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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 05/28/2026 has been entered.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1, 7, 9-13, 16-19, and 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huber (Journal of Chromatography A, 806 (1998) 3–30), in view of Weisburg et al. (US 2009/0048439A1) and Issa et al. (US 2020/0239869A1).
Regarding claims 1, 5 and 27, Huber teaches a method for separating a sample comprising a nucleic acid, the method comprising (abstract, paragraph 2.2):
(a) loading a sample comprising a nucleic acid onto an anion-exchange column (Refer paragraph 2.2 on page 6); and
(b) eluting the sample from the anion-exchange column using a salt gradient (refer paragraph 2.2 disclosing mobile phase comprising electrolyte as eluting salt and a linear gradient of increasing salt concentration is usually applied for elution”), wherein a mobile phase used in the eluting step comprises a mild ion-pairing cation (refer fig. 14 and table 3 disclosing tetramethylammonium chloride), and wherein a column temperature of the anion-exchange column is between ambient to 60 °C (refer paragraph 3.4 disc). In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). Huber discloses that an increase in temperature from ambient to 60°C improved resolution of DNA fragments (refer paragraph 3.4). Huber also discloses that resolution decreased significantly at 45 and 60°C (refer paragraph 3.4). Discovering optimum temperature range would have been obvious to one of ordinary skill in the art since Huber establishes that temperature is a result effective variable. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding the limitation “wherein the salt gradient is produced by a non-ion-pairing salt”, Weisburg teaches isolation of nucleic acid using a combination of NaCl and tetramethylammonium or tetraethylammoniam (refer [0124]). Weisburg further discloses that the addition of tetramethylammonium or tetraethylammoniam provides a function to equalize hydrogen bonding strength of G-C and A-T pairs, thereby reducing the hybridization of contaminating rRNA and mRNA that might normally display advantageous G-C hybridization (refer [0124]). It would have been obvious to one of ordinary skill in the art before the effective filing date of invention to modify the mobile phase to include a combination of NaCl and tetramethylammonium or tetraethylammoniam to equalize hydrogen bonding strength as taught by Weisburg.
Huber discloses that typical eluent concentration is 20 mM or 0.1 M (refer table 3), and discloses 0.5-1.5 M tetramethylammonium chloride (refer table 3).
Modified Huber does not disclose that a concentration of the mild ion-pairing cation remains constant throughout the eluting step.
Issa teaches chromatography method for separating nucleic acid from a complex mixture by using multiple ion pairing agents in the same mobile phase system (refer abstract). Issa discloses that one or more ion pair is a cation selected from a group consisting of: trimethylamine or a salt thereof (e.g., triethylammonium salt), tetrabutylamine or a salt thereof (e.g., tetrabutylammonium salt), hexylamine or a salt thereof (e.g., hexylammonium salt), and dibutylamine or a salt thereof (e.g., dibutylammonium salt)l and one or more ion pairing agent is an inorganic molecule, for example unsubstituted ammonium salts (NH4+ ), lithium
(Li), sodium (Na), potassium (K), etc. (refer [0012], [0014]). Issa further idslcoses that the concentration of each ion pair (e.g., ion pairing agent) in a mobile phase is held constant during elution of the nucleic acid. In some embodiments, the concentration of one or more ion pair (e.g., ion pairing agent) in the mobile phase is not held constant during elution of the nucleic acid (refer [0016]). Refer [0030] also disclosing “In some embodiments of methods described by the disclosure, the eluting step is isocratic or gradient with respect to the concentration of one or more ion pairs (e.g., concentration of one or more ion pairing agents)”. Refer [0061] disclosing “Resolution has been further improved through the novel elution strategy of an ion pair gradient, where the ion pairing agent ratio is manipulated at increasing, constant, or decreasing organic composition to provide high resolution separations. The combination of both classes of ion pairing agents in a single system enhances selectivity based on polynucleotide size and composition where the concentrations and ratios of the ion pairs to each other can be tuned to maximize resolution in specific applications.”
It would have been obvious to one of ordinary skill in the art before the effective filing date of invention to modify the method of modified Huber wherein a concentration of the mild ion-pairing cation remains constant throughout the eluting step to optimize resolution in specific applications as taught by Issa.
Regarding claim 7, modified Huber teaches limitations of claim 1 as set forth above. Applying the method of Huber for sample comprising RNA would have been obvious to one of ordinary skill in the art because Huber discloses that such is known in the art (refer page 26).
Regarding claims 9-11, modified Huber teaches limitations of claim 1 as set forth above. Huber teaches two mobile phases (Refer fig. 3 indicating mobile phase A and B) wherein buffering agent being Tris and pH is adjusted to between 7.5 and 8.5 with hydrochloric acid or boric acid (refer paragraph 3.2) suggesting a basic buffering agent.
Regarding claims 12-13, modified Huber teaches limitations of claim 1 as set forth above. Huber teaches that the mild ion-pairing cation is tetramethylammonium (Refer table 3).
Regarding claim 16-17, modified Huber teaches limitations of claim 1 as set forth above. Huber teaches two mobile phases having pH of 9.0 (Refer fig. 3 indicating mobile phase A and B)
Regarding claims 18-19, modified Huber teaches limitations of claim 1 as set forth above. Huber teaches the method uses two mobile phases comprise mild pairing cation (Refer fig. 3 disclosing sodium chloride; paragraph 3.2 disclosing sodium chloride, potassium chloride, and sodium perchlorate being most commonly used gradient formers; table 3 disclosing tetramethylammonium chloride; paragraph 4.2 disclosing tetraethylammonium acetate). Selection of the mild pairing cations would have been obvious to one of ordinary skill in the art from the known mild pairing cations.
Response to Arguments
Applicant's arguments filed 05/28/2026 have been fully considered but they are not persuasive.
Regarding rejection of claim 1, applicant argued that Huber and Weisburg fail to teach or suggest “a concentration of the mild ion-pairing cation remains constant throughout the eluting step”, however, the limitation is taught by Issa et al. (US 2020/0239869A1). Refer claim rejections above.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Son et. al. (US 2017/0022248A1) teaches performing elution while maintaining salt concentration at a constant level to maintain the polymer content of the immunoglobulin at a low level (Refer abstract).
Patra et al. (US 2015/0065689A1) teaches inding the protein preparation to a strong ion exchange support, and enriching said highly sialylated variants of the protein by using pH gradient elution at a constant salt concentration (Refer [0010]).
Laursen et al. (US 2001/0051708A1) teaches elution can also be performed by step gradient elution. It is contemplated that the elution could also be performed as a constant salt elution, in which the elution buffer applied to the cation exchange column has only one single salt concentration in contrast to the gradient elution (Refer [0059]).
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/PRANAV N PATEL/Primary Examiner, Art Unit 1777
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