Notice of Pre-AIA or AIA Status
The present application is being examined under the first-inventor-to-file provisions of the AIA .
DETAILED ACTION
Claims Status
Claims 1-20 are under examination on their merits.
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. §119 (a)-(d). A certified copy indicates a parent Application No. JP2021-053527, filed on 03/26/2021.
Should applicant desire to maintain the benefit of foreign priority under 35 U.S.C. §119(a)-(d) prior to declaration of an interference, a certified English translation of the corresponding foreign application must be submitted in reply to this action, as required under 37 CFR 41.154(b) and 41.202(e). Failure to provide a certified English translation may result in no benefit being accorded for the non-English application.
Currently, the effective filing date of the instant claims is set at the PCT date of 03/25/2022.
Information Disclosure Statement
Information disclosure statements (IDS) submitted on 12/22/2023, 01/11/2024 and 10/01/2024 are in compliance with the provisions of 37 C.F.R. 1.97. Accordingly, all references cited in these IDSs have been fully considered.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claims 1-6 and 13 are objected to as they contain improper and unclear terminology. Specifically, the claims repeatedly recite the action of “pouring” protein solution and virus solution through two pumps into respective “channels” of protein purification and virus removal. “Pouring” implies gravity-driven transfer or manual handling in an uncontrolled manner, which contradicts the use of pumps to control liquid flowrates (cited as “first constant rate” for protein solution and ”second constant rate” for virus solution). Thus, the term “pouring” or “poured” is inconsistent with the recited method for flow control and is vague as to the manner by which the solutions are introduced through pumping. Applicant is advised to amend these claims by replacing “pouring” with terminology consistent with pump-driven fluid handling, such as “introducing,” “delivering,” “feeding,” or equivalent language.
Claims 8-10, 14 and 20 are objected to as having improper ranges. Specifically, these claims repeatedly recite ranges as “x or more and y or less” that not only interrupts logical flow but also creates confusions. Applicant is advised to amend these claims by replacing “x or more and y or less” with a proper citation of numerical range like “x-y,” “x to y.” Alternatively, “is x or more and y or less” can be replaced by “ranges from x to y.”
Claims 9 and 11 are objected to as containing undefined abbreviations. Specifically, “TCID50 (unit/mL)” as a measure of “virus infectivity” (claim 9) and “LMH” as a measure of flux are not defined in any claims or specification. Examiner recommends the insertion of a full spelling for TCID50 (median tissue culture infectious dose) and LMH (L·m−2 h−1).
Claims 9 and 12 are objected to for improper use of parentheses. Specifically, three unit measures (unit/mL, m2 and mL/min) in these claim are not synonyms of the term immediately before parentheses and could easily be something else (e.g., unit/µL for unit/mL, cm2 for m2 and L/hr for mL/min). Applicant is advised to either omit the unit measures or state the units without parentheses -- “expressed as unit/mL”, “expressed as m2” and “expressed as mL/min”, respectively.
Claim 20 is objected to as having an improper mathematic expression. Specifically, “log” is an inaccurate, colloquial expression of “log10”. Examiner recommends a proper definition of LRV (logarithmic reduction value) in claim 20 to match the use of “Log10” in claim 8.
Claim Rejection under 35 U.S.C. §112(b)
The following is a quotation of 35 U.S.C. §112(b) which forms the basis for indefiniteness rejections set forth in this Office action:
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claim 7, 9 and 12 are rejected under 35 U.S.C. §112(b) or 35 U.S.C. §112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
In the case of claim 7, formula (1) (line 5) defines a relationship of two flow rates – first constant rate for the protein and second constant rate for the virus – to virus concentration before and after the protein solution flows into the virus solution to form a “mixture.” Claim 12 goes on to recite four additional formulae (2-5) (lines 8, 11, 14 and 17) related to “membrane area of the virus removal filter”, “minimum flux of the permeate in the virus removal filter”, “maximum flux of the permeate in the virus removal filter”, as well as “minimum” and “maximum” values of “first” and “second” “constant rate” in sample flows controlled by two pumps.
The formulae of claims 7 and 12 are indefinite since both claims recite “wherein supposing” to set up a hypothetical condition that may or may not be required by the claim. Since it is not clear if the features that follow “wherein supposing” are required or not by the claims, these claims have multiple interpretations and so are rejected here as indefinite. One of ordinary skill in the art would not be reasonably apprised of the scope of the claimed invention in terms of throughput, purification efficiency and virus removal capacity.
Applicant is advised that amendments to claims 7 and 12 need to clearly state the metes and bounds of the claimed invention in the context of virus removal/clearance.
Furthermore, claim 9 recites “unit/mL” in a parenthetical phrase (line 2). Claim 12 recites “m2” (line 1) and “mL/min” (lines 6, 9, 12 and 15) in parenthetical phrases. These units for rates and areas give claims 9 and 12 multiple interpretations. First, the units may be suggestions of a measurement that could be used in the claims, but not necessarily required. Second, the units could be the only units to be used in the claims and so are required for the invention. Such multiple interpretations render claims 9 and 12 indefinite.
The following is a quotation of 35 U.S.C. §112(d) which forms the basis for improper reference/dependency rejections set forth in this Office action:
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 7 and 12 are rejected under 35 U.S.C. §112(d) or pre-AIA 35 U.S.C. §112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
As dependents of claim 1, claim 7 and claim 12 recite formula (1) and formulae (2)-(5), respectively, but these mathematic relationships fail to specify or limit the structure, active step or function of claim 1 beyond the broad intention for “virus clearance test.” Since they do not further limit the claims on which they depend, they are rejected here for improper reference/dependency.
Applicant may cancel the claim(s), amend the claim(s) by placing each in a proper dependent form, rewrite the claim(s) in independent form so long as no duplicates are made, or present a sufficient showing that each dependent claim complies with the 35 U.S.C. §112(d) statutory requirements.
Claim Rejections under 35 USC §103
The following is a quotation of 35 U.S.C. §103, which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. §103 are summarized as the following:
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 1-7, 9, 11-13, 15-16 and 18-20 are rejected under USC §103 as being unpatentable over Johnson et al. 2017 (Biotechnol. Bioeng. 114: 21–32) in view of Li et al. 2020 (Biotechnology and Bioengineering 117: 3379–3389) and Lutz et al. 2011(Biotechnol. Prog. 27(1): 121-128).
Johnson et al. 2017 taught quality control processes suitable for “viral safety assurance” (title) as part of continuous processing (CP) of biological products (abstract). In one “possible scheme for modeling viral clearance” (Figure 1A), their apparatus/system encompasses a “continuous cell culture” unit and a “continuous chromatography” unit, followed by a “continuous virus inactivation” unit (“viral inactivation hold”), with sample flow paths (through interconnected channels/columns) controlled by various valves, along with mixers and “in-line spike and sample grab” steps. Protein and virus solutions flow through the channels at different stages, and the continuous chromatography portion (Figure 1a) is the “protein purification unit”. The continuous viral inactivation is a virus removal unit that works via pH adjustment (Figure 1A and page 24, column 1, fourth paragraph), not filtration, although “viral filtration was also widely claimed as a basis for validating viral clearance, encompassing approximately 30% of reported viral clearance operations” (page 24, column 1, fourth paragraph). Still, Figure 1A-1B in Johnson et al. highlight the continuous flow of a biopharmaceutical product, which goes through a channel up to point 4 in Figure 1A, which Figure 1B describes as an in-line spike (virus solution) with a channel meeting the first channel (protein solution) after the protein purification unit but before the virus inactivation unit. Said in-line spike supplies a virus solution to this second channel to create a mixture of the two solutions through a mixer (Figure 1A) before reaching the virus inactivation (VI) tank (Figure 1A). The “in-line” spiking channel (dotted line in Figure 1B) is separate from the protein flow channel (solid line in Figure 1B).
While these figures show pH-based viral inactivation, Johnson et al. taught that “most viral clearance is achieved by chromatographic separations, low pH or solvent/detergent (S/D) viral inactivation, or viral filtration methods” (page 24, first paragraph under the heading “Viral Clearance”) and that “viral filtration as an alternative encompasses about 30% of viral clearance operations” at the time (page 24, first column, 4th paragraph). These notions, paired with Johnson’s teaching that “method to adapt viral filtration into a continuous process” (page 28, last paragraph on the left) “can easily be integrated into an in-line viral spiking system” (page 28, first paragraph on the right), make it clear that the pH-based viral inactivation unit could have obviously been substituted with a virus filtration unit as they both have the equivalent use in the art of virus clearance, and a person of ordinary skill in the art (PHOSITA) would have had a reasonable expectation of success owing to the shared use of these protein purification and virus inactivation units and the success of both in the prior art. Thus, a virus filtration unit is a mere substitution of one virus clearance unit for another to arrive at predictable results of virus removal.
Johnson et al. also taught that “in CP viral clearance studies, there could be cases where there is no need to assess the capture step and make a claim for viral clearance for that particular step. Usually, protein A chromatography clears 1–2 log10 of virus by separation power... If other downstream steps are sufficiently robust and effective for viral clearance, they can compensate for this claim, thereby omitting the need for protein A viral clearance validation” (page 30, paragraph before “Conclusion”). Apart from the teaching of continuous liquid flow, Johnson et al. further taught that “the integration of an in-line conditioning system between unit operations, such as the one currently offered both in small scale or production size models from GE Healthcare (GE, 2010) offers a way to ensure consistent flow with the constant buffer changes” (page 30, first paragraph on the left). Furthermore, “an automated parallel switch-in and -out system filtration scheme (Fig. 2A) between old and fresh filters before they reach a validated total volumetric throughput … allows for incorporation into the post-polishing feed stream of the continuous process with little to no hold while ensuring less filter fouling with cleaner feed streams. The concept of staging them in parallel to allow switch-out as they foul which enables them to be in a CP process. The input stream will be held and mixed in a surge tank to control the flowrate” (page 28, last paragraph on the left). “After proper mixing, the stream is sent to a three-way valve junction where the feed can be directed into one of three channels”, and “pump placed after the flow meter will direct the feed into the next channel” (page 28, last paragraph on the left), suggesting that flow rates can be controlled by a pump and that the number of channels depends on the column assembly and a pausing step required for filter replacement.
Overall, Johnson et al. not only taught various in-line spiking methods for validating virus clearance/inactivation in a continuous process but also disclosed partial virus clearance (1–2 log10 reduction) by protein A chromatography during protein purification before pH-based virus inactivation, with further teachings about the use of multiple channels and pump-controlled flow direction and flow rate into each channel. These viral testing methods (Abstract) obviously require measure of the virus level contained in the filter permeate in order to truly validate safety and efficiency based on log10 virus reduction value (LRV) (page 27, last paragraph on the left). However, they fell short of teaching a system for continuous virus filtration (not through pH-based inactivation) that has a first constant flow rate for the protein solution and a second constant rate for a virus solution.
The teaching of Johnson and the obviousness conclusion for virus filtration above are further supported by Li et al. 2020 with respect to “novel spiking methods developed for anion exchange chromatography operating in a continuous process” (title), using “spiking and loading conditions… modified to mimic the variance introduced by the transition between two connected columns” (abstract and Figure 1 on page 3382). For clearance of mouse minute virus (MMV) (section 2.2 on page 3381), their teaching “focused on the impact of potential fluctuations in load and viral spike concentrations that might be introduced by connected, continuous processes, on the viral clearance performance” (page 3384, first paragraph on the left). Among five methods used for purification of a monoclonal antibody (IgG1) (page 3381, section 2.1), which is a protein, “Method 1” (Figure 1 on page 3382) had constant “mAb conc.” and constant “Virus conc.”, which “represents the virus-spiked loading for a downscaled model of a batch process, typically used in a viral clearance study, in which a homogeneous mixture of virus spike and mAb feed are loaded” (page 3381, section 2.4) . To assess efficiency of virus clearance, “Log10 reduction values (LRVs) were calculated as the logarithmic reduction of total infectious virus particles as recommended by the International Conference on Harmonization Q5A” (page 3381, section 2.2).
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TABLE 1 from Li et al., 2020 (page 3383). Arrows point to elements pertinent to claim limitations in the instant application.
“Method 1” in Li et al. further disclosed that “100 ml of 8.8 ± 0.5 g/L mAb feed was spiked with 0.33 ml MMV stock, and the resultant MMV concentration was measured to be 6.3 ± 0.3 log10 TCID50/ml (a total of 8.2 log10 TCID50 MMV)” (page 3381, section 2.4), within the range of “virus infectivity titer (Log10 TCID50…) 2 or more and 10 or less” in the instant application (claim 9). In contrast, “the mAb feed was split into two pools (18 CV of pool A, 2 CV of pool B)” in “Method 2”, before MMV stock (0.33 ml) was spiked into pool B only. The total MMV spiked into this small pool was 7.8 ± 0.3 log10 TCID50. In the load phase, 9 CV of pool A was loaded onto the column using System pump A, followed by 2 CV of the spiked pool B by System pump B, and finally, the remainder of unspiked pool A using System pump A” (page 3381, section 2.4). “Loading of spiked and unspiked pools was performed by two separate pumping systems to avoid mixing or contamination of the load materials. This method introduced… MMV at a concentration of 6.9 ± 0.3 log10 TCID50/ml, mid-way through the loading of the mAb” (page 3382, first paragraph on the left). Moreover, “virus can also be spiked and mixed in‐line with the feed stream instead of spiking directly into the mAb pool, and this was evaluated in Method 5” (page 3384, second paragraph on the right). Regardless of the methods, “data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar” (abstract): “~8 log10 TCID50 MMV” was reduced to less than 4.0 log10 in all four fractions (5 CV each), including “a final buffer wash” (fraction 5, also 5 CV) (FIGURE 2 on page 3383). Thus, Li et al. taught various operational procedures for virus spiking and assessment of virus clearance in terms of LRV based on measurements of TCID50, with a clear motivation for “two separate pumping systems to avoid mixing or contamination.” While their teachings “support the use of a constant introduction of (virus) spike using an in-line method” (page 3387, paragraph preceding section 4 | CONCLUSION), their “methods can also be readily adopted in the evaluation of other flow-through unit operations, such as membrane chromatography and virus filtration, that may have fluctuations in feed introduced from a previous unit operation.
The joint teachings of Johnson et al. and Li et al. would have guided a skilled artisan to arrive at an invention that adopts a two-channeled system for protein (MAb) purification and virus clearance, with pump-controlled liquid/stream flow. The pH-based virus inactivation unit in Johnson et al. is readily inter-changeable with the virus filtration unit in Li et al., while the amount and type of spiked virus, as well as the measurement of LRV (the basis for assessing efficiency of virus clearance) could follow the teaching of Li et al. However, neither Johnson et al. nor Li et al. explicitly taught the use of a first pump for maintaining a first constant rate for the protein solution and the use of a second pump for maintaining a second constant rate for the virus solution.
The deficiency left by Johnson et al. and Li et al. is overcome by Lutz et al. 2011, as they taught “a novel inline spiking method for virus filter validation” (title), using a “syringe pump” to supply virus spiking before a “Static Inline Mixer’ and a “Virus Filter” (Figure 2 on page 123), and “the virus spike was continuously injected, mixed with prefiltered feed, sampled, and fed to the virus filter (Inline spiking test system on page 123). A “dual syringe pump… provide accurate and uniform matched flows between the spiking syringe and sampling syringe” (Inline spiking test system on page 123). Their “inline spiking” was an equivalent of a second channel that supplies virus solution. For actual testing of “MAb1” (a monoclonal antibody) spiked with “ΦX-174” virus, spike flow to filtrate flow ratio was either 5% or 10% (TABLE 1 on page 125; TABLE 2 & TABLE 3 on page 126), and virus spike titers (log10 pfu/mL) ranged from 6.1 to 8.8, and spike volumes was set to 5% or 10% (Tables 2-4 on pages 126-127). Residual virus left in the permeate was measured and used to calculate LRV, which was defined as “log10(pooled feed viral load/pooled filtrate viral load” (formula 3 on page 125). Additional “inline” testing involved “MAb2” spiked with “5% MMV and 5% xMuLV” (page 126, last paragraph on the left), with LRV ranging from ≥3.4 to ≥4.4 (Table 4 on page 127).
The dual syringe pump in Lutz and the teaching of “two separate pumping systems to avoid mixing or contamination” by Li would motivate a PHOSITA to use two pumps, with pump 1 controlling the flow rate of protein solution in the first channel devoted to protein purification. Pump 2 then controls the liquid flow rate of the second channel that is placed downstream of the protein purification unit for virus spiking. The two channels then meet in front of an inline mixer so the protein solution and virus solution form a homogenous mixture before passing through a virus removal filter, as taught by Li. The pumps maintain a continuous flow and constant rate for the protein solution and virus solution at each channel, as Lutz emphasized “a constant percentage spiking ratio” (page 123, last paragraph on the right). In this case, the ratio of B/(A+B) is maintained through a constant rate A and a constant rate B, where A and B correspond to protein flow and virus flow, respectively, which is the same as formula (1) in claim 7 of the instant application. Thus, it is clear that constant flow rates are known, functional rates for the methods of the prior art above, and so all flow rates of the instant claims being constant is obvious here.
Of note, Lutz et al. 2011 also taught that “inline spiking mirrors manufacturing operation with inline prefiltration… and maintains high throughputs during spiking” with “a constant flow ratio” (1st paragraph under Summary and Conclusions on page 127). “The volume vs. time data was numerically differentiated to calculate flow rate and divided by virus filter area to determine the LMH flux. “The filtered volume was divided by the virus filter area to determine L/m2 throughput” (page 124, 2nd paragraph on the right and below Figure 3). In testing “two mammalian virus spikes: MMV and xMuLV” (Abstract), “the MMV stock had a starting titer of 7.64 log10 (TCID50/mL) and a protein content <25 µg/mL”, while “the xMuLV stock… had a starting titer of 7.07 log10 (TCID50/mL) with a protein content of 335 µg/mL” (page 122, second paragraph under Virus spikes and assays), so the protein concentrations in the two virus stocks differed by over 13-fold. “Inline MMV” and “Inline xMuLV” also had a broad range of flux (scaled as 0-500 LMH) and throughput (scaled as 0-1000 L/m2) (Figures 7-8 on page 127). A step for “filter flushing” with water and buffer was also included as part of “standard operation” (page 123, last paragraph on the left). Thus, Lutz et al. 2011 also taught operational parameters, including emphasis on fixed spiking ratio, the inclusion of a flushing/washing step, as well as methods for calculating LRV and LMH flux based on virus titer, flow rate; and virus filter area. Moreover, pump-controlled “constant flow ratio” in Lutz et al. 2011 was intended for maintaining “a constant percentage spiking ratio” (page 123, last paragraph on the right).
For a skilled artisan interested in testing virus removal as a priority, starting from the apparatus of Johnson et al. 2017 and simplifying it according to the teachings of Li et al. 2020 and Lutz et al. 2011 would readily arrive at a two-channeled apparatus suitable for continuous purification of protein products and validation of virus clearance for the reasons above, with two constant flow rates controlled by pumps to maintain operational parameters like a fixed virus spiking ratio (5% or 10%) and flux rate (LMH), and measurement of virus titer in the pooled solution (protein and virus mixture) before virus filtration and residual virus in the permeate forms the basis for calculating LRV, meeting all the elements for structure and work flow cited in claim 1.
With respect to the first pump that maintains first constant flow rate of protein solution in claim 2 and a second pump that controls a second constant flow rate of virus solution in claim 3 and, Li used separate pumps for protein solution and virus solution to avoid contamination, while Lutz adopted a dual syringe pump for controlling the flow of protein and virus solution. As Johnson and Lutz both taught the positioning of the in-line spiking channel after the protein purification unit and before the virus clearance unit, it would be obvious to place a pump to control the flow rate of protein solution in first channel and the flow rate of the virus solution in the second channel in order to maintain a fixed spike ratio (5% or 10%) as taught by Lutz. These references taught different options for the use of pump to control feed rate and feed direction, effectively meeting the limitations of claims 2-3 in the instant application.
Regarding claims 4-5, the continuous processing method of Johnson and Lutz and the obvious method above using the combined references will ensure continuous flow or streaming of the protein solution into the protein purification unit. Similarly, the second channel and the continuous method of the obvious method above will allow for the virus solution to be continuously “pumped” into the second channel (Lutz, Figure 2 on page 123). The same argument is made for claim 6, as Lutz taught “a constant flow ratio” for protein solution and virus solution (1st paragraph under Summary and Conclusions on page 127) in order to “maintain a constant percentage spiking ratio” before reaching the virus filtration (page 123, last paragraph on the right). Li et al offered the same teaching: “a homogeneous mixture of virus spike and mAb are loaded onto the column” (page 3381, 2nd paragraph under section 2.4) for anion exchange chromatography.
Claims 7 and claim 12 are also rejected because they both depend on claim 1, and recitation of formula (1) in claim 7 and formulae (2-5) in claim 12 only disclose inherent features of claim 1 and fail to specify or limit the structure, active step or function of claim 1 beyond the broad intention for virus clearance test. All rates and starting viral spike concentrations (before and after mixing with protein) are result-effective variables as they will determine the efficiency of the purification system in the obvious method above as well as the accuracy of the viral clearance validation measurement. Thus, they will be arrived at by routine experimentation. It has long been settled to be no more than routine experimentation for one of ordinary skill in the art to discover an optimum value of a result-effective variable: "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum of workable ranges by routine experimentation." Application of Aller, 220 F.2d 454, 456, 105 USPQ 233, 235-236 (C.C.P.A. 1955). "No invention is involved in discovering optimum ranges of a process by routine experimentation." Id. at 458, 105 USPQ at 236-237. The "discovery of an optimum value of a result-effective variable in a known process is ordinarily within the skill of the art." Application of Boesch, 617 F.2d 272, 276, 205 USPQ 215, 218-219 (C.C.P.A. 1980). The same can be said for the flux of the permeate in claim 11, and for the membrane area and D values and flux values in claim 12, and all these results will all be arrived at by routine experimentation because they are result-effective variables.
For claim 9, “virus infectivity titer” before filtration (“Log10 TCID50… is 2 or more and 10 or less”) was first taught by Li et al and then by Lutz et al. Specifically, Li et al. taught that “MMV concentration was measured to be 6.3 ± 0.3 log10 TCID50/ml (a total of 8.2 log10 TCID50 MMV)” (page 3381, section 2.4), while Lutz et al. used virus spike titers (log10 pfu/mL) at 6.1-8.8 and set spike volumes to 5% or 10% (Tables 2-4 on pages 126-127). While such variations in virus titer between references are within the “Log10 TCID50” range of “2 or more and 10 or less” in claim 9, they also reflect the different choices for work flow. For example, Lutz et al. 2011 tested three viruses (ΦX-174, MMV, xMuLV) at two spike volumes (5% and 10%) (Tables 2-4 on pages 126-127), while Li et al focused on MMV alone and provided different methods for virus spiking (TABLE 1 on page 3383 and shown earlier). Li et al further asserted that “virus needs to be spiked… to provide enough virus to determine viral reduction” (page 3380, first paragraph on the right, lines 13-15).
With respect to claims 13, Lutz et al. 2011 taught that “filter flushing with water and buffer” was a “standard” procedure (page 123, last paragraph on the left). Johnson et al. 2017 also made it clear that “filter needs to be in off-line mode prior to buffer flushing” (page 28, last paragraph on the left), so pausing is necessary before flushing. Li et al. further taught a washing step for “anion exchange chromatography” (section 2.3 on page 3381): “the total mAb load was maintained at 175 ± 10 mg/ml for all runs, and the flow rate was set at 1 ml/min to achieve a contact time…. and four flow-through fractions of 5 CV each were collected. At the end of the load phase, the column was washed with 5 CV of equilibration buffer, and the collection of this wash fraction was terminated when OD280 dropped to <50 mV. This wash was collected and assayed as a fifth fraction, and a percentage of each of the five fractions was pooled and assayed for infectious virus.” Moreover, Li et al. also measured virus remaining in “a final buffer wash” (fraction 5) that was applied to five methods (FIGURE 2 on page 3383), including “Method 1” that maintained constant mAb concentration and constant virus concentration (FIGURE 1 on page 3382 and FIGURE 2 on page 3383. In other words, regardless of the methods for protein flow and virus spiking (constant rate or not), pausing and flushing are routine procedures in the operation of a continuous flow system, and viruses in each step, including washing, can be measured for residual infectious virus to establish LRV. , Thus, all three references taught the washing/flushing step in the context of cleansing and assessment of virus filtration efficiency, with an pausing (off-line) that allows the in-flow of washing buffer. Overall, these teachings meet the limitations of claims 13. The reason for pausing protein and virus flows to allow a “flushing step” was also well-known in the art: “clogging and filter overload” are common issues for continuous virus filtration (Johnson et al. 2017, page 28 – second last paragraph on the left), and “trace quantities of foulants that are difficult to measure by other analytical methods can plug virus filters” (Lutz eta l., page 122, first paragraph on the left). Therefore, washing both the protein purification unit and virus filtration filter are obvious here and this will be done after pausing protein solution entry, which will cause pausing of protein solution into the first channel. Assaying this wash was one in the prior art as stated above and so is equally obvious to do here for the advantage of continuing to verifying purification quality.
For claims 15-16, Lutz et al. 2011 further taught that MMV and xMuLV had protein content <25 µg/mL and 335 µg/mL, respectively (page 122, second paragraph under Virus spikes and assays), so it is also obvious to have protein in the virus solution before spiking. In terms of specific protein content, Li et al (Table 1) simulated “fluctuating conditions that might be introduced from connected, continuous processes”, and for their “Method 4”, “mAb feed was split into two pools at different mAb concentrations (18 CV of pool A at a mAb concentration of 3.1 ± 0.1 g/L and 2 CV of pool B at a mAb concentration of 56 ± 2 g/L)…. MMV stock was spiked into pool B to a measured total 8.1 ± 0.3 log10 TCID50; the concentration of virus was 7.2 ± 0.3 log10 TCID50/ml” (page 3382, last paragraph on the left). In other words, the protein solution and the virus solution for spiking before virus filtration was the same mAb (a protein), meeting the limitations of claims 15-16.
Coming to claim 18, “comparing an amount of the virus contained in the mixture before passing through the virus removal filter with the amount of the virus contained in the permeate of the mixture which has passed through the virus removal filter” is a step for calculating LRV, which is defined in Lutz et al. 2011 as “log10(pooled feed viral load/pooled filtrate viral load” (formula 3 on page 125). When “Viresolve NFP” and “Viresolve Pro” filters were tested, their LRV ranged from 3.42 (Table 4 on Pg. 127) to 6.3 (Table 3 on Pg. 126), depending further on type of proteins (antibody versus bovine serum albumin), type of viruses (ΦX-174, MMV, xMuLV), virus spike titers (log10TCID50/mL = 6.1-8.2) and spike volumes (5% versus 10%) (Tables 2-4 on pages 126-127). Li et al. went further to teach that “Log10 reduction values (LRVs) were calculated as the logarithmic reduction of total infectious virus particles as recommended by the International Conference on Harmonization Q5A” (page 3381, section 2.2), so multiple references taught the LRV methods and range of LRV that meets regulatory requirements, rendering claim 18 obvious.
For two other dependents of claim 1, claim 19 cites a limitation that “wherein the protein purification unit has a virus removal capacity”, while claim 20 extends claim 19 by citing “wherein a log reduction value (LRV) in the protein purification unit [[11]] is 0 or more and 7 or less.” These are obvious parameters from Johnson et al. 2017, as they made it clear that “Protein A chromatography clears 1–2 log10 of virus by separation power” (page 30, paragraph before “Conclusion”). Indeed, use of protein A chromatography in purification of monoclonal antibodies is consistent with the use of antibodies as model proteins in the other two references: mAb in Lin et al 2020; IgG2 in Lutz et al. Thus, partial virus removal in the protein purification unit is a well-known operational parameter cited in multiple references.
Overall, the invention of claims 1-7, 9, 11-13, 15-16 and 18-20 as a whole was rendered prima facie obvious by Johnson et al. 2017, Li et al., 2020 and Lutz et al. 2011, as an application of rationales A and B of MPEP 2143: substituting the virus inactivation unit in Johnson et al. 2017 with a virus filtration unit from Li et al. 2020 and then insert a pump-controlled in-line virus spiking channel from Lutz et al. 2011 between a protein purification unit and a virus clearance filter would arrive at a two-channeled, continuous virus removal/clearance system that mimics manufacturing operation in the production of therapeutic proteins, including monoclonal antibodies. Pump-controlled constant flow of protein solution and virus solution was made obvious by Li et al., 2020 and Lutz et al. 2011 in terms of constant spiking ratio, while the virus titers and protein content before spiking, as used by Li et al., 2020 and Lutz et al. 2011, was broad due to different choices of spiked viruses, as well as other methodological preferences for virus culture, purification and spiking volume, as taught by Li et al. and Lutz et al. The flux can also vary by filter size, while partial virus clearance during protein purification is readily achievable by Protein A chromatography, as disclosed by Johnson et al. 2017. The end result would be a success in a virus clearance system that has a LRV ≥4 (FIGURE 2 in Li et al. 2020), and more specifically between 4.4 and 6.2 in the test of three viruses (Lutz et al. 2010), but other parameters like protein type, virus type, filter type, virus spike volume and amount of spiked virus can all impact the outcome, as taught by Li et al. 2020 and Lutz et al. 2010, so routine optimization/customization is expected. Finally, to reduce clogging and filter overload, pausing the protein and virus flow to allow a “washing/flushing step” is a routine procedure recommended by Johnson et al. 2017 and further supported by Lutz et al. 2011. The method for measuring residual virus in the flushing/washing step was taught by Li et al. 2020.
Claims 13-14 are rejected under USC §103 as being unpatentable over Johnson et al. 2017 (supra) in view of Li et al. 2020 (supra) and Lutz et al. 2011 (supra) as applied to claims 1-7, 9, 11-13, 15-16 and 18-20, further in view of Hongo & Hayashida 2014 (PGPub 2014/0199262 A1, published 07/17/2014).
As discussed supra for claims 1-7, 9, 11-13, 15-16 and 18-20, Johnson et al. 2017, Li et al. 2020 and Lutz et al. 2011 collectively taught all the elements of claims 1 and 13 for a two-channeled, continuous virus removal/clearance apparatus that allows constant (pump-controlled) flow of protein solution and virus solution, and the mixture is filtered for virus removal to achieve LRV of 4 based on the measurement of virus titers (log10TCID50/mL) before and after filtration, including a washing step to reduce “clogging and filter overload” (Johnson et al. 2017, page 28 – second last paragraph on the left). However, the three references fell short of teaching the exact time for pausing between virus clearance mode and washing/flushing mode in the operation.
Hongo & Hayashida, on the other hand, taught “a filtration step of filtering a virus-containing protein solution through a small pore size virus removal membrane to obtain a virus-free protein solution” (Abstract), which “can be performed by a usual method… including… constant-rate filtration in which the rate of filtration is kept constant, etc.” (¶[0082]). The “constant-rate filtration” cited in Hongo & Hayashida is consistent with the concept of “constant spiking ratio” taught by Lutz et al. 2011. To reduce “clogging”, Hongo & Hayashida taught a washing/flushing step using “protein-free buffer solution)” (¶[0005]): “this filtration step is added after the protein filtration and therefore called post-wash or post-filtration. For this postwash, typically, the filtration pressure is temporarily relieved in order to have a switching, at an entrance of solution to be filtered, from a line for protein solutions to a line for washing solutions. If the filtration pressure is not decreased, the solution flows backward to the washing solution side.” (¶[0005]). Specifically, “in the case of performing the post-wash step, a line to which the solution prior to filtration is introduced is switched because the washing solution is filtered instead of the protein-containing solution. If the filtration pressure is kept high during this line switching, the solution flows backward to the washing solution side. Thus, the filtration pressure is temporarily relieved to 0.0 kPa. After the line switching, the filtration pressure is applied again to filter the washing solution. The time period from the pressure drop to zero to the restart of filtration of the washing solution under filtration pressure is not particularly limited. A sufficient pressure drop occurs after, for example, 5 seconds or longer. A more sufficient pressure drop occurs after 1 minute or longer, 5 minutes or longer, or 30 minutes or longer. From the viewpoint of workability, the filtration is often restarted, for example, within 7 days, within 5 days, within 3 days, or within 24 hours” (¶[0122]). Claim 24 in Hongo & Hayashida (page 14) further taught two formulae for calculating LRV before and after washing, with both LRV measurements being “4 or higher” (¶[0040] and claim 24 on page 14).
For reasons discussed supra, a skilled artisan could readily follow the teachings of Johnson et al. 2017, Li et al. 2020 and Lutz et al. 2011 to arrive at a two-channeled apparatus controlled by pumps to facilitate continuous purification of protein products and validation of virus clearance, with a washing/flushing step to reduce/clear clogging or filter overload. The pausing time could follow the detailed teaching of Hongo & Hayashida, ranging from “5 seconds” to “30 minutes or longer”, meeting the limitations of claim 14. The motivation for pausing before washing, according to Hongo & Hayashida is to allow “a sufficient pressure drop” and a “switching” between protein solution and buffer solution (¶[0005] and ¶[0122]) . Thus, the invention of claims 13-14 as a whole was rendered obvious by four references before the time of invention, as an example of rationale A of MPEP 2143: Combining prior art elements according to known methods to yield predictable results.
Claims 8, 10, 15 and 17 are rejected under USC §103 as being unpatentable over Johnson et al. 2017 (supra), Li et al. 2020 (supra) and Lutz et al. 2010 (supra) as applied to claims 1-7, 9, 11-13, 15-16 and 18-20 above, further in view of Yanagida 2011 (PGPub 2011/0166326 A1, published 07/07/2011).
As stated earlier in the rejection of claims 1-7, 9, 11-13, 15-16 and 18-20, Johnson et al. 2017, Li et al. 2020 and Lutz et al. 2011 collectively taught all the elements of claim 1 for a two-channeled, continuous virus removal/clearance apparatus that allows constant (pump-controlled) flow of protein solution and virus solution, and the mixture is filtered for virus removal to achieve LRV of 4 based on the measurement of virus titers (log10TCID50/mL) before and after filtration, but these references did not delve into the details about (i) the ratio of two flow rates as applied separately to protein solution and virus solution, (ii) the size of virus removal filter, and (iii) specifics about the protein composition in virus solution before spiking.
The deficiencies left by Johnson et al. 2017, Li et al. 2020 and Lutz et al. 2011 are overcome by Yanagida’s virus removal system, which was applied to protein/immunolglobulin purification (¶[0016]-¶[0020]). The “effective membrane area of the virus removal membrane is suitably 0.0001 to 0.03 m2” or “preferably 0.0003 to 0.02 m2) (¶[0032]) to allow adequate filtration volume and enough virus for downstream quantification (¶[0032]). Yanagida 2011 also taught that the flowrate of protein solution may change according to “protein concentration” (¶[0030]), and that the flowrate of virus solution could vary according to the shape of virus removal membrane (“flat membrane” in ¶[0033] or “hollow fiber membrane” in ¶[0034]). With a filtration time of “3 to 5 hours” per run (Yanagida 2011, claim 2 on page 7) and “an average protein filtration rate of 1.0 kg/m2/Hr or more” (¶[0049]), the “virus removal rate”, as calculated according to “virus concentration in the solution before filtration, and… virus concentration in the solution after filtration with the virus removal filter” (¶[0045]), and “it is desirable to add the virus solution in an amount equal to or more than the detectable minimal amount of a virus removal rate (LRV) of 3 or more” (¶[0044]). In Yanagida’s tests for three viruses, each virus solutions contained “human immunoglobulin G” at “30 mg/ml” ([0063]), while “human immunoglobulin G concentration of 30 mg/ml” served “as a model of intermediate protein product” for purification (¶[0056]). Moreover, “protein product may be filtered using a virus removal filter” as a pretreatment step “to improve filtration rate (¶[0030]). In one example, the system was used to filter/clear “porcine parvovirus (PPV)” (¶[0056]). The ratio of PPV (virus) solution to human immunoglobulin G (protein) solution was “0.5 vol %” (¶[0056]), but “when the virus solution has not been purified and contains a large amount of impurities, the virus solution is preferably added in a small amount of 1 vol % or less, preferably 0.5 vol % or less, and more preferably 0.1 vol % or less” (¶[0044]). The “virus removal rates (LRV)… obtained after 1, 3 and 5 hour of filtration were 5.6 or more” (¶[0057]), and the flux in Yanagida 2011, when converted to LMH (L·m−2 h−1), ranged from 10 LMH to 50 LMH based on “protein concentration of 20 to 100 mg/ml” (¶[0049]), which is consistent with the teaching of Lutz et al. 2010 (Figures 7-8 on page 127). Li et al recommended “that future studies cover the entire design space… and be tailored to each
specific case” (page 3387, first paragraph on the right) “to simulate other fluctuating conditions, such as pH, buffer composition, salt concentration, multiple peaks of concentrated mAb load or virus spike” (page 3387, first paragraph on the right). Their “methods can also be readily adopted in the evaluation of other flow-through unit operations, such as membrane chromatography and virus filtration, that may have fluctuations in feed introduced from a previous unit operation” (page 3387, first paragraph on the right).
For claim 8, the spiked virus accounts for 0.1% to 20% of the total volume of protein-virus “mixture” (a sum of the first constant rate and the second first constant); Yanagida taught “0.5 vol %” for virus spiking (¶[0056]), while Lutz et al. chose a “5% or 10% fixed rate (Tables 2-3 on page 126). “Method 5” taught by Li et al. “delivered a constant concentration of virus spike”: “0.33 ml (measured 7.9 ± 0.3 log10 total TCID50) of virus stock was diluted with equilibration buffer to a final volume of 4 ml and loaded separately through System pump B at a flow rate of 0.05ml/min” (page 3382, second paragraph on the right). “The total mAb load was maintained at 175 ± 10 mg/ml for all runs, and the flow rate was set at 1 ml/min to achieve a contact time with the resin” (section 2.3 on page 3381), so the constant virus flow through “System pump B” in a continuous mode accounts for 5.25% [0.05ml/min over (0.05 ml/min + 1 ml/min)] of the mixture in Li et al., which fell in between the 0.5% and 10% range set by Yanagida and Lutz et al, substantially overlapping the range cited in claim 8. Furthermore, each rate of the claim is a result effective variable as it affects protein quality and quality testing and so both rates, and thus their ratio, will be arrived at by routine experimentation by a PHOSITA.
As for claim 10, “the virus removal filter has a membrane area of 0.0001 m2 or more and 4 m2 or less”, while Yanagida taught that filter types are known to vary ¶[0032]). In one example Yanagida defined filter size as “suitably 0.0001 to 0.03 m2” or “preferably 0.0003 to 0.02 m2) (¶[0032]). For flux, the end results in Yanagida ranged from 10 LMH to 50 LMH – well within “0.1 LMH or more and 500 LMH or less” being cited in claim 11 (rejected earlier). And by definition, LMH value varies according to “membrane area” (m2) in the virus removal filter “(0.0001 m2 or more and 4 m2 or less)” in claim 10 versus “suitably 0.0001 to 0.03 m2” in Yanagida 2011 (¶[0032]). The use of “human immunoglobulin G” at “30 mg/ml” in virus solutions (¶[0063]) versus “human immunoglobulin G concentration of 30 mg/ml” as a model of intermediate protein product” for purification (¶[0056]) in Yanagida 2011 met the limitations of claims 16-17: the same protein is present in the protein solution and virus solution, even at the same concentration. Of note, maintaining a certain range of virus spiking ratio and protein concentration in both channels has one key motivation: “regulatory guidelines suggest limiting spiking volumes to not more than 10% of the feed solution to minimize the impact on the feed solution properties” (Lutz et al., page 124, fourth paragraph on the left), as protein content is part of the feed solution properties. Having the same protein at the same concentration for both protein solution and virus solution should further mimic the continuous protein purification process that Li et al attempted to accomplish in their Method 5 of “in-line spiking to deliver a constant concentration of virus spike” (page 3383, TABLE 1, FIGURE 2 and second paragraph on the right). Routine experimentation would also allow a range of protein concentrations for the various assays, as “fluctuating conditions… might be introduced from connected, continuous processes (Li et al, TABLE 1 on page 3383). Also see Application of Aller, 220 F.2d 454, 456, 105 USPQ 233, 235-236 (C.C.P.A. 1955). "No invention is involved in discovering optimum ranges of a process by routine experimentation." Id. at 458, 105 USPQ at 236-237.
A skilled artisan could readily follow the teachings of Johnson et al. 2017, Li et al. 2020 and Lutz et al. 2011 to first arrive at a system similar to claim 1 and then follow the teachings of Yanagida 2011 to consider options for the spike ratio and virus filter size before arriving at an acceptable LRV (≥4), as taught by Li et al. (FIGURE 2 on page 3383). Variations in parameters like protein flow, virus flow and filter size are also expected, as “differences in mAb and virus loading patterns… might be observed in a continuous process” (Li et al., page 3384, last paragraph on the right). And “unlike batch processing, each manufacturer may use distinct methods and equipment for continuous processing” (page 3380, first paragraph on the right). In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%) (see MPEP 2144.05). Thus, the operational and consequential elements of claims 8, 10, 15 and 17 are rendered prima facie obvious and unpatentable by four references: as another application of rationales A and B of MPEP 2143.
Conclusion
No claims are allowed.
Additional Prior Art Cited but Not Applied
Maiser et al. 2018 (WO 2018/075716 A1, published 04/26/2018 and of record on ISR and IDS).
This prior art taught “a novel, simple and inexpensive solution for demonstrating that viral contamination is reliably cleared” (page 1, lines 35-36), drawn to a method (Fig. 1) for “validation of continuous viral clearance” (title) in a streamlined operation, “comprising the steps of providing a probe to be validated, spiking the probe in a valid manner, performing viral clearance, sampling the spiked probe and analyzing the sample of the spiked probe” (Abstract), which “allows for a simple and inexpensive validation of continuous viral clearance” (page 2, lines 3-4) and “results in less viral particles being present in the probe…” (page 2, lines 22-23). “The probe comprises at least one component selected from the group consisting of a peptide, protein, a small molecule drug, a nucleic acid” (page 5, lines 9-11), and the amount of spiked virus was recommended at “a factor of 104 or 105 of the original probe” (page 3, lines 2-3)”, which is within the range of “virus infectivity titer (Log10 TCID50…) 2 or more and 10 or less” in the instant application (claim 9). And for “calculation of Reduction Factor (RF)” (page 3, lines 2-3), Maiser et al. 2018 provided a formula “RF = log10 [(V1 x T1)/(V2 x T2)]” (page 3, line 10), which is the equivalent of the method for calculating LRV in the instant application (claim 18 and formula (11) under ¶[0085]).
Wieser et al. 2014 (The Evolution of Down-Scale Virus Filtration Equipment for Virus Clearance Studies. Biotechnology and Bioengineering, Vol. 112, No. 3, pp. 633-637; published online 19 November 2014).
This prior art taught the operational parameters of “virus filtration in assuring the safety of biopharmaceutical products” (Abstract), including set-ups (Table 1 on page 634), as well as filtrate volume and filtrate flow rate (page 635, third paragraph on the right). Wieser et al. also made it clear that virus filtration as part of quality assurance is a well-known art because “in order to demonstrate the virus clearance capacity of any manufacturing step, manufacturers are required to perform… virus clearance studies (ICH, 1999)” (page 633, right column).
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/JIANMING TANG/Examiner, Art Unit 1671
/Michael Allen/Supervisory Patent Examiner, Art Unit 1671
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