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 Claims
Claims 1, 4-5, 7-8, 13-22 and 24-29 are pending following the Reply filed 12/19/2025. Claims 2, 12 and 23 have been cancelled. Claims 1, 4-5, 13-16, 20-22, 24 and 25 have been amended, and claims 26-29 have been added without introducing new matter. Claims 1, 4-5, 7-8, 13-22 and 24-29 have been examined on the merits.
Information Disclosure Statement
The information disclosure statement (IDS) filed 09/12/2025 has been considered by the examiner.
Notice
Claim 20 is presented with the status of “Previously Presented” while the claim contains an amendment by strike-through (“
Since the above-mentioned reply appears to be bona fide, and no other errors or omissions appear in the pending claim set, examination of the claims has proceeded on the merits. Claim 20 is therefore treated as a “Currently Amended” claim.
Withdrawn
The objection to the specification is withdrawn in light of the amendments.
Any objection or rejection of claims 2, 12 or 23 is moot because the claims have been cancelled.
The objections to claims 5, 20 and 21 are withdrawn in light of the amendments.
The rejections of claims 5, 14, 16 and 24 under 35 U.S.C. 112(b) are withdrawn in light of the amendments.
The rejection of claims 1, 4-5, 7-8, 14 and 20 under 35 U.S.C. 102 are withdrawn in light of the amendments. See Response to Arguments below for further discussion.
The rejection of claim 21 under 35 U.S.C. 103 is withdrawn in light of the amendments. See Response to Arguments below for further discussion.
Drawings
The drawings are objected to because:
In FIG. 15A, the various shadings used in the “Color code for recovery” legend are difficult to distinguish. In addition, the arrows above the legend overlap with the legend. There also appears to be an artifact obscuring one of the characters, as shown below:
[AltContent: arrow]
PNG
media_image1.png
103
225
media_image1.png
Greyscale
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.
Claim Objections
Claim 25 is objected to because of the following informalities: it is understood that the phrase “PTMs” in line 6 refers to “post-translational modifications”. When reciting an abbreviated term in a claim, it is proper to define the term upon first use. Please amend the claim to recite “post-translational modifications (PTMs)” in line 6. 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.
Claim 29 is 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 29 recites the limitation “wherein solubility of the digested sample is after said incubating” which renders the claim indefinite, because is not clear what “solubility… is after… incubating” means. On one hand, the phrase could be could be interpreted to mean that the digested sample is insoluble prior to incubating and only soluble after. On the other hand, the limitation may mean that the digested sample is less soluble before and more soluble after incubating. It also appears that a modifier after the term “is” may have been omitted in error, for example, “solubility... is increased… after… incubating”.
Suggestion to obviate the rejection: Applicant may amend the claim to recite, for example: “wherein the digested sample has increased solubility after said incubating”. This suggestion appears to be supported by the instant specification (see pg. 16, paras. [0043]-[0044]).
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim 24 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Galvão et al. (previously cited), hereafter, “Galvao”.
Regarding claim 24, Galvao’s study examined the production of protein hydrolysates, using whey proteins and purified β-lactoglobulin as substrates, and trypsin and α-chymotrypsin as catalysts, at several enzyme concentrations (see Abstract). Galvao teaches that α-chymotrypsin is assumed to hydrolyze peptide bonds where tryptophan is in the carboxyl side of the bond (see pg. 763, para. 5 to pg. 764, para. 1). Galvao teaches that results using α-chymotrypsin allowed for the conclusion that this enzyme is able to attack peptide bonds of β-lactoglobulin protein within its specificity (see pg. 769, para. 2). Figure 2 of Galvao shows the simulated distribution of peptide size after the hydrolysis of β-lactoglobulin with chymotrypsin (see pg. 772, Fig. 2). Here it can be seen that multiple peptides were produced by the digestion of the substrate using chymotrypsin. Thus, Galvao teaches a method of processing (i.e., the production of protein hydrolysates from a substrate) a polypeptide (e.g., β-lactoglobulin) comprising digesting the polypeptide with a protease which cleaves C-terminal to tryptophan (i.e., chymotrypsin) to produce a digested sample comprising at least two peptides.
Regarding the recited result of “wherein at least one of the peptides of the digested sample comprises a C-terminal tryptophan and >20% recovery of this peptide is achieved upon digesting”, Galvao teaches the same method using a protease that cleaves peptide bonds C-terminal to tryptophan, as discussed above. Here, the recited result of the claim is directed to an inherent property that does not affect the structure or steps of the claimed invention. As Galvao teaches the same method comprising digesting a polypeptide with a protease which cleaves C-terminal to tryptophan, the resulting effects of said method are presumed to be inherent and the claimed method is anticipated. See MPEP 2112.01 which states:
Under the principles of inherency, if a prior art device, in its normal and usual operation, would necessarily perform the method claimed, then the method claimed will be considered to be anticipated by the prior art device. When the prior art device is the same as a device described in the specification for carrying out the claimed method, it can be assumed the device will inherently perform the claimed process. In re King, 801 F.2d 1324, 231 USPQ 136 (Fed. Cir. 1986)
In the instant case, the claim does not recite any features (method steps or structures) that achieve the claimed result that are not also recited in the prior art. Hence, Galvao teaches the same method as recited in the claim and the functional limitations of the claim are directed to an inherent property of performing said method.
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 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.
Claim(s) 1, 4-5, 7-8, 13-14, 20 and 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Egeland (previously cited), and further in view of Loziuk et al. (Understanding the role of proteolytic digestion, J Proteome Res. 2013 Dec 6;12(12):5820-9; cited on Form 892), hereafter, “Loziuk”. See also the “Supplemental Data” associated with Egeland’s disclosure (previously cited).
Regarding claim 1, Egeland evaluates the effect of increasing the amount of trypsin in bottom-up based targeted protein analysis (see Abstract). In the disclosure, Egeland systematically evaluates the possibility of speeding up the digestion step in targeted protein analysis by increasing the trypsin-to-protein ratio (see pg. 155, col. 2, para. 3). Egeland discloses that “extreme” trypsin-to-protein ratios (1:1 and 5:1) were compared to the conventional 1:40 ratio (see pg. 157, col. 1, para. 1). Egeland teaches that the peptides used contained varying amounts of “chymotryptic” cleavage sites, including sites that are carboxyl terminal to tyrosine, the idea being that if the increased trypsin amounts result in increased chymotryptic activity, this effect would be apparent when studying the peptides’ digestion profiles (see pg. 156, col. 2, para. 3). Egeland discloses that a follow-up experiment was performed to prove the presence of chymotryptic activity during digestion (see pg. 156, col. 2, para. 4). Egeland teaches that by increasing the trypsin-to-protein ratio, the digestion speed is increased (see pg. 155, col. 1, para. 1) and makes it possible to increase throughput substantially while increasing method sensitivity (see pg. 155, col. 2, para. 2).
Figure S4 of Egeland’s “Supplemental Data” shows that peptides observed and confirmed by MS/MS (denoted with a checkmark below) in samples digested for up to twenty hours included peptides comprising a tyrosine (Y) at the C-terminus, as shown below:
PNG
media_image2.png
25
1043
media_image2.png
Greyscale
As seen in the figure above, the digestion of hCGβ-T5 using trypsin at an E:S ratio of 1:1 produced a peptide with a tyrosine at the C-terminus using unmodified trypsin (VLQGVLPALPQVVCNY) and also using modified trypsin (QGVLPALPQVVCNY), and the digestion of hCGβ-T9 produced a peptide with a tyrosine at the C-terminus under all conditions (GVNPVVSY). For clarity, see the annotated version of the figure below:
PNG
media_image2.png
25
1043
media_image2.png
Greyscale
[AltContent: arrow][AltContent: arrow][AltContent: arrow]
PNG
media_image2.png
25
1043
media_image2.png
Greyscale
Therefore, Egeland teaches a method of processing polypeptide(s) (i.e., hCGβ-T5, hCGβ-T9) to produce a digested sample comprising at least one or two peptides comprising a tyrosine at the C-terminus (e.g., GVNPVVSY), said method comprising digesting the polypeptide(s) with trypsin at an enzyme:substrate (E:S) ratio of 1:1.
Egeland does not explicitly disclose experiments using an enzyme:substrate (E:S) weight ratio of about 1:2 to about 1:10.
However, Egeland does teach that trypsin-to-protein ratios (up to 1:3 ratio) in solution have previously been applied with success in global proteomic workflows, phosphoproteomics, and in targeted proteomics applications (see pg. 155, col. 1, para. 2). Egeland also teaches that tryptic digestion has been reported for overnight digestion using 1:5 ratios, citing Loziuk, et al. (see pg. 155, col. 2, para. 1; pg. 161, Reference 11). In light of Egeland’s teachings, all of these ratios (i.e., 1:1, 1:3, 1:5) use a higher amount of trypsin compared to conventional ratios (i.e., 1:40), as previously discussed.
Loziuk teaches that workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation, where enzymatic digestion is most commonly performed using trypsin. Loziuk teaches that while the structure, specificity, and kinetics are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These parameters include the type of trypsin used, pH during digestion, incubation temperature, as well as enzyme-to-substrate ratio. Loziuk teaches the optimization of these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. See Abstract.
Loziuk discloses that to ensure reproducible complete peptide production an enzyme/substrate ratio of 1:5 was chosen for optimized digestion in targeted proteomics experiments (see pg. 5826, col. 1, para. 1). Loziuk teaches that peptides previously determined to have slow production rates displayed a discrepancy in quantification with the newly optimized conditions, and it was determined that an enzyme/substrate ratio of 1:5 is sufficient for complete production of all surrogate peptides (see Fig. 4). Loziuk also discloses that chymotryptic activity was observed in time course experiments using unmodified trypsin in a 1:5 enzyme:substrate ratio (see pg. 5828, Figure 6).
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Egeland and Loziuk, because both references teach optimized methods for the enzymatic digestion of proteins using trypsin. One would have been particularly motivated to have combined these teachings in order to speed up digestion protocol, increase throughput and method sensitivity, while maximizing control of the desired digestion products. One would have recognized that both references disclose an enzyme-to-substrate ratio of 1:5, which Loziuk further teaches to be optimal for certain proteins under the optimized conditions of the disclosure. One would have recognized that Egeland teaches that the results of the disclosure were achieved using a trypsin-to-substrate ratio of 1:1, which was more “extreme” compared to the conventional ratio of 1:40, while Loziuk, as cited by Egeland, teaches the ratio of 1:5 to be sufficient for the complete digestion of the peptides. As both references teach this ratio to have been a result-effective variable, and Loziuk teaches further considerations, such as the pH during digestion and incubation temperature, a skilled artisan would have recognized that the results of using this ratio would have been predictable and would have had a reasonable expectation of success. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 4, Egeland and Loziuk disclose a trypsin-to-protein ratio of 1:5, as discussed above.
Regarding claim 5, Egeland teaches that all peptides monitored were produced at similar or higher levels after 45 minutes compared to conventional overnight digestion (see Abstract). Hence, it would have been obvious to have digested the polypeptide for less than 12 hours. In addition, Loziuk teaches a digestion buffer comprising CaCl2 (calcium chloride) (see pg. 5821, col. 2, para. 4) and digestion conditions with a pH 7 or 8 (see pg. 5822, col. 1, paras. 1-2).
Regarding claim 7, Egeland teaches that all peptides monitored were produced at similar or higher levels after 45 minutes compared to conventional overnight digestion (see Abstract). Hence, it would have been obvious to have digested the polypeptide for less than 4 hours.
Regarding claim 8, Egeland teaches that after the addition of trypsin, the samples were incubated at a temperature of 37°C (see “Supplemental Data” at pg. S-2, para. 4). Loziuk also discloses that temperature had the greatest influence on the data, with 37°C being the better condition, which also yielded more unique peptides compared to 48°C (see pg. 5823, col. 2, para. 4).
Regarding claim 13, Egeland discloses that the digestion of hCGβ-T9 produced a peptide with a tyrosine at the C-terminus (GVNPVVSY), as discussed above.
Egeland does not explicitly teach the digested sample comprising peptides of HGNFGNSY (SEQ ID NO: 108), HGNFGNSYISY (SEQ ID NO: 109), and/or HGNFGNSYISYWAY (SEQ ID NO: 110).
Examiner notes that the further limitation of the claim does not require any further active steps in the claimed method and is merely directed to a result that is expected to be achieved by said method. Therefore, the method of claim 1 would inherently produce the same results recited in dependent claim 13, because the further limitations of the dependent claims merely recite a result of using the method of claim 1.
Furthermore, in view of Applicant’s Examples, the claimed peptides are the result of the tryptic digestion of a BiTE® molecule (see instant specification at “Example 8”, pg. 67, paras. [00157]-[00158]) which is further disclosed as a “bispecific T-cell engager” molecule (see instant specification at pg. 5, para. [0008]). Therefore, it is understood that polypeptides that do not contain the same subsequences as BiTE® (e.g., HGNFGNSY) will not be expected to produce peptides with the same sequence identity when digested, because these results are dependent on the amino acid sequence of the polypeptide selected for processing. As set forth under Claim interpretation in the previous Office Action, the claimed results are understood to be the effect of digesting a polypeptide which already contains the recited sequences.
Nonetheless, Egeland teaches proteins with predicted chymotryptic cleavage sites comprising a tyrosine residue that produce peptides with a C-terminal tyrosine when following the methods of the disclosure. One of ordinary skill would have recognized from Egeland that the results of any digestion using trypsin would be dependent on the polypeptide selected for said digestion, and that these portions of the protein can be predicted based on the presence of art-recognized cleavage sites in the sequence. Furthermore, one would have expected that the results of the tryptic digestions disclosed by Egeland and Loziuk would be predictable when applying the same techniques to the tryptic digestion of BiTE®, and it would have been within the ordinary skill in the art to have applied these teachings to the digestion of any polypeptide predicted to produce peptides having a C-terminal tyrosine. Here, the only difference between the results of the claimed invention and what is disclosed in the prior art is the selection of the polypeptide for processing, and one of ordinary skill would have expected these results to be dependent on the structure of the polypeptide itself. Further, one would have recognized from Egeland and Loziuk that the resulting digested peptides can be predicted based on the structure of the polypeptide, the specificities of the protease, and the conditions in which the digesting occurs. Hence, the invention of claim 13 would have been obvious in view of Egeland and Loziuk, because the claim merely recites an effect of digesting a polypeptide using trypsin, which the prior art of Egeland teaches will produce peptides with C-terminal tyrosine’s.
Regarding claim 14, Egeland teaches the samples containing the proteins were denaturated, reduced, and alkylated prior to tryptic digestion (see pg. 156, col. 1, para. 4).
Regarding claim 20, Egeland teaches injecting digested sample aliquots into an LC-MS system (see “Supplemental Data” at pg. S-4, paras. 3 and 5; and pg. S-3, para. 4).
Regarding claim 28, Egeland discloses that each protein sample was subjected to tryptic digesting using either unmodified or modified trypsin (see pg. 156, col. 1, para. 4). Therefore, it is understood from Egeland’s disclosure that each protein sample (polypeptide) was digested using only one protease and that protease was trypsin.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Egeland and Loziuk, as applied to claims 1, 4-5, 7-8, 13-14, 20 and 28 above, and further in view of Agilent Technologies (previously cited), hereafter, “Agilent”.
Regarding claim 15, Egeland and Loziuk do not explicitly teach a “buffer exchange” before digesting the polypeptide with trypsin and after denaturing, reducing, and/or alkylating the polypeptide.
However, Loziuk does state that the samples were “washed” with digestion buffer containing 2 M urea and 20 mM CaCl2 after denaturing, reduction and alkylation (see pg. 5821, col. 2, para. 4).
Agilent teaches that peptide mapping, an invaluable tool for biopharmaceuticals, is the most widely used identity test for proteins, particularly those produced by recombinant means, and most commonly involves enzymatic digestion (usually using trypsin) of a protein to produce peptide fragments, followed by separation and identification of the fragments in a reproducible manner (see pg. 2, col. 1, para. 1). Agilent teaches that to provide an optimal pH for the enzymatic cleavage, a buffer is added prior to the addition of trypsin (see pg. 7, col. 1, para. 2). Agilent teaches that pH of the reaction is among several factors that are critical to the effectiveness of the digestion (see pg. 7, col. 1, para. 3) and is empirically determined to ensure the optimization of the performance of a given cleavage agent (see pg. 7, col. 2, para. 1). Agilent also teaches that some of the more common approaches used for sample cleanup prior to digestion include depletion/enrichment dialysis and desalting by gel filtration (see pg. 4, col. 1, para. 1). Agilent further teaches that samples often need dialysis or desalting to ensure they are compatible and optimized for digestion, and that dialysis and desalting products allow for buffer exchange and small molecule removal to prevent interference with downstream processes (see pg. 4, col. 2, para. 2). Agilent teaches that gel filtration, also known as size exclusion chromatography (see pg. 5, col. 1, para. 1), is the most practical procedure for desalting samples, while buffer exchange replaces the sample buffer with a new buffer (see pg. 4, col. 2, para. 4). Hence, Agilent teaches a buffer exchange prior to digestion which is critical to the effectiveness of the digestion.
It would have been obvious at the time of filing for a person of ordinary skill to have arrived at the claimed invention by combining the teachings of Egeland, Loziuk and Agilent, because each reference is directed to methods of digesting proteins using trypsin for the identification of said proteins. One of ordinary skill would have recognized that Agilent teaches the step of buffer exchange prior to trypsin digestion as an effective means to control the pH during digestion, which Agilent teaches to be a critical factor in ensuring an effective digestion of the protein. Hence, one would have been motivated to combine these teachings in order to optimize the conditions of the protein sample during enzymatic digestion and to achieve the best outcome for peptide mapping analysis. As each reference teaches enzymatic digestions using the same protease (i.e., trypsin) to produce peptide fragments for further analysis, one would have recognized the results of the combination would have been predictable. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 16, Agilent teaches depletion/enrichment dialysis and desalting by gel filtration to ensure the sample is compatible and optimized for digestion and to allow for buffer exchange, as discussed above. Agilent teaches that gel filtration, also known as size exclusion chromatography, is the most practical procedure for desalting samples, while buffer exchange replaces the sample buffer with a new buffer, as discussed above. According to the instant specification, “[i]n various aspects, the buffer exchange comprises using a size exclusion cartridge, optionally a NAPTM-5 column with the Sephadex® gel filtration material” (see pg. 7, para. [0009]). It is understood in view of Agilent’s disclosure that size exclusion chromatography requires the use of a column (see, e.g., pg. 5, col. 1, para. 1, to col. 2, para. 1). Hence, it would have been obvious to have carried out the buffer exchange using gel filtration using a size exclusion cartridge (column), because Agilent teaches that gel filtration using size exclusion chromatography during buffer exchange is the most practical way to desalt samples prior to digestion.
Claims 17-19, 22, 26 and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Egeland and Loziuk, as applied to claims 1, 4-5, 7-8, 13-14, 20 and 28 above, and further in view of Movius et al., US 20070021591 A1 (previously cited), hereafter, “Movius”.
Regarding claim 17, Loziuk discloses sample preparation included incubating protein extracts with 8 M urea and 100 mM dithiothreitol in order to denature and reduce, followed by alkylation using 200 mM iodoacetamide, and the samples were then washed three times for 15 minutes with digestion buffer containing 2 M urea and 10 mM CaCl2 (see pg. 5821, col. 2, para. 4). In view of the instant specification, “urea” is a chaotrope (see pg. 4, penultimate line). Hence, Loziuk discloses the samples were incubated and washed with a chaotrope prior to digestion.
Egeland and Loziuk do not explicitly teach incubating the digested sample in the presence of a chaotrope at a mildly acidic pH.
Movius teaches methods for achieving the deaggregation of binding proteins and compositions suitable for the deaggregation of highly concentrated solutions of binding proteins containing one or more chaotrope and formulated at an acidic pH (see Abstract). Movius teaches that protein aggregation is of major importance to the biotechnology industry because of the importance of in vitro production of recombinant proteins (see pg. 1, para. [0007]). Movius teaches that proteins in solution, even highly purified proteins, can form aggregates upon storage, or during production processes, and in vitro aggregation limits protein stability, solubility, and production yields of recombinant proteins (see pg. 1, para. [0007]). Movius teaches that existing methodologies for achieving protein deaggregation commonly employ the step of solubilizing the protein in high concentrations of strong chaotropes, such as guanidine hydrochloride, which results in nearly complete protein unfolding (see pg. 1, para. [0011]). Movius teaches that compositions for deaggregating binding proteins comprise one or more chaotropic agent, including guanidine hydrochloride (see pg. 6, para. [0066]), and are buffered to about pH 5 (see pg. 6, para. [0068]). Movius teaches that the binding proteins include antibody fragments and variable fragment single-chain antibodies (see pg. 1, para. [0002]).
In view of the instant specification, “a mildly acidic pH” refers to an acidic pH of about 4 or higher and below 7 (see pg. 3, para. [0008]), which includes pH 5. The instant specification also states that the BiTE® molecule used in Applicant’s Examples is a bispecific monoclonal antibody which is “the fusion of two single-chain variable fragments” (see pg. 46, para. [00111]; Emphasis added). Hence, it is understood that the “digested sample” of the claimed method includes antibody fragments, which Movius teaches are prone to aggregation upon storage and during production processes, which can be prevented by the presence of a chaotrope at a mildly acidic pH.
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Egeland, Loziuk and Movius, because each reference relates to production processes involving fragmented proteins which are prone to aggregation during storage and production processes. Therefore, one would have been motivated to combine the teachings of Movius with the other references when processing antibody polypeptides (binding proteins), because Movius teaches that in vitro aggregation of binding proteins, including antibody fragments, limits protein stability and solubility which can be detrimental to the digested peptides during storage and to their further use in production processes. Hence, one would have expected that incubating the digested samples in the presence of a chaotrope at a mildly acidic pH would be an effective means to ensure the stability and solubility of the peptides for downstream processes (e.g., LC-MS). Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 18, Movius teaches the chaotrope is guanidine hydrochloride, as discussed above.
Regarding claim 19, Movius teaches the mildly acidic pH is about 5, as discussed above.
Regarding claim 22, it would have been obvious to have incubated the digested sample in the presence of a chaotrope and at a mildly acidic pH for the reasons discussed regarding claim 17. Regarding the limitation that the digested sample comprises “at least two peptides”, Egeland teaches the digestion of polypeptides using trypsin resulted in multiple peptides in the digested samples, as discussed regarding claim 1.
Regarding claim 26, Movius teaches compositions for the deaggregation of binding proteins comprising a chaotrope, wherein the binding proteins include “antibodies” (see Abstract) which meet the limitation of an “antigen binding protein”.
Regarding claim 29, Movius teaches that existing methodologies for achieving protein deaggregation commonly employ the step of solubilizing the protein in high concentrations of strong chaotropes, as discussed above.
Claim(s) 25 is/are rejected under 35 U.S.C. 103 as being unpatentable over Egeland and Loziuk, as applied to claims 1, 4-5, 7-8, 13-14, 20 and 28 above, and further in view of Pascovici, et al. (Clinically Relevant Post-Translational Modification Analyses-Maturing Workflows and Bioinformatics Tools, Int J Mol Sci., 2018 Dec 20;20(1):16), hereafter, “Pascovici”.
Regarding claim 25, Egeland and Loziuk teach the method of claim 1, which meets limitation (a).
Regarding limitation (b), Egeland teaches injecting digested sample aliquots into an LC-MS system (see “Supplemental Data” at pg. S-4, paras. 3 and 5; and pg. S-3, para. 4). Loziuk discloses that the samples were analyzed using LC-MS/MS (see pg. 5823, col. 1, para. 2) and the responses obtained using optimized digestion conditions greatly affected the researchers’ ability to identify proteins and discover potential surrogate peptides that can be used for absolute quantification as well as site-specific identification of post-translational modifications (see pg. 5825, Figure 2).
Regarding limitations (c)-(d), these limitations repeat the steps of (a) and (b) using a second polypeptide. Egeland teaches the monitoring of peptides produced from the proteolytic digestion of multiple polypeptides, i.e., BSA, CytC and hCG (see Fig. 1).
Regarding limitation (e), Loziuk discloses that LC-MS/MS can be used to identify site-specific post-translational modifications, as discussed above.
Egeland and Loziuk do not explicitly teach comparing the PTMs of a first polypeptide in a first sample obtained at a first timepoint to the PTMs of a second polypeptide in a second sample obtained at a second timepoint.
Pascovici’s review focuses on canvassing the options and progress of post-translational modification (PTM) analysis for large quantitative studies, from choosing the platform, through to data analysis, with an emphasis on clinically relevant samples such as plasma and other body fluids, and well-established tools and options for data interpretation (see Abstract). Pascovici teaches that the ability to analyze protein post-translational modifications (PTMs) occurring on a large scale in a biological system yields insight into their roles and relevance to disease states, and the wealth of these changes and their importance in cell signaling and disease have led to modified proteins being the focus of clinical and pharmaceutical research as potential drug targets (see pg. 1, para. 1). Pascovici teaches that recent reviews have illustrated the potential of PTMs as disease biomarkers, surveyed disease associated PTM changes, in cardiovascular disease, cancer, neurodegenerative disease and diabetes, demonstrating a growing interest in characterizing PTMs to answer clinical questions (see pg. 2, para. 2).
Pascovici teaches that many quantitative LC-MS/MS approaches make use of the “bottom-up” proteomics strategy, which involves the digestion of proteins using a proteolytic enzyme such as trypsin, then LC-MS/MS analysis and identification of peptides using a protein sequence database and search algorithm (see pg. 7, para. 4 to pg. 8, para. 1). Pascovici discloses a general workflow illustrating some of the common steps associated with sample preparation, PTM enrichment, MS and bioinformatics analysis, as shown in Figure 1 below:
PNG
media_image3.png
579
720
media_image3.png
Greyscale
Pascovici also discloses examples of PTM studies for various clinically relevant biological fluids, which are shown on page 6, Table 1. The PTM studies listed in Table 1 include, for example: a study showing that plasma samples demonstrated higher phosphorylated α-synuclein levels in Parkinson’s disease compared to healthy controls (see pg. 6, Table 1); and a study evaluating the expression of specific phosphoproteins found in urine during pregnancy in comparison with non-pregnancy (see pg. 7, Table 1). Hence, it is understood in view of Pascovici’s disclosure that PTMs present in a first protein are typically compared to PTMs present in a second protein (e.g., a control or database) in order to use such modifications as a biomarker for determining a disease or health condition. Moreover, Pascovici teaches that previous studies have suggested that glycosylation of proteins acetylcholinesterase and butyrylcholinesterase obtained from antemortem tissues in Alzheimer’s disease patients could be used as markers of disease progression, and glycan changes in breast cancer were shown to mediate disease progression and influence overall survival rates (see pg. 4, para. 2). Hence, it is also clear in view of this disclosure that monitoring disease progression by the sampling and processing of proteins must involve obtaining the proteins at different timepoints in order to monitor the progression of the disease.
It would have been obvious at the time of filing for a person of ordinary skill to have combined the teachings of Egeland, Loziuk and Pascovici because each reference discloses methods for bottom-up proteomics, using trypsin digestion and LC-MS, which Pascovici teaches can be used for PTM studies. One would have been motivated to do so, because Pascovici teaches that the ability to analyze protein post-translational modifications (PTMs) occurring on a large scale in a biological system yields insight into their roles and relevance to disease states, and such modified proteins can be used to identify potential drug targets and to determine and/or monitor disease progression. A person having ordinary skill would have recognized that in order to use such modifications as biomarkers for monitoring disease progression, comparisons between multiple peptides, processed under the same conditions and obtained at different timepoints, would be necessary to make a useful determination. As such disease-related proteins and PTMs are known in the prior art, and Pascovici teaches the same common sample preparation steps as Egeland and Loziuk, one would have had a reasonable expectation of success when combining these methods to compare PTM changes in proteins. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Claim(s) 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Egeland, Loziuk and Movius, as applied to claims 17-19, 22, 26 and 29 above, and further in view of Wu et al. (Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in quantitative proteomics studies, Rapid Commun. Mass Spectrom 2016, 30: 1059–1066; cited on Form 892), hereafter, “Wu”.
Regarding claim 27, Egeland and Loziuk do not explicitly teach the method wherein the trypsin is a recombinant protein.
Wu teaches trypsin is an important digestive enzyme in peptide sample preparation for proteomics, and the majority of commercial products are obtained from animal sources (see pg. 1059, “Rationale”). In Wu’s study, the researchers evaluated whether recombinant trypsin (r-trypsin) and recombinant acetylated trypsin (r-Ac-trypsin) are suitable for proteomics research (see pg. 1059, “Rationale”). Wu discloses that the r-trypsin and r-Ac-trypsin had better efficiency than the commercial products at high protein/enzyme ratios, and both demonstrated similar specificity and efficiency compared to commercial products in typical in-solution digestion of HSA (see pg. 1064, col. 1). While the activity and specificity of the r-Ac-trypsin were similar to that of commercial trypsins, it also demonstrated superior activity and specificity on complicated samples and, more interestingly, was more resistant to autolysis, which enabled more complete digestion of proteomic samples (see pg. 1059, “Results”). Wu concludes that the r-Ac-trypsin studied is a recombinant product, showing similar or superior properties such as stability activity and specificity compared to commercial products, and can be used in peptide sample preparation in proteomics studies (see pg. 1059, “Conclusions”).
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by substituting any of the trypsins used by Egeland or Loziuk with the recombinant trypsin taught by Wu, because Wu teaches recombinant trypsin proteins having better efficiency than animal-derived products. One would have been motivated to do so, because Wu teaches recombinant trypsin proteins to have similar or superior properties compared to non-recombinant products. Furthermore, Wu teaches that both recombinant trypsins had better efficiency than the commercial products at high protein/enzyme ratios. Therefore, a person of skill would have recognized that the substituted component and its functions were known in the art and one could have substituted one known element for another to obtain predictable results. Furthermore, Egeland, Loziuk and Wu all relate to methods for studying proteins comprising proteolytic trypsin digestion, and a person of skill would have recognized that many different types of trypsin may be used to perform such studies. Therefore, as the substituted component was known in the art and has been demonstrated in the art to be suitable for such processes, there would have been a reasonable expectation of success. Hence, the combination would have been readily apparent and deemed to be a mere (B) simple substitution of one known element for another to obtain predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Response to Arguments
Regarding the rejections under 35 U.S.C. 102 in view of Egeland, Applicant argues that Egeland does not disclose a method of processing a polypeptide comprising digesting the polypeptide with trypsin at an enzyme:substrate (E:S) weight ratio of about 1:2 to about 1:10.
Applicant’s arguments have been fully considered and are persuasive.
Accordingly, the rejection of claims 1, 4-5, 7-8, 14 and 20 under 35 U.S.C. 102 has been withdrawn.
Regarding the rejection of claim 24 under 35 U.S.C. 102 as being anticipated by Galvao, Applicant argues that Galvao does not disclose a method wherein at least one of the peptides of the digested sample comprises a C-terminal tryptophan and >20% recovery of this peptide is achieved upon digesting.
Applicant’s arguments have been fully considered but they are not persuasive. In response to applicant's argument that Galvao does not disclose the same results as recited in the claim were achieved, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). In the instant case, Galvao teaches the same method steps as the claim. As Galvao teaches the same method comprising digesting a polypeptide with a protease which cleaves C-terminal to tryptophan, the resulting effects of said method are presumed to be inherent and the claimed method is anticipated. See the present rejection for further discussion.
Regarding the rejections under 35 U.S.C. 103 in view of Egeland, Applicant argues that Egeland provides no pointer or reason to modify its disclosure with any particular enzyme:substrate ratio, but rather, asserts that its results are sample-specific: “Finally, since both the peptide production and the peptide decay vary for different peptides, also within the same protein, optimum digest conditions should always be determined, especially when the most sensitive method is required.” (Egeland at p. 160, RHC, last paragraph) Thus, Egeland provides no reason, much less any reasonable expectation of success for any alleged modification of its disclosure with different ratios.
Applicant’s arguments have been fully considered but they are not persuasive.
Contrary to Applicant’s argument, the evidence presented from Egeland appears to support modification, as Egeland expressly states “since both the peptide production and peptide decay vary for different peptides… optimum digest conditions should always be determined” (see above). It is unclear to the examiner how this could be interpreted to mean that Egeland is teaching away from modifying digest conditions, because it appears that this is in fact what Egeland is suggesting. To a person of ordinary skill, Egeland’s suggestion that optimum digest conditions should always be determined based on the peptides that are being produced appears to be a suggestion for further optimization, rather than a teaching against it.
Regarding the rejections under 35 U.S.C. 103 in view of Egeland and Agilent, Applicant argues that Agilent does not teach the claimed E:S weight ratio, and therefore does not cure the deficiency of Egeland.
Applicant’s arguments with respect to claim(s) 15-16 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. Specifically, the present rejection relies on Loziuk who teaches an E:S ratio within the claimed range.
Regarding the rejection of claim 21 under 35 U.S.C. 103 in view of Egeland and Movius, Applicant argues that the references do not disclose, teach, or suggest a method of processing a polypeptide comprising digesting the polypeptide with a protease to obtain a digested sample, incubating the digested sample in the presence of a chaotrope at a mildly acidic pH, and injecting the digested sample with the chaotrope into a mass spectrometer for analysis, as currently recited in claim 21. Applicant argues that the addition of chaotropes to a sample that is directly injected to a mass spectrometer for analysis was counterintuitive, because many chaotropes are known to negatively impact MS performance and are typically absent from the injected samples. Applicant cites Example 3 of the instant specification:
...samples incubated in the presence of guanidine hydrochloride postdigestion led to improved recovery, compared to the samples incubated in the absence of guanidine at the same pH… These results suggest incubating the digested samples at pH 5 in the presence of 4 M guanidine led to substantially increased recovery of the long peptide. Though the overall recovery of the long peptide was poorer using the MWCO filter buffer exchange (Figure SB), the results obtained using this methodology confirmed the observation that incubating the digested samples at pH 5 in the presence of 4 M guanidine leads to improved recovery of long peptides compared to incubating the digested samples in the absence of guanidine.
The improved recovery of the long peptide when incubated in the presence of guanidine was surprising, given that the chaotrope was present in the mass spectrometer and, many chaotropes are known to negatively impact MS performance and consequently are typically absent from the solution injected into the mass spectrometer.
Thus the inventive method is associated with surprising and unexpected effects.
Applicant’s arguments have been fully considered and are persuasive.
The closest prior art of Egeland, Loziuk and Movius do not teach or suggest the limitation of (c) injecting the digested protein sample with the chaotrope into a liquid-chromatography-mass spectrometry (LC-MS) system for analysis. On the contrary, the prior art of record appears to teach away from this limitation.
For example, Vaisar, T. (Thematic review series: proteomics. Proteomic analysis of lipid-protein complexes. J Lipid Res. 2009 May;50(5):781-6; cited on Form 892) teaches membrane-embedded proteins are most efficiently solubilized with strong ionic detergent, SDS or Triton X-100, chaotropic agents, urea, thiourea or guanidine, or their combination. However, the high concentrations of detergent (0.5–4%) or chaotrope (5–8 M) that is typically required inhibit trypsin. Chaotropic agents also suppress ionization and have to be removed prior to MS analysis. Many commonly used detergents (e.g., Triton X-100 and other polyethers) also generate multiple high-abundance ions that interfere with MS analysis. See page 783, column 1, paragraph 2.
Similarly, Specht, et al. (Automated sample preparation for high-throughput single-cell proteomics, Department of Bioengineering, Northeastern University, Boston, MA, bioRxiv, August 25, 2018; cited on Form 892) teach that techniques using detergents or chaotropic agents like urea are robust but require that the chaotropic agents or detergents be removed before MS analysis since these chemicals are incompatible with MS (see pg. 2, para. 1). The examiner also notes that Agilent (previously cited) teaches that BSA samples in urea and BSA samples in guanidine HCl were cleaned using AssayMAP reversed-phase cartridges prior to LC-MS analysis (see pg. 23, col. 1, para. 1) and teaches the importance of desalting samples prior to LC-MS analysis, as discussed under 35 U.S.C. 103.
After an updated search, the examiner has not found any evidence that it would have been obvious to have performed this method step with any reasonable expectation of achieving the results disclosed by the present inventors. Hence, in the absence of any evidence to the contrary, claim 21 is free of the prior art and nonobvious in view of the closest prior art of record.
Conclusion
Claim 21 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DENNIS ARMATO whose telephone number is (703)756-5348. The examiner can normally be reached Mon-Fri 11:00am-7:30pm EST.
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, Melenie Gordon can be reached at (571) 272-8037. 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.
/DENNIS IGNATIUS ARMATO JR/Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
Prosecution Insights