DETAILED ACTION
Claims 1-3, 5-10, 12-18, 20, and 23-25 are pending.
Withdrawn Claim Objections and/or Rejections
The rejection of claims 1-3, 5-10, 12-18, 20, 23, and 25 under 35 USC 103 as being unpatentable over Fraunhofer and Runyon et al., as set forth on pp. 3-11 of the previous office action (mailed on 12/01/2025) has been withdrawn in view of the amended claims.
The rejection of claim 24 under 35 USC 103 as being unpatentable over Fraunhofer and Orlandi et al., as set forth on pp. 11-14 of the previous office action (mailed on 12/01/2025) has been withdrawn in view of the arguments (filed on 03/02/2026).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-3, 5-10, 12-18, 20, and 23-25 are rejected under 35 U.S.C. 103 as being
unpatentable over Fraunhofer et al., “Asymmetrical flow field-flow-fractionation in
pharmaceutical analytics”, 226 pages, 2003 Ludwig Maximillans University (2003) (IDS filed
on 11/03/2022), in view of Roda et al., “Toward Multianalyte Immunoassays: A Flow-Field Assisted, Solid-Phase Format with Chemiluminescence Detection”, Clinical Chemistry, Volume 51, Issue 10, 1 October 2005, Pages 1993-1995, https://doi.org/10.1373/clinchem.2005.053108, and in view of Rapp et al., Biosensors with label-free detection designed for diagnostic applications. Anal Bioanal Chem. 2010 Nov;398(6):2403-12. doi: 10.1007/s00216-010-3906-2. Epub 2010 Jun 19. PMID: 20563563.
Fraunhofer teaches a label-free method for characterization of a complex liquid sample,
wherein the complex liquid sample is a mammalian body fluid (Pg. 20 “The challenging task to
separate dissolved and undissolved sample components via AF4 was repeatedly performed,
e.g., by examining the distribution of a lipophilic drug within human plasma or when
characterizing lipoproteins originating from human blood.”). Note that AF4 is a known label free technique in the art (instant claim 14). Fraunhofer teaches a first component or a fragment thereof (pg. 60, “In summarizing the results, the applicability of AF4 in protein characterization appears feasible. Protein sample specimen can be separated with sufficient resolution in reproducible way. Furthermore, the coupling of AF4 with MALS, UV- and RI-detection facilitates both data interpretation and evaluation”), said first component and said fragment thereof are capable of being in a monomeric form in an aggregate form (pg. 38, fig. 20 “(A) Simplified representation of a protein monomer and a domain-swapped dimer.”, pg. 72 “G-CSF monomers and dimers could be seized as separated fractions. Additionally, an individual fraction of aggregates eluted when no cross flow was effective (Fig. 41b). According to the data, the unstressed G-CSF solution exhibits only little dimer and no multimers, but a considerable amount of hmw aggregates.”),
wherein the first component is a protein and/or a biomarker, which is amyloid-beta (pg. 42 “Given this background, one candidate with great potential for aggregate quantification is AF4, as this method can analyze both aggregate specimen.”, pg. 29 “Most attention has been focused on a group of diseases where proteins or protein fragments convert from their soluble forms with intact helical folding to insoluble fibrils or plaques. The final forms of these aggregates often reveal a well-defined fibrillar nature, known as amyloids (Fig. 16).”) (instant claim 12), the method comprising:
(a) providing a complex liquid sample comprising the first component or the fragment
thereof; (pg. 20 “The challenging task to separate dissolved and undissolved sample
components via AF4 was repeatedly performed, e.g., by examining the distribution of a lipophilic
drug within human plasma or when characterizing lipoproteins originating from human blood.).
In particular, the potential of AF4 was demonstrated in the separation of lipoprotein particles of
coronary artery disease patients”);
(b) applying a field flow fractionation (FFF) to the complex liquid sample (pg. 20 “The
challenging task to separate dissolved and undissolved sample components via AF4
(asymmetrical flow field flow fractionation) was repeatedly performed”);
(c) obtaining a plurality of fractions of the complex liquid sample of (b) (pg. 72 “G-CSF
monomers and dimers could be seized as separated fractions.”;
(d) applying to the fractions a label-free detection method selective for the first
component or the fragment thereof (pg. 106-107 “once the challenge of particulate matter
separation is performed, the walk through the task of identification is straightforward: one
possibility is to combine AF4 with electrospray mass spectrometry as on- line detector for
analyte identification.”, pg. 54 “The AF4 system was connected to a ternary detection system,
combining MALS, UV-spectrophotometry and RI detection.”),
(e) obtaining an output signal from the label-free detection method (pg. 85 “AF4 was
proved to reveal reliable data when confronted with the task to analyze samples with varying
concentrations”, pg. 106-107 “once the challenge of particulate matter separation is
performed, the walk through the task of identification is straightforward: one possibility is to
combine AF4 with electrospray mass spectrometry as on-line detector for analyte
identification.”), wherein the output signal obtained for the first component or the fragment
thereof is dependent on the form in which the first component or the fragment thereof exists in
the complex liquid sample, and wherein the output signal is an elution time and/or a signal
amplitude of the first component or a fragment thereof (fig. 28, pg. 56) (instant claim 10);
(f) analyzing the output signal to determine presence of the first component or the
fragment thereof within the plurality of fractions, thereby characterizing the relative amounts of
the first component or the fragment thereof as being in the monomeric form and in the
aggregate form (pg. 72 “G-CSF monomers and dimers could be seized as separated fractions.
Additionally, an individual fraction of aggregates eluted when no cross flow was effective (Fig.
41b). According to the data, the unstressed G-CSF solution exhibits only little dimer and no
multimers, but a considerable amount of hmw aggregates.”), wherein said monomeric form
consists of the first component or the fragment thereof, wherein said aggregate form comprises
(a) two or more monomers of the first component or the fragment thereof (pg. 38, fig. 20
“(A) Simplified representation of a protein monomer and a domain-swapped dimer.”), or
(b) the first component or the fragment thereof together with one or more further components of the complex liquid sample (pg. 38, fig. 20 “(C) Illustration of open-ended and close-ended domain swapped oligomers, exemplifying aggregation processes induced by domain.”), wherein any of the one or more further components in each independently a biological moiety and/or a chemical moiety and the one or more further components is a nanoparticle or a peptide (pg. 129 “Whereas AF4 was shown to be able to characterize nanoparticles qualitatively - i.e., to assess size and size distributions – the quantification of nanoparticle concentration in reproducible way is a precedent condition for the monitoring of the nanoparticle loading step.”, pg. 134 “This AF4 application was developed further to render a new way of monitoring the drug loading process of nanoparticles possible. AF4 separation parameters were established which enable the separation of colloidal drug carriers and the designated drug load simultaneously in one single run.”), pg. 2 “Given the sample dimensions in the arena of pharmaceutical protein drugs - varying between 1 – 10 nm by native state peptides and proteins, the two-digit nm range of soluble aggregates and several microns of insoluble aggregates –, FFF may be considered as ideal candidate for analysis of complex protein samples”) (instant claims 13 and 23), and
wherein the field flow fractionation (FFF) is applied using a field-flow fractionation separator (FFF separator) that is coupled, on-line or in-line, to a device used to perform the label-free detection method detection of the first component or the fragment thereof, and FFF is AF4 (pg. 2 “To gain maximum information on the fractionated specimen, AF4 was to be coupled on-line with multi-angle light scattering (MALS).”) (instant claims 1, 17, and 24-25). Fraunhofer teaches a system being capable of performing the method of claim 1, said system comprising a field fraction separator (FFF separator) coupled to a device for the label-free detection of the first component or the fragment thereof, and wherein said label-free detection is selective for the first component or the fragment thereof (pg. 2 “To gain maximum information on the fractionated specimen, AF4 was to be coupled on-line with multi-angle light scattering (MALS).” pg. 20-21 “One is certainly to combine AF4 with absolute detection techniques like MALS or other methods like inductively coupled plasma (ICP) or electronspray mass spectroscopy. Thereby, a convenient identification of the separated analytes by assessment of molar mass, hydrodynamic radius or chemical composition is possible.”, pg. 54 “The AF4 system was connected to a ternary detection system, combining MALS, UV-spectrophotometry and RI detection.”) (instant claim 20).
Fraunhofer teaches the label-free detection method is a quantitative and/or selective
detection method (pg. 42 “one candidate with great potential for aggregate quantification is AF4,
as this method can analyze both aggregate specimen.”) (instant claim 2). Fraunhofer teaches
the label-free detection method is capable of estimating, as a numerical value in appropriate
units, the amount, or the concentration, of a first component present in the complex liquid
sample (pg. 57 “On the other hand (knowledge on dn/dc values of the protein provided), each
peak can be assigned the appropriate analyte molar mass via simultaneous MALS/UV-
detection”, pg. 102, fig. 70) (instant claim 3). Fraunhofer teaches wherein the label-free
detection method is capable of detecting the first component or the fragment thereof without
interference from other components, such as other particles, present in the complex liquid
sample (pg. 45 “Pertaining to that, R0 is proportional to the fraction of incident light that is
scattered by the pure solute without interference.”, pg. 67 “Minimizing the cross flow intensity
rapidly from 70% to zero after 16 min experiment time enables an unimpeded protein elution of
IFN α-2a, visualized by a sharp raise of the UV280 signal, pretending a symmetrical analyte
peak.”) (instant claim 5). Fraunhofer teaches wherein the characterization is a determination
of a multimerization state of the first component of the fragment thereof or a determination of an
aggregation state of the first component or the fragment thereof (pg. 42 “Given this background,
one candidate with great potential for aggregate quantification is AF4, as this method can
analyze both aggregate specimen.”) (instant claim 6). Fraunhofer teaches wherein the
monomeric form consists of the fragment of the first component, wherein said fragment is the
result of an interaction between the first component and one or more further components of the
complex liquid sample (pg. 40 “For instance, newly cloned proteins, especially those generated
subsequent to large-scale sequencing, may exist in solution optionally in monomeric or
oligomeric forms”) (instant claim 7).
Fraunhofer teaches wherein said aggregate form is the result of an interaction between
the two more monomers of the first component or the fragment thereof or between the first
component or the fragment thereof and the one or more further components of the complex
liquid sample and where the interaction between the first component and the one or more
further components is aggregation (pg.32-33 “The formation of soluble and insoluble protein
aggregate specimen is based on either covalent or non-covalent interactions. Covalently linked
protein dimers or specimen of higher order are due to chemical reactions between the protein
molecules. Because the aggregate origin is generally not evident, unfortunately the term
“aggregation” is employed to describe this process, which should rather be termed
“polymerization”. As protein aggregates often represent a potpourri of various covalently and
non-covalently bound components, the term “aggregation” is universalized, pg. 33 “Protein
aggregation can occur from a conformational intermediate or from more extensively unfolded
(completely denatured) protein molecules, where hydrophobic residues are exposed to the
aqueous solvent. The initial stages of aggregation are quite specific in the sense that they
involve the interaction of structural subunits of one molecule with “corresponding” hydrophobic
surface areas of structural subunits of a neighboring molecule. Two sites can be sufficient, in
which case the aggregation most likely propagates in a linear fashion forming long fibres.
Anyhow, the process will yield larger aggregates, whose sizes will eventually exceed the
solubility limit. Hydrophobic interaction, i.e., the reluctance of nonpolar groups to
be exposed to water, is prevalently deemed the primum mobile for protein unfolding and
subsequent aggregation.”) (instant claims 8-9).
Fraunhofer teaches comparing the elution time and/or the signal amplitude obtained for
the first component or the fragment thereof with one or more control values, wherein the one or
more control values are provided reference values of the first component or the fragment
thereof, or wherein the one or more control values are the average elution time values and/or the average signal amplitude values relative to the first component or the fragment thereof,
wherein said control values are obtained by characterizing the first component or the fragment
thereof in a simple liquid sample (pg. 56 “human or bovine serum albumin (HSA/BSA) may be
used, due to its inherence of considerable amount of dimer (~10%) and higher-order
oligomers/aggregates. Additionally, the amount of higher-order specimen can easily be
increased by protein stressing, e.g., via 60 °C storage for 3 d of HSA lyo-philisate, yielding
accretions from 9.3% up to 29% for dimer and from 2.5% to 11.9% for trimer and higher-order
specimen) (instant claim 15). Fraunhofer teaches wherein shear stress exerted on the first
component or fragment thereof during step (b) is not sufficient for shear degradation (pg. 20
“Additionally, the FFF potential for degradation-free separation was applied to provide analytes
with extremely narrow size distributions for subsequent high-precision measurement of analyte
dimensions via MALS. In this respect, the superiority of AF4/MALS over established methods
like transmission electron microscopy (TEM) was shown”, pg. 134 “Bearing in mind the
contribution of hmw components to gelatin nanoparticle stability, the feature of AF4 to analyze
hmw gelatin without inducing shear degradation is deemed a crucial parameter in evaluating
bulk preparation efficacy by desolvation steps”, table 15, pg. 138-139 “In order to verify the
findings above – i.e., shear stress leads to a reduction of particle size, but does not relieve the
particle contamination per se – the vials were subjected to light obscuration analysis (SVSS-
C40, PAMAS GmbH, Rutesheim, Germany) (Table 15). According to Table 15, the findings of
visible inspection are corroborated: vials exhibiting visible particles contain a considerably
greater number of particles with dimensions beyond 1 µm and 2 µm, respectively, than vials
without a visible component. Thereby, the exertion of shear stress via shaking has virtually no
impact on the data, i.e., the extent of particle contamination remains constant.”) (instant claim
16). Fraunhofer teaches wherein the label-free detection method involves the use of a biological and/or chemical moiety which selectively targets the first component, the fragment of the first component, an aggregate comprising the first component, or an aggregate comprising the fragment of the first component, wherein the biological and/or chemical moiety is immobilized on a surface of a detector of any one of the label-free detection methods and/or on a surface of a free-floating particle, wherein said free-floating particle is provided to the fraction obtained in step (c) (pg. 66- 67 “By subjecting IFN α-2a to 300 s focusing and a 80% cross flow, the protein is immobilized upon the membrane, as no UV280 signal can be seized for 30 min elution with PBS.”, pg. 73 “Thus, membrane adsorption/immobilization of larger aggregates can be circumvented.”) (instant claim 18).
Fraunhofer does not teach using FFF with immunoassays or using FFF with SPR.
Roda teaches using FFF with an immunoassay (pg. 1993 “This was the basis for the development of FFF-CL–based solid-phase competitive immunoassays, in which micrometer- sized beads coated with the capture antibody are used as a solid phase and an analyte–HRP conjugate is used as a tracer.”) (instant claims 1 and 24).
Fraunhofer and Roda do not explicitly teach motivation for using label-free detection methods, nor do they teach the use of SPR.
Rapp teaches the use of an SPR sensor for label-free detection (see page 2410) (instant claims 1 and 25). Rapp provides motivation for using label-free detection methods by teaching that label-free ensures only the analyte binding to the corresponding biomolecular recognition element immobilized on the biosensor surface will lead to a significant change in the biosensor signal response, not any unspecific interaction with the sample matrix (see page 2405). Rapp further provides motivation by teaching that labeled compounds implies higher operational costs, including longer assay times, compared to most label-free formats (see page 2404).
It would have been obvious to one of ordinary skill in the art at the time of the instant
application to combine the methods of label free detection using FFF as taught by Fraunhofer, with the methods of using FFF with in immunoassay taught by Roda, with the label-free detection teachings and methods of using SPR taught by Rapp. Fraunhofer teaches that FFF occupies a unique niche in the field of analytical fractionations because it is the only technique being capable to separate material over the entire colloidal size range with high resolution (see page 4). Fraunhofer provides motivation by teaching that AF4 is applied for characterization of complex samples, in order to separate and for determination of size (see page 15). Fraunhofer teaches that the possibility of AF4 to characterize unprepared samples gives AF4 a significant edge. E.g., for the purpose of quality control in pharmaceutical manufacturing this AF4 feature permits the native sample to be analyzed, without ignorantly removing the information sought (see page 85). Roda provides motivation for combining FFF with an immunoassay by teaching that FFF based immunoassays provide several advantages such as kinetics of the immunological reaction on micrometer-sized beads are faster than in the case of conventional microtiter plate assays, multianalyte FFF-based immunoassays are developed by the use of beads of different sizes, each coated with a specific antibody for 1 analyte, and the beads are fractionated by FFF before chemiluminescence (CL) detection using different enzymes (page 1993). Rapp provides motivation for using label-free detection methods by teaching that label-free detection methods are cheaper, quicker and easier to use, as well as provide more accurate results as they do not recognize unspecific interactions (see pages 2404-2405). Rapp further provides motivation by teaching that label-free detection SPR allows multianalyte detection and is sensitive (see page 2410). Thus, one of ordinary skill in the art would have been motivated to use a label-free detection method based on the advantages discussed above. The artisan would have reasonable expectation of success based on the cumulative disclosures of these prior art references.
Response to Arguments
The arguments filed on 03/02/2026 have been considered by the examiner.
On p. 9 applicant argues that the references do not teach SPR sensing with an SPR sensing device. However, Rapp teaches the use of an SPR sensor (see page 2410). On pp. 10-11 applicant argues that Fraunhofer and Roda do not teach the use of label free detection. Applicant argues that Roda teaches chemiluminescent immunoassays that contain a label. However, Rapp provides motivation for modifying Roda’s teachings of an immunoassay with FFF with a label-free method. Rapp teaches that the removal of a label provides more accurate results, quicker assay run times, and is more cost effective. For those reasons, one of ordinary skill in the art would have been motivated to modify Fraunhofer and Roda with label-free detection methods.
Conclusion
No claim is allowed.
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/MCKENZIE A DUNN/Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678
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