WO2023018805A1

WO2023018805A1 - Methods and related aspects of detecting and purifying influenza neuraminidase

Info

Publication number: WO2023018805A1
Application number: PCT/US2022/039964
Authority: WO
Inventors: Mario Barro; Liqun Han; Jianxin Zhang
Original assignee: Sanofi Pasteur Inc.
Priority date: 2021-08-11
Filing date: 2022-08-10
Publication date: 2023-02-16

Abstract

Provided are methods of selectively binding, detecting, and/or purifying a tetrameric neuraminidase (NA) of an influenza virus. Also provided are related binding mixtures, biosensor devices, and systems.

Description

METHODS AND RELATED ASPECTS OF DETECTING AND PURIFYING INFLUENZA NEURAMINIDASE

BACKGROUND

[0001] Influenza is caused by a virus that attacks mainly the upper respiratory tract - the nose, throat and bronchi and rarely also the lungs. The infection usually lasts for about a week. It is characterized by the sudden onset of high fever, myalgia, headache and severe malaise, nonproductive cough, sore throat, and rhinitis. Most people recover within one to two weeks without requiring any medical treatment. However, in the very young, the elderly and people suffering from medical conditions, such as lung diseases, diabetes, cancer, kidney or heart problems, influenza poses a serious risk. In these people, the infection may lead to severe complications of underlying diseases, pneumonia, and death, although even healthy adults and older children can be affected as well. Annual seasonal influenza epidemics are thought to result in between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world.

[0002] Influenza virus is a member of the Orthomyxoviridae family. There are three main subtypes of influenza viruses, designated influenza A, influenza B, and influenza C. The influenza virion contains a segmented negative-sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ionchannel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). The HA, NA, M1, and M2 are membrane associated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsid associated proteins. The HA and NA proteins are envelope glycoproteins, primarily responsible for virus attachment and penetration of the viral particles into the cell and release from the cell, respectively.

[0003] Both HA and NA proteins are the sources of the major immunodominant epitopes for virus neutralization and protective immunity, making them important components for prophylactic influenza vaccines. The genetic makeup of influenza viruses allows frequent minor genetic changes, known as antigenic drift. Thus, the amino acid sequence of the major antigens of influenza, including HA and NA, is highly variable across certain groups, subtypes and/or strains. For this reason, current seasonal influenza vaccines are recommended every year and require yearly surveillance to account for mutations in HA and NA proteins (antigenic drift) and to match rapidly evolving viral strains. [0004] Influenza NA is a homotetrameric type II transmembrane glycoprotein, with each monomer having a globular head domain, a stalk region, a hydrophobic transmembrane region, and a short, N-terminal cytoplasmic tail. Tetramerization of the head domain is important for formation of the enzymatic active site and the sialidase activity that is required for the release of new virus particles from infected cells. Sialidase activity also appears important for the virus to traverse mucus barriers in the host. The head domain is also the most immunologically relevant part of NA. Antibodies against the head region of influenza NA can block NA’s enzymatic activity and interfere with viral pathogenesis, especially cell-to-cell spreading and transmission.

[0005] Recombinant NA or purified NA obtained by proteolysis or solubilization of viral membranes with detergents has been studied for structural, enzymatic, and immunological analysis. If a soluble version of NA is desired for use as an immunogen, then the NA molecule has to be expressed without the anchoring transmembrane region, which frequently results in the loss of stabilizing forces that help to hold the NA molecule in its tetrameric form. Without the transmembrane region, and the ability to embed the HA protein in the membrane, the stability of the tetrameric NA head is compromised, resulting in partial disassembly and loss of immunogenicity. Furthermore, the secondary and tertiary structure of the NA stalk and associated transmembrane domain is unknown, complicating rational protein engineering approaches based on native structure.

[0006] Thus, expression of soluble, tetrameric NA in cells is very challenging, particularly expression of soluble, tetrameric NA in high yield and/or in an appropriate host cell that is compatible with the large-scale production of a therapeutic, recombinant protein. These challenges stem, in part, from impaired assembly of tetrameric NA from recombinant NA constructs, resulting primarily in the expression of inactive, soluble monomers and dimers, or inclusion bodies (large aggregates of protein that pose major challenges for recovery of large- scale production of bioactive protein), and little to no expression of active, tetrameric NA. Mather et al., 1992, Virus Res., 26:127-39; Castrucci et al., J. Virology, 1993, 67(2):759-64; Martinet, Eur. J. Biochem, 1997, 247:332-38; Yongkiettrakul et al., 2011, J. Virological Methods, 156:44-51; Romanik et al., 2012, FEBS Journal, 279(Suppl. l):52-576, 339. In one instance, a recombinant truncated NA was prepared that produced a mixed population of tetrameric NA, dimeric NA and monomeric NA when expressed in insect cells, however, this specific construct generated from the NA of the A/Victoria/3/1975 influenza strain was missing amino acids 1-45 of the NA protein (i.e., the cytoplasmic domain, the transmembrane domain, and only the first several amino acids of the stalk region). DeRoo et al., Vaccine, 1996, 14(6):561-69. Further studies with the A/Victoria/3/1975 influenza strain used by DeRoo et al. showed that when substantially all of the stalk region was removed (i.e., a recombinant construct lacking amino acids 1-78 or 1-79 of the NA protein), only monomeric NA was produced when expressed in insect cells. Martinet, Eur. J. Biochem, 1997, 247:332-38. Viral NAs purified from viral membranes by proteolysis or detergent-solubilization are unstable and quickly lose enzymatic activity. Schmidt et al., PLos ONE, 2011, 6(2):el6284.

[0007] Accordingly, it is apparent that new methods of detecting tetrameric influenza NA proteins, including recombinant influenza NA proteins, as well as methods of screening and purifying such tetrameric NA proteins, are needed to facilitate influenza vaccine research, development, and production.

SUMMARY

[0008] This application discloses methods and related aspects for detecting and/or purifying influenza virus neuraminidase (NA) molecules that form soluble tetrameric neuraminidase when expressed in host cells. The methods and other aspects disclosed in this application allow for the high-throughput screening of vaccine candidates and the large-scale production of desired NA molecules, thereby permitting rapid and flexible responses to newly emerging variant influenza strains.

[0009] A first aspect is directed to a method of selectively binding a tetrameric neuraminidase (NA) of an influenza virus. The method includes contacting a fluidic sample that comprises a mixture comprising the tetrameric NA, such as a mixture comprising tetrameric NA and monomeric NA molecules, with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA. The NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety. The at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. In addition, the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules in the fluidic sample under the conditions, thereby selectively binding the tetrameric NA in the fluidic sample. In some embodiments, the method further comprises detecting the bound tetrameric NA, and/or separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample. Various features and embodiments of this first aspect are described in further detail throughout the disclosure of this application. [0010] A second aspect is directed to a method of detecting a tetrameric neuraminidase (NA) of an influenza virus. The method comprises contacting a fluidic sample that comprises a mixture comprising the tetrameric NA, such as a mixture comprising tetrameric NA and monomeric NA molecules, with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA. The NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, and the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. In addition, the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules in the fluidic sample under the conditions. The method also includes detecting the bound tetrameric NA, thereby detecting the tetrameric NA in the fluidic sample. Various features and embodiments of this second aspect are described in further detail throughout the disclosure of this application.

[0011] A third aspect is directed to a method of purifying a tetrameric neuraminidase (NA) of an influenza virus. The method includes contacting a fluidic sample that comprises a mixture comprising the tetrameric NA, such as a mixture comprising tetrameric NA and monomeric NA molecules, with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA. The NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, and the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. In addition, the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules in the fluidic sample under the conditions. The method also includes separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample, thereby purifying the tetrameric NA. Various features and embodiments of this third aspect are described in further detail throughout the disclosure of this application.

[0012] A fourth aspect is directed to a binding mixture that comprises a tetrameric neuraminidase (NA) of an influenza virus, monomeric NA molecules, and an NA binding agent. The binding mixture provides conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA to produce a bound tetrameric NA. The NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety. Also, the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. In addition, the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules under the conditions. Various features and embodiments of this fourth aspect are described in further detail throughout the disclosure of this application.

[0013] A fifth aspect is directed to a biosensor device that comprises a solid support in contact with a mixture comprising tetrameric neuraminidase (NA), such as a mixture comprising tetrameric NA and monomeric NA molecules of an influenza virus, under a set of conditions, a second recognition moiety attached to a surface of the solid support, an NA binding agent that comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the first recognition moiety is bound to the second recognition moiety, and at least one tetrameric NA is bound to the tetrameric NA binding moiety. The at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. The NA binding agent is structured to substantially only bind to the tetrameric NA under the set of conditions and to remain substantially unbound to monomeric NA molecules under the set of conditions. In some embodiments, an interferometry capture probe comprises the solid support. Various features and embodiments of this fifth aspect are described in further detail throughout the disclosure of this application.

[0014] A sixth aspect is directed to a system that comprises a processing chamber that comprises, or is capable of receiving at least a portion of, a solid support when the solid support is in contact with a mixture comprising tetrameric neuraminidase (NA), such as a mixture comprising tetrameric NA and monomeric NA molecules of an influenza virus, under a set of conditions. The solid support comprises a second recognition moiety attached to a surface of the solid support, an NA binding agent that comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the first recognition moiety is bound to the second recognition moiety, and at least one of the tetrameric NA is bound to the tetrameric NA binding moiety to form a bound tetrameric NA molecule. The at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. The NA binding agent is structured to substantially only bind to the tetrameric NA under the set of conditions and to remain substantially unbound to monomeric NA molecules under the set of conditions. The system also includes a fluidic material handling component that fluidly communicates with the processing chamber and at least one fluidic material source. In addition, the system also includes a controller that is operably connected, or connectable, at least to the fluidic material handling component, wherein the controller comprises, or is capable of accessing, computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least conveying at least one fluidic material from the fluidic material source to the processing chamber using the fluidic material handling component. Various features and embodiments of this sixth aspect are described in further detail throughout the disclosure of this application.

[0015] The foregoing general summary and the following detailed description are exemplary and explanatory and are not restrictive of the claims.

BRIEF DESCRIPTION OF THE DRAWING

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some embodiments, and together with the written description, explain certain principles of the neuraminidase molecules, compositions, and methods disclosed herein.

[0017] FIG. 1 is a flow chart that schematically shows exemplary method steps of detecting and/or purifying a tetrameric neuraminidase (NA) of an influenza virus according to some aspects disclosed herein.

[0018] FIG. 2A schematically shows a biosensor device from a side view according to some aspects disclosed herein.

[0019] FIG. 2B schematically shows a solid support from the biosensor device shown in FIG. 2A from a detailed view.

[0020] FIG. 3 is a schematic diagram of an exemplary system suitable for use with some embodiments.

[0021] FIG. 4 shows a plot (y-axis represents kobs (1/s); x-axis represents NA concentration (nM)) of TAMIFLU® binding to tetrameric recombinant NAs comprising a heterologous tetrabrachion tetramerization domain (tet-NA). As shown, TAMIFLU® broadly binds to tetrameric NA of various influenza strains, including type A (both group 1 and 2) and type B influenza strains, in a dose-dependent manner. As further shown, the linear dynamic range is about 0.16 - 40ug/ml for all strains, while the exact linear equation (slope and intercepts) differs depending on the strain of rNA.

[0022] FIG. 5 are plots (y-axis represents wavelength (nm); x-axis represents time (seconds (sec)), respectively) showing that TAMIFLU® broadly recognizes purified NA tetramers (tetrabrachion-NA; tet-NA) including as shown for type A (both group 1 and 2) and type B (Yamagata and Victoria lineages) influenza NA (i.e., N1, N2, B-NA) with dose response. [0023] FIG. 6 is a plot (y-axis represents binding response; x-axis represents the tetrameric NA) showing TAMIFLU® binding to tetrameric NA (N2 tet-SING16, N2 dTM75_SING16, N1 tet-MICH15), but not binding to monomeric NA (N2 dTM36_SING16, N1 dTM36_MICH15).

[0024] FIG. 7 is a plot (y-axis represents wavelength (nm); x-axis represents time (seconds (s))) showing raw data from NA quantification for ExpiCHO cell culture NA supernatants (a panel of 49 Peth09 NA samples) using Octet-HTX system.

[0025] FIG. 8 is a plot (y-axis represents binding rate; x-axis represents concentration (μg/ml)) with a standard curve showing a standard tet-NA_SG16 (n = 2 for each standard) and unknown samples (n = 2 for each unknown). The lower limit of quantification in this assay is 0.3 μg/mL.

[0026] FIG. 9 is a plot (y-axis represents wavelength (nm); x-axis represents time (seconds (s))) showing that low pH helps to elute the bound NA from SAX biosensor, as only bound NA are eluted from SAX biosensor at those pH levels.

[0027] FIG. 10 are plots (y-axis represents the year and quarter of transfection harvest date of a dTM75_KS17 construct; x-axis represents NA concentration in supernatant (Sup) by TAMIFLU® (μg/ml) and protein yield (mg)) showing NA expression level vs conformational NA protein yield. The supernatants were from three lots of large-scale transfection harvest. NA concentration in supernatant was measured by quantitative TAMIFLU®-NA binding assay. Protein yield was calculated based on the measurement of total protein by absorbance at 280nm.

[0028] FIG. 11 are gel images showing an analysis of purification in-process samples (KS17_dTM75 purified by TAMIFLU® affinity chromatography (TAC) or nickel affinity chromatography (NiAC)) under denaturing or reducing conditions by SDS-PAGE and Westem-blot. Lane M, molecular weight standards. Lane Sup, supernatant. Lane FT, flow through. Lane Wl, wash 1. Elution El and E2 fractions represent the purified product. C3, C4, C5 and C6 are column numbers.

[0029] FIG. 12 are plots (y-axis represents absorbance units (AU); x-axis represents time (minutes (min)), respectively) showing SEC-UPLC profiles of dTM75_KS17 expressed in CHO cells. As shown, TAMIFLU® affinity chromatography (TAC) produces a mixture of NA tetramer and higher-order NA oligomers, while nickel affinity chromatography (NiAC) produces mixture of NA tetramer and monomer.

[0030] FIG. 13 are plots (y-axis represents strain; x-axis represents yield (mg) and tetrameric NA) showing a comparison of NiAC and TAC methods for NA purification. TAC selectively enriches for the tetrameric form of NA from supernatant and produces highly purified tetrameric NA (92-100%).

[0031] FIG. 14 are plots (y-axis represents strain; x-axis represents specific NA activity (nmole/min/pg), ratio of TAMIFLU® binding/total protein, and ratio of mAh binding/total protein, respectively) showing comparisons of NiAC with TAC purified dTM75_KS17 by MUNANA, TAMIFLU®-NA binding assay, and N2 mAb binding assay. The purified NA proteins preserve comparable enzyme activity, TAMIFLU® binding and mAb binding capacity.

[0032] FIG. 15 are gel images showing an analysis of TAC and NiAC purification in-process samples (PerthO9_dTM75) under denaturing and reducing conditions by SDS-PAGE and Westem-blot. Lane M, molecular weight standards. Lane Sup, supernatant. Lane FT, flow through. Lane Wl, wash 1. Elution El and E2 fractions represent the purified product. Cl and C7 are column numbers.

[0033] FIG. 16 are plots (y-axis represents absorbance units (AU); x-axis represents time (minutes (min)), respectively) showing SEC-UPLC profiles of PerthO9_dTM75 expressed in CHO cells. As shown, TAMIFLU® affinity chromatography (TAC) produces predominately tetrameric NA (94.87%), while nickel affinity chromatography (NiAC) produces mixture of NA tetramer and monomer.

DETAILED DESCRIPTION

A. Definitions

[0034] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0035] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0036] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0037] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0038] Artificial nucleic acid molecule'. As used herein, an artificial nucleic acid molecule may typically be understood to be a nucleic acid molecule, e.g. a DNA or an RNA, that does not occur naturally. In other words, an artificial nucleic acid molecule may be understood as a nonnatural nucleic acid molecule. Such nucleic acid molecule may be non-natural due to its individual sequence (which does not occur naturally) and/or due to other modifications, e.g., structural modifications of nucleotides which do not occur naturally. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule or a hybrid-molecule comprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). Further, the term “artificial nucleic acid molecule” is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

[0039] Binding: As used herein, the term “binding”, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; “indirect” binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts — including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). [0040] Binding Mixture'. As used herein, “binding mixture” refers a mixture that comprises molecules that can participate in and/or facilitate a given binding reaction or assay. To illustrate, a binding mixture generally includes a solution containing reagents necessary to carry out a tetrameric NA binding assay and/or purification process, and typically contains at least tetrameric NA molecules, monomeric NA molecules, and NA binding agents under conditions sufficient for the NA binding agents to substantially only bind to the tetrameric NA molecules, while remaining substantially unbound to the monomeric NA molecules in the solution under those same conditions. The binding mixture can also include other oligomeric forms of NA, such as dimeric NA and trimeric NA, under conditions sufficient for the NA binding agents to substantially only bind to the tetrameric NA molecules. The binding mixture can also include higher order oligomeric forms of NA, such as higher order tetrameric NA comprising aggregates or multimers of tetrameric NA. A binding mixture is referred to as complete if it contains all reagents necessary to carry out the assay, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of skill in the art that assay or reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for applicationdependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete binding mixture. Furthermore, it will be understood by one of skill in the art that assay components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction or assay components.

[0041] Conjugate: As used herein, the term “conjugate” in the context of chemical structures refers to two or more chemical compounds or moieties that are covalently linked to one another. Chemical moieties can be directly conjugated with one another or indirectly conjugated with one another via a linker or other spacer moiety.

[0042] Detect: As used herein, “detect,” “detecting,” or “detection” refers to an act of determining the existence or presence of one or more analytes (e.g., tetrameric neuraminidase molecules) in a given sample.

[0043] Expression'. The term “expression”, when used in reference to a nucleic acid herein, refers to one or more of the following events: (1) production of an RNA transcript of a DNA template (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide; and/or (4) post-translational modification of a polypeptide. [0044] Host: The term “host” is used herein to refer to a system (e.g., a cell, organism, etc.) in which a polypeptide of interest is present. In some embodiments, a host is a system that is susceptible to infection with a particular infectious agent. In some embodiments, a host is a system that expresses a particular polypeptide of interest.

[0045] Host cell: As used herein, the phrase “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. For example, host cells may be used to produce the influenza neuraminidase polypeptides (e.g., wild-type, recombinant, or modified NA molecules) referenced herein by standard production techniques. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: Chinese Hamster Ovary or CHO cells (e.g., CHO KI, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa cells, HepG2 cells, W138 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, BHK21 cells, Jurkat cells, Daudi cells, A431 (epidermal) cells, CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, SP2/0 cells, NS-0 cells, MMT 060562 cells, Sertoli cells, BRL 3 A cells, HT1080 cells, myeloma cells, tumor cells, and a cell line derived from an aforementioned cell. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

[0046] Immune response'. As used herein, the term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and/or adaptive immune response. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and/or other white blood cells.

[0047] Immunogen'. As used herein, the term “immunogen” or “immunogenic” refers to a compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, “immunize” means to render a subject protected from an infectious disease.

[0048] In some embodiments: As used herein, the term “in some embodiments” refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0049] Isolated: As used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be “isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an “isolated” polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an “isolated” polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.

[0050] Mixture'. As used herein, “mixture” refers to a combination of two or more different components (e.g., chemical compounds, reagents, or proteins).

[0051] Moiety. As used herein, “moiety” in the context of chemical compounds or structures refers to one of the portions into which the compound or structure is or can be divided (e.g., a functional group, a substituent group, or the like). For example, a neuraminidase binding agent includes tetrameric NA binding and recognition moieties.

[0052] Monomeric NA molecule'. As used herein, “monomeric NA molecule” refers to a NA monomeric polypeptide unit that is not part of a tetrameric or other oligomeric form of a NA polypeptide molecule. In some embodiments, a monomeric NA molecule includes a globular head domain, a stalk region, a hydrophobic transmembrane domain, and a short, N-terminal cytoplasmic domain. In some embodiments, one or more of these domains or regions of a given monomeric NA molecule are truncated, altogether absent, or modified relative to a reference wild-type monomeric NA molecule.

[0053] A7: As used herein, “Nl” refers to an influenza virus subtype 1 neuraminidase (NA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the vims hemagglutinin (HA) and NA. Currently, there are 18 recognized HA subtypes (H1 -H18) and 11 recognized NA subtypes (N1-N11). Nl is thus distinct from the other NA subtypes, N2-N11

[0054] N2: As used herein, “N2” refers to an influenza virus subtype 2 neuraminidase (NA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus hemagglutinin (HA) and NA. Currently, there are 18 recognized HA subtypes (H1-H18) and 11 recognized NA subtypes (Nl-N11). N2 is thus distinct from the other NA subtypes, N1 and N3- N11.

[0055] NB: As used herein, “NB” refers to an influenza B neuraminidase (NA). Influenza B strains are classified into two lineages: B/Y amagata and B/Victoria.

[0056] NA binding agent As used herein, the term “NA binding agent” refers to a chemical compound that includes a tetrameric NA binding moiety. The tetrameric NA binding moiety can be directly or indirectly conjugated to a recognition moiety. In some embodiments, the tetrameric NA binding moiety is oseltamivir phosphate, or a tetrameric NA binding portion thereof.

[0057] Pandemic strain: A “pandemic” influenza strain is one that has caused or has capacity to cause pandemic infection of subject populations, such as human populations. In some embodiments, a pandemic strain has caused pandemic infection. In some embodiments, such pandemic infection involves epidemic infection across multiple territories; in some embodiments, pandemic infection involves infection across territories that are separated from one another (e.g., by mountains, bodies of water, as part of distinct continents, etc.) such that infections ordinarily do not pass between them.

[0058] Pure: As used herein, an agent or entity is “pure” if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0059] Recognition moiety. As used herein, the term “recognition moiety” refers to a portion of a chemical compound or structure that selectively or preferentially binds to another chemical compound or structure. In some embodiments, a NA binding agent includes biotin as a recognition moiety, which selectively or preferentially binds to another recognition moiety, such as streptavidin or avidin.

[0060] Recombinant: As used herein, the term “recombinant” or “modified” is intended to refer to polypeptides (e.g., NA polypeptides as described herein) that are designed, engineered, prepared, expressed, created or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant, combinatorial polypeptide library or polypeptides prepared, expressed, created or isolated by any other means that involves splicing selected sequence elements to one another. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source. In some embodiments, one or more such selected sequence elements results from the combination of multiple (e.g., two or more) known sequence elements that are not naturally present in the same polypeptide (e.g., two epitopes from two separate NA polypeptides).

[0061] Sample'. As used herein, “sample” or “fluidic sample” refers to a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a polypeptide), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells, cell components, or non-cellular fractions.

[0062] Seasonal strain-. A “seasonal” influenza strain is one that has caused or has capacity to cause a seasonal infection (e.g., annual epidemic) of subject populations, such as human populations. In some embodiments, a seasonal strain has caused seasonal infection.

[0063] Selectively binds: The term “selectively binds” in the context of biomolecules refers to a ligand that binds preferentially to a given biomolecule rather than to other biomolecules. In some embodiments, for example, the NA binding agents disclosed herein preferentially bind to tetrameric NA molecules, but not to monomeric NA molecules.

[0064] Solid suppor The term “solid support” refers to a solid material which can be derivatized with, or otherwise attached to, a chemical moiety, such as a recognition moiety. Exemplary solid supports include a microplate, a resin (e.g., an agarose resin), a bead, a microbead, a fiber, a whisker, a ceramic layer, a comb, a membrane, a crystal, and a selfassembling monolayer, among others.

[0065] Spacer moiety. As used herein, the term “spacer moiety” refers to a chemical moiety that covalently or non-covalently (e.g., ionically, etc.) attaches a compound or substituent group to, e.g., a solid support, another compound or group (e.g., a tetrameric NA binding moiety to a recognition moiety in a NA binding agent), or the like. [0066] Stalk region'. As used herein, the “stalk region” of influenza subtype 2 neuraminidase refers to a region of about amino acid 36 to about amino acid 82 of the subtype 2 neuraminidase. As used herein “substantially all of a stalk region” refers to amino acid 36 to amino acid 69 of the stalk region of an influenza virus subtype 2 NA. Thus, a modified N2 lacking the cytoplasmic tail, the transmembrane region, and substantially all of the stalk region may lack amino acids 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, or 1-81 of an influenza virus subtype 2 NA. Put another way, the modified N2 described herein can include up to 13 of the most C-terminal amino acids of the stalk region of the influenza virus subtype 2 NA, where the most C-terminal amino acids of the stalk region typically refer to amino acids 70-82 of the N2. In certain embodiments, the cytoplasmic tail, the transmembrane region, and the entire stalk region (e.g., amino acids 1-82) have been removed from the modified N2.

[0067] Subject As used herein, the term “subject” means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, and/or a clone. In some embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”

[0068] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0069] System: As used herein, "system" in the context of analytical instrumentation refers a group of objects and/or devices that form a network for performing a desired objective.

[0070] Tetrameric NA binding moiety: As used herein, the term “tetrameric NA binding moiety” refers to a portion of a chemical compound or structure that selectively or preferentially binds to tetrameric NA molecules under a given set of conditions, while remaining substantially unbound to monomeric NA molecules under the same set of conditions. In some embodiments, the tetrameric NA binding moiety is oseltamivir phosphate. As used herein, the term “tetrameric NA binding portion thereof’ refers to a portion of a tetrameric NA binding moiety that selectively or preferentially binds to tetrameric NA molecules under a given set of conditions, while remaining substantially unbound to monomeric NA molecules under the same set of conditions.

[0071] Tetrameric NA molecule'. As used herein, the term “tetrameric NA molecule” refers to a compound that includes four NA monomeric polypeptide units. In some embodiments, each monomeric NA molecule in a given tetrameric NA compound includes a globular head domain, a stalk region, a hydrophobic transmembrane domain, and a short, N-terminal cytoplasmic domain. In some embodiments, one or more of these domains or regions of a given monomeric NA molecule are truncated, altogether absent, or modified relative to a reference wild-type monomeric NA molecule. The samples analyzed or purified as described in the present disclosure typically include mixtures of monomeric and tetrameric NA molecules.

[0072] Tetramerization domain'. As used herein, the term “tetramerization domain” refers to an amino acid sequence encoding a domain that causes the tetrameric assembly of a polypeptide or protein. A tetramerization domain that is not native to a particular protein may be termed an artificial or a heterologous tetramerization domain. Exemplary tetramerization domains include, but are not limited to, sequences from Tetrabrachion, GCN4 leucine zippers, or vasodilator-stimulated phosphoprotein (VASP).

[0073] Vaccination: As used herein, the term “vaccination” or “vaccinate” refers to the administration of a composition to generate an immune response, for example to a diseasecausing agent such as an influenza virus. Vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and/or to the development of one or more symptoms, and in some embodiments, before, during, and/or shortly after exposure to the agent. Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. Vaccines may be administered with an adjuvant to boost the immune response. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.

[0074] Wild-type (WT): As is understood in the art, the term “wild-type” generally refers to a normal form of a protein or nucleic acid, as is found in nature. For example, wild-type NA polypeptides are found in natural isolates of influenza virus. A variety of different wild-type NA sequences can be found in the NCBI influenza virus sequence database.

B. Introduction

[0075] This application discloses binding assay methods to detect and quantify substantially only conformationally properly folded tetrameric neuraminidase (NA) or higher order tetrameric NA oligomers in biological systems or mixtures that comprise tetrameric NA and/or higher order tetrameric NA oligomers, such as mixtures comprising tetrameric NA and/or higher order tetrameric NA oligomers, monomeric NA, and/or other oligomeric forms of NA. In some embodiments of this assay, for example, a NA binding agent (e.g., a TAMIFLU®- biotin conjugate, etc.) acts as a ligand to immobilize tetrameric NA on a surface of a biosensor that is coated with streptavidin or another recognition moiety. In some of these embodiments, the NA/ligand interaction is monitored using a biolayer interferometry (BLI) or other detection instrument that facilitates real-time label-free analysis for the determination of kinetics, affinity, and quantitation regarding the tetrameric NA bound to the biosensor tip. In some implementations, the binding assays are used as part of a high-throughput screening process to identify potential influenza virus vaccine candidates. This application also discloses methods for the isolation and purification of conformationally properly folded NA proteins (again, preferably NA tetramers or higher order tetrameric NA oligomers, but not NA monomers or other oligomers of NA) from, for example, cell culture harvests (e.g., Chinese Hamster Ovary (CHO) cells, insect cells, or the like) expressing an encoded target NA polypeptide. The use of these purification methods achieves an NA protein sufficiently pure for influenza vaccine research and development, among other applications. In some embodiments, these purification methods use TAMIFLU®-biotin affinity chromatography (TAC) techniques. Moreover, in some exemplary embodiments, these methods are used as part of large-scale NA purification processes to produce adequate amounts of tetrameric NA molecules at sufficiently high purity levels for influenza virus vaccine commercial production. These and other aspects will be readily apparent upon a complete review of the present disclosure.

C. Nomenclature for Influenza Virus

[0076] All nomenclature used to classify influenza virus is that commonly used by those skilled in the art. Thus, a Type, or Group, of influenza virus refers to the three main ty pes of influenza: influenza Type A, influenza Type B or influenza Type C that infects humans. Influenza A and B cause significant morbidity and mortality each year. It is understood by those skilled in the art that the designation of a virus as a specific Type relates to sequence difference in the respective Ml (matrix) protein or P (nucleoprotein).

[0077] Type A influenza viruses are further divided into group 1 and group 2. These groups are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and NA. Currently, there are 18 recognized HA subtypes (H1-H18) and 11 recognized NA subtypes (N1- N11). Group 1 contains N1, N4, N5, and N8 and H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18. Group 2 contains N2, N3, N6, N7, and N9 and H3, H4, H7, H10, H14, and H15. N10 and N11 have been identified in influenza-like genomes isolated from bats. Wu et al., Trends in Microbiology, 2014, 22(4):183-91. While there are potentially 198 different influenza A subtype combinations, only about 131 subtypes have been detected in nature. Current subtypes of influenza A viruses that commonly circulate in the human population, giving rise to seasonal outbreaks, include: A(H1N1) and A(H3N2). Influenza A subtypes can be further broken down into different genetic " clades" and “sub-clades.” Finally, the term strain refers to viruses within a subtype that differ from one another in that they have small, genetic variations in their genome.

[0078] Influenza B viruses are not divided into subtypes, but instead are classified into two lineages: B/Yamagata and B/Victoria. Like influenza A viruses, influenza B viruses can be further classified into specific clades and sub-clades.

[0079] For convenience, certain abbreviations can be used to refer to protein constructs, and portions thereof, described herein. For example, NA can refer to influenza neuraminidase protein, or a portion thereof. N2 refers to neuraminidase from an influenza subtype 2 strain. The term tet-N A refers to a recombinant NA comprising a heterologous tetramerization domain that forms tetrameric NA when expressed in cells, HA refers to hemagglutinin or a portion thereof.

D. Neuraminidase (NA)

[0080] Neuraminidase (NA), along with hemagglutinin (HA), is one of the two major influenza surface proteins. The functions of both NA and HA involve interactions with sialic acid, a terminal molecule bound to sugar moieties on glycoproteins or glycolipids expressed on the surface of cells. The binding of HA to sialic acid on the cell surface induces endocytosis of the virus by the cell, allowing the virus to gain entry and infect cells. Sialic acid is also added to HA and NA as part of the glycosylation process that occurs within infected cells. NA removes sialic acid from cellular glycoproteins and glycolipids and from newly synthesized HA and NA on nascent virions. The removal of sialic acid by NA promotes the efficient release of viral particles from the surface of infected cells by preventing aggregation of viral particles. It also prevents virus from binding via HA to dying cells that have already been infected, promoting the further spread of the viral infection. a. Wild-type Influenza Virus Neuraminidase

[0081] The methods of the present disclosure can be used to detect and/or purify tetrameric NA polypeptides that comprise four copies of a wild-type monomeric NA molecule. NA is a type II transmembrane glycoprotein that assembles on the virus surface as a tetramer of four identical monomers. The molecular mass of the wild-type monomer is about 55-72 kDa, depending on the influenza subtype; the molecular mass of the tetramer is about 240-260 kDa, depending on the influenza subtype. Each monomer consists of four distinct structural domains: the enzymatic head region, the stalk region, the transmembrane region, and the cytoplasmic tail. The largest domain is the head region, which is tethered to the viral membrane by a stalk region connected to the transmembrane region and finally the N-terminal cytoplasmic domain.

[0082] The stalk region among different influenza A virus subtypes, including N1 and N2, can vary significantly in size and amino acid structure. Blok et al., Biochemistry, 1982, 21:4001- 4007. The differences in stalk length are thought to regulate the distance of the enzymatic head region and impact the ability of NA to access sialic acid on cell surface receptors, with shorter stalk regions correlating with reduced sialidase activity. Da Silva et al., J Biol Chem, 2013, 288(l):644-53; McAuley et al., Frontiers in Microbiology, 2019, 10(39). Notwithstanding the variability among stalk regions of different subtypes, NA stalk regions also share some structural features, including at least one cysteine residue and a potential glycosylation site. The cysteine residue(s) may be involved in the formation of disulfide bonds between NA monomers and assist in the formation of a stabilized NA tetramer, while the glycosylation site may contribute to tetramer stabilization. McAuley et al., Frontiers in Microbiology, 2019, 10(39). For example, a conserved cysteine residue at amino acid position 78 of N2 NA is believed to play a role in the tetramer assembly mechanism. Shtyrya et al., Acta Naturae. 2009; 1(2): 26-32.

[0083] The enzymatic head region is comprised of four monomers. Each monomer in the head forms a conserved six-bladed propeller structure. Each blade has four anti -parallel [3-sheets that are stabilized by disulfide bonds and connected by loops of varying length. McAuley et al., Frontiers in Microbiology, 2019, 10(39). Tetramerization of the monomers is important for the formation of the active site and synthesis of the enzymatically active NA. Dai et al., J. Virology, 2016, 90(20):9457-70. b. Recombinant or Modified Influenza Virus Neuraminidase

[0084] The methods of the present disclosure can be used to detect and/or purify tetrameric NA polypeptides that comprise four copies of a recombinant or modified monomeric NA molecule that forms soluble, tetrameric NA when expressed in a host cell, including recombinant or modified NA from influenza A virus subtypes, such as N1 and N2 and influenza B, as demonstrated herein. In some of these embodiments, a given recombinant or modified monomeric NA molecule includes a head region of an influenza virus NA, but lacks at least a portion of one or more of a cytoplasmic tail, a transmembrane region, and a stalk region of the influenza virus NA. For example, the modified monomeric NA may include a heterologous tetramerization domain that replaces one or more of a cytoplasmic tail, a transmembrane region, and a stalk region of the influenza virus NA. By way of further example, it has been discovered that modified monomeric N2 lacking all or substantially all of the stalk domain can form soluble tetrameric NA when expressed in cells, even without the addition of a heterologous tetramerization domain. Although not all N2 strains lacking all or substantially all of the stalk domain produced soluble tetrameric NA in detectable amounts, the majority of N2 strains tested produced detectable amounts of soluble tetrameric NA, showing that a truncated stalk design strategy, as referenced herein, can be broadly applied to the NA protein from various N2 influenza strains. Depending on the N2 strain used, this modified monomeric NA design strategy may result in the production of predominately tetrameric NA or a mixture of monomeric NA and tetrameric when expressed in a host cell. Thus, certain N2 strains and certain stalk-deleted variants of specific N2 strains produce higher yields of soluble, tetrameric NA when expressed in cells. In either instance, it may be desirable to purify the tetrameric NA produced when such modified NA constructs are expressed in host cells.

[0085] Tetrameric NA molecules formed by these modified monomeric NA are generally substantially soluble in fluidic samples and are also typically catalytically active (e.g., capable of enzymatically cleaving glycosidic linkages of neuraminic acids). However, tetrameric NA molecules may also be catalytically inactive, for example, due to a mutation. The soluble tetrameric NA produced from these different modified monomeric NA constructs can be detected in the cell supernatant and can be purified therefrom in high yields using the TAMIFLU® binding assays described herein. [0086] As shown in the examples, and discussed in further detail herein, high throughput screening can be used to easily identify those strains that produce soluble tetrameric NA, as well as to quantify the amount of soluble tetrameric NA produced. The same high throughput screening can also be used to test modified NA stalk truncated variants with varying lengths of the stalk region to identify the variants producing soluble tetrameric NA or the highest amount of soluble, tetrameric NA, as demonstrated in the examples.

[0087] Most of the soluble tetrameric NA formed from the modified NA constructs referenced herein retain neuraminidase enzymatic activity. Neuraminidase activity can be measured using techniques known in the art, including, for example, a MUNANA assay or an NA-Star® assay (ThermoFisher Scientific, Waltham, MA). In the MUNANA assay, 2'-(4-methylumbelliferyl)- alpha-D-N-acetylneuraminic acid (MUNANA) is used as a substrate. Any enzymatically active neuraminidase contained in the sample cleaves the MUNANA substrate, releasing 4- Methylumbelliferone (4-MU), a fluorescent compound. Thus, the amount of neuraminidase activity in a test sample correlates with the amount of 4-MU released, which can be measured using the fluorescence intensity (RFU, Relative Fluorescence Unit).

[0088] For purposes of determining the neuraminidase activity of a soluble tetrameric NA of the present disclosure, a MUNANA assay should be performed using the following conditions: mix soluble tetrameric NA with buffer [33.3 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 6.5), 4 mM CaCh, 50 mM BSA] and substrate (100 pM MUNANA) and incubate for 1 hour at 37°C with shaking; stop the reaction by adding an alkaline pH solution (0.2M Na2COs); measure fluorescence intensity, using excitation and emission wavelengths of 355 and 460 nm, respectively; and calculate enzymatic activity against a 4MU reference. If necessary, an equivalent assay can be used to measure neuraminidase enzymatic activity.

[0089] As noted, in some implementations, the application discloses methods of binding, detecting, and/or purifying tetrameric NA formed from recombinant influenza virus subtype 2 neuraminidase (N2) proteins that lack the cytoplasmic domain, the transmembrane domain and all or substantially all of the stalk region and do not contain a heterologous tetramerization domain. Recombinant N2 monomeric units of these polypeptides typically form soluble, tetrameric NA when expressed in cells. In some embodiments, for example, a tetrameric NA comprises four copies of a modified influenza virus subtype 2 neuraminidase in which the modified influenza virus neuraminidase comprises a head region of an influenza virus neuraminidase and lacks the cytoplasmic tail, transmembrane region and all or substantially all of the stalk region of the influenza virus neuraminidase. In some of these embodiments, the cytoplasmic tail, transmembrane region and all or substantially all of the stalk region of the influenza virus neuraminidase have been replaced by the signal peptide. The signal peptide is normally cleaved during post-translational processing such that the secreted, NA polypeptide typically does not contain the signal peptide. In some of these embodiments, for example, amino acid 1 to at least amino acid 70-82 of a wild-type N2 influenza virus NA have been replaced by the signal peptide. These modified N2 constructs in which the cytoplasmic domain, the transmembrane domain and all or substantially all of the stalk region are replaced by a signal peptide and which form tetrameric NA when expressed in cells and which can be detected and/or purified according to the methods of the present disclosure are also described in further detail in a separately filed patent application, entitled TRUNCATED INFLUENZA NEURAMINIDASE AND METHODS OF USING THE SAME, which was filed on 11 August 2021, and is hereby incorporated by reference in its entirety.

E. Methods of Binding, Detecting, and Purifying Neuraminidase

[0090] In overview, FIG. 1 is a flow chart that schematically shows exemplary method steps of the present disclosure. As shown in step 102, method 100 includes contacting a fluidic sample that comprises a mixture of tetrameric neuraminidase (NA) and monomeric NA molecules with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA, thereby selectively binding the tetrameric NA. As shown in step 104, method 100 additionally includes detecting the bound tetrameric NA (e.g., when performed as part of a NA binding assay or high-throughput NA screening assay) and/or separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample (e.g., when performed as part of a NA purification method, such as a large-scale NA production process). The binding and screening assays of the present disclosure can include contacting fluidic samples with NA binding agents and detecting bound tetrameric NAs substantially in real-time, which facilitates, for example, high-throughput NA screening implementations. a. NA Binding Agents

[0091] NA binding agents used to perform the methods and other aspects of the present disclosure include various embodiments. Typically, a given NA binding agent includes a tetrameric NA binding moiety conjugated to a recognition moiety (e.g., a first recognition moiety). The NA binding agents utilized in performing the methods disclosed herein are generally unlabeled. In some embodiments, tetrameric NA binding moieties include oseltamivir phosphate (TAMIFLU®), which is an oral prodrug that is converted by endogenous esterase into oseltamivir carboxylate. In this application, the terms oseltamivir phosphate and TAMIFLU® are used interchangeably. Oseltamivir phosphate acts as a competitive inhibitor of influenza's neuraminidase (NA) and binds to NA-active sites on the NA head, causing NA inhibition. Oseltamivir phosphate has the chemical name (3R,4R,5S)-4-acetylamino-5- amino3(1-ethylpropoxy)-1-cyclohexene-l-carboxylic acid, ethyl ester, phosphate (1:1) and chemical formula C₁₆H₂₈N₂O₄ (free base). The molecular weight is 312.4 for oseltamivir free base and 410.4 for oseltamivir phosphate salt. The structural formula is shown in Table 1.

[0092] Salts or tetrameric NA binding portions of oseltamivir phosphate are optionally used as tetrameric NA binding moi eties. A NA binding portion of a referenced tetrameric NA binding moiety (e.g., oseltamivir phosphate) refers to any part of that moiety that retains the capability of preferentially binding to tetrameric NA in a given fluidic sample under a selected set of conditions, while remaining substantially unbound to monomeric NA molecules in that sample under the same selected set of conditions. In some embodiments, the active metabolite of oseltamivir phosphate, namely, oseltamivir carboxylate is used as a tetrameric NA binding moiety in a NA binding agent.

[0093] NA binding agents also include recognition moi eties (e.g., first recognition moieties) that are conjugated to the tetrameric NA binding moieties. These recognition moieties are generally selected for use based upon other recognition moieties (e.g., second recognition moieties) intended for use in a given assay or purification process. Second recognition moieties are typically attached to solid supports, such that NA binding agents are immobilized on the solid supports when associated pairs of recognition moieties (e.g., first recognition moieties of the NA binding agent conjugates bind to the second recognition moieties attached to the solid supports) selectively bind or otherwise pair with one another. In some embodiments, the methods disclosed herein (e.g., binding assays, purification processes, etc.) include binding the first recognition moiety of a NA binding agent to the second recognition moiety after contacting fluidic samples with the NA binding agent. In some embodiments, the methods of the present disclosure include binding the first recognition moiety of a NA binding agent to the second recognition moiety before contacting the fluidic sample with the NA binding agent.

[0094] Exemplary recognition moiety pairs include those selected from compounds, such as biotin (e.g., D-biotin), streptavidin, avidin, an antibody (e.g., an mAb), an antigen, an aptamer, a protein, a peptide, and a carbohydrate, among others. In some embodiments, for example, the first recognition moiety used in a given application comprises biotin, while the corresponding second recognition moiety used in that application comprises streptavidin. In other exemplary embodiments, the first recognition moiety used in a given application comprises streptavidin, while the corresponding second recognition moiety used in that application comprises biotin.

[0095] In some embodiments, first recognition and tetrameric NA binding moieties are directly conjugated with one another in NA binding agents. In some embodiments, first recognition and tetrameric NA binding moieties of NA binding agents are conjugated to one another via spacer moieties. In some embodiments, the spacer moiety is undecaethylene glycol (3,6,9,12,15,18,21,24,27,30-Decaoxadotriacontane-l,32-diol (C₂₂H₄₆O₁₂)) or another poly(ethylene glycol). Generally, a spacer moiety has no specific biological activity other than to, e.g., join chemical species together or to preserve some minimum distance or other spatial relationship between such species. However, the constituents of a linker may be selected to influence some property of the linked chemical species such as three-dimensional conformation, net charge, hydrophobicity, etc. Additional description of linker molecules is provided in, e.g., Trawick et al. (2001) Bioconjugate Chem. 12:900, Shchepino et al. (2001) Nucleosides, Nucleotides, & Nucleic Acids 20:369, Lyttle et al. (1996) Nucleic Acids Res. 24(14):2793, Doronina et al. (2001) Nucleosides, Nucleotides, & Nucleic Acids 20:1007, Olejnik et al. (1998) Methods in Enzymology 291:135, Pljevaljcic et al. (2003) J. Am. Chem. Soc. 125(12):3486, Stavrianopoulos, U.S. Pat. No. 4,707,352, and Stavrianopoulos, U.S. Pat. No. 4,707,440, which are each incorporated by reference. b. NA Binding Assays

[0096] Stabilizing soluble NA protein in a correct, (e.g., catalytically active) tetrameric conformation and rationally designing recombinant NAs for improved production and immunogenicity have been goals of vaccine developers for many years. To facilitate reaching these goals, the binding assays (e.g., TAMIFLU®-NA binding assays) described in the present disclosure provide useful approaches to detect and quantify wild-type and/or recombinant NA tetramers expressed in, for example, any given cell culture harvest. One advantage of these assays is that the ligand (i.e., NA binding agent) used in these assays selectively binds to tetrameric NA and does not substantially bind to monomeric NA. This binding behavior facilitates high throughput screening which accelerates the ability to identify and rank novel NA vaccine designs that form stable tetrameric NA. A second advantage is that the ligand broadly recognizes different NA subtypes. Since there is broad recognition, the binding assay can be used to screen and characterize a diverse set of NAs including Nl, N2, and B-NAs, among others. A third exemplary advantage is that the NA binding agent (e.g., TAMIFLU®- biotin conjugate) strongly and selectively immobilizes NA onto a recognition moiety coated (e.g., streptavidin, avidin, etc.) biosensor surface with a slow off-rate in some embodiments. This provides a compelling case to use these kinetic NA binding assays to screen optimal binding and elution conditions for the purpose of, for example, purification process development for a given wild-type or recombinant NA polypeptide target.

[0097] The NA binding assay disclosed herein may be used to detect tetrameric NA in fluidic samples that include mixtures comprising tetrameric NA, such as mixtures comprising tetrameric NA and monomeric NA molecules. The assays include contacting those mixtures with a NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce bound tetrameric NA molecules. In some embodiments, the tetrameric NA is present in a concentration in a range of about 0.1 μg/ml to about 50 μg/ml (e.g., about 0.15 μg/ml to about 40 μg/ml, about 0.16 μg/ml to about 35 μg/ml, about 0.17 μg/ml to about 30 μg/ml, about 0.18 μg/ml to about 25 μg/ml, or about 0.19 μg/ml to about 20 μg/ml) in the fluidic sample. In some embodiments, the NA binding agent is present in a concentration in a range of about 1 μg/ml to about 100 μg/ml (e.g., about 5 μg/ml to about 75 μg/ml, about 10 μg/ml to about 50 μg/ml, or about 15 μg/ml to about 25 μg/ml). The binding of those tetrameric NA molecules is typically detected using an interferometry technique or another available detection approach. In some embodiments, these methods include using the detected bound tetrameric NA as a measure of tetrameric NA expression in the fluidic sample (e.g., a cell culture harvest or lysate) and/or to predict a probable yield of catalytically active tetrameric NA in the fluidic sample (e.g., a cell culture harvest or lysate). In some embodiments, the methods also include quantifying the tetrameric NA in the fluidic sample and/or the bound tetrameric NA. In some embodiments, these assays include determining a kinetics and/or an affinity property of the tetrameric NA in or from the fluidic samples.

[0098] In some embodiments, the NA binding assays of the present disclosure are used to detect targeted tetrameric NA proteins in substantially real-time. In some embodiments, for example, these tetrameric NA detection methods are used as part of a high-throughput screening process that detects the bound tetrameric NA and/or a catalytic activity of the tetrameric NA within about 5 minutes of contacting the fluidic sample with the NA binding agent. In some embodiments, the high-throughput screening process further comprises screening recombinant NA expressed from a plurality of recombinant NA constructs and identifying particular recombinant NA constructs within the plurality of recombinant NA constructs that produce tetrameric NA when expressed in a host cell.

[0099] In some embodiments, a synthesized phospha-oseltamivir-biotin conjugate (e.g., 5-10 μg/ml in IxKB buffer) is captured on the surface of streptavidin-coated biosensors (e.g., a High Precision Streptavidin (SAX) Dip and Read Biosensor, Cat. No. 18-51182). The binding of NA to TAMIFLU® initiates when the biosensors are dipped into microplate sample wells containing a targeted NA protein (e.g., a 2-fold dilution series of recombinant NA (0.16-40 μg/ml in IxKB buffer (e.g., containing PBS pH [7.4], 0.02% Tween-20, 0.1% albumin, and 0.05% sodium azide)) in some embodiments. Any change in the number of molecules bound causes a measured shift in the pattern at the detector (e.g., an Octet HTX, ForteBio Inc.). The level of binding response is proportional to the concentration of NA. The NA concentration in unknown samples can be calculated against NA reference standards. c. NA Purification

[00100] The present disclosure also provides methods of purifying tetrameric NA molecules. In some embodiments, these include using an affinity chromatography technique, such as a TAMIFLU®-biotin affinity chromatography method (TAC) that is a simple and efficient NA purification process that produces significant advantages when compared to previously established methods. For example, the TAC method reduces multi-step processes into a one- step purification method without sacrificing the yield of sufficiently pure NA proteins. The resulting high yield and purity of tetrameric NA significantly reduces the operational load for upstream protein purification and streamlines outputs to later stage vaccine characterization and animal studies. Moreover, in contrast to NiAC-based approaches, for example, the specific ligand used in TAC method selectively enriches tetrameric NA in supernatant. Moreover, TAC is suitable for purification of both peptide-tagged (e.g., His-tagged) and untagged NA versions. This ability to serve effectively as a standard method across both tagged and untagged NA purification facilitates higher efficiency in protein chemistry related efforts. In addition, the purification set-up with flow-through columns can purify proteins from multiple NA designs in parallel, thereby significantly accelerating the rate and range of purification outcomes. In some embodiments, a TAMIFLU®-biotin affinity chromatography workflow involves the use of manual gravity-flow columns, whereas in others, the workflow is adapted for use with automated chromatography instruments for large-scale NA purification.

[00101] In overview, the purification methods typically include contacting fluidic samples (e.g., a cell culture harvest or lysate) that comprise a mixture of tetrameric NA and monomeric NA molecules with an NA binding agent (e.g., TAMIFLU®-biotin conjugate) under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce bound tetrameric NA molecules. Exemplary binding conditions are described further herein. The bound tetrameric NA molecules are then generally separated from the monomeric NA molecules and other contaminants in the fluidic sample to produce purified tetrameric NA molecules. For examples, these methods of separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample typically yield a purity level of the tetrameric NA of at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or more. In some embodiments, the methods include separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample as part of a large-scale NA purification process that yields between about 0.01 mg and about 25 mg of the tetrameric NA per ml of the fluidic sample. Typically, the methods of separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample yields the tetrameric NA at a purity level that is at least as high as when using a Ni- NTA affinity chromatography purification process (NiAC) to purify tetrameric NA from an identical fluidic sample. In some embodiments, the methods of separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample yields improved preservation of catalytic activity as measured by a MUNANA assay or improved substrate binding site integrity as measured by an oseltamivir-NA binding assay than when using a Ni- NTA affinity chromatography purification process (NiAC) to purify tetrameric NA from an identical fluidic sample. Optionally, the purification methods of the present disclosure further comprise determining a kinetics and/or affinity property of the tetrameric NA.

[00102] In some embodiments, the methods of separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample comprise: washing the bound tetrameric NA one or more times using at least one binding buffer to substantially remove unbound tetrameric NA, the monomeric NA molecules, and/or other reagents or compounds to produce a washed bound tetrameric NA, and eluting the washed bound tetrameric NA from the NA binding agent using at least one elution buffer to produce an eluted tetrameric NA. In some embodiments, the elution buffer comprises glycine-HCl in a range of about 0. IM to about 1 ,0M at pH of about 5.0 or less. In some embodiments, the elution buffer comprises about 0.1M glycine-HCl at pH of about 2.8 (e.g., a pH ofbetween about 3.5 to about 2.5, or a pH of between about 3.0 to about 2.6). In some embodiments, the purification methods further comprise substantially neutralizing the eluted tetrameric NA when the elution buffer comprises a pH of about 5.0 or less.

[00103] In some embodiments, TAMIFLU®-biotin is used as a ligand for NA purification by simple pull-down of the NA protein with the conjugate on the surface of streptavi din-resin particles, or by affinity chromatography (TAC). An exemplary description of a TAC method includes the following steps:

[00104] A) Initiate purification process by removing cells and cell debris from the production bioreactor harvest containing secreted NA protein through centrifugation.

[00105] B) Pack streptavidin resin (e.g., Pierce® Streptavidin Agarose Resins, part# 20361) into an empty column (e.g., 10 ml ZEBA Spin Column, Fisher Part# PI89898). Equilibrate the resin with five column bed volumes of binding buffer (e.g., IxDPBS buffer, Gibco part#14190- 14).

[00106] C) Capture TAMIFLU®-biotin onto the streptavidin resin by adding TAMIFLU®- biotin solution to the column (e.g., 250 pg TAMIFLU®-biotin per 1ml resin). Incubate the resin along with TAMIFLU®-biotin for 60 minutes at 22-25°C using a rotating platform. Remove the un-bound TAMIFLU®-biotin from the resin by washing the resin with 15 column bed volumes of binding buffer.

[00107] D) Add the captured TAMIFLU® resin in a conical centrifuge tube (e.g., 50 ml conical centrifuge tube) containing the clarified supernatant (15-45 ml supernatant per 1 ml of resin depending on protein expression levels) and incubate the mixture for 40 minutes at 22- 25 °C using a rotating platform.

[00108] E) Load the mixture of resin and supernatant in an empty column such as the ones used above in Step B, let settle, and collect the flow through.

[00109] F) Wash the column with binding buffer equivalent to 10 times the column bed volume and collect the flow through. This should remove any unbound proteins from the resin.

[00110] G) Elute the target NA protein using 5-10 times the column bed volume of an appropriate elution buffer (e.g., 0.1M glycine-HCl, pH2.8) and collect the flow through. If using a low pH elution buffer, preemptively add a neutralizing buffer in each fraction collection tube (1/10^th the volume of elute fraction) in order to adjust the pH. Store the eluted fractions at 4°C.

[00111] H) Monitor protein recovery by measuring the absorbance of each elution fraction at 280nm. Further analyze protein purity by SDS-PAGE.

[00112] I) Pool elution fractions containing desired target protein into a single tube.

[00113] J) Concentrate and dialyze the eluted fractions into a buffer suitable for the downstream application using an ultra-centrifugal filter unit (e.g., MilliporeSigma™ Amicon™ Ultra Centrifugal Filter Units, 10 kD MWCO, Fisher part# UFC901024).

[00114] K) Sterilize the purified protein by filtering through a syringe filter (e.g., Fisherbrand™ Syringe Filters - Sterile, 0.2pm, 13mm, Fisher part# 09-720-3).

[00115] L) Monitor protein recovery by measuring the absorbance of each elution fraction at 280nm. Store the purified protein at 4°C (for short term storage) and -80°C (for long term storage).

[00116] As it was found that recombinant monomeric NA does not bind to TAMIFLU®, the TAMIFLU® purification method provides a convenient way to purify soluble tetrameric NA in a mixture comprising soluble tetrameric NA, such as a mixture comprising tetrameric NA and monomeric NA. The mixture can also include other oligomeric forms of NA, including dimeric NA, trimeric NA, or higher order oligomeric NA. In some embodiments, a TAMIFLU® binding conjugate is used to purify soluble, tetrameric NA from cell culture supernatants. Typically, the fluidic sample to be purified comprises a supernatant from a cell culture in which the cell culture comprises a host cell (e.g., a mammalian cell, such as a CHO cell) that includes an artificial nucleic acid molecule that encodes the NA molecules (e.g., monomeric NA molecules, such as wild-type, recombinant or modified monomeric NA molecules). The NA molecules are expressed from the artificial nucleic acid in the host cell, which secretes tetrameric NA or a mixture of tetrameric NA and monomeric NA molecules into the cell culture supernatant. In some embodiments, for example, the NA molecule is an influenza A (e.g., a subtype 1 or subtype 2 influenza A neuraminidase) or influenza B neuraminidase. In some embodiments, the tetrameric NA represents less than about 1%, less than about 5%, less than about 10%, or less than about 15% of the recombinant protein that is secreted upon expression of the recombinant or modified monomeric NA molecule in the host cell, as measured by size exclusion chromatography (SEC). In some embodiments, the cell culture supernatants are subjected to a first purification method, such as ion exchange chromatography or SEC, to obtain a partially purified peak or pool containing a mixture of monomeric and tetrameric NA, followed by a second purification method, wherein the second purification method is a TAMIFLU®-based purification method comprising a step of incubating the mixture of monomeric and tetrameric NA with a TAMIFLU® conjugate and isolating the tetrameric NA bound to the TAMIFLU® conjugate to purify the tetrameric NA. [00117] In some embodiments, the method of producing the soluble, tetrameric NA uses large- scale production conditions. As used herein, “large-scale production conditions” refer to cultivating host cells that express an artificial nucleic acid molecule encoding a targeted NA protein in a culture vessel, typically a bioreactor, with a working volume of between 10 and 10,000 liters, between 25 and 5000 liters, between 25 and 2000 liters, between 50 liters and 1000 liters, between 50 and 500 liters, between 50 and 250 liters, between 50 and 200 liters, between 100 and 200 liters, between 100 liters and 5000 liters, between 500 liters and 8000 liters, between 1500 liters and 6500 liters, about 1500-1600 liters, about 3000-3200 liters, about 6000-6400 liters, greater than or equal to 25 liters, such as greater than or equal to 100 liters, such as at least 100 liters and less than or equal to 10,000 liters, such as at least 100 liters and less than or equal to 8000 liters, such as at least 100 liters and less than or equal to 4000 liters or such as at least 100 liters and less than or equal to 2000 liters.

[00118] In some embodiments, following purification, a purified composition is obtained, the composition comprising at least 90% tetrameric NA. In some embodiments, the purified sample comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% tetrameric NA. In some embodiments, following purification, a purified composition is obtained, the composition comprising tetrameric NA at a concentration of at least 0.01, 0.2, 0.5, 1, 2, 3, 4, or 5 mg/mL, such as 0.01 mg/mL to about 25 mg/mL, 0.2 mg/mL to about 25 mg/mL, such as 0.5 mg/mL to 10 mg/mL, 2 mg/mL to 10 mg/mL, 0.5 mg/mL to 20 mg/mL, 2 mg/mL to 20 mg/mL, 0.5 mg/mL to 15 mg/mL, or 2 mg/mL to 15 mg/mL.

[00119] F. Binding Mixtures

[00120] The present disclosure also provides binding mixtures that are useful for binding, detecting, and/or purifying targeted wild-type, recombinant, or modified tetrameric influenza virus neuraminidase proteins. The mixtures comprise tetrameric NA molecules (and in some embodiments, other oligomeric forms of NA proteins), monomeric NA molecules, and an NA binding agent. The binding mixture comprises conditions, as described herein, that are sufficient for the NA binding agent to substantially only bind to the tetrameric in the mixture NA so as to produce a bound tetrameric NA. The NA binding agent includes a tetrameric NA binding moiety (e.g., oseltamivir phosphate or a tetrameric NA binding portion thereof) conjugated to a first recognition moiety, as described herein. The NA binding agent is structured to remain substantially unbound to the monomeric NA molecules under the conditions.

[00121] In some embodiments, for example, NA binding is performed using the following conditions: capture an oseltamivir-phosphate-biotin conjugate (e.g., 5-10μg/ml in IxKB buffer, as described further herein) on the surface of streptavidin-coated biosensors; dip the biosensors into wells containing serial 2-fold dilutions of a sample of recombinant NA (e.g., 0.16-10μg/ml in IxKB); and measure the binding kinetics of the recombinant NA to oseltamivir-phosphate using the Bio-Layer Interferometry (BLI) technique on an Octet instrument (ForteBio, Molecular Devices, LLC). If necessary, an equivalent assay can be used to measure TAMIFLU® binding.

[00122] G. Biosensor Devices and Systems

[00123] The present disclosure also provides various biosensor devices and systems that can be used to bind, detect, and/or purify targeted tetrameric influenza virus NA proteins. In some embodiments, for example, biosensor devices are provided that can be used with a biolayer interferometry apparatus to effect real-time, label-free detection of tetrameric NA molecules. In some embodiments, systems are configured to perform affinity chromatography to effect purification of targeted tetrameric NA molecules.

[00124] To illustrate, FIG. 2A schematically shows an exemplary biosensor device from a side view, while FIG. 2B schematically shows a solid support from the biosensor device shown in FIG. 2A from a detailed view. As shown, biosensor device 200 (shown as a biolayer interferometry biosensor) includes solid support 202 (shown as comprising an optic fiber) that is in contact with a mixture that comprises tetrameric influenza virus NA molecules 208 and monomeric influenza virus NA molecules 214 under a set of conditions. As also shown, second recognition moiety 204 is attached to a surface (e.g., a thin glass disc tip) of solid support 202. NA binding agent 206 is shown as including tetrameric NA binding moiety 212 (e.g., oseltamivir phosphate or a tetrameric NA binding portion thereof) conjugated to first recognition moiety 210. FIG. 2B schematically shows an example of a first recognition moiety bound to second recognition moiety 204, and tetrameric NA molecule 208 bound to a tetrameric NA binding moiety that is attached to the first recognition moiety of a NA binding agent. NA binding agent 206 is structured to substantially only bind to tetrameric NA molecule 208 and to remain substantially unbound to monomeric NA molecules 214 under the set of conditions.

[00125] When biosensor device 200 is operably connected to a biolayer interferometry apparatus, white light shines down the optic fiber which terminates at its tip in the thin glass disc from which light reflects both from its internal and external surface. Reflection from the internal surface constitutes the reference beam while reflection from the external surface constitutes the signal beam. The phase of the reflected signal beam is modulated by the number of tetrameric NA molecules 208 that bind to the tip of biosensor device 200. Thus, the reference and signal reflections show constructive and destructive interference at different wavelengths. This interference pattern is captured by a spectrometer (not within view) coupled to a photodetector (not within view). A change in the number of tetrameric NA molecules 208 bound to the tip of biosensor device 200 causes a shift in the interference pattern which is reported as a wavelength shift (nm). The magnitude of the wavelength shift is a direct measure of the number of tetrameric NA molecules 208 bound to the tip of biosensor device 200. Also, examples of biolayer interferometry apparatus, including biolayer interferometry biosensors and related aspects, are described in U.S. Pat. Nos. 7,394,547 and 8,512,950, the contents of which are herein incorporated by reference in their entirety. Suitable biolayer interferometry apparatus or systems that are optionally used to detect tetrameric NA molecule binding with the biosensor devices as described herein are commercially available from vendors, such as Sartorius AG (Goettingen, Germany). Additional systems are described further herein.

[00126] The present disclosure also provides various systems. In some aspects, for example, the methods described herein are optionally performed or facilitated at least in part using systems, distributed computing hardware and applications (e.g., cloud computing services), electronic communication networks, communication interfaces, computer program products, machine readable media, electronic storage media, software (e.g., machine-executable code or logic instructions) and/or the like. To illustrate, FIG. 3 provides a schematic diagram of an exemplary system suitable for use with implementing at least aspects of the methods disclosed in this application. As shown, system 300 includes at least one controller or computer, e.g., server 302 (e.g., a search engine server), which includes processor 304 and memory, storage device, or memory component 306, and one or more other communication devices 314, 316, (e.g., client-side computer terminals, telephones, tablets, laptops, other mobile devices, etc. (e.g., for sending and/or receiving data/instructions, etc.)) positioned remote from processing chamber 318, fluidic material handling component 320, and detection component 322, and in communication with the remote server 302, through electronic communication network 312, such as the Internet or other internetwork. Communication devices 314, 316 typically include an electronic display (e.g., an internet enabled computer or the like) in communication with, e.g., server 302 computer over network 312 in which the electronic display comprises a user interface (e.g., a graphical user interface (GUI), a web-based user interface, and/or the like) for displaying results upon implementing the methods described herein. In certain aspects, communication networks also encompass the physical transfer of data from one location to another, for example, using a hard drive, thumb drive, or other data storage mechanism.

[00127] Processing chamber 318 (e.g., one or more wells of a microplate used as part of a NA binding assay, one or more columns in an affinity chromatography configuration used a part of a tetrameric NA protein purification application, etc.) comprises, or is capable of receiving at least a portion of, a solid support (e.g., a surface of a microplate well, an affinity chromatography resin, etc.) when the solid support is in contact with a mixture that comprises tetrameric neuraminidase (NA) and monomeric NA molecules of an influenza virus under a set of conditions. In some embodiments, for example, the solid support comprises: a second recognition moiety attached to a surface of the solid support; an NA binding agent that comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the first recognition moiety is bound to the second recognition moiety; and at least one of the tetrameric NA bound to the tetrameric NA binding moiety to form a bound tetrameric NA molecule. In some embodiments, the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof. As described herein, the NA binding agent is structured to substantially only bind to the tetrameric NA and to remain substantially unbound to monomeric NA molecules under the set of conditions. Fluidic material handling component 320 fluidly communicates with processing chamber 318 and at least one fluidic material source, such as a fluid reservoir (not within view). Detection component 322 (operably connected, or connectable, to the controller) typically comprises electromagnetic radiation source 324 configured to convey electromagnetic radiation at least to the solid support, and electromagnetic radiation detector 326 configured to detect one or more interference patterns, or changes therein, of the electromagnetic radiation conveyed to the solid support.

[00128] System 300 also includes program product 308 (e.g., related to implementing a method of detecting and/or purifying tetrameric NA proteins as described herein) stored on a computer or machine readable medium, such as, for example, one or more of various types of memory, such as memory 306 of server 302, that is readable by the server 302, to facilitate, for example, a guided search application or other executable by one or more other communication devices, such as 314 (schematically shown as a desktop or personal computer). In some aspects, system 300 optionally also includes at least one database server, such as, for example, server 310 associated with an online website having data stored thereon searchable either directly or through search engine server 302. System 300 optionally also includes one or more other servers positioned remotely from server 302, each of which are optionally associated with one or more database servers 310 located remotely or located local to each of the other servers. The other servers can beneficially provide service to geographically remote users and enhance geographically distributed operations.

[00129] Memory 306 of the server 302 optionally includes volatile and/or nonvolatile memory including, for example, RAM, ROM, and magnetic or optical disks, among others. Although illustrated as a single server, the illustrated configuration of server 302 is given only by way of example and other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 302 shown schematically in FIG. 3, represents a server or server cluster or server farm and is not limited to any individual physical server. The server site may be deployed as a server farm or server cluster managed by a server hosting provider. The number of servers and their architecture and configuration may be increased based on usage, demand and capacity requirements for the system 300. Other user communication devices 314, 316 in these aspects, for example, can be a laptop, desktop, tablet, personal digital assistant (PDA), cell phone, server, or other types of computers. Network 312 can include an internet, intranet, a telecommunication network, an extranet, or world wide web of a plurality of computers/servers in communication with one or more other computers through a communication network, and/or portions of a local or other area network.

[00130] Exemplary program product or machine readable medium 308 is optionally in the form of microcode, cloud computing formats, programs, routines, and/or symbolic languages that provide one or more sets of ordered operations that control the functioning of the hardware and direct its operation. Program product 308, according to an exemplary aspect, also need not reside in its entirety in volatile memory, but can be selectively loaded, as necessary, according to various methodologies as known and understood by those of ordinary skill in the art.

[00131] The term “computer-readable medium” or “machine-readable medium” refers to any medium that participates in providing instructions to a processor for execution. To illustrate, the term “computer-readable medium” or “machine-readable medium” encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 308 implementing the functionality or processes of various aspects of the present disclosure, for example, for reading by a computer. A “computer-readable medium” or “machine-readable medium” may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks. Volatile media includes dynamic memory, such as the main memory of a given system. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications, among others. Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.

[00132] Program product 308 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 308, or portions thereof, are to be run, it is optionally loaded from their distribution medium, their intermediate storage medium, or the like into the execution memory of one or more computers, configuring the computer(s) to act in accordance with the functionality or method of various aspects. [00133] To further illustrate, in certain aspects, this application provides systems that include one or more processors, and one or more memory components in communication with the processor. The memory component typically includes one or more instructions that, when executed, cause the processor to provide information that causes data (e.g., targeted tetrameric NA detection and/or purification data), processing information, and/or the like to be displayed (e.g., upon being received from processing chamber 318, fluidic material handling component 320, and detection component 322 and/or via communication devices 314, 316 or the like) and/or receive information from other system components and/or from a system user (e.g., via processing chamber 318, fluidic material handling component 320, and detection component 322 and/or via communication devices 314, 316, or the like).

[00134] In some aspects, program product 308 includes non-transitory computer-executable instructions which, when executed by electronic processor 304 perform at least: conveying at least one fluidic material from a fluidic material source to processing chamber 318 using fluidic material handling component 320. In some embodiments, the instructions further perform at least: detecting the bound tetrameric NA molecule; conveying at least one binding buffer from at least a first fluidic material source to processing chamber 318 one or more times to substantially remove any unbound tetrameric NA, the monomeric NA molecules, and/or other reagents or compounds through at least one outlet of processing chamber 318 to produce a washed bound tetrameric NA molecule in processing chamber 318; and/or conveying at least one elution buffer from at least a second fluidic material source to processing chamber 318 one or more times to elute the washed bound tetrameric NA molecule from the NA binding agent through the outlet of processing chamber 318 to produce a purified tetrameric NA. Other exemplary executable instructions that are optionally performed are described further herein.

[00135] H. Nucleic Acids, Cloning, and Expression Systems.

[00136] The present disclosure further relates to artificial nucleic acid molecules encoding the wild-type, recombinant, or modified NA proteins detected and/or purified according to the methods disclosed herein. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise. [00137] The present disclosure also relates to constructs in the form of a vector (e.g., plasmids, phagemids, cosmids, transcription or expression cassettes, artificial chromosomes, etc.) comprising an artificial nucleic acid molecule encoding a given NA molecule. The disclosure further relates to a host cell which comprises one or more of these constructs.

[00138] Also provided are methods of making the NA proteins encoded by these artificial nucleic acid molecules. The NA polypeptides may be produced using recombinant techniques. The production and expression of recombinant proteins is well known in the art and can be carried out using conventional procedures, such as those disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Ed. 2012), Cold Spring Harbor Press. For example, expression of the NA polypeptide may be achieved by culturing under appropriate conditions host cells containing the artificial nucleic acid molecule encoding the NA polypeptide. Thus, a method for producing tetrameric NA may comprise culturing host cells in a cell culture medium, wherein the host cells contain an artificial nucleic acid encoding the NA, and expressing the NA in the host cells, wherein the NA is secreted from the host cells as soluble, tetrameric NA. Following production by expression, the tetrameric NA may be isolated and/or purified as described herein or using any other suitable technique, then used as appropriate.

[00139] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known in the art. Any protein expression system (e.g., stable or transient) compatible with a given NA encoding construct may be used to produce that protein.

[00140] Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[00141] A further aspect of the disclosure relates to a host cell comprising an artificial nucleic acid molecule that encodes a targeted NA protein. A still further aspect provides a method comprising introducing such artificial nucleic acid molecules into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia, alphavirus, etc. or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. These techniques are well known in the art. (See, e.g., “Current Protocols in Molecular Biology,” Ausubel et al. eds., John Wiley & Sons, 2010). DNA introduction may be followed by a selection method (e.g., antibiotic resistance) to select cells that contain the vector.

[00142] The host cell may be a plant cell, a yeast cell, or an animal cell. Animal cells encompass invertebrate (e.g., insect cells), non-mammalian vertebrate (e.g., avian, reptile and amphibian) and mammalian cells. In one embodiment, the host cell is a mammalian cell. Examples of mammalian cells include, but are not limited to COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO) cells; mouse sertoli cells; African green monkey kidney cells (VERO-76); human cervical carcinoma cells (e.g., HeLa); canine kidney cells (e.g., MDCK), and the like. In one embodiment, the host cells are CHO cells.

[00143] The present disclosure will be more fully understood by reference to the following Examples.

EXAMPLES

[00144] Example 1. Tetrameric NA Dose Dependent Binding

[00145] Tetrameric NA from different influenza strains, including influenza A and B, was produced using recombinant constructs comprising a tetrabrachion tetramerization domain (Dai et al., 2016, J. Virology, 90(20):9457-70; Schmidt et al., PLos ONE, 2011, 6(2):el6284). More specifically, recombinant constructs comprising modified NA with a tetrabrachion tetramerization domain was expressed in cells to produce soluble tetrameric NA (tet-NA). For influenza A, NA from subtype 1 and 2 strains (N1 and N2) were used to make the tet-NA constructs. The subtype 1 strain used to make the tet-NA construct was A/Michigan2015 (Nl). The subtype 2 strains used to make the tet-NA constructs were A/Singapore2016 (N2), A/Switzerland2013 (N2), and A/Texas2012 (N2). For influenza B, NA from Victoria and Yamagata strains were used to make the tet-NA constructs. The Victoria influenza B strain used to make the tet-NA construct was B/Colorado2017. The Yamagata influenza B strain used to make the tet-NA construct was B/Phuket2013.

[00146] To measure the production of tetrameric NA from these recombinant constructs, a TAMIFLU®-NA binding assay was developed on an Octet-Red96, an Octet BLI Detection System (ForteBio, Sartorius). BLI is an optical analytic technique that utilizes the changing interference patterns of white light shown over a reflective biosensor surface (immobilized with a ligand) to detect interactions with an analyte (NA) in solution. In brief, TAMIFLU®-biotin conjugates (5-10 μg/ml in IxKB buffer (0.1% BSA + 0.02% Tween 20 in PBS, pH 7.4)) were first captured on a streptavidin-coated biosensor (e.g., a High Precision Streptavidin (SAX) Dip and Read Biosensor, Cat. No. 18-51182). The interaction between NA and TAMIFLU® was then initiated by dipping TAMIFLU® bound biosensors into sample wells containing a 2- fold dilution series of recombinant NA (0.16-40 μg/ml in IxKB buffer). The binding between TAMIFLU® and NA produced a measured shift in the interference pattern via the detector. The TAMIFLU®-binding assay successfully detected the binding of tetrameric NA for each of the constructs tested. The level of TAMIFLU® binding response was proportional to the concentration of NA (FIG 4). As shown in FIG. 4 and FIG. 5, TAMIFLU® broadly recognizes NA tetramers from both Type A and Type B influenza. SEC-MALS was used to confirm the presence of tetrameric NA.

[00147] Example 2. Characterization of purified recombinant NA vaccine candidates using TAMIFLU®-NA binding assay: quantitation of NA tetramers

[00148] Tetrameric NA from different N2 strains was also produced using stalk deletion variants in which all or substantially all of the stalk region was deleted, as described herein. For example, amino acids 1-74 of the N2 were replaced by a signal peptide (also referred to as dTM75). When expressed in cells, the dTM75 variants form tetrameric NA. NA variants lacking amino acids 1-35 (also referred to as dTM36) were also constructed. When expressed in cells, dTM36 forms monomeric NA. FIG. 6 shows that TAMIFLU® selectively binds to tetrameric NA from tet-NA constructs (tet-NA_SING16, tet-NA_MICH15) and dTM75 constructs (dTM75_SG16), but does not bind to monomeric NA (dTM36_SG16, dTM36_MICH15). Therefore, the TAMIFLU®-NA binding assay can be used to evaluate the integrity of substrate binding sites on the NA head.

[00149] Example 3. High throughput screening of novel NA vaccine designs

[00150] The TAMIFLU®-NA binding assay’s ability to detect the presence of NA tetramers has been demonstrated in unpurified CHO cell supernatants (7.5ml-scale of transfection). CHO cells were transfected with plasmid constructs encoding modified NA proteins that form tetramers when expressed in cells. Unknown analyte concentrations were calculated against a generated standard curve (FIGS. 7 and 8). Adapting the TAMIFLU®-NA binding assay from Octet Red-96 (8-channel) to Octet-HTX (96-channel) further enables high throughput screening (e.g., for NA vaccine designs), allowing up to 96 samples with 4 dilutions to be processed within 3 hours. This increase in throughput significantly accelerates the ability to screen and rank novel NA vaccine designs.

[00151] Example 4. Screening binding and elution conditions for the development of TAMIFLU® affinity NA purification method

[00152] After discovering that TAMIFLU® can be used as a ligand to immobilize tetrameric NA, a TAMIFLU® based affinity chromatography method was developed that was aimed at purifying NA from cell transfection supernatants. During the development of the TAMIFLU® affinity NA purification method, various steps of the kinetic TAMIFLU®-NA binding assay process were utilized to screen for binding and elution conditions pertaining to NA purification (FIG. 9). Specifically, optimal binding conditions were screened during the association step which involved immobilizing NA onto the TAMIFLU®-biotin-streptavidin coated biosensor. In addition, optimal elution conditions were screened during the disassociation step which involved eluting NA from the complex bound to the biosensor post initial association. The screened buffer conditions were subsequently successfully adapted as the binding and elution conditions for the TAMIFLU® affinity purification column methodology.

[00153] Example 5. Monitoring NA transfection efficiency during the preparation of material for large scale NA purification

[00154] It was discovered that large scale NA transfection efficiency can vary from lot to lot during expression optimization studies. These varying expression levels in transfection supernatant significantly impact the tetrameric NA yield (FIG. 10). Prior to NA purification, the TAMIFLU®-NA binding assay can be used as a quantitative means to measure NA expression amongst other information regarding transfection efficiency. It can further predict the functional conformational NA yield resulting from any individual lot of NA supernatant. Therefore, TAMIFLU®-NA binding assay can play an important role in NA vaccine research and as a control assay during the CMC process (manufacturing) of NA vaccine products.

[00155] Example 6. One-step TAMIFLU®-biotin affinity chromatography method (TAC)

[00156] The one-step TAMIFLU®-biotin affinity chromatography method (TAC) described in this disclosure was used to isolate and purify tetrameric NA from more than 10 His-tagged and untagged recombinant NA constructs derived from influenza N2 A/Perth/16/2009 and N2 A/Kansas/14/2017 strains, including tet-NA and dTM75 constructs. Tet-NA_KS17 forms predominately tetrameric NA but also produces a certain amount of monomeric NA, while dTM75_KS17 forms both monomeric and tetrameric NA. Under denaturing and reducing conditions, where the disulfide bonds that help to maintain the integrity of the conformational, tetrameric NA structure are broken, the eluates of the tet-NA and dTM75 constructs purified using either the NiAC or the TAC method, contained a single, predominant band around 55 kDa, corresponding to monomeric NA (FIG. 11 and FIG. 15).

[00157] The TAC method was used to purify tetrameric NA obtained from CHO cells transfected with the Tet-NA_KS17, dTM75_KS17, Tet-NA_PerthO9, and dTM75_PerthO9 constructs and produced substantially pure tetrameric NA (about 90-100%), as shown in FIG. 13. When the constructs were purified using the NiAC method, the percentage of tetrameric NA was reduced, particularly for the dTM75_KS17 constructs (FIG. 13). Compared to the broadly used Ni-NTA affinity chromatography purification method (NiAC) for His-tagged protein purification that copurifies NA tetramers and monomers, TAC is uniquely able to selectively enrich tetrameric NA from CHO cell culture harvests, as shown in FIG. 12 (KS17_dTM75) and FIG. 16 (PerthO9_dTM75). In FIG. 12, Peak 1 and 2 in the NiAC method correspond to tetrameric and monomeric NA, respectively and Peak 1 and 2 in the TAC method correspond to higher-order oligomeric NA and tetrameric NA, respectively. In FIG. 16, the Peak 1 and 2 in the NiAC method correspond to tetrameric and monomeric NA, respectively and Peak 1 (94.87%) in the TAC method corresponds tetrameric NA. Furthermore, the TAC method was efficient regarding the preservation of enzyme activity (measured by MUNANA assay), substrate binding site integrity (TAMIFLU®-NA binding assay), and N2 epitope integrity (N2-mAb binding assay), as shown in FIG. 14.

[00158] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the protein constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations.

[00159] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., some embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[00160] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number, if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated.

Claims

We claim:

1. A method of selectively binding a tetrameric neuraminidase (NA) of an influenza virus, the method comprising: contacting a fluidic sample that comprises a mixture of the tetrameric NA and monomeric NA molecules with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA, wherein the NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof, and wherein the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules in the fluidic sample under the conditions, thereby selectively binding the tetrameric NA in the fluidic sample.

2. The method of claim 1 , further comprising: detecting the bound tetrameric NA; and/or, separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample.

3. A method of detecting a tetrameric neuraminidase (NA) of an influenza virus, the method comprising: contacting a fluidic sample that comprises a mixture of the tetrameric NA and monomeric NA molecules with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA, wherein the NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof, and wherein the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules in the fluidic sample under the conditions; and, detecting the bound tetrameric NA, thereby detecting the tetrameric NA in the fluidic sample.

4. A method of purifying a tetrameric neuraminidase (NA) of an influenza virus, the method comprising: contacting a fluidic sample that comprises a mixture of the tetrameric NA and monomeric NA molecules with an NA binding agent under conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA in the fluidic sample to produce a bound tetrameric NA, wherein the NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof, and wherein the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules in the fluidic sample under the conditions; and, separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample, thereby purifying the tetrameric NA.

5. The method of any one of the preceding claims, wherein the fluidic sample comprises a supernatant from a cell culture, wherein the cell culture comprises a host cell comprising an artificial nucleic acid molecule that encodes the NA molecules, wherein the NA molecules are expressed from the artificial nucleic acid in the host cell, and wherein the host cell secretes tetrameric NA or a mixture of tetrameric NA and monomeric NA molecules into the cell culture supernatant.

6. The method of any one of the preceding claims, comprising using the detected bound tetrameric NA as a measure of tetrameric NA expression in the fluidic sample and/or to predict a probable yield of catalytically active tetrameric NA in the fluidic sample.

7. The method of any one of the preceding claims, wherein host cell is a mammalian cell.

8. The method of any one of the preceding claims, wherein the tetrameric NA is substantially soluble in the fluidic sample.

9. The method of any one of the preceding claims, wherein the tetrameric NA is catalytically active.

10. The method of any one of the preceding claims, wherein the tetrameric NA comprises four copies of a monomeric NA molecule.

11. The method of any one of the preceding claims, wherein the monomeric NA molecule is a wild-type, recombinant or modified monomeric NA molecule.

12. The method of any one of the preceding claims, wherein the NA molecule is an influenza A or influenza B neuraminidase.

13. The method of claim 12, wherein the influenza A neuraminidase is a subtype 1 or subtype 2.

14. The method of any one of the preceding claims, wherein the tetrameric NA comprises four copies of a modified influenza virus subtype 1 or 2 neuraminidase or neuraminidase from influenza B virus, wherein the modified influenza virus neuraminidase comprises: a signal peptide; and a head region of an influenza virus neuraminidase, wherein the cytoplasmic tail, transmembrane region and at least a portion of the stalk region of the influenza virus neuraminidase have been replaced by the signal peptide, wherein the modified influenza virus neuraminidase optionally comprises a heterologous oligomerization domain, and wherein expression of the modified influenza virus neuraminidase in a cell results in the secretion of the tetrameric neuraminidase.

15. The method of any one of the preceding claims, wherein the tetrameric NA represents less than about 1%, less than about 5%, less than about 10%, or less than about 15% of the recombinant or modified monomeric NA molecule that is secreted upon expression of the recombinant or modified monomeric NA molecule in the host cell, as measured by size exclusion chromatography-multiple angle light scattering (SEC-MALS).

16. The method of any one of the preceding claims, wherein amino acid 1 to at least amino acid 70-74 of a wild-type N2 influenza virus NA have been replaced by the signal peptide.

17. The method of any one of the preceding claims, wherein the monomeric NA molecules comprise a head region of the NA of the influenza virus, but lack at least a portion of one or more of a cytoplasmic tail, a transmembrane region, and a stalk region of the NA of the influenza virus.

18. The method of any one of the preceding claims, wherein the tetrameric NA binding moiety comprises oseltamivir carboxylate.

19. The method of any one of the preceding claims, wherein the tetrameric NA binding moiety is conjugated to the first recognition moiety via a spacer moiety.

20. The method of any one of the preceding claims, wherein the spacer moiety comprises an undecaethylene glycol spacer moiety.

21. The method of any one of the preceding claims, wherein the first recognition moiety comprises biotin.

22. The method of any one of the preceding claims, wherein the first recognition moiety is a compound selected from the group consisting of: streptavidin, avidin, an antibody, an antigen, an aptamer, a protein, a peptide, and a carbohydrate.

23. The method of any one of the preceding claims, wherein the first recognition moiety selectively binds to a second recognition moiety.

24. The method of any one of the preceding claims, wherein the second recognition moiety is a compound selected from the group consisting of: biotin, streptavidin, avidin, an antibody, an antigen, an aptamer, a protein, a peptide, and a carbohydrate.

25. The method of any one of the preceding claims, wherein the tetrameric NA and/or the NA binding agent is unlabeled.

26. The method of any one of the preceding claims, comprising binding the first recognition moiety to the second recognition moiety after contacting the fluidic sample with the NA binding agent.

27. The method of any one of the preceding claims, comprising binding the first recognition moiety to the second recognition moiety before contacting the fluidic sample with the NA binding agent.

28. The method of any one of the preceding claims, wherein the second recognition moiety is attached to a solid support.

29. The method of any one of the preceding claims, further comprising quantifying the tetrameric NA in the fluidic sample and/or the bound tetrameric NA.

30. The method of any one of the preceding claims, wherein the NA binding agent is present in a concentration in a range of about 1 μg/ml to about 100 μg/ml.

31. The method of any one of the preceding claims, wherein the NA binding agent is present a concentration in a range of about 5 μg/ml to about 10 μg/ml.

32. The method of any one of the preceding claims, comprising contacting the fluidic sample with the NA binding agent and detecting the bound tetrameric NA substantially in real-time.

33. The method of any one of the preceding claims, comprising detecting the tetrameric NA as part of a high-throughput screening process that detects the bound tetrameric NA and/or a catalytic activity of the tetrameric NA within about 5 minutes of contacting the fluidic sample with the NA binding agent.

34. The method of claim 33, wherein the high-throughput screening process further comprises screening recombinant NA expressed from a plurality of recombinant NA constructs and identifying recombinant NA constructs within the plurality of recombinant NA constructs that produce tetrameric NA when expressed in a host cell.

35. The method of any one of the preceding claims, wherein separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample yields a purity level of the tetrameric NA of at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or more.

36. The method of any one of the preceding claims, comprising separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample as part of a large-scale NA purification process that yields between about 0.2 mg and about 25 mg of the tetrameric NA per ml of the fluidic sample.

37. The method of any one of the preceding claims, wherein separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample yields the tetrameric NA at a purity level that is at least as high as when using a Ni-NTA affinity chromatography purification process (NiAC) to purify tetrameric NA from an identical fluidic sample.

38. The method of any one of the preceding claims, wherein separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample yields improved preservation of catalytic activity as measured by a MUNANA assay or improved substrate binding site integrity as measured by an oseltamivir-NA binding assay than when using a Ni-NTA affinity chromatography purification process (NiAC) to purify tetrameric NA from an identical fluidic sample.

39. The method of any one of the preceding claims, comprising detecting the bound tetrameric NA using an interferometry technique.

40. The method of any one of the preceding claims, comprising separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample using an affinity chromatography technique.

41. The method of any one of the preceding claims, further comprising determining a kinetics and/or affinity property of the tetrameric NA.

42. The method of any one of the preceding claims, wherein the tetrameric NA is present in a concentration in a range of about 0.16 μg/ml to about 50 μg/ml in the fluidic sample.

43. The method of any one of the preceding claims, wherein the tetrameric NA is present in a concentration in a range of about 0.16 μg/ml to about 40 μg/ml in the fluidic sample.

44. A purified tetrameric NA produced using the method of any one of the preceding claims.

45. The method of any one of the preceding claims, wherein separating the bound tetrameric NA from at least the monomeric NA molecules in the fluidic sample comprises: washing the bound tetrameric NA one or more times using at least one binding buffer to substantially remove unbound tetrameric NA, the monomeric NA molecules, and/or other reagents or compounds to produce a washed bound tetrameric NA; and, eluting the washed bound tetrameric NA from the NA binding agent using at least one elution buffer to produce an eluted tetrameric NA.

46. The method of claim 45, wherein the elution buffer comprises glycine-HCl in a range of about 0. IM to about 1.0M at pH of about 5.0 or less.

47. The method of claim 45, wherein the elution buffer comprises about 0. IM glycine- HCl at pH of about 2.8.

48. The method of any one of the preceding claims, further comprising substantially neutralizing the eluted tetrameric NA when the elution buffer comprises a pH of about 5.0 or less.

49. A binding mixture, comprising: a tetrameric neuraminidase (NA) of an influenza virus, monomeric NA molecules, and an NA binding agent, wherein the binding mixture comprises conditions sufficient for the NA binding agent to substantially only bind to the tetrameric NA to produce a bound tetrameric NA, wherein the NA binding agent comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof, and wherein the NA binding agent is structured to remain substantially unbound to the monomeric NA molecules under the conditions.

50. A biosensor device, comprising: a solid support in contact with a mixture that comprises tetrameric neuraminidase (NA) and monomeric NA molecules of an influenza virus under a set of conditions; a second recognition moiety attached to a surface of the solid support; an NA binding agent that comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the first recognition moiety is bound to the second recognition moiety; and, at least one tetrameric NA bound to the tetrameric NA binding moiety, wherein the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof, wherein the NA binding agent is structured to substantially only bind to the tetrameric NA under the set of conditions, and wherein the NA binding agent is structured to remain substantially unbound to monomeric NA molecules under the set of conditions.

51. The biosensor device of claim 50, comprising a biolayer interferometry biosensor that comprises the solid support.

52. A system, comprising: a processing chamber that comprises, or is capable of receiving at least a portion of, a solid support when the solid support is in contact with a mixture that comprises tetrameric neuraminidase (NA) and monomeric NA molecules of an influenza virus under a set of conditions, which solid support comprises: a second recognition moiety attached to a surface of the solid support; an NA binding agent that comprises at least one tetrameric NA binding moiety conjugated to at least a first recognition moiety, wherein the first recognition moiety is bound to the second recognition moiety; and at least one of the tetrameric NA bound to the tetrameric NA binding moiety to form a bound tetrameric NA molecule, wherein the at least one tetrameric NA binding moiety comprises oseltamivir phosphate or a tetrameric NA binding portion thereof, wherein the NA binding agent is structured to substantially only bind to the tetrameric NA under the set of conditions, and wherein the NA binding agent is structured to remain substantially unbound to monomeric NA molecules under the set of conditions; a fluidic material handling component that fluidly communicates with the processing chamber and at least one fluidic material source; and, a controller that is operably connected, or connectable, at least to the fluidic material handling component, wherein the controller comprises, or is capable of accessing, computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: conveying at least one fluidic material from the fluidic material source to the processing chamber using the fluidic material handling component.

53. The system of claim 52, further comprising: a detection component operably connected, or connectable, to the controller, wherein the instructions further perform at least: detecting the bound tetrameric NA molecule.

54. The system of claim 52 or 53, wherein the detection component comprises: an electromagnetic radiation source configured to convey electromagnetic radiation at least to the solid support; and, an electromagnetic radiation detector configured to detect one or more interference patterns, or changes therein, of the electromagnetic radiation conveyed to the solid support.

55. The system of any one of claims 52-54, wherein the instructions further perform at least: conveying at least one binding buffer from at least a first fluidic material source to the processing chamber one or more times to substantially remove any unbound tetrameric NA, the monomeric NA molecules, and/or other reagents or compounds through at least one outlet of the processing chamber to produce a washed bound tetrameric NA molecule in the processing chamber; and, conveying at least one elution buffer from at least a second fluidic material source to the processing chamber one or more times to elute the washed bound tetrameric NA molecule from the NA binding agent through the outlet of the processing chamber to produce a purified tetrameric NA.