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 .
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.
Status of the Claims
Claims 1, 3-7, 9-12, 14, 16-18, 20, 21, 23, 25, 27, 29 are pending and examined herein.
Priority
This application, 18/681,198, filed 02/05/2024, is a 371 of PCT/CA2022/051194 filed on 08/05/2022, and claims benefit of provisional application 63/260,027 filed on 08/06/2021. This priority is acknowledged and the claims examined herein are treated as having an effective filing date of 08/06/2021.
Information Disclosure Statement
The Information Disclosure Statements filed on 08/05/2022 and 02/05/2024 are acknowledged and have been considered.
Claim Objections
Claim 3 is objected to because of the following informalities:
Claim 3 is objected to due the recitation of “a chromatography” rather than “chromatography”.
Appropriate correction is required.
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 1, 3-7, 9-12, 14, 16-18, 20, 21, 23, 25, 27, 29 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.
Claim 1 recites “…to obtain fractions of isolated antibodies or antibody fragments…”.
The claims are indefinite because there are multiple possible interpretations based on the claim language. For example, it is unclear if the claim language is to be interpreted as obtaining fractions of isolated antibodies or fractions of antibody fragments, or if it is to be interpreted as obtaining fractions of isolated antibodies or antibody fragments non-specific to fractions.
Claim 1 also recites “…analyzing the antibody or antibody fragment peptides; and (d) determining heavy chain and light chain pairing of the antibody or antibody fragments based on the analysis…”. However, the recited language of “analyzing” and “determining” is indefinite because it is unclear how and what kind of analyzing is performed to determine the heavy chain and light chain pairing given the information in the claim. The claims are indefinite because there are multiple possible conflicting interpretations based on the claim language. For example, some analysis methods would require a treatment of antibody samples which would render other forms of analysis incompatible. Similarly regarding “determining” it is unclear how this could be done given that the body of the claim does not teach the “analyzing” in a way that is definite and allows a determination for pairing to be made.
Additionally, claim 20 recites “…quantifying peptide abundance or intensity of peptide signature from the heavy and light chain…”. The claim is indefinite because the relationship between peptide signature determining heavy and light chain pairing is unclear. What the peptide “signature” entails is not clearly defined, and therefore it is unclear how it could be used to determine heavy and light chain pairing is unclear.
Appropriate correction is required.
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.
Claims 1, 3, 4, 5-7, 11, 12, 16-18, 20, and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Cebe et al. (WO 2018/158719 A1), (herein referred to as Cebe) in view of Rogers et al. (2015). “Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics”. In MAbs (Vol. 7, No. 5, pp. 881-890), (herein referred to as Rogers).
Regarding claims 1, 3, and 4, Cebe teaches heterodimeric antibodies and fragments thereof and methods for their preparation, wherein the pairing of heavy and light chains has been improved (abstract). Cebe teaches using liquid mass-spectrometry analysis to determine the extent of correct heavy chain and light chain pairing, and comparing the results with a parental antibody or antibody fragment (“Example 3”, page 23, 1st paragraph). Cebe teaches that the antibody can be a mixture of polyclonal or monoclonal antibodies (page 9, 1st paragraph). Cebe also teaches that an antibody can comprise an antigen-binding fragment of an antibody, such as a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, and that these antibody fragments are obtained using conventional techniques known to those with skill in the art (page 9, 1st full paragraph). The heterodimerization of the antibodies or fragments thereof is determined using liquid chromatography-mass spectrometry (page 25, 1st paragraph). Additionally, Cebe teaches a separation step using SDS-polyacrylamide gel electrophoresis under non-reducing conditions (page 8, 1st full paragraph, Fig. 1). Cebe also teaches that heterodimeric antibodies can be produced by antibody chemical cross-linking to generate bi-specific structures using a heterobifunctional reagent having an amino-reactive group and a sulfhydryl reactive group (page 15, 1st paragraph).
However, Cebe does not specifically teach digesting the isolated antibodies or antibody fragments in the fractions to obtain antibody or antibody fragment peptides in the method.
Rogers teaches a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics (abstract). Rogers teaches that the multi-attribute method (MAM) uses a combination of high mass accuracy / high resolution MS data generated by Orbitrap technology and automated identification and relative quantification of PQAs with dedicated software (abstract). Additionally, Rogers teaches that in depth characterization of these antibody bio-therapeutics is essential before they can be used in clinical trials, and that a panel of separation techniques such as capillary electrophoresis (CE), ion exchange chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) or hydrophobic-interaction chromatography (HIC) can be typically utilized on intact molecules to monitor consistency of the process and identify product variants and impurities (page 882, column 1, 1st full paragraph). Rogers teaches that the antibody samples of Mab 1 (IgG1) and anti-streptavidin IgG2 were denatured and reduced using (10 mM) DTT at pH 8.3 in a solution of 7.5 M guanidine at ambient temperature for 30 minutes followed by alkylation with (20 mM) IAA for 20 min, and that trypsin was added to ~100 ug of reduced-alkylated antibody at a 1:10 enzyme: substrate ratio and the mixture was incubated at 37 C for 30 minutes (page 886, column 2, 4th paragraph). Rogers teaches that the tryptic peptides were separated and monitored by RP-HPLC coupled to MS (page 887, column 1, 2nd full paragraph). Rogers also teaches the sequencing of antibody heavy and light chains, and that the primary sequence coverage for the light chain and heavy chain, of the anti-streptavidin IgG2 molecule, were 100% and 96.8%, respectively (page 882, column 2, 2nd full paragraph).
It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of determining the light and heavy chain pairing of antibodies and fragments thereof, as taught by Cebe, to digest the isolated antibodies or antibody fragments in the fractions to obtain peptides for analysis, as disclosed by Rogers, as a matter using a known technique to improve a similar method in the same way. Both references are in the same field of characterizing antibodies or antibody fragments using mass spectrometry, and Rogers demonstrates that a digestive step further into antibody peptides still allows for more in-depth characterization of the antibodies, and that such a digestion step could be performed on antibodies commonly used in the art.
A skilled artisan would have been motivated to make these modifications to the method taught by Cebe, because digestion to produce antibody fragments allows peptide mapping which provides a more in-depth characterization of the antibody. Additionally, a digestion step allows for the use of various digestion enzymes with different cleavage specificities to produce different antibody fragments as needed for analysis and sequencing. A person of ordinary skill would have had a reasonable expectation of success in making these modifications because: digestion of antibodies or antibody fragments into peptides is a well-understood, routine, and conventional activity in the art, Rogers demonstrates that such digestive enzymes are commercially available, and because Rogers demonstrates that the peptides can be analyzed to characterize heavy and light chains.
Additionally, it would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the separation step under non-reducing conditions, as taught by Cebe, to use hydrophobic interaction chromatography (HIC), as disclosed by Rogers, as a matter of simple substitution within inventions in the same field. Both references are in the same field of characterizing antibodies or antibody fragments using mass spectrometry, and both electrophoresis and HIC are used in the art for the same purpose of protein separation.
A skilled artisan would have been motivated to make these modifications to the method taught by Cebe, because the use of HIC allows for higher resolution which can be useful by allowing for more accurate molecular weights. A person of ordinary skill would have had a reasonable expectation of success in making these modifications because: the use of HIC for separation is a well-understood, routine, and conventional activity in the art, and Rogers shows it can be used together with mass spectrometry for antibody characterization.
Regarding claims 5-7, Cebe teaches a separation step using SDS-polyacrylamide gel electrophoresis under non-reducing conditions (page 8, 1st full paragraph, Fig. 1).
Regarding claims 11 and 12, Cebe teaches a step of substituting an amino acid at a specific position in the wildtype sequence with an amino acid of a different charge, to ensure that the resulting antibody formed has an interface that is electrostatically favorable to heterodimerization, wherein a positively-charged amino acid residues is changed to be neutral or negatively-charged (page 2, 4th paragraph – page 3, first paragraph).
Regarding claim 16, Rogers teaches wherein the method further comprises reducing and alkylating the cysteine residues prior to said digesting. “Solutions used in the reduction, alkylation and trypsin digestion of the antibody samples were Tris pH 7.5 and pH 8.4 (Teknova), guanidine hydrochloride and trifluoroacetic acid solutions (TFA) (Thermo Scientific), hydrogen chloride (HCl), dithiothreitol (DTT) and sodium iodoacetate (IAA)” (page 886, column 2, 2nd full paragraph).
Regarding claim 17, Cebe teaches that the antibody can be a mixture of polyclonal or monoclonal antibodies (page 9, 1st paragraph).
Regarding claim 18, Rogers teaches the use of enzymatic digestion to obtain antibody fragments using trypsin (page 886, column 2, 2nd full paragraph).
Regarding claim 20, Cebe teaches the evaluation of correct heavy chain-light chain pairing of Fab variants was done by liquid chromatography-mass spectrometry (LC-UV-ESI-MS) (page 23, 1st paragraph). Eluted Fab variants were detected by UV (210-450nm) and ionized by electrospray ionization (ESI) before analysis of their mass by QTOF (Xevo G2-S QTof, Waters), then the relative composition of the injected Fab mixture was determined by double integration of UV signal and mass intensity (page 23, 1st paragraph; Table 3).
Regarding claim 21, Rogers teaches determining the amino acid sequence of at least a portion of the light and heavy chains of the antibodies or antibody fragments present in the mixture (Figure 1A).
Claims 9 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Cebe in view of Rogers as applied to claim 1 above, and further in view of Wu et al. (1998). “Optimizing separation conditions for proteins and peptides using imaged capillary isoelectric focusing”. Journal of Chromatography a, 817(1-2), 163-171, (herein referred to as Wu).
The teachings of Cebe in view of Rogers are incorporated herein.
Regarding claims 9 and 10, Cebe in view of Rogers recites all of the limitations of claim 1 of the application but does not recite wherein the separation step comprises submitting the antibody and/or antibody fragment mixture to imaged capillary isoelectric focusing (iCIEF).
Wu teaches optimized separation conditions for proteins and peptides using imaged capillary isoelectric focusing (abstract). More specifically, Wu teaches that iCIEF was applied to the quantitation of monoclonal antibodies including human IgG, monoclonal anti-alpha1-acid glycoprotein, monoclonal anti-alpha1-antitrypsin (page 165, column 1, 2nd full paragraph). Wu also teaches that capillary isoelectric focusing offers many advantages over gel-based isoelectric focusing (IEF), such as ease of automation, quantitation and faster analysis speed (page 163, column 1, 1st paragraph), and that iCIEF is shown to be a useful tool for fast quantitation of monoclonal antibodies (page 164, column 1, 3rd paragraph). Wu also teaches that two commercially available capillary coatings, polyacrylamide and fluorocarbon, were found to provide reproducible results for cIEF separations (abstract).
It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the separation step under non-reducing conditions, as taught by Cebe, to use imaged capillary isoelectric focusing (iCIEF), as disclosed by Wu, as a matter of simple substitution. Wu teaches that iCIEF for separation of antibodies provides advantages over gel-based methods such as those used by Cebe, though both methods ultimately have the same function of separating proteins, and the substitution of iCIEF in the method of Cebe would have predictable accomplished that.
A skilled artisan would have been motivated to make these modifications to the method taught by Cebe, because the use of iCIEF provides advantages such as ease of automation, quantitation and faster analysis speed. A person of ordinary skill would have had a reasonable expectation of success in making these modifications because: the use of iCIEF for separation is a well-understood, routine, and conventional activity in the art, and Wu demonstrates that the reagents for successful use of the technique are commercially available and can be performed on commercially available antibodies.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Cebe in view of Rogers as applied to claim 1 above, and further in view of Pimenova et al. (2008). “Epitope mapping on bovine prion protein using chemical cross‐linking and mass spectrometry”. Journal of mass spectrometry, 43(2), 185-195, (herein referred to as Pimenova).
The teachings of Cebe in view of Rogers are incorporated herein.
Regarding claim 14, Cebe in view of Rogers recites all of the limitations of claim 1 of the application, as well as the use of chemical cross-linking agents to generate heterodimeric antibodies (page 15, 1st paragraph). However, Cebe in view of Rogers does not teach that the cross-linking agent comprises two N-hydroxysulfosuccinimide (NHS) ester groups.
Pimenova teaches epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry (abstract). More specifically, Pimenova teaches epitope mapping of an antigen/antibody complex, allowing for rapid data collection of the parts on the protein surface involved in the interaction (page 186, column 1, 1st full paragraph). Pimenova teaches that the epitope of the immunocomplex [2bPrP·mAb3E7] with a total molecular weight of 195 kDa was analyzed (page 186, column 1, 1st full paragraph). Additionally, Pimenova teaches the use of light cross-linking reagents DSS-d0 and DSG-d0, and the heavy cross-linking reagents DSS-d12 and DSG-d (page 186, column 2, 2nd full paragraph).
It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Cebe in view of Rogers, to use DSS (agent comprises two N-hydroxysulfosuccinimide (NHS) ester groups), as taught by Pimenova, as a matter of simple substitution. Cebe recites the use protocols for generating heterodimeric antibodies that use amine-reactive chemical cross-linking (page 15, 1st paragraph), and Pimenova recites that the cross-linkers disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6) are amine reactive.
A skilled artisan would have been motivated to make these modifications to the method taught by Cebe in view of Rogers to use DSS cross-linkers because Pimenova teaches that these cross-linking reagents generate patterns that are easily observed by MS, which is very helpful in distinguishing cross-linker-modified peptides from unmodified peptides after proteolytic digestion, and allows for high resolution and mass accuracy necessary to assign the cross-linked peptides without ambiguity (page 186, column 2, 1st full paragraph). A person of ordinary skill would have had a reasonable expectation of success in making these modifications because Pimenova teaches that: these cross-linking agents are commercially available, amines are the commonly used reactive functional groups targeted by commercially available cross-linkers, and chemical cross-linking can be successfully used in combination with mass spectrometry for analysis of antibody characterization.
Claims 23, 25, 27, and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Cebe et al., in view of Rogers as applied to claim 1 above, and further in view of Park et al. (2017). “Proteomic analysis of host cell protein dynamics in the culture supernatants of antibody-producing CHO cells”. Scientific reports, 7(1), 44246, (herein referred to as Park).
The teachings of Cebe in view of Rogers are incorporated herein.
Regarding claim 23, Cebe in view of Rogers recites all of the limitations of claim 1 of the application, and additionally recites submitting the antibody or antibody fragment peptides to mass spectrometry (MS) and sequencing the peptides, but does not recite using tandem MS (MS/MS).
However, Park teaches identifying and quantifying host cell proteins (HCPs) accumulated extracellularly at different growth phases of a mAb-producing rCHO cell line using nanoflow liquid chromatography-tandem mass spectrometry LC-MS/MS (abstract, page 2, 2nd paragraph). Additionally, Park teaches analyzing the quality attributes of the monoclonal antibodies (mAbs) (aggregation, charge variation, and N-glycosylation) to understand the effects of HCPs present in the culture supernatants on their quality (page 2, 2nd paragraph). Park teaches a cluster analysis of quantified HCPs, which was performed manually according to their concentration profiles during the cultures (page 7, 1st full paragraph). Park also teaches that the MS/MS data were queried against the Chinese hamster database (UniProt-CHO) using the following parameters: MS accuracy, 10 ppm; MS/MS accuracy, 0.8 Da for HCD; trypsin digestion with 2 missed cleavages allowed; fixed carbamidomethyl modification of cysteine, + 57.0215 Da; and variable modification of oxidized methionine, + 15.9949 Da, and also that the number of peptides and proteins in the protein groups was estimated using Scaffold program (page 11, 7th full paragraph).
It would have been obvious to person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Cebe in view of Rogers, to use tandem MS, as taught by Park, as a matter of using a known technique to improve a similar device in the same way. Cebe, Rogers, and Park all use some form of mass spectrometry for the analysis of antibodies, fragments, or various proteins. It is known by one of ordinary skill in the art that tandem mass spectrometry provides advantages over mass spectrometry such as the capability to determine molecular structure and sequence.
A skilled artisan would have been motivated to make these modifications to the method taught by Cebe in view of Rogers in order to acquire data with higher specificity and accuracy, and the ability to perform more complex analysis. A person of ordinary skill would have had a reasonable expectation of success in making these modifications because Park demonstrates that the devices to perform MS/MS analysis are commercially available, and protocols for performing the analysis are well-understood, routine, and conventional activity in the field.
Regarding claim 25, Park teaches that the MS/MS data were queried against the Chinese hamster database (UniProt-CHO) (page 11, 7th full paragraph).
Regarding claim 27, Park teaches a cluster analysis of proteins (page 7, 1st full paragraph).
Regarding claim 29, Cebe teaches that in one embodiment, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods (page 10, 1st full paragraph).
Conclusion
For all the reasons discussed above, claims 1, 3-7, 9-12, 14, 16-18, 20, 21, 23, 25, 27, 29 are rejected and therefore no claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ALEXANDER JOSEPH HOFFMAN whose telephone number is (571)272-9080. The examiner can normally be reached 10:00-6:30 M-F.
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/ALEXANDER J. HOFFMAN/ Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/ Supervisory Patent Examiner, Art Unit 1677 July 13, 2026
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