Compositions and methods for detection of contaminant enzymes

Abstract

Compositions for the detection of contaminant enzymes are provided. In particular, oligonucleotide primers are provided, wherein the primer is conjugated to a blocking moiety at its 3'end, wherein the blocking moiety comprises a cleavage site for an enzyme, and wherein cleavage at the cleavage site releases the blocking moiety and produces a 3' hydroxyl group on the oligonucleotide primer. Also provided is an oligonucleotide primer comprising a cleavable moiety at its 5' end, wherein the cleavable moiety is also conjugated to an oligonucleotide tail. Also provided are methods for making the disclosed oligonucleotide primers and methods for using the oligonucleotide primers to detect a contaminant enzyme in a sample.

Description

COMPOSITIONSAND METHODS FOR DETECTION OF CONTAMINANT ENZYMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application Serial No. 63 / 631,847, filed April 9, 2024, the content of which is incorporated herein by this reference as if fully set forth herein.BACKGROUND

[0002] Antibody therapies have emerged as an important class of therapeutics, exhibiting both efficacy and safety in the treatment of diseases, such as cancer. As of 2021, more than 125 antibodies have been approved, and five of the top ten selling drugs in 2019 were antibodies, with an additional two drugs being fusion proteins. Biotherapeutic protein formulations require non-ionic detergents to stabilize their formulations. The most common non-ionic detergents are Polysorbate-20 and Polysorbate-80, commonly referred to as Tween- 20 and Tween-80. Although polysorbates stabilize formulations, these non-ionic detergents are also known to be degraded, both chemically and enzymatically, which negatively impacts product quality and / or the shelf life of the biotherapeutic protein. Enzymatic hydrolysis of polysorbates has been identified as a significant problem in biotherapeutic formulations. The primary enzymes responsible for the enzymatic degradation of polysorbates in biotherapeutic formulations have been identified as a series of lipases and esterases. The high activity of these enzymes, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. Detecting the presence of these enzymes is therefore crucial to determining the stability and shelf-life of a biotherapeutic. Currently available methods for detection of these contaminating enzymes, which can be expensive and time- and labor- intensive, include one or more of proteomic analysis, high performance liquid chromatography (HPLC), reverse phase high performance liquid chromatography (RP- HPLC), enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS), bicinchoninic acid assay (BCA). and assays using fluorescence micelle-based approaches or thiocyanate complexation.BRIEF SUMMARY OF THE DISCLOSURE

[0003] Oligonucleotide primers conjugated to a blocking moiety at its 3’ end are provided. The blocking moiety comprises a cleavage site for an enzy me, wherein cleavage at the cleavage site releases the blocking moiety and produces a 3?hydroxyl group on the oligonucleotide primer. In some embodiments, the blocking moiety comprises structure I:wherein R comprises a phospholipid or hy drocarbon chain. In some embodiments, the cleavage site for the enzyme comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site. In some embodiments, the oligonucleotide primer is conjugated to the blocking moiety by an ester linkage. In some embodiments, the blocking moiety comprises polyethylene glycol. In certain embodiments, the blocking moiety comprises cholesterol-triethylene glycol (TEG). In some embodiments, the oligonucleotide primer further comprises an oligonucleotide tail. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise anon-standard base. In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5’ end. In some embodiments, the first capture moiety comprises a first affinity tag. In certain embodiments, the first affinity tag comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the blocking moiety comprises a second capture moiety at its 3’ end. In some embodiments, the second capture moiety comprises a second affinity tag. In certain embodiments, the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

[0004] Also provided is a reaction mixture comprising a target nucleic acid and an oligonucleotide primer conjugated to a blocking moiety at its 3’ end. In some embodiments, the oligonucleotide tail comprises a nucleic acid sequence that is not complementary to the target nucleic acid, and the oligonucleotide primer comprises a nucleic acid sequence that is complementary' to the target nucleic acid. In some embodiments, the reaction mixture comprises two or more of the disclosed oligonucleotide primers, wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a first contaminantenzyme, and wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a second contaminant enzyme.

[0005] Methods for detecting a contaminant enzyme in a sample are also provided. The methods comprise: providing a sample having or suspected of having the contaminant enzyme: producing a reaction mixture by mixing the sample with a target nucleic acid and an oligonucleotide conjugated to a blocking moiety’ at its 3’ end, wherein the oligonucleotide primer comprises at its 5’ end a first nucleic acid sequence that is not complementary' to the target nucleic acid and at its 3’ end a second nucleic acid sequence that is complementary to a portion of the target nucleic acid, wherein the blocking moiety7comprises a cleavage site for the contaminant enzyme, wherein cleavage at the cleavage site releases the blocking moiety and produces an oligonucleotide primer having a 3’ hydroxyl group, and wherein the blocking moiety comprises structure I:(I) wherein R is a phospholipid or hydrocarbon chain; extending the oligonucleotide primer with a polymerase using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid, and extending a reverse primer that anneals to the reverse complement of the target nucleic acid to produce a second strand product; amplifying the first and second strand products using a forward primer and the reverse primer, wherein the forward primer comprises the first nucleic acid sequence of the oligonucleotide; and detecting the presence of the first or second strand product; wherein the presence of the first or second strand product indicates that the contaminant enzyme is present in the sample.

[0006] In some embodiments, the reaction mixture comprises two or more of the oligonucleotide primers, wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a first contaminant enzyme, and wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a second contaminant enzyme. In some embodiments, the contaminant enzy me is a lipase, phospholipase, or esterase. In some embodiments, the forward primer comprises at least one synthetic nucleotide that increases the Tm of the forw ard primer. In some embodiments, the concentration of theforward primer in the amplification reaction is higher than the concentration of the oligonucleotide primer. In other embodiments, the forward primer comprises at least one synthetic nucleotide that increases the Tm of the forward primer, and the concentration of the forward primer in the amplification reaction is higher than the concentration of the oligonucleotide primer. In some embodiments, the first or second strand product is detected with a probe. In some embodiments, the first or second strand product is detected with an intercalating dye. In some embodiments, the method detects a single molecule of the contaminant enzyme. In some embodiments, the amplifying step comprises a PCR reaction. In other embodiments, the amplifying step comprises isothermal amplification. In some embodiments, the amplifying step is performed in a droplet. In some embodiments, the cleavage site for the enzyme comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site. In some embodiments, the oligonucleotide primer is conjugated to the blocking moiety by an ester linkage. In some embodiments, the blocking moiety comprises polyethylene glycol. In certain embodiments, the blocking moiety comprises cholesterol-triethylene glycol (TEG). In some embodiments, the oligonucleotide primer further compnses an oligonucleotide tail. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise anon-standard base. In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5’ end. In some embodiments, the first capture moiety comprises a first affinity tag. In certain embodiments, the first affinity tag comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the blocking moiety comprises a second capture moiety at its 3’ end. In some embodiments, the second capture moiety comprises a second affinity tag. In certain embodiments, the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

[0007] Also provided are methods for producing an oligonucleotide primer conjugated to a blocking moiety at its 3’ end. The methods comprise: providing an oligonucleotide primer comprising a first nucleic acid sequence that is not complementary to a target nucleic acid and a second nucleic acid sequence that is complementary to a portion of the target nucleic acid;conjugating the oligonucleotide primer with a blocking moiety comprising a cleavage site for an enzyme, wherein cleavage at the cleavage site releases the blocking moiety and produces an oligonucleotide primer having a 3’ hydroxyl group, and wherein the blocking moiety of the oligonucleotide primer comprises structure I:wherein R is a phospholipid or hydrocarbon chain.

[0008] In some embodiments, the cleavage site for the enzyme compnses a lipase cleavage site, phospholipase cleavage site, or esterase cleavage site. In some embodiments, the oligonucleotide primer is conjugated to the blocking moiety by an ester linkage. In some embodiments, the blocking moiety comprises polyethylene glycol. In certain embodiments, the blocking moiety comprises cholesterol-triethylene glycol (TEG). In some embodiments, the oligonucleotide primer further comprises an oligonucleotide tail. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA. In other embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise anon-standard base. In some embodiments of the methods, the conjugated oligonucleotide primers are purified from unconjugated oligonucleotides and unconjugated blocking moieties. In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5’ end. In some embodiments, the first capture moiety comprises a first affinity' tag. In certain embodiments, the first affinity tag comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the blocking moiety comprises a second capture moiety at its 3’ end. In some embodiments, the second capture moiety comprises a second affinity tag. In certain embodiments, the second affinity tag comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the methods further comprise purifying conjugated oligonucleotide primers by, after the conjugating step, adding a bead conjugated to a second binding moiety that binds the second capture moiety, allowing the binding of the second binding moiety and second capture moiety, isolating the beads, and releasing the oligonucleotide primers bound to the beads with or without the capture moiety. In some embodiments, the methods further comprise purifying conjugated oligonucleotide primers by after the conjugating step, adding a bead conjugated to a first binding moiety that binds the first capture moiety, allowing the binding of the first bindingmoiety and first capture moiety, removing blocking moieties not bound to the beads, removing the beads from the reaction mixture, and releasing the oligonucleotide primers bound to the beads with or without the capture moiety.

[0009] Also provided is an oligonucleotide primer conjugated to a cleavable moiety7at its 5’ end, wherein the cleavable moiety is further conjugated to an oligonucleotide tail. The cleavable moiety comprises a cleavage site for an enzyme, wherein cleavage at the cleavage site releases the oligonucleotide primer from the cleavable moiety. In some embodiments, the cleavable moiety comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site. In some embodiments, the oligonucleotide tail, the oligonucleotide primer, or both are conjugated to the cleavable moiety by an ester linkage. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise a nonstandard base. In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5' end. In some embodiments, the first capture moiety comprises a first affinity tag. In certain embodiments, the first affinity tag comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the oligonucleotide tail is attached to a bead by binding of the first affinity tag with a binding moiety conjugated to the bead. In some embodiments, the oligonucleotide primer comprises a second capture moiety at its 3’ end. In some embodiments, the second capture moiety comprises a second affinity tag. In certain embodiments, the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

[0010] Also provided is a reaction mixture comprising a target nucleic acid and an oligonucleotide primer conjugated to a cleavable moiety at its 5’ end, wherein the cleavable moiety is further conjugated to an oligonucleotide tail, and wherein the oligonucleotide tail is attached to a bead. In some embodiments, the oligonucleotide primer comprises a nucleic acid sequence that is complementary to the target nucleic acid, and the oligonucleotide tail comprises a nucleic acid sequence that is not complementary to the target nucleic acid.

[0011] Methods for detecting a contaminant enzyme in one or more samples are also provided. The methods comprise:providing one or more samples having or suspected of having the contaminant enzyme; producing a first reaction mixture for each sample by mixing the sample with a target nucleic acid and an oligonucleotide primer conjugated to a cleavable moiety at its 5’ end, wherein the cleavable moiety is further conjugated to an oligonucleotide tail, wherein the oligonucleotide tail is attached to a bead, wherein the oligonucleotide tail comprises a nucleic acid sequence that is not complementary to the target nucleic acid, wherein the oligonucleotide primer comprises a nucleic acid sequence that is complementary to a portion of the target nucleic acid, wherein the cleavable moiety comprises a cleavage site for the contaminant enzy me, wherein cleavage at the cleavage site releases the oligonucleotide primer; extending the oligonucleotide primer with a polymerase using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid, thereby producing a double stranded target nucleic acid attached to the beads; washing the beads and discarding the supernatant; optionally producing a second reaction mixture by adding the target nucleic acid to each first reaction mixture and, if the oligonucleotide primer was not extended in the first reaction mixture, extending the oligonucleotide primer with a polymerase using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid, thereby producing double stranded target nucleic acid attached to the beads; washing the beads and discarding the supernatant; combining the washed beads from the first or second reaction mixtures for one or more samples; cleaving the cleavage moiety7at the cleavage site, releasing the double stranded target nucleic acid from the beads; amplifying the double stranded target nucleic acid using a forward primer and reverse primer, thereby producing an amplification product; and detecting the presence or concentration of the amplification product; wherein the presence of or concentration of the amplification product that is lower than a control sample indicates that the contaminant enzy me is present in one or more of the samples.

[0012] In some embodiments, the contaminant enzyme is a lipase, phospholipase, or esterase. In some embodiments, the amplification product is detected with a probe. In someembodiments, the amplification product is detected with an intercalating dye. In some embodiments, the method detects a single molecule of the contaminant enzyme. In some embodiments, the amplifying comprises a PCR reaction. In other embodiments, the amplify ing comprises isothermal amplification. In some embodiments, the amplifying is performed in a droplet. In some embodiments, the cleavage site for the enzyme comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site. In some embodiments, the oligonucleotide tail, oligonucleotide primer, or both are conjugated to the cleavable moiety by an ester linkage. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both comprise anon-standard base. In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5' end. In some embodiments, the first capture moiety comprises a first affinity tag. In certain embodiments, the first affinity tag comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the oligonucleotide tail is attached to a bead by binding of the first affinity tag with a binding moiety conjugated to the bead. In some embodiments, the oligonucleotide primer comprises a second capture moiety at its 3’ end. In some embodiments, the second capture moiety comprises a second affinity tag. In certain embodiments, the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

[0013] Also provided are methods for producing an oligonucleotide primer conjugated to a cleavable moiety at its 5’ end, wherein the cleavable moiety is further conjugated to an oligonucleotide tail. The methods comprise: providing an oligonucleotide primer comprising a nucleic acid sequence that is complementary to a portion of the target nucleic acid; providing an oligonucleotide tail attached to a bead, wherein the oligonucleotide tail comprises a nucleic acid sequence that is not complementary to the target nucleic acid; conjugating the oligonucleotide primer at its 5’ end to a cleavable moiety comprising a cleavage site for an enzyme; conjugating the oligonucleotide tail at its 3’ end to the cleavable moiety, wherein cleavage at the cleavage site releases the oligonucleotide primer.In some embodiments, the cleavable moiety comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a schematic illustration of an embodiment of the oligonucleotide primer with a blocking moiety having a lipase cleavage site. The middle panel provides an enlarged view of the 3’ region of the primer from the left panel, showing the oligonucleotide conjugated to a blocking moiety. The R group may be for example, a phospholipid or hydrocarbon chain. The right panel provides an enlarged view of the primer after cleavage by a lipase enzyme, showing that cleavage of the blocking moiety results in a free hydroxyl group at the 3’ end.

[0015] FIGS. 2A-2B provide a schematic diagram of methods of detecting a contaminating enzyme in a sample, using the DNA conjugate of FIG. 1. FIG 2A shows the first steps of the methods, including the binding of the primer produced in the right panel of FIG. 1A. upon cleavage of its blocking moiety, and amplification of a target nucleic acid. FIG. 2B starts with the product from the bottom panel of FIG. 2A and shows additional amplification steps with a forward primer that binds to a portion of the primer sequence and the reverse primer. The amplification product is detected with a probe or beacon or by use of an intercalating dye. In some embodiments, a probe may, as shown in this figure, include a quencher (Q) and fluorophore (F).

[0016] FIGS. 3A-3B are schematic diagrams depicting the conjugation of the blocking moiety to an oligonucleotide. The figures use lipase as an example of a contaminating enzyme. FIG. 3A shows the successful conjugation, which results in a primer that is unable to initiate amplification unless a contaminating enzyme is present and cleaves the blocking moiety to produce a free hydroxyl group on the 3’ end. FIG. 3B shows an unsuccessful conjugation, which leaves the OH on the 3’ end even when a contaminating enzy me is not present.

[0017] FIGS. 4A-4B are schematic diagrams depicting embodiments of methods to conjugate an oligonucleotide to a blocking moiety and to purify the conjugated oligonucleotides using a single tag system. The figures use biotin as an example of a tag that may be used. FIG. 4 A shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the blocking moiety. FIG 4B shows that oligonucleotides thathave not been conjugated will not have the tag for selection because the blocking moiety is not present.

[0018] FIGS. 5 A-5B are schematic diagrams for methods for using a two tag system to purify the conjugated oligonucleotides. Different tags are used on the oligonucleotide and on the blocking moiety (substrate). FIG. 5A shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the blocking moiety to remove free unconjugated oligonucleotides. FIG 5B shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the oligonucleotide to remove free blocking moieties.

[0019] FIGS. 6A-6B are schematic diagrams for methods for detecting a contaminating enzyme in a sample in a bulk reaction. The top panel of FIG. 6A shows a representative bulk design primer attached to a magnetic bead, with an oligonucleotide tail, a cleavable moiety, and an oligonucleotide primer on its 3’ end with a template binding sequence (reporter tag). In the shown exemplary embodiment, the oligonucleotide tail includes a biotin tag (b) at its 5’ end and is attached to the magnetic bead by interaction of the biotin with a streptavidin moiety (SA) on the magnetic bead. The middle and bottom panels of FIG. 6A show the first steps of the bulk detection methods, including binding of the primer to a target molecule (T’, complement sequence), washing, generating template (T) attached to the bead, and washing. The top panel of FIG. 6B shows an additional step of binding the primer (any primer that was not extended in the previous step) to a target molecule, washing, and generating template attached to the bead. The middle panel of FIG. 6B shows the bulk cleavage of the cleavable moiety and release of the double stranded target nucleic acid from the magnetic bead. The bottom panel of FIG. 6B shows the amplification and quantification of the target nucleic acid (e.g., via ddPCR), including amplification steps with a forward primer (that binds downstream of the primer's binding sequence) and a reverse primer. The amplification product is detected with a probe or beacon or by use of an intercalating dye.

[0020] FIGS. 7A-7C demonstrate that the assay, DNA conjugate primers (structure described in FIG. 1), and template nucleic acid result in productive amplification signal in a detection assay. FIG. 7A shows results for a positive control using rare mutation detection standard oligonucleotide primers and Taqman probe. FIGS. 7B-7C show results obtained in the presence of an inner primer that was not blocked (FIG. 7B) or in the absence of an inner primer (FIG. 7C).

[0021] FIGS. 8A-8D show the amplification signal observed in the detection assay using a DNA conjugate primer. FIGS. 8A-8B demonstrate that background signal increases with higher concentrations of a DNA conjugate primer. FIG. 8C provides results of the assay in the presence of the enzyme that cleaves the blocking moiety (alkaline phosphatase), and FIG. 8D show results for the negative control (no enzyme).

[0022] FIG. 9 is a graph showing results of methods of detecting a contaminating enzyme with a DNA conjugate over time. The results are shown for detection assays conducted in the presence of varying known concentrations of phospholipase D (PLD). The lines from top to bottom in the figure represent results obtained in the presence of PLD at a concentration of 0.1 U / well PLD (solid top line), 0.01 U / well PLD (dashed line), 0.001 U / well PLD (dotted line), and no PLD (solid bottom line), respectively.

[0023] FIG. 10 demonstrates the ability of the assay to detect the presence of 0. 1 U PLD in a sample (top panel) over time. The lower panel shows the background signal in the absence of PLD.

[0024] FIGS. 11 A- 1 IB are graphs demonstrating that cleavage of a phospholipase specific inner primer (structure described in FIG. 1) generates an increase in overall signal in the detection assay. FIG. 11A shows results for the titration of the primer in the absence (left bar in each pair) or presence of PLD (right bar in each pair). FIG. 1 IB shows that background signal increases with the concentration of the inner primer.

[0025] FIGS. 12A-12B are schematic diagrams showing an embodiment for the targeted elimination of free inner primer (primer that has not been conjugated successfully to the blocking moiety (R)) during a pre-amplification reaction. FIG. 12A depicts that in the absence of an enzyme that cleaves the blocking moiety from the DNA conjugate inner primer, extension from the primer is blocked (left panel). For free inner primer (not conjugated to the blocking moiety ), extension from the primer is not blocked, creating background signal in the detection assay. FIG. 12B demonstrates the use of a scavenger template to remove free inner primer in a pre-amplification reaction. The scavenger template binds and extends the free inner primer on a template that cannot bind the detection probe, resulting in the consumption of the free inner primer before the amplification with the target DNA template.

[0026] FIG. 13 is a graph showing that the concentration of inner primer impacted the amount of background in the assay’. The concentration of inner primer is shown, ranging from50-2200 nM. The number of scavenger template copies used in each assay is also shown on the x axis (0, 30, 50, or 70 copies).

[0027] FIGS. 14A-14B demonstrate the result of different purification methods of exemplary oligonucleotide conjugate primers.

[0028] FIG. 15 demonstrates that washing the beads bound with 5 ’-biotinylated primers before the detection assay resulted in decreased background noise. The beads were washed with Tris-EDTA buffer (left panel) or high salt buffer (right panel).

[0029] FIG. 16 shows the effect of different primer designs on the background observed in the detection assay. The results are shown for an unblocked primer (right panel), a blocked primer (FIG. 1) with no additional nucleotide bases added downstream of the blocking moiety (middle panel), or a blocked primer that includes two non-complementary bases downstream of the blocking moiety.DETAILED DESCRIPTIONIntroduction

[0030] Compositions and methods for detecting contaminant enzy mes in biotherapeutic protein formulations are provided. Biotherapeutic protein formulations require non-ionic detergents (e.g., Polysorbate-20 and Polysorbate-80, commonly referred to as Tween-20 and Tween-80) to stabilize their formulations (See, e.g., Strickley & Lambert, 2021, J. Pharm. Sci. 110:2590-2608). However, degradation of polysorbates by contaminating enzy mes, including host cell proteins (HCPs), such as lipases and esterases, can lead to particle formation and can negatively impact the shelf life of a biotherapeutic (Dwivedi et al., 2018, Int’l. J. Pharmaceutics 552:422-436; Kishore et al., 2011, Pharm. Res. 28: 1194-1210; Wuchner et al. , 2022, J. Pharm. Sci. Il l : 1280-1291). Therefore, detecting the presence of these enzymes in a biotherapeutic protein formulation (during development and commercialization) is crucial to determining the stability and shelf-life of the biotherapeutic. Current detection methods for detecting contaminant enzymes can be expensive and timeconsuming. Moreover, the current methods are not sensitive enough to detect the enzymes when present at lower concentrations (Li et al., 2021, Anal. Chem. 93:8161-8169).

[0031] The present disclosure provides primers that include an oligonucleotide conjugated to a substrate for a targeted enzyme. A system was developed in which the oligonucleotide is cleaved from the substrate by a contaminating enzyme, thus enabling the oligonucleotide toact as a primer to generate and amplify a signal for detection when the enzyme is present in a sample. In this way, the system is extremely sensitive and only requires a single active enzyme in the sample to initiate a cascade reaction in which a sufficient signal can be generated for detection. The methods disclosed herein provide a faster, higher throughput, less expensive, and more sensitive analysis of shelf-life stability of samples than what is possible with currently available methods.Terminology

[0032] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory' Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document.

[0033] The terms “a.” “an.” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a primer” includes a plurality' of such primers and reference to “the sequence” includes reference to one or more sequences known to those skilled in the art. and so forth.

[0034] An “oligonucleotide” is a polynucleotide. Generally, oligonucleotides will have fewer than 250 nucleotides, in some embodiments, between 5-200, e.g., 10-100 nucleotides or between 30 and 50 nucleotides. A “polynucleotide” or “nucleic acid” includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA); DNA molecules produced by amplification; or synthetically produced DNA or RNA molecules. The terms include chimeric molecules and molecules comprising non-standard bases, modifications, or nucleotide analogs. For example, an oligonucleotide may contain naturally occurring nucleotides and / or analogs thereof. Polynucleotides may be single-stranded or double-stranded, or have both single-stranded and double-stranded regions (e.g., hairpins).

[0035] A “primer’" refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths and are often less than 150 nucleotides in length, for example 10-50 nucleotides, in length. The length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art, see, e.g., PCR Protocols: A Guide to Methods and Applications (Innis et al., eds.. 1990). Primers can be DNA, RNA, or a chimera of DNA and RNA portions. In some cases, primers can include one or more modified or nonnatural nucleotide bases. In some cases, primers are labeled.

[0036] A nucleic acid, or a portion thereof, “hybridizes” or “anneals” to another nucleic acid under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mM chloride salt). In some cases, a nucleic acid, or portion thereof, hybridizes to a conserved sequence shared among a group of target nucleic acids. In some cases, a primer, or portion thereof, can hybridize to a primer binding site if there are at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous complementary nucleotides, including “universal” nucleotides that are complementary to more than one nucleotide partner. Alternatively, a primer, or portion thereof, can hybridize to a primer binding site if there are fewer than 1 or 2 complementarity mismatches over at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous complementary nucleotides. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C. In some embodiments, the defined temperature at which specific hybridization occurs is 37, 40. 42. 45. 50, 55, 60, 65, 70, 75, or 80 °C.

[0037] A “template” or “target nucleic acid” refers to a polynucleotide sequence that comprises the polynucleotide to be copied or amplified, flanked by or a pair of primer hybridization sites. Thus, a “target template” comprises the target polynucleotide sequence adjacent to at least one hybridization site for a primer. In some cases, a “target template” comprises the target polynucleotide sequence flanked by a hybridization site for a “forward” primer and a "‘reverse” primer.

[0038] As used herein, “nucleic acid” means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates). 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3’ and 5’ modifications including but not limited to capping with a blocking moiety, a fluorophore (e.g., quantum dot), or another moiety. As used herein, the terms “reverse complement” or “reverse complementary sequence” of a particular nucleic acid refer to a sequence that has the complementary nucleotide at all or substantially all positions of the nucleic acid and, therefore, specifically binds to the nucleic acid.

[0039] The term “blocking moiety ” refers to a molecule that is conjugated to the 3’ end of an oligonucleotide and blocks the 3 ’ hydroxyl group of the oligonucleotide. The blocking moiety, when present, prevents the oligonucleotide from initiating polymerization of a nucleic acid strand. Any method known in the art for conjugating a nucleic acid to a blocking moiety or to a label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego. The term “cleavable moiety” refers to a moiety that includes a target site that is recognized and cleaved by an enzyme.

[0040] The term “sample” refers to a solution that includes or is suspected of including a contaminant enzyme or chemical. For example, a sample may be a biotherapeutic protein formulation. As used herein, a “biotherapeutic protein,” biotherapeutic,” “therapeutic protein,” or the like refers to a protein that may be administered to a subject to achieve a therapeutic benefit to the subject. A biotherapeutic protein may be, for example, an antibody, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, Fc fusion protein, growth factor, hormone, interferon, interleukin, or thrombolytic. An “enzyme” refers to a catalytic protein that facilitates a biochemical reaction such as, butnot limited to, bond cleavage, bond formation, oxidation-reduction, group transfers within the molecules or between the molecules, hydrolysis, isomerization, and ligation. In some cases, the biotherapeutic protein formulation includes a contaminant host cell enzyme that affects the stability of the biotherapeutic.

[0041] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which at least one amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L- amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0042] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0043] The term “antibody,” as used herein, includes antibody fragments that retain binding specificity. For example, there are a number of well characterized antibody fragments. Thus, for example, pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)52, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab’ty dimer into an Fab’ monomer. The Fab’ monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed.. Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.

[0044] The term “amplification reaction” refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid in a linear or exponential manner. Such methods include but are not limited to polymerase chain reaction (PCR); DNA ligase chain reaction(see U.S. Pat Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds.. 1990)) (LCR); QBeta RNA replicase and RNA transcriptionbased amplification reactions (e.g, amplification that involves T7, T3, or SP6 primed RNA polymerization), such as the transcription amplification system (TAS), nucleic acid sequence based amplification (NASBA), and self-sustained sequence replication (3SR); isothermal amplification reactions (e.g.. single-primer isothermal amplification (SPIA)); as well as others known to those of skill in the art.

[0045] '‘Amplifying” refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide if all of the components of the reaction are intact. Components of an amplification reaction include, e.g., primers, a polynucleotide template, polymerase, nucleotides, and the like. The term “amplifying” typically refers to an “exponential” increase in target nucleic acid. However, “amplifying” as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, such as is obtained with cycle sequencing or linear amplification. In an exemplary embodiment, amplifying refers to PCR amplification using a first and a second amplification primer (e.g., a forward and a reverse primer). In some embodiments, an amplification primer introduces a heterologous sequence to the amplification product. In an exemplary embodiment, the amplification primer introduces a universal adapter sequence to the amplification product to facilitate sequencing of the amplification product. In some embodiments, multiple pairs of forward and reverse amplification primers are used and may be referred to as outer and inner primers, depending on the location of the binding site for the primer with respect to each other.

[0046] “Polymerase chain reaction” or “PCR” refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications. Innis et al., eds, 1990). Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by ahybridization / elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

[0047] A “polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides, e.g., DNA and / or RNA. The term encompasses both the full-lengthpolypeptide and a domain that has polymerase activity. DNA polymerases are well-known to those skilled in the art. including but not limited to DNA polymerases isolated or derived om Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerase enzymes include, but are not limited to: Klenow fragment (New England Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9 °N™ DNA polymerase (New England Biolabs® Inc.), Deep Vent™ DNA polymerase (New England Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNA polymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (New England Biolabs® Inc.). Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA- dependent and RNA-dependent.

[0048] The term “about’7refers to the usual error range for the respective value that is know n by a person of ordinary’ skill in the art for this technical field, for example, a range of ± 10%, ± 5%, or ± 1% can encompass the recited value, even if the recited value is not modified by the term “about.”

[0049] It w ill be understood that any range of numerical values disclosed herein can include the endpoints of the range, and any values or sub-ranges in between the endpoints. For example, a range of 1 to 10 includes a range from 2 to 9, 3 to 8, 4 to 7, 5 to 6, 1 to 5, 2 to 5, 2 to 10, 3 to 10, and so on. The values typically include one significant digit.A. Oligonucleotide Primers

[0050] In some embodiments, the disclosure provides oligonucleotide primers that facilitate detection of a contaminating enzyme or chemical in a sample. The oligonucleotide primers include an oligonucleotide conjugated to a blocking moiety at its 3’ end, and the blocking moiety has a cleavage site for a contaminating enzyme (FIG. 1). The oligonucleotide primers also may be referred to herein as nucleic acid conjugate primers or oligonucleotide conjugate primers. When the contaminating enzy me is present in a sample, it produces a primer capable of initiating polymerization, by cleaving the blocking moiety from the oligonucleotide and liberating a hydroxyl group at the 3’ end of the primer.

[0051] In some embodiments, the oligonucleotide is between about 5 to 200 nucleotides in length. Therefore, the oligonucleotide is about 5, 10, 15, 20, 25, 30. 35. 40. 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides, or any length in between. In certain embodiments, the oligonucleotide is about 10 to 100 nucleotides. In certain embodiments, the oligonucleotide is about 30 to 50 nucleotides. In some embodiments, the oligonucleotide primer further comprises an oligonucleotide tail. In some embodiments, the oligonucleotide tail is between about 5 to 200 nucleotides in length. Therefore, the oligonucleotide tail is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides, or any length in between. In certain embodiments, the oligonucleotide tail is about 10 to 100 nucleotides. In certain embodiments, the oligonucleotide tail is about 30 to 50 nucleotides.

[0052] In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both include one or more DNA nucleotides. In certain embodiments, the oligonucleotide primer, oligonucleotide tail, or both consist solely of DNA nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both include one or more RNA nucleotides. In certain embodiments, the oligonucleotide primer, oligonucleotide tail, or both consist solely of RNA nucleotides. In some embodiments, the oligonucleotide primer, oligonucleotide tail, or both include a non-standard base.

[0053] The blocking moiety of the oligonucleotide primer includes a cleavage site for an enzyme or chemical. For example, the blocking moiety comprises structure I:(I) wherein R comprises a phospholipid or hydrocarbon chain.

[0054] In some embodiments, the enzyme that cleaves the blocking moiety is a lipase, phospholipase, or esterase. An esterase cleaves esters into an acid and an alcohol by hydrolysis of an ester bond. A lipase is a serine hydrolase enzyme that breaks down triglycerides into free fatty acids and glycerol by catalyzing the hydrolysis of the ester bonds in triglycerides. An ester is a compound derived from an acid in which the hydrogen atom (H) of at least one acidic hydroxyl group (-OH) of that acid is replaced by an organyl group or hydrocarbon chain ( R). The bond that forms between the oxygen and carbon atoms is referred to as an ester bond or ester linkage. A phospholipase is an enzy me that hydrolyzes phospholipid substrates at specific ester bonds.

[0055] A phospholipid is a lipid molecule that has a hydrophilic head containing a phosphate group and two hydrophobic tails derived from fatty acids, joined by an alcohol residue (e.g., a glycerol molecule). Examples of phospholipids include, but are not limited to phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA), cardiolipin (CL), sphingomyelin (SM), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2). and phosphatidylinositol trisphosphate (PIP3).

[0056] As used herein, a hydrocarbon chain refers to a compound consisting entirely of hydrogen and carbon. A hydrocarbon chain may include saturated, unsaturated, or aromatic hydrocarbons, or combinations thereof. For example, a hydrocarbon chain may include an alkane, alkene, alkyne, cycloalkane, or alkadiene. In some embodiments, the R group includes a linear or branched alkyl group comprising one or more carbon atoms, which is a monovalent group derived by removing an arbitrary hydrogen atom from an aliphatic hydrocarbon consisting of one or more carbons. Non-limiting examples of an alkyl group include a methyl group, an ethyl group, a 1 -propyl group, a 2-propyl group, a 2-methyl-l- propyl group, a 2-methyl-2-propyl group, a I -butyl group, a 2-butyl group, a 1 -pentyl group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-l -butyl group, a 3-methyl-l -butyl group, a 2- methyl-2-butyl group, a 3-methyl-2-butyl group, a 2,2-dimethyl-l -propyl group, a 1 -hexyl group, a 2-hexyl group, a 3-hexyl group, a 2-methyl-l -pentyl group, a 3-methyl-l -pentyl group, a 4-methyl-l -pentyl group, a 2-methyl-2-pentyl group, a 3-methyl-2-pentyl group, a 4- methyl-2-pentyl group, a 2-methyl-3 -pentyl group, a 3-methyl-3-pentyl group, a 2.3- dimethyl-I -butyl group, a 3,3-dimethyl-l-butyl group, a 2,2-dimethyl-l -butyl group, a 2- ethyl-l-butyl group, a 3,3-dimethyl-2-butyl group, or a 2,3-dimethyl-2-butyl group. In some embodiments, the R group includes a linear or branched alkenyl group comprising two or more carbons. Non-limiting examples of an alkenyl group include a vinyl group, an allyl group, a 1 -propenyl group, a 2-propenyl group, a 1-butenyl group, a 2-butenyl group, a 3- butenyl group, a pentenyl group, or a hexenyl group. In some embodiments, the R group includes a linear or branched alkynyl group comprising two or more carbons. Specifically, the R group may include, for example, an ethynyl group, a 1-propynyl group, a 2-propynyl group, a butynyl group, a pentynyl group, or a hexynyl group. In some embodiments, the R group includes an oxy group to which an alkyd group is linked. Specifically, the alkoxy group includes, for example, a methoxy group, an ethoxy group, a 1 -propyloxy group, a 2- propyloxy group, a 2-methyl-l -propyloxy group, a 2-methyl-2-propyloxy group, a 1 -butyloxygroup, a 2-butyloxy group, a 1 -pentyloxy group, a 2-pentyloxy group, a 3-pentyloxy group, a 2-methyl-l -butyloxy group, a 3-methyl- 1 -butyloxy group, a 2-methyl-2 -butyloxy group, a 3- methyl-2-butyloxy group, a 2, 2-dimethy 1-1 -propyloxy group, a 1 -hexyloxy group, a 2- hexyloxy group, a 3-hexyloxy group, a 2-methyl-l -pentyloxy group, a 3-methyl- 1 -pentyloxy group, a 4-methyl-l -pentyloxy group, a 2-methyl-2-pentyloxy group, a 3-methyl-2-pentyloxy group, a 4-methyl-2-pentyloxy group, a 2-methyl-3-pentyloxy group, a 3-methyl-3-pentyloxy group, a 2,3-dimethyl-l -butyloxy group, a 3, 3-dimethyl-l -butyloxy group, a 2.2-dimethyl-l- butyloxy group, a 2-ethyl- 1 -butyloxy group, a 3,3-dimethyl-2-butyloxy group, or a 2,3- dimethyl-2-butyloxy group.

[0057] In some embodiments, the oligonucleotide primer is conjugated to the blocking moiety by an ester linkage. In some embodiments, the blocking moiety comprises polyethylene glycol. In certain embodiments, the blocking moiety comprises cholesteroltriethylene glycol (TEG).

[0058] In some embodiments, the oligonucleotide tail and / or the blocking moiety include an affinity tag, for example, to facilitate purification of the conjugated oligonucleotide primers by interactions of the affinity tag with its ligand. Examples of suitable combinations of affinity purification tags and their ligands include, but are not limited to. biotin: streptavidin, desthiobiotin: streptavidin polyhistidine:nickel, polyarginine: carboxypeptidase B, 1D4 sequence: 1D4 antibody, Maltose Binding Proteins: Amylose, Glutathione-S-Transferase:Glutathione, SUMO Tagsmickel, c-Myc:anti-myc antibody, Hemaglutinin antigen: HA antibody, FLAG: FLAG antibody. Caladium, Native or Engineered Protein A:IgG (See, e.g., Kimple et al. (2013, Curr. Protoc. Protein Sci. 73:Unit 9-9) for a review of affinity tags available for protein purification). In some embodiments, tandem affinity purification may be used to increase purity, including use of two tags with a cleavage site between them. In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5’ end. In some embodiments, the first capture moiety comprises an affinity tag. In certain embodiments, the first capture moiety comprises biotin, desthiobiotin, or streptavidin. In some embodiments, the blocking moiety comprises a second capture moiety at its 3‘ end. In some embodiments, the second capture moiety comprises a different affinity tag. In certain embodiments, the second capture moiety comprises biotin, desthiobiotin, or streptavidin.

[0059] Also provided is a reaction mixture comprising a target nucleic acid and an oligonucleotide primer conjugated to a blocking moiety at its 3’ end. The target nucleic acid may be any nucleic acid that is not cross-reactive to other components in the samples or methods. In some embodiments, the oligonucleotide tail comprises a nucleic acid sequence that is not complementary7to the target nucleic acid, and the oligonucleotide primer comprises a nucleic acid sequence that is complementary to the target nucleic acid. In some embodiments, the reaction mixture comprises two or more of the oligonucleotide primers, wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a first contaminant enzyme, and wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a second contaminant enzyme.

[0060] In another embodiment, provided is a bulk design oligonucleotide primer that includes a cleavable moiety conjugated to its 5’ end, and the cleavable moiety is further conjugated to a 3’ end of an oligonucleotide tail (FIG. 6A). The cleavable moiety comprises a cleavage site for an enzyme, wherein cleavage at the cleavage site releases the oligonucleotide primer. In some embodiments, the cleavage site for the enzyme comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site. In some embodiments, the oligonucleotide, oligonucleotide tail, or both are conj ugated to the blocking moiety by an ester linkage. The oligonucleotide, oligonucleotide tail, and cleavage site are as described earlier in this section.

[0061] Also provided is a reaction mixture comprising a target nucleic acid and an oligonucleotide primer conjugated to a cleavable moiety at its 5’ end, wherein the cleavable moiety is further conjugated to an oligonucleotide tail, and wherein the oligonucleotide tail is attached to a bead. The bead is described further below in Section C. In some embodiments, the oligonucleotide primer comprises a nucleic acid sequence that is complementary to the target nucleic acid, and the oligonucleotide tail comprises a nucleic acid sequence that is not complementary7to the target nucleic acid.B. Methods for Detecting Contaminant Enzymes

[0062] In some embodiments, the disclosure provides methods for detecting a contaminant enzyme by using the disclosed oligonucleotide primers. In some embodiments, the methods are for detecting two or more contaminant enzymes. In some embodiments, the sample to be analyzed using the oligonucleotide primer is a solution that includes or is suspected of including a contaminant enzy me. For example, a sample may be a biotherapeutic proteinformulation. A biotherapeutic protein may be, for example, an antibody, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, chimeric enzyme, fusion protein, Fc fusion protein, grow th factor, hormone, interferon, interleukin, or thrombolytic.

[0063] In some embodiments, the biotherapeutic protein is an antibody (e.g., a monoclonal antibody). Non-limiting examples of therapeutic monoclonal antibodies include, but are not limited to Datopotamab deruxtecan, Nemolizumab, Zanidatamab, Linvoseltamab, Axatilimab, Patritumab deruxtecan, Tarlatamab, Marstacimab, Garadacimab, Vilobelimab, Zolbetuximab, Odronextamab, Crovalimab, Camrelizumab, Serplulimab, Sugemalimab, Concizumab, Cosibelimab, Trastuzumab duocarmazine, Donanemab, Sintilimab, Narsoplimab, Pozelimab, Elranatamab, Rozanolixizumab, Talquetamab, Epcoritamab, Lebrikizumab. Glofitamab, Mirikizumab, Tislelizumab. Toripalimab. Retifanlimab, Lecanemab, Teplizumab, Ublituximab, Mirvetuximab soravtansine, Nirsevimab, Tremelimumab, Spesolimab, Teclistamab, Mosunetuzumab, Tixagevimab, cilgavimab, Relatlimab, Tebentafusp, Faricimab, Sutimlimab, Sotrovimab, Regdanvimab, Casirivimab + imdevimab, Tezepelumab. Tisotumab vedotin, Amivantamab, Anifrolumab, Loncastuximab tesirine, Bimekizumab, Tralokinumab, Evinacumab, Aducanumab, Dostarlimab, Ansuvimab, Margetuximab, Naxitamab, Atoltivimab, Maftivimab, and Odesivimab-ebgn, Belantamab mafodotin, Tafasitamab, Satralizumab, Inebilizumab, Sacituzumab govitecan, Teprotumumab, Isatuximab, Eptinezumab, [fam] -trastuzumab deruxtecan, Enfortumab vedotin, Crizanlizumab, Brolucizumab, Polatuzumab vedotin, Risankizumab, Romosozumab, Caplacizumab, Ravulizumab, Emapalumab, Cemiplimab, Fremanezumab, Moxetumomab pasudotox, Galcanezumab, Lanadelumab, Mogamulizumab, Erenumab, Tildrakizumab, Ibalizumab, Burosumab, Durvalumab. Emicizumab, Benralizumab. Ocrelizumab, Guselkumab. Inotuzumab. ozogamicin, Sarilumab, Dupiluma, Avelumab, Brodalumab, Atezolizumab, Bezlotoxumab, Olaratumab, Reslizumab, Obiltoxaximab, Ixekizumab, Daratumumab, Elotuzumab, Necitumumab, Idarucizumab, Alirocumab, Mepolizumab, Evolocumab, Dinutuximab, Secukinumab, Nivolumab, Blinatumomab, Pembrolizumab, Ramucirumab, Vedolizumab, Siltuximab, Obinutuzumab. Ado-trastuzumab emtansine, Raxibacumab, Pertuzumab, Brentuximab vedotin, Behmumab, Ipilimumab, Denosumab, Tocilizumab, Ofatumumab, Canakinumab, Golimumab, Ustekinumab, Certolizumab pegol, Catumaxomab, Eculizumab, Ranibizumab, Panitumumab, Natalizumab, Bevacizumab, Cetuximab, Efalizumab, Omalizumab, Tositumomab-1131, Ibritumomab tiuxetan,Adalimumab, Alemtuzumab, Gemtuzumab, ozogamicin, Trastuzumab, Infliximab, Palivizumab. Basiliximab, Daclizumab, Rituximab, Abciximab, Edrecolomab, Nebacumab. In some embodiments, the biotherapeutic protein is fusion protein. Non-limiting examples of therapeutic fusion proteins include etanercept, alefacept, rilonacept, anakinra, abatacept, romiplostim, and belatacept.

[0064] Biotherapeutic protein formulations are often high concentration liquid protein solutions and must be stable under pharmaceutically relevant storage conditions. These formulations typically contain at least a biotherapeutic protein (e.g., an antibody), an excipient to adjust tonicity or osmolality of the formulation, one or more buffers, and a surfactant. A surfactant is included in the formulation to inhibit aggregation, and to minimize surface adsorption at the air-water interface, the packaging container, and upon dilution into intravenous fluids. Examples of surfactants used in biotherapeutic protein formulations include, but are not limited to polysorbate-20, polysorbate-80, and polaxamer 188. Polysorbates are a heterogeneous mixture of partial esters of fatty acids with sorbitol and its anhydrides along with approximately 20 polymerized ethylene oxide moieties, ethoxylated to sorbitol and its anhydrides. Accordingly, polysorbates are targets for degradation by host cell proteins, such as lipases and esterases. In some embodiments, the contaminant enzyme in a sample (e.g., sample from a biotherapeutic protein formulation) is a lipase, phospholipase, or esterase.

[0065] In these detection methods, a sample that includes or is suspected of including a contaminant enzyme is mixed with a target nucleic acid and an oligonucleotide primer as described in the previous Section A or elsewhere herein. In some embodiments, the sample is mixed with the target nucleic acid and two or more of the disclosed oligonucleotide primers, wherein at least one oligonucleotide primer includes a cleavage site for a first contaminant enzyme, and wherein at least one oligonucleotide primer includes a cleavage site for a second contaminant enzyme. The target nucleic acid may comprise any nucleotide sequence. The oligonucleotide primer includes a nucleic acid sequence that is complementary to the target nucleic acid and a nucleic acid sequence that is not complementary to the target nucleic acid (oligonucleotide tail). If the contaminant enzyme is present in the sample, it will produce an unblocked primer by cleaving the blocking moiety and liberating a hydroxyl group on the 3’ end of the oligonucleotide. The blocked oligonucleotide primer and / or the unblocked oligonucleotide primer are allowed to bind to the target nucleic acid at the complementary region. Only the unblocked oligonucleotide primer is extended with a polymerase, using thetarget nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid (FIG. 2A). The two strands are denatured, and a reverse primer is allowed to bind to the first strand product and is extended to produce a second strand product that includes the target nucleic acid and a reverse complement of the oligonucleotide primer, including the oligonucleotide tail (FIG. 2A). The first and second strand products are denatured and amplified using a forward primer and the reverse primer (FIG. 2B). The forward primer comprises the nucleic acid sequence of the oligonucleotide tail. The first or second strand product is then detected by binding and subsequent degradation of a fluorescent probe or detecting the presence of an intercalating dye in the first or second strand product (FIG. 2B). The detection of the presence of the first or second strand product indicates that the contaminant lipase is present in the sample.

[0066] In some embodiments, the forward primer comprises at least one synthetic nucleotide that increases the Tm of the forw ard primer. In some embodiments, the forward primer includes at least one synthetic nucleotide selected from a group consisting of a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2’-O-methyl-RNA, nucleotides with phosphorothioate bond, bridged nucleic acid (BNA), propynyl-modified nucleotide, 2’- fluoro-RNA, morpholino, glycol nucleic acid (GNA), threose nucleic acid (TNA), and hexitol nucleic acid (HNA). In some embodiments, the forward primer when bound to its respective target sequence has an increased Tm that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more degrees higher when compared to a primer that does not include a synthetic nucleotide.

[0067] In some embodiments, the concentration of the forw ard primer in the amplification reaction is higher than the concentration of the oligonucleotide primer. In some embodiments, concentration of the forw ard primer is at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more times greater than the concentration of the oligonucleotide primer.

[0068] In some embodiments, the forward primer comprises at least one synthetic nucleotide that increases the Tm of the forward primer, and the concentration of the forw ard primer in the amplification reaction is higher than the concentration of the oligonucleotide primer.

[0069] In some embodiments, the amplification product is detected w ith a probe or beacon. The probe may include a nucleic acid sequence that is complementary to the first or second strand products. The probe may include an optically detectable label such as a fluorescentlabel. Numerous agents (e.g., dyes, probes, or indicators) are known in the art and can be used. See, e.g, Invitrogen, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition (2005). Fluorescent agents can include a variety of organic and / or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof. In some embodiments, the agent is a fluorophore. Many fluorophores have been reported in the literature, and many are readily available from commercial suppliers to the biotechnology industry. Literature sources for fluorophores include Cardullo et al., Proc. Natl. Acad. Set. USA 85: 8790-8794 (1988); Dexter, D.L., J. of Chemical Physics 21 : 836- 850 (1953); Hochstrasser et al. , Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40: 83- 114 (1971); Stryer, L. Ann. Rev. Biochem.. 47: 819-846 (1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); Wang et al., Anal. Chem. 67: 1197-1203 (1995). Non-limiting examples of fluorophores include cyanines, fluoresceins (e.g., 5’-carboxyfluorescein (FAM), Oregon Green, and Alexa 488), HEX, rhodamines (e.g., N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA). tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC)). eosin, coumarins, pyrenes, tetrapyrroles, arylmethines, oxazines, polymer dots, and quantum dots.

[0070] In some embodiments, the amplification product is detected with an intercalating dye. Intercalating agents produce a signal when intercalated in double stranded nucleic acids. Exemplary intercalating agents include e.g., 9-aminoacridine. ethidium bromide, a phenanthridine dye, EvaGreen, PICO GREEN (P-7581. Molecular Probes), EB (E-875 L Sigma), propidium iodide (P-4170, Sigma), Acridine orange (A-6014, Sigma), thiazole orange, oxazole yellow, 7-aminoactinomycin D (A-1310, Molecular Probes), cyanine dyes (e.g., TOTO, YOYO, BOBO, and POPO). SYTO. SYBR Green I (U.S. Pat. No. 5,436,134: N',N'-dimethyl-N-[4-[(E)-(3-methyl-l,3-benzothiazol-2-ylidene)methyl]-l-phenylquinolin-l- ium-2-yl]-N-propylpropane-l,3-diamine), SYBR Green II (U.S. Pat. No. 5,658,751), SYBR DX, OliGreen, CyQuant GR, SYTOX Green, SYTO9, SYTO10, SYTO17, SYBR14, FUN-1, DEAD Red, Hexidium Iodide, ethidium bromide, Dihydroethidium, Ethidium Homodimer, 9- Amino-6-Chloro-2-Methoxyacridine, DAPI, DIPI, Indole dye, Imidazole dye, Actinomycin D, Hydroxystilbamidine, LDS 751 (U.S. Pat. No. 6,210,885), and the dyes described in dyes described in Georghiou, Photochemistry and Photobiology, 26:59-68, Pergamon Press (1977); Kubota, et al., Biophys. Chem., 6:279-284 (1977); Genest, et al., Nuc. Ac. Res.,13:2603-2615 (1985); Asseline, EMBO J., 3: 795-800 (1984); Richardson, et. al., U.S. Pat.No. 4,257,774; and Letsinger, et. al., U.S. Pat. No. 4,547,569.

[0071] In some embodiments, the method detects a single molecule of the contaminant enzyme. The amplification step of the methods may include, for example, a PCR reaction. In some embodiments, the amplification step includes isothermal amplification. The amplification step may be performed, for example, in a droplet or microcell. In some embodiments, the droplet is a mixture of immiscible fluids (e.g., water and oil). In some embodiments, the droplet is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil). The methods may be performed on any suitable droplet digital PCR platform (e.g., Bio-Rad QX200, QX600, QX One, QXC, etc.).

[0072] In another embodiment, the detection method is performed on one or more samples and includes mixing each of the one or more samples with a target nucleic acid and a bulk design oligonucleotide primer as described herein that is conjugated to a cleavable moiety at its 5’ end. The cleavable moiety is conjugated to an oligonucleotide tail which is attached to a bead. The oligonucleotide primer is extended with a polymerase to produce a double stranded target nucleic acid attached to the bead. The beads are washed and the supernatant is discarded. If a contaminant enzyme is present in the sample, a cleavage site in the cleavable moiety is cleaved for at least a portion of the oligonucleotide primer conjugates. Therefore, in the presence of the contaminant enzyme, at least a portion of the produced double stranded target nucleic acids would not be attached to the bead and would be discarded with the supernatant. Optionally, target nucleic acid is added, and a second reaction is performed to produce additional double stranded target nucleic acids attached to the beads. The beads are washed, and the supernatant is discarded. The beads attached to double stranded target nucleic acids may then be combined with beads with double stranded target nucleic acids produced for one or more different samples. The cleavable moiety may be cleaved at the cleavage site, releasing the double stranded target nucleic acids from the beads. The released double stranded target nucleic acids may be amplified using a forward primer and reverse primer to produce an amplification product, and the presence or concentration of the amplification product can be detected. The presence of or concentration of the amplification product that is lower than a control sample indicates that the contaminant enzyme is present in one or more of the samples.C. Methods for Producing Oligonucleotide Conjugate Primers

[0073] The disclosure also provides methods for producing an oligonucleotide primer conjugated to a blocking moiety at its 3’ end (as described in Section A or elsewhere herein). An oligonucleotide primer is designed to include a first nucleic acid sequence that is not complementary' to a target nucleic acid and a second nucleic acid sequence that is complementary' to a portion of the target nucleic acid. The oligonucleotide primer is conjugated with a blocking moiety with a cleavage site for an enzyme. To reduce background in the detection assays, the conjugated oligonucleotide primer is purified from oligonucleotides and blocking moi eties that have not been conjugated.

[0074] To facilitate this purification, capture moieties may be added to the oligonucleotide and / or the blocking moiety (FIGS. 4A-4B, 5A-5B). In some embodiments, the oligonucleotide tail comprises a first capture moiety at its 5‘ end. In some embodiments, the blocking moiety comprises a second capture moiety' at its 3’ end. In some embodiments, the second capture moiety' comprises a second affinity' tag. In certain embodiments, the first or second affinity tag or both comprise biotin, desthiobiotin, or streptavidin. Examples of suitable combinations of affinity purification tags and their ligands include, but are not limited to, biotin: streptavidin, desthiobiotimstreptavidin polyhistidinemickel, polyarginine: carboxypeptidase B, 1D4 sequence: 1D4 antibody, Maltose Binding Proteins: Amylose, Glutathione-S-Transferase:Glutathione, SUMO Tagsmickel, c-Myc:anti-myc antibody, Hemaglutinin antigen: HA antibody, FLAG: FLAG antibody. Caladium, Native or Engineered Protein A:IgG (See, e.g.. Kimple et al. (2013. Curr. Protoc. Protein Sci. 73:Unit 9-9) for a review of affinity tags available for protein purification).

[0075] In some embodiments, the methods further include purifying conjugated oligonucleotide primers by adding a bead conjugated to a binding moiety (ligand) that binds a capture moiety (affinity tag) on the conjugated oligonucleotide primer, allowing the binding of the binding moiety and capture moiety, isolating the beads, and releasing the conjugated oligonucleotide primers bound to the beads. In some embodiments, the methods for purifying the conjugated oligonucleotides use a single tag system (FIGS. 4A-4B). The figures use biotin as an example of an affinity tag that may be used, and its ligand is shown as conjugated to a bead for purification. FIG. 4A shows successful conjugation, and purification of the conjugated oligonucleotide from unconjugated oligonucleotides, using a tag on the blocking moiety. FIG 4B shows that oligonucleotides that have not been conjugated will not have the tag for selection and purification because the blocking moiety is not present. The conjugatedoligonucleotide primers bound to the beads may be released from the beads with or without the capture moiety.

[0076] In some embodiments, the methods for purifying the conjugated oligonucleotides use a two tag system (FIGS. 5A-5B). Different affinity tags are used on the oligonucleotide and on the blocking moiety (substrate). FIG. 5A shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the blocking moiety to remove free unconjugated oligonucleotides. FIG 5B shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the oligonucleotide to remove free blocking moieties. The conjugated oligonucleotide primers bound to the beads may be released from the beads with or without the capture moiety.

[0077] The disclosure also provides methods for producing an oligonucleotide primer conjugated to a cleavable moiety at its 5?end. wherein the cleavable moiety is further conjugated to an oligonucleotide tail. The methods include providing an oligonucleotide primer having a nucleic acid sequence that is complementary to a portion of the target nucleic acid and providing an oligonucleotide tail attached to a bead, wherein the oligonucleotide tail has a nucleic acid sequence that is not complementary to the target nucleic acid. The oligonucleotide primer is conjugated at its 5’ end to a cleavable moiety comprising a cleavage site for an enzy me, wherein cleavage at the cleavage site releases the oligonucleotide primer. The oligonucleotide tail is conjugated at its 3’ end to the cleavable moiety. In some embodiments, the cleavable moiety comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site.

[0078] A bead, as used in any of the disclosed methods described above or elsewhere herein, is a small particle or other solid support. A non-limiting example of a bead for use in purification is a magnetic bead. In some embodiments, the magnetic beads are about 1-4 pm in diameter and are solid (non-porous), providing sufficient surface area-to-volume ratio needed for effective ligand immobilization and affinity purification. Magnetic beads are produced as superparamagnetic iron oxide particles that are covalently coated with silane derivatives. The coating makes the beads inert (to minimize nonspecific binding) and provides the particular chemical groups needed for attaching ligands of interest. Affinity purification with magnetic particles is performed by mixing the beads with a sample as a loose slurry, allowing affinity interactions to occur with the immobilized ligand, collecting and separating the beads from the sample using a powerful magnet, and then releasing the bound molecules.D. Kits

[0079] In some embodiments, the disclosure provides kits for any of the disclosed methods for using the disclosed oligonucleotide primers. In certain embodiments, the kits include an oligonucleotide conjugated to a blocking moiety or cleavage moiety that has an enzyme cleavage site as described above or elsewhere herein; a target nucleic acid that is not cross- reactive to other components in the samples or methods; optionally, reagents for amplification; and instructions for using the components to detect a contaminant enzyme in a sample.

[0080] In some embodiments, the disclosure provides kits for use in any of the disclosed methods for producing the disclosed oligonucleotide conjugate primers. In certain embodiments, the kits include a blocking moiety that has an enzyme cleavage site and an affinity tag as described above or elsewhere herein; a different affinity tag to conjugate to a desired oligonucleotide; beads conjugated to the ligand for the first affinity tag; beads conjugated to the ligand for the second affinity tag; and instructions for using the components to produce and purity' the conjugated oligonucleotide.EXAMPLES

[0081] The following examples are offered to illustrate, but not to limit the claimed invention.Example 1 — Production of oligonucleotide conjugate primers

[0082] The utilization of a blocked oligonucleotide for enzyme detection enables rapid detection of enzyme activity for pharmaceutical quality assurance and quality' control. An embodiment of the disclosed oligonucleotide primer is shown in FIG. 1. The oligonucleotide primer has a blocking moiety' having a lipase cleavage site. The middle panel is an enlarged view of the 3’ region of the primer from the left panel, showing the lipase cleavable blocking moiety. The right panel is an enlarged view of the primer after cleavage by a lipase enzyme, showing that cleavage of the blocking moiety results in a free hydroxyl group at the 3’ end. Because conjugation of the oligonucleotide to the blocking moiety' is not absolute, some unconjugated oligonucleotides may remain in the mixture and would cause false positives in downstream detection methods (FIGS. 3A-3B). To reduce this background in the detection assays, the conjugated oligonucleotide primer may be purified from oligonucleotides and blocking moieties that have not been conjugated. The purification of the oligonucleotideprimer is facilitated by the addition of affinity tags to the 5’ end of the oligonucleotide and / or the 3’ end of the blocking moiety.

[0083] The methods for purifying the conjugated oligonucleotides use a single tag system (FIGS. 4A-4B) or two tag system (FIGS. 5A-5B). Biotin as an example of an affinity' tag that may be used, and its ligand streptavidin is shown as conjugated to a bead for purification in FIGS. 4A-4B. FIG. 4A shows successful conjugation, and purification of the conjugated oligonucleotide from unconjugated oligonucleotides, using a tag on the blocking moiety. FIG 4B shows that oligonucleotides that have not been conjugated will not have the tag for selection and purification because the blocking moiety is not present. The conjugated oligonucleotide primers bound to the beads may be released from the beads with or without the capture moiety. For the two tag system, different affinity tags are used on the oligonucleotide and on the blocking moiety (substrate). FIG. 5A shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the blocking moiety to remove free unconjugated oligonucleotides. FIG 5B shows successful conjugation, and purification of the conjugated oligonucleotide using a tag on the oligonucleotide to remove free blocking moi eties. The conjugated oligonucleotide primers bound to the beads may be released from the beads with or without the capture moiety.Example 2 - DNA Conjugate Primers Can Detect Presence of Different Enzymes

[0084] Standard oligonucleotide primers (unblocked primers) for amplification of a target nucleic acid were designed and tested. The positive and negative controls produced the expected amplification products (FIGS. 7A-7C). The assay and target nucleic acid were shown to generate a productive amplification signal using rare mutation detection (RMD) standard oligonucleotide primers (FIG. 7 A). An expected amplification signal also was detected using the assay with the target nucleic acid and outer and inner unblocked forw ard and reverse primers and Taqman probe (FIG. 7B). The signal was not produced when the inner forward primer was not included in the assay (FIG. 7C).

[0085] The inner forward primer was used to produce two different DNA conjugate primers (a DNA oligonucleotide primer conjugated to a blocking moiety at its 3’ end) for additional analysis of the assay. The DNA conjugate primers were designed to have a blocking moiety that includes either a phospholipase cleavage site or a phosphatase cleavage site.

[0086] A DNA conjugate primer with a phospholipase D cleavage site was HPLC purified and included in an amplification reaction at a high concentration (FIG. 8A) or low concentration (FIG. 8B). The data demonstrate that background signal increases with higher concentrations and purity of the DNA conjugate primer is important to reduce the false positive rate.

[0087] To determine if the DNA conjugate primer design can be used for detection of a different enzyme in a sample, a blocked inner primer was produced with a phosphate group at the 3' end of the standard inner primer. Alkaline phosphatase (ALP) can cleave accessible phosphate groups from DNA. This DNA conjugate primer was used in an amplification reaction with the target nucleic acid in the presence (FIG. 8C) or absence (FIG. 8D) of alkaline phosphatase. The enz matic cleavage of the blocking moiety (the 3‘ phosphate group) from the DNA conjugate inner primer results in productive amplification signal (FIG. 8C). The results demonstrate that the DNA conjugate primer design may be used for different enz matic targets by including a cleavage site for the enzyme in the blocking moiety of the DNA conjugate primer.

[0088] The initial assay conditions used for the detection of ALP resulted in a positive signal over the negative control, but a striation pattern was observed. Increasing the amplification cycle number or template (target nucleic acid) concentration in the assay reduced the striation pattern (data not shown).Example 3 - DNA Conjugate Primers Can Detect Presence of Enzyme over Time

[0089] The previous example demonstrated that use of the disclosed DNA conjugate primers in an amplification reaction allows for the detection of an amplification signal (over background) in samples that include a contaminant enzyme. To determine whether this effect was possible over time, different concentrations of enzy me w ere used in the assay over a 2 hour window (FIGS. 9-10). For these assays, the DNA conjugate primer that was used included a PLD cleavage site and a pair of non-template-binding bases (O=C-TT). The data lines from top to bottom of FIG. 9 summarize results obtained for the detection assays for samples that included PLD at a concentration of 0.1 U / well PLD (solid top line), 0.01 U / well PLD (dashed line), 0.001 U / well PLD (dotted line), and no PLD (solid bottom line), respectively. The underlying data for the 0.01 U PLD / well assays are shown in FIG. 10. Enzymatic activity as low as 0.001 U of PLD was observed over that of the background (approximately 5 million enzymes / droplet). A consistent level of background noise wasobserved with no PLD present, while over time PLD was able to cleave the primer end and enable more efficient PCR as seen in the 0. 1 U PLD sample. The data suggest the lipasespecific DNA conjugate primer is successfully cleaved in the standard 2x ddPCR Supermix (no dUTP) and activated, enabling it to amplify the template and generate an amplification signal. Therefore, the DNA conjugate primer assay system has demonstrated the ability to identify PLD activity in a sample over time.

[0090] To determine the effect of the primer concentration on background noise, additional experiments were performed with varying dilutions of the DNA conjugate primer (FIGS.11 A-l IB). As discussed above, PCR with the DNA conjugate primer is inhibited if the carboxy group is not cleaved because the blocking moiety prevents extension from the 3’ end of the primer. The DNA conjugate primers are capable of being cleaved at the enzymatic cleavage site to free the 3’ end of the primer and generating an increase in overall signal. A dilution of the phospholipase specific DNA conjugate primer shows statistically significant differences in signal with and without PLD. FIG. 11 A shows the titration of the primer in the absence (left bar in each pair) or presence of PLD (right bar in each pair). Background signal increases as a function of the concentration of the inner primer (FIG. 1 IB). The current limit of detection is approximately IxlO8enzyme molecules, similar to the range of detection possible with the currently available fluorescent probe-based detection methods.Example 4 - Methods for Reduction of Background Signal

[0091] Conjugation of the blocking moiety to the inner primer is not 100% efficient. Therefore, after conjugation, two populations of primers exist in the mixture - conjugated primer and non-conjugated or free primer. The conjugated primer is blocked and, therefore, unable to participate in amplification in the absence of an enzyme to cleave the blocking moiety (FIG. 12A). However, free primer, when present, can participate in amplification in the absence of the enzy me, leading to background and limiting the sensitivity of the assay. In one embodiment of a method to reduce background noise, a scavenger template is used to remove free inner primer in a pre-amplification reaction (FIG. 12B). The scavenger template (dashed line) binds and extends the free inner primer on a template that cannot bind the detection probe, resulting in the consumption of the free inner primer before the amplification with the target DNA template. The remaining inner primer is conjugated inner primer, which is only capable of producing an amplification signal if the enzyme that cleaves the blocking moiety is present in the sample. Another method to reduce background noise involveslowering the concentration of the conjugated inner primer used in the assay. The data demonstrate that lowering the concentration of the inner primer decreased the background signal observed (FIG. 13). The concentration of inner primer is shown on the x axis, ranging from 50-2200 nM. The number of scavenger template copies used in each assay is also shown on the x axis (0, 30, 50, or 70 copies). Different inner primer concentrations have statistically significantly different signal intensities via Tukey -Kramer analysis.

[0092] Another method to reduce background noise is to purify the conjugated primer with a tag on the blocking moiety (FIG. 4A). By attaching a biotin tag to the blocking moiety, successfully conjugated primers may be purified with a streptavidin-tagged bead. The normalized dirtiness of the conjugated primers obtained with different purification methods is shown in FIG. 14 A. Biotin purification reduced false positives by about 20% (FIG. 14B).

[0093] The background noise may be further reduced by washing the beads (FIG. 15). Washing the beads bound with 5 ’-biotinylated primers with Tris-EDTA buffer (left panel) or high salt buffer (right panel) reduced background. The high salt wash maintained approximately half the amount of DNA attached to beads after washing as compared to the simple Tris-EDTA w ash.

[0094] To determine if the design of the ends of the conjugated primer have an effect on the background noise observed in the detection assay (e.g., if the polymerase is able to bypass the blocked end of the primer and initiate extension from the primer), different primer designs w ere tested in the detection assay (FIG. 16). Amplification signals observed in a detection assay are shown for an unblocked primer (right panel), a blocked primer (as shown in FIG. 1) with no additional nucleotide bases added downstream of the blocking moiety (middle panel), or a blocked primer that includes two non-complementary bases dow nstream of the blocking moiety. The results show' that the inclusion of non-complementary bases downstream of the blocking moiety reduced background noise.Example 5 - Contaminant Enzyme Detection in Bulk Reaction

[0095] Methods for detecting a contaminating enzyme in one sample or in two or more samples in a bulk reaction are provided (FIGS. A-6B). The top panel of FIG. 6A shows a representative bulk design primer attached to a magnetic bead; however, these primers also may be used for analysis of a single sample. The primer includes an oligonucleotide tail, a cleavable moiety, and an oligonucleotide primer on its 3’ end with a template binding sequence (reporter tag). In the shown exemplary embodiment, the oligonucleotide tailincludes a biotin tag (b) at its 5 ’ end and is attached to the magnetic bead by interaction of the biotin with a streptavidin moiety (SA) on the magnetic bead. The first steps of the bulk detection methods include binding the primer to a target molecule (T\ complement sequence), washing, generating template (T) attached to the bead, and washing the bead with attached template (FIG. 6A). If a contaminant enzy me is present in the sample, it cleaves at the cleavage site of the cleavable moiety, releasing the oligonucleotide primer from the bead. In that case, the template produced with the oligonucleotide primer would not be attached to the bead and would be removed from the reaction during the wash step. Optionally, the methods may include an additional step of binding the primer (any primer that was not extended in the previous step) to a target molecule, washing, and generating template attached to the bead (top panel of FIG. 6B).

[0096] The templates may be released from the bead in a bulk reaction by cleaving the cleavable moiety (middle panel of FIG. 6B). The amplification and quantification of the amplification product (e.g., via ddPCR) includes amplifying with a forward primer (that binds downstream of the primer's binding sequence) and a reverse primer (bottom panel of FIG. 6B). The amplification product is detected with a probe or beacon or by use of an intercalating dye. The disclosed bulk assay method allows for the exponential amplification of signal, providing a high degree of sensitivity.Example 6 - Contaminant Enzyme Detection

[0097] A therapeutic antibody is formulated for storage and later use for the treatment of a subject in need thereof. The formulation includes polysorbate-20 to stabilize the formulation. The formulation is suspected of also including a host cell lipase that may degrade the polysorbate-20. A sample is taken from the formulation for analysis.

[0098] The sample is mixed with a target nucleic acid and an oligonucleotide conjugate primer as described herein. The oligonucleotide conjugate primer includes a first nucleic acid sequence that is not complementary to the target nucleic acid and a second nucleic acid sequence that is complementary to a portion of the target nucleic acid. A lipase in the sample cleaves the oligonucleotide primer at the cleavage site in the blocking moiety7, producing a hydroxyl group at the 3’ end. The blocked and / or unblocked oligonucleotide primers bind to the target nucleic acid. The unblocked oligonucleotide primer is extended with a polymerase, using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid (FIG. 2A). Thetwo strands are denatured, and a reverse primer is allowed to bind to the first strand and is extended to produce a second strand product (FIG. 2A). The first and second strand products are denatured and amplified using a forward primer and the reverse primer (FIG. 2B). The forward primer comprises the first nucleic acid sequence of the oligonucleotide. The first or second strand product is then detected by the detecting the binding of a fluorescent probe or detecting the presence of an intercalating dye in the first or second strand product (FIG. 2B). The detection of the presence of the first or second strand product indicates that the contaminant lipase is present in the sample.

[0099] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein, including patents, patent applications, non-patent literature, and GenBank accession numbers, is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

WHAT IS CLAIMED IS:

1. An oligonucleotide primer conjugated to a blocking moiety at its 3’ end, wherein the blocking moiety comprises a cleavage site for an enzyme, wherein cleavage at the cleavage site releases the blocking moiety and produces a 3’ hydroxyl group on the oligonucleotide primer, and wherein the blocking moiety comprises structure I:(I) wherein R comprises a phospholipid or hydrocarbon chain.

2. The oligonucleotide primer of claim 1, wherein the cleavage site for the enzyme comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site.

3. The oligonucleotide primer of claim 1 or 2, wherein the oligonucleotide primer is conjugated to the blocking moiety7by an ester linkage.

4. The oligonucleotide primer of any one of claims 1-3. wherein the blocking moiety comprises polyethylene glycol.

5. The oligonucleotide primer of any one of claims 1-4, wherein the blocking moiety comprises cholesterol-triethylene glycol (TEG).

6. The oligonucleotide primer of any one of claims 1-5, further comprising an oligonucleotide tail.

7. The oligonucleotide primer of any one of claims 1-6, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides.

8. The oligonucleotide primer of any one of claims 1-7, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides.

9. The oligonucleotide primer of any one of claims 1-8. wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA.

10. The oligonucleotide primer of any one of claims 1-8, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA.

11. The oligonucleotide primer of any one of claims 1-10, wherein the oligonucleotide tail comprises a first capture moiety at its 5‘ end.

12. The oligonucleotide primer of claim 11 , wherein the first capture moiety comprises a first affinity tag.

13. The oligonucleotide primer of claim 12, wherein the first affinity tag comprises biotin, desthiobiotin, or streptavidin.

14. The oligonucleotide primer of any one of claims 1-13, wherein the blocking moiety comprises a second capture moiety at its 3’ end.

15. The oligonucleotide primer of claim 14, wherein the blocking moiety comprises a second affinity tag at its 3’ end.

16. The oligonucleotide primer of claim 15, wherein the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

17. A reaction mixture, comprising a target nucleic acid and the oligonucleotide primer of any one of claims 6-16, wherein the oligonucleotide tail comprises a nucleic acid sequence that is not complementary to the target nucleic acid, and wherein the oligonucleotide primer is complementary to the target nucleic acid.

18. The reaction mixture of claim 17, wherein the reaction mixture comprises two or more oligonucleotide primers, wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a first enzyme, and wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a second enzyme.

19. A method for detecting a contaminant enzyme in a sample, comprising: providing a sample having or suspected of having the contaminant enzyme; producing a reaction mixture by mixing the sample with a target nucleic acid and an oligonucleotide primer conjugated to a blocking moiety at its 3’ end, wherein the oligonucleotide primer comprises at its 5’ end a first nucleic acid sequence that is notcomplementary to the target nucleic acid and at its 3’ end a second nucleic acid sequence that is complementary to a portion of the target nucleic acid, wherein the blocking moiety comprises a cleavage site for the contaminant enzyme, wherein cleavage at the cleavage site releases the blocking moiety and produces an oligonucleotide primer having a 3’ hydroxyl group, and wherein the blocking moiety7comprises structure I:(I) wherein R is a phospholipid or hy drocarbon chain; extending the oligonucleotide primer with a polymerase using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid, and extending a reverse primer that anneals to the reverse complement of the target nucleic acid to produce a second strand product; amplifying the first and second strand products using a forward primer and the reverse primer, wherein the forward primer comprises the second nucleic acid sequence of the oligonucleotide; and detecting the presence of the first or second strand product; wherein the presence of the first or second strand product indicates that the contaminant enzyme is present in the sample.

20. The method of claim 19, wherein the reaction mixture comprises two or more of the oligonucleotide primers, wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a first contaminant enzyme, and wherein at least one of the two or more oligonucleotide primers comprises a cleavage site for a second contaminant enzyme.

21. The method of claim 19 or 20, wherein the contaminant enzyme is a lipase, phospholipase, or esterase.

22. The method of any one of claims 19-21, wherein the forward primer compnses at least one synthetic nucleotide that increases the Tm of the forward primer.

23. The method of any one of claims 19-22, wherein the concentration of the forward primer in the amplification reaction is higher than the concentration of the oligonucleotide primer.

24. The method of any one of claims 19-23, wherein the amplification product is detected with a probe.

25. The method of any one of claims 19-24, wherein the amplification product is detected with an intercalating dye.

26. The method of any one of claims 19-25, wherein the method detects a single molecule of the contaminant enzyme.

27. The method of any one of claims 19-26, wherein the amplifying comprises a PCR reaction.

28. The method of any one of claims 19-26, wherein the amplifying comprises isothermal amplification.

29. The method of any one of claims 19-28, wherein the amplify ing is performed in a droplet or microcell.

30. The method of any one of claims 19-29, wherein the cleavage site for the enzyme comprises a lipase cleavage site, phospholipase cleavage site, or esterase cleavage site.

31. The method of any one of claims 19-30, wherein the oligonucleotide primer is conjugated to the blocking moiety by an ester linkage.

32. The method of any one of claims 19-31, wherein the blocking moiety of the oligonucleotide primer comprises polyethylene glycol.

33. The method of any one of claims 19-32, wherein the blocking moiety of the oligonucleotide primer comprises cholesterol-triethylene glycol (TEG).

34. The method of any one of claims 19-33, wherein the first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of 10-100 nucleotides.

35. The method of any one of claims 19-34, wherein the first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of 30-50 nucleotides.

36. The method of any one of claims 19-35, wherein the first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of DNA.

37. The method of any one of claims 19-35, wherein the first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of RNA.

38. The method of any one of claims 19-37, wherein the first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise a non-standard base.

39. The method of any one of claims 19-38, wherein the oligonucleotide tail comprises a first capture moiety at its 5’ end.

40. The method of claim 39, wherein the first capture moiety comprises a first affinity' tag.

41. The method of claim 40, wherein the first affinity tag comprises biotin, desthiobiotin, or streptavidin.

42. The method of any one of claims 19-41, wherein the blocking moiety compnses a second capture moiety at its 3‘ end.

43. The method of claim 42, wherein the second capture moiety' comprises a second affinity tag.

44. The method of claim 43, wherein the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

45. A method for producing an oligonucleotide conjugated to a blocking moiety at its 3 ’ end, comprising: providing an oligonucleotide primer comprising a first nucleic acid sequence that is not complementary to a target nucleic acid and a second nucleic acid sequence that is complementary to a portion of the target nucleic acid; conjugating the oligonucleotide primer with a blocking moiety comprising a cleavage site for an enzyme, wherein cleavage at the cleavage site releases the blockingmoiety and produces an oligonucleotide primer having a 3’ hydroxyl group, and wherein the blocking moiety of the oligonucleotide primer comprises structure I:(I) wherein R is a phospholipid or hydrocarbon chain.

46. The method of claim 45, wherein the cleavage site for the enzy me comprises a lipase cleavage site, phospholipase cleavage site, or esterase cleavage site.

47. The method of claim 45 or 46, wherein the oligonucleotide primer is conjugated to the blocking moiety by an ester linkage.

48. The method of any one of claims 45-47, wherein the blocking moiety of the oligonucleotide primer comprises polyethylene glycol.

49. The method of any one of claims 45-48, wherein the blocking moiety of the oligonucleotide primer comprises cholesterol-triethylene glycol (TEG).

50. The method of any one of claims 45-49, wherein first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of 10-100 nucleotides.

51. The method of any one of claims 45-50, wherein first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of 30-50 nucleotides.

52. The method of any one of claims 45-51, wherein first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of DNA.

53. The method of any one of claims 45-51, wherein first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise or consist of RNA.

54. The method of any one of claims 45-53, wherein first nucleic acid sequence of the oligonucleotide primer, the second nucleic acid sequence of the oligonucleotide primer, or both comprise a non-standard base.

55. The method of any one of claims 45-54, wherein the oligonucleotide tail comprises a first capture moiety at its 5’ end.

56. The method of claim 55, wherein the first capture moiety comprises a first affinity tag.

57. The method of claim 56, wherein the first affinity tag comprises biotin, desthiobiotin, or streptavidin.

58. The method of any one of claims 45-57, wherein the blocking moiety comprises a second capture moiety at its 3’ end.

59. The method of claim 58, wherein the second capture moiety comprises a second affinity tag.

60. The method of claim 59, wherein the second affinity tag comprises biotin, desthiobiotin, or streptavidin.

61. The method of any one of claims 58-60, further comprising purifying conjugated oligonucleotide primers by: after the conjugating step, adding a bead conjugated to a second binding moiety that binds the second capture moiety, allowing the binding of the second binding moiety and second capture moiety, isolating the beads, and releasing the oligonucleotide primers bound to the beads.

62. The method of any one of claims 55-61, further comprising , further comprising purityring conjugated oligonucleotide primers by: after the conjugating step, adding a bead conjugated to a first binding moiety that binds the first capture moiety, allowing the binding of the first binding moiety and first capture moiety, removing blocking moieties not bound to the beads, removing the beads from the reaction mixture, and releasing the oligonucleotide primers bound to the beads.

63. An oligonucleotide primer conjugated to a cleavable moiety at its 5’ end, wherein the cleavable moiety is further conjugated to a 3’ end of an oligonucleotide tail, wherein the cleavable moiety comprises a cleavage site for an enzyme, wherein cleavage at the cleavage site releases the oligonucleotide primer from the cleavable moiety.

64. The oligonucleotide primer of claim 63, wherein the cleavage site for the enzy me comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site.

65. The oligonucleotide primer of claim 63 or 64, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides.

66. The oligonucleotide primer of any one of claims 63-65, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides.

67. The oligonucleotide primer of any one of claims 63-66, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA.

68. The oligonucleotide primer of any one of claims 63-66, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA.

69. The oligonucleotide primer of any one of claims 63-68, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise a non-standard base.

70. The oligonucleotide primer of any one of claims 63-69, wherein the oligonucleotide tail comprises a first capture moiety at its 5' end.

71. The oligonucleotide primer of claim 70, wherein the first capture moiety7comprises a first affinity7tag.

72. The oligonucleotide primer of claim 71 , wherein the first affinity7tag comprises biotin, desthiobiotin, or streptavidin.

73. The oligonucleotide primer of claim 71 or 72, wherein the oligonucleotide tail is attached to a bead by binding of the first affinity tag to a binding moiety conjugated to the bead.

74. A reaction mixture comprising a target nucleic acid and an oligonucleotide primer conjugated to a cleavable moiety at its 5‘ end. wherein the cleavable moiety is further conjugated to an oligonucleotide tail, and wherein the oligonucleotide tail is attached to a bead.

75. A method for detecting a contaminant enzyme in one or more samples comprising: providing one or more samples comprising or suspected of comprising the contaminant enzyme; producing a first reaction mixture for each sample of the one or more samples by mixing the sample with a target nucleic acid and an oligonucleotide primer conjugated to a cleavable moiety at its 5’ end, wherein the cleavable moiety is further conjugated to a 3’ end of an oligonucleotide tail, wherein the oligonucleotide tail is attached at its 5’ end to a bead, wherein the oligonucleotide tail comprises a nucleic acid sequence that is not complementary to the target nucleic acid, wherein the oligonucleotide primer comprises a nucleic acid sequence that is complementary to a portion of the target nucleic acid, wherein the cleavable moiety comprises a cleavage site for the contaminant enzyme, and wherein cleavage at the cleavage site releases the oligonucleotide primer; extending the oligonucleotide primer with a polymerase using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid, thereby producing a double stranded target nucleic acid attached to the bead in the first reaction mixtures; washing the beads and discarding the supernatant; optionally producing a second reaction mixture by adding the target nucleic acid to each first reaction mixture and. if the oligonucleotide primer was not extended in the first reaction mixture, extending the oligonucleotide primer with a polymerase using the target nucleic acid as a template to form a first strand product comprising the oligonucleotide primer and a reverse complement of the target nucleic acid, thereby producing a double stranded target nucleic acid attached to the bead in the second reaction mixtures; washing the beads and discarding the supernatant; combining the washed beads from the first or second reaction mixtures for one or more samples; cleaving the cleavage moiety at the cleavage site, releasing the double stranded target nucleic acids from the beads; amplifying the double stranded target nucleic acids using a forward primer and reverse primer, wherein the forward primer comprises a sequence that is located 3’ to the oligonucleotide primer, thereby producing an amplification product; anddetecting the presence or concentration of the amplification product; wherein the presence of or concentration of the amplification product that is lower than a control sample indicates that the contaminant enzyme is present in one or more of the samples.

76. The method of claim 75, wherein the contaminant enzy me is a lipase, phospholipase, or esterase.

77. The method of claim 75 or 76, wherein the amplification product is detected with a probe.

78. The method of claim 75 or 76, wherein the amplification product is detected with an intercalating dye.

79. The method of any one of claims 75-78, wherein the amplifying comprises a PCR reaction.

80. The method of any one of claims 75-78, wherein the amplifying comprises isothermal amplification.

81. The method of any one of claims 75-80, wherein the amplify ing is performed in a droplet.

82. The method of any one of claims 75-81, wherein the cleavage site for the enzyme comprises a lipase cleavage site, a phospholipase cleavage site, or an esterase cleavage site.

83. The method of any one of claims 75-82, wherein the oligonucleotide tail is conjugated to the cleavable moiety by an ester linkage.

84. The method of any one of claims 75-83, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 10-100 nucleotides.

85. The method of any one of claims 75-84, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of 30-50 nucleotides.

86. The method of any one of claims 75-85, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of DNA.

87. The method of any one of claims 75-85, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise or consist of RNA.

88. The method of any one of claims 75-85, wherein the oligonucleotide primer, oligonucleotide tail, or both comprise a non-standard base.

89. The method of any one of claims 75-88, wherein the oligonucleotide tail comprises a first capture moiety at its 5’ end.

90. The method of claim 89, wherein the first capture moiety comprises a first affinity tag.

91. The method of claim 90, wherein the first affinity tag comprises biotin, desthiobiotin, or streptavidin.

92. The method of claim 90 or 91, wherein the oligonucleotide tail is attached to a bead by binding of the first affinity tag with a binding moiety7conjugated to the bead.

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