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
Claims 1-20 are pending and under consideration in this action.
Priority
The instant claims are entitled to an effective filing date of 03/31/2022.
Information Disclosure Statement
The listing of references in paragraph [0076] of the specification filed on 03/31/2023 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The use of the terms Qiagen (twice in paragraph [0030]), Cytiva (in paragraph [0035]) and Tris (three times in paragraph [0048]) which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 1, 6, 13 and 15 are objected to because of the following informalities:
Claims 1 and 13 recite “-“, which is either a strikethrough indicative of a claim amendment or an em dash. See line 11 of claim 1 and line 3 of claim 13. If the dash intends to indicate an amendment, then claims 1 and 13 should include the status identifier (Currently Amended). If the dash is an em dash, then it should be deleted because the wherein clause is already introduced with a comma, such that an em dash is unnecessary.
Claims 6 and 15 recite “Fast Protein Liquid Chromatography”, which should be lowercased to “fast protein liquid chromatography” because it is not a proper noun.
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 1-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 step (e), claim 6, and claim 10 step (e) all recite “at least a portion of the resin mixture”, which renders these claims indefinite because there are multiple reasonable interpretations for this limitation. In the first reasonable interpretation, “at least a portion of the resin mixture” in part (e) references at least a portion of the resin mixture formed in part (c). In the second reasonable interpretation, “at least a portion of the resin mixture” in part (e) references the portion of the resin mixture that is introduced into the at least one purification receptacle in part (d) before it goes through the purification receptacle; such that step (e) requires the portion of resin mixture to travel through the purification receptacle of part (d) to thereby form the purification protein amplification substrate. In the third reasonable interpretation, “at least a portion of the resin mixture” in part (e) references the portion of the resin mixture that has traveled through the at least one purification receptacle of step (d).
Dependent claims 2-9 and 11-16 are rejected for the same reason as above.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claims 1, 7, 10 and 16-18 recite the broad recitation “protein”, and the claims also recite “prion protein” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claims 2-6, 8-9, 11-15 and 19-20 depend from either claim 1, 10 or 17 and are rejected for the reason set forth above.
Claim 11 recites “the protein amplification substrate” in line 1. There is insufficient antecedent basis for this limitation in the claim because it is unclear whether “the protein amplification substrate” intends to refer to the purified protein amplification substrate of part (e) of claim 10, or the prion protein amplification substrate of part (f) of claim 10.
Claim Interpretation
Claim 1 requires 5 active method steps (a-e) in order to extract and purify a prion protein amplification substrate for the non-limiting intended purpose of being used for RT-QuIC testing. As such, the protein aggregate recited throughout the claim is reasonably interpreted as being a prior protein (PrP) aggregate. In part (b), the denaturant buffer encompasses TRIS, SDS, urea, guanidine hydrochloride or a combination thereof. See [0023] of the instant specification. In part (c), the protein affinity resin can comprise Qiagen™ ni-nitrilotriacetic acid (NTA) superflux resin. See [0030] of the instant specification. Part (d) requires introducing at least a portion of the resin mixture into at least one purification receptacle, e.g. column(s). The dimensions of each purification receptacle depend on the portion of the resin mixture introduced into it. If the introduced portion of resin mixture includes less than 30 mL (<30mL) of solubilized protein aggregate, then the inner diameter of the purification receptacle is required to be less than 25 mm and the ratio of the length to the inner diameter is required to be at least 27:1. If the introduced portion of resin mixture includes at least 30 mL (≥30mL) of solubilized protein aggregate, then the inner diameter of the purification receptacle is required to be at least 25 mm and the ratio of the length to the inner diameter of the purification receptacle is required to be less than 27:1. See paragraph [0038] of the instant specification for the definition of “maximum transverse interior dimension” and “maximum longitudinal interior dimension”.
In claims 4-5, “the resin mixture” is interpreted as the formed resin mixture of part (c) of claim 1.
Claim 6 requires subjecting a resin mixture to FPLC, for the intended result of forming the eluted protein fraction and spent affinity resin. Eluted protein and spent affinity resin are considered inherent to the FPLC step, because, for example, the specification discloses that subjecting the resin mixture to FPLC thereby forms an eluted protein fraction and spent affinity resin. See [0047].
Claim 7 requires treating at least a portion of the eluted protein fraction with a dialysis buffer. However, mode of treatment is not limited. The specification teaches diluting the eluted protein fraction with dialysis buffer, and the specification teaches a dialysis treatment in dialysis tubing. See [0050] and [0057].
Claim 17 recites “contacting”, which encompasses indirect and direct contact. For example, part (f) requires contacting at least a portion of the eluted protein fraction with at least 2.5 mL of dialysis buffer. This contact step encompasses mixing at least a portion of the eluted protein fraction with at least 2.5 mL of dialysis buffer or contacting via dialysis tubing at least a portion of the eluted protein fraction with at least 2.5 mL.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-17 and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Minikel (Recombinant prion protein, Rocky Mountain-style, last updated 2019-04-10, Cureffi) in view of Orrù, (Prions: methods and protocols, 2017, (pp. 185-203)), with evidence from GE Healthcare Life Sciences (2006)(hereafter GE Healthcare™).
Regarding claim 1, Minikel teaches preparing recombinant prion protein (PrP) substrate for an RT-QuIC assay. See the first paragraph on page 1. Minikel teaches expressing prion protein (PrP) in E. coli. See the third paragraph on page 1. Minikel teaches pouring a 1L culture into four 250 mL conical tubes and centrifuging them to arrive at a bacterial solid pellet [one for each 250 mL portion]. See the first paragraph on page 7. The pellet is transferred to a 50 mL tube, and ground up with a lysis buffer. See page 8. From the mostly lysed cells, PrP aggregates pellet out (relevant to instant part (a)). See the first two paragraphs on page 10. Minikel teaches purifying PrP protein from 750 mL worth of bacterial culture. See the first paragraph on page 16. For the three pellets [one PrP pellet per 250 mL bacterial culture], Minikel teaches mixing each with 14 mL of an 8M guanidine hydrochloride (GdnHCl) solution (e.g. denaturant), grinding them up on a homogenizer and setting them on a rotisserie for 50 minutes. However, a note added 2019-04-10 suggests that for best yields PrP can be left in 8M GdnHCl at 4˚C for at least one week. See the paragraph spanning pages 14-15. Thus, Minikel suggests that accumulatively the three PrP pellets are contacted with a total of 42 mL of 8M guanidine (3 pellets x 14 mL), which is at least 20 mL as required by the instant claim 1. Minikel teaches pouring denaturing buffer over 18g of semi-dry Ni-NTA beads to 30 mL. See page 16. Minikel teaches pouring the supernatant [i.e. the 14 mL homogenized PrP pellet+GdnHCl] into the tubes with the [30 ml] Ni-NTA (e.g. protein affinity resin) (relevant to instant part (c)). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of the column (e.g. purification receptacle). See the first two paragraphs on page 18. Since Minikel teaches combining the three tubes in the column, Minikel teaches a mixture of 42 mL of homogenized PrP in GdnHCl and 90mL Ni-NTA resin. Minikel teaches running fast protein liquid chromatography (FPLC). See the second paragraph on page 19 and the image on page 21 for the PrP purification curve. Minikel discloses that the protocol usually gives a PrP concentration in the 0.4-0.6 mg/mL range (e.g. purified protein amplification substrate) (relevant to instant part (e)). See the fifth paragraph on page 23.
Minikel does not teach contacting at least a portion of the protein aggregate with at least one protein denaturant for at least one hour (relevant to instant part (b)), because Minikel explicitly teaches setting the PrP and GdnHCl mixture on the rotisserie for 50 minutes, however, Minikel also suggests leaving PrP in the GdnHCl for at least one week.
Minikel is silent regarding the maximum transverse interior dimension and the maximum longitudinal interior dimension of the purification receptacle. As such, Minikel does not teach (i) the maximum transverse interior dimension is less than 25 mm and the ratio of the maximum longitudinal interior dimension to the maximum transverse interior dimension is at least 27:1, when the resin mixture introduced into the purification receptacle comprises less than 30 mL of the solubilized protein aggregate, and (ii) the maximum transverse interior dimension is at least 25 mm and the ratio of the maximum longitudinal interior dimension to the maximum transverse interior dimension is less than 27:1, when the resin mixture introduced into the purification receptacle comprises at least 30 mL of the solubilized protein aggregate (relevant to instant part (d)).
Orrù teaches RT-QuIC protocols as well as factors that can affect the implementation, improvement and adaption of RT-QuIC assays to various prion strains and specimens. See the first paragraph on page 187. Orrù teaches splitting a 1L culture in 4x250mL portions. See section 3.2.1 number 7. To an obtained inclusion body pellet, Orrù teaches adding 14 mL of 8M GdnHCl and homogenizing it. See section 3.2.2 and section 3.2.3 numbers 3-4. Orrù teaches bringing the volume of 18g of Nickel-NTA bead slurry to 30 mL. The supernatant [of the homogenized inclusion body pellet +GdnHCl] is transferred to the tube with the Nickel-NTA and incubated to allow protein binding to the beads. Then, Orrù teaches loading the protein-bound beads into a column. See numbers 6-12 of section 3.2.3 on page 193. Orrù teaches using XK16/40 for 1X preparation or XK 26/20 for a 3X preparation. See number 7 of section 2.2.3.
Evidentiary refence GE Healthcare™ teaches a XK 26/20 column and indicates that the inner diameter (i.d.) of the column is 26 mm and the length is 20 cm or 200mm. See the table and the “Chromatographic tube” section on page 1. Thus, the XK 26/20 column has an i.d. that is at least 25 mm, and has a length to i.d. ratio of 7.7:1 (i.e. 200:26mm or 20:1.6cm), which is less than the instantly claimed 27:1 ratio.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the 50 min rotisserie step of Minikel with leaving the PrP in 8M GdnHCl for one week as taught by Minikel, and to further use the XK 26/20 column of Orrù as the column in the preparation method of Minikel (relevant to parts (b) and (d)(ii) of instant claim 1). Doing so is mere substitution of one known protein purification column for another. One would be motivated to leave the PrP in 8M GdnHCl for one week because Minikel suggests that it may result in better yields, as compared to the 50 min rotisserie step. There would be a reasonable expectation of success because Minikel references other published papers that specifically call for leaving PrP in guanidine for two or more days. See the first paragraph on page 15 of Minikel. One would be further motivated to use the XK 26/20 column of Orrù because Orrù suggests that the XK 26/20 column is appropriate for 3X preparations. There would be a reasonable expectation of success because Orrù suggests that the XK 26/20 column can be used for preparations with the same volume taught by Minikel, i.e. 3X(14mL PrP and GdnHCl + 30 mL of Ni-NTA resin).
Regarding claim 2, Minikel discloses that the protocol usually gives a PrP concentration in the 0.4-0.6 mg/mL range, which overlaps with the instantly claimed 0.2 to 0.45 mg/mL range. See the fifth paragraph on page 23.
Orrù teaches that the protein concentration of the final purification product is typically around 0.4-0.6 mg/ml. See note 25 on page 199.
Regarding claim 3, Minikel teaches pouring the supernatant [i.e. the 14 mL homogenized PrP pellet+GdnHCl] into the tubes with the [30 ml] Ni-NTA (e.g. protein affinity resin). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of the column (e.g. purification receptacle). See the first two paragraphs on page 18.
Orrù teaches loading the protein-bound beads into a column (e.g. purification receptacle). See number 12 of section 3.2.3. Orrù teaches using XK16/40 for 1X preparation or XK 26/20 for a 3X preparation. See number 7 of section 2.2.3.
Regarding claims 4-5, Minikel teaches pouring denaturing buffer over 18g of semi-dry Ni-NTA beads to bring the volume to 30 mL. See page 16. Minikel teaches pouring the supernatant [i.e. the 14 mL homogenized PrP pellet+GdnHCl] into the tubes with the [30 ml] Ni-NTA (e.g. protein affinity resin). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of the column (e.g. purification receptacle). See the first two paragraphs on page 18. As such, Minikel teaches a mixture of 42 mL of homogenized PrP in GdnHCl and 90mL Ni-NTA resin.
Regarding claim 6, Minikel teaches running FPLC. See the second paragraph on page 19. Minikel teaches the FPLC curve from the PrP purification. See the image on page 21.
Orrù teaches loading the protein-bound beads into a column. Orrù teaches attaching the column to AKTA FPLC and running a gradient. See numbers 12-13 of section 3.2.3.
Regarding claim 7, Minikel teaches collecting rPrP fractions as they elute. See the image on page 21. Minikel teaches pouring the PrP solution into dialysis tubing, clipping it shut and setting it to float in 3.6L of dialysis buffer (e.g. treatment). See the last paragraph and the image on page 22.
Orrù teaches pooling elution fractions, pouring the pooled protein into dialysis tubing and dialyzing in 3.6 L of prechilled dialysis buffer overnight. See numbers 17-19 of section 3.2.3 on page 194.
Regarding claim 8, Minikel teaches collecting rPrP fractions as they elute. See the image on page 21. Minikel teaches pouring the PrP solution into dialysis tubing, clipping it shut and setting it to float in 3.6L of dialysis buffer. See the last paragraph and the image on page 22. Thus, Minikel teaches adding 3.6 L dialysis buffer, which is at least 2.5 mL as required, to at least a portion of the eluted protein fraction.
Orrù teaches pouring pooled protein into dialysis tubing and dialyzing in 3.6 L dialysis buffer overnight. See number 19 on page 194.
Minikel and Orrù both teach adding dialysis buffer to at least a portion of the eluted protein fraction.
Regarding claim 9, Minikel teaches dividing rPrP [after dialysis] into 1 mL aliquots to be frozen at -80˚C. See the last paragraph on page 24.
Minikel does not teach storing at least a portion of the dialyzed protein fraction at a temperature greater than 0˚C and less than 10˚C for at least one hour.
Orrù teaches filtering [protein] after dialysis. Following filtration, Orrù teaches making 1 ml aliquots of the protein and immediately storing at -80˚C. See numbers 21-24 of section 3.2.3 on page 195. Orrù suggests that while freezing and eventually thawing rPrPsen may result in some level of protein precipitation, the practice provides consistent results from one batch to the next over an extended period of time, months and even years. Alternatively, storing a batch of protein at 4˚C for weeks or months (e.g. at least one hour) may result in loss of rPrPsen due to precipitation or degradation. See note 26 on page 199.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the 4˚C storage technique of Orrù to the 1 mL aliquots of Minikel. One would be motivated to do so because Orrù suggests that freezing and eventually thawing may result in some level of protein precipitation. There would be a reasonable expectation of success because Orrù suggests that aliquots can be stored at 4˚C for weeks or months (e.g. at least one hour) as an alternative to freezing 1 mL aliquots at -80˚C.
Regarding claim 10, Minikel teaches preparing recombinant PrP substrate for an RT-QuIC assay. See the first paragraph on page 1. Minikel teaches expressing prion protein (PrP) in E. coli. See the third paragraph on page 1. Minikel teaches obtaining PrP aggregate pellets from lysed cells (relevant to instant part (a)). See the first two paragraphs on page 10. Minikel teaches mixing each pellet with 14 mL of an 8M GdnHCl solution (e.g. denaturant), grinding them up on a homogenizer and setting them on a rotisserie for 50 minutes. However a note added 2019-04-10 suggests that for best yields PrP can be left in 8M GdnHCl at 4˚C for at least one week, which is at least eight hours as instantly claimed. See the paragraph spanning pages 14-15. Minikel teaches pouring the supernatant [i.e. PrP pellet+GdnHCl] into the tubes with the Ni-NTA (e.g. protein affinity resin) (relevant to instant part (c)). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of a column (e.g. purification receptacle) (relevant to instant part (d)). See the first two paragraphs on page 18. Minikel teaches running FPLC. See page 21. Minikel teaches collecting fractions from the FPLC and filtering them. Minikel suggests that these fractions make for a substrate that is most sensitive for detecting prions in RT-QuIC. See the paragraph spanning pages 21-22. However, Minikel suggests that that the imidazole still needs to be removed from the PrP. So Minikel teaches pouring the PrP in dialysis tubing and floating it in dialysis buffer. See page 22. Minikel discloses that the protocol typically gives results in the 0.4-0.6 mg/mL range (e.g. purified PrP protein amplification substrate) (relevant to instant part (e)). See page 22 and paragraphs 1 and 4 on page 23. Minikel teaches dividing rPrP [after dialysis] into 1 mL aliquots to be frozen at -80˚C. See the last paragraph on page 24.
Minikel does not teach contacting at least a portion of the protein aggregate with at least one protein denaturant for at least eight hours (relevant to instant part (b)) in the main scheme, because Minikel explicitly teaches setting the PrP and GdnHCl mixture on the rotisserie for 50 minutes, however Minikel also suggests leaving PrP in the GdnHCl for at least one week.
Minikel does not teach storing at least a portion of the dialyzed protein fraction at a temperature greater than 0˚C and less than 10˚C for at least one hour (relevant to instant part (f)).
Orrù teaches filtering protein after dialysis. Following filtration, Orrù teaches making 1 ml aliquots of the protein and immediately storing at -80˚C. See numbers 21-24 of section 3.2.3 on page 195. Orrù suggests that while freezing and eventually thawing rPrPsen may result in some level of protein precipitation, the practice provides consistent results from one batch to the next over an extended period of time, months and even years. Alternatively, storing a batch of protein at 4˚C for weeks or months (e.g. at least one hour) may result in loss of rPrPsen due to precipitation or degradation. See note 26 on page 199.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the 50 min rotisserie step of Minikel with leaving the PrP in 8M GdnHCl for one week as taught by Minikel, and to further apply the 4˚C storage technique of Orrù to the 1 mL aliquots of Minikel. One would be motivated to leave the PrP in 8M GdnHCl for one week as taught by Minikel because Minikel suggests that doing so may result in better yields, as compared to 50 min rotisserie step. There would be a reasonable expectation of success because Minikel references other published papers that specifically call for leaving PrP in guanidine for two or more days. See the first paragraph on page 15 of Minikel. One of ordinary skill in the art would be further motivated to apply the 4˚C storage technique of Orrù because Orrù suggests that freezing and eventually thawing may result in some level of protein precipitation. There would be a reasonable expectation of success because Orrù suggests that aliquots can be stored at 4˚C for weeks or months (e.g. at least one hour) as an alternative to freezing 1 mL aliquots at -80˚C.
Regarding claim 11, Minikel discloses that the protocol usually gives a PrP concentration in the 0.4-0.6 mg/mL range, which overlaps with the instantly claimed 0.2 to 0.45 mg/ml. See the fifth paragraph on page 23.
Orrù teaches that the protein concentration of the final purification product is typically around 0.4-0.6 mg/ml. See note 25 on page 199.
Regarding claim 12, Minikel teaches pouring the contents of the three tubes into the top of a column (e.g. purification receptacle). See the first two paragraphs on page 18.
Orrù teaches loading the protein-bound beads into a column (e.g. purification receptacle). See number 12 of section 3.2.3. Orrù teaches using XK16/40 for 1X preparation or XK 26/20 for a 3X preparation. See number 7 of section 2.2.3.
Regarding claim 13, Minikel teaches pouring the supernatant [i.e. the 14 mL homogenized PrP pellet+GdnHCl] into the tubes with the [30 ml] Ni-NTA (e.g. protein affinity resin). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of the column (e.g. purification receptacle). See the first two paragraphs on page 18. Since Minikel teaches combining the three tubes in the column, Minikel teaches a mixture of 42 mL of homogenized PrP in GdnHCl, which is at least 30 mL.
Minikel is silent regarding the maximum transverse interior dimension and the maximum longitudinal interior dimension of the purification receptacle. As such, Minikel does not teach (i) the maximum transverse interior dimension is less than 25 mm and the ratio of the maximum longitudinal interior dimension to the maximum transverse interior dimension is at least 27:1, when the resin mixture introduced into the purification receptacle comprises less than 30 mL of the solubilized protein aggregate, and (ii) the maximum transverse interior dimension is at least 25 mm and the ratio of the maximum longitudinal interior dimension to the maximum transverse interior dimension is less than 27:1, when the resin mixture introduced into the purification receptacle comprises at least 30 mL of the solubilized protein aggregate.
Orrù teaches using XK16/40 for 1X preparation or XK 26/20 for a 3X preparation. See number 7 of section 2.2.3. Evidentiary reference GE Healthcare™ discloses that the XK 26/20 column has an i.d. that is at least 25 mm, and has a length to interior diameter ratio of 7.7:1 (i.e. 200:26mm or 20:1.6cm). See the table and the “Chromatographic tube” section on page 1.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to use the XK 26/20 column of Orrù as the column in the preparation method of Minikel (relevant to instant part (ii)). Doing so is mere substitution of one known protein purification column for another. One would be motivated to use the XK 26/20 column of Orrù because Orrù suggests that the XK 26/20 column is appropriate for 3X preparations. There would be a reasonable expectation of success because Orrù teaches using the XK 26/20 column to hold the same volume taught by Minikel, i.e. 3X(14mL PrP&GdnHCl + 30 mL of Ni-NTA resin).
Regarding claim 14, Minikel teaches mixing each of three PrP pellets with 14 mL of an 8M guanidine hydrochloride (GdnHCl) solution (e.g. denaturant), grinding them up on a homogenizer. See the paragraph spanning pages 14-15. Minikel teaches pouring denaturing buffer over 18g of semi-dry Ni-NTA beads to bring the volume to 30 mL. See page 16. Minikel teaches pouring the supernatant [i.e. the 14 mL homogenized PrP pellet+GdnHCl] into the tubes with the [30 ml] Ni-NTA (e.g. protein affinity resin). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of a column. See the first two paragraphs on page 18. Thus, Minikel teaches mixture that comprises 90 mL of Ni-NTA resin and 42 mL of the homogenized PrP+GdnHCl, which meets the instantly claimed at least 15 mL of protein affinity resin and at least 20 mL of solubilized protein aggregate limitation.
Regarding claim 15, Minikel teaches pouring the contents of the three tubes into the top of a column. See the first two paragraphs on page 18. Minikel teaches placing the column onto the machine and running FPLC. See page 21. Minikel discloses that PrP elutes from the column. Minikel teaches collecting fractions. See page 21.
Regarding claim 16, Minikel teaches collecting rPrP fractions as they elute. See the image on page 21. Minikel teaches pouring the PrP solution into dialysis tubing, clipping it shut and setting it to float in 3.6L of dialysis buffer. See the last paragraph and the image on page 22. Thus, Minikel teaches treating at least a portion of the eluted PrP with dialysis buffer, specifically the eluted PrP in the dialysis tubing is added to the 3.6 L of dialysis buffer; and 3.6 L is at least 2.5 mL as instantly claimed.
Orrù teaches pouring pooled protein into dialysis tubing and dialyzing in 3.6 L dialysis buffer overnight. See number 19 on page 194.
Minikel and Orrù both teach adding dialysis buffer to at least a portion of the eluted protein fraction.
Regarding claim 17, Minikel teaches preparing recombinant PrP substrate for an RT-QuIC assay. See the first paragraph on page 1. Minikel teaches expressing prion protein (PrP) in E. coli. See the third paragraph on page 1. Minikel teaches obtaining PrP aggregate pellets from the lysed cells (relevant to instant part (a)). See the first two paragraphs on page 10. Minikel teaches mixing each pellet with 14 mL of an 8M GdnHCl solution (e.g. denaturant), grinding them up on a homogenizer and setting them on a rotisserie for 50 minutes. However, a note added 2019-04-10 suggests that for best yields PrP can be left in 8M GdnHCl at 4˚C for at least one week, which is at least eight hours as instantly claimed. See the paragraph spanning pages 14-15. Minikel teaches pouring the supernatant [i.e. PrP pellet+GdnHCl] into the tubes with the Ni-NTA (relevant to instant part (c)). See the first passage on page 18. Minikel teaches pouring the contents of the three tubes into the top of a column (relevant to instant part (d)). See the first two paragraphs on page 18. Minikel teaches running FPLC. See page 21. Minikel teaches collecting fractions from the FPLC and filtering them (relevant to instant part (e)). Minikel suggests that these fractions make for a substrate that is most sensitive for detecting prions in RT-QuIC (e.g. purified protein amplification substrate). See the paragraph spanning pages 21-22. However, Minikel suggests that that the imidazole still needs to be removed from the PrP. So Minikel teaches pouring the PrP in dialysis tubing and floating it in 3.6 L dialysis buffer at 4˚C overnight (relevant to instant part (f)). See page 22 and the first passage on page 23. Minikel teaches dividing rPrP [after dialysis] into 1 mL aliquots to be frozen at -80˚C. See the last paragraph on page 24.
Minikel does not teach contacting at least a portion of the protein aggregate with at least one protein denaturant for at least nine hours (relevant to instant part (b)) ipsis verbis, however Minikel explicitly teaches setting the PrP and GdnHCl mixture on the rotisserie for 50 minutes, and Minikel also suggests leaving PrP in the GdnHCl for at least one week.
Minikel does not teach storing at least a portion of the dialyzed protein fraction at a temperature of at least 1˚C and less than 10˚C for at least one hour (relevant to instant part (g)).
Orrù teaches filtering [protein] following dialysis. Following filtration, Orrù teaches making 1 ml aliquots of the protein and immediately storing at -80˚C. See numbers 21-24 of section 3.2.3 on page 195. Orrù suggests that while freezing and eventually thawing rPrPsen may result in some level of protein precipitation, the practice provides consistent results from one batch to the next over an extended period of time, months and even years. Alternatively, storing a batch of protein at 4˚C for weeks or months (e.g. at least one hour) may result in loss of rPrPsen due to precipitation or degradation. See note 26 on page 199.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the 50 min rotisserie step of Minikel with leaving the PrP in 8M GdnHCl for one week as taught by Minikel, and to further apply the 4˚C storage technique of Orrù to the 1 mL aliquots of Minikel. One would be motivated to do so because Minikel suggests that doing so may result in better yields, as compared to 50 min rotisserie step. There would be a reasonable expectation of success because Minikel references other published papers that specifically call for leaving PrP in guanidine for two or more days. See the first paragraph on page 15 of Minikel. One would be further motivated to apply the 4˚C storage technique of Orrù because Orrù suggests that freezing and eventually thawing may result in some level of protein precipitation. There would be a reasonable expectation of success because Orrù suggests that aliquots can be stored at 4˚C for weeks or months as an alternative to freezing 1 mL aliquots at -80˚C.
Regarding claim 19, Minikel teaches collecting rPrP fractions as they elute. See the image on page 21. Minikel teaches pouring the PrP solution into dialysis tubing, clipping it shut and setting it to float in 3.6L of dialysis buffer. See the last paragraph and the image on page 22. Thus, Minikel indicates that the eluted PrP in the dialysis tubing is added to the 3.6 L of dialysis buffer; and 3.6 L is at least 3.0 mL as instantly claimed.
Orrù teaches pouring pooled protein into dialysis tubing and dialyzing in 3.6 L dialysis buffer overnight. See number 19 on page 194.
Minikel and Orrù both teach adding dialysis buffer to at least a portion of the eluted protein fraction.
Regarding claim 20, Minikel teaches mixing each of three PrP pellets with 14 mL of an 8M GdnHCl solution, and grinding them up on a homogenizer. See the paragraph spanning pages 14-15. Minikel teaches pouring denaturing buffer over 18g of semi-dry Ni-NTA beads to 30 mL. See page 16. Minikel teaches pouring the supernatant [i.e. the 14 mL homogenized PrP pellet+GdnHCl] into the tubes with the [30 ml] Ni-NTA (e.g. protein affinity resin). See the first passage on page 18.
Orrù teaches adding 14ml of 8M GdnHCl to an inclusion body pellet and homogenizing it [to arrive at a solubilized inclusion body pellet]. Orrù teaches weighing 18 grams of Nickel-NTA beads and bringing it up to a volume of 30 ml. Orrù teaches spinning the solubilized inclusion body pellet and transferring the supernatant to the tube containing the Nickel-NTA equilibrated beads. See numbers 3-91 of section 3.2.3 on page 193.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine the Ni-NTA resin with the solubilized PrP in any order. MPEP 2144.04(IV)(C) which states that “selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results”, and “Selection of any order of mixing ingredients is prima facie obvious”.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Minikel (Recombinant prion protein, Rocky Mountain-style, last updated 2019-04-10, Cureffi) and Orrù, (Prions: methods and protocols, 2017, (pp. 185-203)), as applied to claims 1-17 and 19-20 above, and further in view of Shi (Acta neuropathologica communications, 2013, 1(1), 44).
The teachings of Minikel and Orrù with respect to instant claim 17 are discussed above.
Regarding claim 18, Minikel teaches pouring the PrP in dialysis tubing and floating it in 3.6 L dialysis buffer overnight. See page 22-23. Minikel discloses that the protocol typically gives results in the 0.4-0.6 mg/mL range. See paragraph 5 on page 23. Minikel teaches dividing rPrP [after dialysis] into 1 mL aliquots to be frozen at -80˚C. See the last paragraph on page 24.
Orrù teaches that the protein concentration of the final purification product is typically around 0.4-0.6 mg/ml following dialysis. Orrù suggests not storing rPrPsen at concentrations higher than 0.8 mg/ml since it may cause higher rates of protein precipitation at such high concentrations. Orrù teaches freezing 0.5-1 ml aliquots of ~0.5 mg/ml rPrPSen. See notes 25-26 on page 199.
Minikel and Orrù do not teach diluting at least a portion of the dialyzed prion protein fraction to a protein concentration of 0.3 to 0.5 mg/mL.
Shi teaches loading rPrPsen into dialysis tubing and dialyzing. The dialyzed solutions are sterilized and the concentration of the rPrPsen is calculated. The concentration of each fraction is adjusted to 0.5 mg/ml by adding (e.g. diluting) sterilized and chilled dialysis buffer. The rPrPsen solution is aliquoted and frozen, followed by a transfer to a freezer for long-term storage. See the paragraph spanning the left and right column on page 7.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the dialysis buffer addition step of Shi to the dialyzed PrP protein of Minikel in the method of Minikel and Orrù. One would be motivated to add sterilized and chilled dialysis buffer as taught by Shi before storage, because Orrù suggests that higher concentration may cause higher rates of protein precipitation. There would be a reasonable expectation of success because Minikel and Orrù teach yielding a protein concentration between 0.4-0.6 mg/ml, which overlaps with the instantly claimed 0.3 to 0.5 mg/mL range, and Shi demonstrates adding dialysis buffer to rPrPsen to adjust the concentration to 0.5 mg/ml before storage.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657