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
Applicant’s amendment filed on 13 August 2025 is entered. Claims 25, 32, 35, and 37 are amended, and claims 26 and 31 are canceled. Claims 25, 32, 35, and 37 are pending.
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.
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 25, 32, 35 and 37 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.
(Maintained) Claim 25 recites “…conducting a buffer exchange of the antibacterial protein solution containing the mixture of the first antibacterial protein and the second antibacterial protein with a buffer comprising poloxamer 188, D-sorbitol, and L-histidine to obtain the solution…”. The claim does not recite the antibacterial protein solution comprises a buffer prior to this limitation. It is unclear what buffer is being exchanged for the buffer comprising poloxamer 188, D-sorbitol, and L-histidine.
(New necessitated by amendment) Claim 25 recites the limitation "culture medium" in line 15. There is insufficient antecedent basis for this limitation in the claim. The limitation in line 15 can be amended to “incubating host cells at 37oC in a culture medium, adding arabinose to the culture medium…”.
Claims 32, 35 and 37 are dependent on claim 25 and are indefinite for the same reasons.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
(Maintained) Claims 25, 32, 35, and 37 are rejected under 35 U.S.C. 103 as being unpatentable over Yoon et al. (US8232370B2; previously cited) in view of Liao et al. (Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase, 2004, Protein Science, 13:1802-1810; previously cited and attached in Office Action mailed 06 March 2023), Yoon et al. (US8377866B2; hereinafter referred to as “Yoon ‘866” to avoid confusion; previously cited), Pohle et al.( Robust Batch Cultivation Process for Recombinant Protein Production with Escherichia coli BL21 in a BIOSTAT® B Reactor, 2013, Sartorius Stedim Biotech Application Note, v11: 1-3; previously cited and attached in Office Action mailed 19 April 2024), Kang et al. (WO2013180316A1; previously cited and attached in Office Action mailed 06 March 2023), Andya et al. (WO9704801A1; previously cited and attached in Office Action mailed 06 March 2023), and Phillips et al. (Unit 4.4 Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration, Current Protocols in Protein Science (2004) 4.4.1-4.4.15).
Claim 25 recites the limitation “the mixture having killing activity against all of the following Staphylococcus species: Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus carnosus, Staphylococcus carprae, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus delphini, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus hemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus muscae, Staphylococcus pasteuri, Staphylococcus saprophyticus, Staphylococcus warneri, and Staphylococcus xylosus”, which recites inherent characteristics of the mixture of a first antibacterial protein consisting of the amino acid sequence of SEQ. ID. NO: 1 and a second antibacterial protein consisting of the amino acid sequence of SEQ ID. NO: 2. Additionally, claim 25 recites “wherein the mixture includes an intact protein amount of 99.8% after 6 months and a difference in biological activity of less than 5%”, which recites an intended characteristic of the freeze-dried antimicrobial protein composition that results from practicing the instant method, but does not recite any additional active steps necessary to practice the instant method. Where the active steps of the instant method and structural components of the resultant freeze-dried antimicrobial formulation are found in the art, the recited limitations describing the inherent characteristics of the mixture of antibacterial proteins are considered necessarily met.
Regarding claim 25, Yoon teaches an antimicrobial protein having killing activity against Staphylococcus aureus and having the amino acid sequence represented by SEQ. ID. NO: 19 and a gene encoding the same (Yoon Col. 2, lines 54-57). Yoon’s SEQ ID NO:19 has 100% sequence identity with instant SEQ ID NO:1 (Sequence alignment previously attached in Office Action mailed 19 April 2024). The gene encoding the antimicrobial protein has the nucleotide sequence represented by SEQ. ID. NO: 18 (Yoon Col. 3, lines 62-65). SEQ ID NO:18 has 100% sequence identity with instant SEQ ID NO:3 (Sequence alignment previously attached in Office Action mailed 19 April 2024). Yoon teaches E. coli host cells were successfully transformed with a plasmid comprising the antimicrobial protein, which was overexpressed using an L-arabinose-mediated induction expression system based on pBAD-TOPO vector, which is favorable in the expression of toxic protein. The transformant was inoculated in a TSA medium, and when the cell concentration reached 0.8-1 of OD600, L-arabinose was added to induce the expression of the antimicrobial protein. The cells were then cultured for 4 more hours after induction (Yoon Col. 12, lines 51-66). Yoon further teaches a pharmaceutical composition of the present invention can include a pharmaceutically acceptable carrier such as sorbitol (Yoon Col. 4, lines 32-35), and can additionally include a stabilizing agent (Yoon Col. 4, lines 66-67). The pharmaceutical formulation can also be in dried solid forms such as powders, granules, tablets or capsules (Yoon Col. 4, lines 63-66). Yoon also teaches the centrifugation of the purified protein solution to remove insoluble substances, and filtering the resulting supernatant through a 0.2 µm filter (Yoon col. 15 lns. 34-37).
Yoon does not teach the specific conditions of incubating at 37°C, adding arabinose at an OD600 of 2.0, culturing the production host cells for 10 hours at 19°C, subjecting the solution to freeze-drying wherein the mixture includes 25-29 mole% of the first antibacterial protein and 71-75 mole% of the second antibacterial protein, buffer exchange of the mixture of first and second antimicrobial protein mixture with a buffer comprising poloxamer 188, L-histidine, and sorbitol, concentrating the antimicrobial proteins in the solution by using a centrifugation filter, adjusting the concentration of the first and second antibacterial proteins in the solution to 18mg/mL, nor a protein having the sequence of SEQ ID NO: 2.
Instant SEQ ID NOs: 1 and 2 differ only in that SEQ ID NO: 2 has the N-terminal methionine deleted. Every other amino acid residue of SEQ ID NO: 2 is identical to the those of SEQ ID NO: 1.
Liao teaches the removal of N-terminal translation initiator Met by enzyme methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins (Liao Abstract lines 1-2).
One of ordinary skill in the art would have been motivated to remove the N-terminal methionine from the antimicrobial protein of Yoon because one of ordinary skill in the art would recognize that doing so could enhance the function and stability of the antimicrobial protein. One of ordinary skill in the art would have had reasonable expectations of success because Liao taught that removal of that N-terminal translation initiator methionine is crucial for the function and stability of proteins (Liao Abstract lines 1-2).
Yoon and Liao do not teach the specific conditions of incubating at 37°C, adding arabinose at an OD600 of 2.0, culturing the production host cells for 10 hours at 19°C, subjecting the solution to freeze-drying wherein the mixture includes 25-29 mole% of the first antibacterial protein and 71-75 mole% of the second antibacterial protein, buffer exchange of the mixture of first and second antimicrobial protein mixture with a buffer comprising poloxamer 188, L-histidine, and sorbitol, concentrating the antimicrobial proteins in the solution by using a centrifugation filter, nor adjusting the concentration of the first and second antibacterial proteins in the solution to 18mg/mL.
Yoon ‘866 discloses a host cell producing the same antimicrobial protein having killing activity against Staphylococcus aureus as taught by Yoon (Yoon ‘866 Abstract, lines 1-3). The host cell is inoculated in growth medium and cultured overnight at 37°C. The culture broth was the re-inoculated in fresh growth medium and was cultured again at 37°C. When the OD600 of the culture broth reached 0.5, L-arabinose was added to induce the expression of the antimicrobial protein. Then, the culture temperature was adjusted to 23°C, followed by low temperature culture for 12 hours (Yoon ‘866, Col. 14, lines 22-33).
Pohle teaches that recombinant protein production is often done with well-known strains of E. coli (Pohle Pg. 1, 1. Introduction, [1]), that production of a recombinant GFP in an E. coli host cell culture was induced at an OD600 of 19±2 with 2, 6, or 10 g/L of L-arabinose (Pohle Pg. 2, left column, [2]).
Routine optimization within prior art conditions disclosed by Yoon, Yoon ‘866 and Pohle would have led to the claimed conditions of incubating the host cells at 37°C, adding arabinose at an OD600 of 2.0, and subsequently culturing the host cells for 10 hours at 19°C, because the instantly claimed values fall between values disclosed by the prior art for induction of proteins in E. coli host cells. The claimed value of the cell concentration of OD600 at 2.0 falls between the OD600 values disclosed by Yoon (0.8-1) and Pohle (19±2). The claimed value of 10 hours of culturing time falls between the culturing times disclosed by Yoon (4 hours) and Yoon ‘866 (12 hours). One of ordinary skill in the art would have had reasonable expectations of success in optimizing these values to fall between these prior art values because the prior art taught that a range of values are satisfactory. MPEP §2144.05(II)(A) states “[g]enerally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[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)”. Therefore, there is a prima facie case of obviousness to optimize host cell culture conditions.
Yoon, Liao, Yoon ‘866, and Pohle do not teach subjecting the solution to freeze-drying wherein the mixture includes 25-29 mole% of the first antibacterial protein and 71-75 mole% of the second antibacterial protein, buffer exchange of the mixture of first and second antimicrobial protein mixture with a buffer comprising poloxamer 188, L-histidine, and sorbitol, concentrating the antimicrobial proteins in the solution by using a centrifugation filter, nor adjusting the concentration of the first and second antibacterial proteins in the solution to 18mg/mL.
Kang teaches a method of stabilizing a composition comprising antimicrobial protein SAL-1 having killing activity against Staphylococcus aureus represented by the amino acid sequence of SEQ ID NO: 1 comprising the step of adding a poloxamer to the composition (Kang Claim 1 and Description [5], lines 3-4). Kang’s SEQ ID NO:1 has 100% sequence identity with instant SEQ ID NO:1. Kang further teaches SAL-1 was purified to 95% purity and was then concentrated to 20 mg/mL to prepare a SAL-1 solution which was subjected to buffer exchange with L-Histidine buffer comprising 10 mM L-Histidine and 5%(w/v) sorbitol, pH 6.0 (Kang Example 1, lines 3-8). Various surfactants, including poloxamer, were added to the histidine buffered SAL-1 to investigate the suitability of particular surfactants on composition stability (Kang Example 1, lines 11-13). Kang found that adding 0.1-0.5% poloxamer 188 could be selected as the most suitable surfactant both in terms of enhancing stability and maintaining biological activity of the histidine buffered SAL-1 (Kang Example 1, last paragraph).
One of ordinary skill in the art would have been motivated to buffer exchange of the mixture antimicrobial proteins of Yoon with Kang’s buffer comprising poloxamer 188, L-histidine, and sorbitol in order to enhance the stability of the antimicrobial proteins during long term storage and transportation. One of ordinary skill in the art would have had reasonable expectations of success because Kang found that a solution comprising antimicrobial protein SAL-1, L-histidine and sorbitol containing buffer, and poloxamer 188 was capable of enhancing stability and maintaining biological activity of the antimicrobial proteins (Kang Example 1, last paragraph and Fig. 6).
Yoon, Liao, Yoon ‘866, Pohle, and Kang do not teach subjecting the solution to freeze-drying wherein the mixture includes 25-29 mole% of the first antibacterial protein and 71-75 mole% of the second antibacterial protein, concentrating the antimicrobial proteins in the solution by using a centrifugation filter, nor adjusting the concentration of the first and second antibacterial proteins in the solution to specifically 18mg/mL.
Andya teaches a method for preparing a protein formulation comprising lyophilizing a mixture of a protein and a lyoprotectant (Andya Claim 10 step a). Andya teaches that a lyophilized protein formulation is stable upon storage and delivery (Andya Pg.1, lines 27-28). A "lyoprotectant" is a molecule which, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage, and Andya teaches exemplary lyoprotectants include histidine and sorbitol (Andya Pg. 9, lines 9-15). Andya further teaches it is desirable to add a surfactant, such as a poloxamer (e.g. poloxamer 188), to the pre-lyophilized formulation or reconstituted formulation (Andya Pg. 15, lines 22-25).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to modify Yoon, Liao, Yoon ‘866, and Pohle’s method of making an antibacterial formulation by adding Kang’s poloxamer 188, L-histidine, and sorbitol, and freeze-drying the formulation as taught by Andya in order to enhance the long term stability and storage/transportation of the antimicrobial protein formulation.
One of ordinary skill in the art would have been motivated to subject a composition comprising a mixture of antibacterial proteins with amino acid sequences identical to instant SEQ ID Nos: 1 and 2, poloxamer 188, sorbitol, and L-histidine by subjecting the composition to freeze-drying because doing so would enhance the stability and storage shelf life of the composition. There would have been a reasonable expectation of success because Andya teaches freeze-drying compositions comprising proteins, poloxamer, sorbitol, and L-histidine significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage and/or delivery.
Routine experimentation would have led one of ordinary skill in the art to optimize, with reasonable expectations of success, the concentrations of the first and second antibacterial protein of the mixture to include the ranges of 25-29 mole% of the first antibacterial protein and 71-75 mole% of the second antibacterial protein, and the concentration of combined mixture of antimicrobial proteins in the solution to be 18mg/mL because Kang teaches antimicrobial protein SAL-1 concentrations of 1, 13, and 20 mg/mL were effective and long-term stable when combined with L-histidine buffer and surfactant poloxamer 188 (Kang Example 1, lines 3-8, Example 5, [2] and [4]). MPEP §2144.05(II)(A) states that “[g]enerally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical.
Yoon, Liao, Yoon ‘866, Pohle, Kang, and Andya do not teach concentrating the antimicrobial proteins in the solution by using a centrifugation filter.
Philips teaches concentration and/or desalting of protein solutions using centrifugation through ultrafilters (Phillips pg. 10 Alternate Protocol 2).
One of ordinary skill in the art would have been motivated to concentrate the first and second antimicrobial protein in the solution further comprising poloxamer 188, sorbitol, and L-histidine by centrifugation filtration in order to control and/or optimize the antimicrobial protein concentrations in the resultant freeze-dried composition. One of ordinary skill in the art would have had reasonable expectations of success because Phillips taught one such protein concentration method is by means of centrifugation filtration. While purifying the antimicrobial protein SAL-1, Yoon taught that the protein was concentrated in its solution, as well as centrifugation of said solution to remove impurities, but did not teach the method employed to concentrate the protein. By combining the teachings of Yoon and Phillips, one of ordinary skill in the art would have had reasonable expectations of success in concentrating the antimicrobial proteins in solution.
Regarding claim 32, Kang teaches poloxamer 188 was added in the range of 0.1-0.5%(w/v) (0.1% -0.5 % w/v = 1-5 g/L). There was no difference in significant stability levels according to poloxamer concentration (Kang Example 2, [2], Table 1). Sufficient stabilization effect would be expected with addition of 1-5 g/L of poloxamer 188 (Kang Example 2, [4]).
Regarding claims 35 and 37, the L-histidine buffer of Kang comprises 10 mM L-Histidine and 5%(w/v) sorbitol, pH 6.0 (Kang Example 1, lines 3-8). Notably, 10 mM L-histidine = 1.55 g/L and 5% w/v sorbitol = 50 g/L, which falls within the ranges of instant claims 37 and 35, respectively.
Response to Arguments
Applicant's arguments filed 13 August 2025 have been fully considered but they are not persuasive.
Regarding Applicant’s arguments that prior art references Yoon, Liao, Yoon ‘866, Pohle, Kang, Andya, and Phillips fail to teach or suggest a preparation method without the use of methionine aminopeptidase and without a process of preparing individual purified antibacterial proteins and mixing them (Remarks pg. 7 para. 2 through pg. 8 para. 1), the claims do not limit the instant method to exclude the use of methionine aminopeptidase, nor any process of purifying individual antibacterial proteins and mixing them.
Regarding Applicant’s arguments that the hypothetical method of the prior art would be labor intensive and time consuming compared to the instant invention, and practicing the instant invention would be more simple and economically beneficial (Remarks pg. 7 last para. through pg. 8 para. 1), no evidence has been submitted that shows the obvious method of Yoon, Liao, Yoon ‘866, Pohle, Kang, Andya, and Phillips would be more labor intensive and time consuming as compared to the instant invention. Regardless, any differences in the amount of labor and time required by one of ordinary skill in the art to practice the obvious method of Yoon, Liao, Yoon ‘866, Pohle, Kang, Andya, and Phillips is not a convincing argument to overcome the 103 rejection at least because there is no recited claim limitation requiring specific labor or time requirements.
Regarding Applicant’s arguments that the freeze-dried formulation of prepared by the instant invention is active against 22 kinds of Staphylococcus species, whereas Yoon’s is only active against Staphylococcus aureus (Remarks pg. pg. 8 para. 2), the composition produced by Yoon’s method comprises the same antibacterial proteins recited in the present invention, so the composition of Yoon would inherently have a killing activity against the same 22 Staphylococcus species recited in claim 25. Regardless, any alleged differences between the capabilities of the composition created by Yoon’s method and the composition created by the instant invention are not sufficient to overcome the 103 rejection because the instant invention is drawn to a method of preparing a freeze-dried formulation, not a freeze-dried composition itself. Since the prior art taught the same active steps and structural components recited in the instant claims, the instant invention would have been obvious to one of ordinary skill in the art prior to the effective filing date of the present invention.
Regarding Applicant’s arguments that the alleged differences between the compositions produced by Yoon’s method and the instant invention may come from differences in the protein expression and host cell incubation steps (Remarks pg. 8 last para. through pg. 9 para. 1), any alleged differences between the capabilities of the composition created by Yoon’s method and the composition created by the instant invention are not sufficient to overcome the 103 rejection because the instant invention is drawn to a method of preparing a freeze-dried formulation, not a freeze-dried composition itself. Since the prior art taught the same active steps and structural components recited in the instant claims, the instant invention would have been obvious to one of ordinary skill in the art prior to the effective filing date of the present invention. Additionally, speculation that any alleged functional differences between the composition produced by Yoon’s method versus the composition produced by the instant invention is not convincing to overcome the 103 rejection because there is no evidence that any differences in the protein expression and host cell incubation steps do result in any antibacterial spectrum differences.
Regarding Applicant’s argument that the claimed invention achieves superior and unexpected stability improvement because it is very rare that stability can be improved when freeze-drying is performed on a solution state composition that was effective in improving stability of protein in solution, and Kang does not disclose any stability implications of freeze-drying, and Andya only provides general guidance regarding excipients for protein stabilization and does not disclose the specific combinations involving the antibacterial proteins having SEQ ID NOs: 1 and 2 (Remarks pg. 9-10 bridging para.), there is no evidence presented that demonstrates the rarity that stability can be improved when freeze-drying is performed on a solution that is effective in improving stability of protein in solution. Kang was not used within the rejection to describe any stability implications for freeze-drying, although Kang did investigate the long-term storage stability of a composition comprising various concentrations of SAL-1 (the antimicrobial protein of instant SEQ ID NO: 1) and poloxamer 188 (Kang example 5 and Fig. 6). Notably, Kang does not freeze-dry their composition. However, Andya teaches that freeze-drying is a known technique for stabilizing protein containing compositions, particularly when paired with lyoprotectants such as histidine, sorbitol, and poloxamers (Andya claim 10, pg. 1 lns. 27-28, pg. 9 lns. 9-15, pg. 15 lns. 22-25). As described in the above 103 rejection, the combination of Yoon, Liao, Yoon ‘866, Pohle, Kang, Andya, and Phillips shows that it would have been obvious to freeze-dry a composition comprising the antimicrobial proteins recited in the instant claims. Therefore, it would not be unexpected to one of ordinary skill in the art that the obvious freeze-dried formulation would have improved intact protein concentrations, stability, and extended shelf life as compared the already storage stable composition of Kang.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Alexander M Duryee/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
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