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 .
Withdrawal of Rejections
The response and amendments filed on 03/20/2026 are acknowledged. Any previously applied minor objections and/or minor rejections (i.e., formal matters), not explicitly restated here for brevity, have been withdrawn necessitated by Applicant’s formality correction and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, and/or maintaining, if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner’s Response to Arguments section.
Briefly, the previous claim rejections under 35 U.S.C. 112(b) for indefiniteness have been withdrawn necessitated by Applicant’s amendments. The previous claim rejections under 35 U.S.C 103 for obviousness have been withdrawn necessitated by Applicant’s amendments; however, new grounds of rejection are set forth below.
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
New Grounds of Rejection Necessitated by Amendments
Claim Rejections - 35 USC § 103, Obviousness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-2, 6, 8, 10, 12-13, 16, 21, 52, 55, 56-57, 59, 61, 64, 67, 81, and 84 are rejected under 35 U.S.C. 103 as being unpatentable over Davis (U.S. Patent No. 8,586,713; Date of Publication: November 19, 2013 – previously cited) in view of Angelo (WO 2020/214792; Date of Publication: November 22, 2020 – cited in the IDS filed on 05/09/2023 – previously cited), and Chollangi (Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies; 2015 – previously cited).
Davis’ general disclosure relates to “A bispecific antibody format providing ease of isolation is provided, comprising immunoglobulin heavy chain variable domains that are differentially modified in the CH3 domain, wherein the differential modifications are non-immunogenic or substantially non-immunogenic with respect to the CH3 modifications, and at least one of the modifications results in a differential affinity for the bispecific antibody for an affinity reagent such as Protein A, and the bispecific antibody is isolable from a disrupted cell, from medium, or from a mixture of antibodies based on its affinity for Protein A” (see, e.g., Davis, abstract). Moreover, Davis discloses “ a novel format that combines a common light chain strategy with an implementation of a selective Protein A purification scheme that can be used with human antibody components” (see, e.g., Davis, pg. 18, col. 12, lines 17-20).
Regarding claims 1, 12, 81, and 84 pertaining to the method of purifying a heterodimeric protein or bispecific antibody, Davis teaches a method of purifying a bispecific antigen-binding protein, wherein “at least one amino acid difference results in an improved ability to isolate the protein, because the difference results in a differential ability of the CH3 domain sequences to bind an affinity agent” (see, e.g., Davis, “Summary”, pg. 14, col. 3, lines 61-67). Moreover, Davis teaches “a binding protein comprising an Fc, wherein the Fc comprises a first CH3 domain that is modified as described herein and a second CH3 that is not modified, so as to form a heterodimeric Fc” (see, e.g., Davis, pg. 16, col. 7, lines 46-49). Additionally, Davis teaches “a bispecific antigen-binding protein is provided that comprises a first specificity that binds an antigen and a second specificity that activates a receptor, wherein the bispecific antigen-binding protein comprises a first polypeptide comprising a first IgG1, IgG2, or IgG4 CH3 domain that comprises a Protein A-binding determinant, and a second polypeptide comprising a second IgG1, IgG3, or IgG4 CH3 domain that lacks the Protein A-binding determinant (see, e.g., Davis, pg. 15, col. 6, lines 41-48). Davis teaches that the chromatography column was washed with a wash buffer having a pH of 7.2 (see, e.g., Davis, Example 10, pg. 26, col. 28, lines 45-52). Furthermore, Davis teaches eluting the heterodimeric protein at a pH of 4.2 (see, e.g., Davis, pg. 16, col. 7, lines 54-55). Davis teaches “the bispecific antibody is isolated using a Protein A affinity support, wherein the bispecific antibody elutes at a pH between about 3.9 to about 4.4, about 4.0 to about 4.3, about 4.1 to about 4.2, or at about pH 4.2. In one embodiment, the bispecific antibody elutes at a pH of about 4, 4.1, 4.2, 4.3, 4.4, or 4.5” (see, e.g., Davis, pg. 16, col. 7, lines 24-29).
Regarding claim 13 pertaining to the reference level of binding impurity, Davis teaches ”In one embodiment, the bispecific antibody comprising the heterodimeric IgG CH3 domain elutes from the Protein A support in one or more fractions substantially free of non-heterodimeric IgG. In a specific embodiment, the eluted bispecific antibody fraction(s) comprise less than about 1%, 0.5%, or 0.1% of total protein by weight that is non-heterodimeric antibody” (see, e.g., Davis, pg. 24, col. 23, lines 12-18). Therefore, based on this teaching, one of ordinary skill in the art would readily understand that the level of impurities is about 1%, when compared to the overall protein levels (see, e.g., MPEP 2144.05(I)).
Regarding claims 2, 6, 16, 67, and 84 pertaining to the preliminary cycles, Davis teaches chromatography methods for purifying a heterodimeric protein (see, e.g., Davis, “Summary”, pg. 14, col. 3, lines 61-67). Regarding the cycles, the Examiner has interpreted this to simply be repetition of the chromatography steps; therefore, the simple repetition of a known step to achieve an art recognized outcome is “merely the logical result of common sense application to the maxim ‘try, try again’ (see, e.g., Perfect Web Technologies, Inc v. InfoUSA, Inc. 587 F.3d 1324 (Fed Cir. Dec. 2, 2009)). In the instant case, an ordinary artisan would understand that repeating the chromatographic cycles would predicably lead to higher purification of the heterodimeric protein, and therefore, would readily appreciate that such steps would be repeated to logically achieve a desired outcome. Additionally, measuring the level of impurities within the eluate after a certain number of cycles will allow the ordinary artisan to merely identify if the level of impurities is increasing or decreasing after each chromatographic cycle.
Regarding claims 8, 10, 21, and 84 pertaining to the preliminary and subsequent pH levels, Davis teaches Davis teaches “the bispecific antibody is isolated using a Protein A affinity support, wherein the bispecific antibody elutes at a pH between about 3.9 to about 4.4, about 4.0 to about 4.3, about 4.1 to about 4.2, or at about pH 4.2. In one embodiment, the bispecific antibody elutes at a pH of about 4, 4.1, 4.2, 4.3, 4.4, or 4.5” (see, e.g., Davis, pg. 16, col. 7, lines 24-29).
Regarding claims 52 and 55 pertaining to the elution buffer salt, Davis teaches CaCl2 as an ionic modifier, wherein the ionic modifier can be present at a concentration of about 0.5 molar to about 1.0 molar, or about 0.15 molar to about 0.5 molar (see, e.g., Davis, pg. 23, col. 22, lines 61-67).
Regarding claim 56, 81, and 84 pertaining to the first and second polypeptides, Davis teaches “a bispecific antigen-binding protein is provided that comprises a first specificity that binds an antigen and a second specificity that activates a receptor, wherein the bispecific antigen-binding protein comprises a first polypeptide comprising a first IgG1, IgG2, or IgG4 CH3 domain that comprises a Protein A-binding determinant, and a second polypeptide comprising a second IgG1, IgG3, or IgG4 CH3 domain that lacks the Protein A-binding determinant (see, e.g., Davis, pg. 15, col. 6, lines 41-48). Moreover, Davis teaches that “bispecific antibodies are provided that are non-immunogenic or substantially non-immunogenic in a human, with respect to their heavy chain constant domains, but nonetheless bear one or more differential modifications of the heavy chain constant domain, including a modification that results in a differential affinity of the heavy chain constant domains with respect to an affinity reagent (e.g., Protein A). The modifications comprise those disclosed herein. In a specific embodiment, the bispecific antibody that is non-immunogenic or substantially non-immunogenic in a human with respect to its CH3 domain, yet having differentially modified heavy chains is a human IgG1, IgG2, or IgG4 comprising a CH3 domain that comprises one of the following modifications (or, in another embodiment, consists essentially of one of the following modifications): H95R, or H95R and Y96F (IMGT numbering)” (see, e.g., Davis, pg. 20, col. 15, lines 17-32).
Regarding claim 57, 81, and 84 pertaining to the first and second polypeptides, Davis teaches a human IgG1 isotype bispecific antibody having the H435 and Y436F modifications to a variety of human Fc receptors (see, e.g., Davis, Example 8, pg. 25, col. 26, lines 38-41).
Regarding claims 59, 81, and 84 pertaining to the first pH, Davis teaches that the chromatography column was washed with a wash buffer having a pH of 7.2 (see, e.g., Davis, Example 10, pg. 26, col. 28, lines 45-52).
Regarding claim 61, 81, and 84 pertaining to the heterodimeric protein, Davis teaches that the heterodimeric protein is a bispecific antigen-binding protein (see, e.g., Davis, pg. 15, col. 6, lines 41-48).
However, Davis does not teach: introducing impurities to the heterodimeric protein (claims 1, 12, 81, and 84); or wherein at least one impurity binds and one impurity does not bind to the protein-binding ligand (claims 1, 12, 81, and 84); or washing the affinity matrix with a second wash buffer at a third pH of less than 4 to remove binding impurities (claims 1, 12, 81, and 84); or wherein the second pH is at a preliminary pH and the second pH is raised to a subsequent pH higher than the preliminary pH during a subsequent series of cycles, wherein the preliminary pH and subsequent pH are within the range of 4.0 and 5.2 (claims 1 and 12); or measuring a level of binding impurity in an eluate (claim 12); or wherein the second pH is increased to a range of 4.3 to 4.7 from a range of 4.0 to 4.2 if the measured level of binding impurity exceeds the reference level of binding impurity (claim 21 and 84); or wherein at least 85% of the heterodimeric protein is recovered in each cycle within the series of chromatographic cycles (claim 64).
Angelo’s general disclosure relates to “improved methods of regenerating and using affinity chromatography resin, in particular Protein A affinity chromatography resins” (see, e.g., Angelo, abstract). Moreover, Angelo discloses “a method for cleaning a chromatography resin, comprising contacting the chromatography resin with a first buffer comprising acetic acid and benzyl alcohol and a second buffer comprising sodium hydroxide, sodium citrate, and benzyl alcohol” (see, e.g., Angelo, [0006]).
Regarding claims 1, 12, 81, and 84 pertaining to the impurities, Angelo teaches “Chromatography resins are used in the purification of a desired protein from other impurities in a sample solution. Such resins are often used for purification of biopharmaceuticals, e.g., monoclonal antibodies (Mab) and other Fc-containing proteins by affinity chromatography” (see, e.g., Angelo, [0003]). Moreover, Angelo teaches “the term "cleaning" refers to a step during the process of purifying a target protein (e.g., an immunoglobulin or another Fc-containing protein) which entails removing impurities and foulants left on an affinity chromatography resin, for example, in a column (e.g., a Protein A column) in order to retain the performance of the resin” (see, e.g., Angelo, [0043]). Furthermore, Angelo teaches “Protein A chromatography resin is typically exposed to the clarified cell culture which contains higher level of impurities. Therefore, some residual impurities can bind to the Protein A resin, thereby leading to loss of binding capacity or increase of elution pool impurity upon re-use of the resin” (see, e.g., Angelo, [0045]).
Chollangi’s general disclosure relates to a “high throughput screening to evaluate three areas of separation: (i) Harvest treatment; (ii) Protein-A Chromatography; and (iii) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein–protein interaction resulted in further and more pronounced reduction in impurity levels” (see, e.g., Chollangi, abstract). Furthermore, Chollangi discloses “that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification” (see, e.g., Chollangi, abstract).
Regarding claims 1, 12, 81, and 84 pertaining to washing with a pH less than 4 to remove binding impurities, Chollangi teaches “The resin was then subjected to PBS wash followed by an acidic pH wash (pH 5.0–6.0) before eluting the mAb using buffers at pH < 4” (see, e.g., Chollangi, “Protein A Chromatography”, pg. 2294). Moreover, Chollangi teaches that HCP impurities increase in the eluate when an acidic pH wash buffer is used (see, e.g., Chollangi, Figure 2B); therefore, one of ordinary skill in the art would readily understand to wash the Protein A chromatography column with an acidic was buffer around a pH of 4.0 in order to remove all impurities from the column after elution
Regarding claims 12 and 21 pertaining to measuring the level of binding impurity, Chollangi teaches measuring the amount of impurity (e.g., Host Cell Protein; HCP) in the eluate at various pH washes (see, e.g., Chollangi, Figure 2). In order to obtain the percent recovery, one of ordinary skill in the art would understand that measured level of binding impurity would need to be compared to a reference level.
Regarding claims 64 pertaining to the recovery of the heterodimeric protein, Chollangi teaches that at a basic pH, approximately 80% of the protein is recovered; therefore, the amount of binding impurities is 20% or less (see, e.g., Chollangi, Figure 2) (see, e.g., MPEP 2144.05(I)).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify a heterodimeric protein according to the methods set forth by Davis, wherein the method includes removing impurities that are within the heterodimeric protein mixture, as taught by Angelo. One would have been motivated to do so because Angelo teaches cleaning of the affinity matrix “during the process of purifying a target protein (e.g., an immunoglobulin or another Fc-containing protein) which entails removing impurities and foulants left on an affinity chromatography resin, for example, in a column (e.g., a Protein A column) in order to retain the performance of the resin” (see, e.g., Angelo, [0043]). Moreover, Davis teaches methods of purifying heterodimeric antibodies, wherein these antibodies can be easily purified from their mixture because they are modified at their CH3 regions to have enhanced binding properties to protein A, which is different from their parental antibodies (see, e.g., Davis, pg. 13, col. 2, lines 37-39). Therefore, based on the teachings of Davis and Angelo, it would have been obvious to remove impurities from the antibody mixtures in order to obtain a purified antibody product and retain the performance of the Protein A chromatography resin. One would have expected success because Davis and Angelo both teach methods of purifying antibodies using Protein A chromatography.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify a heterodimeric protein according to the methods set forth by Davis, wherein the method includes washing the affinity matrix with a was buffer at a pH less than 4, as taught by Chollangi. One would have been motivated to do so because Chollangi teaches that when the wash pH about 4, that there is an increased amount of impurities in the eluate (see, e.g., Chollangi, Figure 2B), which signifies that the impurities bound to the column elute at acidic pH, compared to a basic pH. Moreover, Davis teaches washing of the Protein A chromatography columns before and after elution of a heterodimeric antibody (see, e.g., Chollangi, Examples 10 and 11). Therefore, based on the teachings of Davis and Chollangi, it would have been obvious to wash the Protein A chromatography column with an acidic wash buffer at a pH less than 4 in order to remove impurities from the column. One would have expected success because Davis and Chollangi both teach Protein A chromatography methods for purifying antibodies.
Regarding claim 1, 8, 10, 12, 21, 59, 68, 81, and 84’s pH limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., concentrations, percentages, pH levels, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan (see, e.g., MPEP 2144.05). For example, Davis teaches “the bispecific antibody is isolated using a Protein A affinity support, wherein the bispecific antibody elutes at a pH between about 3.9 to about 4.4, about 4.0 to about 4.3, about 4.1 to about 4.2, or at about pH 4.2. In one embodiment, the bispecific antibody elutes at a pH of about 4, 4.1, 4.2, 4.3, 4.4, or 4.5” (see, e.g., Davis, pg. 16, col. 7, lines 24-29). Additionally, as it pertains to the washing steps, Chollangi teaches contacting the chromatography columns with basic pH solutions at a pH of 10, which results in decreased HCP impurities in the eluate (see, e.g., Chollangi, Figure 2); therefore, as the pH increases, the amount of impurities in the eluate decreases. Therefore, this is motivation for someone of ordinary skill in the art to practice or test the parameter widely in order to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the pH levels, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Regarding claims 13 and 64’s percentage limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., concentrations, percentages, pH levels, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan (see, e.g., MPEP 2144.05). For example, Davis teaches ” In one embodiment, the bispecific antibody comprising the heterodimeric IgG CH3 domain elutes from the Protein A support in one or more fractions substantially free of non-heterodimeric IgG. In a specific embodiment, the eluted bispecific antibody fraction(s) comprise less than about 1%, 0.5%, or 0.1% of total protein by weight that is non-heterodimeric antibody” (see, e.g., Davis, pg. 24, col. 23, lines 12-18). Moreover, Chollangi teaches that HCP impurities increase in the eluate when an acidic pH wash buffer is used (see, e.g., Chollangi, Figure 2B); therefore, one of ordinary skill in the art would readily understand to wash the Protein A chromatography column with an acidic was buffer around a pH of 4.0 in order to remove all impurities from the column after elution. Additionally, Chollangi teaches that at a basic pH, approximately 80% of the protein is recovered; therefore, the amount of binding impurities is 20% or less (see, e.g., Chollangi, Figure 2) (see, e.g., MPEP 2144.05(I)). Therefore, this is motivation for someone of ordinary skill in the art to practice or test the parameter widely in order to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the percentages, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Davis, Angelo, and Chollangi as applied to claims 1-2, 6, 8, 10, 12-13, 16, 21, 52, 55, 56-57, 59, 61, 64, 67, 81, and 84 above, and further in view of Li (US 2021/0380638; Date of Publication: December 9, 2021 – previously cited).
The teachings of Davis, Angelo, and Chollangi, herein referred to as modified-Davis-Angelo-Chollangi, are discussed above as it pertains to purifying a heterodimeric protein.
However, modified-Davis-Angelo-Chollangi does not teach: wherein the level of binding impurity in the eluate is measured in a combined pool collected from a series of cycles (claim 20).
Li’s general disclosure relates to “a combination and a method that can significantly improve Protein A's aggregate removal capability. The combination comprises polyethylene glycol (PEG) and a salt (chaotropic or kosmotropic) as wash and elution buffer additives. The synergistic effect of salt and PEG results in almost complete separation of monomer from aggregates. For the case used for demonstration, in comparison with the control run the optimized procedure reduces aggregates in elution pool from 20% to 3-4%. This new method, by facilitating aggregate removal at the capture step, improves the overall robustness of downstream process” (see, e.g., Li, abstract).
Regarding claim 20 pertaining to the eluate pool, Li teaches generating elution pools following Protein A chromatography (see, e.g., Li, abstract, [0039], [0056], Table 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify a heterodimeric protein according to the methods set forth by modified-Davis-Angelo-Chollangi, wherein the method includes measuring the level of binding impurity in a combined eluate pool. One would have been motivated to do so because Li teaches that measuring impurities in a combined eluate pool allows one to identify and understand the percentage of impurities throughout all chromatography runs (see, e.g., Li, Table 2). Additionally, Li teaches methods to reduce aggregates in elution pools at the capture step so that impurities can be removed from a combined pool at one time instead of individual chromatography runs, thereby improving the overall robustness of the downstream process (see, e.g., Li, abstract & [0066]). Moreover, modified-Davis-Angelo-Chollangi teaches that “Protein A chromatography resin is typically exposed to the clarified cell culture which contains higher level of impurities. Therefore, some residual impurities can bind to the Protein A resin, thereby leading to loss of binding capacity or increase of elution pool impurity upon re-use of the resin” (see, e.g., Angelo, [0045]). Therefore, based on the teachings of modified-Davis-Angelo-Chollangi and Li, it would be obvious to remove impurities from a combined eluate pool since Protein A chromatography resin often contains residual impurities resulting in a loss of binding capacity.
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Davis, Angelo, and Chollangi as applied to claims 1-2, 6, 8, 10, 12-13, 16, 21, 52, 55, 56-57, 59, 61, 64, 67, 81, and 84 above, and further in view of Jorgensen (US 2021/0355215; Date of Publication: November 18, 2021 – previously cited).
The teachings of Davis, Angelo, and Chollangi, herein referred to as modified-Davis-Angelo-Chollangi, are discussed above as it pertains to purifying a heterodimeric protein.
However, modified-Davis-Angelo-Chollangi does not teach: wherein the impurities comprise homodimeric species of the first and second polypeptides (claim 43).
Jorgensen’s general disclosure relates to “methods for purifying heterodimeric, multispecific antibodies from solution” (see, e.g., Jorgensen, abstract). Moreover, Jorgensen discloses “methods for purifying a multispecific IgG antibody from a mixture by affinity chromatography, the methods comprising immobilizing the multispecific IgG antibody from said mixture on a first affinity chromatography column having binding specificity to a heavy chain constant domain of said IgG antibody; and eluting the multispecific antibody from the first affinity chromatography column with an elution buffer comprising an anti-aggregation composition to purify the multispecific antibody from the mixture, wherein the anti-aggregation composition comprises one or more polyols” (see, e.g., Jorgensen, [0004]).
Regarding claim 43 pertaining to the impurities, Jorgensen teaches that purification of heterodimeric multispecific antibodies results in the removal of “unwanted Fc-containing product variants (e.g., unwanted homodimer species)” within the protein mixture (see, e.g., Jorgensen, [0080]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify a heterodimeric protein according to the methods set forth by modified-Davis-Angelo-Chollangi, wherein the method includes removal of unwanted homodimer species within the protein mixture, as taught by Jorgensen. One would have been motivated to do so because Jorgensen teaches that in Protein A chromatography, multimeric proteins, such as antibodies, form unwanted homodimer species, which have a higher tendency to aggregate, contributing to significantly increased impurity levels (see, e.g., Jorgensen, [0080]). Moreover, modified-Davis-Angelo-Chollangi teaches “Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein–protein interaction resulted in further and more pronounced reduction in impurity levels” (see, e.g., Chollangi, abstract). Furthermore, modified-Davis-Angelo-Chollangi discloses “that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities” (see, e.g., Chollangi, abstract). Therefore, based on the teachings of modified-Davis-Angelo-Chollangi and Jorgensen, it would have been obvious to remove impurities, such as unwanted homodimer species, during Protein A chromatography. One would have expected success because modified-Davis-Angelo-Chollangi and Jorgensen teach Protein A chromatography for purifying antibodies.
Claims 44 and 46 are rejected under 35 U.S.C. 103 as being unpatentable over Davis, Angelo, and Chollangi, as applied to claims 1-2, 6, 8, 10, 12-13, 16, 21, 52, 55, 56-57, 59, 61, 64, 67, 81, and 84 above, and further in view of Scheffel (Zca: A Protein A-Derived Domain with Calcium-Dependent Affinity for Mild Antibody Purification; 2021 – previously cited).
The teachings of Davis, Angelo, and Chollangi, herein referred to as modified-Davis-Angelo-Chollangi, are discussed above as it pertains to purifying a heterodimeric protein.
Regarding claim 46 pertaining to the substrate, modified-Davis-Angelo-Chollangi teaches that the chromatography resin can be agarose or polymethacrylate (see, e.g., Angelo, [0011]).
However, modified-Davis-Angelo-Chollangi-Bolton does not teach: wherein the protein-binding ligand is an engineered Protein A comprising a Z-domain tetramer (claim 44).
Scheffel’s general disclosure relates to “a method for neutral and selective purification of antibodies by utilizing an engineered affinity ligand, Zca, derived from Protein A. This domain displays a calcium-dependent binding of antibodies and has been multimerized and immobilized to a chromatography resin to achieve an affinity matrix with high binding capacity. IgG antibodies can be eluted from the tetrameric Zca ligand at pH 7 with the addition of EDTA, or at pH 5.5 with EDTA for purification of monoclonal IgG1, which is significantly milder than the low pH (3–4) required in conventional Protein A affinity chromatography” (see, e.g., Scheffel, abstract).
Regarding claim 44 pertaining to the protein-binding ligand, Scheffel teaches an engineered affinity ligand, ZCa, derived from Protein A with high binding capacity (see, e.g., Scheffel, abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify a heterodimeric protein according to the methods set forth by modified-Davis-Angelo-Chollangi, wherein the method includes chromatography comprising an engineered Protein A comprising a Z-domain tetramer, as taught by Scheffel. One would have been motivated to do so because Scheffel teaches “Furthermore, ZCa was shown to display a calcium-dependent IgG binding, and by coupling the ligand to a chromatography resin, antibodies could be captured in the presence of calcium and eluted at neutral pH by removal of the calcium ions with a chelator, such as ethylenediaminetetraacetic acid (EDTA). Antibodies of subclass IgG2 and IgG4 can be eluted at neutral pH, while antibodies of subclass IgG1 require a slight decrease in pH for complete elution, although not lower than pH 5.5. This is a large improvement over conventional Protein A processes, where pH 3.2 is usually needed” (see, e.g., Scheffel, Introduction, pg. 246). Additionally, Scheffel teaches “For an enhanced binding capacity of the chromatography resin, the protein domain was multimerized. A tetrameric version of the ligand was coupled to a chromatography resin using a cysteine incorporated C-terminally, reducing the risk of disrupting the binding sites. The produced matrix was shown to have both high specificity and capacity” (see, e.g., Scheffel, Introduction, pgs. 246-247). Moreover, modified-Davis-Angelo-Chollangi teaches purification of IgG antibodies with affinity reagents, such as Protein A (see, e.g., Davis, abstract). Furthermore, in regards to Protein-A affinity chromatography, modified-Davis-Angelo-Chollangi teaches “The reduction in resin performance and binding capacity is often due to fouling of the resin by a build-up of impurities on the resin from both the product and the process” (see, e.g., Angelo, [0003]). Therefore, based on the teachings of modified-Davis-Angelo-Chollangi and Scheffel, it would have been obvious to purify a heterodimeric protein according to the methods set forth by modified-Davis-Angelo-Chollangi, wherein the method includes chromatography comprising an engineered Protein A comprising a Z-domain tetramer, as taught by Scheffel. One would have expected success because modified-Davis-Angelo-Chollangi and Scheffel both teach purification of antibodies using protein A chromatography.
Claims 68, 72, 76, and 81 are rejected under 35 U.S.C. 103 as being unpatentable over Davis, Angelo, and Chollangi, as applied to claims 1-2, 6, 8, 10, 12-13, 16, 21, 52, 55, 56-57, 59, 61, 64, 67, 81, and 84 above, and further in view of Mahajan (WO 2015/035180; Date of Publication: March 12, 2015 – previously cited).
The teachings of Davis, Angelo, and Chollangi, herein referred to as modified-Davis-Angelo-Chollangi, are discussed above as it pertains to purifying a heterodimeric protein.
Regarding claim 81 pertaining to the recovery of the heterodimeric protein, modified-Davis-Angelo-Chollangi teaches that at a basic pH, approximately 80% of the protein is recovered; therefore, the amount of binding impurities is 20% or less (see, e.g., Chollangi, Figure 2) (see, e.g., MPEP 2144.05(I)).
However, modified-Davis-Angelo-Chollangi do not teach: wherein the affinity matrix is contacted with a basic solution having a pH of at least 11 following every two cycles (claim 68); or wherein the pH of the basic solution is at least 12, and wherein the basic solution is NaOH at a concentration of 0.1N to 0.5 N (claims 72 and 77); or wherein each cycle further comprises cleaning the affinity matrix by contacting the affinity matrix with a basic solution having a pH of at least 11 (claim 76).
Mahajan’s general disclosure relates to “methods to clean or regenerate a chromatography material, e.g., a chromatography resin, for reuse. The chromatography material may be cleaned and/or regenerated for use with the same product or with different product” (see, e.g., Mahajan, [0006]). Moreover, Mahajan discloses “methods to clean a chromatography material for reuse comprising the steps of a) passing about two material volumes of elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid and is about pH 2.9, b) statically holding the material in elution buffer for about 30 minutes, c) passing about two material volumes of elution buffer through the material, and d) passing about two and one- half material volumes of regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and is about pH 13, e) statically holding the material in regeneration buffer for about 30 minutes, f) passing about two and one-half material volumes of regeneration buffer through the material” (see, e.g., Mahajan, [0009]).
Regarding claims 68, 72, and 76 pertaining to the basic pH solution, Mahajan teaches “passing about two or more material volumes of regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and is about pH 13” (see, e.g., Mahajan, [0007]). Moreover, Mahajan teaches that passing 0.1N NaOH at a pH of at least 13 allows for the chromatography material to be cleaned for reuse (see, e.g., Mahajan, [0007]-[0009]).
Regarding claim 68 pertaining to contacting the affinity matrix with a basic solution every number of cycles, Mahajan teaches that contacting the chromatography material with a basic solution at a pH of 13 results in cleaning of the chromatography material for reuse (see, e.g., Mahajan, [0007]-[0009]). Regarding the cycles, the Examiner has interpreted this to merely be repetition of the chromatography steps; therefore, the simple repetition of a known step to achieve an art recognized outcome is “merely the logical result of common sense application to the maxim ‘try, try again’ (see, e.g., Perfect Web Technologies, Inc v. InfoUSA, Inc. 587 F.3d 1324 (Fed Cir. Dec. 2, 2009)). In the instant case, an ordinary artisan would understand that repeating the number of washes with the basic pH would lead to cleaning of the affinity matrix and allow for reuse of the matrix, and therefore, would readily appreciate that such steps would be repeated to logically achieve a desired outcome.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify a heterodimeric protein according to the methods set forth by modified-Davis-Angelo-Chollangi, wherein the method includes contacting the affinity matrix with a basic solution having a pH of 13, as taught by Mahajan. One would have been motivated to do so because Mahajan teaches that contacting an affinity matrix with a 0.1N NaOH solution at a pH of 13 allows for the matrix to be cleaned for reuse (see, e.g., Mahajan, [0007]-[0009]). Moreover, modified-Davis-Angelo-Chollangi teaches that contacting the chromatography columns with basic pH solutions at a pH of 10, which results in decreased host cell protein impurities in the eluate (see, e.g., Chollangi, Figure 2); therefore, as the pH increases, the amount of impurities in the eluate decreases since basic pH solutions result in cleaning of the chromatography columns. Therefore, based on the teachings of modified-Davis-Angelo-Chollangi and Mahajan, it would have been obvious to wash the affinity chromatography matrix columns with a basic NaOH solution at a pH of 13 because this allows for cleaning of the matrix for reuse and results in a decrease in impurities in the eluate. One would have expected success because modified-Davis-Angelo-Chollangi and Mohajan teach purification of proteins using affinity chromatography.
Regarding claims 72 and 76’s pH limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., concentrations, percentages, pH levels, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan (see, e.g., MPEP 2144.05). For example, Davis teaches “the bispecific antibody is isolated using a Protein A affinity support, wherein the bispecific antibody elutes at a pH between about 3.9 to about 4.4, about 4.0 to about 4.3, about 4.1 to about 4.2, or at about pH 4.2. In one embodiment, the bispecific antibody elutes at a pH of about 4, 4.1, 4.2, 4.3, 4.4, or 4.5” (see, e.g., Davis, pg. 16, col. 7, lines 24-29). Additionally, as it pertains to the washing steps, Chollangi teaches contacting the chromatography columns with basic pH solutions at a pH of 10, which results in decreased HCP impurities in the eluate (see, e.g., Chollangi, Figure 2); therefore, as the pH increases, the amount of impurities in the eluate decreases. Therefore, this is motivation for someone of ordinary skill in the art to practice or test the parameter widely in order to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the pH levels, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Regarding claim 81’s percentage limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., concentrations, percentages, pH levels, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan (see, e.g., MPEP 2144.05). For example, Davis teaches ” In one embodiment, the bispecific antibody comprising the heterodimeric IgG CH3 domain elutes from the Protein A support in one or more fractions substantially free of non-heterodimeric IgG. In a specific embodiment, the eluted bispecific antibody fraction(s) comprise less than about 1%, 0.5%, or 0.1% of total protein by weight that is non-heterodimeric antibody” (see, e.g., Davis, pg. 24, col. 23, lines 12-18). Moreover, Chollangi teaches that HCP impurities increase in the eluate when an acidic pH wash buffer is used (see, e.g., Chollangi, Figure 2B); therefore, one of ordinary skill in the art would readily understand to wash the Protein A chromatography column with an acidic was buffer around a pH of 4.0 in order to remove all impurities from the column after elution. Additionally, Chollangi teaches that at a basic pH, approximately 80% of the protein is recovered; therefore, the amount of binding impurities is 20% or less (see, e.g., Chollangi, Figure 2) (see, e.g., MPEP 2144.05(I)). Therefore, this is motivation for someone of ordinary skill in the art to practice or test the parameter widely in order to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the percentages, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Examiner’s Response to Arguments
Applicant's arguments filed 03/20/2026 have been fully considered but they are not persuasive.
Regarding Applicant’s arguments pertaining to the claimed invention pertaining to extending the useful lifetime of a Protein A chromatography column (remarks, pages 10-11), this argument is not persuasive because the instantly claimed invention pertains to purifying a heterodimeric protein, not extending the useful lifetime of a Protein A chromatography column. Moreover, nowhere in the instantly claimed invention does it claim methods pertaining to extending the lifetime of chromatography columns, in general, and as it pertains to Protein A chromatography columns. Furthermore, the claims are examined based on their Broadest Reasonable Interpretation (BRI) and the BRI of the instantly claimed invention pertains to purifying a heterodimeric protein from impurities using Protein A chromatography. Based on the BRI, the instantly claimed invention does not pertain to extending the useful lifetime of a Protein A chromatography column.
Regarding Applicant’s arguments pertaining to Davis not teaching changes in elution pH as a function of cycles, or measuring the level of binding impurities (remarks, page 10), this argument is not persuasive because, as discussed in the 103 rejection above, Davis was not used to teach these limitations. Instead, Angelo teaches “the term "cleaning" refers to a step during the process of purifying a target protein (e.g., an immunoglobulin or another Fc-containing protein) which entails removing impurities and foulants left on an affinity chromatography resin, for example, in a column (e.g., a Protein A column) in order to retain the performance of the resin” (see, e.g., Angelo, [0043]). Chollangi teaches “The resin was then subjected to PBS wash followed by an acidic pH wash (pH 5.0–6.0) before eluting the mAb using buffers at pH < 4” in order to remove impurities (see, e.g., Chollangi, “Protein A Chromatography”, pg. 2294). Moreover, as stated above, the repetition of the number of cycles is the simple repetition of a known step to achieve an art recognized outcome is “merely the logical result of common sense application to the maxim ‘try, try again’ (see, e.g., Perfect Web Technologies, Inc v. InfoUSA, Inc. 587 F.3d 1324 (Fed Cir. Dec. 2, 2009)). In the instant case, an ordinary artisan would understand that repeating the chromatographic cycles would predicably lead to higher purification of the heterodimeric protein, and therefore, would readily appreciate that such steps would be repeated to logically achieve a desired outcome. Additionally, measuring the level of impurities within the eluate after a certain number of cycles will allow the ordinary artisan to merely identify if the level of impurities is increasing or decreasing after each chromatographic cycle. Additionally, as discussed above pertaining to the pH limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., concentrations, percentages, pH levels, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan (see, e.g., MPEP 2144.05). Applicant has not provided evidence that the pH limitations are critical to the instantly claimed invention; therefore, it is prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Regarding Applicant’s arguments pertaining to routine optimization and pH (remarks, pages 11-12), this argument is not persuasive because, as discussed above, Applicant has not provided evidence that the changes in pH’s, or the pH’s in general, are critical to the instantly claimed invention. For example, Davis teaches “the bispecific antibody is isolated using a Protein A affinity support, wherein the bispecific antibody elutes at a pH between about 3.9 to about 4.4, about 4.0 to about 4.3, about 4.1 to about 4.2, or at about pH 4.2. In one embodiment, the bispecific antibody elutes at a pH of about 4, 4.1, 4.2, 4.3, 4.4, or 4.5” (see, e.g., Davis, pg. 16, col. 7, lines 24-29). Additionally, as it pertains to the washing steps, Chollangi teaches contacting the chromatography columns with basic pH solutions at a pH of 10, which results in decreased HCP impurities in the eluate (see, e.g., Chollangi, Figure 2); therefore, as the pH increases, the amount of impurities in the eluate decreases. Therefore, this is motivation for someone of ordinary skill in the art to practice or test the parameter widely in order to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the pH levels, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Conclusion
Claims 1-2, 6, 8, 10, 12-13, 16, 20-21, 43-44, 46, 52, 55-57, 59, 61, 64, 67-68, 72, 76, 81 and 84 are rejected.
No claims are allowed.
THIS ACTION IS MADE FINAL. 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.
Correspondence Information
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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