(12) Patent Application Publication (10) Pub. No.: US 2015/0293.086 A1 MESSMER Et Al

Home , Motavizumab, Nebacumab

US 20150293O86A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0293.086 A1 MESSMER et al. (43) Pub. Date: Oct. 15, 2015

(54) LATERAL FLOW IMMUNOASSAY Publication Classification (71) Applicant: ABREOS BIOSCIENCES, INC., San (51) Int. Cl. Diego, CA (US) GOIN33/543 (2006.01) GOIN33/68 (2006.01) (72) Inventors: Bradley Todd MESSMER, San Diego, GOIN33/94 (2006.01) CA (US); Peter HABERZ, San Diego, GOIN33/558 (2006.01) CA (US) (52) U.S. Cl. CPC ...... G0IN33/54386 (2013.01); G0IN33/558 (21) Appl. No.: 14/686,578 (2013.01); G0IN33/6854 (2013.01); G0IN 33/94 (2013.01) (22) Filed: Apr. 14, 2015 (57) ABSTRACT Disclosed is a test device for determining the presence or Related U.S. Application Data integrity of a biologic comprising a sample pad for receiving the biologic, a conjugate pad, and a test membrane compris (60) Provisional application No. 61/979,123, filed on Apr. ing at least one test line comprising an immobilized mime 14, 2014. tope, and methods of using the same.

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LATERAL FLOW IMMUNOASSAY bilized mimetope, (b) determining whether at least one test line undergoes a color change, (c) determining, based on the RELATED APPLICATIONS color change, the activity of the biologic. 0001. The present application claims the benefit of U.S. 0009 Disclosed herein is a kit comprising (a) a test device Provisional Application Ser. No. 61/979,123, filed on Apr. 14, for determining the integrity of a biologic, the test device 2014, by Bradley T. MESSMER et al. and entitled “LAT comprising a sample receiving pad, a conjugatepad, and a test ERAL FLOW IMMUNOASSAY, the entire disclosure of membrane, the test membrane further comprising at least one which is incorporated herein by reference, including the test line and at least one control line, and (b) instructions for drawings. use thereof. BRIEF DESCRIPTION OF THE DRAWINGS GOVERNMENT RIGHTS 0010 FIG. 1 illustrates one embodiment of the lateral flow 0002 The subject matter disclosed herein was made with assay disclosed herein. The top panel illustrates a schematic government support under grants 1R41CA192697-01 and of the prototype. A drop of the biologic sample is placed on a 1R43CA183241-01 awarded by the National Institutes of sample pad and is wicked through the device. After binding Health Small Business Innovation Research (NIH-SBIR). the secondary detection antibody, conjugated to gold, the The government has certain rights in the disclosed subject sample encounters the peptide, anti-light chain, and anti matter. secondary capture agents. The bottom panel illustrates pos sible outcome matrix for an antibody that is known to possess FIELD OF THE INVENTION a kappa light chain. 0003. The present invention is in the field of test devices, 0011 FIG. 2 (A) illustrates one embodiment of the lateral and more specifically in the field of lateral flow test devices. flow assay validating that rituximab binding mimetope pep tide function in immunoassays and competes with the native BACKGROUND OF THE DISCLOSURE antigen (CD20). FIG. 2 (A) is a standard curve for rituximab by peptide based ELISA. Biotinylated peptides are bound 0004 Biologics are valuable therapeutics for treating can onto neutravidin coated ELISA plates. Rituximab is diluted in cers and other diseases. As they are among the most expensive TBST. Each value shows the mean (S.D.) of triplicates. The pharmaceuticals produced and increasingly prescribed, there solid line indicates the mean of the buffer control and the have been a growing number of incidents involving counter dashed line represents the mean +10 times the SD of the feit biologics, such as counterfeit antibodies. These econom buffer control. ics create a considerable incentive for counterfeit or other (0012 FIG. 2 (B) illustrates peptide inhibition of Chronic deceptions. The problem of counterfeit medications is grow Lymphocytic Leukemia (CLL) cell staining. Fluorescently ing rapidly. The Center for Medicines in the Public Interest labeled rituximab is incubated with primary CLL cells and estimates that the worldwide trade for counterfeit medicines evaluated by flow cytometry (solid line). Weak staining for exceeds S75 billion. This number is S25 billion more than the CD20 was observed because CLL cells have low levels of illicit drug trade. Therefore, there is a concern that organized CD20. When peptide RTX-10 was added at a large molar crime will become more active in this area. excess (dashed lines), the cell labeling was largely abrogated. 0005. Furthermore, biologics are sensitive to environmen Control peptides had no effect (not shown). The shaded his tal conditions and improper storage or handling at any point in togram represents CLL cells incubated with fluorescently the Supply chain. Improper handling of a biologic. Such as an labeled normal human IgG. antibody, could reduce or inactivate their ability to bind the 0013 FIG.3 illustrates one embodiment of the lateral flow target antigen and mitigate their therapeutic effect. At present, immunoassay design and development path. The flow chart there are no routine safeguards in place at the time of admin istration to ensure that a patient is receiving the correct, active shows the design process for a lateral flow immunoassay. therapy. 0014 FIG. 4 illustrates one embodiment of mimetope LFA for bevacizumab, trastuzumab, and rituximab. The 0006 Thus, a need remains to develop simple, rapid sample comprising an antibody flows through a conjugatepad assays that Verifies both the presence and integrity of appro loaded with goat anti-human IgG attached to colloidal gold, priate antibody in the sample and that are amenable for use at then onto a membrane striped with the appropriate mimetope any point in the Supply chain, including bedside. Such an peptide, and finally an assay control. A false positive on one assay will provide patients and their physicians an extra anti-lambda test line is shown by an asterisk. degree of confidence that the correct, active therapeutic is (0015 FIG.5 illustrates that the lateral flow assay disclosed administered. herein has wide dynamic range. Samples tested were Ritux imab at 0.0 mg/ml (negative control), 0.01 mg/ml, 0.1 mg/ml. SUMMARY OF THE INVENTION 1.0 mg/ml, and 10 mg/ml (vial concentration). With increas 0007 Disclosed herein is a test device for determining the ing Rituximab concentration, the test line gets more intense. presence or integrity of a biologic comprising a sample pad 0016 FIG. 6 further illustrates that the lateral flow assay for receiving the biologic, a conjugate pad, and a test mem disclosed herein has a wide dynamic range. FIG. 6 shows one brane comprising at least one test line comprising an immo embodiment of the sandwich lateral flow assay with two test bilized mimetope. lines. The test lines comprise peptide mimetope striped at two 0008 Disclosed herein is a method of determining the different concentrations on the membrane. Samples tested are integrity of a biologic comprising (a) contacting the biologic Rituximab at 0.0 mg/ml (negative control), 0.01 mg/ml, 0.1 with a test device, wherein the test device comprises a sample mg/ml, 1.0 mg/ml, and 10 mg/ml (vial concentration). With pad for receiving the biologic, a conjugate pad, a test mem increasing Rituximab concentration, the lower test line gets brane comprising at least one test line comprising an immo less intense whereas the upper test line gets more intense. US 2015/029.3086 A1 Oct. 15, 2015

DETAILED DESCRIPTION OF THE (IgA1 and IgA2), Igl), IgE. IgM, and IgG (IgG1, IgG3 and EMBODIMENTS IgG4) etc. In some embodiments, the antibody is polyclonal 0017. The terminology used herein is for the purpose of or monoclonal. In some embodiments, the antibody is from describing particular embodiments only and is not intended to any origin, such as mouse or human, including a chimeric be limiting of the invention. As used herein, the singular antibody thereof. In some embodiments, the antibody is forms “a,” “an,” and “the' are intended to include the plural humanized. forms as well, unless the context clearly indicates otherwise. (0023 Examples of antibodies include, but are not limited Furthermore, to the extent that the terms “including.” “includes,” “having.” “has.” “with.” or variants thereof are to, bevacizumab, trastuzumab, rituximab, abciximab, adali used in either the detailed description and/or the claims, such mumab, alemtuzumab, basiliximab, belimumab, brentux terms are intended to be inclusive in a manner similar to the imab vedotin, canakinumab, cetuximab, certolizumab pegol, term “comprising.” daclizumab, denosumab, eculizumab, efalizumab, gemtu 0018. The term “about' or “approximately” means within Zumab, golimumab, ibritumomab tiuxetan, infliximab, ipili an acceptable error range for the particular value as deter mumab, muromonab-CD3, natalizumab. ofatumumab, oma mined by one of ordinary skill in the art, which will depend in lizumab, palivizumab, panitumumab, ranibizumab. part on how the value is measured or determined, i.e., the raxibacumab, tocilizumab, to situmomab and ustekinumab. limitations of the measurement system. For example, "about Other examples of antibodies include, but are not limited to, can mean within one or more than one standard deviation, per 3F8, abagovomab, abatacept, acz885, adecatumumab, afeli the practice in the art. Alternatively, “about can mean a range momab, aflibercept, afutuzumab, alacizumab, altumomab, of up to 20%, of a given value. Where particular values are anatumomab, anrukinzumab, apolizumab, arcitumomab, described in the application and claims, unless otherwise aselizumab, atlizumab, atorolimumab, bapineuZumab, bavi stated the term “about' meaning within an acceptable error tuximab, bectumomab, belatacept, bertilimumab, besileso range for the particular value should be assumed. mab, biciromab, bivatuzumab, blinatumomab, cantuzumab. 0019. Unless otherwise defined, all technical and scien capromab, catumaxomab, cedelizumab, citatuZumab, cixutu tific terms used herein have the same meaning as commonly mumab, clenoliximab, cinto 1275(-ustekinumab), cinto 148 understood by one of ordinary skill in the art to which this (golimumab), conatumumab, dacetuZumab, detumomab, invention belongs. dorlimomab, dorlixiZumab, ecromeximab, edobacomab, 0020. Unless otherwise stated, the following terms used in edrecolomab, efungumab, elsilimomab, enlimomab, epitu this application, including the specification and claims, have momab, epratuzumab, erlizumab, ertumaxomab, etanercept, definitions given below. etaracizumab, exbivirumab, fanolesomab, faralimomab, 0021. The terms “protein” as used herein refers to at least felvizumab, figitumumab, fontolizumab, foravirumab, galix one sequence of amino acids linked by sequential peptide imab, gantenerumab, gavilimomab, gomiliximab, ibali bonds, and is generally synonymous with "polypeptide. In Zumab, igovomab, imciromab, inolimomab, inotuZumab one embodiment, the amino acid sequence is one that occurs ozogamicin, iratumumab, keliximab, labetuZumab, lebrili in nature. In other embodiments, the amino acid sequence is Zumab, lemalesomab, lerdelimumab, lexatumumab, libiviru engineered by man. The term protein includes, but is not rnab, lintuzumab, lucatumumab, lumiliximab, mapatu limited to, proteins having pharmaceutical, diagnostic, agri mumab, maslimomab, matuZumab, mepolizumab, cultural, and/or any of a variety of other properties that are metelimumab, milatuzumab, minretumomab, mitumomab, useful in commercial, experimental, and/or other applica morolimumab, motavizumab, myo-029, nacolomab, naptu tions. In some embodiments, the protein is a protein thera momab, nebacumab, necitumumab, nerelimomab, nimotu peutic. The term “protein therapeutic' (or “therapeutic pro Zumab, nofetumomab, ocrelizumab, odulimomab, oportu tein') as used herein contemplates a protein that has a Zumab, oregovomab, otelixizumab, pagibaximab, biological effect in the body, or on a region in the body on panobacumab, pascolizumab, pemtumomab, pertuzumab, which it directly acts, or on a region of the body on which it pexelizumab, pintumomab, priliximab, pritumumab, pro remotely acts via intermediates, etc. Examples of therapeutic 140, rafivirumab, ramucirumab, regavirumab, reslizumab, proteins are, but is not limited to, pharmaceutically or com rilonacept, robatumumab, rovelizumab, roZrolimupab, rupli mercially relevant enzymes, receptors, receptor fusions, Zumab, satumomab, sevirumab, sibrotuzumab, siltuximab, soluble receptors, soluble receptor fusions, antibodies (e.g., siplizumab, solanezumab, Sonepcizumab, Sontuzumab, sta monoclonal and/or polyclonal antibodies), antigen-binding mulurnab, sulesomab, tacatuZumab, tadocizumab, tali fragments of an antibody, Fc fusion proteins, SMIPs5 cytok Zumab, tanezumab, tapliturnomab, tefibazumab, tellimomab, ines, hormones, regulatory factors, growth factors, coagula tenatumomab, teneliximab, teplizumab, tp.nl 412, ticili tion/clotting factors, and antigen-binding agents. The above mumab (=tremelimumab), tigatuzumab, tinx-355 (ibali list of proteins is merely exemplary in nature, and is not Zumab), tinx-650, tinx-901 (=talizumab), toralizumab, trem intended to be a limiting recitation. elimumab, tucotuzumab, tuvirumab, urtoxazumab. 0022. The term “antibody” as used herein contemplates a vapaliximab, vedolizumab. Veltuzumab. Vepalimomab, polypeptide or a protein complex that specifically binds an visilizumab, Volociximab. Votumumab, Zalutumumab, Zano epitope of an antigen or mimetope thereof. An antibody limumab, Ziralimumab, and Zolimomab. includes an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and 0024. The term “antibody” as used herein also contem includes chimeric, humanized, fully human, and bispecific plates the biosimilar or second generation version of the antibodies. Binding fragments include, but are not limited to, monoclonal antibodies described herein. Fab, Fab', F(ab')2, Fv, and single-chain antibodies. In some 0025. The term “biological fluid” as used herein contem embodiments, an antibody is referred to as an immunoglobu plates a liquid with biomolecules, bioparticles, blood, Sweat, lin and include the various classes and isotypes, such as IgA saliva, amniotic fluid, lacrimal fluid, or urine. Examples of US 2015/0293O86 A1 Oct. 15, 2015

biomolecules are, but not limited to, nucleic acids, peptides, sample pad. In some embodiments, the conjugatepad is of the and enzymes. Examples of bioparticles are, but not limited to, same or different material as the sample pad. In some embodi cells, organelles etc. ments, the conjugate pad further comprises an antibody. In 0026. The term “mimetope' as used herein contemplates a one of these embodiments, the antibody is an immunoglobu compound that is recognized by the same binding molecule, lin antibody. Such as an antibody, as a particular epitope but which has a 0030 The term “test line' as contemplated herein, refers different composition from the epitope. In one embodiment, to a band or Zone on the test membrane that contains at least the binding molecule is an antibody which recognizes (i.e., one mimetope peptide. The mimetope peptide is usually binds to) an epitope comprising a linear sequence of amino immobilized in abandor Zone such that after reaction with the acids. A mimetope of this epitope comprises a different linear antibody-detectable marker complex, the band or Zone pro sequence of amino acids but which is still recognized by the duces an observable or measurable signal reflecting the pres same antibody. In one embodiment, mimetope includes a ence or amount of antibody present in the sample. For exem peptide epitope that is able to mimic the ability of an epitome plary purposes only, if the test device is intended to detect the to bind to an antibody. presence or the amount of bevacizumab in a sample, then at 0027. The terms “polypeptide' and “peptide' are used least one test line will contain a mimetope peptide sequence broadly to refer to macromolecules comprising linear poly specific for bevacizumab immobilized on the test membrane. mers of natural or synthetic amino acids. In some embodi In some embodiments, the test membrane contains one, two, ments, polypeptides are derived naturally or synthetically by three, four, or more test lines. In some embodiments, the test standard methods known in the art. While the terms polypep lines contain the same or different immobilized mimetopes. tide and peptide are synonymous, the term polypeptide, as In some embodiments, more than one test line is applied for used herein, generally refers to molecules of greater than 40 multi-analyte testing or for semi-quantitative evaluation. In amino acids, while the term peptide generally refers to mol Some embodiments, the peptides in the test lines are conju ecules of 2 to 40 amino acids. gated to a protein. In an exemplary embodiment, the protein is 0028. The term “sample pad' as used herein generally a globular protein, Such as but not limited to, fibronectin, refers to a hydrophilic element, such as a membrane that albumin, recombinant capsid protein VP1 of the foot-and receives the antibody. In some embodiments, the sample pad mouth-disease virus (rVP1), recombinant capsid protein VP2 is part of the conjugate pad, or a discrete pad positioned of the foot-and-mouth-disease virus (rVP2), recombinant downstream of and in fluid communication with the conju capsid protein VP3 of the foot-and-mouth-disease virus gate pad. In some embodiments, the sample pad is useful for (rVP3), or precursor protein P1 of VP1,VP2, VP3, and VP4. promoting the even and controlled distribution of the anti In other embodiments, the protein is a chimeric protein, a body onto the conjugatepad, controlling the rate at which the naturally occurring protein or a non-naturally occurring pro antibody enters the conjugate pad, or preventing flooding of tein. the test device. In some embodiments, the sample pad mate 0031. The term “control line' as used herein contemplates rial comprise of, but not limited to, for example, woven mesh aband or Zone on the test membrane used for, but not limited or cellulose filters, glass fiber, mixed glass fiberand cellulose, to, confirming negative test results, for comparison with at man-made fiber, mixed fiber, Surface modified plastic (poly least one test line, determining incorrect antibody, inactive ester, polypropylene, or polyethylene), nitrocellulose, or antibody, wrong antibody, non-antibody protein, or conclud graded density polyethersulfone (PES). In some embodi ing failed experiments. In some embodiments, the controlline ments, the sample pad is of the same or different material as comprises an antibody. In one of these embodiments, the the conjugate pad. In some embodiments, the sample pad is antibody is an immunoglobulin antibody. The immunoglobu impregnated with agents to influence the flow rate of the lin antibody include various classes and isotypes, such as IgA sample, including, for example, protein detergents or Surfac (IgA1 and IgA2), Ig|D, IgE. IgM, and IgG (IgG1, IgG3 and tants, viscosity enhancers, signal enhancers, or buffer salts. In IgG4) etc. The antibody may be a human antibody or a non Some embodiments, agents are added to the sample pad to human antibody. The antibody may be naturally occurring or prevent the detectable marker and antibody from binding non-naturally occurring. nonspecifically to any downstream materials or to modify the chemical nature of the antibody so that it is compatible to 0032. The term “wicking pad” as used herein contem complex at the test line. In some embodiments, the sample plates an absorbent pad attached at the distal side of the test pad further comprises a buffer, pH calibrator, peptide, or device. The wicking pad aids to maintain the flow of liquid to antibody. the end of the strip. In some embodiments, the wicking pad 0029. The term “conjugate pad' as contemplated herein, comprises, but not limited to, an absorbent material, cellulose generally refers to a hydrophilic element, such as a mem filter, or any other material which acts to maintain the flow of brane, containing a conjugate reagent Such as colloidal gold, liquid to the end of the strip. latex particles, enzymes, colored dyes, or paramagnetic or 0033. It is a general object of the present disclosure to fluorescent particles. In some embodiments, the conjugate provide methods, devices and kits which is used to visually pad acts to ensure uniform transfer of the detectable marker determine the presence or absence of a specific, biologic and the antibody onto the test membrane. In some embodi and/or to visually quantify or semi-quantify an amount of a ments, the conjugatepad comprises, but not limited to, woven specific, biologic. It is also a general object of the present mesh or cellulose filters, glass fiber, mixed glass fiber and disclosure to provide methods, devices and kits which is used cellulose, man-made fiber, mixed fiber, surface modified to visually determine the presence or absence of a specific plastic (polyester, polypropylene, or polyethylene), nitrocel biologic and/or to visually quantify or semi-quantify an lulose, or graded density PES. In some embodiments, the amount of a specific biologic. In some embodiments, it will be conjugate pad is part of the sample pad, or a discrete pad readily appreciated that other detection systems, optical or positioned upstream of, and in fluid communication with the otherwise, is used in Such tests. US 2015/0293O86 A1 Oct. 15, 2015

0034. In some embodiments, the term “biologic” as used eases while at the same time creating a barrier to the illicit herein refers to a virus, therapeutic serum, toxin, antitoxin, profitability of counterfeit therapeutic proteins. vaccine, blood, blood component or derivative, allergenic 0040. In one embodiment, the disclosed test device deter product, protein, or analogous product, or arsphenamine or mines the integrity of a therapeutic protein through a lateral derivative of arsphenamine (or any other trivalent organic flow assay. Lateral flow assay is an immunoassay that is used arsenic compound), applicable to the prevention, treatment, to detect various chemical or biological agents. In a typical or cure of a disease or condition of human beings. In other lateral flow assay, capillary action draws fluid sample towards embodiments, the term biologic has the definition of biologi a Zone where specific immobilized reagents reside. If the cal product set forth by the Food and Drug Administration target molecule is present in the sample, the target binds to the (http://www.fda.gov/Drugs/DevelopmentApprovalProces immobilized reagent and is visualized with a detectable S/%20HowDrugsareDevelopedandApproved/ApprovalAp marker. Control lines function to confirm that the test is plications/TherapeuticBiologic Applications/ucm113522. functional or valid, independently of whether the sample htm). binds to the immobilized reagent. 0035. In an exemplary aspect, the biologic is a protein. In 0041. In one aspect, the test device for determining the those aspects, the present disclosure relates to detection of presence or integrity of a therapeutic protein comprises a proteins. Proteins are typically used in therapeutic applica sample pad for receiving a sample comprising the therapeutic tions, such as in the treatment of cancer. In some embodi protein, a conjugate pad, and a test membrane comprising at ments, as used herein, the protein is from any origin, Such as least one test line comprising an immobilized mimetope. In mouse, rabbit, pig, horse, dog, or human, including a chi Some embodiments, the sample pad, conjugate pad, and test meric protein thereof. In some embodiments, the protein is a membrane are in fluid flow contact with one another. The monoclonal antibody. sample pad absorbs the sample into the membrane. This is 0036. In one embodiment of the present disclosure, the test illustrated in FIG. 1. device includes qualitative readout (e.g., presence/absence of 0042. In one embodiment, the test device is a lateral flow a specific protein). Semi-quantitative or quantitative results immunoassay. In one embodiment, the lateral flow immu are also contemplated. In some embodiments, semi-quantita noassay format is chosen from antigen Sandwich assay, anti tive or quantitative results is achieved by including a series of body assay, or competitive hapten assay. test lines of mimetopes of increasing concentration. In some 0043. In one embodiment, the sample pad of the test embodiments, the test device of the present disclosure detects device further comprises a buffer for pH stabilization, a pH active protein at least about 30, 40, 50, 60, 70, 80,90, or 95% calibrator, a surfactant to guarantee a uniform wetting, a activity. In certain embodiments, evaluation of the signal stabilizing polymer, and a blocker. The position of the sample intensity is also performed. In some embodiments, the signal pad often varies. is digitized and evaluated using a flatbed scanner or a CCD 0044. In one embodiment, the protein received by the camera and appropriate Software. In some embodiments, sen sample pad is a protein therapeutic molecule. In another sitivity is increased by use of enhancement agents such as, for embodiment, the protein received by the sample pad is example, a silver enhancer. More sensitive chemiluminescent selected from a group consisting of pharmaceutically or or fluorescent labels are optionally used to increase the sen commercially relevant enzymes, receptors, receptor fusions, sitivity as well. Free mimetope peptide, to compete for bind soluble receptors, soluble receptor fusions, antibodies (e.g., ing with the protein, is optionally included in the conjugate monoclonal and/or polyclonal antibodies), antigen-binding pad to threshold the assay, when decreased sensitivity is fragments of an antibody, Fc fusion proteins, SMIPs5 cytok desired. In some embodiments, sensitivity is also controlled ines, hormones, regulatory factors, growth factors, coagula by adjusting, for example, the bed Volume of the membrane, tion/clotting factors, and antigen-binding agents. In another dimensions of the test membrane, porosity of the test mem embodiment, the protein received by the sample pad is brane, position and width of the test line and control line. selected from a group consisting of Etanercept, Pegfilgrastim, 0037. In one embodiment, the disclosed test device and Interferon B1a, and Erythropoietin. methods are related to determining the integrity of a protein. 0045. In one embodiment, the protein is introduced to the Proteins are increasingly used as therapeutics to treat human sample pad using a dipstick format and contacting one end of diseases. With their high cost, protein therapeutics are also the test device with the protein. In another embodiment, the extremely attractive targets for counterfeiting or illegal dis protein is introduced onto the sample pad using an applicator tribution. Furthermore, being large molecules, these proteins Such as, for example, a pipette, a Syringe, a dropper, a spray, are temperature and light sensitive, and require proper han and others known in the art. The amount of protein received dling and storage. With a complex pharmaceutical distribu on the sample pad is preferably between about 1 and 200 uL. tion chain making adequate oversight difficult, and increasing more preferably between about 3 and 100 u, and most pref the likelihood that such problems will persist, it is imperative erably between about 5 and 50 uL. that a simple, inexpensive assay be developed to determine 0046. In one embodiment, the sample pad of the test the integrity of a protein therapeutic prior to use. device receives a protein in a fluid selected from the group 0038. In one embodiment disclosed herein, the test device consisting of buffer, saline solution, pharmaceutical compo allows the rapid determination of the presence of a correct sition, and biological fluid. In another embodiment, the bio protein, presence of incorrect or inactive protein, contami logical fluid received by the sample pad is selected from a nated protein, no protein, and/or quantity of protein present in group consisting of blood, urine, lacrimal fluid, Sweat, saliva, a sample in a single assay format. and amniotic fluid. In another embodiment, the biological 0039. In another embodiment disclosed herein, are device fluid received by the sample pad is blood. and methods for improved assessment of the therapeutic pro 0047. In one embodiment, the conjugate pad of the test tein integrity that provides an additional level of safety and device comprises a detectable marker. In another embodi reassurance for patients fighting serious life-threatening dis ment, the detectable marker in the conjugate pad is capable of US 2015/0293O86 A1 Oct. 15, 2015 binding the protein that the sample pad receives. In one embodiment, the secondary protein is an antibody. In another embodiment, the conjugate pad acts to ensure uniform trans embodiment of the method disclosed herein, the secondary fer of the detectable marker and the protein onto the test protein is conjugated to gold. membrane. In one embodiment, the detectable marker com 0053. In one embodiment of the method disclosed herein, prises, but not limited to, particles, luminescent labels, calo the test membrane comprises at least one test line and at least rimetric labels, fluorescent labels, chemical labels, enzymes, one control line. In another embodiment of the method dis radioactive labels, metal colloids, and chemiluminescent closed herein, the test membrane comprises two or more test labels. In one embodiment, gold colloidal spheres are used. In lines. In another embodiment of the method disclosed herein, another embodiment, other metal sols and latex micropar at least one test line comprises an immobilized mimetope. In ticles are used as well. In other embodiments, photostable, another embodiment of the method disclosed herein, eachtest color tunable nanoparticles such as carbon, selenium, or line contains an immobilized mimetope different from quantum dots are used as detectable markers. These detect another test line. In another embodiment of the method dis able markers provide colorimetric indicators for reporting closed herein, the immobilized mimetope mimics the native whether the target molecule is present. The size of the detect antigen epitope recognized by an antibody. In another able markers are related to the porosity of the membrane. The embodiment of the method disclosed herein, the immobilized markers are preferably sufficiently small to be transported mimetope mimics the native antigen epitope recognized by an along the membrane by the capillary action of the fluid. In one antibody. embodiment, the amount of detectable marker present varies 0054. In one embodiment of the method disclosed herein, depending on the size and composition of the detectable at least one test line is upstream of at least one control line. In marker, the composition of the membrane, and the level of another embodiment of the method disclosed herein, at least sensitivity of the assay. The detectable marker will bind to the one test line is downstream of at least one control line. protein to form a protein-detectable marker complex. In one 0055. In one embodiment of the method disclosed herein, embodiment, the detectable marker comprises gold colloidal at least one control line comprises a light chain antibody. In spheres. In another embodiment, the detectable marker com another embodiment of the method disclosed herein, the light prises a secondary protein. In some embodiments, the sec chain antibody is an anti-kappa light chain antibody. In ondary protein is an antibody. In some embodiments, the another embodiment of the method disclosed herein, the light secondary protein is conjugated to gold. chain antibody is an anti-lambda light chain antibody. In 0048. In one aspect, the present disclosure relates to meth another embodiment of the method disclosed herein, at least ods of determining the presence or integrity of a protein one control line comprises an antibody that binds directly to comprising (a) contacting an protein with a test device, the secondary antibody of the conjugate pad. wherein the test device comprises: (i) a sample pad for receiv 0056. One embodiment of the method disclosed herein, ing the protein, (ii) a conjugate pad, (iii) a test membrane further comprises a wicking pad. comprising at least one test line comprising an immobilized 0057. One embodiment of the present disclosure com mimetope, (b) determining whether at least one test line prises a kit comprising (a) a test device for determining the undergoes a color change, and (c) determining, based on the presence or integrity of a protein, as described above, and (b) color change, the activity of the protein. An illustration of instructions for use thereof. In another embodiment, the kit such a method is shown in FIG. 3. further comprising a protein sample. In another embodiment, the kit further comprises a protein applicator. In another 0049. In one embodiment, the method of determining the embodiment, the protein applicator is, for example, a pipette, presence or integrity of a protein comprises a test device a syringe, a dropper, or others known in the art. wherein the test device is a lateral flow assay. In another 0058. The disclosure provided herein produces a synergis embodiment the sample pad in the method further comprises tic effect and results in better recognition and binding a buffer, pH calibrator, peptide, or antibody. between the protein and the mimetope. For exemplary pur 0050. In one embodiment of the method disclosed herein, poses only, when a Gly-Gly-Gly-Serlinker, reactive chemis the sample pad of the test device receives a protein in a fluid try, or biotin is added to the C-terminal serine of the mime selected from the group consisting of buffer, Saline solution, tope, the peptide is displayed in Such a way that is available pharmaceutical composition, and biological fluid. In another for antibody binding. Attaching the mimetope to the mem embodiment of the method disclosed herein, the biological brane that way allows for accurate determination of the pres fluid is selected from a group consisting of blood, urine, ence and purity of a sample protein or protein therapeutic. lacrimal fluid, Sweat, saliva, and amniotic fluid. In a preferred This synergism in binding was unexpected based on prior embodiment of the method disclosed herein, the biological knowledge in the art. The results of this synergism are being fluid is blood. employed in the present disclosure to determine the integrity ofan antibody. These unexpected and Surprising results are of 0051. In one embodiment of the method disclosed herein, significant, practical advantage to patients because adminis the conjugate pad comprises a detectable marker. In another tering the wrong or inactive protein results in otherwise embodiment of the method disclosed herein, the detectable avoidable disease progression, permanent injury, or even marker is selected from a group consisting of particles, lumi death. nescent labels, calorimetric labels, fluorescent labels, chemi 0059. In an exemplary aspect, the present disclosure cal labels, enzymes, radioactive labels, metal colloids, and relates to detection of proteins. Proteins are typically used in chemiluminescent labels. In another embodiment of the therapeutic applications, such as in the treatment of cancer. method disclosed herein, the detectable marker comprises 0060. In one embodiment, if trace amounts of a protein gold colloidal spheres. gives a positive result, free mimetope peptides are added to 0.052. In one embodiment of the method disclosed herein, the conjugate pad. Those free mimetopes compete for the the detectable marker comprises a secondary protein. In one protein in the sample and its concentration will determine the US 2015/0293O86 A1 Oct. 15, 2015

threshold amount required for detection. The precise thresh of non-antibody protein, contaminated antibody, no antibody, old will ultimately depend on the point of use, in particular and/or quantity of antibody present in a sample in a single whether the test is to be performed on the antibody as pack assay format. aged in the shipping vials or after mixing for infusion. 0067. In another embodiment disclosed herein, are device 0061. In another exemplary aspect, the present disclosure and methods for improved assessment of the antibody integ relates to detection of antibodies. Antibodies are typically rity that provides an additional level of safety and reassurance used in therapeutic applications, such as in the treatment of for patients fighting serious life-threatening diseases while at cancer. In some embodiments, as used herein, the antibody is the same time creating a barrier to the illicit profitability of from any origin, Such as mouse, rabbit, pig, horse, dog, or counterfeit antibodies. human, including a chimeric antibody thereof. 0068. In one embodiment, the disclosed test device deter mines the integrity of an antibody through a lateral flow assay. 0062. In some embodiments, the antibody is a monoclonal Lateral flow assay is an immunoassay that is used to detect antibody. In some embodiments, the monoclonal antibody is various chemical or biological agents. In a typical lateral flow a bispecific monoclonal antibody. In other embodiments, the assay, capillary action draws fluid sample towards a Zone antibody is biosimilar or second generation versions of where specific immobilized reagents reside. If the target mol monoclonal antibodies or similar antibodies. ecule is present in the sample, the target binds to the immo 0063. In some embodiments of the present disclosure, the bilized reagent and is visualized with a detectable marker. monoclonal antibody is a bispecific monoclonal antibody Control lines function to confirm that the test is functional or such as, for example, catumaxomab, ertumaxomab, FBTA05 valid, independently of whether the sample binds to the and TRBS07. The term “bispecific monoclonal antibody’ as immobilized reagent. used herein refers to an antibody that has binding sites for two 0069. In one aspect, the test device for determining the different antigens within a single antibody molecule. It will presence or integrity of an antibody comprises a sample pad be appreciated by those skilled in the art that other molecules for receiving a sample comprising the antibody, a conjugate in addition to the canonical antibody structure may be con pad, and a test membrane comprising at least one test line structed with two binding specificities. comprising an immobilized mimetope. In some embodi 0064. In one embodiment of the present disclosure, the test ments, the sample pad, conjugate pad, and test membrane are device includes qualitative readout (e.g., presence/absence of in fluid flow contact with one another. The sample pad a specific antibody). Semi-quantitative or quantitative results absorbs the sample into the membrane. This is illustrated in are also contemplated. In some embodiments, semi-quantita FIG 1. tive or quantitative results is achieved by including a series of 0070. In one embodiment, the test device is a lateral flow test lines of mimetopes of increasing concentration. In some immunoassay. In one embodiment, the lateral flow immu embodiments, the test device of the present disclosure detects noassay format is chosen from antigen Sandwich assay, anti active antibody at least about 30, 40,50, 60, 70,80,90, or 95% body assay, or competitive hapten assay. activity. In certain embodiments, evaluation of the signal 0071. In one embodiment, the sample pad of the test intensity is also performed. In some embodiments, the signal device further comprises a buffer for pH stabilization, a pH is digitized and evaluated using a flatbed scanner or a CCD calibrator, a surfactant to guarantee a uniform wetting, a camera and appropriate Software. In some embodiments, sen stabilizing polymer, and a blocker. The position of the sample sitivity is increased by use of enhancement agents such as, for pad often varies. example, a silver enhancer. More sensitive chemiluminescent 0072. In one embodiment, the antibody received by the or fluorescent labels are optionally used to increase the sen sample pad is a monoclonal antibody. In another embodi sitivity as well. Free mimetope peptide, to compete for bind ment, the monoclonal antibody received by the sample pad is ing with the antibody, is optionally included in the conjugate selected from a group consisting of bevacizumab, trastu pad to threshold the assay, when decreased sensitivity is Zumab, rituximab, abciximab, adalimumab, alemtuzumab, desired. In some embodiments, sensitivity is also controlled basiliximab, belimumab, brentuximab, vedotin, canaki by adjusting, for example, the bed Volume of the membrane, numab, cetuximab, certolizumab pegol, daclizumab, denos dimensions of the test membrane, porosity of the test mem umab, eculizumab, efalizumab, gemtuzumab, golimumab, brane, position and width of the test line and control line. ibritumomab, tiuxetan, infliximab, ipilimumab, muromonab CD3, natalizumab. ofatumumab, omalizumab, palivizumab, 0065. In one embodiment, the disclosed test device and panitumumab, ranibizumab, raxibacumab, tocilizumab, tosi methods are related to determining the integrity of an anti tumomab, and ustekinumab. In another embodiment, the body. Antibodies are increasingly used as therapeutics to treat monoclonal antibody received by the sample pad is a bispe human diseases such as cancer and autoimmunity. With their cific monoclonal antibody. In another embodiment, the bispe high cost, antibodies are also extremely attractive targets for cific monoclonal antibody received by the sample pad is counterfeiting or illegal distribution. Furthermore, being selected from a group consisting of catumaxomab, ertumax large proteins, antibodies are temperature and light sensitive, omab, FBTA05, and TRBS07. and require proper handling and storage. With a complex 0073. In one embodiment, the antibody is introduced to pharmaceutical distribution chain making adequate oversight the sample pad using a dipstick format and contacting one end difficult, and increasing the likelihood that such problems of the test device with the antibody. In another embodiment, will persist, it is imperative that a simple, inexpensive assay the antibody is introduced onto the sample pad using an be developed to determine the integrity of an antibody prior to applicator Such as, for example, a pipette, a syringe, a drop SC. per, a spray, and others known in the art. The amount of 0.066. In one embodiment disclosed herein, the test device antibody received on the sample pad is preferably between allows the rapid determination of the presence of a correct about 1 and 200 uL, more preferably between about 3 and 100 antibody, presence of incorrector inactive antibody, presence uL, and most preferably between about 5 and 50 uL. US 2015/0293O86 A1 Oct. 15, 2015

0074. In one embodiment, the sample pad of the test of three control lines. It is also contemplated that the test device receives an antibody in a biological fluid. In another device consists of more than three control lines. embodiment, the biological fluid received by the sample pad 0080. In some embodiments, the test device further com is selected from a group consisting of blood, urine, lacrimal prises a wicking pad. fluid, Sweat, saliva, and amniotic fluid. In another embodi I0081. In another aspect, the present disclosure relates to ment, the biological fluid received by the sample pad is blood. methods of determining the presence or integrity of an anti 0075. In one embodiment, the conjugate pad of the test body comprising (a) contacting an antibody with a test device comprises a detectable marker. In another embodi device, wherein the test device comprises: (i) a sample pad for ment, the detectable marker in the conjugate pad is capable of receiving the antibody, (ii) a conjugate pad, (iii) a test mem binding the antibody that the sample pad receives. In one brane comprising at least one test line comprising an immo embodiment, the conjugate pad acts to ensure uniform trans bilized mimetope, (b) determining whether at least one test fer of the detectable marker and the antibody onto the test line undergoes a color change, and (c) determining, based on membrane. In one embodiment, the detectable marker com the color change, the activity of the antibody. An illustration prises, but not limited to, particles, luminescent labels, calo of such a method is shown in FIG. 3. rimetric labels, fluorescent labels, chemical labels, enzymes, I0082 In one embodiment, the method of determining the radioactive labels, metal colloids, and chemiluminescent presence or integrity of an antibody comprises a test device labels. In one embodiment, gold colloidal spheres are used. In wherein the test device is a lateral flow immunoassay. In another embodiment, other metal sols and latex micropar another embodiment the sample pad in the method further ticles are used as well. In other embodiments, photostable, comprises a buffer, pH calibrator, peptide, or antibody. color tunable nanoparticles such as carbon, selenium, or 0083. In another embodiment of the method disclosed quantum dots are used as detectable markers. These detect herein, the antibody is a monoclonal antibody. In some able markers provide colorimetric indicators for reporting embodiments, the monoclonal antibody in this method is whether the target molecule is present. The size of the detect selected from a group consisting of bevacizumab, trastu able markers are related to the porosity of the membrane. The Zumab, rituximab, abciximab, adalimumab, alemtuzumab, markers are preferably sufficiently small to be transported basiliximab, belimumab, brentuximab vedotin, canaki along the membrane by the capillary action of the fluid. In one numab, cetuximab, certolizumab pegol, daclizumab, denos embodiment, the amount of detectable marker present varies umab, eculizumab, efalizumab, gemtuzumab, golimumab, depending on the size and composition of the detectable ibritumomab tiuxetan, infliximab, ipilimumab, muromonab marker, the composition of the membrane, and the level of CD3, natalizumab. ofatumumab, omalizumab, palivizumab, sensitivity of the assay. The detectable marker will bind to the panitumumab, ranibizumab, raxibacumab, tocilizumab, tosi antibody to form an antibody-detectable marker complex. In tumomab, and ustekinumab. one embodiment, the detectable marker comprises gold col 0084. In another embodiment of the method disclosed loidal spheres. In another embodiment, the detectable marker herein, the monoclonal antibody is a bispecific monoclonal comprises a secondary antibody. In some embodiments, the antibody. In some embodiments of the method disclosed secondary antibody is conjugated to gold. herein, the bispecific monoclonal antibody is selected from a 0076. In one embodiment, the test membrane in the test group consisting of catumaxomab, ertumaXomab, FBTA05, device comprises at least one test line and at least one control and TRB SO7. line. In another embodiment, the test membrane comprises 0085. In one embodiment of the method disclosed herein, two or more test lines. In another embodiment, at least one the sample pad receives an antibody in a biological fluid. In test line comprises an immobilized mimetope. In another another embodiment of the method disclosed herein, the bio embodiment, each test line contains an immobilized mime logical fluid is selected from a group consisting of blood, tope different from another test line. In some embodiments, urine, lacrimal fluid, Sweat, saliva, and amniotic fluid. In a the immobilized mimetope mimics the native antigen epitope preferred embodiment of the method disclosed herein, the recognized by an antibody. biological fluid is blood. 0086. In one embodiment of the method disclosed herein, 0077. In one embodiment, at least one test line is upstream the conjugate pad comprises a detectable marker. In another of at least one control line. In another embodiment, at least embodiment of the method disclosed herein, the detectable one test line is downstream of at least one control line. marker is selected from a group consisting of particles, lumi 0078. In one embodiment, at least one control line com nescent labels, calorimetric labels, fluorescent labels, chemi prises a light chain antibody. In some embodiments, the light cal labels, enzymes, radioactive labels, metal colloids, and chain antibody is an anti-kappa light chain antibody. In chemiluminescent labels. In another embodiment of the another embodiment, the light chain antibody is an anti method disclosed herein, the detectable marker comprises lambda light chain antibody. In other embodiments, the light gold colloidal spheres. chain antibody is a light chain antibody known in the art. 0087. In one embodiment of the method disclosed herein, 0079. In one embodiment, the at least one control line of the detectable marker comprises a secondary antibody. In the disclosed test membrane comprises an antibody that binds another embodiment of the method disclosed herein, the sec directly to the secondary antibody of the conjugate pad Such ondary antibody is conjugated to gold. as, for example, anti-IgG. In some of these embodiments, the 0088. In one embodiment of the method disclosed herein, anti-IgG acts as a non-specific control and turns positive the test membrane comprises at least one test line and at least regardless of the presence or absence of antibody within the one control line. In another embodiment of the method dis test sample. In some embodiments, the test device consists of closed herein, the test membrane comprises two or more test only one control line. In one embodiment, the test device of lines. In another embodiment of the method disclosed herein, the present disclosure consists of two control lines. In another at least one test line comprises an immobilized mimetope. In embodiment, the test device of the present disclosure consists another embodiment of the method disclosed herein, eachtest US 2015/0293O86 A1 Oct. 15, 2015

line contains an immobilized mimetope different from displayed on the solid substance in such a way to be available another test line. In another embodiment of the method dis for antibody binding. In one embodiment, the linker is about closed herein, the immobilized mimetope mimics the native 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In other antigen epitope recognized by an antibody. In another embodiments, reactive chemistry or biotin is added to the embodiment of the method disclosed herein, the immobilized C-terminal serine. If amine reactive chemistries are used to mimetope mimics the native antigen epitope recognized by an attach the mimetopes to the Solid Substance and there are no antibody. amine containing side chains within the peptide sequence, the 0089. In one embodiment of the method disclosed herein, N-terminus is acetylated and a C-terminal lysine added to at least one test line is upstream of at least one control line. In provide the reactive amine. Additionally, linear and disulfide another embodiment of the method disclosed herein, at least constrained peptide libraries are also used. In one embodi one test line is downstream of at least one control line. ment, mimetopes are expressed as a fusion with a carrier 0090. In one embodiment of the method disclosed herein, protein such as KLH. at least one control line comprises a light chain antibody. In 0096. The disclosure provided herein produces a synergis another embodiment of the method disclosed herein, the light tic effect and results in better recognition and binding chain antibody is an anti-kappa light chain antibody. In between the antibody and the mimetope. For exemplary pur another embodiment of the method disclosed herein, the light poses only, when a Gly-Gly-Gly-Serlinker, reactive chemis chain antibody is an anti-lambda light chain antibody. In try, or biotin is added to the C-terminal serine of the mime another embodiment of the method disclosed herein, at least tope, the peptide is displayed in Such a way that is available one control line comprises an antibody that binds directly to for antibody binding. Attaching the mimetope to the mem the secondary antibody of the conjugate pad. brane that way allows for accurate determination of the pres 0091. One embodiment of the method disclosed herein, ence and purity of a sample antibody. This synergism in further comprises a wicking pad. binding was unexpected based on prior knowledge in the art. 0092. One embodiment of the present disclosure com The results of this synergism are being employed in the prises a kit comprising (a) a test device for determining the present disclosure to determine the integrity of an antibody. presence or integrity of an antibody, as described above, and These unexpected and Surprising results are of significant, (b) instructions for use thereof. In another embodiment, the practical advantage to patients because administering the kit further comprising an antibody sample. In another wrong or inactive antibody results in otherwise avoidable embodiment, the kit further comprises an antibody applicator. disease progression, permanent injury, or even death. In another embodiment, the antibody applicator is, for I0097. In an exemplary aspect, the present disclosure example, a pipette, a Syringe, a dropper, or others known in relates to detection of monoclonal antibodies. Monoclonal the art. antibodies are typically used in therapeutic applications. Such 0093. In some embodiments, the test device of the present as in the treatment of cancer. For exemplary purposes only, disclosure has a protective cover. In some embodiments, the Table 1 lists some common monoclonal antibodies and the protective cover is formed of any material which is impervi corresponding mimetope peptides. ous to water, and is preferably translucent or transparent. In certain embodiments, the protective covering comprises a TABLE 1. single or multiple layers. Preferable materials for use in the protective covering include optically transmissive materials Mimetope peptide sequence for some antibody Such as polyamide, polyester, polyethelene, acrylic, glass, or similar materials. The protective covering is optionally clear Target antibody Mimetope sequence (i.e., see-through) or not clear (i.e., opaque) depending on the Rituximab ACPYSNPSLC method of detection used. In a preferable embodiment, a protective covering is optically clear polyester. Alemtuzumab ACGSLSPSSC 0094. In some embodiments, the present disclosure Bevacizumab ACFLRSGLPC include mimetope peptides comprising about 5 to 15 amino acids or about 7 to 12 amino acids, that bind to the antigen Trastuzumab ACWDHHLDHC binding site of the antibody and are competed by the natural ligand. 0098. In one embodiment, if trace amounts of antibody 0095. In one embodiment, the mimetope peptides dis gives a positive result, free mimetope peptides are added to closed herein are generated as described in WO 2009/121024, the conjugate pad. That free mimetope competes for the anti the disclosure of which is incorporated herein by reference. body in the sample and its concentration will determine the The mimetope peptides are selected using phage displayed threshold amount required for detection. The precise thresh peptide libraries. These mimetopes are then modified for use old will ultimately depend on the point of use, in particular in, for example, lateral flow assays. In one embodiment, whether the test is to be performed on the antibody as pack mimetopes contain additional amino acids to alter or modify aged in the shipping vials or after mixing for infusion. mimetope characteristics. In some embodiments, mimetopes contain cysteines flanking the mimetope sequence to increase 0099 Embodiments of the disclosure are further described stability of the mimetope through disulfide bond formation. with reference to the accompanying drawings. In one embodiment, mimetopes are attached to a solid Sub 0100. The following examples are provided to further stance Such as, for example, the test membrane. In one illustrate the embodiments of the present disclosure, but are embodiment, mimetopes are attached to the test membrane not intended to limit the scope of the disclosure. While they directly or synthesized directly on the test membrane. In other are typical of those that might be used, other procedures, embodiments, linkers such as, for example, Gly-Gly-Gly-Ser methodologies or techniques known to those skilled in the art linkers, are added to mimetopes to ensure the mimetope are may alternatively be used. US 2015/0293O86 A1 Oct. 15, 2015

Example 1: Identification and Testing of Mimetopes Fluorescently labeled rituximab was incubated with primary CLL cells and evaluated by flow cytometry (solid line). Weak 0101 While the following assay reagent protocol is staining for CD20 was observed since CLL cells are known to described using specifically identified reagents, such as spe have low levels of CD20. When peptide RTX-10 was added at cific phage-displayed libraries and generation of specifically a large molar excess (dashed lines), the cell labeling is largely identified peptides, the methods described herein is utilized to abrogated. Control peptides had no effect (not shown). The generate peptides including mimetopes that interact with any shaded histogram represents CLL cells incubated with fluo antibody. rescently labeled normal human IgG. 0102 Peptide mimetopes were identified and generated as described in the disclosure of WO2009/121024 and in Example 3: Validation of Etanercept and Sanchez et al., Cancer Chemther: Pharmacol. (2010) 66:919 Erythropoetin Binding Mimetope Peptide in the 925, which is incorporated herein by reference in its entirety. Phage-display libraries of peptide mimetopes were screened Lateral Flow Assay against the monoclonal antibodies, including, but not limited 0106 Peptide mimetopes are identified and generated as to, bevacizumab, tratuZumab, and rituximab. Short peptides described in Example 1. Phage-display libraries of peptide of 7 to 12 amino acids were screened and were selected from mimetopes are screened against protein molecules, including, a library that contain cysteines flanking the peptide mimetope but not limited to, protein therapeutics such as Etanercept and sequence. To ensure that the peptide was displayed on the test Erythropoetin. The identified peptide mimetope is displayed membrane in such a way as to be available for antibody on the test membrane in such a way as to be available for binding, a glycine-glycine-glycine-serine linker was added protein binding, Such as by adding a glycine-glycine-glycine and the reactive chemistry or biotin added to the C-terminal serine linker, and/or adding the reactive chemistry or biotinto serine. If amine reactive chemistries were used for conjuga the C-terminal serine. If amine reactive chemistries are used tion and there were no amine containing side chains within for conjugation and there are no amine containing side chains the peptide sequence, the N-terminus was acetylated and a within the peptide sequence, the N-terminus is acetylated and C-terminal lysine added to provide the reactive amine. a C-terminal lysine is added to provide the reactive amine. 0103) To confirm that a synthetic peptide captures the 0107 To confirm that a synthetic peptide captures the antibody, ELISAs were performed. Peptides biotinylated at protein Etanercept and/or Erythropoetin, Sandwich type the C-terminus were coated onto streptavidin coated plates assays, lateral flow or otherwise, are performed. and then incubated with a dilution serious of antibody. Bind ing was detected via a peroxidase labeled secondary anti-Fc Example 4: Validation of Test Device antibody. Specificity was confirmed by lack of signal from either total Ig or using a different antibody. Native antigen 0.108 FIG. 4 shows the results of an independent double competition assays were performed to confirm that the vali blinded test was performed on the test device. Samples of dated mimetope peptide was binding to the antibody binding antibodies, including, but not limited to, bevacizumab, tratu site and thus detecting only active antibody. For antigens that Zumab, rituximab, and total human IgG (IVIG), were pre were cell membrane bound, competition was shown by flow pared at 0.1 mg/ml and control Saline sample. 240 numbered cytometry. The antibody was fluorescently labeled and the LFA tests and samples were provided such that each mab cell staining evaluated with or without a molar excess of the assay was tested with 20 samples of each mab and 10 presumed mimetope as well as control peptides. samples of human IgG and buffer. This was a double-blinded 0104 Validated peptides detects >100 ng/ml of the anti test—the laboratory technicians who ran the assays knew body. For example, the sensitivity was 4 ug/ml for rituximab. neither the sample's identity nor the type of LFA, both of Specificity was confirmed by ensuring that there was no sig which were randomly ordered, but recorded the results they nal from either total human Igor a different antibody. Com observed. The test lines were accurate when the tests ran petition for antibody binding between the peptide and the properly. Only two bevacizumab assays failed to run. There native antigen confirmed that the mimetope peptide was bind was one false positive on the kappa line of a rituximab LFA ing the antibody-antigenbinding site and thus detecting only but several false positive lambda lines, suggesting that the active antibody. As another example, bevacizumab, whose anti-lambda antibody used was not optimal. No false nega target antigen is the soluble protein VEGF, competitive ELI tives other than the two defective assays were observed. SAS were used in which the peptide of VEGF was coated to the plate, and the other was used in Solution to compete for Example 5: Range of the Lateral Flow Assay antibody binding. 0109 FIG. 5 and FIG. 6 illustrate two embodiments that Example 2: Validation of Rituximab Binding the lateral flow assay disclosed herein has wide dynamic Mimetope Peptide in the Lateral Flow Assay range. FIG.5 shows that samples tested were Rituximab at 0.0 mg/ml (negative control), 0.01 mg/ml, 0.1 mg/ml, 1.0 mg/ml. 0105 FIG. 2 illustrates one embodiment of the lateral flow and 10 mg/ml (vial concentration). With increasing Ritux assay validating that rituximab binding mimetope peptide imab concentration, the test line gets more intense. FIG. 6 function in immunoassays and competes with the native anti illustrate one embodiment of the sandwich lateral flow assay gen (CD20). (A) is a standard curve for rituximab by peptide with two test lines. The test lines comprise peptide mimetope based ELISA. Biotinylayed peptides were bound onto neu striped at two different concentrations on the membrane. travidin coated ELISA plates. Rituximab was diluted in Samples tested were Rituximab at 0.0 mg/ml (negative con TBST. Each value shows the mean (S.D.) of triplicates. The trol), 0.01 mg/ml, 0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml (vial solid line indicates the mean of the buffer control and the concentration). With increasing Rituximab concentration, the dashed line represents the mean +10 times the SD of the lower test line gets less intense whereas the upper test line buffer control. (B) Peptide inhibition of CLL cell staining. gets more intense. US 2015/0293O86 A1 Oct. 15, 2015

0110. While the present disclosure has been described and 20. The test device of claim 18, wherein at least one test line illustrated herein, it is intended that the specification and comprises an immobilized mimetope. examples be considered as exemplary only, with the true 21. The test device of claim 20, wherein each test line scope and spirit of the invention being indicated by the fol contains an immobilized mimetope different from another lowing claims. test line. What is claimed is: 22. The test device of claim 21, wherein the immobilized 1. A test device for determining the presence or integrity of mimetope mimics the native antigen epitope recognized by an a biologic comprising antibody. a. a sample pad for receiving the biologic, 23. The test device of claim 18, wherein the at least one test b. a conjugate pad, and line is upstream of the at least one control line. c. a test membrane comprising at least one test line com 24. The test device of claim 18, wherein the at least one test prising an immobilized mimetope. line is downstream of the at least one control line. 2. The test device of claim 1, wherein the test device is a 25. The test device of claim 18, wherein at least one control lateral flow immunoassay. line comprises a light chain antibody. 3. The test device of claim 1, wherein the sample pad 26. The test device of claim 25, wherein the light chain further comprises a buffer, pH calibrator, peptide, or anti antibody is an anti-kappa light chain antibody. body. 27. The test device of claim 25, wherein the light chain 4. The test device of claim 1, wherein the biologic is a antibody is an anti-lambda light chain antibody. protein. 28. The test device of claim 18, wherein the at least one 5. The test device of claim 4, wherein the protein is an control line comprises an antibody that binds directly to the antibody. secondary antibody of the conjugate pad. 6. The test device of claim 5, wherein the antibody is a 29. The test device of claim 1, further comprising a wicking monoclonal antibody. pad. 7. The test device of claim 6, wherein the monoclonal 30. A method of determining the integrity of a biologic antibody is selected from a group consisting of bevacizumab, comprising trastuzumab, rituximab, abciximab, adalimumab, alemtu (a) contacting the biologic with a test device, wherein the Zumab, basiliximab, belimumab, brentuximab vedotin, test device comprises: (i) a sample pad for receiving the canakinumab, cetuximab, certolizumab pegol, daclizumab, biologic, (ii) a conjugatepad, (iii) a test membrane com denosumab, eculizumab, efalizumab, gemtuzumab, goli prising at least one test line comprising an immobilized mumab, ibritumomab tiuxetan, infliximab, ipilimumab, mimetope, muromonab-CD3, natalizumab. ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, raxibacumab, (b) determining whether at least one test line undergoes a tocilizumab, to situmomab, and ustekinumab. color change, 8. The test device of claim 6, wherein the monoclonal (c) determining, based on the color change, the activity of antibody is a bispecific monoclonal antibody. the biologic. 9. The test device of claim 8, wherein the bispecific mono 31. The method of claim 30, wherein the test device is a clonal antibody is selected from a group consisting of catu lateral flow immunoassay. maxomab, ertumaxomab, FBTA05, and TRBS07. 32. The method of claim 30, wherein the sample pad fur 10. The test device of claim 1, wherein the sample pad ther comprises a buffer, pH calibrator, peptide, or antibody. receives an antibody in a fluid selected from the group con 33. The test device of claim 30, wherein the biologic is a sisting of buffer, saline Solution, pharmaceutical composi protein. tion, and biological fluid. 34. The test device of claim 33, wherein the protein is an 11. The test device of claim 10, wherein the biological fluid antibody. is selected from a group consisting of blood, urine, Saliva, 35. The method of claim 34, wherein the antibody is a lacrimal fluid, Sweat, and amniotic fluid. monoclonal antibody. 12. The test device of claim 11, wherein the biological fluid 36. The method of claim 35 wherein the monoclonal anti is blood. body is selected from a group consisting of bevacizumab, 13. The test device of claim 1, wherein the conjugate pad trastuzumab, rituximab, abciximab, adalimumab, alemtu comprises a detectable marker. Zumab, basiliximab, belimumab, brentuximab vedotin, 14. The test device of claim 13, wherein the detectable canakinumab, cetuximab, certolizumab pegol, daclizumab, marker is selected from a group consisting of particles, lumi denosumab, eculizumab, efalizumab, gemtuzumab, goli nescent labels, calorimetric labels, fluorescent labels, chemi mumab, ibritumomab tiuxetan, infliximab, ipilimumab, cal labels, enzymes, radioactive labels, metal colloids, and muromonab-CD3, natalizumab. ofatumumab, omalizumab, chemiluminescent labels. palivizumab, panitumumab, ranibizumab, raxibacumab, 15. The test device of claim 13, wherein the detectable tocilizumab, to situmomab, and ustekinumab. marker comprises gold colloidal spheres. 37. The method of claim 35, wherein the monoclonal anti 16. The test device of claim 13, wherein the detectable body is a bispecific monoclonal antibody. marker comprises a secondary antibody. 38. The method of claim 37, wherein the bispecific mono 17. The test device of claim 16, wherein the secondary clonal antibody is selected from a group consisting of catu antibody is conjugated to gold. maxomab, ertumaxomab, FBTA05, and TRBS07. 18. The test device of claim 1, wherein the test membrane 39. The method of claim 30, wherein the sample pad comprises at least one test line and at least one control line. receives an antibody in a fluid selected from the group con 19. The test device of claim 18, wherein the test membrane sisting of buffer, Saline Solution, pharmaceutical composi comprises two or more test lines. tion, and biological fluid. US 2015/0293O86 A1 Oct. 15, 2015

40. The method of claim39, wherein the biological fluid is 51. The method of claim 49, wherein the immobilized selected from a group consisting of blood, urine, saliva, mimetope mimics the native antigen epitope recognized by an lacrimal fluid, Sweat, and amniotic fluid. antibody. 41. The method of claim 40, wherein the biological fluid is 52. The method of claim 47, wherein the at least one test blood. line is upstream of the at least one control line. 53. The method of claim 47, wherein the at least one test 42. The method of claim 30, wherein the conjugate pad line is downstream of the at least one control line. comprises a detectable marker. 54. The method of claim 47, wherein at least one control 43. The method of claim 42, wherein the detectable marker line comprises a light chain antibody. is selected from a group consisting of particles, luminescent 55. The method of claim 54, wherein the light chain anti labels, calorimetric labels, fluorescent labels, chemical body is an anti-kappa light chain antibody. labels, enzymes, radioactive labels, metal colloids, and 56. The method of claim 54, wherein the light chain anti chemiluminescent labels. body is an anti-lambda light chain antibody. 44. The method of claim 42, wherein the detectable marker 57. The method of claim 47, wherein at least one control comprises gold colloidal spheres. line comprises an antibody that binds directly to the second ary antibody of the conjugate pad. 45. The method of claim 42, wherein the detectable marker 58. The method of claim 30, further comprising a wicking comprises a secondary antibody. pad. 46. The method of claim 45 wherein the secondary anti 59. A kit comprising: (a) a test device for determining the body is conjugated to gold. integrity of a biologic, the test device comprising: (i) a sample 47. The method of claim 30, wherein the test membrane receiving pad, (ii) a conjugate pad, (iii) a test membrane, the comprises at least one test line and at least one control line. test membrane further comprising at least one test line and at 48. The method of claim 47, wherein the test membrane least one control line; and (b) instructions for use thereof. comprises two or more test lines. 60. The kit of claim 59, further comprising a biologic 49. The method of claim 47, wherein the at least one test sample. line comprises an immobilized mimetope. 61. The kit of claim 59, further comprising a biologic 50. The method of claim 48, wherein each test line contains applicator. an immobilized mimetope different from another test line.