allowable over the prior art.
Regarding the arguments pertaining to the rejection of Claim 9 under 35 U.S.C. 103 and the rejection of Claim 33 under 35 U.S.C. 103, Applicant’s arguments have been addressed as above for the rejection of Claim 3.
Regarding the arguments pertaining to the rejections of Claim(s) 5, 6, 12-15, and 30 under 35 U.S.C. 103 as being unpatentable over STANEK in view of ANON and KREMER, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In this case, STANEK had already disclosed the use of AEX with loading, washing, and elution steps for the capture of target protein and removal of unwanted contaminants. KREMER simply further teaches that after an AEX step, a hydrophobic interaction chromatography step can be utilized to further purify a target by removing other unwanted analytes not removable by AEX. Advantageously, while the AEX treatment removes negatively charged impurities, the subsequent HIC treatment removes any remaining large molecular impurities (e.g., aggregates; pg. 8, lines 7-16). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to include a subsequent hydrophobic interaction chromatography step taught by KREMER after the AEX step of the method made obvious by modified STANEK.
While it is acknowledged that KREMER teaches a flowthrough process, the Office’s rejection relied upon KREMER’s disclosure of the combination of AEX followed by HIC to advantageously remove more types of contaminants (i.e., the AEX treatment removes negatively charged impurities, and the subsequent HIC treatment removes any remaining large molecular impurities (e.g., aggregates; pg. 8, lines 7-16)). One of ordinary skill in the art in reading KREMER would appreciate that HIC can be used in any mode, whether flowthrough or bind-elute, and still provide the same advantage. Such an operation of an HIC process, i.e., in a bind-elute mode rather than a flowthrough, would be obvious because one of ordinary skill in the art has good reason to pursue the known options within his or her technical grasp; if this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense (MPEP §2143.01 E).
Furthermore, regarding Applicant’s argument pertaining to one of ordinary skill as not being motivated “to multiply the steps in a purification because it would increase the cost to run said purification”, 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, sufficient motivation was certainly provided by KREMER for including another chromatography purification step. It is acknowledged that adding additional steps will certainly increase costs to a purification; however, the most obvious advantage of these additional steps is that the end product will be more purified. While there may be an increasing cost-benefit ratio for further purification steps, such a consideration is not being evaluated for patentability; the claimed invention is simply a method for purifying a protein of interest, not a method for optimizing the cost-benefit of achieving a certain degree of purification of a protein of interest.
Regarding the arguments pertaining to the rejections of Claims 10 and 32 under 35 U.S.C. 103 as being unpatentable over STANEK in view of ANON, KREMER, and ISLAM, the cited references by ISLAM nevertheless offer the added advantage of increased purity—however minor—which would be sufficient motivation for one of ordinary skill in the art despite the other supposed disadvantages. 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). While such disclosed advantages, especially by FONSECA, may be “unacceptable for an industrial point of view”, the prior art nevertheless discloses an advantage to the use of osmotic shock, which is considered sufficient motivation for one of ordinary skill in the art to incorporate into modified STANEK. Whether a teaching by the prior art is considered economical or difficult to implement has no bearing on the patentability of the claimed method.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claim 33 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding Claim 33, there is insufficient antecedent basis for “the solution obtained at the elution step”. The elution step of Claim 3 is only associated with “a composition” (see Step (c): “filtering a composition obtained at the elution step”), and no “solution” is explicitly or indirectly referenced in context of the elution step.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim(s) 3, 7, 8, 11, 17, 29, and 31 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021) and ZHANG et al. (US 2021/0370199 A1).
Regarding Claim 3, STANEK discloses a method for purifying a protein of interest (e.g., repeat in toxin leukotoxin, RTX) from an E. coli culture (§4.1, par. 2). Briefly, the proteins are produced in E. coli; the bacterial cells are lysed and cells are lysed in a buffer to collect cell contents containing the protein of interest and contaminants (e.g., lipopolysaccharides and other endotoxins; i.e., step (a) conditioning said protein of interest in a solution; §4.1; §abstract). Lysed contents are loaded onto DEAE-Sepharose chromatography resin beds (i.e., wherein the series of purification steps comprises capture chromatography using an anion exchange resin and/or hydrophobic interaction chromatography), and trapped LPS and other endotoxins are washed away using solutions containing 10 column volumes of non-ionic detergents Triton X-100 or Triton X-114 (i.e., wherein the removal of contaminants comprises removing endotoxins and comprises a step of adding a solution comprising [a detergent]; at least two column volumes of the washing solution comprising [the detergent] are applied to said support; §2.1, par. 1; §4.2). Subsequently, the detergents are removed from the resins with washes using 5 bed volumes of buffer without detergent, (i.e., step (b) a series of purification steps to recover said protein of interest in a fraction to be retained and contaminants in one or more fractions to be removed, said contaminants comprising endotoxins; at least two column volumes of a washing solution free of [the detergent] are applied before elution; each comprising a loading step, a washing step, and an elution step; §2.1, par. 1; §4.2) and the proteins of interest are collected via a separate eluate wash (i.e., wherein, for the washing step of each chromatography… are applied before elution of said protein of interest; §4.2)
STANEK is deficient in disclosing using polyoxyethylene isooctylcyclohexyl ether as the detergent.
ANON discloses the use of detergents in purifying nucleic acids of endotoxins by anion exchange chromatography (e.g., a DEAE column; §Background, pg. 1-2). The testing included comparisons of the effectiveness in nucleic acid yield and reduction in endotoxin levels of detergents including Triton X-100, Triton X-114, and reduced Triton X-100 (§Materials and Methods, pg. 6). Diagram 2 (pg. 8) shows negligible differences among these detergents in DNA yield; Diagram 7 (pg. 11) shows similar levels of reduced endotoxin concentrations in the DNA eluates. ANON concludes that Triton X-100 reduced “would be a suitable replacement” for Triton X-100 and Triton X-114 (§Discussion; pg. 12). Thus, given the statistically same results (i.e., predictable results), prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to utilize Triton X-100 reduced (i.e., polyoxyethylene (10) isooctylcyclohexyl ether) as a substitute detergent for Triton X-100 or Triton X-114 as taught by ANON in the method of purifying proteins disclosed by STANEK. The claim would have been obvious to one of ordinary skill in the art because the substitution of one known element for another would have yielded predictable results (MPEP §2143.01 B). Furthermore, it is recognized that although STANEK discloses protein purification and ANON discloses nucleic acid purification, the methods and protocols used for DNA separations are obvious to apply to protein separations and vice versa because both separations exploit similar separation mechanisms. In the instant case, both ANON and STANEK utilize charge-based separations, i.e., anion exchange chromatography, to bind DNA and proteins to the anion exchange resins thereby allowing for the wash removal of unwanted impurities (e.g., endotoxins).
Modified STANEK is deficient in disclosing filtering the composition obtained at the elution step through an anion exchange membrane.
ZHANG discloses the purification of a protein vector using anion exchange chromatography (abstract; p0284); the method comprises purification on an AEX media comprising, e.g., DEAE ligand (p0220), followed by downstream processing to further purify the protein vector, i.e., eluates from the AEX purification are further processed by an anion exchange chromatography step (p0286). ZHANG lists such an AEX step as using Sartobind® Q AEX media (p0309), which Applicant has identified to be applicable for the claimed membrane filtration step (see Specification, pg. 4, par. 9). Because the prior art teaches the same AEX filtration membrane for the downstream purification of a target sample as that disclosed by the Applicant, the requirement that “any remaining endotoxins in the composition are fixed on the membrane” is necessarily expected (i.e., step (c) filtering the composition obtained at the elution step through an anion exchange membrane wherein any remaining endotoxins in the composition are fixed on the membrane; p0286). The claiming of a new use, new function or unknown property which is inherently present in the prior art does not necessarily make the claim patentable (In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977)). Advantageously, the practice of providing these additional downstream processing steps ensures an efficient separation of the target protein from contaminants (p0003). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to include a step of filtering an obtained eluate through an anion exchange membrane as taught by ZHANG for the method made obvious by modified STANEK.
Regarding Claim 7, modified STANEK makes obvious the method of Claim 3. STANEK further discloses no detergents in the subsequent washes (i.e., no phenolic solvent is used during the step of removing endotoxins; §2.1, par. 1; §4.2). STANEK also discloses 1% (v/v) Triton X-100 (§4.2); while this concentration is outside the claimed range of between 0.1% and 0.75% (w:v), it is noted that ANON discloses the preparation of a 0.5 % v/v reduced TX-100 (i.e., wherein the endotoxins are removed using a concentration of polyoxyethylene isooctylcyclohexyl ether between 0.1% and 1% (w:v); pg. 5). Thus, the claimed range of 0.1 to 0.75% (w:v) is recognized as part of the capabilities of one of ordinary skill in the art prior to the effective filing date of the claimed invention, and thus, the claimed range would have been obvious absent evidence of non-obviousness and criticality.
Regarding Claim 8, modified STANEK makes obvious the method of Claim 3. STANEK further discloses endotoxins were washed away using solutions containing 10 column volumes of non-ionic detergents Triton X-100 or Triton X-114 (i.e., wherein endotoxins… are removed with a volume between 1 and 10 times the column volume; §2.1, par. 1; §4.2).
Regarding Claim 11, modified STANEK makes obvious the method of Claim 3. While STANEK discloses a DEAE-Sepharose anion exchange column and, similarly, ANON and ZHANG both disclose DEAE columns, such resins are considered weak anion exchange resins. ZHANG does disclose that both a strong anion exchange media and a weak anion exchange media can be used in the purification of protein targets (p0212), including strong AEX resins as CIMMULTUSTM, POROS XQTM, etc. (p0213). The claim would have been obvious because one of ordinary skill in the art has good reason to pursue the known options within his or her technical grasp; if this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense (MPEP §2143.01 E). The choice of whether a strong AEX resin or a weak AEX resin is utilized is dependent on a number of factors, including the target protein of interest; one of ordinary skill in the art would be capable of determining whether a strong or a weak AEX resin would be appropriate for the isolation/purification of the desired protein of interest. Such a choice is not innovative but is merely common sense.
Regarding Claim 17, modified STANEK makes obvious the method of Claim 3. ZHANG further discloses the downstream processing includes multiple steps (i.e., “at least 2”), including sterile filtration (i.e., further comprising a sterile filtration step; p0286).
Regarding Claim 31, modified STANEK makes obvious the method of Claim 3. The instant limitations “wherein the removing endotoxins is performed during the washing step of the anion exchange chromatography and/or the hydrophobic interaction chromatography” and “wherein the anion exchange chromatography and/or the hydrophobic interaction chromatography comprise binding the protein of interest” are directed toward inherent properties resulting from the practice of claimed purification. Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed. A “whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003); MPEP §2111.04). Where a reference discloses the terms of the recited method steps, and such steps necessarily result in the desired and recited effect, the fact that the reference does not describe the recited effect in haec verba is of no significance because the reference meets the claim under the doctrine of inherency. No actual steps are required in these claims; and the prior art discloses or makes obvious all limitations of parent Claim 3. One of ordinary skill in the art in practicing the steps of Claim 3 will necessarily expect similar results as those claimed in Claim 31.
Claim(s) 9 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021) and ZHANG et al. (US 2021/0370199 A1), as applied to Claim 3 above, and further in view of BLATTNER et al. (US 2017/0073379 A1).
Regarding Claim 9, modified STANEK makes obvious the method of Claim 3. Modified STANEK is deficient in disclosing the protein of interest is SEQ ID NO:1. (It is noted that SEQ ID NO:1 has been identified by Applicant to be the protein CRM197; pg. 5, bottom paragraph of the disclosure.)
BLATTNER discloses the production of CRM197 via E. coli expression (p0010) and further discloses the need to reliably and inexpensively produce high amounts of therapeutically useful CRM197 (p0009). The method entails harvesting E. coli cells from culture media, lysing the cells, and purifying the lysate via anion exchange chromatography and hydrophobic interaction chromatography to obtain CRM197 (p0018). The nature of the problem to be solved would have led one of ordinary skill in the art to combine the elements as claimed by known methods with no change in their respective, individual functions, and the combination would have yielded nothing more than predictable results (MPEP §2143.01 A). Thus, one of ordinary skill in the art prior to the effective filing date of the claimed invention would have found it obvious to apply the AEX/HIC method for purifying a protein of interest made obvious by modified STANEK for purifying CRM197 via AEX/HIC as disclosed by BLATTNER.
Claim(s) 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021) and ZHANG et al. (US 2021/0370199 A1), as applied to Claim 3 above, and further in view of KREMER et al. (WO 2011/098526 A1).
Regarding Claim 29, modified STANEK makes obvious the method of Claim 3. Modified STANEK is deficient in disclosing the series of purification steps comprises hydrophobic interaction chromatography.
KREMER discloses a method for purifying antibody proteins produced from cell culture to remove residual impurities, e.g., endotoxins (pg. 1, line 29-pg. 2, line 6). The method entails the use of serial, in-line anion exchange chromatography (AEX) followed by hydrophobic interaction chromatography (HIC) (pg. 4, lines 18-23). KREMER discloses that the chromatographic purification of proteins entails loading and washing using buffers having specific pH and conductivity (pg. 1, lines 32-35). Advantageously, while the AEX treatment removes negatively charged impurities, the subsequent HIC treatment removes any remaining large molecular impurities (e.g., aggregates; pg. 8, lines 7-16). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to include a hydrophobic interaction chromatography step taught by KREMER after the AEX step of the method made obvious by modified STANEK.
Claim(s) 33 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021), ZHANG et al. (US 2021/0370199 A1), and BLATTNER et al. (US 2017/0073379 A1), as applied to Claim 9 above, and further in view of KREMER et al. (WO 2011/098526 A1).
Regarding Claim 33, modified STANEK makes obvious the method of Claim 9. Modified STANEK is deficient in disclosing the solution/composition obtained at the elution step has a pH of 8.0±0.2 and a conductivity between 7 and 10 mS/cm.
As claimed, it is interpreted that the composition obtained at the elution step primarily comprises the eluting buffer and the target protein of interest, i.e., collectively known as the composition.
KREMER discloses a method for purifying antibody proteins produced from cell culture to remove residual impurities, e.g., endotoxins (pg. 1, line 29-pg. 2, line 6). The method entails the use of serial, in-line anion exchange chromatography (AEX) followed by hydrophobic interaction chromatography (HIC) (pg. 4, lines 18-23). KREMER discloses that the chromatographic purification of proteins entails loading and washing using buffers having specific pH and conductivity (pg. 1, lines 32-35). All claimed elements were known in the prior art and one of ordinary skill in the art could have combined the elements as claimed by known methods with no change in their respective, individual functions, and the combination would have yielded nothing more than predictable results (MPEP §2143.01 A). In this case, one of ordinary skill in the art would find it obvious to control the pH and conductivity of a loading or washing solution used in chromatography—such as the AEX or HIC made obvious by modified STANEK— because such variables have significant impact on the effectiveness of the purification as taught by KREMER.
KREMER is deficient in explicitly disclosing a conductivity of the elution buffer (but does disclose a 5 mS conductivity for the loading and washing buffers; see pg. 11, lines 29-30) or a pH of loading/washing buffers (but does disclose slightly alkaline pH of 7.4; see pg. 11, lines 29-32). However, KREMER discloses the conductivity of the washing and elution buffers are optimized, i.e., “optimum conditions in terms of pH and conductivity” (pg. 5, lines 27-28). 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 absent unexpected results or evidence indicating such optimum or workable ranges are critical (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); MPEP§2144.05). Thus, the limitations wherein the solution has a pH of 8.0 ± 0.2 and a conductivity between 7 and 10 mS/cm would be obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claim(s) 35 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021) and ZHANG et al. (US 2021/0370199 A1), as applied to Claim 3 above, and further in view of BLANK et al. (US 2017/0198008 A1).
Regarding Claim 35, modified STANEK makes obvious the method of Claim 3. Modified STANEK is deficient in disclosing the loading step of the ion exchange chromatography is carried out with a solution having a conductivity between 5.0 mS/cm and 10.0 mS/cm and the elution step is carried out with a solution having an electrical conductivity greater than two times that of the loading step.
BLANK discloses chromatographic methods for the purification of proteins from a sample (abstract). Briefly, BLANK discloses sample loading onto a column under a conductivity of about 9.5 to about 14 mS/cm, which overlaps with the claimed range of between 5.0 mS/cm and 10.0 mS/cm and therefore, establishes a case of prima facie obviousness (MPEP 2144.05), and eluting under a conductivity of 80 mS/cm or more (i.e., an electrical conductivity greater than two times that of the loading step; p0032). Advantageously, such a loading and elution method provides for the purification of a protein from a sample without the need to significantly alter the characteristics of the sample and with minimal adjustment to the chromatography column (p0016). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to load a protein sample at a relatively low electrical conductivity between 5.0 mS/cm and 10.0 mS/cm and subsequently elute with a solution having an electrical conductivity greater than two times that of the loading step as suggested by BLANK for the method of modified STANEK.
Claim(s) 5, 6, 15, 30, and 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021), KREMER et al. (WO 2011/098526 A1), and BLANK et al. (US 2017/0198008 A1).
Regarding Claim 5, STANEK discloses a method for purifying a protein of interest (e.g., repeat in toxin leukotoxin, RTX) from an E. coli culture (§4.1, par. 2). Briefly, the proteins are produced in E. coli; the bacterial cells are lysed and cells are lysed in a buffer to collect cell contents containing the protein of interest and contaminants (e.g., lipopolysaccharides and other endotoxins; i.e., step (a) conditioning said protein of interest in a solution; §4.1; §abstract).
Lysed contents were loaded onto DEAE-Sepharose chromatography resin beds (i.e., loading the conditioned protein in a solution on a capture chromatography using an ion exchange resin), and trapped LPS and other endotoxins were washed away using solutions containing 10 column volumes of non-ionic detergents Triton X-100 or Triton X-114 (i.e., washing the said ion exchange resin with at least two column volumes of a solution comprising [a detergent]; §2.1, par. 1; §4.2). Subsequently, the detergents are removed from the resins with washes using 5 bed volumes of buffer without detergent, (i.e., washing said ion exchange resin with a solution free of [the detergent]; §2.1, par. 1; §4.2) and the proteins of interest are collected via a separate eluate wash (i.e., eluting the said protein of interest from the said ion exchange resin; each comprising a loading step, a washing step, and an elution step; §4.2)
STANEK is deficient in disclosing using polyoxyethylene isooctylcyclohexyl ether as the detergent.
ANON discloses the use of detergents in purifying nucleic acids of endotoxins by anion exchange chromatography (e.g., a DEAE column; §Background, pg. 1-2). The testing included comparisons of the effectiveness in nucleic acid yield and reduction in endotoxin levels of detergents including Triton X-100, Triton X-114, and reduced Triton X-100 (§Materials and Methods, pg. 6). Diagram 2 (pg. 8) shows negligible differences among these detergents in DNA yield; Diagram 7 (pg. 11) shows similar levels of reduced endotoxin concentrations in the DNA eluates. ANON concludes that Triton X-100 reduced “would be a suitable replacement” for Triton X-100 and Triton X-114 (§Discussion; pg. 12). Thus, given the statistically same results (i.e., predictable results), prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to utilize Triton X-100 reduced (i.e., polyoxyethylene (10) isooctylcyclohexyl ether) as a substitute detergent for Triton X-100 or Triton X-114 as taught by ANON in the method of purifying proteins of interest disclosed by STANEK. The claim would have been obvious to one of ordinary skill in the art because the substitution of one known element for another would have yielded predictable results (MPEP §2143.01 B). Furthermore, it is recognized that although STANEK discloses protein purification and ANON discloses nucleic acid purification, the methods and protocols used for DNA separations are obvious to apply to protein separations and vice versa because both separations exploit similar separation mechanisms. In the instant case, both ANON and STANEK utilize charge-based separations, i.e., anion exchange chromatography, to bind DNA and proteins to the anion exchange resins thereby allowing for the wash removal of unwanted impurities (e.g., endotoxins).
Modified STANEK is deficient in disclosing the series of purification further includes hydrophobic interaction chromatography.
KREMER discloses a method for purifying antibody proteins produced from cell culture to remove residual impurities, e.g., endotoxins (pg. 1, line 29-pg. 2, line 6). The method entails the use of serial, in-line anion exchange chromatography (AEX) followed by hydrophobic interaction chromatography (HIC) (i.e., a hydrophobic interaction chromatography is performed; pg. 4, lines 18-23). The liquid flowthrough in the system entails in-line loading of the protein extract in a buffer A, a washing buffer, and an elution buffer B (i.e., a loading step, a washing step and an elution step; pg. 9, lines 13-20). Advantageously, while the AEX treatment removes negatively charged impurities, the subsequent HIC treatment removes any remaining large molecular impurities (e.g., aggregates; pg. 8, lines 7-16). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to include a second hydrophobic interaction chromatography step taught by KREMER after the AEX step of the method made obvious by modified STANEK. Although KREMER is deficient in explicitly specifying the HIC process involves initially washing with a solution comprising polyoxyethylene isooctylcyclohexyl ether and subsequently washing with a solution free of polyoxyethylene isooctylcyclohexyl ether, STANEK in view of ANON discloses such a serial washing for AEX as summarized earlier. Such serial washing is considered a duplication of parts or process steps has no patentable significance unless a new and unexpected result is produced (In re Harza, 274 F.2d 669, 124 USPQ 378 (CCPA 1960); MPEP §2144.04). In this case, the claimed serial wash is the same or broader as the claimed earlier serial wash in all ways except for the chromatography resin being washed. However, in both serial washes, the chromatography methods are performed such that endotoxins are removed. Performance of such a repeated step regardless of the chromatography resin on which it was performed would expectedly result in a lower endotoxin level in the fraction containing the protein of interest. Therefore, no new and unexpected result is produced from a subsequent serial wash with an initial washing with a solution comprising polyoxyethylene isooctylcyclohexyl ether followed by a washing with a solution free of polyoxyethylene isooctylcyclohexyl ether.
Modified STANEK is deficient in disclosing the elution step of the hydrophobic interaction chromatography is performed with a solution with an electrical conductivity of greater than 40 mS/cm and less than 105 mS/cm.
BLANK discloses chromatographic methods for the purification of proteins from a sample (abstract). Briefly, BLANK discloses sample elution from onto a column under a conductivity of between 40 mS/cm and 100 mS/cm (i.e., the elution step of the hydrophobic interaction chromatography is performed with a solution with an electrical conductivity of greater than 40 mS/cm and less than 105 mS/cm; p0028). Advantageously, such an elution method provides for the purification of a protein from a sample without the need to significantly alter the characteristics of the sample and with minimal adjustment to the chromatography column (p0016). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to elute with a solution having an electrical conductivity greater than 40 mS/cm and less than 105 mS/cm as suggested by BLANK for the method of modified STANEK.
The limitations “so that the protein is bound to the ion exchange resin” in step (i) and “so that the protein is bound to a hydrophobic interaction chromatography support” in step (v) are directed toward inherent properties expected from the practice of the claimed method. Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed. A “whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003); MPEP §2111.04). Where a reference discloses the terms of the recited method steps, and such steps necessarily result in the desired and recited effect, the fact that the reference does not describe the recited effect in haec verba is of no significance because the reference meets the claim under the doctrine of inherency. Here, because the prior art discloses the same claimed protein of interest being loaded onto the same claimed ion exchange resin and HIC support, the instant limitations whereby the protein is bound to the ion exchange resin and the HIC support are necessarily expected.
Regarding Claim 6, modified STANEK makes obvious the method of Claim 5. STANEK further discloses that endotoxins were washed away using solutions containing 10 column volumes of non-ionic detergents Triton X-100 or Triton X-114 (i.e., wherein removing endotoxins is carried out during the washing step (ii) of the ion exchange chromatography; §2.1, par. 1; §4.2). KREMER further discloses serial chromatography with AEX followed by HIC (pg. 4, lines 18-23) and that each chromatography flowthrough entails in-line loading of the protein extract in a buffer A, a washing buffer, and an elution buffer B (i.e., wherein removing endotoxins is carried out during the washing step (ii) of the ion exchange chromatography and during the washing step (vi) of the hydrophobic interaction chromatography; pg. 9, lines 13-20).
Furthermore, one of ordinary skill in the art would inherently expect that the washing step of each chromatography process entails the partial or complete removal of impurities, such as endotoxins. Thus, such limitations are further inherent in the prior art.
Even further, the instant limitations are directed toward an inherent property resulting from the practice of the claimed method. Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed. A “whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003); MPEP §2111.04). Where a reference discloses the terms of the recited method steps, and such steps necessarily result in the desired and recited effect, the fact that the reference does not describe the recited effect in haec verba is of no significance because the reference meets the claim under the doctrine of inherency.
Regarding Claim 15, modified STANEK makes obvious the method of Claim 5. KREMER further discloses a loading of 4.37 g protein in 362 mL volume, i.e., approximately 12 g/L load ratio (pg. 12, lines 32-33). While such a load ratio is outside the instantly claimed range of between 2.5 and 3.5 g/L, one of ordinary skill would find it obvious that protein load ratios can be optimized depending on a number of factors including the specific target protein, the specific column resin, and the desired purification efficiency. Applicant has broadly claimed a generic protein being purified on a generic AEX column with no limitations regarding desired purification. Therefore, because the prior art describes an exemplary chromatography meeting the broadly claimed chromatography, the general conditions of a claim are disclosed or obvious over the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation absent unexpected results or evidence indicating such optimum or workable ranges are critical (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); MPEP§2144.05). Thus, such a claimed load ratio of between 2.5 and 3.5 g/L would be obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Regarding Claim 30, modified STANEK makes obvious the method of Claim 5. STANEK further discloses after AEX treatment of an E. coli culture, the level of endotoxins in the eluting fraction decreased by three to four orders of magnitude to, e.g., 8 EU/mg (Table 1, §2.1, par. spanning pg. 3-4). While this level is above the claimed range of a level of endotoxins in the eluting fraction after the chromatography using an anion exchange resin and/or hydrophobic interaction chromatography is at or below 5 EU/mg, such claimed lower levels can be capably achieved by one of ordinary skill in the art by optimizing or adjusting various factors during purification, e.g., starting endotoxin load, buffer composition, identifying the specific endotoxin, etc. 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 absent unexpected results or evidence indicating such optimum or workable ranges are critical (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); MPEP§2144.05).
Furthermore, the instantly claimed limitation is also directed toward an inherent property resulting from the practice of claimed purification. Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed. A “whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003); MPEP §2111.04). Where a reference discloses the terms of the recited method steps, and such steps necessarily result in the desired and recited effect, the fact that the reference does not describe the recited effect in haec verba is of no significance because the reference meets the claim under the doctrine of inherency. No actual steps are required in these claims; and the prior art discloses or makes obvious all limitations of parent Claim 5. One of ordinary skill in the art in practicing the steps of Claim 5 will necessarily expect similar results as those claimed in Claim 30.
Regarding Claim 34, modified STANEK makes obvious the method of Claim 5. BLANK further discloses sample loading onto a column under a conductivity of about 9.5 to about 14 mS/cm, which overlaps with the claimed range of between 5.0 mS/cm and 10.0 mS/cm and therefore, establishes a case of prima facie obviousness (MPEP 2144.05), and eluting under a conductivity of 80 mS/cm or more (i.e., an electrical conductivity greater than two times that of the loading step; p0032).
Claim(s) 10 and 32 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021), KREMER et al. (WO 2011/098526 A1), and ISLAM et al. (Micromachines, 2017, 8, 83).
Regarding Claim 10, STANEK discloses a method for purifying a protein of interest (e.g., repeat in toxin leukotoxin, RTX) from an E. coli culture (§4.1, par. 2). STANEK discloses the protein of interest is produced in E. coli cultures (i.e., wherein the protein of interest is secreted into the periplasmic space of a Gram-negative bacterium; §4.1, par. 2). Briefly, the proteins are produced in E. coli; the bacterial cells are lysed and cells are lysed in a buffer to collect cell contents containing the protein of interest and contaminants (e.g., lipopolysaccharides and other endotoxins; i.e., step (a) conditioning said protein of interest… in a solution; §4.1; §abstract).
Lysed contents were loaded onto DEAE-Sepharose chromatography resin beds (i.e., wherein the series of purification steps comprises capture chromatography using an anion exchange resin and/or hydrophobic interaction chromatography), and trapped LPS and other endotoxins were washed away using solutions containing 10 column volumes of non-ionic detergents Triton X-100 or Triton X-114 (i.e., wherein the removal of contaminants comprises removing endotoxins and comprises a step of adding a solution comprising [a detergent]; at least two column volumes of the washing solution comprising [the detergent] are applied to said support; §2.1, par. 1; §4.2). Subsequently, the detergents are removed from the resins with washes using 5 bed volumes of buffer without detergent, (i.e., step (b) a series of purification steps to recover said protein of interest in a first fraction to be retained and contaminants in one or more fractions to be removed, said contaminants comprising endotoxins; at least two column volumes of the washing solution with a solution free of [the detergent] are applied before elution; each comprising a loading step, a washing step, and an elution step; §2.1, par. 1; §4.2) and the proteins of interest are collected via a separate eluate wash (i.e., wherein, for the washing step of each chromatography… are applied before elution of said protein of interest; §4.2)
STANEK is deficient in disclosing using polyoxyethylene isooctylcyclohexyl ether.
ANON discloses the use of detergents in purifying nucleic acids of endotoxins by anion exchange chromatography (e.g., a DEAE column; §Background, pg. 1-2). The testing included comparisons of the effectiveness in nucleic acid yield and reduction in endotoxin levels of detergents including Triton X-100, Triton X-114, and reduced Triton X-100 (§Materials and Methods, pg. 6). Diagram 2 (pg. 8) shows negligible differences among these detergents in DNA yield; Diagram 7 (pg. 11) shows similar levels of reduced endotoxin concentrations in the DNA eluates. ANON concludes that Triton X-100 reduced “would be a suitable replacement” for Triton X-100 and Triton X-114 (§Discussion; pg. 12). Thus, given the statistically same results (i.e., predictable results), prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to utilize Triton X-100 reduced (i.e., polyoxyethylene (10) isooctylcyclohexyl ether) as a substitute detergent for Triton X-100 or Triton X-114 as taught by ANON in the method of purifying proteins of interest disclosed by STANEK. The claim would have been obvious to one of ordinary skill in the art because the substitution of one known element for another would have yielded predictable results (MPEP §2143.01 B).
Modified STANEK is deficient in explicitly disclosing a preliminary step of causing an osmotic shock in the Gram-negative bacterium.
However, the use of osmotic shock is a well-known technique for recovering periplasmic proteins from E.coli. For instance, ISLAM discloses osmotic shock as one of the more common physical disruption methods (§3.2.1 “Osmotic Shock”). In the method, cells are lysed by permeabilizing the cell membrane via osmosis due to a sudden change in osmotic pressure. ISLAM specifically identifies that “periplasmic proteins may be released in the case of gram-negative bacteria” and cites references discovering improved protein recovery when compared with other physical methods of lysing. Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to cause an osmotic shock to the Gram-negative bacterium as disclosed by ISLAM for the method made obvious by modified STANEK.
Regarding Claim 32, modified STANEK makes obvious the method of interest of Claim 10. STANEK further discloses after AEX treatment of an E. coli culture, the level of endotoxins in the eluting fraction decreased by three to four orders of magnitude to, e.g., 8 EU/mg (Table 1, §2.1, par. spanning pg. 3-4). While this level is above the claimed range of a level of endotoxins in the eluting fraction after the chromatography using an anion exchange resin and/or hydrophobic interaction chromatography is at or below 5 EU/mg, such claimed lower levels can be capably achieved by one of ordinary skill in the art by optimizing or adjusting various factors during purification, e.g., starting endotoxin load, buffer composition, identifying the specific endotoxin, etc. 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 absent unexpected results or evidence indicating such optimum or workable ranges are critical (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); MPEP§2144.05).
Furthermore, the instantly claimed limitation is also directed toward an inherent property resulting from the practice of claimed purification. Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed. A “whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Id. (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003); MPEP §2111.04). Where a reference discloses the terms of the recited method steps, and such steps necessarily result in the desired and recited effect, the fact that the reference does not describe the recited effect in haec verba is of no significance because the reference meets the claim under the doctrine of inherency. No actual steps are required in these claims; and the prior art discloses or makes obvious all limitations of parent Claim 10. One of ordinary skill in the art in practicing the steps of Claim 10 will necessarily expect similar results as those claimed in Claim 32.
Claim(s) 14, 12, and 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over STANEK et al. (Toxins, 2019, 11, pg. 336-347) in view of ANON (Research Disclosure, 682031, 07 January 2021), KREMER et al. (WO 2011/098526 A1), and BLANK et al. (US 2017/0198008 A1).
Regarding Claim 14, as described in the rejection of Claim 5, STANEK in view of ANON and KREMER disclose or make obvious the claimed steps (i)-(viii). The prior art is deficient in disclosing wherein the elution step of the hydrophobic interaction chromatography is performed with a solution having an electrical conductivity of greater than 40 mS/cm and less than 105 mS/cm.
BLANK discloses a method for purifying a protein using chromatography and high concentration salt buffers (abstract). BLANK discloses such a method is used for hydrophobic interaction chromatography (p0038) and that the elution step is carried out using a very high conductivity buffer of between about 40 mS/cm and 100 mS/cm (p0028). Advantageously, the use of such a high conductivity range precludes any significant alteration of the sample containing the target protein with minimal adjustment to the chromatographic column (p0016). Thus, prior to the effective filing date of the claimed invention, one of ordinary skill in the art would have found it obvious to perform the HIC elution with a buffer having an electrical conductivity of greater than 40 mS/cm and less than 105 mS/cm as taught by BLANK for the method made obvious by modified STANEK.
Regarding Claim 12, modified STANEK makes obvious the method of Claim 14. KREMER further discloses the HIC unit includes hydrophobic ligands include aromatic phenyl groups (i.e., the hydrophobic interaction chromatography includes a support with exposed phenyl groups; pg. 7, lines 24-28).
Regarding Claim 13, modified STANEK makes obvious the method for purifying a protein of interest of Claim 12. BLANK further discloses sample containing the target protein is loaded at a pH of about 8.0 or about 9.0 (i.e., wherein the loading and/or washing step of the hydrophobic interaction chromatography using the support with exposed phenyl groups is performed at a pH between 8 and 9 and/or in the presence of a concentration of salts between 2 and 3M and/or has a load ratio between 0.l g/L and 5 g/L; p0021).
Allowable Subject Matter
Claims 16 and 36 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter: Claim 16 requires that the eluate fraction obtained from the elution step during hydrophobic interaction chromatography be subject to an ion exchange membrane treatment. Such a limitation is distinguished from an ion exchange chromatography treatment. While the prior art of record, e.g., ZHANG, does disclose upstream pretreatment steps prior to AEX, including hydrophobic interaction chromatography, the primary treatment using AEX utilizes chromatographic separation, not the instantly claimed membrane separation. It is acknowledged that ZHANG does disclose the use of Sartobind® Q resins for downstream processing steps, which has been identified by the Applicant to be suitable for AEX membrane treatment; however, there is no teaching explicit or suggested whereby an eluate fraction collected from a HIC process is passed through an ion exchange membrane as claimed.
Further search and consideration did not yield any prior art that discloses or suggests the use of an ion exchange membrane treatment of the eluate fraction obtained from hydrophobic interaction chromatography. One of the closest prior art, NORTON et al. (US 20180334493 A1), suggests the use of a Sartobind Q membrane absorber step as a downstream process to reduce contaminants (e.g., DNA, HCP, viruses) in a pharmaceutical product purified by a POROS XS column (i.e., cation exchange chromatography; p0209); NORTON earlier suggested upstream purification using AEX, CEX, and HIC (p0031) but fails to explicitly disclose treating the eluate of an HIC purification with an ion exchange membrane treatment.
Claim 36 requires a loading step with a solution comprising between 2 M and 3 M salts on a hydrophobic support. None of the prior art referenced teaches or suggests a salt concentration of between 2 M and 3 M.
Conclusion
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/Ryan B Huang/Primary Examiner, Art Unit 1777
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