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. Status of the Application 1. Claims 1-16 are pending and subject to examination on the merits. Claims 7-16 are withdrawn from consideration as being drawn to non-elected subject matter with traverse (see answer below). Election/Restrictions 2. Applicant's election with traverse of Group II in the reply filed on 04 December 2025 is acknowledged. The traversal is on the ground(s) that “claims 1 and 7 share a specific technical feature over Arbour et al. as the reduced ribosome binding of claims 1 or 7 infers a lower production rate of SEQ ID NO: 12, which is explicitly stated in claim 7 ‘“to synthesize and secrete SEQ ID NO: 12 in the periplasmic space in a slow and controlled manner”’ (p. 8, paragraph 3). This is not found persuasive because Unity of Invention was broken due to claim 1’s recitation of having “(iii) a medium strength ribosome binding site, said medium strength ribosome binding site corresponds to an mRNA sequence modified to have a reduced ribosome binding,” where Arbour et al. teaches a ribosome binding site to facilitate translation of the polypeptide ( paragraph 0034). The examiner contends that the modulation of ribosomal binding is well-known in the art and thus qualifies as routine optimization. Additionally, routine optimization to determine the optimal working conditions of an invention is not non-obvious. The MPEP states, "[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). See MPEP Section 2144.05. it is unclear from the claim construction of claim 1 if only one of the five are required, or if all of (i)-(v) are required and thus they do have different technical features which was also broken by Arbour et al The requirement is still deemed proper and is therefore made FINAL. Priority 3. Acknowledgement is made of applicant’s claim for foreign priority based on an application filed in BE (BE2021/5137) on 28 February 2022 . Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Information Disclosure Statement 4. The information disclosure statement (IDS) submitted on 24 August 2023 ha s been considered by the examiner. See initialed and signed PTO/SB/08’s. Drawings 5. The drawings are objected to because they are not in English . Additionally, Fig. 3 is illegible and there is no description for the differing bar colors . Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance. Specification 6. The disclosure is objected to because of the following informalities: Table 2 is illegible Table 3 is illegible Table 4 is illegible Table 5 is illegible. Appropriate correction is required. Claim Objections 7. Claim 7 is objected to because of the following informalities: “, and a constant pH under conditions” should be amended to “strain, at a constant temperature and pH, under…” to improve grammar. Appropriate correction is required. 8 . Claim 13 is objected to because of the following informalities: “ wherein the expression vector is a copy number defined by a plasmid copy number of 20 per cell or less. ” should be amended to “ wherein the expression vector has a plasmid copy number of 20 per cell or less ” to improve grammar. Appropriate correction is required. Claim Rejections - 35 USC § 112(b) 9 . 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. 10 . Claim 7-1 4 and 16 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. 11 . The term “medium strength” in part (i ii ) of claim 7 renders the claim indefinite because there is no baseline comparison. It is unclear what qualifies as “medium strength,” since there is no quantification or reference to which “medium strength” is interpreted, i.e. how much reduced is “medium strength?” Claims 8- 14 and 16 are included in the instant rejection, since they do not remedy this deficiency. For examination purposes, “medium strength” will be interpreted in the broad but reasonable interpretation to be anything below a ribosomal binding site meant to enhance/increase ribosomal binding. 12. The term “ reduced ” in part (i ii ) of claim 7 renders the claim indefinite because there is no baseline comparison. It is unclear what qualifies as “ reduced ,” since there is no quantification or reference to which “reduced ” is interpreted, i.e. how much qualifies as reduced ? Claims 8-14 and 16 are included in the instant rejection, since they do not remedy this deficiency. For examination purposes, “ reduced ” will be interpreted in the broad but reasonable interpretation to be anything below a ribosomal binding site meant to enhance/increase ribosomal binding. 1 2 . Regarding claim 7, the phrase "substantially" in part (ii) renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Claims 8- 14 and 16 are included in the instant rejection, since they do not remedy this deficiency. 1 3 . Regarding claim 7, the term “slow” in part (iii) renders the claim indefinite. It is unclear what qualifies as slow as there is no reference to compare slow to, i.e. what constitutes as normal, or a specific timing to quantify slow. Claims 8- 14 and 16 are included in the instant rejection, since they do not remedy this deficiency. For examination purposes, “slow” will be interpreted in the broad but reasonable interpretation to be any production of the peptide that is not enhanced/increased. 1 4 . Regarding claim 7, the phrase “and after cooling” in part ( i v) renders the claim indefinite. It is unclear if the cooling is a part of the induction phase in part (iii) or an entirely different step. Claims 8 -14 and 16 are included in the instant rejection, since they do not remedy this deficiency. 1 5 . Claim 7 recites the limitation "the culture" in part (iv). There is insufficient antecedent basis for this limitation in the claim. It is suggested to amend the claim to “a culture” or “a culture comprising the E. coli strain.” Claims 8 -14 and 16 are included in the instant rejection, since they do not remedy this deficiency. 1 7 . Claim 7 recites the limitation "the bacteria" in part (vi). There is insufficient antecedent basis for this limitation in the claim. Claims 8 -14 and 16 are included in the instant rejection, since they do not remedy this deficiency. It is suggested to change “the bacteria” to “the E. coli ”. 1 8 . Claim 7 recites the limitation "said protein" in part (vii). There is insufficient antecedent basis for this limitation in the claim. Claims 8- -14 and 16 are included in the instant rejection, since they do not remedy this deficiency. It is suggested to either change “said protein” to “said peptide”; or to change the preamble to recite “A method for producing in the periplasmic space a protein having at least 95% identity to SEQ ID NO: 12….” Claim Rejections - 35 USC § 103 19. 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. 20. 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. 21. Claims 7-1 3 are rejected under 35 U.S.C. 103 as being unpatentable over Arbour et al ( Arbour et al., 2017, EP3444269A1—cited on the Information Disclosure Statement filed 24 August 2023) , as evidenced by Qiagen (Qiagen “Growth of Bacterial Cultures,” 2013, downloaded 18 December 2025 from < https://www.qiagen.com/us/knowledge-and- support/knowledge-hub/technology-and-research/plasmid-resource-center/growth-of-bacterial-cultures > and provided as a PDF), and in view of Jin and Bum (Jin and Bum, 2019, KR20190113440 A —cited herein ) as evidenced by NovoPro labs (“ NovoPro labs,” 2024, downloaded 18 December 2025 from < https://www.novoprolabs.com/plus/novobuilder/?vid=V0 https://www.novoprolabs.com/plus/novobuilder/?vid=V01285112851> and provided as a PDF) . Regarding claim s 7 -1 0 , drawn to a method for producing a peptide with at least 95% identity to SEQ ID NO: 12 in the periplasmic space, comprising (i) transforming E. coli , specifically BL21 (claim 8), with a vector to express SEQ ID NO: 12 in the periplasmic space, which is provided by a targeting signal peptide (claim 9), inducible by an exogenous agent, and comprising a medium strength ribosome binding site for reduced ribosome binding, (ii) culturing the E. coli strain at constant reduced temperatures , such as 23 o C + 0.5 o C (claim 10) and pH where SEQ ID NO: 12 is not synthesized, (iii) inducing for a predetermined time period in the presence of an exogenous agent at a reduced temperature to synthesize and secrete SEQ ID NO: 12 in the periplasmic space in a slow and controlled manner, (iv) harvesting the culture, (v) collecting the cultured E. coli , (vi) extracting SEQ ID NO: 12 by separating a solid pellet with bacteria and a supernatant with the components of periplasmic space and SEQ ID NO: 12, and (vii) purifying SEQ ID NO: 12 , Arbour et al. teaches the production of diphtheria toxin polypeptides, such as the carrier protein for conjugate vaccine CRM 197 (paragraph 0001). Specifically, Arbour et al. teaches the transformation of an E. coli BL21 strain with an expression vector for the expression of Dip h theria toxin polypeptide, SEQ ID NO: 2 , which is 99.2% identical to SEQ ID NO: 12 of the instant application ( “Sequence Alignment CRM197” downloaded 26 September 2025 from <abss.uspto.gov/abss4examiners/> as a PDF—cited previously ) , with a periplasmic secretion signal comprising SEQ ID NO: 1 , which is 100% identical to SEQ ID NO: 2 of the instant application ( See “Sequence Alignment OmpC ” downloaded 29 September 2025 from <https://abss.uspto.gov/abss4examiners/> a PDF—cited previously ) at a temperature between 20 o C-30 o C at pH of 6.8 for 26-30 hours by inducing expression by adding rhaminose to the culture, where the culture is first grown for 24-32 hours and then induced for 4-8hrs, where the culture is harvested in a bioreactor and the Diphtheria toxin polypeptide is purified and collected, where the purification is performed by ion-exchange chromatography and/or hydrophobic interaction chromatography, and/or mixed-mode chromatography (p. 2-4; paragraph 0010). Regarding claim 1 2 , drawn to the culturing and induction steps being antibiotic free, Arbour et al. teaches the culture medium comprising a carbon source, such as glucose during culturing with the addition of rhaminose during induction, and an iron source (p. 2-4, paragraph 0010). Regarding claim 13, drawn to the expression vector being a low copy number plasmid, Arbour et al. teaches that the vector may be any vector capable of mediating expression of a heterologous protein in E. coli B strain , where specifically “Low-copy” plasmids may be used (p. 6, paragraph 0024), where Qiagen evidences that low-copy plasmids are typically 10-20 copies (p. 3, table, entries 5-6). Arbour et al. does not teach a medium strength ribosome binding site comprising reduced ribosome binding (claim 7) and induction of protein expression by isopropyl-beta-D-1-thiogalactopyranside (IPTG) (claim 11) . Jin and Bum . teaches the cloning, optimization of periplasmic expression and purification of a recombinant CRM197 as a soluble form in E. coli (abstract ). Specifically, Jin and Bum . teaches the utilization of pET22b to express CRM197 in the periplasmic space, which is inducible by IPTG ( Example 1, paragraph 7 ) and further contains a ribosomal binding site (RBS) as evidenced by NovoPro labs (See plasmid ma p, p . 2) . Therefore, it 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 to combine the teachings of Arbour et al., as evidenced by Qiagen, in view of Jin and Bum ., as evidenced by NovoPro labs, to produce SEQ ID NO: 12 in the periplasmic space of E. coli with a medium strength RBS because CRM197 is a carrier protein for conjugate vaccine as taught by Arbour et al. One would motivated to combine these teachings to arrive at the instant claims to produce CRM197 in the p eriplasmic space , since the protein is soluble in the periplasmic space as taught by Jin and Bum. There would be a reasonable expectation of success, yielding no surprising results when combining the teachings of Arbour et al., as evidenced by Qiagen, in view of Jin and Bum , as evidenced by NovoPro labs, and in view of Minshull and Theodorou to produce a periplasmic CRM197, since Arbour et al. and Jin and Bum teach periplasmic production of CRM197 and subsequent isolation thereof. 2 2 . Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Arbour et al ( Arbour et al., 2017, EP3444269A1—cited on the Information Disclosure Statement filed 24 August 2023), as evidenced by Qiagen (Qiagen “Growth of Bacterial Cultures,” 2013, downloaded 18 December 2025 from < https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/technology-and-research/plasmid-resource-center/growth-of-bacterial-cultures > and provided as a PDF), in view of Jin and Bum ( Jin and Bum , 2019, KR20190113440 A-- cited herein) as evidenced by NovoPro labs (“ NovoPro labs,” 2024, downloaded 18 December 2025 from < https://www.novoprolabs.com/plus/novobuilder/?vid=V0 https://www.novoprolabs.com/plus/novobuilder/?vid=V01285112851> and provided as a PDF), as applied to claims 7-13 above, and further in view of Lee et al (Lee et al., 2021, US 20210032284 A1—cited herein) as evidenced by Medx ( Medx , 2025, downloaded 18 December 25 from < Does Ultrafiltration Remove Endotoxins? An Expert Guide> and provided as a PDF) and Canaud et al ( Canaud et al., 2000, Nephrol Dial Transplant —cited herein) . The teachings of Arbour et al., as evidenced by Qiagen, in view of Jin and Bum as evidenced by NovoPro labs, are discussed above and incorporated into the instant rejection. Regarding claim 14, drawn to the successive steps of protein purification from the periplasmic space, comprising (i) clarifying the supernatant with one or more filtration steps and recovering the filtrate, (ii) a series of chromatographic steps to recover SEQ ID NO: 12, where the contaminants are removed, (iii) removing residual endotoxin, and (iv) sterilizing the endotoxin-free fraction comprising SEQ ID NO: 12 , Lee et al. teaches that the CRM197 protein may be purified in a convention method, utili zing a purification method by salting, solvent precipitation, dialysis, gel filtration, column chromatography, such as ion exchange and reversed phase column chromatography, and ultrafiltration, where these methods may be performed alone or in combination (paragraph 0047). E. coli BL21 is utilized to express DNA sequence of CRM197, where related genes are removed to minimize the effects of endotoxins on purification (paragraph 0035). Ultrafiltration in particular is utilized to remove endotoxins because it effectively removes endotoxins by employing membranes as evidenced by Medx (p. 2, Quick Summary). Additionally, ultrafiltration can be utilized to sterilize as evidenced by Canaud et al (p. 25, Fig. 5). Therefore, it 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 to combine the teachings of Arbour et al., as evidenced by Qiagen, in view of Taherian et al., as evidenced by NovoPro labs, and in view of Minshull and Theodorou , and in further view of Lee et al., as evidenced by Medx and Canaud et al., to purify the protein utilizing a combination of filtration, chromatography, and sterilization because of its use in medical applications, such as vaccine adjuvants, as taught by Canaud (paragraph 0003). One would be motivated to combine the teachings to arrive at the instant claims to purify a CRM197 protein because it also demonstrates a potential in antitumor activity, since it can bind soluble HB-EGF, which is highly expressed in some human cancers as taught by Canaud et al (paragraph 0003). There would be a reasonable expectation of success, yielding no surprising results when combining the teachings of Arbour et al., as evidenced by Qiagen, in view of Taherian et al., as evidenced by NovoPro labs, and in view of Minshull and Theodorou , and in further view of Lee et al., as evidenced by Medx and Canaud et al., since Lee et al. teaches the filtration, chromatography, endotoxin removal, and sterilization of CRM197. 23. Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Arbour et al ( Arbour et al., 2017, EP3444269A1—cited on the Information Disclosure Statement filed 24 August 2023), as evidenced by Qiagen (Qiagen “Growth of Bacterial Cultures,” 2013, downloaded 18 December 2025 from < https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/technology-and-research/plasmid-resource-center/growth-of-bacterial-cultures > and provided as a PDF), and in view of Jin and Bum (Jin and Bum, 2019, KR20190113440 A—cited herein) as evidenced by NovoPro labs (“ NovoPro labs,” 2024, downloaded 18 December 2025 from < https://www.novoprolabs.com/plus/novobuilder/?vid=V0 https://www.novoprolabs.com/plus/novobuilder/?vid=V01285112851> and provided as a PDF).and further in view of Minshull and Theodorou (Minshull and Theodorou 2014, US 2014/0178914 A1—cited herein). The teachings of Arbour et al., as evidenced by Qiagen, in view of Jin and Bum as evidenced by NovoPro labs, are discussed above and incorporated into the instant rejection. Arbour et al. as evidenced by Qiagen, in view of Jin and Bum as evidenced by NovoPro labs, does not teach a medium strength ribosome binding site comprising reduced ribosome binding (claim 7), where SEQ ID NO: 6 is said ribosome binding site (claim 16), Minshull and Theodorou teach the utilization of various RBS sequences for an artisan to select an RBS based on strength, where specifically, non-natural fluorescent protein DasherGFP was introduced to E. coli in plasmids bearing different RBS’s in SEQ ID NOs: 405-456 (paragraphs 0072 and 0238); further, Minshull and Theodorou teach that different RBS’s yielded very different expression levels, where the RBS is not dependent on the exact sequence of the gene expressed (paragraph 0239). SEQ ID NOs: 426 and 452 of Minshull and Theodorou contain the exact RBS encoded by SEQ ID NO: 6 of the instant claim (See Supplemental file: 20250922_134145_us-18-278-778a-6.rng; results 3-4). Therefore, it 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 to combine the teachings of Arbour et al., as evidenced by Qiagen, in view of Jin and Bum as evidenced by NovoPro labs, and in further view of Minshull and Theodoroou to produce CRM197 in the periplasmic space with a medium strength RBS, since the modulation of RBS is a variable to optimize in molecular biology as taught by Minshull and Theodorou . One would be motivated to combine these teachings to utilize an RBS with reduced binding, since RBS selection can influence protein production as taught by Minshull and Theodorou . There would be a reasonable expectation of success, yielding no surprising results when combing the teachings of Arbour et al., as evidenced by Qiagen, in view of Jin and Bum as evidenced by NovoPro labs, and in further view of Minshull and Theodoroou to produce a periplasmic CRM197 with a reduced binding RBS, since Minshull and Theodorou teach the utilization of the same RBS of the instant claim. Conclusion 2 4 . All claims are rejected. Any inquiry concerning this communication or earlier communications from the examiner should be directed to FILLIN "Examiner name" \* MERGEFORMAT CIARA A MCKNIGHT whose telephone number is FILLIN "Phone number" \* MERGEFORMAT (703)756-4791 . The examiner can normally be reached FILLIN "Work Schedule?" \* MERGEFORMAT M-F 8:00am-4:30pm . Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath Rao can be reached on (571) 272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /CIARA A MCKNIGHT/ Examiner, Art Unit 1656 /SUZANNE M NOAKES/ Primary Examiner, Art Unit 1656