WO2017161345A1 - Improved cell based assay - Google Patents

Abstract

Methods and compositions are provided for rapid and efficient removal of interferents from analyte containing solutions, where such methods and compositions are effective in removing such interferents to concentrations that do not interfere with cell-based assays. Affinity-based phase separation is employed to quantitatively harvest analyte from an interferent-containing solution, followed by quantitative recovery of the analyte from the affinity-based phase in active form. Recovery can be performed at reduced volumes relative to harvesting to provide for concentration of the analyte and extension of the dynamic range of a cell-based assay.

Description

IMPROVED CELL BASED ASSAY

[0001] This application claims priority to United States Provisional Application No. 62/310,434, filed on March 18, 2016. This and all other referenced extrinsic materials are incorporated herein by reference in their entirety. Where a definition or use of a term in a reference that is incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be controlling.

Field of the Invention

[0002] The field of the invention is cell based assays for pharmaceutical compounds. Background

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] The gold standard for characterization of molecules of biological interest is animal testing. Such testing, however, requires the use of a large number of subject animals in order to provide statistically useful data. Since the processes involved are not readily automatable such studies necessarily require the use of highly trained staff. In addition, the use of animals in such studies is facing increasing regulatory restriction and negative pressure from a public that, nevertheless, demands high levels of safety and efficacy from consumed products. As a result considerable effort is under way to identify suitable alternatives to animal research.

[0005] While in vitro biochemical assays have proven invaluable for characterization and quantitation of proteins, drugs, hormones, and other compounds of biological interest, they have a number of inherent shortcomings. Nucleic acid-based tests (such as PCR, real time PCR, and reverse transcription PCR), while extraordinarily sensitive, do not provide accurate quantitation and are limited to genetic materials. Immunochemical assays (such as EIA) are highly quantitative. Such immunochemical tests, however, do not provide information regarding the functionality of the analyte. For example, the vast majority of EIAs fail to distinguish between active and inactive (for example partially denatured) forms of a protein of interest. While antibodies can be selected for use in EIA that provide a degree of selectivity between active an inactive forms of the analyte, they only provide data related to concentration and do not provide insight into functionalities such as absorption, cellular internalization, and cellular processing of the analyte. As a result, while such in vitro techniques are invaluable tools they often are inadequate for characterization of many analytes of biochemical and biomedical interest.

[0006] One approach has been to utilize cultured cells in place of living animals. Cultured cells, which are frequently immortalized cell lines derived from tissues representing target tissues of the compound being tested, are exposed to the analyte of interest. The compound can interact with cell surface receptors, be internalized, and be processed using pathways that are presumably identical to those of an animal subject. The response of the exposed cells can then be characterized, for example by direct observation or by lysing the cells and characterizing their contents.

[0007] While such cell based assays are useful tools, they have significant shortcomings. For example, an analyte of interest may be present in concentrations that are significant in a living animal but that are too low to be reliably characterized by cultured cells. Alternatively, the analyte of interest may be present in a sample that contains contaminating substances that are toxic to the cultured cells. For example, many pharmaceutical compounds are provided for use with excipients that improve stability and help retain activity during processing (for example, lyophilization) and during storage. While such pharmaceutical excipients (for example, salts, proteins, surfactants, reducing agents, polysaccharides, and/or synthetic organic polymers) can be relatively benign when the pharmaceutical preparation is used therapeutically, they can have cytotoxic activity that directly interferes with such cell based assays. For example, International Patent Application Publication No. WO 2011/008713, to Ton et al, describes the preparation of a highly purified botulinum neurotoxin to which excipients are added following purification for pharmaceutical use. All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0008] While the effects of excipients can be moderated to some extent by dilution, this approach is necessarily limited by the sensitivity of the assay used. In some instances, such contaminating substances can be removed, for example by dialysis or through the use of specialty resins. In many circumstances, however, excipients may not be removed adequately or the removal process can lead to excessive loss of the analyte. This is particularly true when the contaminant has a molecular weight similar to that of the pharmaceutical compound, and/or when the pharmaceutical analyte of interest is present in low (e.g. less than 1 nM)

concentrations.

[0009] Thus, there is still a need for compositions and methods that provide for removal of toxic contaminating substances and/or provides quantitative detection of a molecule of interest using a cell based assay.

Summary of The Invention

[0010] The inventive subject matter provides compositions and methods in which an analyte is harvested from solution in a quantitative manner using an affinity partner for the analyte coupled to an insoluble (e.g. solid) phase. Following removal of the supernatant, which can contain compounds that interfere with cell-based assays, the analyte is harvested quantitatively from the insoluble phase and characterized using a cell-based assay. Surprisingly, Inventors have found that such an affinity solid phase can have sufficient affinity to provide quantitative harvesting of an analyte from solution at low (i.e. less than 1 nM) concentrations and also release analyte so harvested quantitatively under relatively mild conditions that retain the analyte' s structure and/or function.

[0011] One embodiment of the inventive concept is a method for reducing interference effects in a cell based assay, in which an analyte specific antibody coupled to an insoluble phase (for example, a solid phase) is brought into contact with a sample that includes an analyte (such as a botulinum neurotoxin) and an interfering substance thereby forming an analytednsoluble phase antibody complex in quantitative manner. Substances that can interfere with subsequent analysis can be removed by separating the analytednsoluble phase from the interferent-containing supernatant. In some embodiments the analytednsoluble phase can be subsequently rinsed or washed with an interferent-free buffer. The insoluble phase is recovered from the sample by quantitatively dissociating the analytednsoluble phase antibody complex (for example, using an elution buffer) to provide a purified analyte. The purified analyte is then brought into contact with a cell used in a cell based assay for the analyte. In some embodiments the interfering substance is a cytotoxic substance, such as a surfactant. In some embodiments the insoluble phase is magnetically responsive. In some embodiments the elution buffer has a pH between 3 and 9, and/or can have an ionic strength of 50mM to 500mM.

[0012] In some embodiments of the inventive concept the sample is provided in a first volume and the purified analyte is recovered in a second volume that is smaller than the first volume, thereby providing a concentration effect. In such embodiments the second volume can less than or equal to 20%, 15%, 10%, or 5% of the first volume.

[0013] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

Brief Description of The Drawings

[0014] FIG. 1 schematically depicts use of a fluorescent reporting construct in cell-based detection of a serotype A botulinum neurotoxin (BoNT/A).

[0015] FIG. 2 schematically depicts a method of the inventive concept, where an analyte of interest is captured quantitatively from solution, unwanted (e.g. cytotoxic) contaminants are removed, and the analyte of interest is quantitatively released for subsequent cell-based analysis.

[0016] FIGs. 3A and 3B schematically depict steps of an exemplary cell-based assay performed on analyte recovered quantitatively from a solution containing cytotoxic contaminants. FIG. 3A depicts an exemplary cell containing an exemplary reporting construct, prior to exposure to the analyte. FIG. 3B depicts an exemplary cell containing an exemplary reporting construct upon exposure to an analyte quantitatively recovered from a solution containing cytotoxic

contaminants and reactivity of the analyte with the exemplary reporting construct. [0017] FIG. 4 shows typical results from characterization of a BoNT/A holotoxin in an excipient-free buffer and a commercial BoNT/A pharmaceutical in an excipients -containing solution in a cell based assay.

[0018] FIG. 5 shows typical results of attempts to remove excipients from a commercial BoNT/A pharmaceutical preparation by exhaustive dialysis.

[0019] FIGs. 6A, 6B, and 6C show typical results from studies of HiPPR™ Detergent Removal Resin treatment of samples of BoNT/A in cell culture media supplemented with typical excipients. FIG. 6A shows results from treated and untreated BoNT/A samples containing humans serum albumin (HSA) and polysorbate 80 (P80) excipients. FIG. 6B shows results from treated and untreated BoNT/A samples containing HSA, P80, and NaCl excipients. FIG. 6C compares results of a BoNT/A sample prepared with supplemental HSA to a BoNT/A sample containing HSA and P80 excipients after treatment with HiPPR™ Detergent Removal Resin.

[0020] FIGs. 7A, 7B, and 7C show typical results from studies of DetergentOUT™ Tween treatment of samples of BoNT/A in cell culture media supplemented with typical excipients. FIG. 7A shows results from treated and untreated BoNT/A samples containing HSA and P80 excipients. FIG. 7B shows results from treated and untreated BoNT/A samples containing HSA, P80, and NaCl excipients. FIG. 7C compares results of a BoNT/A sample prepared with supplemental HSA to a BoNT/A sample containing HSA and P80 excipients after treatment with DetergentOUT™ Tween.

[0021] FIGs. 8A, 8B, 8C, and 8D show typical results from studies of harvesting and recovery of BoNT/A from cell culture media supplemented with typical excipients for different antibody- coated magnetic bead preparations. FIG. 8A shows typical results for excipients effects and BoNT/A recovery for four different antibody-magnetic bead preparations using 0.7 mL of sample. FIG. 8B shows typical results for excipients effects and BoNT/A recovery for an IgY- Dynabead™ antibody-magnetic bead preparation using 4 mL of sample. FIG. 8C shows typical results for excipients effects and BoNT/A recovery for a first murine IgG-Dynabead™ antibody- magnetic bead preparation using 4 mL of sample. FIG. 8D shows typical results for excipients effects and BoNT/A recovery for a second murine IgG-Dynabead™ antibody-magnetic bead preparation using 4 mL of sample. [0022] FIGs. 9A, 9B, and 9C show typical results from studies of harvesting and recovery of BoNT/A from different volumes of media containing typical excipients using a previously optimized antibody-magnetic bead preparation. FIG. 9A shows typical results obtained from a 2 mL BoNT/A sample. FIG. 9B shows typical results obtained from a 7 mL BoNT/A sample. FIG. 9C shows typical results obtained from a 10 mL BoNT/A sample.

[0023] FIG. 10 shows typical results from studies of harvesting and recovery of BoNT/A from cell culture media supplemented with typical excipients using a previously optimized antibody- magnetic bead preparation, where the elute BoNT/A was characterized using a second cell-based assay.

Detailed Description

[0024] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0025] The inventive subject matter provides apparatus, systems and methods in which an analyte of interest is collected from a solution containing a cytotoxic substance using one or more antibodies that are specific for the analyte of interest and are coupled to a solid phase, as an antigen: antibody complex. Such antibodies can be monoclonal antibodies, polyclonal antibodies, a predetermined blend of monoclonal antibodies, and/or a mixture of polyclonal and monoclonal antibodies. The solid phase is mixed with a sample of the solution, and the analyte of interest binds quantitatively to the solid phase. Such quantitative capture or harvesting can be essentially complete (e.g. greater than 90% or more of the available analyte in the sample) capture of the analyte of interest, or can be a consistent portion or fraction of the available analyte of interest in the sample (e.g. greater than 40%, 50%, 60%, 70%, 80%, 85% or more of the available analyte in the sample). In some embodiments the consistent portion of fraction that is captured is sufficiently reproducible to permit accurate quantitation (e.g. providing a CV of less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%). The solid phase is collected, and optionally washed. The analyte of interest is then eluted in a quantitative fashion from the solid phase using an elution buffer that disrupts the antigen: antibody complex while retaining the biological activity of the analyte of interest. Such quantitative elution can be essentially complete (i.e. greater than 90%) release of the harvested or captured analyte of interest, or can be a consistent portion or fraction of the harvested or captured analyte of interest in the sample (e.g. greater than 40%, 50%, 60%, 70%, 80%, 85% or more of the harvested or captured analyte). In some embodiments the consistent portion of fraction that is eluted is sufficiently reproducible to permit accurate quantitation (e.g. providing a CV of less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%). Such an elution buffer can be non-denaturing and/or non-cytotoxic. The elution buffer or a sample thereof can then be characterized using a cell-based assay. In some embodiments the analyte of interest can be collected from a large volume of sample solution and released using a small volume of elution buffer, thereby providing a concentration effect.

[0026] Surprisingly, Inventors have found that such an affinity solid phase can have sufficient affinity to provide quantitative harvesting of an analyte from solution at low (i.e. less than 1 nM) concentrations and also release analyte so harvested quantitatively under relatively mild conditions that retain the analyte' s structure and/or function. Typically capture of low

concentrations of analyte on a high affinity antibody-derived solid phase (e.g. a coated microwell plate) is a feature in stepwise enzyme immunoassays, however subsequent analysis occurs without release of the analyte from the solid phase. Conventional practice holds that such elution requires harsh conditions that are not compatible with subsequent activity of the analyte. While affinity media can be used in a preparative fashion for certain analytes, such affinity media utilize high specificity, low-affinity antibodies with (consequently) relatively high concentrations of the molecule to be purified. Harvesting and recovery from such processes are notoriously inefficient, and as such they are not suitable for quantitative work.

[0027] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain

embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are

approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0028] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0029] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0030] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the

specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0031] One should appreciate that the disclosed techniques provide many advantageous technical effects including characterization of compounds from cytotoxic samples using a cell-based assay and detection of low concentrations of such compounds by concentration.

[0032] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0033] In one embodiment of the inventive concept, a compound that is to be characterized using a cell-based assay is present in a solution that includes a cytotoxic contaminant. Within the context of this application the term "cytotoxic" refers to a substance or substances in

combination that result in the death, injury, or a loss or reduction of function that impairs assay performance in cells utilized in a cell based assay. Examples of cytotoxic contaminants includes surfactants, antibiotics, antifungal compounds, biological contaminants (such as bacteria, viruses, and/or fungi), metabolic poisons, cellular waste products, etc. In methods of the prior art, attempts to characterize an analyte of interest in such a solution would not be possible due to the presence of the cytotoxic substance. It should be appreciated that in many instances (for example, low CMC surfactants, proteins, soluble organic polymers) the cytotoxic substances cannot be removed from the sample by conventional means without significant or total loss of the analyte of interest. This is particularly true when the analyte of interest is present in low (i.e. sub nanomolar) concentrations).

[0034] The Inventors have found that excipients provided for improved stability and/or retention of activity during processing and storage of pharmaceutical compounds can have a significant cytotoxic effect on cells of cell-based assays utilized in characterizing the processed

pharmaceutical. Typical excipients used include albumin, human serum albumin, recombinant human serum albumin, gelatin, sucrose, trehalose, hydroxyethyl starch, dextrans, cyclodextrins, collagen, lactose, sodium chloride, polysaccharides, caprylic acid and caprylate salts,

polyvinylpyrrolidone, reducing agents, ionic surfactants, and non-ionic surfactants. While such excipients can serve to reduce oxidation, aggregation, and other phenomena that destabilize pharmaceuticals during processing and storage, they can be present in amounts that prove cytotoxic when pharmaceuticals so prepared are subjected to analysis. It should be appreciated that many of these excipients (particularly low-CMC non-ionic surfactants) can be extremely difficult to remove to concentrations that are compatible with cell-based assays, despite the availability of commercial resins specifically designed for their removal. This problem is compounded when the pharmaceutical to be characterized is present at low concentrations.

[0035] In methods of the inventive concept the analyte of interest can be collected or harvested from such cytotoxic solutions using an analyte-specific antibody coupled to a solid phase. Such analyte- specific antibodies can be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single-chain antibodies, and/or antibody fragments (e.g. F(ab), F(ab'), and/or F(ab)₂ fragments). In some embodiments the antibody utilized is a monoclonal antibody. In other embodiments a population of antibodies can be used. For example, in some embodiments the antibodies utilized can be controlled mixtures of two or more isolated monoclonal antibodies. In other embodiments the antibodies used can be a polyclonal population of antibodies derived from immunization of an animal. In still other embodiments the antibodies used can be a mixture of monoclonal and polyclonal antibodies. In still other embodiments, non-antibody binding species can be utilized with the solid phase. Examples of suitable species include dendrimers, receptors, and small molecule (i.e. having a molecular weight of less than 5,000Da) ligands.

[0036] Binding characteristics of such antibodies or other analyte-binding species are selected to provide efficient, quantitative harvesting of the analyte from the contaminated solution while providing for efficient, quantitative release. Suitable binding constants can range from 10^~6 to 10^~15 to provide efficient, quantitative harvesting. Such antibodies or other analyte-binding species can be selected by screening for efficient and quantitative release under relatively mild elution conditions that preserve analyte activity necessary for a subsequent cell-based assay. Such elution conditions can include low pH (e.g. pH 2 to 6), increased or decreased ionic strength, use of chaotropic agents (e.g. urea, guanidinium) at concentrations compatible with retention of analyte activity, and use of a competing analog of the analyte that lacks activity in the cell-based assay. In some embodiments the analyte can be released by cleavage of the antibody or other analyte-binding species, for example through the use of a reducing agent, protease, and/or nuclease.

[0037] As noted above, the antibodies utilized in methods of the inventive concept are coupled to a solid phase. This permits separation from the cytotoxic solution following harvesting of the analyte of interest through the formation of antigen: antibody complexes. Suitable solid phases include microparticles, magnetically responsive microparticles, chromatography media, membranes, hollow fibers, and polymer surfaces (for example, wells of microwell plates, walls of polymeric test tubes, etc.). For example, antibodies can be coupled to magnetically responsive microparticles for use in methods of the inventive concept. Such antibody coated microparticles can be conveniently recovered from suspension in a cytotoxic solution using a magnetic field. In another example, antibodies coupled to chromatography media can be recovered from

suspension in a cytotoxic solution by settling or centrifugation. Alternatively such antibody coupled chromatography media can be held within a column or pipette tip, permitting the cytotoxic solution to be flowed over the fixed chromatography media. Antibodies can be coupled to the solid phase by any suitable means. Such coupling methods can include nonspecific binding, direct chemical coupling (for example, via tosyl, triazine, epoxide, N- hydroxysuccinimide, N-ethylmaleimide, hydrazide, aldehyde, and/or cyanogen bromide coupling chemistries), or indirect coupling (for example, via coupling of biotinylated antibodies to avidin or streptavidin coupled to the solid phase, via coupling of avidin or streptavidin conjugated antibodies to a biotin or biotin-analog derivatized solid phase, via interaction with a protein A conjugated solid phase, via interaction with a protein G conjugated solid phase, via interaction with an anti-species antibody conjugated solid phase, etc.).

[0038] Inventors have found that selection of the solid phase can impact the degree of harvesting and release of analyte from solution. Without wishing to be bound by theory, Inventors believe that local interactions with the base material of the solid phase, antibody presentation, and/or antibody density can impact efficient and quantitative capture and efficient and quantitative release of the analyte. As such, one or more of solid phase composition, coupling chemistry, and/or antibody coupling density can be optimized to provide a suitable solid phase. Such suitable solid phases can be identified by incubation with analyte provided at one or more concentrations in a suitable solution (e.g. one containing cytotoxic excipients), release of the analyte using a suitable mild (i.e. one that retains analyte activity under eluting conditions), and characterization of the released analyte (e.g. using a cell-based assay).

[0039] In methods of the inventive concept, such an antibody conjugated solid phase is brought into contact with a cytotoxic solution containing the analyte of interest. The analyte of interest is subsequently harvested from the solution through formation of an antigen: antibody complex with the conjugated antibody. The antibody conjugated solid phase, carrying the harvested analyte of interest, is then separated from the cytotoxic solution. The method by which this separation is carried out is dependent on the nature of the solid phase, as noted above. Following separation of the antibody conjugated solid phase can, in some embodiments, be rinsed or washed one or more times by contact with a wash buffer (for example, phosphate buffered saline, tris buffered saline, cell culture media, etc.). Such a wash buffer can be selected to maintain the

antigen: antibody complex.

[0040] The analyte of interest can be released from the antibody conjugated solid phase by exposure to an elution buffer. Such an elution buffer is selected to disrupt the antigen: antibody complex without irreversibly denaturing or otherwise damaging the analyte of interest. This disruption can be accomplished using non-neutral pH (i.e. mildly acidic or mildly basic pH), elevated ionic strength, reduced ionic strength, protein destabilizing compounds (such as a chaotrope), or a combination of these. In some embodiments, an analyte of interest can be displaced from an antigen: antibody complex using an analog of the analyte of interest that is not active in a cell based assay. Such eluting conditions and/or species should be selected to have minimal to no negative impact on the cells of a cell based assay under normal assay conditions (sample dilution, etc.), and to retain the activity of the eluted analyte of interest. In some embodiments the eluting condition of the elution buffer can be modified following release of the analyte of interest from the solid phase. For example, a mildly basic or mildly acidic elution buffer can be neutralized following release of the analyte of interest and separation from the antibody conjugated solid phase. After the analyte of interest has been recovered the solid phase can be removed. In some embodiments of the inventive concept the antibody conjugate solid phase is discarded after use. In other embodiments the antibody conjugated solid phase can be re-used. [0041] In some embodiments of the inventive concept the analyte of interest can be harvested from a relatively large volume of cytotoxic solid phase and eluted using a relatively small volume of elution buffer to provide a concentration effect that has utility in detecting low concentrations of the analyte of interest. For example an analyte of interest can be harvested from a 10 mL volume of cytotoxic sample and, recovered in a ΙΟΟμί volume of elution buffer, leading to a 100-fold increase in the concentration of the analyte of interest in the elution buffer relative to the cytotoxic solution (assuming complete recovery from the solid phase). This can advantageously permit quantitation of an analyte of interest that is present at concentrations in the cytotoxic sample that are normally below the limit of detection of a cell based assay. Such concentration can also permit dilution of non-analyte compounds to acceptable, non-cytotoxic levels prior to testing. In some embodiments of the inventive concept, this concentration effect can be utilized with sample(s) that do not contain cytotoxic components, thereby providing a method that extends the measuring range of a cell based assay by effectively improving sensitivity and/or dynamic range of the cell based assay.

[0042] As noted above, following release from the antibody conjugated solid phase the analyte of interest can be characterized using a cell based assay. It should be appreciated that such assays have distinctive operating requirements, including retention of full biological function of the analyte of interest. For example, while a denatured protein antigen is likely to be at least partially recognized in a conventional immunoassay such a denatured antigen is highly unlikely to be correctly identified by cell surface receptors, internalized by normal processes, undergo normal processing, and demonstrate normal activity within a living cell. Such cell based assays can utilize observation of cell growth, morphology, and/or behavior (for example, reaction to a defined stimulus) in order to indicate the presence of the eluted analyte of interest following addition to a cell culture. Such responses can be quantified and compared to a standard curve of known compound concentrations in order to derive a concentration of the analyte of interest in the sample solution (with appropriate correction for dilution or concentration during processing). In such embodiments the cells can be modified (for example, through the use of expression vectors) to include reporting constructs that respond to the presence of the analyte of interest. For example, a reporting construct can be expressed within a cell used in cell based assay that is designed to show changes in fluorescence emission or luminescence on exposure to an analyte of interest. In other embodiments, the cells can be exposed to the eluted analyte of interest for a characteristic period of time, then lysed and their contents analyzed for cellular products known to be affected by the presence of the compound. Examples of such cellular products include mRNA, proteins, peptides, glycoproteins, lipids, carbohydrates, hormones, and metabolic products. For example, characteristic proteins or protein fragments generated within the cell in response to an analyte of interest can be identified by Western blotting or other mass-based techniques. In some embodiments such cellular products can be quantified and the results compared to the amounts produces by cells exposed to a set of concentration standards produced using the analyte of interest in order to provide quantitation of the analyte of interest.

[0043] It should be appreciated that the compositions and methods described above can be applied to a wide variety of analytes. Suitable analytes include drugs, hormones, metabolites, nucleic acids, peptides, proteins, lipids, and carbohydrates. In some embodiments the analyte is a protein or peptide that is present at low concentrations (i.e. less than 1 nM) in a solution or suspension that includes a substance that interferes with a cell based assay for the analyte. In some embodiments the interfering substance can be a cytotoxic substance, for example a surfactant. In a preferred embodiment the interfering substance is a low CMC surfactant, which is not effectively removed (i.e. reduced to non-interfering concentrations) by dialysis, ultrafiltration, size exclusion chromatography, and/or treatment with commercial surfactant removing solid phases. In a preferred embodiment the analyte is a pharmaceutical protein or peptide product, for example a Clostridial neurotoxin such as a botulinum neurotoxin or a derivative thereof.

[0044] Botulinum neurotoxin serotype A and other pharmaceutical proteins are often provided in a form that includes excipients that interfere with cell based assays that can be used to quantify their potency. Typical excipients include polysorbate surfactants, such as polysorbate 20™ and/or polysorbate 80™. Such surfactants can have very low CMCs that make removal by prior art methods to levels that are not cytotoxic and/or that do not interfere with cell based assays impractical, particularly for protein analytes that are present at low concentrations.

[0045] An example of a cell based assay for a botulinum toxin is shown schematically in FIG. 1. Cells used in the assay (for example, cells from an adherent murine neuroblastoma cell line) are genetically modified to express a detecting construct that exhibits fluorescence. Exposure of the cells to an appropriate botulinum neurotoxin (for example botulinum neurotoxin serotype A, or BoNT/A) results in receptor mediated uptake of intact and active BoNT/A, followed by intracellular processing that results in the generation of a highly specific proteolytic activity. This proteolytic activity results in cleavage of the detecting construct and release of a fluorescent moiety into the cytoplasm, where degradation results in a loss of observed fluorescence that is proportional to the initial concentration of BoNT/A in the sample being tested.

[0046] In many instances protein/peptide pharmaceuticals, such as BoNT/A, are provided mixed with excipients that improve stability, but can be cytotoxic. Some excipients can be removed relatively easily by processes such as dialysis or gel filtration. Such processes can be

problematic, however, when the analyte is present at low concentrations as it may be lost due to nonspecific binding to the membrane to gel filtration media. Other excipients are not readily removed from solution. Such excipients include surfactants, particularly surfactants with low critical micellar concentrations (such as a polysorbate 80).

[0047] In studies performed using the above described cell-based assay, polysorbate 80 provided in concentrations as low as 0.1% in the sample material was found to strongly interfere with detection of BoNT/A. This interference was correlated with changes in cellular morphology, indicating cytotoxic activity. This interference and the associated changes in cellular

morphology persisted despite pre-treatment of the sample by conventional methods for surfactant removal, including dialysis, gel filtration (for example, using HiPPR Detergent Removal Resin™, obtained from Thermo Fisher), and treatment with a commercial chromatography media commonly used for removal of surfactants (DetergentOUT Tween™, obtained from G- Biosciences). Attempts to eliminate surfactant from the material to be tested by capture of the BoNT/A using anion exchange media (for example, Q Sepharose™) followed by elution of the analyte resulted in irretrievable loss of active BoNT/A.

[0048] Inventors have surprisingly found that analytes (such as BoNT/A) present in cytotoxic solutions can be selectively bound to an antibody conjugated to a solid phase and eluted in an active form to provide a solution that is substantially free of cytotoxic compounds (i.e. at least reduced in concentration to an extent that cytotoxic activity and/or cell based assay interference is no longer evident) and suitable for use in cell based assays. For example, Matrix A™ (from BioSentinel) is a preparation of magnetic microparticles conjugated with antibodies directed to BoNT/A. Other magnetically responsive microparticles, for example Dynabeads™ (obtained from Thermo Fisher) were also found to provide a suitable solid phase. Such solid phase antibody preparations can be readily incorporated into a testing protocol for use with BoNT/A containing samples that include cytotoxic compounds, as shown in FIGs. 2, 3 A, and 3B.

[0049] FIG. 2 provides a schematic depiction of an example of a method of the inventive concept. A solid phase having coupled antibody (130) specific for an analyte of interest (110) is brought into contact with a solution that contains the analyte (110) and one or more cytotoxic contaminants (120). Analyte (110) present in the solution forms an antigen: antibody complex with the solid phase (130), whereas unwanted contaminants (120) do not. Such capture can be essentially complete (i.e. capturing greater than or equal to about 90% of the available analyte) in some embodiments, for example when the amount of solid phase (130) used represents an excess of binding capacity over the amount of analyte (110) present. In other embodiments a portion of the analyte (110) is captured on the solid phase (130), that portion being sufficiently consistent to provide accurate quantitation. Following capture of the analyte (110) the solid phase (130) can be isolated and washed (140), for example by rinsing and/or resuspending with a suitable buffer (e.g. PBS, TBS, cell culture media, etc.). The solid phase can be captured by any suitable means, including settling and decantation, centrifugation, filtration, size exclusion chromatography, and/or exposure to a magnetic field. If necessary such wash steps (140) can be repeated. In some embodiment a wash step (140) is not necessary, and isolation of the solid phase followed by resuspension is sufficient to reduce the concentration of cytotoxic contaminants to acceptable levels. Subsequently, analyte (110) can be eluted (150) from the solid phase (130), for example by exposure to an elution buffer. The elution buffer provides quantitative release of the analyte (110). Such quantitative release can be essentially complete (i.e. greater than or equal to 90%) release of the bound analyte (110) or partial but sufficiently consistent to provide quantitative results on subsequent testing.

[0050] FIGs. 3A and 3B provide a schematic depiction of an exemplary cell based assay suitable for use in a method of the inventive concept. The upper portion of FIG. 3 A schematically depicts a cell (210) that includes a reporting construct (215). The reporting construct (215) is shown in greater detail in the lower portion of FIG. 3A. The reporting construct (215) includes a reporting tag (220) (in this instance a fluorophore that is excited at 505 nm and emits at 527 nm) and a region (230) that is sensitive to the analyte (110). As shown, the reporting construct can bind to cell membrane (depicted as a lipid bilayer), and in some embodiments can include a secondary tag (240). In some embodiments the secondary tag can be utilized for data

normalization (e.g. to expression level of the reporting construct and/or cell density in a test well). In other embodiments the secondary tag (240) can interact with the reporting tag (220) to provide a signal (e.g. FRET) or lack of signal (e.g. fluorescence quenching) that indicates the presence of an intact reporting construct (215).

[0051] FIG. 3B schematically depicts events on exposure to an analyte (110) prepared as in FIG. 2. Events at the cellular level are show in the upper portion of FIG. 3B. Events at the reporting construct level are shown in the lower portion of FIG. 3B. Contact of the analyte (110) with the cell (210) results in internalization of the analyte (for example, through interaction with specific cell surface receptors) into a vesicle (250). Following internalization the analyte or a portion of the analyte is translocated from the vesicle (250) and can access the reporting construct (215). Interaction with the reporting construct results in cleavage of the sensitive region (230) and subsequent release of the reporting tag (220). In some embodiments this release can be assessed by observation of a loss of interaction (e.g. FRET) between the secondary tag (240) and the reporting tag (220). In other embodiments this release can be assessed by observing a loss of signal from the reporting tag (220) due to intracellular degradative events (260). For example, if the reporting tag (220) is selected to be green fluorescent protein peptide or mutant green fluorescent protein peptide that is susceptible to intracellular degradation a loss of fluorescence corresponding to the peptide will be observed in the presence of the analyte.

[0052] It should be appreciated that in such cell based assays the presence of cytotoxic compounds in a test sample can lead to loss of the reporting construct, which in turn would provide an incorrect result. Utilization of a solid phase that provides capture and release of the analyte in an active form and in a quantitative fashion advantageously permits the use of cell based assays in characterization of such samples, which otherwise would require live animal testing. [0053] In another embodiment of the inventive concept a solid phase having a coupled antibody specific for an analyte of interest (for example, element 130 of FIG. 2) can be exposed to a sample of an analyte-containing solution to be analyzed, and subsequently isolated from the sample (for example by decantation, centrifugation, filtration, etc.). Such a solid phase, now loaded with harvested analyte, can be divided into two or more portions. One such portion can be treated as described above to elute the bound analyte and characterize it using a cell-based assay. Another portion of the solid phase can be utilized in a separation-based immunoassay, for example by exposing the portion of the solid phase to a second analyte- specific antibody in solution and allowing it to bind to unoccupied sites on the analyte captured by the solid phase. Binding of such a second analyte- specific antibody to the solid phase can be detected by washing to remove excess second analyte- specific antibody and either detection of a label (e.g. a fluorescent, enzymatic, luminescent, and/or phosphorescent moiety) coupled to the second analyte specific antibody or to a species-specific antibody selective for the second analyte- specific antibody. Such embodiments advantageously permit both mass detection (e.g. the immunoassay result) and activity detection (e.g. the cell-based assay result) from the same sample and from common preliminary steps, permitting a user to gain information regarding both mass and activity yield in a highly efficient manner that is less subject to variation than if performed as independent processes.

Examples

[0054] Effects of excipients included in a commercial BoNT/A pharmaceutical preparation (BoNT/A comm.) on results obtained from a cell-based assay can be seen in FIG. 4, which shows normalized fluorescence data from both a BoNT/A holotoxin in an excipient-free buffer and a commercial BoNT/A pharmaceutical in an excipients -containing solution in a cell based assay as shown in FIGs. 3 A and 3B. Serial dilutions of stock solutions of the BoNT/A preparations having similar BoNT/A activity were prepared in corresponding buffers. As shown, BoNT/A holotoxin provided in an excipient-free solution provides a typical sigmoidal dose response curve, indicating an EC50 of 0.99 pM for the BoNT/A preparation. The

pharmaceutical preparation, however, provides no useful results from the same cell based assay, despite having significant proven activity in live animal studies. Photomicrographs of cells exposed to the commercial BoNT/A pharmaceutical preparation showed significant morphological changes relative to those exposed to BoNT/A provided in an excipient-free solution.

[0055] FIG. 5 shows typical results of attempts to remove excipients from the commercial BoNT/A pharmaceutical preparation by exhaustive dialysis. Studies were performed as those shown in FIG. 4, with the exception of dialysis of the commercial BoNT/A pharmaceutical preparation against the excipients -free buffer and subsequent serial dilution in same. Retention of BoNT/A in the dialyzed sample was verified using an in vitro enzyme immunoassay, which showed a mass concentration of BoNT/A of 5.9 pM for the dialyzed commercial BoNT/A pharmaceutical preparation and 4.7 pM for the excipient-free BoNT/A holotoxin preparation. As shown dialysis does not give a useful result, as considerable inhibition of the cell based assay is still evident. Photomicrographs of cells exposed to the dialyzed commercial BoNT/A

pharmaceutical preparation also showed significant morphological changes relative to those exposed to BoNT/A provided in an excipient-free solution.

[0056] Polysorbate 80 (P80) and/or human serum albumin (HSA) are typical excipients used in pharmaceutical preparation, and various commercial resins are available that are purported to remove or significantly reduce the concentrations of these substances. In initial studies, BoNT/A holotoxin was prepared at about 10 pM (similar to concentrations provided in pharmaceutical preparations) in BAM2 culture media supplemented with 0.1% (w/v) P80 or with 0.1% (w/v) P80 and 0.5% (w/v) HSA. Samples were treated with various commercial resins commonly utilized for removal of surfactants and/or proteins, including HiPPR™ Detergent Removal Resin (Thermo Fisher), Q Sepharose™ (GE Healthcare), and DetergentOUT™ Tween (G-Biosciences) per manufacturer's directions. HiPPR™ Detergent Removal Resin was washed with either BAM2 media or BAM2 media supplemented with 0.5% (w/v) HSA prior to exposure to the BoNT/A samples. Results from an in vitro immunoassay are shown in Table 1.

BAM2 + 0.1% P80 + Treated BAM2 + 0.5% HSA 11.7

0.5% HSA

BAM2 + 0.1% P80 Untreated N/A 12.8

BAM2 + 0.1% P80 + Untreated N/A 12.1

0.5% HSA

BAM2 + 0.1% P80 + Untreated N/A 11.6

0.5% HSA

BAM2 Untreated N/A 12.1

Table 1

As shown, treatment with HiPPR™ Detergent Removal Resin had no apparent effect on the recovery of BoNT/A from any of the excipients mixtures tested when characterized by an in vitro immunoassay.

[0057] FIGs. 6A, 6B, and 6C show typical results from studies of HiPPR™ Detergent Removal Resin treatment of samples of BoNT/A in BAM2 media supplemented with 0.1% (w/v) P80, 0.5% (w/v) HSA, and/or 140 mM NaCl using a cell-based assay as shown in FIGs. 3 A and 3B. In these figures samples labeled "column" have been treated with HiPPR™ Detergent Removal Resin per the manufacturer's directions. FIG. 6A shows results from treated and untreated BoNT/A samples containing HSA and P80 excipients. FIG. 6B shows results from treated and untreated BoNT/A samples containing HSA, P80, and NaCl excipients. FIG. 6C compares results of a BoNT/A sample prepared with supplemental HSA to a BoNT/A sample containing HSA and P80 excipients after treatment with HiPPR™ Detergent Removal Resin. It is evident that this resin fails to remove P80 from such samples to an extent that is effective in reducing interference with a cell based assay.

[0058] Table 2 shows the results of attempts to remove excipients from a BoNT/A containing solution using Q Sepharose™ (GE Healthcare), a strong anion exchange resin. As described above, BoNT/A holotoxin was prepared at about 10 pM (similar to concentrations provided in pharmaceutical preparations) in BAM2 culture media supplemented with 0.1% (w/v) P80 or with 0.1% (w/v) P80 and 0.5% (w/v) HSA. Samples were treated with Q Sepharose per the manufacturer's instructions. Following exposure of the ion exchange resin to the BoNT samples the resin was washed with 25 mM phosphate + 50 mM NaCl buffer, pH 8.0, then eluted with either 25 mM phosphate, pH 4, or 25 mM phosphate + 200 mM NaCl, pH 8. The resulting eluates were tested for BoNT/A content using an immunoassay that is not sensitive to the excipients tested.

Table 2

Attempts to further optimize the use of Q Sepharose™ (e.g. resin volume, pre-incubation of the resin with HSA, etc.) failed to improve the observed low recovery of BoNT/A from the ion exchanger.

[0059] Table 3 shows the results of attempts to remove excipients from a BoNT/A containing solution using DetergentOUT™ Tween, a surfactant removal resin. As described above, BoNT/A holotoxin was prepared at about 10 pM (similar to concentrations provided in pharmaceutical preparations) in BAM2 culture media supplemented with 0.1% (w/v) P80 or with 0.1% (w/v) P80 and 0.5% (w/v) HSA. Samples of the BoNT preparations were incubated with the resin in accordance with the manufacturer's instructions, and the solutions collected after 15 minutes at ambient temperature. BoNT/A content of these solutions and control solutions not exposed to DetergentOUT™ Tween was measured using an immunoassay that is not sensitive to the excipients tested.

Table 3

As shown, mass recovery of BoNT/A from the various test matrices was unaffected by

DetergentOUT™ Tween treatment.

[0060] FIGs. 7A, 7B, and 7C show typical results from studies of DetergentOUT™ Tween treatment of samples of BoNT/A in BAM2 media supplemented with 0.1% (w/v) P80, 0.5% (w/v) HSA, and/or 140 mM NaCl using a cell-based assay as shown in FIGs. 3 A and 3B. In these figures samples labeled "column" have been treated with HiPPR™ Detergent Removal Resin per the manufacturer's directions. FIG. 7A shows results from treated and untreated BoNT/A samples containing HSA and P80 excipients. FIG. 7B shows results from treated and untreated BoNT/A samples containing HSA, P80, and NaCl excipients. FIG. 7C compares results of a BoNT/A sample prepared with supplemental HSA to a BoNT/A sample containing HSA and P80 excipients after treatment with DetergentOUT™ Tween. It is evident that this resin fails to remove P80 from such samples to an extent that is effective in reducing interference with a cell based assay. [0061] In light of the failure of traditional methods to provide adequate removal of common excipients for characterization using cell based assays, Inventors surprisingly found that affinity media (e.g. antibody coated particles or beads) can both bind an analyte from a solution containing solution in a quantitative fashion and release the bound analyte in a quantitative fashion and in a functional, nondenatured form useful in cell based assays. It should be recognized that quantitative binding (i.e. binding of essentially all or a highly reproducible majority) of an analyte present in a solution at low concentrations (i.e. 10^~9 M or less) is generally recognized as a function of high affinity binding, which in turn has generally been thought to require harsh conditions in order to provide quantitative recovery of the bound species. Such harsh conditions would, in turn, be expected to be denaturing and therefore not compatible with subsequent analysis using a cell based assay.

[0062] In some embodiments of the inventive concept the affinity media can be magnetically responsive beads that are covalently coated with antibodies (e.g. monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single chain antibodies, and/or antibody fragments) developed against BoNT/A. Typical results of excipients removal (i.e. BoNT/A quantitative capture and release) for four different antibody-coated beads are shown in FIG. 8A. In these studies BoNT/A was provided in 0.7 mL of BAM2 media containing 0.1% (w/v) P80 and 0.5% (w/v) HSA. Following incubation with the antibody coated bead preparation, the antibody coated beads were eluted with a small volume of 50 mM glycine + 200 mM NaCl, pH 3.2 and brought back to the original sample volume with BAM2 media. The eluted and volume adjusted samples were then characterized using a cell based assay as described in FIGs. 3A and 3B. Antibody coated bead preparation included an IgY antibody coated particle used in a commercial BoNT/A immunoassay (Bead 1), Dynabeads™ (ThermoFisher) coupled to the same BoNT/A specific IgY (Bead 2), Dynabeads™ (ThermoFisher) coupled to a BoNT/A murine IgG typically used as an immunoassay detection antibody (Bead 3), and Dynabeads™

(ThermoFisher) coupled to a BoNT/A specific murine IgG typically used as an immunoassay capture antibody (Bead 4). As shown, detectable BoNT/A was eluted from all Dynabead™ preparations.

[0063] FIGs. 8B, 8C, and 8D show results from similar studies performed using 4mL of 10 pM BoNT/A preparations with dilution of the eluted fraction to 0.7 mL using BAM2 media. FIG. 8B shows results with Dynabeads™ (ThermoFisher) coupled to the same BoNT/A specific IgY (Bead 2). FIG. 8C shows results with Dynabeads™ (ThermoFisher) coupled to a BoNT/A murine IgG typically used as an immunoassay detection antibody (Bead 3). FIG. 8D shows results with Dynabeads™ (ThermoFisher) coupled to a BoNT/A specific murine IgG typically used as an immunoassay capture antibody (Bead 4). As shown, all of the antibody coated bead preparations show typical sigmoidal dose/response curves with no discernible negative effect from residual excipients. Inventors believe that this is due, at least in part, to a concentration effect provided by highly efficient capture and release of the BoNT/A analyte in native form useful in a cell based assay.

[0064] Further characterization of the starting BoNT/A solution and the supernatant remaining following extraction with the antibody coated beads shows that approximately 70% of the BoNT/A in the starting solution is captured, with approximately 30% remaining in the supernatant following extraction. Similar studies performed using 2 mL, 7 mL, and 10 mL volumes of the starting BoNT/A solution have shown that approximately 93%, 78%, and 60% of the BoNT/A in the starting solution is captured, respectively, when the capture step is performed for an extended time (e.g. 16 hours or more) at reduced temperature (e.g. 2°C to 8°C). Typical results obtained from such 2 mL, 7 mL, and 10 mL starting volumes are shown in FIGs. 9 A, 9B, and 9C respectively. In all cases the use of beads coated with analyte-specific antibody permitted quantitative capture and elution of the analyte and elimination of excipient effects.

[0065] Additional studies show that the removal of excipients using compositions and methods of the inventive concept is effective across different cell based assays. Results of excipient removal from a BoNT/A preparation containing P80, HSA, and NaCl excipients and subsequent characterization using a cell based assay employing a different cell line and reporting construct from the cell line utilized in FIGs. 7A, 7B, 7C, 8A, 8B, 8C, 8D, 9A, 9B, and 9C are shown in FIG. 10. Results are not shown as a fluorescence ratio due to the presence of only a single fluorophore species in this reporting construct. As shown, interference from excipients present in the pre-treatment sample is apparent and is completely resolved in the post-treatment eluted BoNT/A sample provided by methods and compositions of the inventive concept. [0066] While a number of different solid phase antibody preparations are suitable for use in methods of the inventive concept, it should be appreciated that magnetically responsive microparticles (i.e. magnetic microparticles, paramagnetic microparticles, diamagnetic microparticles, etc.) provide rapid binding and elution kinetics and are readily adaptable to automation. The rapid kinetics permitted by use of such microparticle preparations can substantially reduce the time required for assay performance (for example, relative to dialysis and/or chromatographic steps), which can be advantageous for labile species of analyte.

[0067] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMS What is claimed is:

1. A method for reducing interference effects in a cell based assay, comprising:

providing an analyte specific affinity partner coupled to an insoluble phase;

contacting a sample comprising an analyte and an interfering substance with the insoluble phase, thereby quantitatively forming an analytednsoluble phase affinity partner complex;

recovering the insoluble phase from the sample;

quantitatively dissociating the analytednsoluble phase affinity partner complex to provide a purified analyte; and

contacting the purified analyte with a cell used in the cell based assay.

2. The method of claim 1, wherein the interfering substance is a cytotoxic substance.

3. The method of claim 2, wherein the interfering substance is a surfactant.

4. The method of one of claims 1 to 3, wherein the insoluble phase is a solid phase.

5. The method of one of claims 1 to 4, further comprising the step of separating the insoluble phase from the purified analyte.

6. The method of one of claims 1 to 5, wherein the step of dissociating the analytednsoluble phase affinity partner complex comprises contacting the analytednsoluble phase affinity partner complex with an elution buffer.

7. The method of claim 6, wherein the elution buffer has a pH between 3 and 9.

8. The method of one of claims 6 and 7, wherein the elution buffer has an ionic strength of 50mM to 500mM.

9. The method of one of claims 6 to 8, wherein the elution buffer comprises an analog of the analyte.

10. The method of one of claims 1 to 9, wherein the sample is provided in a first volume and the purified analyte is recovered in a second volume, and wherein the second volume is less than the first volume.

11. The method of claim 10, wherein the second volume is less than or equal to 20% of the first volume.

12. The method of claim 10 or 11, wherein the second volume is less than or equal to 5% of the first volume.

12. The method of one of claims 1 to 11, wherein the analyte is a botulinum neurotoxin.

13. The method of one of claims 1 to 12, wherein the affinity partner is selected from the group consisting of an antibody, a recombinant antibody, a single chain antibody, an antibody fragment, an ap tamer, a receptor, and a ligand.

14. The method of one of claims 1 to 13, wherein the insoluble phase comprises particles.

15. The method of claim 14, wherein the particles are magnetically responsive.

16. The method of one of claims 1 to 15, comprising the additional steps of:

following recovery of the insoluble phase, separating the insoluble phase into a first portion and a second portion;

submitting the first portion of the insoluble phase to the dissociation step;

contacting the second portion of the insoluble phase with a soluble second affinity

partner; and

detecting a signal related to the formation of a second complex comprising the

analytednsoluble phase affinity partner complex and the second affinity partner, wherein the signal is proportional to mass of analyte present in the sample.

17. The method of claim 16, wherein the signal is selected from the group consisting of enzyme activity, fluorescence, luminescence, and phosphorescence.