DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group I in the reply filed on 6/18/2026 is acknowledged. The traversal is on the ground(s) that “the examiner's statement that "each group of invention involves a technical feature not required by the other groups" is incorrect with regard to Groups I and III” (response, p.6, para 5, line 1-3).
This is not found persuasive because “Groups I-III (including Group I and Group III) lack unity of invention because even though the inventions of these groups require the technical feature of producing saponins by culturing plant cells, this technical feature is not a special technical feature as it does not make a contribution over the prior art…”, as described in the Office action dated 3/27/2026 (bridging paragraph between p.3-4).
The requirement is still deemed proper and is therefore made FINAL.
Claim Status
Claims 1-16 and 18-28 are pending.
Claims 16 and 22-27 are withdrawn from examination as being part of non-elected groups.
Claim 1-15 and 18-21 are being examined along with the newly added claim 28.
Claim Objections
Claim 5 is objected to because of the following informalities: It is suggested that the word “replacing” in line 3 be substituted by “replaced”.
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 7, 10-11 and 15 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.
Claims 7 and 10-11 directly or indirectly depend from claim 1, which recites, “… ii) depleting the culture medium of the nitrogen source…”. Now, for example, claim 7 recites, “…ii) is from 1 to 9 days”.
It is not clear to the examiner if claims 7 and 10-11 require depleting (as a singular event or as a continuous process in which depletion is occurring) the culture medium on continuous and/or gradual basis of any nitrogen source for a specific period (days).
The same issue is with claim 15 which also depends on claim 1 that recites the process of “eliciting” in step (iii).
To provide better customer service and compact prosecution, the Examiner interprets the claims as maintaining the cells in the culture medium with depleted nitrogen source (for claims 7 and 10-11) and maintaining the cells in the culture medium comprising the elicitor (for claim 15) for specific days.
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.
Claims 1-15, 18-21, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Dalsgard et al. (US 5,716,848) in view of Mathur et al. (In vitro Saponin Production in Plant Cell and Tissue Cultures, 2010, In vitro saponin production in plant cell and tissue cultures. In Medicinal plant biotechnology (pp. 115-137). Wallingford UK: CABI), Gangwar, A. (Morphogenetic studies and in vitro secondary metabolite production in Panax species. 2003, PhD thesis, Lucknow University, India), and Zhang et al. (Integrated analysis on biochemical profiling and transcriptome revealed nitrogen-driven difference in accumulation of saponins in a medicinal plant Panax notoginseng, 2020, Plant Physiology and Biochemistry, 154:564–580), and in evidence of Guerra et al. (Saponin production from Quillaja genus species. An insight into its applications and biology, 2021, Sci. Agric., 78:e20190305).
Claim 1 is drawn to a method for producing saponins comprising a quillaic acid triterpenoid aglycone by culturing cells of plants belonging to Quillaja genus in a culture medium comprising a nitrogen source, depleting the culture medium from the nitrogen source, eliciting the production of the saponins with at least one elicitor, and recovering the saponins produced.
Dalsgard et al. teaches producing saponins (which comprise quillaic acid) from either a callus culture or a suspension cell culture (which reads on to “suspension cell lines”, as recited in claim 2) while the plant cells are derived from any of the 3 plant species including Quillaja Saponaria (as recited in claim 18) (besides Q. smegmademws and Quillaja brasiliesis) belonging to the Quillaja genus (abstract). The callus is subsequently subcultured in a liquid medium having the composition comprising a nitrogen source (as described in Table 1) to obtain suspension cell culture (column 3, line 32-35). It is known in the art that quillaic acid is the most common triterpene aglycon saponin (as recited in claim 1) produced by Quillaja spp. (Guerra et al.; p.4, right column, para 2, line 6-11). Dalsgard et al. also describes producing different active substances from Quillaja sp., various products comprising the active substances comprising various adjuvants including the adjuvant QS-21 (column 1, line 6-14; column 1, line 24; column 2, line 11-13; Fig. 5; column 8, Example 6), as recited in claims 19-20 and 28.
The culture media used comprise several nitrogen sources including potassium nitrate, calcium nitrate, sodium nitrate and ammonium nitrate (column 2, line 30-31; Table 1). Combined concentration of nitrogen sources, as described by Dalsgard et al., is (39.40 mM) which comprise 1650 mg/l (i.e., 20.61 mM) of ammonium nitrate (NH4NO3) and 1900 mg/l (i.e., 18.79 mM) Potassium Nitrate (KNO3) in both Murashige and Skoog's medium (MS) and in Linsmaier and Skoog's medium (LS) (Table 1) and is within the recited (in claim 3) range of 10-50 mM. All the culture media described by Dalsgard et al. comprise sucrose (Table 1), as recited in claim 4.
Dalsgard et al. describes a method for preparing (reads on to “recovering” as recited in claim 1) active substances from Quillaja sp. (column 4, line 9-10; column 6, bridging paragraph between column 6-77, Examples 2-3).
The Applicant defines “depleting” as ”depleting the culture medium from any source of nitrogen", in the context of the invention, means reducing the level of any source of nitrogen which has been included in the culture medium in step i) and maintaining the cells in such culture medium having a reduced level of nitrogen source” (Spec, p.10, para 7, line 34-37). Applicant asserts that “Nitrogen depletion may be performed either (i) by letting the cells naturally consume the source of nitrogen included in the culture medium in step i) down to a residual level, with no further replenishment of the culture medium with any nitrogen source (or "natural depletion")” (Spec, p.10, last para, last 3 lines).
It is inherent that the growing plant cells in any cell culture including in a suspension culture, as described by Dalsgard et al., are continuously consuming nitrogen (and other nutrients) present in the medium and, thus, the total concentration of nitrogen source is being continuously reduced or depleted, which reads on to “depleting the culture medium from any of the nitrogen source”, as recited in claims 1 and 8. Even replenishing the culture medium and maintaining the plant cells in the nitrogen depleted culture medium for certain period of time would not stop (further) depleting the culture medium of its nutrients including nitrogen.
However, Dalsgard et al. does not explicitly teach any elicitor to elicit the production of saponins. Dalsgard et al. does not describe a culture medium containing no nitrogen source.
Mathur et al. teaches that plants are valued for its storage roots, which are the source of a group of triterpene saponins, collectively called as “ginsenosides” (p.117, para 1, line 9-10). It describes that accumulation of saponins in plant cell cultures including in suspension and root cell cultures is stimulated by using different elicitors including methyl jasmonate (MJ) (as recited in claim 13) (p.126, para 2, line 1-5; p.126, para 3, line 1-3) by adding it directly to the culture medium (Table 8.3), as recited in claim 14. Methyl jasmonate is a well-known monocarboxylic compound (as recited in claim 12) containing one carboxyl group (-COOH) in its structure and part of its inherent property (Human metabolome database), as shown below.
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Mathur et al. also describes that total nitrogen concentration in the media significantly affects the cell growth and saponin production and/or accumulation in cell cultures (p.123, para 5, line 6-8). Manipulation of media composition including NO3 concentration (as a nitrogen source) has been shown to affect ginsenoside (saponin) production (p.123, para 5, line 12-17) besides culture age and medium replenishment/exchange strategy (p.123, para 5, line 17-19).
Gangwar, A. describes testing four levels (concentrations) of KNO3 (0, 9.39, 18.78 and 28.17 mM) (as recited in claims 3 and 9) (p.75, para 4, line 3) while KNO3 at a concentration of 18.78 mM is used as control (p.75, para 4, line 4-5). The cells are maintained in the culture medium including the one without any nitrogen source (as recited in claim 8) for 7 weeks to study the influence of NO3 (in form of KNO3) (p.75, para 4, line 1-2). Eliminating KNO3 altogether from the culture medium increased (or elicited) saponins (ginsenoside) accumulation to 0.891% than in the control (0.68%) while the concentration of saponins (ginsenoside) is significantly reduced from 0.80% to 0.14% at the elevated level of the nitrogen source KNO3 (Gangwar, A.; p.110, para 1, line 12-16). It is noted, however, that the Applicant also uses KNO3 as the nitrogen source (spec, p.66, Experiment 1, table 1).
Zhang et al. describes that saponins content in the roots of the Chinese medicinal plant Panax notoginseng is increased under low nitrogen condition (abstract, line 1-5), where the plants grown under “low nitrogen” (LN) condition comprising no nitrogen (0 kg/ha) (i.e., “less than 10 mM”, as recited in claim 9) (p.565, right column, para 5, line 21-22), as recited in claims 5 and 9.
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to produce saponins including quillaic acid triterpenoid aglycone in cell cultures (including suspension culture) of plants belonging to Quillaja genus, as described by Dalsgard et al., and increase the yield of saponins by adding an elicitor like methyl jasmonate in the culture, as described by Mathur et al., while depleting or reducing nitrogen source (e.g., NO3) in the growth environment, as taught by Gangwar A. and as indicated by Zhang et al.
sing any specific medium including a medium having 1.25 to 5 mM nitrogen (which is lower nitrogen content than the “control medium” having 18.78 mM KNO3, as described by Gangawr, A.), as recited in claim 6, would have been an experimental design choice of the artisan.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to increase the yield of saponins including quillaic acid by adding methyl jasmonate in the culture while depleting nitrogen source in the growth environment including in the cell culture derived from the plants belonging to Quillaja genus.
Regarding claims 7, 10-11 and 15; It is well known in the art that production of secondary metabolites including saponins (ginsenosides) depends on various factors including genotype and plant age (of the explant) (Mathur et al., p.117, para 2, line 3). Optimization of a culture-based production system for a plant product varies significantly and requires routine standardization of various techniques ranging from cell line selection to media manipulation, pathway elicitation and precursor feeding (Mathur et al, p.123, para 1, line 1-3) which include duration of a specific step in a specific cultural condition and media composition used in the process. Preponderance of evidence shows that the saponins (ginsenosides) in hairy roots of P. ginseng is present at day 1 and its production increased by 1.85-9.7-fold within 7 days (as recited in claims 7, 10-11 and 15) of elicitor (MJ) treatment.
It is noted that claim 1 “comprises” four steps (i-iv). However, the sequence and/or duration of each step is not specified by the claim. Thus, depleting the culture medium of any nitrogen source [i.e., step (ii)] can be done and/or continue simultaneously with the elicitation process using an elicitor [i.e., step (iii)] and still fulfill all the claim limitations in claim 1.
However, the Applicant is reminded that specific range and the differences in time (days) and/or media composition will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such ranges are critical for the invention. "[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; see also Peterson,315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages."); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969). See MPEP § 2144.
Regarding claim 21, The Applicant describes, “"QS-21 saponin family" as used herein means (i) the xylose chemotype QS-21 species having a monoisotopic molecular weight (m/z) of 1988 ("QS-21 1988") with negative mode electrospray mass spectrometry (which exist as A and B isomers, and V1 apiose and V2 xylose isomers)” (spec, p.15, line 8-12). The structures of Quillaja saponaria saponins comprising QS-21 family of saponins (as discussed by Dalsgard et al. and as described above) including QS-21 Xyl (xylose) and QS-21Api (apiose) saponins having molecular weight of about 1988 is well known in the art before the effective filing date (evidence for the inherent structure is provided by Flake et al. Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, 2019, Molecules, 24:171; p.5, S4/S6 in Table 1) and reads on to “QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1, or QS-21 1988 B V2” (as recited in claim 21), absent evidence to the contrary.
Conclusion
No claim is allowed.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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J.C.
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Unit 1661 & 1662
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