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 .
Election/Restrictions
Applicant’s election without traverse of Group 1: claims 1-33 and 51-52, drawn to methods of reducing host cell protein content in a protein preparation, of claims filed 03/29/2023, in the reply filed on 12/03/2025 is acknowledged.
After reconsideration, the restriction requirement between inventions of Group I and Group II, as set forth in the Office action mailed on 10/28/2025, is hereby withdrawn and claims 34-50 and 55-56 are hereby rejoined and fully examined for patentability under 37 CFR 1.104. Once the restriction requirement is withdrawn, the provisions of 35 U.S.C. 121 are no longer applicable. See In re Ziegler, 443 F.2d 1211, 1215, 170 USPQ 129, 131-32 (CCPA 1971). See also MPEP § 804.01.
Claim Status
Claims 1-58 are pending.
Claims 53-54 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected groups of invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/03/2025.
Therefore, claims 1-52 and 55-56 are under examination.
Priority
The instant application is a 371 of PCT/US21/53318, filed 10/04/2021, which claims priority to Provisional application 63086915, filed 10/02/2020. The effective filing date is 10/02/2020, which is the filing date of the provisional application.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 03/29/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - The Incorporation by Reference paragraph required by 37 CFR 1.821(c)(1) is missing or incomplete. See item 1) a) or 1) b) above.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Rejections - 35 USC § 103
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.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 7-10, 12-17, 19-23, 51-52 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017).
Regarding claim 1, Hickman et al teaches:
a method of reducing host cell protein content in a protein preparation (Example 1 paragraph 247 and Table 1)
comprising a protein of interest recombinantly produced in a host cell (paragraphs 72-73 and Example 1 paragraph 244),
the method comprising the steps of:
a. subjecting the protein preparation to an affinity chromatography column (paragraph 226 teaches MabSelect use in Example 1; paragraph 247 teaches MabSelect is a Protein A affinity column);
b. eluting the protein of interest from the chromatography column using acid to obtain an eluate comprising the protein of interest (Example 1 paragraph 227) and that this acid could be acetic acid (e.g. Example 1 Table 5), citrate acid, or other suitable acids (claim 2);
c. raising pH of the eluate to above about pH 6.0 (Example 1 paragraph 230); and
d. subjecting the eluate to a depth filter and obtaining a filtered protein preparation (Example 1 paragraph 251).
Regarding the limitation for strong and weak acids, by the definitions provided by the instant specification, citrate acid is a strong acid and acetic acid is a weak acid (page 17).
Hickman et al does not explicitly teach eluting the protein of interest from the chromatography column with a combination of acids comprising of a weak acid and a strong acid, as is relevant to claim 1.
However, Hickman et al further teaches that elution of antibodies off of Protein A affinity chromatography resin occurs at an acidic pH, with known optimal pH ranges (Example 1 paragraph 227). Hickman et al further teaches that pH and conductivity can be used to differentially precipitate and separate out host cell protein from antibodies using a depth filter (paragraph 251). Particularly, increasing the pH from pH 5 to about 8.0 (or about 6.0-about 8.0, paragraph 9) and lowering the conductivity of the protein solution led to precipitation of protein that was mostly not antibody and led to HCP reduction after depth filtration (paragraph 251 and Table 2).
EMD Millipore teaches the exact pH and choice of acid as an eluant are factors to consider for protein elution and downstream steps (Introduction). EMD Millipore teaches that the inherent properties of the acid used to elute antibodies, such as the acid's pKa, determine the pH, buffering capacity, volatility, and conductivity of the eluate (Conclusions and Recommendations). EMD Millipore teaches the pros and cons of using eluants instantly defined as a "weak acid", such as acetic acid, versus instantly defined as a "strong acid", such as citric acid (entire document). It thus follows that by the inherent properties of acids, a mixture of these acids would modulate these eluant and downstream properties; for example the properties of a strong acid and weak acid can balance each other's effects.
Reference Kornecki et al is cited to provide relevant inherent properties for the record. Kornecki et al teaches that there is differential acid-induced precipitation of proteins depending on the isoelectric point (pI) of the protein (page 8), which is the pH where a protein has neutral charges and effectively is the pH where a protein is leas soluble. This is an inherent property of each protein. Proteins that have different isoelectric points therefore precipitate at different pH (pages 8-9). The protein amino acid sequence generally allows calculation of its isoelectric point. Therefore, the pH of the eluant can be chosen to determine the precipitation properties of the host cell proteins and antibodies.
The instant specification discloses removal of HCP after depth filtration (instant Specification, e.g. Table 1), implying that the HCP was a precipitant, as depth filtration is used to remove particulates. The instant disclosure is silent on the mechanism underlying differential HCP removal as disclosed in the instant Tables and how the results are different from those already known in the art. The methods and results instantly disclosed can be explained by the technique of differential precipitation of HCP from antibodies and optimization of antibody elution, as taught by Hickman et al, utilizing the properties of different acids, as taught by EMD Millipore, that was known to one skilled in the art, before the effective filing date of the instant application.
HCP and antibodies are coeluted from affinity chromatography at low pH, leading to relatively similar HCP ppm after Protein A elution (Table 2 and Table 3). The eluate is eventually neutralized to about pH 7.0-7.25 before depth filtration (Example 1 and 2). Table 6 can be explained by the difference in isoelectric points of host cell proteins and antibodies leading to differential precipitation. At pH 5, HCP is still slightly soluble, as taught by Hickman et al and so not removed by depth filtration. However, upon increasing the pH to neutral, the HCP reaches and exceeds its isoelectric point and precipitates or becomes positively charged, which may lead to increased electrostatic interactions with the also positively charged antibodies, leading to aggregation. As such, these precipitants and aggregates can be removed by depth filtration.
All combinations of weak and strong acids as elution buffer reduces the HCP, but the instant application does not compare the results to elution with acetic acid alone. The reason for the difference in HCP ppm in Tables 2-3 between the different combinations of weak and strong acids could be explained by combinations of reasons related to the difference in acid strength, buffering capacity, and volatility of the acids. Acetic acid used in each elution buffer is a volatile weak acid with a low buffering capacity. Citric acid is a weaker acid than lactic acid and phosphoric acid. Starting with an eluate comprising of weaker acids with lower buffering capacity, the eluate will have a higher pH, and so lowering the pH of the eluate for viral inactivation will therefore require more HCl, a very strong acid, and subsequently more base to neutralize it, leading to a higher ionic strength and higher conductivity mixture applied to the depth filter than if starting with an eluate comprising of stronger acids. Higher ionic strength buffers help prevent electrostatic protein-protein interactions, leading to increased solubility of proteins, and therefore less precipitated HCP and less HCP removal by depth filtration. This is also consistent with the results of instant Table 5.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, to harness the desired inherent properties of both a strong acid and weak acid for an eluant as taught by EMD Millipore to optimize desired antibody elution conditions and precipitation of host cell protein for removal from the antibody eluate, as taught by the method of Hickman et al and supported by Kornecki et al, which teaches the inherent properties and resultant effects of the acid and proteins that underly this method of host cell reduction in protein purification. It would be obvious to one skilled in the art, before the effective filing date, that the pH, conductivity, and ionic strength of the eluant can be modulated using both strong acids or weak acids and that there are pros and cons for both weaker and stronger acids, as taught by EMD Millipore. It would be obvious to one skilled in the art, before the effective filing date, that host cell protein can be differentially precipitated from antibody eluate by modulating pH and conductivity, which is related to the acid concentration, as taught by Hickman et al with background on inherent properties from Kornecki et al. As such, the acid selection during the elution step impacts both the elution and the subsequent precipitation step by its impact on pH, conductivity, and ionic strength, and modulating these properties can be done by both a strong acid and weak acid.
One skilled in the art, before the effective filing date of the instant application, would be motivated to reduce the host cell protein content using the known method of differential precipitation because removing host cell protein is vital to antibody purification and precipitation is a relatively simple method of helping to achieve this. One skilled in the art, before the effective filing date of the instant application, would be motivated to utilize strong and weak acids for the known properties they impart to the elution and eluate, as taught by EMD Millipore. Because the acid plays a large role in both the antibody elution step and the host cell protein precipitation step, one skilled in the art, before the effective filing date of the instant application, would be motivated to choose acids that enabled and optimized both steps.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success of reducing host cell protein content in a protein preparation based on the teachings of precipitation of host cell proteins by Hickman et al as it concurs with the expected properties one could harness from strong acids and weak acids to optimize elution or precipitation conditions, as taught by EMD Millipore, informed by the inherent properties of the acids and proteins taught by Kornecki et al.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that combining two previously unintegrated prior art elements of similar devices according to known methods to yield predictable results is obvious. In this case, the two unintegrated prior art elements are the strong acid and weak acid in the elution step, and the similar "device" they are a part of is the method of antibody elution from affinity chromatography and subsequent HCP reduction. The prior art teaches antibody elution with a strong acid and antibody elution with a weak acid (EMD Millipore). The prior art teaches subsequent differential precipitation of HCP from antibodies based on pH and conductivity. Therefore, the prior art includes each element claimed in the appropriately same device, although not necessarily from a single reference, with the difference being the lack of actual combination of the elements in a single reference. One skilled in the art, before the effective filing date of the instant application, could have combined the elements, and in combination, each element performs the function as it does separately - that is, both strong acid and weak acid work to modulate pH and elute the antibody as expected and subsequent titration of the acid to a specific pH leads to precipitation of host cell protein, as expected by the inherent properties of the relevant acids and proteins. The strong acid and weak acid would still have their inherent and calculatable effects on the pH, buffering capacity, volatility, and conductivity of the eluate depending on their concentration. Therefore, the antibody and host cell protein would be expected to co-elute in acid and the host cell protein would be expected to precipitate at a specific pH and conductivity, regardless of whether it was a combination of acids or not, so long as the final pH and conductivity were controlled. One skilled in the art, before the effective filing date of the instant application, would have recognized the results of the combination were predictable. The addition of a strong acid to a weaker acid could create a desired balance of pH, conductivity, and buffering capacity that enables effective eluting the antibody of interest from the affinity chromatography and subsequent precipitation of the specific host cell protein of interest. Subsequent depth filtration, which removes precipitants and aggregates, would remove the host cell protein precipitant, better than if the host cell protein had remained soluble.
Claims 2-3, 7-10, 12-17, 19-23, and 51-52 depend on claim 1. The teachings of the prior art regarding claim 1 are incorporated in its entirety here and further described below.
Regarding claim 2, Hickman et al further teaches the chromatography column is a Protein A affinity chromatography column (paragraph 247 teaches MabSelect is a Protein A affinity column, paragraph 226 teaches use in Example 1 purification).
Regarding claim 3, Hickman et al teaches elution by weak acids or elution by strong acids (paragraph 95). Hickman et al does not explicitly teach the pKa of the acids used in elution, as is relevant to claim 3. However, EMD Millipore teaches elution with a strong acid (citric acid) and elution with a weak acid (acetic acid), which inherently have pKa values that meet the limitations of claim 3, which is also consistent with the definition provided by the instant specification on page 17.
Regarding claims 7-10, Hickman et al further teaches the method further comprising a step of viral inactivation of the eluate, which could be through low pH since it is a very efficient and widely used in antibody purification processes (paragraph 281). Hickman teaches holding the eluate for about 0 to 180 minutes hours at pH 2.5-3.8 for low pH treatment did not significantly affect antibody aggregation (paragraph 283), and specifically taught adjusting the pH of the eluate from said step of eluting the protein from the chromatography column, to below about pH 4.0, specifically about pH 3.3 to about pH 3.7, or about pH 3.5 (paragraph 95) and wherein the eluate is maintained at below about pH 4.0 for about 0 minutes to about 180 minutes (paragraph 283).
Regarding claims 12-15, Hickman et al further teaches the step of raising the pH of the eluate comprises raising the pH to about pH 6.5 to about pH 7.5 (paragraph 9) or neutral (paragraph 95) by adding Tris (paragraph 9) (and e.g. Example 1 paragraph 230).
Further regarding claim 15, Hickman et al does not explicitly teach the ionic strength of the solution applied to the depth filter, but teaches the solution is diluted with water so that the conductivity is reduced to a particular value or range, depending on the antibody being purified (paragraphs 251 and 260). Hickman et al teaches the close relationship between ionic strength and conductivity (paragraph 111). Hickman et al teaches that once conductivity started to reach a certain value, precipitation started to occur over time, and this precipitation was found to not be antibody (paragraph 251). It can be understood by one skilled in the art, before the effective filing date of the instant application, that the conductivity, and therefore ionic strength, is optimized to differentially precipitate the host cell protein and minimize precipitating the antibody. The specific ionic strength, conductivity, and pH combination required to achieve this differential precipitation differs depending on the particular antibody and host cell protein, and their isoelectric points and antibody-HCP structural interactions. As such, it can be understood that the conductivity, and relatedly ionic strength, is a result-effective variable that a person of ordinary skill in the art would routinely optimize to differentially precipitate antibody and host cell proteins for reduction of host cell protein in antibody purification preparations. As such, differences in ranges of result-effective variables between the art and instant application are not novel and are a matter of routine optimization. It would have been obvious to one of ordinary skill in the art, before the effective filing date of the instant application, to routinely screen for the conductivity or ionic strength needed to precipitate out host cell protein but not antibody, for the particular antibody-HCP pair, as disclosed by Hickman et al. One of ordinary skill in the art would be motivated to choose values of conductivity or ionic strength that minimize antibody loss and maximize host cell protein precipitation for the particular antibody preparation of interest, instead of using a set value for every antibody preparation. One of ordinary skill in the art would have had a reasonable expectation of success for the reasons underlying this method of purification, which is the basis of the isoelectric focusing separation system taught by Hickman et al (paragraph 141).
Regarding claim 16, Hickman et al further teaches a step of subjecting the depth filtered protein preparation to ion exchange chromatography (Example 1 paragraph 251).
Regarding claim 17, Hickman et al further teaches the host cell protein content in the filtered protein preparation is reduced to less than 100 ppm (Example 1 paragraph 255 and Table 1). Hickman et al teaches that host cell protein ng/mg antibody is equivalent to host cell protein ppm of antibody (paragraph 255), and that the host cell protein was reduced to about 11 ng/mg antibody after Phenyl HP purification (Table 1), which is equivalent to 11 ppm.
Regarding claim 19, Hickman et al further teaches the protein preparation comprises a harvested cell culture fluid, a capture pool, or a recovered protein pool (Example 1 paragraphs 227-228).
Regarding claim 20-23, Hickman et al further teaches the protein is a therapeutic or diagnostic protein, specifically a IgG1 monoclonal antibody (paragraph 142).
Regarding claim 51-52, Hickman et al further teaches that the host cell is a mammalian cell, which is a CHO cell (Example 1, paragraph 244).
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Tee et al (Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay, bioRxiv preprint; published 08/02/2020).
Hickman et al does not explicitly teach that the protein is an anti-SARS-COV-2 antibody, as is relevant to claim 24. Hickman et al provides an anti-IL2 IgG1 antibody as an example but does not limit the antibody that can be purified by what the antibody targets. Hickman et al discloses that the choice of affinity chromatography resin, such as Protein A, Protein G, or other commonly used resin, can be changed according to their affinity to subclass of the IgG of interest.
However, Tee et al teaches a method of antibody purification by affinity chromatography of an anti-SARS-COV-2 antibody recombinantly expressed in CHO cells wherein host cell proteins are reduced (Abstract and page 6, section: Protein purification of CR3022 mAb). Tee et al also teaches pH-mediated differential precipitation of host cell protein that can be filtered away from the antibody (page 6, section: Protein purification of CR3022 mAb).
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the antibody purification method taught by Hickman et al is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, as taught by Tee et al. One skilled in the art, before the effective filing date of the instant application, would be motivated to purify anti-SARS-COV-2 antibodies to develop diagnostic strategies for SARS-COV-2, which is the causative virus of the COVID-19 global pandemic, as taught by Tee et al (Introduction, paragraph 1).
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the purification techniques taught by Hickman et al in view of Tee et al and further in view of EMD Millipore because Hickman et al and Tee et al discloses similar methods of antibody purification by affinity chromatography, albeit Hickman with more details, and Tee et al more explicitly teaching the method could be applied to an anti-SARS-COV-2 antibody. As such, the anti-SARS-COV-2 antibody is compatible with the affinity chromatography and has a higher precipitation pH than the host cell protein, and therefore, would be expected to be compatible with the method from Hickman et al.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific antibodies being purified. The prior art as taught by Hickman et al provides an affinity chromatography method for purifying antibodies, but does not explicitly teach an anti-SARS-COV-2 antibody. Tee et al provides a similar affinity chromatography method for purifying antibodies, explicitly teaching an anti-SARS-COV-2 antibody. The substituted components, the anti-SARS-COV-2 antibody from Tee et al and the anti-IL2 IgG1 from Hickman et al, were known in the art before the effective filing date of the instant application to have similar relevant properties for antibody elution and HCP precipitation. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A or Protein G columns. Prior art teaches host cell proteins and both antibodies precipitate at different pH's. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable.
Claims 4-6 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Wang et al (WO2016149088A1, Use of alkaline washes during chromatography to remove impurities; filed 03/11/2016) and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012).
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012).
Claims 4-6 and 11 depend on claim 1. The teachings of the prior art regarding claim 1 are incorporated in its entirety here and further described below.
EMD Millipore further teaches 20 mM acetic acid as a potential antibody elution buffer, as is relevant to claims 4-6.
Regarding claims 4-6, Hickman et al in view of EMD Millipore do not explicitly teach phosphoric acid or lactic acid, nor the approximate concentrations of these acids.
However, Wang et al teaches an elution with 10 mM phosphoric acid, pH 3.0 buffer, as is relevant to claim 4 and 5. Additionally, Golken et al teaches that in steps that use an organic acid, such as during elution, phosphoric acid and lactic acid, could be used in the elution buffer, as is relevant to claim 4 and 6.
Claim 11 depends on claims 1 and 8. The teachings of the prior art regarding claims 1 and 8 are incorporated in its entirety here and further described below.
Regarding claims 11, Hickman et al does teach a nonlimiting list of example acids for low pH treatment, but also teaches that other suitable acids could be added to the sample to adjust the pH (paragraph 95).
Hickman et al in view of EMD Millipore do not explicitly teach the claimed acids used to adjust the pH of the eluate for low pH treatment.
However, Golken et al teaches that in the low pH treatment step, after protein recovery, HCl can be used to titrate the solution (page 13, line 26-30 and Figure 1), meeting the limitation of claim 11. Hickman et al and Golken et al both teach low pH treatment at 3.5, meeting the limitation of claim 11.
Regarding claims 4-6, it would have been obvious to one skilled in the art, before the effective filing date of the instant application, to substitute the strong acids taught in the art for protein purification with other strong acids known in the art for protein purification, guided by the teachings of Wang et al and Golken et al which teaches that antibody elution can occur with a variety of acids, including phosphoric acid and lactic acid . Regarding claim 11, it would have been obvious to one skilled in the art, before the effective filing date of the instant application, to substitute the acid taught in Hickman et al with the HCl acid taught by Golken et al because it can be understood the acid is used to adjust the eluate pH to the desired pH and conductivity and that the choice of acids would depend on its compatibility with the achieving the desired effect. The art teaches that HCl and other acids can achieve pH titration, thus the choice is at the discretion of one skilled in the art, guided by the desired pH, conductivity, and ionic strength.
The common teachings amongst these protein purification references is that pH and acid concentration is optimized for the desired effects on the protein, such as elution efficiency, prevention of permanent protein denaturation, and host cell protein removal. As such, pH and acid concentration are result-effective variables.
Regarding claims 4-6, one skilled in the art, before the effective filing date of the instant application, would be motivated to substitute citrate acid with the stronger phosphoric acid or lactic acid to decrease the pH and increase the conductivity to enable efficient antibody elution and host cell protein precipitation. Regarding claim 11, one skilled in the art, before the effective filing date of the instant application, would be motivated to substitute the acetic acid with stronger acids, like HCl, taught in the art to also titrate protein eluate for low pH treatment because strong acids like HCl inherently can achieve and maintain the low pH more effectively than weaker acids.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because the acids are all taught as useable in protein elution and would function to precipitate host cell proteins as expected according to their inherent properties based on the acid's strength. One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because the ability for HCl and other strong acids to achieve and maintain a low pH is an inherent property of the acid.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific strong acids, which serve the same purpose of reducing the pH and buffering the pH more effectively than a weaker acid could. The prior art differ from the instant application by the substitution of strong acids phosphoric acid or lactic acid for the strong acid citrate acid taught in the base protein purification method, but additional prior art references teach the use of these phosphoric acid or lactic acid in protein purification methods. The prior art differ from the instant application by the substitution of HCl for weaker acids, but additional prior art teaches HCl in low pH treatments. The substituted components, phosphoric acid or lactic acid, or HCl and their functions were known in the art before the effective filing date of the instant application. Prior art teaches that phosphoric and lactic acid can be used in protein elution buffers for affinity chromatography at the approximate concentrations and low pH's disclosed. Prior art teaches HCl can be used for low pH treatment. One skilled in the art, before the effective filing date of the instant application, could have substituted one known element for another known element in the base protein purification method taught by Hickman et al and the results of the substitution would have been predictable, specifically regarding the calculatable pH of the eluate and the subsequent elution and precipitation of host cell protein, as described for claim 1 above.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Tran et al (Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography, Journal of Chromatography A; published 1/22/2016).
Claim 18 depends on claim 1. The teachings of the prior art regarding claims 1 are incorporated in its entirety here and further described below.
Hickman et al further teaches the host cell protein content in the filtered protein preparation is reduced to less than 100 ppm (paragraph 255 and Table 1). Hickman et al teaches that host cell protein ng/mg antibody is equivalent to ppm of host cell protein (HCP) (paragraph 255), and that the host cell protein was reduced to about 11 ng/mg antibody (Table 1), which is equivalent to 11 ppm. Hickman et al also teaches that the protein was recombinantly expressed in CHO cells and contains CHO host cell proteins in the eluate (e.g. paragraph 247).
Hickman et al and EMD Millipore do not explicitly teach that the host cell protein content comprises PLBL2 and that the PLBL2 is reduced to less than 100 ppm.
However, Tran et al teaches that PLBL2 (Phospholipase B-like 2) is host cell protein derived from Chinese Hamster Ovary (CHO) cells (Introduction, paragraph 3), which are the same host cells used in all Hickman et al experiments. Hickman et al, Tran et al, and the instant application all use LC-MS and ELISA to measure total host cell protein and PLBL2, leading to PLBL2 being a population encompassed by the total host cell protein measurement. In other words, although Hickman et al does not explicitly teach PLBL2, the total host cell protein taught by Hickman et al encompasses the total PLBL2. As such, if the total host cell protein taught by Hickman et al is less than 100 ppm, then the total PLBL2 is also less than 100 ppm.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the total host cell protein from CHO cells would inherently contain PLBL2. Further, it would have been obvious to one skilled in the art, before the effective filing date of the instant application, that host cell protein reduction is desired because it impacts both product quality and patient safety (Tran et al, Introduction paragraph 1), and PLBL2 is specifically of interest because is difficult to remove sufficiently (Tran et al, Introduction paragraph 3).
One skilled in the art, before the effective filing date of the instant application, would be motivated to reduce host cell proteins, and particularly PLBL2, to increase product safety and patient safety.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because of the prior success in removal of total host cell proteins, which encompasses PLBL2 when protein was recombinantly expressed by CHO cells, as is relevant to the method taught by Hickman et al.
Claims 25-27 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Westendorf '828 (US11370828B2, Anti-coronavirus antibodies and methods of use; effectively filed 03/09/2020).
Claim 25-27 depend on claim 1 and are related to claim 24 as they recite anti-COV-SARS-2 antibodies. The teachings of the prior art regarding claims 1 and 24 are incorporated in its entirety here and further described below.
Hickman et al in view of EMD Millipore does not explicitly teach the particular antibody or sequences from instant claims 25-27.
However, Westendorf '828 teaches an anti-SARS-COV-2 antibody with VH sequence encoded by SEQ ID NO: 1255 paired with VL sequence encoded by SEQ ID NO: 1256, which matches SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and teaches that this claimed antibody is an IgG1 antibody (Claim 1). Westendorf et al also teaches an anti-SARS-COV-2 antibody with HC sequence encoded by SEQ ID NO: 5363 paired with VL sequence encoded by SEQ ID NO: 5364, which matches SEQ ID NO: 3 and SEQ ID NO: 4, respectively (Claim 7 and Column 141, lines 30-35). Westendorf '828 teaches that the SARS-COV-2-binding antibodies disclosed is compatible with Protein A affinity chromatography (column 132, lines 50-52).
The specifications of the instant application define bamlanivimab as an antibody encoded by paired VH and VL sequences SEQ ID NO: 1 and 2, which is encompassed by the HC and LC paired sequences encoded by SEQ ID NO: 3 and 4. Therefore, it can be understood that Westendorf et al also teaches bamlanivimab as an anti-SARS-COV-2. It also follows that claims 25-27 are directed to an IgG1 antibody.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the methods taught by Hickman et al in view of Tee et al and further in view of EMD Millipore is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, and the particular anti-SARS-COV-2 antibodies taught by Westendorf '828, especially since both antibodies from Hickman et al and Westendorf et al are IgG1 isotypes. Additionally, Westendorf '828 teaches that this SARS-COV-2-binding antibody is compatible with Protein A affinity chromatography (column 132, lines 50-52).
One skilled in the art, before the effective filing date of the instant application, would be motivated to purify the specific anti-SARS-COV-2 antibody taught by Westendorf ' 828 because it targets SARS-COV-2 (Abstract), which is the causative virus of the COVID-19 global pandemic.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the substitution of a specific anti-SARS-COV-2 antibody taught by Westendorf '828 for the anti-SARS-COV-2 purification method taught by Hickman et al in view of EMD Millipore, especially since both antibodies from Hickman et al and Westendorf '828 are IgG1 isotypes and Westendorf '828 discloses purification by Protein A affinity chromatography.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific anti-SARS-COV-2 antibodies being purified. The prior art as taught by Hickman et al in view of Tee et al and further in view of EMD Millipore provides an affinity chromatography method for purifying antibodies, specifically an IgG1 antibody and specifically an anti-SARS-COV-2 antibody as an example. Westendorf '828 teaches the specific anti-SARS-COV-2 antibody instantly disclosed. Therefore the substituted components, the anti-SARS-COV-2 antibodies from Westendorf '828, were known in the art before the effective filing date of the instant application. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A or Protein G columns. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable due to shared compatibility with affinity chromatography purification.
Claims 28-29 rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Walker et al (US11192940B2, Compounds specific to coronavirus S protein and uses thereof, effectively filed 04/10/2020).
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Walker et al (US11192940B2, Compounds specific to coronavirus S protein and uses thereof, effectively filed 04/10/2020) and further in view of Chiu et al (Antibody Structure and Function: The Basis for Engineering Therapeutics, published 2019).
Hickman et al in view of EMD Millipore does not explicitly teach the particular antibody or sequences from instant claims 28-30.
However, Walker et al teaches an anti-SARS-COV-2 antibody with VH sequence encoded by SEQ ID NO: 82 paired with VL sequence encoded by SEQ ID NO: 91, which matches instant SEQ ID NO: 5 and SEQ ID NO: 6, respectively, as is relevant to claim 29. Walker et al also teaches an anti-SARS-COV-2 antibody with LC sequence encoded by SEQ ID NO: 91, which matches with instant SEQ ID NO: 8, as is relevant to claim 30.
The specifications of the instant application define etesevimab as an antibody encoded by paired VH and VL sequences SEQ ID NO: 5 and 6. Therefore, it can be understood that Walker et al also teaches etesevimab as an anti-SARS-COV-2, as is relevant to claim 28.
Hickman et al in view of EMD Millipore in view of Walker et al does not explicitly teach a sequence matching 100% to SEQ ID NO: 7, as is relevant to claim 30.
However, regarding claim 30, Walker et al teaches an anti-SARS-COV-2 antibody with HC sequence encoded by SEQ ID NO: 81 paired with VL sequence encoded by SEQ ID NO: 91, which has a 99.6% sequence homology to SEQ ID NO: 7, differing by 3 conservative residue substitutions outside of the variable region.
However, Chiu et al teaches that the Fc region sequence is shared between antibody subtype, and thus does not define an individual antibody. The Fc region does not have antigen binding properties but may be engineered to modulate effector function activities, half-life, and Protein A binding affinity (section 2, paragraph 1 and Table 2). The instant specification also defines etesevimab by its variable sequence. Therefore a few conservative amino acid substitutions outside of the variable region of an antibody is an obvious variant of an antibody with the same variable region sequence, but with routine residue optimization in the Fc region. As such, the few conservative substitution outside the variable region is an obvious variant of the etesevimab antibody as disclosed by Walker et al.
Hickman et al does not limit the antibody that can be purified using their respective antibody purification method based on the target antigen. Hickman et al discloses that the choice of known affinity chromatography resin, such as Protein A, Protein G, or other commonly used resin, can be changed according to their affinity to subclass of the IgG of interest.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the methods taught by Hickman et al in view of EMD Millipore is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, and the particular anti-SARS-COV-2 antibodies taught by Walker et al, especially since both antibodies from Hickman et al and Walker et al are taught to be purified using Protein A affinity chromatography (Walker et al, column 92, lines 51-55). One skilled in the art, before the effective filing date of the instant application, would be motivated to purify the specific anti-SARS-COV-2 antibody taught by Walker et al because it targets SARS-COV-2 (Abstract), which is the causative virus of the COVID-19 global pandemic.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the substitution of a specific anti-SARS-COV-2 antibody taught by Walker et al for the anti-SARS-COV-2 purification method taught by Hickman et al in view of EMD Millipore, especially since both antibodies from Hickman et al and Walker et al are taught to be purified using Protein A affinity chromatography (Walker et al, column 92, lines 51-55).
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, to routinely optimize the Fc region of antibody with conservative mutations that may impart Fc effector function improvements without changing the binding target of the antibody, as taught by Chau et al, and that this variant would be an obvious variant of an antibody with the same variable sequence.
One skilled in the art, before the effective filing date of the instant application, would be motivated to screen for and optimize specific residues outside of the binding region to conserve the main antibody binding function but potentially improve half-life and effector functions.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success of retaining the same antigen-targeting function and sequence that defines the antibody since the conservative mutations are outside the variable region.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific anti-SARS-COV-2 antibodies being purified. The prior art as taught by Hickman et al in view of EMD Millipore provides an affinity chromatography method for purifying IgG antibodies. Walker et al teaches the specific anti-SARS-COV-2 IgG antibody instantly disclosed. Therefore the substituted components, the anti-SARS-COV-2 antibodies from Walker et al and the IgG antibody from Hickman et al, were known in the art before the effective filing date of the instant application. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A or Protein G columns. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable due to shared compatibility with affinity chromatography purification.
Furthermore, KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses applying a known technique to a known device (method, or product) ready for improvement to yield predictable results is obvious. Conservative mutations outside the variable region to stabilize or modulate effector function while retaining the targeting and variable sequence that defines an antibody is a known improvement to base antibodies. Furthermore, the results of these conservative mutations are predictable to one skilled in the art before the effective filing date of the instant application because the effects of conservative mutations are understood in the art.
Claims 31-33 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017), as applied to claims 1-2, 7-10, 12-17, 19-23 above, and further in view of Westendorf '541 (US11447541B1, Anti-coronavirus antibodies and methods of use; effectively filed 03/09/2020).
Hickman et al in view of EMD Millipore does not explicitly teach the particular antibody or sequences from instant claims 31-33.
However, Westendorf '541 teaches an anti-SARS-COV-2 antibody with VH sequence encoded by SEQ ID NO: 4949 paired with VL sequence encoded by SEQ ID NO: 4950, which matches instant SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and teaches that this claimed antibody is an IgG1 antibody (Claim 1). Westendorf et al also teaches an anti-SARS-COV-2 antibody with HC sequence encoded by SEQ ID NO: 5735 paired with VL sequence encoded by SEQ ID NO: 5736, which matches instant SEQ ID NO: 11 and SEQ ID NO: 12, respectively (Claim 6).
The specifications of the instant application define bebtelovimab as an antibody encoded by paired VH and VL sequences SEQ ID NO: 9 and 10, which is encompassed by the HC and LC paired sequences encoded by SEQ ID NO: 11 and 12. Therefore, it can be understood that Walker et al also teaches bebtelovimab as an anti-SARS-COV-2.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the methods taught by Hickman et al in view of EMD Millipore is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, and the particular anti-SARS-COV-2 antibodies taught by Westendorf '541, especially since both antibodies from Hickman et al and Westendorf '541 can be affinity purified by Protein A (Westendorf '541 column 259, line 40).
One skilled in the art, before the effective filing date of the instant application, would be motivated to purify the specific anti-SARS-COV-2 antibody taught by Westendorf '541 because it targets SARS-COV-2 (Abstract), which is the causative virus of the COVID-19 global pandemic.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the substitution of a specific anti-SARS-COV-2 antibody taught by Westendorf '541for the anti-SARS-COV-2 purification method taught by Hickman et al in view of EMD Millipore, especially since both antibodies from Hickman et al and Westendorf '541 are taught to be purified by protein A, and thus the antibody from Westendorf '541 would be reasonable compatible with the methods of Hickman et al.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific antibodies being purified. The prior art as taught by Hickman et al in view of EMD Millipore provides an affinity chromatography method for purifying antibodies via Protein A affinity chromatography. Westendorf '541 teaches the specific anti-SARS-COV-2 antibody instantly disclosed. Therefore the substituted components, the antibodies, were known in the art before the effective filing date of the instant application. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable due to shared compatibility with affinity chromatography purification.
Claim 34 is rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017) and further in view of Wang et al (WO2016149088A1, Use of alkaline washes during chromatography to remove impurities; filed 03/11/2016) and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012), and further in view of Tee et al (Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay, bioRxiv preprint; published 08/02/2020).
Regarding claim 34, Hickman et al teaches:
a method of reducing host cell protein content in an antibody preparation (Example 1 paragraph 247 and Table 1) recombinantly produced in a host cell comprising cell (paragraphs 72-73 and Example 1 paragraph 244):
a. subjecting the antibody preparation recombinantly produced in a host cell to a Protein A affinity chromatography column (paragraph 226 teaches MabSelect use in Example 1; paragraph 247 teaches MabSelect is a Protein A affinity column);
b. eluting the antibody from the chromatography column using acid to obtain an eluate comprising the antibody (Example 1 paragraph 227) and that this acid could be acetic acid (e.g. Example 1 Table 5), citrate acid, or other suitable acids (claim 2);
c. wherein the pH is lowered to about pH 3.3 to about pH 3.7 (paragraph 95), and wherein the eluate is maintained at about pH 3.3 to about pH 3.7 for about 0 minutes to about 180 minutes (paragraph 224 and 283);
d. raising the pH of the eluate comprising the antibody, wherein the pH is raised to about pH 6.5 to about pH 7.5 (Example 1 paragraph 230); and
e. subjecting the eluate comprising the antibody to a depth filter, and obtaining a filtered antibody preparation (Example 1 paragraph 251),
wherein host cell protein content in the filtered anti-SARS-COV-2 antibody preparation is reduced to about 0 ppm to about 20 ppm (Example 1 paragraph 255 and Table 1),
and wherein the antibody is an IgGI antibody (paragraph 142).
Regarding the host cell protein content levels, Hickman et al teaches that host cell protein ng/mg antibody is equivalent to host cell protein ppm of antibody (paragraph 255), and that the host cell protein was reduced to about 11 ng/mg antibody after Phenyl HP purification (Table 1), which is equivalent to 11 ppm.
Hickman et al does not explicitly teach eluting the antibody with a combination of acids comprising of acetic acid and phosphoric acid or a combination of acetic acid and lactic acid.
However, Hickman et al further teaches that elution of antibodies off of Protein A affinity chromatography resin occurs at an acidic pH, with known optimal pH ranges (Example 1 paragraph 227). Hickman et al further teaches that pH and conductivity can be used to differentially precipitate and separate out host cell protein from antibodies using a depth filter (paragraph 251). Particularly, increasing the pH from pH 5 to about 8.0 (or about 6.0-about 8.0, paragraph 9) and lowering the conductivity of the protein solution led to precipitation of protein that was mostly not antibody and led to HCP reduction after depth filtration (paragraph 251 and Table 2).
EMD Millipore teaches the exact pH and choice of acid as an eluant are factors to consider for protein elution and downstream steps (Introduction). EMD Millipore teaches that the inherent properties of the acid used to elute antibodies, such as the acid's pKa, determine the pH, buffering capacity, volatility, and conductivity of the eluate (Conclusions and Recommendations). EMD Millipore teaches the pros and cons of using eluants instantly defined as a "weak acid", such as acetic acid, versus instantly defined as a "strong acid", such as citric acid (entire document). It thus follows that by the inherent properties of acids, a mixture of these acids would modulate these eluant and downstream properties; for example the properties of a strong acid and weak acid can balance each other's effects.
Reference Kornecki et al is cited to provide relevant inherent properties for the record. Kornecki et al teaches that there is differential acid-induced precipitation of proteins depending on the isoelectric point (pI) of the protein (page 8), which is the pH where a protein has neutral charges and effectively is the pH where a protein is leas soluble. This is an inherent property of each protein. Proteins that have different isoelectric points therefore precipitate at different pH (pages 8-9). The protein amino acid sequence generally allows calculation of its isoelectric point.
Therefore, the pH of the eluant can be chosen to determine the precipitation properties of the host cell proteins and antibodies.
Furthermore, Wang et al teaches an elution with 10 mM phosphoric acid, pH 3.0 buffer. Additionally, Golken et al teaches that in steps that use an organic acid, such as during elution, phosphoric acid and lactic acid, could be used in the elution buffer.
The instant specification discloses removal of HCP after depth filtration (instant Specification, e.g. Table 1), implying that the HCP was a precipitant, as depth filtration is used to remove particulates. The instant disclosure is silent on the mechanism underlying differential HCP removal as disclosed in the instant Tables and how the results are different from those already known in the art. The methods and results instantly disclosed can be explained by the technique of differential precipitation of HCP from antibodies and optimization of antibody elution, as taught by Hickman et al, utilizing the properties of different acids, as taught by EMD Millipore, that was known to one skilled in the art, before the effective filing date of the instant application.
HCP and antibodies are coeluted from affinity chromatography at low pH, leading to relatively similar HCP ppm after Protein A elution (Table 2 and Table 3). The eluate is eventually neutralized to about pH 7.0-7.25 before depth filtration (Example 1 and 2). Table 6 can be explained by the difference in isoelectric points of host cell proteins and antibodies leading to differential precipitation. At pH 5, HCP is still slightly soluble, as taught by Hickman et al and so not removed by depth filtration. However, upon increasing the pH to neutral, the HCP reaches and exceeds its isoelectric point and precipitates or becomes positively charged, which may lead to increased electrostatic interactions with the also positively charged antibodies, leading to aggregation. As such, these precipitants and aggregates can be removed by depth filtration.
All combinations of weak and strong acids as elution buffer reduces the HCP, but the instant application does not compare the results to elution with acetic acid alone. The reason for the difference in HCP ppm in Tables 2-3 between the different combinations of weak and strong acids could be explained by combinations of reasons related to the difference in acid strength, buffering capacity, and volatility of the acids. Acetic acid used in each elution buffer is a volatile weak acid with a low buffering capacity. Citric acid is a weaker acid than lactic acid and phosphoric acid. Starting with an eluate comprising of weaker acids with lower buffering capacity, the eluate will have a higher pH, and so lowering the pH of the eluate for viral inactivation will therefore require more HCl, a very strong acid, and subsequently more base to neutralize it, leading to a higher ionic strength and higher conductivity mixture applied to the depth filter than if starting with an eluate comprising of stronger acids. Higher ionic strength buffers help prevent electrostatic protein-protein interactions, leading to increased solubility of proteins, and therefore less precipitated HCP and less HCP removal by depth filtration. This is also consistent with the results of instant Table 5.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, to harness the desired inherent properties of both a strong acid and weak acid for an eluant as taught by EMD Millipore to optimize desired antibody elution conditions and precipitation of host cell protein for removal from the antibody eluate, as taught by the method of Hickman et al and supported by Kornecki et al, which teaches the inherent properties and resultant effects of the acid and proteins that underly this method of host cell reduction in protein purification. It would be obvious to one skilled in the art, before the effective filing date, that the pH, conductivity, and ionic strength of the eluant can be modulated using both strong acids or weak acids and that there are pros and cons for both weaker and stronger acids, as taught by EMD Millipore. It would be obvious to one skilled in the art, before the effective filing date, that host cell protein can be differentially precipitated from antibody eluate by modulating pH and conductivity, which is related to the acid concentration, as taught by Hickman et al with background on inherent properties from Kornecki et al. As such, the acid selection during the elution step impacts both the elution and the subsequent precipitation step by its impact on pH, conductivity, and ionic strength, and modulating these properties can be done by both a strong acid and weak acid.
One skilled in the art, before the effective filing date of the instant application, would be motivated to reduce the host cell protein content using the known method of differential precipitation because removing host cell protein is vital to antibody purification and precipitation is a relatively simple method of helping to achieve this. One skilled in the art, before the effective filing date of the instant application, would be motivated to utilize strong and weak acids for the known properties they impart to the elution and eluate, as taught by EMD Millipore. Because the acid plays a large role in both the antibody elution step and the host cell protein precipitation step, one skilled in the art, before the effective filing date of the instant application, would be motivated to choose acids that enabled and optimized both steps.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success of reducing host cell protein content in a protein preparation based on the teachings of precipitation of host cell proteins by Hickman et al as it concurs with the expected properties one could harness from strong acids and weak acids to optimize elution or precipitation conditions, as taught by EMD Millipore, informed by the inherent properties of the acids and proteins taught by Kornecki et al.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that combining two previously unintegrated prior art elements of similar devices according to known methods to yield predictable results is obvious. In this case, the two unintegrated prior art elements are the strong acid and weak acid in the elution step, and the similar "device" they are a part of is the method of antibody elution from affinity chromatography and subsequent HCP reduction. The prior art teaches antibody elution with a strong acid and antibody elution with a weak acid (EMD Millipore). The prior art teaches subsequent differential precipitation of HCP from antibodies based on pH and conductivity. Therefore, the prior art includes each element claimed in the appropriately same device, although not necessarily from a single reference, with the difference being the lack of actual combination of the elements in a single reference. One skilled in the art, before the effective filing date of the instant application, could have combined the elements, and in combination, each element performs the function as it does separately - that is, both strong acid and weak acid work to modulate pH and elute the antibody as expected and subsequent titration of the acid to a specific pH leads to precipitation of host cell protein, as expected by the inherent properties of the relevant acids and proteins. The strong acid and weak acid would still have their inherent and calculatable effects on the pH, buffering capacity, volatility, and conductivity of the eluate depending on their concentration. Therefore, the antibody and host cell protein would be expected to co-elute in acid and the host cell protein would be expected to precipitate at a specific pH and conductivity, regardless of whether it was a combination of acids or not, so long as the final pH and conductivity were controlled. One skilled in the art, before the effective filing date of the instant application, would have recognized the results of the combination were predictable. The addition of a strong acid to a weaker acid could create a desired balance of pH, conductivity, and buffering capacity that enables effective eluting the antibody of interest from the affinity chromatography and subsequent precipitation of the specific host cell protein of interest. Subsequent depth filtration, which removes precipitants and aggregates, would remove the host cell protein precipitant, better than if the host cell protein had remained soluble.
Regarding the specific combination of acids, it would have been obvious to one skilled in the art, before the effective filing date of the instant application, to substitute the strong acids taught in the art for protein purification with other strong acids known in the art for protein purification, guided by the teachings of Wang et al and Golken et al which teaches that antibody elution can occur with a variety of acids, including phosphoric acid and lactic acid
The common teachings amongst these protein purification references is that pH and acid concentration is optimized for the desired effects on the protein, such as elution efficiency, prevention of permanent protein denaturation, and host cell protein removal. As such, pH and acid concentration are result-effective variables.
Regarding the exact combination of acids, one skilled in the art, before the effective filing date of the instant application, would be motivated to substitute citrate acid with the stronger phosphoric acid or lactic acid to decrease the pH and increase the conductivity to enable efficient antibody elution and host cell protein precipitation.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because the acids are all taught as useable in protein elution and would function to precipitate host cell proteins as expected according to their inherent properties based on the acid's strength.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific strong acids, which serve the same purpose of reducing the pH and buffering the pH more effectively than a weaker acid could. The prior art differ from the instant application by the substitution of strong acids phosphoric acid or lactic acid for the strong acid citrate acid taught in the base protein purification method, but additional prior art references teach the use of these phosphoric acid or lactic acid in protein purification methods. The substituted components, phosphoric acid or lactic acid and their functions were known in the art before the effective filing date of the instant application. Prior art teaches that phosphoric and lactic acid can be used in protein elution buffers for affinity chromatography at the approximate concentrations and low pH's disclosed.
Hickman et al does not explicitly teach that the protein is an anti-SARS-COV-2 antibody. Hickman et al provides an anti-IL2 IgG1 antibody as an example but does not limit the antibody that can be purified by what the antibody targets. Hickman et al discloses that the choice of affinity chromatography resin, such as Protein A, Protein G, or other commonly used resin, can be changed according to their affinity to subclass of the IgG of interest.
However, Tee et al teaches a method of antibody purification by affinity chromatography of an anti-SARS-COV-2 antibody recombinantly expressed in CHO cells wherein host cell proteins are reduced (Abstract and page 6, section: Protein purification of CR3022 mAb). Tee et al also teaches pH-mediated differential precipitation of host cell protein that can be filtered away from the antibody (page 6, section: Protein purification of CR3022 mAb).
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the antibody purification method taught by Hickman et al is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, as taught by Tee et al. One skilled in the art, before the effective filing date of the instant application, would be motivated to purify anti-SARS-COV-2 antibodies to develop diagnostic strategies for SARS-COV-2, which is the causative virus of the COVID-19 global pandemic, as taught by Tee et al (Introduction, paragraph 1).
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the purification techniques taught by Hickman et al in view of Tee et al and further in view of EMD Millipore because Hickman et al and Tee et al discloses similar methods of antibody purification by affinity chromatography, albeit Hickman with more details, and Tee et al more explicitly teaching the method could be applied to an anti-SARS-COV-2 antibody. As such, the anti-SARS-COV-2 antibody is compatible with the affinity chromatography and has a higher precipitation pH than the host cell protein, and therefore, would be expected to be compatible with the method from Hickman et al.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific antibodies being purified. The prior art as taught by Hickman et al provides an affinity chromatography method for purifying antibodies, but does not explicitly teach an anti-SARS-COV-2 antibody. Tee et al provides a similar affinity chromatography method for purifying antibodies, explicitly teaching an anti-SARS-COV-2 antibody. The substituted components, the anti-SARS-COV-2 antibody from Tee et al and the anti-IL2 IgG1 from Hickman et al, were known in the art before the effective filing date of the instant application to have similar relevant properties for antibody elution and HCP precipitation. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A or Protein G columns. Prior art teaches host cell proteins and both antibodies precipitate at different pH's. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable.
Hickman et al does not explicitly teach adjusting the pH of the eluate comprising the antibody by addition of about 20 mM HCl. However, Hickman et al does teach a nonlimiting list of example acids for low pH treatment, but also teaches that other suitable acids could be added to the sample to adjust the pH (paragraph 95). Furthermore, Golken et al teaches that in the low pH treatment step, after protein recovery, 100 mM HCl can be used to titrate the solution (page 13, line 26-30 and Figure 1). Hickman et al (paragraph 224) and Golken et al (Figure 1) both teach low pH treatment at pH 3.5.
Hickman et al in view of Golken et al do not explicitly teach the concentration of HCl is 20 mM.
Hickman et al teaches using 1M Tris to increase the pH of the eluate (paragraph 251).
Hickman et al does not explicitly teach the specific concentration of Tris to be 250 mM.
In regards to the choice of HCl, Hickman et al in view of EMD Millipore, as evidenced by Kornecki, and further in view of Wang et al and further in view of Golken et al, teaches that HCl and other acids can achieve pH titration, thus the choice is at the discretion of one skilled in the art, guided by, for example, the desired pH, conductivity, and ionic strength. A common teaching amongst these prior art references is that pH, conductivity, and ionic strength, which derives from acid concentration, is optimized for the desired effects on the protein, such as elution efficiency, prevention of permanent protein denaturation, and host cell protein removal via precipitation. As such, pH and acid concentration are result-effective variables, driven by the reduction of host cell protein content for the particular host cell protein and antibody. The desired pH, conductivity, and ionic strength needed to selectively precipitate the host cell protein while keeping the protein of interest soluble depends on the specific host cell protein and protein of interest. The claim is directed towards generic host cell proteins and generic antibodies and therefore, the acid concentration is optimized
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, to substitute the acid taught in Hickman et al with the HCl acid taught by Golken et al because it can be understood the acid is used to adjust the eluate pH to the desired pH and conductivity and that the choice of acids would depend on its compatibility with the achieving the desired effect.
One skilled in the art, before the effective filing date of the instant application, would be motivated to substitute the acetic acid with stronger acids, like HCl, taught in the art to also titrate protein eluate for low pH treatment because strong acids like HCl inherently can achieve and maintain the low pH more effectively than weaker acids.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success because the ability for HCl and other strong acids to achieve and maintain a low pH is an inherent property of the acid.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific strong acids, which serve the same purpose of reducing the pH and buffering the pH more effectively than a weaker acid could. The prior art differ from the instant application by the substitution of HCl for weaker acids, but additional prior art teaches HCl in low pH treatments. HCl acid and its titrating functions were known in the art before the effective filing date of the instant application. Prior art teaches HCl can be used for low pH treatment. One skilled in the art, before the effective filing date of the instant application, could have substituted one known element for another known element in the base protein purification method taught by Hickman et al and the results of the substitution would have been predictable, specifically regarding the calculatable pH of the eluate and the subsequent elution and precipitation of host cell protein, as described.
Further, regarding the specific concentrations of HCl and Tris, ionic strength and conductivity are determined by the concentration and composition of ions in a solution. Hickman et al teaches that once conductivity started to reach a certain value, precipitation started to occur over time, and this precipitation was found to not be antibody (paragraph 251). It can be understood by one skilled in the art, before the effective filing date of the instant application, that the conductivity, and therefore the concentration of ions is optimized to differentially precipitate the host cell protein and minimize precipitating the antibody. The specific ion concentration, and therefore, conductivity combination required to achieve this differential precipitation differs depending on the particular ions used, the antibody and host cell protein, and their isoelectric points and antibody-HCP structural interactions. The concentration of the acid and base used to titrate the antibody eluate before depth filtration determines the final conductivity. However, Hickman et al teaches diluting their eluate with water until the conductivity reached a certain value or range, depending on the antibody being purified (paragraphs 251 and 260). As such, it could also be possible to dilute the acid and base used to titrate the eluate individually. In other words, one skilled in the art before the effective filing date, in light of the teachings of Hickman et al in view of Golken et al teaching HCl and Tris as titrating agents, could understand that instead of starting at high HCl and Tris concentrations and then diluting with water at the end, one could dilute the HCl and Tris to lower concentrations first, and not have to dilute the resultant eluate. As such, it can be understood that the ion concentration is a result-effective variable that a person of ordinary skill in the art would routinely optimize to differentially precipitate antibody and host cell proteins for reduction of host cell protein in antibody purification preparations, and that the concentration of the ions could be diluted before addition to the protein eluate or after addition to the protein eluate. As such, differences in ranges of result-effective variables between the art and instant application are not novel and are a matter of routine optimization. It would have been obvious to one of ordinary skill in the art, before the effective filing date of the instant application, in determining the conductivity needed to precipitate out host cell protein but not antibody, to determine the corresponding concentration of ions. One of ordinary skill in the art, before the effective filing date, would be motivated to choose ion concentration values that minimize antibody loss and maximize host cell protein precipitation for the particular antibody preparation of interest. One of ordinary skill in the art, before the effective filing date would have reasonable expectation of success considering the addition of diluted HCl and Tris to the eluate or dilution of the HCl and Tris after titration of the eluate would have the same effect on the resultant conductivity.
Claim 35-50 and 55-56 depend on claim 34. The teachings of the prior art regarding claim 34 are incorporated in its entirety here and further described below.
Regarding claim 35, Wang et al further teaches an elution with 10 mM phosphoric acid, pH 3.0 buffer. EMD Millipore further teaches 20 mM acetic acid as a potential antibody elution buffer (Experimental Methods and Figure 4B). The motivation to combine acetic acid and phosphoric acid or lactic acid has already been described for claim 34.
Hickman et al (paragraphs 95 and 224) and Golken et al (Figure 1) both teach adjusting the pH of the eluate to about pH 3.5 for low pH treatment, as is relevant to claim 36, and that this pH adjustment achieves viral inactivation (Hickman et al, paragraph 95), as is relevant to claim 37.
Regarding claim 38, Hickman et al teaches neutralizing the pH before removing particulates by filtration (paragraph 95).
Regarding claim 39, Hickman et al does not explicitly teach the ionic strength of the solution applied to the depth filter, but teaches the solution is diluted with water so that the conductivity is reduced to a particular value or range, depending on the antibody being purified (paragraphs 251 and 260). Hickman et al teaches the close relationship between ionic strength and conductivity (paragraph 111). Hickman et al teaches that once conductivity started to reach a certain value, precipitation started to occur over time, and this precipitation was found to not be antibody (paragraph 251). It can be understood by one skilled in the art, before the effective filing date of the instant application, that the conductivity, and therefore ionic strength, is optimized to differentially precipitate the host cell protein and minimize precipitating the antibody. The specific ionic strength, conductivity, and pH combination required to achieve this differential precipitation differs depending on the particular antibody and host cell protein, and their isoelectric points and antibody-HCP structural interactions. As such, it can be understood that the conductivity, and relatedly ionic strength, is a result-effective variable that a person of ordinary skill in the art would routinely optimize to differentially precipitate antibody and host cell proteins for reduction of host cell protein in antibody purification preparations. As such, differences in ranges of result-effective variables between the art and instant application are not novel and are a matter of routine optimization. It would have been obvious to one of ordinary skill in the art, before the effective filing date of the instant application, to routinely screen for the conductivity or ionic strength needed to precipitate out host cell protein but not antibody, for the particular antibody-HCP pair, as disclosed by Hickman et al. One of ordinary skill in the art would be motivated to choose values of conductivity or ionic strength that minimize antibody loss and maximize host cell protein precipitation for the particular antibody preparation of interest, instead of using a set value for every antibody preparation. One of ordinary skill in the art would have had a reasonable expectation of success for the reasons underlying this method of purification, which is the basis of the isoelectric focusing separation system taught by Hickman et al (paragraph 141).
Regarding claim 40, Hickman et al further teaches a step of subjecting the depth filtered protein preparation to ion exchange chromatography (Example 1 paragraph 251).
Regarding claim 50, Hickman et al further teaches the depth filter is Zeta Plus (paragraph 260).
Regarding claim 55-56, Hickman et al further teaches that the host cell is a mammalian cell, which is a CHO cell (Example 1, paragraph 244).
Claims 41-43 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017) and further in view of Wang et al (WO2016149088A1, Use of alkaline washes during chromatography to remove impurities; filed 03/11/2016) and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012), and further in view of Tee et al (Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay, bioRxiv preprint; published 08/02/2020), as applied to claim 34 above, and further in view of Westendorf '828 (US11370828B2, Anti-coronavirus antibodies and methods of use; effectively filed 03/09/2020).
Claim 41-43 depends on claim 34. The teachings of the prior art regarding claim 34 are incorporated in its entirety here and further described below.
Hickman et al in view of EMD Millipore does not explicitly teach the particular antibody or sequences from instant claims 41-43.
However, Westendorf '828 teaches an anti-SARS-COV-2 antibody with VH sequence encoded by SEQ ID NO: 1255 paired with VL sequence encoded by SEQ ID NO: 1256, which matches instant SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and teaches that this claimed antibody is an IgG1 antibody (Claim 1). Westendorf et al also teaches an anti-SARS-COV-2 antibody with HC sequence encoded by SEQ ID NO: 5363 paired with VL sequence encoded by SEQ ID NO: 5364, which matches instant SEQ ID NO: 3 and SEQ ID NO: 4, respectively (Claim 7 and Column 141, lines 30-35). Westendorf '828 teaches that the SARS-COV-2-binding antibodies disclosed is compatible with Protein A affinity chromatography (column 132, lines 50-52).
The specifications of the instant application define bamlanivimab as an antibody encoded by paired VH and VL sequences SEQ ID NO: 1 and 2, which is encompassed by the HC and LC paired sequences encoded by SEQ ID NO: 3 and 4. Therefore, it can be understood that Westendorf et al also teaches bamlanivimab as an anti-SARS-COV-2. It also follows that claims 25-27 are directed to an IgG1 antibody.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the methods taught by Hickman et al in view of Tee et al and further in view of EMD Millipore is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, and the particular anti-SARS-COV-2 antibodies taught by Westendorf '828, especially since both antibodies from Hickman et al and Westendorf et al are IgG1 isotypes. Additionally, Westendorf '828 teaches that this SARS-COV-2-binding antibody is compatible with Protein A affinity chromatography (column 132, lines 50-52).
One skilled in the art, before the effective filing date of the instant application, would be motivated to purify the specific anti-SARS-COV-2 antibody taught by Westendorf ' 828 because it targets SARS-COV-2 (Abstract), which is the causative virus of the COVID-19 global pandemic.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the substitution of a specific anti-SARS-COV-2 antibody taught by Westendorf '828 for the anti-SARS-COV-2 purification method taught by Hickman et al in view of EMD Millipore, especially since both antibodies from Hickman et al and Westendorf '828 are IgG1 isotypes and Westendorf '828 discloses purification by Protein A affinity chromatography.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific anti-SARS-COV-2 antibodies being purified. The prior art as taught by Hickman et al in view of Tee et al and further in view of EMD Millipore provides an affinity chromatography method for purifying antibodies, specifically an IgG1 antibody and specifically an anti-SARS-COV-2 antibody as an example. Westendorf '828 teaches the specific anti-SARS-COV-2 antibody instantly disclosed. Therefore the substituted components, the anti-SARS-COV-2 antibodies from Westendorf '828, were known in the art before the effective filing date of the instant application. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A or Protein G columns. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable due to shared compatibility with affinity chromatography purification.
Claims 44-45 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017) and further in view of Wang et al (WO2016149088A1, Use of alkaline washes during chromatography to remove impurities; filed 03/11/2016) and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012), and further in view of Tee et al (Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay, bioRxiv preprint; published 08/02/2020), as applied to claims 34 above, and further in view of Walker et al (US11192940B2, Compounds specific to coronavirus S protein and uses thereof, effectively filed 04/10/2020).
Claim 46 is rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017) and further in view of Wang et al (WO2016149088A1, Use of alkaline washes during chromatography to remove impurities; filed 03/11/2016) and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012), and further in view of Tee et al (Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay, bioRxiv preprint; published 08/02/2020), as applied to claims 34 above, and further in view of Walker et al (US11192940B2, Compounds specific to coronavirus S protein and uses thereof, effectively filed 04/10/2020) and further in view of Chiu et al (Antibody Structure and Function: The Basis for Engineering Therapeutics, published 2019).
Hickman et al in view of EMD Millipore does not explicitly teach the particular antibody or sequences from instant claims 44-46.
However, Walker et al teaches an anti-SARS-COV-2 antibody with VH sequence encoded by SEQ ID NO: 82 paired with VL sequence encoded by SEQ ID NO: 91, which matches instant SEQ ID NO: 5 and SEQ ID NO: 6, respectively, as is relevant to claim 45. Walker et al also teaches an anti-SARS-COV-2 antibody with LC sequence encoded by SEQ ID NO: 91, which matches with instant SEQ ID NO: 8, as is relevant to claim 46.
The specifications of the instant application define etesevimab as an antibody encoded by paired VH and VL sequences SEQ ID NO: 5 and 6. Therefore, it can be understood that Walker et al also teaches etesevimab as an anti-SARS-COV-2, as is relevant to claim 44.
Hickman et al in view of EMD Millipore in view of Walker et al does not explicitly teach a sequence matching 100% to SEQ ID NO: 7, as is relevant to claim 46.
However, regarding claim 30, Walker et al teaches an anti-SARS-COV-2 antibody with HC sequence encoded by SEQ ID NO: 81 paired with VL sequence encoded by SEQ ID NO: 91, which has a 99.6% sequence homology to SEQ ID NO: 7, differing by 3 conservative residue substitutions outside of the variable region.
However, Chiu et al teaches that the Fc region sequence is shared between antibody subtype, and thus does not define an individual antibody. The Fc region does not have antigen binding properties but may be engineered to modulate effector function activities, half-life, and Protein A binding affinity (section 2, paragraph 1 and Table 2). The instant specification also defines etesevimab by its variable sequence. Therefore a few conservative amino acid substitutions outside of the variable region of an antibody is an obvious variant of an antibody with the same variable region sequence, but with routine residue optimization in the Fc region. As such, the few conservative substitution outside the variable region is an obvious variant of the etesevimab antibody as disclosed by Walker et al.
Hickman et al does not limit the antibody that can be purified using their respective antibody purification method based on the target antigen. Hickman et al discloses that the choice of known affinity chromatography resin, such as Protein A, Protein G, or other commonly used resin, can be changed according to their affinity to subclass of the IgG of interest.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the methods taught by Hickman et al in view of EMD Millipore is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, and the particular anti-SARS-COV-2 antibodies taught by Walker et al, especially since both antibodies from Hickman et al and Walker et al are taught to be purified using Protein A affinity chromatography (Walker et al, column 92, lines 51-55). One skilled in the art, before the effective filing date of the instant application, would be motivated to purify the specific anti-SARS-COV-2 antibody taught by Walker et al because it targets SARS-COV-2 (Abstract), which is the causative virus of the COVID-19 global pandemic.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the substitution of a specific anti-SARS-COV-2 antibody taught by Walker et al for the anti-SARS-COV-2 purification method taught by Hickman et al in view of EMD Millipore, especially since both antibodies from Hickman et al and Walker et al are taught to be purified using Protein A affinity chromatography (Walker et al, column 92, lines 51-55).
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, to routinely optimize the Fc region of antibody with conservative mutations that may impart Fc effector function improvements without changing the binding target of the antibody, as taught by Chau et al, and that this variant would be an obvious variant of an antibody with the same variable sequence.
One skilled in the art, before the effective filing date of the instant application, would be motivated to screen for and optimize specific residues outside of the binding region to conserve the main antibody binding function but potentially improve half-life and effector functions.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success of retaining the same antigen-targeting function and sequence that defines the antibody since the conservative mutations are outside the variable region.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific anti-SARS-COV-2 antibodies being purified. The prior art as taught by Hickman et al in view of EMD Millipore provides an affinity chromatography method for purifying IgG antibodies. Walker et al teaches the specific anti-SARS-COV-2 IgG antibody instantly disclosed. Therefore the substituted components, the anti-SARS-COV-2 antibodies from Walker et al and the IgG antibody from Hickman et al, were known in the art before the effective filing date of the instant application. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A or Protein G columns. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable due to shared compatibility with affinity chromatography purification.
Furthermore, KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses applying a known technique to a known device (method, or product) ready for improvement to yield predictable results is obvious. Conservative mutations outside the variable region to stabilize or modulate effector function while retaining the targeting and variable sequence that defines an antibody is a known improvement to base antibodies. Furthermore, the results of these conservative mutations are predictable to one skilled in the art before the effective filing date of the instant application because the effects of conservative mutations are understood in the art.
Claims 47-49 are rejected under 35 U.S.C. 103 as being unpatentable over Hickman et al (US20100135987A1, Isolation and Purification of Antibodies Using Protein A Affinity Chromatography; filed 10/20/2009) in view of EMD Millipore (Understanding the impact of elution buffer on mAb purification processes, published 2015), as evidenced by Kornecki et al (Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly, Antibodies; published 09/18/2017) and further in view of Wang et al (WO2016149088A1, Use of alkaline washes during chromatography to remove impurities; filed 03/11/2016) and further in view of Golken et al (WO2012135415A1, Buffer system for protein purification; effectively filed 03/29/2012), and further in view of Tee et al (Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay, bioRxiv preprint; published 08/02/2020), as applied to claims 34 above, and further in view of Westendorf '541 (US11447541B1, Anti-coronavirus antibodies and methods of use; effectively filed 03/09/2020).
Hickman et al in view of EMD Millipore does not explicitly teach the particular antibody or sequences from instant claims 47-49.
However, Westendorf '541 teaches an anti-SARS-COV-2 antibody with VH sequence encoded by SEQ ID NO: 4949 paired with VL sequence encoded by SEQ ID NO: 4950, which matches instant SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and teaches that this claimed antibody is an IgG1 antibody (Claim 1). Westendorf et al also teaches an anti-SARS-COV-2 antibody with HC sequence encoded by SEQ ID NO: 5735 paired with VL sequence encoded by SEQ ID NO: 5736, which matches instant SEQ ID NO: 11 and SEQ ID NO: 12, respectively (Claim 6).
The specifications of the instant application define bebtelovimab as an antibody encoded by paired VH and VL sequences SEQ ID NO: 9 and 10, which is encompassed by the HC and LC paired sequences encoded by SEQ ID NO: 11 and 12. Therefore, it can be understood that Walker et al also teaches bebtelovimab as an anti-SARS-COV-2.
It would have been obvious to one skilled in the art, before the effective filing date of the instant application, that the methods taught by Hickman et al in view of EMD Millipore is also applicable to any antibody that binds the affinity column, which includes anti-SARS-COV-2 antibodies, and the particular anti-SARS-COV-2 antibodies taught by Westendorf '541, especially since both antibodies from Hickman et al and Westendorf '541 can be affinity purified by Protein A (Westendorf '541 column 259, line 40).
One skilled in the art, before the effective filing date of the instant application, would be motivated to purify the specific anti-SARS-COV-2 antibody taught by Westendorf '541 because it targets SARS-COV-2 (Abstract), which is the causative virus of the COVID-19 global pandemic.
One skilled in the art, before the effective filing date of the instant application, would have reasonable expectation of success in the substitution of a specific anti-SARS-COV-2 antibody taught by Westendorf '541for the anti-SARS-COV-2 purification method taught by Hickman et al in view of EMD Millipore, especially since both antibodies from Hickman et al and Westendorf '541 are taught to be purified by protein A, and thus the antibody from Westendorf '541 would be reasonable compatible with the methods of Hickman et al.
KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, (2007), discloses that a simple substitution of one known element for another, when swapping similar features that serve the same purpose, to obtain predictable results is obvious. In this case, the similar elements being substituted are the specific antibodies being purified. The prior art as taught by Hickman et al in view of EMD Millipore provides an affinity chromatography method for purifying antibodies via Protein A affinity chromatography. Westendorf '541 teaches the specific anti-SARS-COV-2 antibody instantly disclosed. Therefore the substituted components, the antibodies, were known in the art before the effective filing date of the instant application. Prior art teaches that both antibodies can bind and elute from affinity purification columns, such as Protein A. One skilled in the art, before the effective filing date of the instant application, could have substituted one known antibody for another known antibody in the base antibody purification method taught by Hickman et al and the results of the substitution would have been reasonably predictable due to shared compatibility with affinity chromatography purification.
Conclusion
No claims are allowed.
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/B.C./Examiner, Art Unit 1645
February 18, 2026
/DANIEL E KOLKER/Supervisory Patent Examiner, Art Unit 1645