DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on July 22nd, 2025 has been entered.
Status of the Claims
Claim 1 has been amended. Claims 12-13 have been canceled with claim 20 previously canceled. Claims 1-11 and 14-19 are currently examined herein.
Status of the Rejection
All U.S.C. § 103 and U.S.C. § 112 rejections from the previous office action are withdrawn in view of the amendments.
New grounds of rejection under 35 § U.S.C 103 are necessitated by the Applicant’s amendments.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-7, 9-11, and 14-15 are rejected under 35 U.S.C. 103 as being unpatentable over ProteinSimple iCE3 System User Guide (ProteinSimple iCE3 System User Guide, Revision 12 July 2018) in view of Bo (, Characterization of phospholipid–protein interactions by capillary isoelectric focusing with whole-column imaging detection, Analytical Biochemistry, 250: (2006), 91-98), Valiante (US 2018/0318409 A1), and Smith (US 2018/0243230 A1). Horiba (Isoelectric Point Determination 2018, pages 1-3) is used as an evidence reference for claim 1.
Regarding Claim 1, ProteinSimple teaches a method of separating molecules according to their isoelectric points (molecule stops at a place in the pH gradient where the pH value is equal to the molecule’s pI value in Chapter 2: IEF Principles, page 12), the method comprising: applying a separating voltage (applied voltage in Chapter 2: IEF Principles, page 12) to a separation matrix comprising carrier ampholytes (carrier ampholytes in Chapter 2: IEF Principles, page 15) and the molecules for a sufficient time to separate the molecules according to their isoelectric points (focusing time in Chapter 2: IEF Principles, page 17).
ProteinSimple is silent on separating lipid nanoparticles (LNPs) according to their isoelectric points, wherein the LNPs are empty and comprise, individually, one or more cationic lipid species, one or more non-cationic species, cholesterol, and one or more PEG-lipids.
Bo teaches a method of capillary isoelectric focusing targeted at separating various proteins in the presence of a zwitterionic phosphatidylcholine (a phospholipid, abstract) to study phospholipid-protein interactions based on changes in pI (abstract). Zwitterionic phosphatidylcholine (abbreviated ‘PC’) vesicles were prepared (PC vesicle preparation, page 92) for use in capillary isoelectric focusing. For various proteins, changes in the isoelectric profile were observed when exposed to the PC vesicles due to phospholipid-acidic protein interactions (Phospholipid-acidic protein interactions, first sentence, page 93).
ProteinSimple and Bo are considered analogous art to the claimed invention because they are in the same field of isoelectric focusing. It would have been obvious to one of ordinary skill in the art, before the effective filing date of this application, to substitute the peptides and proteins in ProteinSimple with LNP samples, such as the PC-vesicle protein conjugate samples, as taught by Bo, since it would allow for an analytical approach to characterize LNP samples, for example to study the influences of PC concentration, incubation time, or incubation temperature, using isoelectric focusing (Bo abstract).
Modified ProteinSimple is silent on wherein the LNPs are empty and comprise, individually, one or more cationic lipid species, one or more non-cationic species, cholesterol, and one or more PEG-lipids.
Valiante teaches RNA vaccines and compositions for vaccines (abstract); with lipid nanoparticles being utilized as excipients for cancer RNA vaccines [para. 0330]. Regarding lipid nanoparticle composition, Valiante teaches lipid nanoparticle composition of cationic lipids, neutral lipids, cholesterol, and PEG-modified lipid [para. 0356].
Modified ProteinSimple and Valiante are considered analogous art to the claimed invention because they are in the same field of lipid nanoparticles. It would have been obvious to one of ordinary skill in the art, before the effective filing date of this application, to modify the LNPs of modified ProteinSimple to be composed of one or more cationic lipid species, one or more non-cationic lipid species, cholesterol, and one or more PEG-lipids, as taught by Valiante, as lipid nanoparticles of this composition can be used for cancer vaccines (Valiante, [para. 0004-0005, 0007]).
Modified ProteinSimple is silent on wherein the LNPs are empty.
Smith teaches methods for preparing particles, such as lipid nanoparticles [para. 0092], and teaches wherein the LNPs are empty (“Empty” lipid nanoparticles are formed and their zeta potentials are measured, as illustrated in Figure 5 [para. 0144]). Note that in Figure 5 of Smith the isoelectric point occurs when the zeta potential of the target particle is zero (see abstract of evidence reference Horiba for more information on how isoelectric point is determined using zeta potential).
It would be obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the LNPs of modified ProteinSimple to be empty LNPs, as taught by Smith, as measuring the charge of LNPs using zeta potential or by isoelectric point helps to characterize the LNPs (Smith, [paras. 0025 and 0144]).
Regarding Claim 2, modified ProteinSimple teaches the method of claim 1.
ProteinSimple teaches wherein the separation matrix further comprises a stabilizer (sorbitol, sucrose, and glycerol in section: “I cannot get reproducible peaks due to sample precipitation, what should I do?” page 251).
Regarding Claim 3, modified ProteinSimple teaches the method of claim 2.
ProteinSimple teaches wherein the stabilizer is glycerol (glycerol in section: “I cannot get reproducible peaks due to sample precipitation, what should I do?” page 251).
Regarding Claim 4, modified ProteinSimple teaches the method of claim 2.
ProteinSimple is silent to wherein the stabilizer is present in the separation matrix at 5-15% w/v or v/v.
ProteinSimple does teach wherein stabilizers sorbitol, sucrose, and glycerol may be present up to 25% w/v (section: “I cannot get reproducible peaks due to sample precipitation, what should I do?”, page 251), and the disclosed range overlaps with the claimed range.
It would have been obvious to have selected and utilized the stabilizer in the separation matrix up to 25% w/v, as taught by ProteinSimple, including those amounts that overlap within the claimed range, since one of ordinary skill in the art would reasonably expect any value within the taught range to be suitable given that ProteinSimple specifically teaches the range to be suitable for the stabilizer since it would help to get reproducible peaks (section: “I cannot get reproducible peaks due to sample precipitation, what should I do?” page 251). It has been held that obviousness exists where the claimed ranges overlap or lie inside ranges disclosed by the prior art. See MPEP 2144.05 (I).
Regarding Claim 5, modified ProteinSimple teaches the method of claim 1.
ProteinSimple teaches that the separation matrix further comprises methylcellulose (methylcellulose in Additives and Initial Assay and Focus Settings, page 192).
Regarding Claim 6, modified ProteinSimple teaches the method of claim 5,
ProteinSimple does not teach the methylcellulose is present in the separation matrix at 0.2%-0.25% w/v.
Bo teaches the methylcellulose is present in the separation matrix at 0.25% w/v (0.25% methylcellulose used in sample preparation, preconditioning, and washing steps in section Isoelectric focusing on page 93, whole section).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to use a 0.25% methylcellulose concentration as shown in Bo to modify the method of modified ProteinSimple for separating LNPs, since Bo teaches 0.25% methylcellulose is a suitable concentration of methylcellulose that would enhance resolution for isoelectric focusing experiments (for example, Figure 2 in Bo).
Regarding Claim 7, modified ProteinSimple teaches the method of claim 1.
ProteinSimple teaches the carrier ampholytes form a linear pH gradient (linear pH gradient observed in Figure 2-1, page 13).
Regarding Claim 9, modified ProteinSimple teaches the method of claim 1.
ProteinSimple teaches the separation matrix is in a capillary (capillary in System Specifications, page 72).
Regarding Claim 10, modified ProteinSimple teaches the method of claim 9.
ProteinSimple teaches that the capillary is coated with a fluorocarbon, but does not explicitly state that the capillary is silica (FC-coated cIEF cartridges in FC Cartridge Guidelines page 222).
Bo teaches the capillary is coated with a fluorocarbon (separation cartridge is a silica capillary with inner wall coated by fluorocarbon in Instrumentation, page 92, second paragraph in section).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to modify the method of modified ProteinSimple to use a silica capillary coated with fluorocarbon to ensure successful separation of LNPs by their isoelectric point as Bo successfully demonstrates (Figures 1 and 4 in Bo on pages 93 and 94, respectively).
Regarding Claim 11, modified ProteinSimple teaches the method of claim 1.
ProteinSimple is silent on wherein the one or more cationic lipid species, one or more non-cationic lipid species, cholesterol, and one or more PEG-lipids are in a molar ratio of 50-58:30-38:10:1-2.
Valiante teaches the lipid nanoparticle formulations consist of a lipid mixture in molar ratios of 20-70% cationic lipid, 5-45% neutral lipid, 20-55% cholesterol, and 0.5-15% PEG-modified lipid [para. 0356]. In addition, in some embodiments, the lipid nanoparticle formulation can be 5-50% cholesterol on a molar basis [para. 0346].
It would be obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the LNP composition of modified ProteinSimple to be wherein the one or more cationic lipid species, one or more non-cationic lipid species, cholesterol, and one or more PEG-lipids are in a molar ratio of 20-70:5-45:5-50:0.5-15, as taught by Valiante, as lipid nanoparticle formulation, such as for RNA vaccines, can be influenced by the ratio of all components (Valiante, [para. 0338-0339]. In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).
Regarding Claim 14, modified ProteinSimple teaches the method of claim 1.
ProteinSimple teaches imaging the separation matrix after sufficient time to produce an electropherogram (raw data electropherogram Figure 7-4 on page 170).
Regarding Claim 15, modified ProteinSimple teaches the method of claim 14,
ProteinSimple teaches imaging the separation matrix comprises detecting UV absorbance at 280 nm (whole column light adsorption detection at 280 nm in System Specifications, page 72).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over ProteinSimple in view of Bo, Valiante, and Smith, as applied to claim 1, and in further view of Caspers (Separator Isoelectric Focusing: An Improved Method of Protein Analysis and Purification, Analytical Biochemistry, 79: (1977), 166-180).
Regarding Claim 8, modified ProteinSimple teaches the method of claim 1.
ProteinSimple teaches that the pH gradient of carrier ampholytes can be adjusted to obtain a higher resolution separation in a pH sub-range that includes the isoelectric points of target molecules (a mixture of narrow pH range and wide pH range in various proportions can be used to improve resolution in ProteinSimple section: “Can I use narrow pH range carrier ampholytes to improve the resolution for my sample?” pages 252-253).
Modified ProteinSimple does not explicitly teach that the carrier ampholytes form a sigmoidal pH gradient capable of higher resolution separation in a pH sub-range that includes the isoelectric points of the LNP.
Caspers teaches a gel isoelectric focusing technique that utilizes small amphoteric substances and carrier ampholytes (abstract) to improve the separation of proteins. More specifically, Caspers was motivated to seek better resolution by producing a relatively flat pH region in the gel using carrier ampholytes and amphoteric substances to enable better resolution of proteins within the target pH region (paragraph that extends from page 166-168). Many figures show various shapes of pH gradients. Figure 4, for instance, illustrates the pH profile of gels with constant ampholyte concentration and a varied amount of 5-aminovaleric acid (Figure 4 and paragraph on page 171). From Figure 4, the shape of these pH gradients is sigmoidal in nature.
Modified ProteinSimple and Caspers are considered analogous art to the claimed invention because they are in the same field of isoelectric focusing. It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the carrier ampholytes of modified ProteinSimple with separator amphoteric substances (Caspers page 166, last sentence) that would result in a sigmoidal shaped pH gradient, allowing for an increased resolution of target molecules (paragraph that extends from page 166-168 in Caspers in the pH region of interest.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over ProteinSimple in view of Bo, Valiante, and Smith, as applied to claim 14, and in further view of Zarabadi (Capillary isoelectric focusing with whole column imaging detection (iCIEF): A new approach to the characterization and quantification of salivary α-amylase, Journal of Chromatography B, 1053 (2017), 65-71).
Regarding Claim 16, modified ProteinSimple teaches the method of claim 14. ProteinSimple does not teach measuring the peak area corresponding to the LNPs having a selected pI in the electropherogram and comparing the peak area to a calibration curve to calculate a total lipid concentration.
Zarabadi teaches a capillary isoelectric focusing method using a protein, salivary α-amylase (abstract). In particular, calibration curves for α-amylase are generated using known concentrations, a reference, of standard human salivary α-amylase (Section 3.3 Quantitative analysis, page 68; Figure 4 page 69). Calibration curves are created by measuring the peak area from the capillary isoelectric focusing experiment (Figure 4; page 69). Zarabadi used the generated calibration curve to calculate a total protein concentration (determining α-amylase concentration found in saliva in Section 3.3 Quantitative analysis, page 68).
Modified ProteinSimple and Zarabadi are considered analogous art to the claimed invention because they are in the same field of isoelectric focusing. It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method in modified ProteinSimple by constructing a calibration curve, as taught by Zarabadi, since it would allow to calculate lipid concentrations for LNP samples. Although Zarabadi used a protein sample to generate calibration curves for isoelectric focusing, there is a reasonable expectation that substituting LNPs instead of proteins will yield a predictable result of successfully generating a calibration curve. The use of a known technique to improve similar devices (methods or products) in the same way is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143 (I)(C)).
Claims 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over ProteinSimple, in view of Bo, Valiante, and Smith, as applied to claim 14, and in further view of Cao (Charge profiling and stability testing of biosimilar by capillary isoelectric focusing, Electrophoresis, 35 (2014), 1461-1468).
Regarding Claim 17, modified ProteinSimple teaches the method of claim 14.
ProteinSimple does not teach comparing an electropherogram to a reference electropherogram produced under identical conditions.
Cao teaches the capillary isoelectric focusing of a trastuzumab biosimilar. Capillary isoelectric runs were performed with the same conditions (method of prepping capillary outlined in Method Section 2.3, pages 1462-1463; specificity conditions for conditions during separation outlined in Section 3.3.1 Specificity, page 1465). Runs using identical conditions can be found in Figure 8a, where the protein sample with pI markers is plotted against reference electropherograms ‘blank’ and ‘pI markers’ (Figure 8a on page 1465).
Modified ProteinSimple and Cao are considered analogous art to the claimed invention because they are in the same field of isoelectric focusing. It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method in modified ProteinSimple by adding a comparison of the electropherogram to a reference electropherogram produced under identical conditions, as taught by Cao, since it would allow effective discrimination between the target peaks and other interference peaks from the pI markers and sample mix components (Cao, Section 3.3.1 Specificity, Page 1465). The use of a known technique to improve similar devices (methods or products) in the same way is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143 (I)(C)).
Regarding Claim 18, modified ProteinSimple teaches the method of claim 17, and does not teach the reference electropherogram produced from the same batch of LNPs as the LNPs in the electropherogram, and wherein an acidic shift or altered peaks in the electropherogram indicate a change in LNP stability.
Bo teaches using the same batch of LNPs to generate electropherograms (liposome stock solution created and used within 1 week in PC vesicle preparation, pages 92-93). Bo also discusses stability of protein-phospholipid interactions (Figure 8, page 96), but does not teach acidic shifts or altered peaks in the electropherogram indicate a change in LNP stability.
Cao teaches that the stability of trastuzumab biosimilar can be studied using isoelectric focusing (Section 3.4.1 Stability, page 1466). From Table 3, the stability of trastuzumab biosimilar was determined using a reference electropherogram (the control sample) against other samples subjected to various conditions (Table 3, page 1466). The observation of altered peaks and acidic or basic peak shifts indicates a change in protein stability (Table 3 and Section 3.4.1 Stability, page 1466).
Modified ProteinSimple and Cao are considered analogous art to the claimed invention because they are in the same field of isoelectric focusing. It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the modified ProteinSimple method by using a batch of LNPs having the same lipid composition as the LNPs in the electropherogram, as taught by Bo, and providing a reference electropherogram where an acidic shift or altered peaks in the electropherogram indicate a change in LNP stability, as taught by Cao, since stability criteria are important in downstream processing, storage, functionality, and quality (Cao Section 3.4.1 Stability, page 1466). Although Cao assesses the stability of protein samples by comparing the reference electropherogram with the stability samples using the electropherogram profile and peak pI shifts (Table 3 and Section 3.4.1 Stability, page 1466), there is a reasonable expectation that substituting LNPs will yield a similar outcome, as only the sample is being modified. The use of a known technique to improve similar devices (methods or products) in the same way is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143 (I)(C)).
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over ProteinSimple, in view of Bo, Valiante, Smith, and Cao, as applied to claim 17, and in further view of Janini et al. (Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system, Electrophoresis, 23 (2002), 1605-1611).
Regarding Claim 19, modified ProteinSimple teaches the method of claim 17, Modified ProteinSimple is silent on the reference electropherogram was produced from a reference batch of LNPs having the same lipid composition as the LNPs in the electropherogram, and wherein a difference between the electropherograms indicates a manufacturing problem for the LNPs.
Bo teaches using the same batch of LNPs to generate electropherograms (liposome stock solution created and used within 1 week in PC vesicle preparation, pages 92-93).
Bo does not teach a significant shift between the electropherograms indicates a manufacturing problem for the LNPs.
Janini teaches advances using capillary isoelectric focusing. Specifically, Janini teaches an imaging capillary isoelectric focusing system for a murine monoclonal antibody (abstract), and that capillary isoelectric focusing can be used for pI profiling for monitoring structural changings during the manufacturing process, such as a manufacturing problem (abstract).
ProteinSimple, Bo, Cao, and Janini are considered analogous art to the claimed invention because they are in the same field of isoelectric focusing. It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method in modified ProteinSimple by using LNPs having the same lipid composition to produce the reference electropherogram, as taught by Bo, and use a difference in electropherograms to indicate a manufacturing problem, as taught by Janini, since capillary isoelectric focusing separations are commonly used to assess identity and manufacturing problem for quality control laboratories (Janini Section 3 Results and Discussion, page 1606). Although Janini assesses the manufacturing problem of protein samples there is a reasonable expectation that substituting LNPs will yield a similar outcome, as only the sample is being modified. The use of a known technique to improve similar devices (methods or products) in the same way is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143 (I)(C)).
Response to Arguments
Applicant's arguments, see Remarks pgs. 5-10, filed 05/14/2025, with respect to the 35 U.S.C 103 rejections and amended claims have been fully considered.
Applicant’s Argument #1:
Applicant argues on pages 5-6 that for claims 1-7 and 9-15 that primary reference ProteinSimple and secondary references Bo and Valiante do not teach or suggest the amended limitation the LNPs are “empty”.
Examiner’s Response #1:
Applicant’s arguments have been fully considered, but are moot in view of the new grounds of rejection.
Applicant’s Argument #2:
Applicant argues on pages 7-10 that dependent claims 8 and 16-19, which depend on independent claim 1, are allowable as the secondary references used do not teach or suggest the LNPs are “empty”.
Examiner’s Response #2:
Applicant’s arguments have been fully considered, but are moot in view of the new grounds of rejection.
Conclusion
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/R.L.G./Examiner, Art Unit 1795
/LUAN V VAN/Supervisory Patent Examiner, Art Unit 1795