Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Claims/Application
Claims 4, 8, 10 and 12 are canceled. Claims 1, 7, 9 and 18 are currently amended. Claims 1-3, 5-7, 9, 11 and 13-20 are currently pending and are under examination on the merits herein.
Withdrawn Rejections
Rejections Under - 35 USC § 112(b)
Applicant’s amendments/arguments, see applicants remarks page 6 lines 18-23, filed on 10/17/2025, with respect to claims 7, 12 and 18 have been fully considered and are persuasive. The rejections of claims 7, 12 and 18 have been withdrawn.
Response to Arguments
Applicant’s arguments, see applicant remarks, filed on 10/17/2025, with respect to the rejections of claims 1-3, 5-7, 9, 11 and 13-20 under 35 USC 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new grounds of rejection is made in view of the amended claims to include a recited limitation of “wherein washing of the cation exchange column in step (a) consist of two washing steps”, and newly found prior art references.
As per the applicants argument of the removal of host cell protein and residual DNA and cell growth factors and that the examiner did not establish prima facie case for obviousness (Remarks pg. 7 para. 4 – pg. 8 para. 1), the argument is not persuasive because the examiner rejected the claim based on the recitation thought by US’2384 and US’9988 that this step is commonly performed in the art to further purify the antibody as indicated by US’2384 para. 0023. Therefore, it would have been a motivation by an ordinary skilled artisan to further purify the antibody using ultrafiltration/diafiltration as thought by US’2384 and US’9988. As indicated in MPEP 2145 (X)(C), a teaching, suggestion, or motivation to combine references that is found in the prior art is an appropriate rationale for determining obviousness.
The applicant further argues that the examiner fails to establish prima facie case of obvious why an ordinary skilled artisan would modify the teachings of US’3012 in view of US’2384 or US’9988 to yield a predictable result. The applicant argues that the result would not have been predictable because the instant claim recites two washing steps as compared to the four washing steps of US’3012, but instant claim has a higher yield (Remarks, pg. 8 para 2 – pg. 9). The argument is not persuasive because:
As indicated by US’3012, the pH and composition of the buffer solution for the washing step(s) can be adjusted depending on the type of antibody isoforms to be removed or the type of column used in the purification process (US’3012 Col. 8 ln 53-57). As indicated in MPEP 2145 (X) (C), a teaching, suggestion, or motivation to combine references that is found in the prior art is an appropriate rationale for determining obviousness. KSR, 550 U.S. at 418, 82 USPQ2d at 1396. However, it is just one of a number of valid rationales for doing so. The Court in KSR identified several exemplary rationales to support a conclusion of obviousness which are consistent with the proper "functional approach" to the determination of obviousness as laid down in Graham. KSR, 550 U.S. at 415-21, 82 USPQ2d at 1395-97. See MPEP § 2141 and § 2143. As indicate in MPEP 2145 (I)(C), which refers to a finding that the prior art contained a "base" device (method, or product) upon which the claimed invention can be seen as an "improvement;" is another rationale for obviousness. Therefore, a skilled artisan would have been able to modify the washing steps by performing routine optimization steps to adjust the wash buffer pH, composition, conductivity etc. so as to obtain a desire yield from the CEX. It would have been obvious to an ordinary skill artisan that altering the buffer pH and composition is a determining factor in the washing steps in a CEX as it commonly known in the art to affects the yield, therefore an ordinary artisan would have been motivated to adjust the wash buffers in order to obtain a better yield.
Furthermore, claim 12 as filed on 8/17/2022 recites, recites “Step(a) comprises:”, which is a transitional term and is inclusive or open-ended and does not exclude additional, unrecited elements or methods (MPEP 2111.03). Therefore, in the plain meaning of the claims filed on 08/17/2022, an ordinary skilled artisan would not have been able to clearly determine if the washing steps consisted of only two washing steps or there are additional steps such as those thought by US’3012.
New Rejections
Rejections Under - 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.
Claims 1-3, 6-7, and 13-19 are rejected under 35 U.S.C. 103 as being unpatentable over US9683012, and further in view of Millipore, Development Strategy for Cation Exchange (CEX) Chromatography Step in a Monoclonal Antibody (mAb) Process. 2014 (Ver. 1.0), Zhang et al. Improving pH gradient cation-exchange chromatography of monoclonal antibodies by controlling ionic strength. J Chromatogr A. 2013 Jan 11;1272:56-64, He et al. NuviaTM S Media: A High-Capacity Cation Exchanger for Process Purification of Monoclonal Antibodies, (2010) Bio-Rad Lab. tech note 5984, and US20150139988A1 as evidenced by Adalimumab – PubChem Retrieved from <pubchem.ncbi.nlm.nih.gov/compound/Adalimumab#section=Isoelectric-Point&fullscreen=true> on 02/20/2026, here in after referred to as US’3012, Millipore, Zhang, HeUS’9988 and PubChem respectively.
Regarding claim 1, US'3012 teaches a method of preparing a population of antibody comprising:
a step for removing a host cell protein (HCP) and an isomeric antibody from a sample comprising mixed solution of antibodies, by loading the sample of mixed solution of antibodies into an equilibrated cation exchange column (CEX) comprising a carboxylate (COO-) functional group preequilibrated with an equilibrium buffer comprising 20 to 30 mM sodium acetate (ph 4.5 to 5.5) and 35 to 45 mM sodium chloride, wherein washing the CEX in step (a) comprises:
(a1.1) a step of washing the column with a buffer having a pH of 4.5 to 5 comprising 20 to 30 mM sodium acetate and 35 to 45 mM sodium chloride (US'3012 Col. 9 In 44-46);
(a1.2) washing the column with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5);
(a1.3) another step for washing the column with a buffer having a pH of 5.5 to 6.5 comprising 25 to 35 mM sodium acetate and 55 mM sodium chloride (US'3012 Col. 9 In 48-50);
(a.1.4) washing the column with a buffer comprising 25 to 35 sodium acetate (pH 5.5 to 6.5),
a step for eluting the antibody to the column with elution buffer comprising;
(a2) an elution step of eluting an antibody with a buffer having comprising 25-35 mM sodium acetate and 70 to 90 mM sodium chloride (US'3012 Col. 9 In 52-54).
a step for removing the host cell protein (HCP) and the residual DNA from the antibody elute by loading a sample prepared by mixing salt with the eluate of step (a) above to a hydrophobic interaction column (HIC), and eluting the antibodies bound to the column with an elution buffer (US'3012 Col 3 In 29-33 and Col. 8 In 4-11);
a step for removing the host cell protein (HCP) and the residual DNA by loading the eluate of step (b) above to an anion exchange column and collecting the flow-through (US'3012 Col 3 In 34-35 and Col. 8 In 4-11);
a step of removing viruses by loading the eluate and allowing the flow-through in "step (с)" to pass through a virus filter (US'3012 Sht.8 Fig. 11 and Col. 4 In 27-28); and
a step for secondary ultrafiltration/diafiltration (US'3012 Fig. 11) and a population of antibodies after Step (d) with a concentration of obtain the concentration of host cell protein at 0.0001 ppm to 10 ppm, or more preferably 0.001 to 5 ppm (US'3012 Col. 11 In 60-67), where in the antibody has an isoelectric point of 7 to 11 US'3012 Col. 5 In 51-54).
US'3012 does not specifically that the washing steps consist of two washing steps. Rather US’3012 teaches two additional washing steps wherein the column was washed with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5). US’3012 does not teach a step for concentrating the antibody eluate eluted in Step(d), performing buffer exchange and a 10 ppb or less of residual DNA concentration. US’3012 does not also teach a three elution step method.
Millipore teaches that a CEX can be washed with the same buffer as the loading buffer until the absorbance reaches a baseline (Millipore pg. 5 col. 1), and if there are still impurities bound to the column after washing to the baseline, then an additional wash buffer (wash buffer 2) with a conductivity below that of the elution buffer can be used to further wash the column. An that the conductivity of the wash buffer can be adjusted so as to obtain the desired purity and yield (Millipore pg. 6 col. 1 para 2). Millipore further teaches that the first wash step is usually conducted with the equilibration buffer and in the case where elution step(s) is utilized, an intermediate wash with a buffer conductivity between that of the equilibrium and elution buffers can be utilized to remove loosely bound species (Millipore pg. 6 col.2 para 4).Zhang teaches an exemplary mAb2 having a pI of 8.2 that showed a better full-width at half-maxima (FWHM, a measure of a columns efficiency, selectivity and separation) when the salt (NaCl) concentration was 8 mM and for a mAb3 with a pI of 9.4 having an optimal FWHM when the NaCl was 32 mM compared to when the NaCl concentration is 0 mM in both cases. Zhang further teaches that the effect of ionic strength on pH-gradient IEC of mAbs also depends on the mAb’s pI and that mAb3 with pI of 9.4 required 32 mM of salt to achieve optimal resolution (Zhang Sec 3.3.2). Zhang further teaches that the ionic strength in the pH gradient can be tuned for each mAb to achieve optimal resolution and that low-pI mAbs prefer low ionic strength and high-pI mAbs prefer higher ionic strength (Zhang pg. 63 sec 4).
He, also teaches a two-step washing in a CEX column (Nuvia S) where the first wash buffer comprise of 20 mM sodium acetate and 20 mM sodium chloride and the second wash buffer comprised of 20 mM sodium acetate and 76 mM sodium chloride in other to remove degradation fragments (He pg. 1 col 2 para 3 – pg. 2 col 1 para 1). Li also teaches that the pI value of the antibody, the pH and conductivity are factors that determines the antibody’s interaction with the CEX (He pg. 2 col 2 para 1).
US'9988 teaches different elution buffers with varying pH and/or salt content can be used in a stepwise fashion to elute the antibody from an ion exchange column (US'9988 pg. 20 para. 0167 In 4-20).
Therefore, it would have been obvious before the effective filing date for a skilled artisan to modify the teachings of US’3012 in view of Millipore, Zhang, He and US’9988 with a reasonable high degree of predictable success to use a two-step washing approach rather than 4 step washing approach to purify the antibody. Furthermore, He thought a two steps washing process in the CEX for removing degradation fragments of mAbR using buffers A and B comprising sodium acetate and sodium chloride. As indicated by Millipore’s protocol, the first wash can be done with the same equilibration buffer and the second wash using a buffer that has a conductivity between that of the equilibration buffer and the elution buffer(s) will enable loosely bound impurities to be washed off, an as thought by Zhang, mAbs with higher pI prefer buffers with higher ionic strengths indicating that using buffers with an optimized NaCl concentration for the specific antibody with consideration to the other properties of the antibody would yield a better FWHM which is an indication of the columns efficiency and selectivity for that particular antibody. As thought by US’3012, the equilibration buffer used was the same as the first wash buffer, the buffer used in wash 3 has a conductivity between the equilibration buffer and the elution buffer (US’3012 Tab. 6). As indicated by Millipore, the number of washing is determined when absorbance reaches a baseline. Therefore, a skilled artisan would have been able to follow the suggestions of US’3012 in view of Millipore, Zhang, He and US’9988 to reduce the washing steps of US’3012 to two washing steps and using the buffers of US’3012 containing NaCl concentrations, wherein the equilibration buffer (washing 1 buffer) is used as the first washing buffer and the third buffer (buffer in washing step 3) as the second washing buffer of the instant claim as indicated by Millipore, and given that the antibody has a pI between 7-10 which falls in the range of antibodies that require NaCl concentration to optimized the column’s efficiency, selectivity and separation performance as suggested by Zhang. A skilled artisan would have been able to follow the suggests and calculate the necessary salt concentration for the given antibody requires to optimize the column when applying a two-step washing process. Furthermore, the presence of NaCl in the buffers will enable removal of any loosely bound impurity that may still be present after the first wash but not strong enough to wash off the antibody or perform routing optimization to determine the optimal buffer mixtures of sodium acetate and sodium chloride that will achieve a baseline absorbance after two washes so as to improve operation efficiencies and cost of protein purification (as a result of buffer consumption). Also, a skilled artisan would have been able to apply the methods of US’9988 to calculate several elution buffers in the stepwise or gradient method by changing the concentration of NaCl required in the buffer mixture to elute the antibody so as to obtain a desire yield.
Regarding claim 2, an incorporating the analysis of claim 1, US'3012 further teaches a mixed solution of antibodies used in "step (a)" was prepared by steps including a step for removing precipitated precipitate by adjusting the pH of a culture supernatant to 5 (US'3012 Col. 12 In 30-40 and Table 1).
Regarding claim 3, an incorporating the analysis of claim 1, US'3012 further teaches the conditions of the sample of a mixture of solution of antibodies in "Step (a)" can be adjusted to have a conductivity of 5 mS/cm to 7 mS/cm (US'3012 Col. 7 In 40-43 and Col. 14 In 29-34).
Regarding claim 6, an incorporating the analysis of claim 1, US'3012 further teaches that the antibody eluate eluted in "Step (a)" for the COO- column is 68.7% of active antibody, 23.8% acidic antibody isoforms and 7.5% basic antibody isoforms (US'3012 Col. 17 Table 7).
Regarding claim 7, an incorporating the analysis of claim 1, US'3012 further teaches a step of loading the mixed solution of antibodies into an equilibrated cation exchange comprises loading the mixed solution of antibodies into a COO- exchange column equilibrated with an equilibrium buffer comprising 20 to 30 mM sodium acetate (pH 4.5 to 5.5) and 35 to 45 mM sodium chloride (US'3012 Col. 9 In 39-44).
Regarding claim 13, an incorporating the analysis of claim 1, US'3012 further teaches that Step (b) elute the antibody by a linear-concentration gradient (US'3012 Col. 10 In 18-22).
Regarding claim 14, an incorporating the analysis of claim 13, US'3012 further teaches a method of loading a sample in which the antibody eluate eluted from Step (a) is adjusted to sodium citrate concentration that is the same as the equilibration buffer of the hydrophobic interaction column equilibrated with an equilibration buffer comprising 25 mM to 35mM sodium acetate (pH 5.5 to 6.5) and 0.3 to 1.0 M sodium citrate and applying an elution buffer comprising 25 mM to 35 mM sodium acetate (pH 5.5 to 6.5) in a linear gradient (US'3012 Col. 10 In 60-67 and Col. 11 In 1-2).
Regarding claim 15, an incorporating the analysis of claim 1, US'3012 further teaches that the hydrophobic interaction column in Step (b) can be phenyl Sepharose column (US'3012 Col. 10 In 55-58).
Regarding claim 16, an incorporating the analysis of claim 1, US'3012 further teaches that the anion exchange column in Step (c) is equilibrated with an equilibration buffer having a pH of 7.0 to 8.0 before injection of the sample (US'3012 Col. 11 In 41-48).
Regarding claim 17, an incorporating the analysis of claim 1, US'3012 further teaches an equilibration buffer comprising Tris-HCI having a pH of 7.0 to 8.0 (US'3012 Col. 11 In 41-43).
Regarding claim 18, an incorporating the analysis of claim 1, US'3012 further teaches an anion exchange column in Step (c) is a Q Fast Flow column (US'3012 Col. 11 In 43-47).
Regarding claim 19, an incorporating the analysis of claim 1, US'3012 further teaches a population of antibodies prepared by the method of claim 1, where the population of antibodies comprises 65% of active antibodies.
Claims 5 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over US’3012, and further in view of Millipore, Zhang, He, US’9988 and US20160122384A1, here in after referred to as US’2384.
Regarding claim 5, an incorporating the analysis of claim 1, US'3012 further teaches that any antibody with a therapeutical potential which is commonly used in the art (US'3012 Col. 5 In 53-61). US'3012 further teaches that the method described can be used to purify any antibody with an isoelectric point between 7 to 11 and more preferably 8 to 10 (US'3012 Col. 5 In 51-54).
US'3012 does not specifically teach that the antibody is adalimumab.
US'2384 teaches a method similar to the method described in the instant application for the purification of adalimumab (US'2384 pg. 4 para. 0050).
Therefore, it would have been obvious before the effective filing date of the instant application for one with ordinary skill in the art to modify the teachings of US'3012 in view of Millipore, Zhang, He, US’9988 and US'2384 with a high degree of predictable success to purify adalimumab using the methods described in US'3012. Adalimumab has an isoelectric point (pl) of about 8.25 (PubChem) which falls within the range of pI for antibodies that can be purified by the methods of US'3012. Due to the pI of adalimumab being about 8.25, adalimumab becomes cationic in a pH buffer having an isoelectric point lower than 8.25 allowing for cation exchange between adalimumab and the cation exchange resin thereby increasing the purity of adalimumab during the purification process (US'2384 pg. 3 para. 0024).
Regarding claim 20, an incorporating the analysis of claim 1, US'3012 teaches a step for
secondary ultrafiltration/diafiltration, virus filtration (US'3012 Fig. 11) and a population of antibodies with a concentration of 0.001 to 5 ppm of host cell protein (US'3012 Col. 11 In 60-67).
US'3012 does not specifically teach a concentration of 0.1 ppb or less of residual DNA or the
measurement of residual DNA.
US'2384 teaches a removal of host cell proteins means removal of the host cell protein itself, residual DNA and cell growth factors derived from the host cells (US'2384 pg. 3 para. 0023 and pg. 4 para. 0046).
Therefore, it would have been obvious before the effective filling date to one with ordinary skill in the art to modify the teachings of US'3012 in view of Millipore, Zhang, He, US’9988 and US'2384 with a reasonably degree of predictable success to obtain the concentration of host cell protein at 0.0001 to 10 ppm, or more preferably 0.001 to 5 ppm (US'3012 Col. 11 In 60-67). Suggesting that the concentration of residual DNA was equivalently reduced even though it was not specifically measured by the prior art references. Furthermore, 10 ppb of residual DNA is equivalent to 0.01 ppm residual DNA, indicating that the claimed concentration of residual DNA (10 ppb or 0.01 ppm) is within the concentration of host cell protein (including residual DNA, and other contaminants concentrations) of 0.0001 ppm to 10 ppm as taught by US'3012.
Claims 9 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over US’3012 as applied to claim 1 above, and further in view of Millipore, Zhang, He, US’9988 and GE Principles and Methods, Ion Exchange Chromatography and Chromatofocusing, 2010, here in after referred to as GE.
Regarding claims 9 and 11, and incorporating the analysis of claim 1 above, Millipore teaches that several parameter requires for the CEX purification method for the desired antibody can be established during the design process (Millipore pg. 5 Tab. 1).
GE teaches a method for formulating the wash and elution buffers based on the desired pH and ionic strengths suitable to wash the column without eluting the bound protein, as well as elution buffer for gradient or stepwise elution of the protein from the column. Han teaches that a buffer concentration that is sufficient to maintain buffering capacity and constant pH typically between 20-50 mM should be chosen (GE pg. 37). GE teaches that adjusting the parameters can be achieved by altering the concertation of the salt added to the buffer solution. For step elution, GE illustrates in an exemplary gradient and step elution chart various predetermined salt (NaCl) concentration that was used to make the elution buffer for a column (GE pg. 44 Fig 24).
Therefore, a skilled artisan would have been able to modify the teaching US’3012 in view of Millipore, Zhang, He and GE with a reasonable high degree of predictable success for a skilled artisan to determine during the purification method design process as indicated by Millipore to determine the concentrations of the buffers ingredients to be mixed so as to obtained a predetermined NaCl concentration for the final buffer needed for the washing and/or elution steps.
Double Patenting Rejections
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-3, 6-7, 13-18 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 5, 8 and 10-18 of U.S. Patent No. 9683012 B2 in view of Millipore, Zhang, He and US’9988.
Regarding claim 1 of the instant claim, US'3012 claims 1 and 11 recites a method of preparing a population of antibody comprising:
a step for removing a host cell protein (HCP) and an isomeric antibody from a sample comprising mixed solution of antibodies, by loading the sample of mixed solution of antibodies into an equilibrated cation exchange column (CEX) comprising a carboxylate (COO-) functional group, preequilibrated with an equilibrium buffer comprising 20 to 30 mM sodium acetate (ph 4.5 to 5.5) and 35 to 45 mM sodium chloride, wherein washing the CEX in step (a) comprises:
(a1.1) a step of washing the column with a buffer having a pH of 4.5 to 5 comprising 20 to 30 mM sodium acetate and 35 to 45 mM sodium chloride (US'3012 Col. 9 In 44-46);
(a1.2) washing the column with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5);
(a1.3) another step for washing the column with a buffer having a pH of 5.5 to 6.5 comprising 25 to 35 mM sodium acetate and 55 mM sodium chloride (US'3012 Col. 9 In 48-50);
(a.1.4) washing the column with a buffer comprising 25 to 35 sodium acetate (pH 5.5 to 6.5),
a step for eluting the antibody to the column with elution buffer comprising;
(a2) an elution step of eluting an antibody with a buffer having comprising 25-35 mM sodium acetate and 70 to 90 mM sodium chloride (US'3012 Col. 9 In 52-54).
(b) a step for removing the host cell protein (HCP) and the residual DNA from the antibody elute by loading a sample prepared by mixing salt with the eluate of step (a) above to a hydrophobic interaction column (HIC), and eluting the antibodies bound to the column with an elution buffer (US'3012 Col 3 In 29-33 and Col. 8 In 4-11);
(c) a step for removing the host cell protein (HCP) and the residual DNA by loading the eluate of step (b) above to an anion exchange column and collecting the flow-through (US'3012 Col 3 In 34-35 and Col. 8 In 4-11);
(d) a step of removing viruses by loading the eluate and allowing the flow-through in "step (с)" to pass through a virus filter (US'3012 Sht.8 Fig. 11 and Col. 4 In 27-28); and
(e) a step for secondary ultrafiltration/diafiltration (US'3012 Fig. 11) and a population of antibodies after Step (d) with a concentration of obtain the concentration of host cell protein at 0.0001 ppm to 10 ppm, or more preferably 0.001 to 5 ppm (US'3012 Col. 11 In 60-67), where in the antibody has an isoelectric point of 7 to 11 US'3012 Col. 5 In 51-54).
US'3012 does not specifically that the washing steps consist of two washing steps. . Rather US’3012 teaches two additional washing steps wherein the column was washed with a buffer comprising 25 to 35 mM sodium acetate (pH 5.5 to 6.5). US’3012 does not teach a step for concentrating the antibody eluate eluted in Step(d), performing buffer exchange and a 10 ppb or less of residual DNA concentration. US’3012 does not also teach a three elution step method.
Millipore teaches that a CEX can be washed with the same buffer as the loading buffer until the absorbance reaches a baseline (Millipore pg. 5 col. 1), and if there are still impurities bound to the column after washing to the baseline, then an additional wash buffer (wash buffer 2) with a conductivity below that of the elution buffer can be used to further wash the column. An that the conductivity of the wash buffer can be adjusted to obtain the desired purity and yield (Millipore pg. 6 col. 1 para 2). Millipore further teaches that the first wash step is usually conducted with the equilibration buffer and in the case where elution step(s) is utilized, an intermediate wash with a buffer conductivity between that of the equilibrium and elution buffers can be utilized to remove loosely bound species (Millipore pg. 6 col.2 para 4).
Zhang teaches an exemplary mAb2 having a pI of 8.2 that showed a better full-width at half-maxima (FWHM, a measure of a columns efficiency, selectivity and separation) when the salt (NaCl) concentration was 8 mM and for a mAb3 with a pI of 9.4 having an optimal FWHM when the NaCl was 32 mM compared to when the NaCl concentration is 0 mM in both cases. Zhang further teaches that the effect of ionic strength on pH-gradient IEC of mAbs also depends on the mAb’s pI and that mAb3 with pI of 9.4 required 32 mM of salt to achieve optimal resolution (Zhang Sec 3.3.2). Zhang further teaches that the ionic strength in the pH gradient can be tuned for each mAb to achieve optimal resolution and that low-pI mAbs prefer low ionic strength and high-pI mAbs prefer higher ionic strength (Zhang pg. 63 sec 4).
He, also teaches a two-step washing in a CEX column (Nuvia S) where the first wash buffer comprise of 20 mM sodium acetate and 20 mM sodium chloride and the second wash buffer comprised of 20 mM sodium acetate and 76 mM sodium chloride in other to remove degradation fragments (He pg. 1 col 2 para 3 – pg. 2 col 1 para 1).
US'9988 teaches different elution buffers with varying pH and/or salt content can be used in a stepwise fashion to elute the antibody from an ion exchange column (US'9988 pg. 20 para. 0167 In 4-20).
Therefore, it would have been obvious before the effective filing date for a skilled artisan to modify the teachings of US’3012 in view of Millipore, Zhang, He and US’9988 with a reasonable high degree of predictable success to use a two-step washing approach rather than 4 step washing approach to purify the antibody. Furthermore, He thought a two steps washing process in the CEX for removing degradation fragments of mAbR using buffers A and B comprising sodium acetate and sodium chloride. As indicated by Millipore’s protocol, the first wash can be done with the same equilibration buffer and the second wash using a buffer that has a conductivity between that of the equilibration buffer and the elution buffer(s) will enable loosely bound impurities to be washed off, an as thought by Zhang, mAbs with higher pI prefer buffers with higher ionic strengths indicating that using buffers with an optimized NaCl concentration for the specific antibody with consideration to the other properties of the antibody would yield a better FWHM which is an indication of the columns efficiency and selectivity for that particular antibody. As thought by US’3012, the equilibration buffer used was the same as the first wash buffer, the buffer used in wash 3 has a conductivity between the equilibration buffer and the elution buffer (US’3012 Tab. 6). As indicated by Millipore, the number of washing is determined when absorbance reaches a baseline. Therefore, a skilled artisan would have been able to follow the suggestions of US’3012 in view of Millipore, Zhang, He and US’9988 to reduce the washing steps of US’3012 to two washing steps and using the buffers of US’3012 containing NaCl concentrations, wherein the equilibration buffer (washing 1 buffer) is used as the first washing buffer and the third buffer (buffer in washing step 3) as the second washing buffer of the instant claim as indicated by Millipore, and given that the antibody has a pI between 7-10 which falls in the range of antibodies that require NaCl concentration to optimized the column’s efficiency, selectivity and separation performance as suggested by Zhang. A skilled artisan would have been able to follow the suggests and calculate the necessary salt concentration for the given antibody requires to optimize the column when applying a two-step washing process. Furthermore, the presence of NaCl in the buffers will enable removal of any loosely bound impurity that may still be present after the first wash but not strong enough to wash off the antibody or perform routing optimization to determine the optimal buffer mixtures of sodium acetate and sodium chloride that will achieve a baseline absorbance after two washes so as to improve operation efficiencies and cost of protein purification (as a result of buffer consumption). Also, a skilled artisan would have been able to apply the methods of US’9988 to calculate several elution buffers in the stepwise or gradient method by changing the concentration of NaCl required in the buffer mixture to elute the antibody so as to obtain a desire yield.
Regarding the instant claim 2, an incorporating the analysis of claim 1 above, US'3012 claim 2 further teaches a mixed solution of antibodies used in "step (a)" was prepared by steps including a step for removing precipitated precipitate by adjusting the pH of a culture supernatant to 5 (US'3012 Col. 12 In 30-40 and Table 1).
Regarding instant claim 3, an incorporating the analysis of claim 1, US'3012 claim 1 further teaches the conditions of the sample of a mixture of solution of antibodies in "Step (a)" can be adjusted to have a conductivity of 5 mS/cm to 7 mS/cm (US'3012 Col. 7 In 40-43 and Col. 14 In 29-34).
Regarding instant claim 6, an incorporating the analysis of claim 1 above, US'3012 claim 5 further teaches that the antibody eluate eluted in "Step (a)" for the COO- column is 68.7% of active antibody, 23.8% acidic antibody isoforms and 7.5% basic antibody isoforms (US'3012 Col. 17 Table 7).
Regarding instant claim 7, an incorporating the analysis of claim 1 above, US'3012 claim 10 further teaches a step of loading the mixed solution of antibodies into an equilibrated cation exchange comprises loading the mixed solution of antibodies into a COO- exchange column equilibrated with an equilibrium buffer comprising 20 to 30 mM sodium acetate (pH 4.5 to 5.5) and 35 to 45 mM sodium chloride (US'3012 Col. 9 In 39-44).
Regarding instant claim 13, an incorporating the analysis of claim 1 above, US'3012 claim 12 further teaches that Step (b) elute the antibody by a linear-concentration gradient (US'3012 Col. 10 In 18-22).
Regarding instant claim 14, an incorporating the analysis of claim 13, US'3012 claim 13 further teaches a method of loading a sample in which the antibody eluate eluted from Step (a) is adjusted to sodium citrate concentration that is the same as the equilibration buffer of the hydrophobic interaction column equilibrated with an equilibration buffer comprising 25 mM to 35mM sodium acetate (pH 5.5 to 6.5) and 0.3 to 1.0 M sodium citrate and applying an elution buffer comprising 25 mM to 35 mM sodium acetate (pH 5.5 to 6.5) in a linear gradient (US'3012 Col. 10 In 60-67 and Col. 11 In 1-2).
Regarding instant claim 15, an incorporating the analysis of claim 1 above, US'3012 claim 14 further teaches that the hydrophobic interaction column in Step (b) can be phenyl Sepharose column (US'3012 Col. 10 In 55-58).
Regarding instant claim 16, an incorporating the analysis of claim 1 above, US'3012 claim 15 further teaches that the anion exchange column in Step (c) is equilibrated with an equilibration buffer having a pH of 7.0 to 8.0 before injection of the sample (US'3012 Col. 11 In 41-48).
Regarding instant claim 17, an incorporating the analysis of claim 1 above, US'3012 claim 16 further teaches an equilibration buffer comprising Tris-HCI having a pH of 7.0 to 8.0 (US'3012 Col. 11 In 41-43).
Regarding instant claim 18, an incorporating the analysis of claim 1 above, US'3012 claim 17 further teaches an anion exchange column in Step (c) is a quaternary amine functional group column (US'3012 Col. 11 In 43-47).
Conclusion
No Claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMMANUEL LED YOUTCHOM PENDIE whose telephone number is (571)272-6313. The examiner can normally be reached Mon - Fri: 8AM - 5PM CST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Joanna Hama can be reached at (571) 272-2911. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/EMMANUEL LED YOUTCHOM PENDIE/ Examiner, Art Unit 1647
/JOANNE HAMA/ Supervisory Patent Examiner, Art Unit 1647