WO2007133495A1

WO2007133495A1 - Mixture for decreasing protein interference

Info

Publication number: WO2007133495A1
Application number: PCT/US2007/010992
Authority: WO
Inventors: Marta Corcoran; James Bingham
Original assignee: Human Genome Sciences, Inc.
Priority date: 2006-05-10
Filing date: 2007-05-07
Publication date: 2007-11-22

Abstract

A sample treatment mixture is provided for decreasing protein interference in a protein-containing sample. The sample treatment mixture includes at least three chaotropic agents and a reducing agent that denatures and reduces protein in a sample to allow for analysis for non-proteinaceous components in the sample without protein interference. Additionally, a method of detecting DNA in a protein-containing sample, which includes a pretreatment step using a sample treatment mixture which includes at least three chaotropic agents and a reducing agent to decrease protein interference, is provided.

Description

MIXTURE FOR DECREASING PROTEIN INTERFERENCE

FIELD OF THE INVENTION

[0001] The invention relates to a sample treatment mixture for decreasing protein interference in a protein-containing sample. The sample treatment mixture is useful as a pretreatment step for decreasing protein interference with non-proteinaceous sample components prior to assaying the sample. In one embodiment, the sample treatment mixture includes at least three denaturants and a reducing agent. According to this embodiment, the sample treatment mixture is useful for denaturing and reducing protein in the protein-containing sample. In one embodiment, the sample treatment mixture also contains an endopeptidase. In one embodiment, the sample treatment mixture is useful for pretreating a protein-containing sample prior to assaying for DNA in the sample.

BACKGROUND OF THE INVENTION

[0002] Regulatory agencies such as the Food and Drug Administration (FDA) and the World Health Organization (WHO) require biological drug products to be well characterized. As a result, manufacturers of biological products are required to fully characterize the protein agent in the product as well as identify and/or quantify non- proteinaceous components in the product. Biological drug products, in addition to the protein agent, often include proteins and non-proteinaceous materials including, but not limited to, host cell derived proteins and non-proteinaceous components such as DNA, leachables from processing material, and small molecules "which may be introduced during the production and purification processes.

[0003] In order to meet the requirements of the regulatory agencies, analytical assays must be performed on in-process and final drug product samples for characterization of the protein agent as well as identification and/or quantification of the various non-proteinaceous components that make up the biological drug. Assays are performed, for example, for determining general properties of the biological drug product (e.g., pH, appearance), safety (e.g., bioburden, endotoxin, sterility), activity (e.g., potency), identity, purity (e.g., aggregation, fragments, deamidation, oxidation), primary, secondary, and tertiary structure, and post-translational modifications (e.g., carbohydrate). Moreover, assays are used for quantitating and/or detecting non-proteinaceous components, for example, PCR and ELISA assays for host cell derived DNA and proteins, capillary electrophoresis and GC-MS for identifying leachables from processing material and additives during the production and purification processes.

[0004] In many instances, a sample taken from a final biological drug product has protein quantities that are greater than the quantities of other non-proteinaceous components in the sample. Fox example, while protein is generally present at the level of milligrams per milliliter (mg/ml), DNA is generally present at the level of picograms per milliliter (pg/mϊ). Furthermore, protein in the sample may be incompatible with the analytical method used. As a result, in many assays, protein present in a sample may interfere with the detection and. quantification of one or more non-proteinaceous components.

[0005] Additionally, analysis of non-proteinaceous sample components is often necessary for in-process samples during the research, production and purification stages of biological drug products. During production of the biological drug product, it is often important to determine the amount of drug being produced, as well as the quantities of other components present in the product, for example, additives such as, but not limited to, antifoams. Moreover, during purification, it is important to determine the presence and/or quantities of non-proteinaceous components, for example, residual DNA, that are being removed during a particular step. As with final biological drug product samples, the amount of protein in an in-process sample can be greater than that of non-proteinaceous components in the sample. Thus, protein in an in-process sample may interfere with the ability of an assay to detect non-proteinaceous sample components.

[0006] Unlike final drug product samples, in-process samples may vary in their buffer conditions and the amount of protein and DNA present in the sample at different stages of production and purification. Therefore, there is a need for a sample treatment mixture that is capable of decreasing protein interference in both final and in-process biological drug product samples and which is capable of being used with protein containing samples having different buffer conditions and protein concentrations. [0007] Protein interference is preferably decreased to improve the ability of an assay to identify and/or quantify non-proteinaceous sample components present with the biological drug product. Examples of assays which may require reduction of protein interference include, but are not limited to, Northern blots, Southern blots, nucleic acid hybridization, HPLC, residual DNA, and small molecule analysis (e.g., antifoams, benzyl alcohol). [0008] Frequently, to address protein interference in an assay, a sample treatment mixture may be added to the sample during a pretreatment step to decrease the protein in the protein-containing sample. The sample treatment mixture may act by denaturing and reducing the protein in the sample, which may facilitate digestion and separation of the protein from other non-proteinaceous components in the sample, for example, impurities such as residual DNA and small molecules, which require analysis. For example, in an assay for measuring DNA, a possible pretreatment step may be included to denature and reduce the protein in the sample to enable the protein to be digested and separated from the protein in the sample.

[0009] Various sample treatment mixtures, which include denaturants and detergents, which can be used in a pretreatment step for decreasing protein interference in a protein-containing sample, are known. For example, the Threshold^® Total DNA assay describes a sample treatment mixture for decreasing protein interference that includes 0.05% SDS and proteinase K. In another example, a sample treatment mixture is described that includes 8M buffered urea, 50 mM Tris-HCl, and 0.2 mM CaCl₂ (See, e.g., Iwaki et ah, Infection and Immunity, 68:3727-3730 (2000) and Pulvermuller et al, Biochemistry, 36:9253-9260 (1997), which are herein incorporated by reference). [0010] While sample treatment mixtures such as those described above may be effective in decreasing protein interference in protein-containing samples having protein concentrations less than 10 mg/ml, they are generally ineffective in decreasing protein interference in samples having high protein concentrations (i.e., >10 mg/ml). See, for example, Rung et ah, Analytical Biochemistry, 187:220-227 (1997), which discusses the problems that interference of the protein in the sample causes with the performance of the Threshold^® Total DNA assay, the disclosure of which is herein incorporated by reference. Consequently, when one of skill in the art encounters a sample with a high protein concentration (i.e., >10 mg/ml), the skilled artisan has generally resorted to dilution of the sample to decrease the protein concentration in the sample to a level below 10 mg/ml. By diluting the sample to a level less than 10 mg/ml, the known sample treatment mixtures described above can then be used to decrease protein interference in the sample. [0011] However, sample dilution creates several additional problems, such as, for example, reduced assay sensitivity. In one example, when using the Threshold^® Total DNA assay, samples having high protein concentrations (i.e., > 10 mg/ml) but low concentrations of DNA (i.e., picograms/ml) may be diluted to such an extent that the DNA may no longer be detected by the assay. Moreover, the ability to dilute the sample may be limited by the requirement of a particular sample tube size or well plate for use in a particular analytical assay. Thus, there exists a need for a sample treatment mixture capable of decreasing protein interference, which is useful for pretreating samples having high protein concentrations (i.e., >10 mg/ml) without the need for sample dilution.

SUMMARY OF THE INVENTION

[0012] In accordance with one aspect of the present invention, there is provided a sample treatment mixture for decreasing protein interference in a protein-containing sample. In one embodiment, the sample treatment mixture includes at least three denaturants and a reducing agent. In one embodiment, the three denaturants are chaotropic agents. In another embodiment, the chaotropic agents include guanidine hydrochloride, urea, and sodium iodide. Typically, the denaturants in the sample treatment mixture can be included at a total concentration between about 1.0 M and about 10 M, between about 2.0 M and about 8.0 M, or between about 3.0 M and about 5.0 M. As used herein, the term "about" is used to refer to a range within the standard margin of error.

[0013] Reducing agents that may be used in the sample treatment mixture are known. Examples of reducing agents include, but are not limited to, dithiothreatol (DTT), dithioerythritol, glutathione, cysteine, cystamine, monothioglycerol (MTG) and 2- mercaptoethanol. In one embodiment, the reducing agent comprises dithiothreatol. The reducing agent can be included in the sample treatment mixture at a concentration between about 1.0 mM and about 1.0 M, between about 10 mM and about 5.00 mM, or between about 10 mM and about 100 mM.

[0014] It is envisioned that the sample treatment mixture of the present invention can be used in any situation where there is a need to decrease protein interference in a protein-containing sample. In accordance with one aspect of the present invention, the sample treatment mixture can be used as a pretreatment step in any assay for analyzing a molecule of interest, such as non-proteinaceous components present in a protein- containing sample. In one embodiment, the invention includes a method for detecting DNA. According to this embodiment, the DNA detection method includes the use of a sample treatment mixture having at least three denaturants and a reducing agent as a pretreatment step. More particularly, the invention provides a method for detecting DNA comprising the following steps: pretreatment of the protein-containing sample with a sample treatment mixture having at least three denaturants and a reducing agent; digestion of the protein in the sample; extraction of the DNA in the sample; and detection of the DNA in the sample. According to the invention, the pretxeatment step in the DNA assay uses a sample treatment mixture which includes at least three chaotropic agents and a reducing agent.

[0015] For the protein digestion step, any known protein digestion method compatible with DNA and the precipitation of DNA can be used. In one embodiment, the protein digestion step is performed by adding a protease mixture to the protein-containing sample. In one embodiment, the protease mixture includes an endopeptidase. In another embodiment, the endopeptidase is a serine protease, for example, proteinase K. The protease is generally included in the mixture at a concentration between about 0.1 mg/ml and about 10 mg/ml, between about 0.2 mg/ml and about 5 mg/ml, or between about 1 mg/ml and about 3 mg/ml. According to the invention, the protease mixture can be added following the addition of the sample treatment mixture or concurrently with the sample treatment mixture.

[0016] The DNA extraction step used in the method of detecting DNA can be performed using any methods known in the art compatible with the denaturants and reducing agents in the sample treatment mixture. For example, the DNA extraction step can be performed using a DNA Extraction Kit (commercially available from Wako Chemicals, Richmond, VA) which is a phenol-free DNA extraction method. [0017] The DNA detection step used in the method of detecting DNA can be performed using any methods known in the art. Examples of DNA detection methods include, but are not limited to, fluorescent, for example PicoGreen^® (Molecular Probes, Eugene, OR), ELISA, Threshold^® and PCR methods. In one embodiment, the DNA detection step is performed using the Threshold^® Total DNA assay. According to this embodiment, the assay protocol can be found in the Threshold^® System User Manual. See Chapters 14-18, Part #0112-0046, Molecular Devices Corp. In another embodiment, the DNA detection is performed using a PCR method, for example, but not limited to, the method described in U.S. Pat. No. 5,393,657.

[0018] In one embodiment, the present invention comprises a kit for decreasing protein interference in a protein-containing sample. According to this embodiment, the kit includes at least three denaturing agents and a reducing agent. In another embodiment, the kit additionally includes a protein digestion agent. BRIEF DESCRIPTION OF THE FIGURES

{0019] The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims. [0020] FIGURE 1 is a silver stain gel showing supernatant (a) and pellet (b) results for protein-containing samples treated with the Threshold^® treatment mixture and samples treated with a mixture which includes urea.

[0021] FIGURE 2 is a silver stain showing supernatant (a) and pellet (b) results for

100 mg/ml protein-containing samples treated with sample treatment mixtures including either One (urea) (Lane 1), two (urea and guanidine hydrochloride) (Lane 2) or three (urea, guanidine hydrochloride, and sodium iodide) (Lane 3) chaotropic agents, in addition to a reducing agent (dithiothreitol) and a protease (proteinase K).

[0022] FIGURE 3 is a silver stain gel comparing treatment of a 100 mg/ml protein sample with increasing concentrations of Proteinase K in a sample treatment mixture with three (urea, guanidine hydrochloride and sodium iodide) chaotropic agents and DTT. [0023] FIGURE 4 is a silver stain gel showing supernatant and pellet results for 40 mg/ml fusion protein samples treated with sample treatment mixtures having either one (urea), or three (urea, guanidine hydrochloride, and sodium iodide) chaotropic agents, in addition to a reducing agent (dithiothreitol) and a protease (proteinase K). [0024] FIGURE 5 is a silver stain gel showing supernatant and pellet results of treating in-process samples with a sample treatment mixture having three chaotropic agents, a reducing agent, and a protease.

[0025] FIGURE 6 is a schematic of the GC-MS process for the detection of benzyl alcohol.

[0026] FIGURE 7 is a silver stain showing the results for 100 mg/ml protein- containing samples treated with no chaotropic agent (control) (Lane 1), GnHCl (Lane 2), Urea (Lane 3), WAKO treatment method (Lane 4), and three chaotropic agents (GnHCl, NaI and Urea) (Lane 5).

DETAILED DESCRIPTION OF THE INVENTION

[0027] The invention provides a sample treatment mixture for decreasing protein interference in a protein-containing sample. In one embodiment, the sample treatment mixture includes at least three denaturants and a reducing agent. According to this embodiment, the sample treatment mixture can be added to a protein-containing sample to denature and reduce the protein in the sample. Once the protein in the sample is denatured and reduced, the protein can be digested and separated from other non-proteinaceous components in the sample, which may include impurities such as, but not limited to, residual DNA and small molecules. In another embodiment, the sample treatment mixture additionally contains an endopeptidase.

[0028] Sample treatment mixtures, which include denaturants and/or reducing agents used in pretreatment steps for decreasing protein interference in protein-containing samples, are known^'; For example, in DNA assays, these sample treatment mixtures can - be used to decrease protein interference with DNA by denaturing and reducing the protein in the sample, which facilitates digestion of the protein and subsequent extraction of the DNA from the sample. As used herein, "protein interference" refers to the fact that protein present in a sample can interfere with the ability of the assay to detect a molecule of interest. Furthermore, although not wishing to be limited by theory, it is believed that both precipitated and soluble forms of protein can interfere with a non-proteinaceous component in the sample. For example, precipitated protein in the mixture may inhibit DNA precipitation during the extraction step, prevent immobilization of labeled DNA, and/or lead to artificially high signals linked to interaction with denaturants, such as, for example, urea. Soluble protein may bind to DNA in the sample and therefore inhibit detection of DNA leading to artificially low signals. For these reasons, reduction of protein interference from high protein containing samples may be important for successfully identifying and/or quantifying non-proteinaceous components in a sample. [0029] If effective, the DNA can be detected under conditions of decreased protein interference. However, the sample treatment mixtures may result in protein precipitation and interference with non-proteinaceous sample components. Furthermore, while these known sample treatment mixtures may be effective for decreasing protein interference in samples with protein concentrations less than 10 mg/ml, they are generally ineffective for treating samples with high protein concentrations. The term "high protein concentration" as used herein refers to a protein-containing sample with a protein concentration greater than about 10 mg/ml, greater than about 50 mg/ml, greater than about 100 mg/ml, greater than about 150 mg/ml, or greater than about 200 mg/ml. Generally, to treat samples with high protein concentrations using the known sample treatment mixtures, the samples are diluted to a protein concentration below 10 mg/ml. As previously disclosed, sample dilution results in reduced assay sensitivity and is limited by sample tube size or well-plate limitations for a particular assay.

[0030] Advantageously, the sample treatment mixture of the present invention is capable of decreasing protein interference for a wide range of protein containing samples, including high protein concentration samples, without the need for sample dilution. For example, the sample treatment mixture of the present invention may be used to decrease protein interference in samples having protein concentrations greater than about 0.1 mg/ml, greater than about 1.0 mg/ml, greater than about 10 mg/ml, greater than about 50 mg/ml, greater than about 100 mg/ml, greater than about 150 mg/ml, or greater than about 200 mg/ml.

[0031] Additionally, the sample treatment mixture of the present invention is capable of decreasing protein interference without decreasing assay sensitivity. The term "assay sensitivity" as used herein refers to the ability of an assay to detect and quantitate a particular amount of a non-proteinaceous component in a sample. For example, for a DNA assay, it is not uncommon to be able to detect and quantitate on the order of at least 10 pg of DNA per mg of protein, at least 5 pg of DNA per mg of protein, at least 1 pg of DNA per mg of protein, at least 0.1 pg of DNA per mg of protein, or at least 0.01 pg of DNA per mg of protein. Currently, FDA guidelines specify that residual DNA amounts should be less than 100 pg per dose. Accordingly, final drug product specification limits should be set such that the quantity of DNA in the final drug product is below the 100 pg limit. Furthermore, it is suggested that a method of detecting DNA have a sensitivity or ability to detect 10 pg. Thus, if the dose volume for a particular drug product is, for example, 1 ml, then the assay should have a sensitivity level low enough to detect an amount of DNA below 10 pg/ml. In situations where the concentration of protein in the dose is greater than 10 mg/ml, for example 100 mg/ml, the sample would have to be diluted 10 fold if known treatment mixtures are used. However, dilution to decrease protein concentration will have the effect of decreasing the DNA concentration in the sample. Accordingly, where a sample containing 100 mg/ml of protein has, for example, a DNA concentration of 10 pg/ml prior to dilution, a 10 fold dilution will result in a DNA concentration of 1 pg/ml. For a DNA assay having a lower limit of quantitation at 10 pg/ml, the sample dilution may result in an inability of the DNA assay to detect the DNA in the sample. Thus, having a sample treatment mixtare that is capable of decreasing protein interference would be beneficial by not decreasing assay sensitivity through sample dilution.

[0032] As an additional advantage, pretreating a protein-containing sample with the sample treatment mixture of the present invention generally results in decreased sample related assay failures. As discussed previously, protein interference in the sample is often a cause of sample related failures. Assay failures can be attributed to both errors in the actual dilution of the sample during the pretreatment step and conversion of final results to account for dilution of the sample.

[0033] As referred to herein, "sample related failures" are assay failures that result from the interference of protein with the ability of the assay to detect a molecule of interest, such as a non-proteinaceous component in a sample. To monitor assay performance, assays generally incorporate several controls, for example, spike recoveries, positive controls and negative controls. In a DNA assay, sample related assay failures may be determined by using a DNA spike recovery. For the DNA spike recovery control, successful assay performance requires that the DNA spiked into the control sample be recovered within acceptable limits. A "control sample" as used herein is a sample that has similar buffer and component conditions as a test sample but has an additional known amount of DNA added, i.e., spiked, into the control sample. Acceptable recovery of the DNA spike from the control sample can be indicative of successful elimination of protein interference and generally helps assure that results accurately reflect the amount of DNA in the test sample. A recovered DNA spike result which lies outside of the acceptable limits indicates assay failure. A sample related assay failure can occur, for example, if the detected quantity of DNA spiked into the control sample is not within + 10%, + 20%, or + 30% of the expected quantity. For example, a sample related assay failure would occur for a spiked control sample if the detected final DNA quantity were not within the range of 7 pg DNA to 13 pg DNA (i.e, ±30%) for a sample spiked with 10 pg DNA. [0034] Traditionally, it is not uncommon for DNA detection assays to have sample related assay failure rates greater than 50%. For example, out of 107 DNA assays performed using the recommended treatment protocol in the Threshold^® Total DNA assay {See Chapters 14-18, Threshold System Operator's Manual #0112-0046, Molecular Devices Corp.), assay failures occurred 62 times, correlating to a sample related failure rate of 58% (Data not shown). Surprisingly, the sample treatment mixture of the present invention can decrease assay failure rates and therefore can decrease the need to repeat assays. For example, when using the sample treatment mixture in a DNA assay in which detection is performed using the Threshold^® Total DNA assay, in 89 assays performed, no sample related failures occurred (as determined by spiked recovery). Furthermore, the ability to treat samples having a wide range of protein concentrations, for example, from less than about 1 mg/ml to greater than about 200 mg/ml, allows the samples to be tested without dilution and thereby maintains assay sensitivity. Sample Treatment Mixture

Protein Denaturants

[0035] The terms "denature," "denatured," and "denaturing" as used herein refer to structural changes in a protein such that it is no longer in its' native state, i.e., the structure of the protein as found in nature. For example, a protein may be considered "denatured" when it loses its' three-dimensional structure or conformation and/or its' characteristic folded structure. Protein denaturation can occur at the secondary, tertiary and quaternary structure levels of proteins, but generally not at the primary structural level. {0036] In many instances, proteins are less soluble in a native state than in a denatured state. Typically, protein solubilization by denaturation involves a process in which interactions involved in protein intra- and inter- molecular bonds and interactions are broken, for example, but not limited to, disulfide bonds, hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic interactions. These types of interactions are generally responsible for protein aggregation and/or precipitation. [0037] Denaturation of proteins can be accomplished by various processes including, but not limited to, exposure to heat, pH, acids, bases, detergents, alcohols, salts, chaotropic agents and/or reducing agents. However, in an assay for the detection of molecule of interest, such as a non-proteinaceous component in a protein-containing sample, the purpose of the denaturant is to decrease protein interference without removing or interfering with the non-proteinaceous component in the sample. Consequently, many of the known denaturation methods may be ineffective due to the fact that they may interfere with or remove the non-proteinaceous components in the sample. Furthermore, although many of these known protein denaturation methods may be effective in denaturing protein-containing samples with low protein concentrations, they may be ineffective in denaturing protein-containing samples with high protein concentrations, e.g., greater than 10 mg/ml.

Detergents [0038] "Detergents" as used herein refers to a water-soluble surface-active agent that includes a hydrophobic portion, usually a long alkyl chain, attached to hydrophilic or water solubility enhancing functional groups. Detergents generally denature proteins by disrupting hydrophobic interactions between and/or within the protein. The addition of a detergent in a sample treatment mixture may be useful in decreasing protein interference by solubilizing otherwise insoluble proteins. Solubilized proteins may be separated from other sample components, for example, by selectively precipitating the sample components. Furthermore, the solubilized protein tends to bind less to the other non- proteinaceous sample components. Detergents can be classified according to their charge in solution. They may be anionic, cationic, neutral, or zwitterionic. Examples of detergents include, but are not limited to, octyl glucoside and Triton X-100 (neutral), CHAPS and ASB-14 (zwitterionic), and sodium dodecyl sulfate (SDS) (anionic). Detergents used for denaturing proteins can be used at concentration ranges from about 0.5% to about 10%, about 1% to about 7%, or about 1% to about 4%. [0039] An example of a known sample treatment mixture which uses a detergent to decrease protein interference is described in the Threshold^® Total DNA assay. The Threshold^® sample treatment mixture includes 10% SDS. Typically, a detergent, such as 10% SDS, is capable of denaturing protein-containing samples at concentrations less than 1 mg/ml. However, the ability of a detergent to denature a protein, as well as the ability of other denaturants to denature a protein, can be limited by the solubility of the detergent or denaturant in the sample. Denaturation of high protein concentrations is generally not possible using a detergent because the concentration of detergent necessary to denature the protein may not be soluble in the sample.

Chaotropic Agents

[0040] Chaotropic agents generally denature proteins by disrupting hydrogen bonds that hold the proteins in their native structure. Chaotropic agents may also disrupt hydrophobic interactions in proteins by promoting the solubility of hydrophobic residues in aqueous solutions. Chaotropic agents can therefore be used to decrease protein aggregation and formation of secondary structure, which can alter protein mobility. According to one embodiment, the sample treatment mixture of the present invention includes at least three chaotropic agents. Various chaotropic agents are known and include, but are not limited to urea, guanidine hydrochloride, guanidine thiocyanate, sodium iodide, and potassium iodide. In one embodiment, the chaotropic agents in the sample treatment mixture include urea, guanidine hydrochloride, and sodium iodide. Typically, single chaotropic agents are used at a concentration at least about 1 M, at least about 2 M, at least about 4 M, at least about 6 M, or at least about 8 M to denature proteins. According to another embodiment of the invention, the total concentration of multiple chaotropic agents in the sample treatment mixture of the present invention is between about 1.0 mM and about 1.0 M, between about 10 mM and about 500 mM, or between about 10 mM and about 100 mM. Actual concentrations higher than those listed above may be possible; however, the concentration will be limited by solubility of the chaotropic agent(s) in the sample.

[0041] High concentrations of single chaotropic agents may disrupt secondary protein structure and help solubilize proteins in samples that are otherwise insoluble. However, the use of a high concentration of a single chaotropic agent, for example, at a concentration greater than 4M, is generally limited by the ability of the chaotropic agent to go into solution. At the same time, the ability of a chaotropic agent to denature protein in a sample may be limited by the concentration or the amount of protein present. For example, while buffered urea is generally effective in denaturing sample protein concentrations at or below 10 mg/ml, it is generally ineffective at high protein concentrations (i.e., protein concentrations greater than 10mg/ml). Additionally, because concentrations of buffered urea greater than 4M are unlikely to be soluble in the sample and therefore cannot be used, single chaotropic agents may not be effective to decrease protein interference for protein containing samples having high protein concentrations. See e.g., the silver stain gel in Figure 2 (from Experiment 2), which shows a precipitated protein band in lane 1 indicating ineffective reduction of protein interference by the single chaotropic agent urea to treat a 100 mg/ml protein-containing sample. Similarly, the use of two chaotropic agents, for example urea and guanidine, may also be limited by the solubility of these chaotropic agents in the sample and thus may be ineffective in denaturing samples containing protein concentrations greater than 10 mg/ml. See e.g., the silver stain gel in Figure 2 (from Experiment 2), which shows a precipitated protein band in lane 2 indicating ineffective protein interference reduction by the two chaotropic agents urea and guanidine hydrochloride to treat a 100 mg/ml protein-containing sample. [0042] It was discovered that treating a sample containing protein at a high protein concentration, for example, a sample greater than 10 mg/ml protein, with a sample treatment mixture containing at least three chaotropic agents resulted in effective reduction of protein interference without sample dilution. Surprisingly, the three chaotropic agents remained soluble in the sample despite the high concentrations of each chaotropic agent. Furthermore, the combination of multiple chaotropic agents in the sample treatment mixture was able to decrease protein interference in high protein concentration samples, whereas the uses of sample treatment mixtures containing one and two chaotropic agents were not. Reducing Agents

[0043] According to one embodiment, the sample treatment mixture of the present invention also includes a reducing agent. As used herein, a "reducing agent" is an element or compound which causes a redox (reduction-oxidation) reaction in which electrons are transferred from the element or compound (the reducing agent) to the protein (the oxidizing agent). Reducing agents can be used to cleave disulfide bond crosslinks within proteins and between protein subunits. Examples of reducing agents include, but are not limited to, sulfhydryl reducing agents such as dithiothreitol (DTT), dithioerythritol (DTE), and β-mercaptoethanol and phosphine reducing agents such as tributylphosphine (TBP) and tris-carboxyethylphosphine (TCEP). In one embodiment the reducing agent included in the sample treatment mixture can be either a phosphine or a sulfhydryl reducing agent. Although phosphine reducing agents can be used at lower concentrations than sulfhydryl reductants and are generally active over a wider pH range, sulfhydryl reducing agents are more commonly used. In one embodiment, the reducing agent is a sulfhydryl reductant. In another embodiment the sulfhydryl reducing agent is dithiothreitol. In another embodiment, the reducing agent is included in the sample treatment mixture at a concentration between about 1 mM and about 1 M, between about 100 mM and about 750 mM, or between about 400 mM and about 600 mM. Sample Types

[0044] The term "protein-containing sample" or "sample" as used herein refers to any biological fluid that contains protein, which includes any fluid derived from cells, cell components, or cell products. Biological fluids include, but are not limited to, fluids from fermentation broth, cell cultures supernatants, conditioned cell culture medium, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood, plasma, serum, sputum, semen, mucus, milk, and fractions thereof that contain protein.

[0045] The invention is suitable for use with fluid derived from a variety of cell types, including, but not limited to, animal, insect, microbial, fungal and/or plant cells. Examples of animal cells include, but are not limited to mammalian cells, for example, human (including 293, WI38, PER.C6 and Bowes melanoma cells), mouse (including 3T3, NSO, NSl, Sp2/0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), chicken (CEF, CAM), and hybridoma cell lines. Bacterial cells include, but are not limited to E. coli, Streptomyces and Salmonella typhimurium cells. Fungal cells include molds (e.g. Aspergillus spp.) and yeast cells such as Saccharomyces cerevisiae and Pichia pastori. Insect cells include, but are not limited to Drosophila S2 and Spodoptexa Sf9 and Sf21 cells.

[0046] The protein-containing sample can be any sample taken from the beginning, intermediate, in-process, or final step of any process. Examples of processes include, but are not limited to, bench top experiments, cell culture or fermentation processes performed using a plate, roller bottle, shaker flask, T-flask, spinner flask, cell culture bag, bioreactor, fermentor, separation and purification processes using chromatography, filtration (both static and tangential flow filtration), centrifugation, formulation, lyophilization and fill processes. In one embodiment, the sample is an in- process sample taken during the production stage of a protein. In one embodiment, the sample is an in-process sample taken during the purification of a protein. In another embodiment, the sample is taken from a bulk.

[0047] The term "protein" as used herein refers to an organic macromolecule made up of 2 or more amino acids, which can be branched or unbranched, including host cell protein and recombinantly produced protein, such as, but not limited to, a therapeutic protein (e.g., a biological drug product), or any protein or combination of proteins present in the sample resulting from any process described above or known in the art. The term "therapeutic protein" as used herein refers to any protein that may be administered to a human and/or animal for treatment. The term "protein" can refer to both antibody and non-antibody proteins. Antibodies can include both monoclonal and polyclonal antibodies, antibody fragments, chimeric antibodies, human or humanized antibodies. Antibody fragments are known and include, but are not limited to, single chain antibodies, such as ScFv, Fab fragments, Fab' or F(ab')₂ fragments, etc. Non-antibody proteins include, but are not limited to, proteins such as secreted proteins, enzymes, receptors, and fragments or variants thereof. The term "protein" can also include proteins fused to a heterologous protein, for example, fusion proteins or chimeric proteins. According to one embodiment, the protein is fused to albumin. The protein may or may not be glycosylated. The term "protein" may also include multimeric proteins, such as hetero- or homo-dimers, trimers, etc. In one embodiment, the protein-containing sample has a protein concentration greater than about 0.1 mg/ml, 1 mg/ml, 10 mg/ml, 50 mg/ml, 100 mg/ml, 150 mg/ml, or 200 mg/ml.

[0048} The term "non-proteinaceous sample components" or "sample components" as used herein refers to any component in the sample other than protein. Examples of such non-proteinaceous components include, but are not limited to, molecules of interest, residual host cell DNA, buffer components, contaminants due to microbial (e.g., bacterial), fungal (e.g., mold and yeast), and viral sources, and production and purification impurities and leachables, such as, for example, residual small molecule contaminants (elg., antifoams, benzyl alcohol). '

DNA Detection Assay

[0049] Another aspect of the invention provides a method for detecting DNA in a protein-containing sample wherein the method includes a pretreatment step using a sample treatment mixture having at least three denaturants and a reducing agent. More particularly, the invention provides a method for detecting DNA which includes the following steps: pretreatment of the protein-containing sample with the sample treatment mixture; digestion of the protein in the sample; extraction of the DNA in the sample; and detection of the DNA in the sample.

[0050] Biological drug products have the potential to contain host cell DNA as well as contaminating DNA from other sources such as, for example, microbial (e.g., bacterial), fungal (e.g., mold and yeast), and viral DNA. Due to safety concerns, the FDA and WHO require that final drug products contain very low levels of host cell components, for example, picograms of DNA per dose. Current analytical assays for measuring DNA are known and include, but are not limited to, the Threshold^® Total DNA assay which is performed using the Threshold^® system (Molecular Devices Corporation, Menlo Park, Calif.). The Threshold^® system is also commonly used to detect and quantitative other non-proteinaceous components in biopharmaceutical products. For example, components commonly analyzed with the Threshold^® system include total DNA, host cell proteins, bovine contaminants, Proteins A and G, or any protein that can be bound by antibodies. The Threshold^® system is commonly used in assay development, process development, quality control applications, and in in-process and final drug product analysis. [0051] The Threshold^® Total DNA assay protocol generally includes the following steps: extraction of the DNA, labeling of the DNA, immobilization of labeled DNA onto a filter, and electrochemical detection of labeled DNA. Optionally, a sample pretreatment and proteolytic digestion step can be performed prior to the DNA extraction step. The labeling, immobilization, and detection steps of the assay protocol are performed using the Threshold^® system.

[0052] The detection of DNA using the Threshold^® Total DNA assay protocol is performed using two DNA-binding proteins that have high affinity for single-stranded DNA. One protein, a monoclonal anti-DNA antibody is conjugated to a urease enzyme. The other protein, E.coli single stranded DNA-binding protein, is conjugated to biotin. Single stranded DNA generated in samples via a snap cooling step, and is incubated with a cocktail of these proteins and streptavidin. The resulting protein-DNA complex is concentrated on the surface of a biotinylated membrane and an automated reader is used to quantitate the DNA by detecting the rate of pH change in enzyme-bound DNA samples. [0053] PCR methods are also commonly used to detect and quantitate non- proteinaceous components in biopharmaceutical products. PCR assays for detecting DNA in samples are known. Methods of detecting DNA using polymerase chain reaction (PCR) include, for example, the method described in U.S. Pat. No. 5,393,657, the disclosure of which is incorporated by reference herein. PCR methods for detecting DNA in a protein- containing sample generally include the following steps: (1) optional sample pretreatment and proteolytic digestion, (2) denaturing the intact nucleic acids to reveal single stranded DNA, (3) annealing the single stranded DNA to one or more primers, (4) extending the primers with a polymerase capable of copying a DNA template, and (5) repeating steps 2- 4 multiple times sufficient to generate and identify any detectable DNA. PCR methods are advantageous in that they are generally low cost and highly sensitive assays. PCR methods are capable of detecting DNA on the order of about 0.01 picograms of DNA. However, PCR methods are generally disadvantageous in that they are designed to specifically detect host cell DNA (based on primers used to perform PCR) and not other possible DNA sources such as, for example, those derived from contaminating microbial (e.g., bacterial), fungal (e.g., mold and yeast), and viral DNA.

[0054] While the Threshold^® Total DNA assay is generally considered the industry standard in determining DNA levels in biopharmaceuticals, it also considered labor- intensive, for example, due to requirements for sample preparation, sample handling, and overall design of the instrumentation. Additionally, the Threshold^® Total DNA assay has generally been considered problematic due to high assay failure rates. Assay failures can be grouped into two categories: system suitability failures and sample related failures. As used herein, the term "system suitability failure(s)" refers to problems with the assay system such as failure of the sample reader, reference electrode, or accessory equipment such as pipettes and operator error. In addressing system suitability failure rates, operator training can be an effective solution. As used herein, the term "sample related failure(s)" refers to an assay failure due to interference by one or more components in the sample. Frequently, sample related failures can be attributed to interference of protein with the detection of a non-proteinaceous sample component. In fact, when assaying for DNA using the Threshold^® Total DNA assay, "sample related failure" rates greater than 50% are not uncommon as a result of protein interference with the DNA in a protein-containing sample.

Pretreatment Step

[0055] As previously stated, protein in a sample may interfere with the ability of a

DNA assay to detect the DNA in the sample. The protein in the sample may interfere with DNA extraction, the labeling of the DNA in an assay, and/or may cause artificially high signals during DNA detection. Thus, a sample pretreatment step that decreases protein interference in a sample can improve DNA assay performance.

[0056] One embodiment of the invention provides a pretreatment step which uses a sample treatment mixture having three denaturing agents and a reducing agent. According to one embodiment, the three denaturing agents are chaotropic agents. The pretreatment step denatures and reduces protein in a protein-containing sample. A protein that has been denatured and reduced, can be digested more readily by digested by known methods, such as, for example, the use of an endopeptidase. The pretreatment and digestion steps increase solubility of the protein in the sample, which facilitates extraction and detection of the DNA in the sample. Known methods, such as, a qualitative analysis using SDS- PAGE, can be used to confirm that the amount of interfering protein in the sample is decreased.

[0057] Surprisingly, the pretreatment step is capable of decreasing protein interference for protein containing samples up to 0.1 mg/ml, up to 1.0 mg/ml, up to 10 mg/ml, up to 50 mg/ml, up to 100 mg/ml, up to 150 mg/ml, or up to 200 mg/ml. Although not wishing to be limited by theory, the pretreatment step is believed to decrease protein interference by keeping protein solubilized in the sample. Accordingly, this allows for precipitation of the DNA in the sample under conditions in which the protein does not co- precipitate, bind to or otherwise inhibit precipitation of DNA. As a result of decreased protein interference, sample related assay failures are significantly decreased, samples are capable of being assayed without the need for dilution, and sensitivity of the DNA assay is retained.

Protein Digestion

[0058] In one embodiment, the method of detecting DNA in a sample also includes a protein digestion step. Methods of digesting protein are known. According to one aspect of the invention, digestion of the protein-containing sample is performed in conjunction with a protein denaturation and reduction steps. According to this aspect of the invention, a protein digestion solution is added to the sample treatment mixture of the present invention. Alternately, the digestion step can be performed separately from the denaturation and reduction pretreatment step. In one embodiment, the digestion solution includes a protease. In one embodiment the protease is an endopeptidase. "Endopeptidases" as used herein refers to enzymes that are capable of catalyzing the cleavage of peptide bonds within a polypeptide or protein. Peptidase refers to the fact that the enzyme acts on peptide bonds and "endo" refers to the fact that these are internal bonds. In one embodiment, the endopeptidase is proteinase K.

[0059] Proteinase K is a non-specific serine protease isolated from the fungus

Tritirachium album Limber. Proteinase K cleaves peptide bonds adjacent to the carboxylic group of aliphatic, hydrophobic, and aromatic amino acids. Proteinase K can digest both native and denatured proteins and can exhibit increased proteolytic activity when the protein is treated with a chaotxopic agent and a reducing agent such as, for example, dithiothreitol. Additionally, activation of proteinase K requires two bound calcium ions, and therefore calcium chloride (CaCl₂) may be added to the protein digestion solution. According to the invention, calcium chloride is added at a final concentration about 0.1 mM to about 100 mM, about 1 mM to about 50 mM, or about 1 mM to about 10 mM.

[0060] Proteinase K can retain its activity over a wide range of temperatures, for example, between about 20⁰C and about 60⁰C, between about 20⁰C and about 40⁰C, or between about 20 ⁰C and about 30⁰C. Proteinase K can also retain its activity over a wide range of pH values, for example, between about pH 4 and about pH 12. Additionally, proteinase K can remain active in the presence of chaotropic protein denaturing reagents such as, guanidine thiocyanate (GTC), guanidine hydrochloride (GuHCI), and urea and thus makes it useful for the digestion of proteins in a sample in conjunction with or following a protein denaturation and reduction pretreatment step.

[0061] Typically, the specific activity of commercially available supplies of proteinase K used for protein digestion is about 30 units/mg of protein. The protocol outlined in the Threshold System Operator's Manual specifies the addition of 25μl proteinase K at 2mg/ml followed by an overnight incubation at 55°C. However, for samples containing high protein concentrations, sufficient digestion to decrease protein interference may not be achieved using the quantities recommended in the Threshold^® System Operator's Manual. Thus, proteinase K levels may need to be increased for protein digestion. According to one embodiment of the present invention, the Proteinase K is added at a final concentration of about 0.1 mg/ml to about 10 mg/ml, about 0.2 mg/ml to about 7 mg/ml, or about 0.2 mg/ml to about 5 mg/ml. According to this embodiment, the proteinase K would allow protein digestion for samples having eitiαer low or high protein concentrations. See e.g., Figure 3, which shows the results of treating 100 mg/ml protein samples with increasing Proteinase K concentrations present in sample treatment mixtures having three chaotropic agents and a reducing agent.

DNA Extraction

[0062] In one embodiment of the invention, the method for detecting DNA in a protein-containing sample includes a DNA extraction step. One purpose of the extraction step is to separate the DNA in the sample from protein in the sample that may interfere with the detection of the DNA. DNA extraction procedures that can be used in the present invention are known. In one embodiment, the extraction procedure is performed using the commercially available Wako DNA extraction kit (Wako Chemicals, VA, Catalog #296- 60501). See, phenol-free DNA extraction method described in the Threshold^® DNA Application Note, Part #0120-0304D, Molecular Devices Corp. See also Ishizawa et ai, Simple procedure of DNA isolation from human serum. Nucleic Acids Research, 19: 5792 (1991).

[0063] The Wako DNA extraction procedure is particularly effective in instances where the protein in the sample may have a high isoelectric point (pi) and thus may tend to bind the DNA. The sample pH can be increased to or above the pi of the protein prior to the extraction so as to inhibit DNA binding. Treating the protein-containing sample with the sample treatment mixture of the present invention prior to the Wako DNA extraction step can reduce binding and interference with the DNA. The DNA can then be extracted by precipitation and pelleted by centrifugation resulting in decreased protein interference. Once extracted, the DNA can be resuspended in a buffer, for example, water for injection (WFI) for subsequent DNA quantification using known detection methods. [0064] One benefit of the Wako extraction procedure is that it decreases safety and waste disposal concerns such as those associated with the use of organic solvents, such as phenol and/or chloroform. Additionally, for soluble proteins that do not bind DNA, the extraction procedure separates the DNA from the soluble protein by selective precipitation of the DNA. The Wako DNA extraction procedure describes the use of a chaotropic agent (sodium iodide), an anionic detergent (sodium N-lauroyl sarcosinate), and isopropanol to co-precipitate nucleic acids with a polysaccharide carrier, glycogen. The co-precipitated DNA is then pelleted by centrifugation allowing for separation of the DNA from interfering protein that may interfere with the DNA detection step.

DNA Detection

[0065] The DNA detection step can be performed using any known detection method. In one embodiment, the detection step is performed using a PCR method, such as described in U.S. Pat. No. 5,393,657. In another embodiment, the DNA detection step is performed using the Threshold^® Total DNA procedure described in the Threshold^® System User Manual (See Chapters 14-18, Part #0112-0046, MoleculaT Devices Corp., which is herein incorporated by reference).

[0066] When detection is performed using the Threshold^® Total DNA procedure the following steps are performed. Following DNA precipitation, the DNA is incubated with a labeling reagent. The DNA pellet is solubilized in DNA free buffer, thermally denatured to separate DNA strands and snap-cooled to produce single-stranded DNA molecules (ssDNA). The single-stranded DNA is labeled using a mixture, which includes biotinylated single-stranded binding protein (SSB), streptavidin and a urease conjugated anti-DNA antibody. Incubation of the ssDNA with these reagents promotes the formation of a DNA complex between all of these proteins. Following DNA labeling, the DNA is vacuum filtered through a biotinylated membrane. Immobilization occurs through free biotin binding sites on streptavidin. Following immobilization, the DNA is then electrochemically detected. The DNA-protein complexes are immobilized onto a biotinylated filter membrane (Threshold^® filter stick) using a vacuum filtration assembly. Each stick accommodates one sample in triplicate with controls or a single standard curve. The filter membrane is placed between a solution of urea and an ammonia sensitive silicon sensor (LAPS-light-addressable potentiometer sensor) unit. An electrical current proportional to the amount of ammonia in contact with the LAPS is produced. Urease immobilized on the filter membrane with the DNA produces ammonia from urea and the level of DNA present is correlated to the rate of change in μVolts per seconds (slope) or the change in the surface potential at the membrane surface is measured by the LAPS. The quantity of DNA in the sample is interpolated relative to a generated DNA standard curve during each assay. Under the current method, rather then using the zero calibrator buffer as the zero control sample as suggested in the Threshold^® Total DNA procedure, one can optionally use WFI. The DNA detection step can be performed using known any known methods, such as, but not limited to Southern Blot analysis using either a random or specific probe conjugated to the DNA to facilitate visualization

[0067] For ease of discussion, the disclosure has focused on the use of the sample treatment mixture for treating a sample used in a DNA assay. However, the invention is not limited to use in a DNA assay. The sample treatment mixture of the present invention can be used as a pretreatment step in any assay, known by one of skill in the art, for assaying a non-proteinaceous component present in a sample taken from a final or in- process biological drug product. According to this embodiment, the sample treatment mixture is used as a pretreatment step for removing protein interference prior to assaying for a non-proteinaceous sample component.

[0068] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting. The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures) in the disclosure is hereby incorporated by reference herein.

EXAMPLES

Example 1 - Protein Denaturation and Digestion Using a Single Denaturant [0069] An evaluation was performed on two sample treatment mixtures. The results for the sample treatment mixtures were qualitatively evaluated by SDS-PAGE for their ability to denature, reduce and digest samples containing varying protein concentrations. Materials

[0070] The first sample treatment mixture was recommended for use as a pretreatment step in the Threshold Total DNA assay. The Threshold^® treatment mixture includes 0.05% SDS and 100 μg/ml proteinase K and CaCl₂- The second treatment mixture was a single chaotropic solution, which included 10 mM TRIS (pH 8), 1.5 M buffered urea, 91 mM EDTA, 8 mM DTT₅ 0.2 mg/ml Proteinase K, and 2 mM CaCl₂. EDTA was included in the buffered urea solution to inhibit potential DNAses that may be present in the samples. The samples tested were diluted to 5, 10, 20, or 40 mg/ml of protein and were spiked with 50pg calf thymus DNA. All protein samples were in a citrate buffer containing sucrose, glycine, and polysorbate-80.

Methods

[0071] The methods used for treatment mixtures are described in the Threshold^®

Total DNA assay protocol. Initially, 500 μl samples treated with the Threshold^® treatment mixture and the single chaotropic agent treatment methods were incubated overnight at 55°C. Following the 55°C overnight digestion for both mixtures, the samples were chilled on ice and centrifuged to pellet any precipitated protein. The supernatant was removed from each sample and precipitates were resuspended with sample buffer to equal the starting volume. Nu-PAGE sample buffer was added to 10 μg equivalent of the supernatant. Samples were loaded onto a 4-20% Nu-PAGE gel (Invitrogen, Carlsbad, CA). The gels were overloaded with a higher concentration of protein normally required for silver stains in order to monitor subtle differences in the level of proteolysis of the protein. Molecular weight markers (Mark 12) were also included on the gel. The proteins were separated using the Nu-PAGE system (Invitrogen, Carlsbad, CA) as recommended by the manufacturer and the protein was visualized using a silver staining kit (Owl Scientific, Wobum, MA).

Results

[0072] The efficiency of the sample treatment mixtures were qualitatively determined using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). An effective treatment is achieved if the protein in the sample tested remains solubilized and no precipitation is visualized in the SDS-PAGE gels for both the supernatant and pellet. As shown in Figure 1, a concentration-dependent protein precipitation is visible in pellet samples for both sample treatment methods (i.e., 5 mg/ml (lanes 1 and 2), 10 mg/ml (lanes 3 and 4), 20 mg/ml (lanes 5 and 6), and 40 mg/ml (lanes 7 and 8). Specifically, as shown by the significant protein precipitation in lanes 2, 4, 6 and 8 of Figure l(b), the Threshold^® treatment mixture was ineffective in treating the protein. Notably, the Threshold^® treatment mixture was ineffective even at the lowest level tested (5 mg/ml — lane 2). As shown in lanes 1, 3, 5 and 7 of Figure l(b), the buffered urea treatment mixture was more effective as indicated by less visible protein precipitation for these protein samples. Very little precipitation was observed in the 5 mg/ml (lane 1) and 10 mg/ml (lane 3) samples. Precipitation slightly increased in the 20 mg/ml (lane 5) and 40 mg/ml (lane 7) samples but the levels of precipitation were visually significantly less than the Threshold^® treatment mixture samples.

[0073] Overall, the Threshold^® treatment mixture was ineffective at each of the protein concentration ranges tested. Accordingly, use of the Threshold^® treatment mixture as an initial treatment step for samples containing concentrations of protein greater than 5 mg/ml in an assay would likely result in protein interference with the assay. In contrast, the buffered urea treatment mixture was effective in denaturing protein concentrations at 10 mg/ml or less. However, the buffered urea treatment mixture was ineffective at concentrations greater than 10 mg/ml. Thus, similar to the Threshold^® treatment mixture, use of the buffered urea treatment mixture for treating protein containing samples having protein concentrations greater than 10 mg/ml would also likely result in protein interference in an assay.

Example 2 - Evaluation of Sample Treatment Mixtures

[0074] Sample treatment mixtures having one, two, or three chaotropic agents, were tested for their ability to denature, reduce and digest samples containing higher concentrations of protein (100 mg/ml).

Materials

[0075] Samples with 100 mg/ml of protein in a citrate buffer containing sucrose, glycine, and polysorbate-80 were treated with either one denaturant (urea, guanidine or NaI), two denaturants (guanidine and urea) or three denaturants (guanidine, urea, and sodium iodide). The sample treatment mixture included reducing agent (dithiothreatol), calcium chloride, and proteinase K as indicated in Table 1.

Table 1. Sample Treatment Mixtures

Methods

[0076] Samples (50μl at 100mg/ml) were treated with sample treatment mixtures as indicated in Table 1 and incubated overnight at 55° C. Following the 55°C overnight treatment, samples were chilled on ice and then centrifuged for 10 minutes at 16,000 x g to pellet any precipitated protein. For each sample, the supernatant was separated from the pellet and the^{^} pellet was resuspended in an equivalent volume to the amount of supernatant removed.

[0077] The efficiency of each treatment step was then qualitatively determined using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were loaded onto a 4-20% Nu-PAGE gel (Invitrogen, Carlsbad, CA)). The gels were overloaded with a higher concentration of protein normally required for silver stains in order to monitor subtle differences in the level of proteolysis of the protein. Molecular weight markers (Mark 12) were also included on the gel. The proteins were separated using the Nu-PAGE system (Invitrogen, Carlsbad, CA) as recommended by the manufacturer and the protein was visualized using a silver staining kit (Owl Scientific, Woburn, MA).

Results [0078] Similar to the analysis in Example 1, successful sample treatment is achieved if the protein in the sample is solubilized and thus no precipitation is observed using SDS-PAGE. As indicated by the protein precipitation in lanes 1 and 2 of Figure 2(a), the treatment mixtures used in treating samples 1 and 2, which represent samples treated with buffered urea and buffered urea and guanidine hydrochloride, respectively, were ineffective. In contrast, as shown in lane 3 of Figure 2, a treatment mixture which included buffered urea, guanidine hydrochloride and sodium iodide, was effective in decreasing protein precipitation.

[0079] In addition to the effective protein treatment provided by the three chaotropic agents, increased Proteinase K concentrations in the sample treatment mixtures further improved the effectiveness of the treatment mixture. As shown in lanes 1-4 of Figure 3, as the Proteinase K concentration increased from 0.2 mg/ml to 2 mg/ml, a corresponding decrease in the amount of precipitated protein was observed. As shown in lane 4, a treatment mixture which included 1.1M buffered urea, 1.1M Guanidine Hydrochloride, 1.1M Sodium iodide, 19mM DTT, 175mM EDTA, 2 mg/ml Proteinase K, and 6mM calcium chloride resulted in the most effective reduction in protein precipitation.

Example 3

Comparison of sample treatment methods on samples containing over 40 mg/ml of a fusion protein

[0080] An experiment was performed on samples containing approximately 47 mg/ml of a protein fused to human serum albumin (HSA) to compare a sample treatment method including one chaotropic agent with a sample treatment method including three chaotropic agents.

Materials and Methods

[0081] Samples containing a fusion protein at a concer>tration of 47 mg/ml were treated with either a single chaotropic agent or a sample treatment mixture including three chaotropic agents. AU samples were initially diluted to 10 mg/ml. For the sample treated with a single chaotropic sample treatment mixture (referred to hereafter as the buffered urea treatment mixture), 1OmM TRIS^" (pH8), 1.5M urea, 9ImM EDTA, 8mM DTT, 0.2mg/ml Proteinase K, 2mM CaC^ was added to the sample. For the sample treated with a mixture containing three chaotropic agents containing a final concentration of 1.1 M urea, LlM Guanidine, 1.1 M NaI, 49mM EDTA, 36mM DTT, 2mg/ml Proteinase K, 6mM CaCl₂ was added to the sample. All samples were incubated overnight (approximately 14-16 hours) at 55° C. Samples were then chilled on ice and centrifuged for 10 minutes at 16,000 x g to pellet any precipitated protein. For each sample, the supernatant and pellet were separated and the pellet was resuspended in an equivalent volume to the amount of supernatant removed.

[0082] The efficiency of each sample treatment mixture was then qualitatively determined using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 6 μg of protein equivalents for each sample, both precipitate and supernatant, were loaded onto a 4-20% Nu-PAGE gel (Invitrogen). Molecular weight markers (Mark 12) were also included on the gels. The proteins were separated using the Nu-PAGE system (Invitrogen, Carlsbad, CA) as recommended by the manufacturer and the protein was visualized using the silver staining kit (Owl Scientific, Woburn, MA).

Results

[0083] The results, shown in the silver stained gel of Figure 4, indicate that the sample treatment mixture containing three chaotropic agents was more successful in treating the protein sample when compared to the buffered urea treatment mixture. A successful result is achieved if the protein sample tested remained solubilized and thus little protein precipitation is visualized using SDS-PAGE. As indicated by the dark protein band in lane 2 of Figure 4, the buffered urea treatment mixture was ineffective in denaturing and digesting the protein and thus protein precipitation occurred. In contrast, the pellet for the sample treatment mixture containing three chaotropic agents showed no visual precipitated protein, as indicated by lane 1 of the gel; and, the respective supernatant load showed only a small amount of protein present in the sample (lane 5). Thus, these results indicate that the sample treatment mixture containing three chaotropic agents effectively denatured and digested protein in a sample containing high concentrations of protein.

Example 4

Use of a sample treatment mixture in a DNA Assay

[0084] An experiment was performed to determine whether a sample treatment method having three chaotropic agents are capable of decreasing protein interference in samples containing high protein concentrations to allow for effective measurement of

DNA. Sample Pretreatment

[0085] 500 μl samples containing either 30 mg/ml of a monoclonal antibody, 44 mg/ml of a fusion protein, 105 mg/ml of a monoclonal antibody, or 19 mg/ml of a monoclonal antibody were spiked with various picogram amounts of DNA. A sample treatment mixture containing three chaotropic agents (4M urea, 4M guanidine hydrochloride, 4M sodium iodide) in addition to DTT, CaCl₂ and Proteinase K was added to each of the undiluted samples. After adding the sample treatment mixture, the samples had final concentrations of LlM Urea, 1.1 guanidine hydrochloride, 1.1M sodium iodide, 36 mM DTT, 6 mM CaCl₂, and 2 mg/ml Proteinase K. All samples were then incubated overnight (approximately 14-16 hours) at 55° C.

DNA Extraction

[0086] Following the overnight incubation, DNA was extracted from each of the samples following the procedures described in the WAKO DNA Extractor Kit (WAKO Chemicals, Richmond, VA). Initially, 280 μl of an extraction buffer made up of 8M sodium iodide in 13 mM EDTA was added to each sample. Additionally, 3 μl of glycogen solution provided in the DNA Extractor Kit from Wako Chemicals was added to each sample. The samples were then incubated for fifteen minutes in a 37° C water bath. Following the incubation, 800 ul of isopropyl alcohol (HPLC grade, Sigma Aldrich Catalog) was added to each sample. After letting the samples stand at room temperature for fifteen minutes, 800 μl of Wash B from the DNA Extractor Kit was added to each sample. The samples were then centrifuged at 16,000xg for fifteen minutes to pellet the precipitated DNA. After removal of the supernatant, the pellet for each sample was resuspended in 500 μl of water for injection (WFI).

DNA Detection

[0087] After resuspending the samples in WFI, DNA detection was performed using the Threshold^® system. The procedures followed for applying the Threshold^® system for use in a DNA assay are described in Chapter 15 of Threshold System User Manual (Threshold^® System Operator's Manual #0112-0046, Molecular Devices Corp.).

Results

[0088] DNA values for each of the samples were determined using the Threshold^®

Total DNA assay. Based on these values, the percent recovery of the spike was determined. A spiked DNA recovery value within 30% of the expected concentration indicates a successful elimination of protein interference and indicates an absence of a sample related assay failure. Percent recoveries of spiked samples were determined as follows:

((amount of spiked DNA recovered — zero calibrator)/amount spiked) x 100 = % recovery.

A zero calibrator, having the same conditions as other test samples hut without DNA was run concurrently with the samples in each assay. Percent recoveries for each of the samples tested are shown in Tables 3-6. Table 3. Spike Recovery of DNA in 30 mg/ml monoclonal antibody samples.

* Not Determined

[0089] As shown in Table 3, each of the spiked DNA ranges from 6 pg to 180 pg were recovered within 30% of the expected concentration. Additionally, the 30 mg/ml monoclonal antibody samples were run three times and each of the runs was also within the acceptable recovery limitation of 30%.

Table 4. Spike Recovery of DNA in 44 mg/ml fusion protein samples.

[0090] As shown in Table 4, DNA sample spikes of 6, 12, and 50 picograms for samples containing 44 mg/ml of a fusion protein were recovered within the 30% recovery limit. As a result, the sample treatment mixture of the present invention was effective in decreasing protein interference with the detection of DNA for a sample containing 44 mg/ml.

Table 5. Spike Recovery of DNA in 105 mg/ml monoclonal antibody samples.

[0091] As shown in Table 5, each of the spiked DNA ranges from 6 pg to 180 pg for triplicate samples containing 105 mg/ml of a monoclonal antibody was recovered within 30% of the expected concentration. Similarly, as shown in Table 6, each of the spiked DNA ranges from 6-180 pg were recovered within 30% of the expected concentration for each of the three assay runs performed for the 19 mg/ml monoclonal antibody samples.

[0092] Overall, the results show that the sample pretreatment method was effective in decreasing protein interference in samples with protein concentrations ranging from 19 to 105 mg/ml without the need for sample dilution. Therefore, the sample pretreatment method would be effective in treating a wide variety of proteins, as indicated by its ability to treat both monoclonal antibodies and fusion proteins.

Table 6. Spike Recovery of DNA in 19 mg/ml monoclonal antibody samples.

Example 5 - Evaluation of the Sample Treatment Mixture for In-Process Samples in a DNA assay.

[0093] Biological drug products are typically purified using purification processes having multiple chromatography, filtration, and viral inactivation and removal steps. Most purification processes incorporate orthogonal chromatography column steps (e.g., anion, cation, affinity chromatography). For optimal performance and purification of the biological drug product, each step in the purification process typically requires the use of different buffers and operating parameters. As a result, in-process samples taken from one step during the purification process will have sample component compositions that vary from in-process samples taken from a different column purification step in the process. [0094] To assess purification performance for each column step, in-process samples are typically assayed for the presence and/or quantity of various non- proteinaceous components present in the sample in addition to the biological drug product. For evaluating in-process non-proteinaceous sample components, assays must be performed without protein interference. As previously discussed, protein interference affects the sensitivity of the assay and also contributes to sample related assay failures. [0095] Accordingly, an experiment was performed to test the sample treatment mixture with three chaotropic agents and a reducing agent for its' ability to decrease protein interference in in-process samples having different protein concentrations. Following a qualitative performance assessment of the sample treatment mixture using SDS-PAGE, the in-process samples were evaluated to determine if the sample treatment mixture was effective in decreasing the effect that various components (e.g., protein concentration and buffer conditions) in the in-process samples typically have on measuring picogram (pg) amounts of DNA.

Table 7. In-process sample buffer compositions and typical protein and DNA concentrations.

Materials

[0096] In-process samples were taken from the pools of four chromatography steps used in the purification process for a monoclonal antibody. A description of each in- process sample taken during the purification process is shown in Table 7. Typical values for protein and DNA concentrations for each sample are also provided in table 7. The typical values were generated from historical data (not shown) for the purification of the monoclonal antibody using the in-process columns. A sample treatment mixture including three chaotropic agents and a reducing agent was added to each sample. After adding the sample treatment mixture, the samples had final concentrations of 2M Urea, 2M guanidine hydrochloride, 2M sodium iodide, 100 mM DTT, 10 mM CaCl₂, and 2 mg/ml Proteinase K.

Methods

[0097] Qualitative evaluation of the pretreatrnent mixture was performed using 4-

20% Nu-PAGE gels (Invitrogen, Carlsbad, CA). Sample proteins were separated using the Nu-PAGE system as recommended by the manufacturer and the proteins were visualized using an Owl silver staining kit (Owl Scientific, Woburn, MA) as per the manufacturer's instructions. The DNA provided in the Threshold kit was diluted using WFI and spiked into each of the samples. Extraction of the DNA for each in-process sample was performed using the DNA Extractor Kit from Wako Chemicals. Detection of the DNA levels was determined using the Threshold^® system. The procedures followed for applying the DNA assay to the Threshold^® system are described in Chapter 15 of Threshold System User Manual (Threshold^® System Operator's Manual #0112-0046, Molecular Devices Corp.).

In-process sample pretreatment

[0098] Four 500 μl aliquots from each in-process column pool sample were tested without dilution. Four additional 500 μl aliquots from the column 1 load were diluted 10,000 fold with water for injection (WFI) in order for the DNA value to be interpolated within the range of the assay. Two of the four aliquots from each in-process sample were then spiked with either 6 or 180 pg/test of DNA to target the top and bottom end of the Threshold^® DNA assay working range (6-180 pg/test DNA). 150 μl of the sample treatment mixture containing three chaotropic agents, DTT, CaCl₂ and proteinase K was then added to each of the samples. After adding the sample treatment mixture, the samples had final concentrations of 2M Urea, 2M guanidine hydrochloride, 2M sodium iodide, 100 mM DTT₃ 10 mM CaCl₂, and 2 mg/ml proteinase K. A control sample for each of the in-process samples, which was not pretreated with the sample treatment mixture, was also used in the experiment. AU samples were incubated overnight (approximately 14-16 hours) at 55° C. Following incubation, an aliquot, equivalent to 6 μg, was taken from one of each of the non-spiked samples and placed in an eppendorf tube and centrifuged at 16,000 x g for 15 minutes. Following centrifugation, the supernatant and pellet were separated and the pellet was resuspended in an equivalent volume to the amount of supernatant removed.

[0099] The efficiency of the sample treatment mixture for the non-spiked in- process samples was qualitatively determined using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 6 μg protein equivalents for each sample, both precipitate and supernatant, were loaded onto a 4-20% Nu-PAGE gel (Invitrogen). Molecular weight markers (Mark 12) were also included on the gels. The proteins were separated using the Nu-PAGE system as recommended by the manufacturer and the protein was visualized using the silver staining kit.

DNA spike recovery from in-process samples

[0100] The three remaining aliquots (un-spiked, 6 μg DNA spike, and 180 μg

DNA spike) for each in-process sample were evaluated to determine the accuracy of detecting the spiked DNA and thus indicating the efficiency of the sample treatment mixture for decreasing protein interference.

DNA Extraction

[0101] Following the overnight incubation, DNA was extracted from each of the three aliquots taken from each sample. Initially, 280 μl of an extraction buffer made up of 8M sodium iodide in 13 mM EDTA was added to each sample. Additionally, 3 μl of glycogen solution provided in the DNA Extractor Kit from Wako Chemicals was added. The samples were then incubated for fifteen minutes in a 37° C water bath. Following the incubation, 800 μl of isopropyl alcohol (HPLC grade, Sigma Aldrich Catalog) was added to each sample. After letting the samples stand at room temperature for fifteen minutes, 800 μl of Wash B from the DNA Extractor Kit was added to each sample. The samples were then centrifuged at 16,000xg for fifteen minutes to pellet the precipitated DNA. After removal of the supernatant, the pellet for each aliquot was resuspended in 500 μl of water for injection (WFI).

DNA Detection

[0102] After resuspending the samples in WFI, DNA levels were determined using the Threshold^® system. The procedures followed for applying the DNA assay to the Threshold^® system are described in Chapter 15 of Threshold System User Manual (Threshold^® System Operator's Manual #0112-0046, Molecular Devices Corp.). Results

Evaluation of sample pretreatment mixture using SDS-PAGE

[0103] A sample treatment mixture which included three denaturants, a reducing agent, and proteinase-K was qualitatively assessed by SDS-PAGE for its' effectiveness in decreasing protein interference in in-process samples. The level of performance of the sample treatment mixture was determined by the amount of protein precipitation and/or digestion of the sample relative to the undigested control. As shown in Figure 5, there was no evidence of protein precipitation after treating with the sample treatment mixture in any of the in-process samples as indicated by the absence of bands for treated in-process samples. Furthermore, considering the column 1 load sample, regardless of whether this sample was diluted, the sample treatment mixture significantly decreased possible protein interference as indicated by the absence of nearly all of the silver stainable bands compared to the undigested sample (one weak band remains as indicated by the arrow in lane 4).

Evaluation of DNA spike recovery from in-process samples

[0104] The sample treatment mixture was tested for its' ability to decrease protein interference in in-process samples and thus allow for accurate determination of spiked DNA values using the Threshold DNA assay. Prior data, shown in Table 7, indicates that after the column 1 load is processed and collected in the column 1 pool, the levels of DNA for the in-process column pools are near or below the lower detection limit for the assay range (i.e., 3 pg/test). The efficiency of the sample treatment mixture to decrease protein interference and the accuracy of recovering the spiked DNA were evaluated using undiluted in-prbcess samples spiked with DNA at the low end (6 pg/test) and at the high end (180 pg/test) of the curve.

[0105] DNA results were determined for each in-process sample from the average of all results, within the range of the assay, and expressed in pg/test. To allow for meaningful spike recovery data, DNA concentrations were also calculated from non- spiked samples below the limit of detection. As shown in Table 8, DNA spiked at 6 pg/test in each in-process sample, was consistently recovered within the acceptable recovery limitation of 30%. Additionally, the DNA spiked at 180 pg/test into each in- process sample, was consistently recovered for all four in-process samples. As expected, based on the historical data, values for several of the non-spiked in-process samples were below the lower detection limit as shown by the value in parenthesis and thus were indicated as not determinable (ND). Table 8

Example 6 Use of the Sample Treatment Mixture in a Benzyl Alcohol Assay

Introduction

[0106] Small molecules are often introduced into protein containing mixtures during production and purification processes. When storing chromatography columns used in the purification process, buffers are used to maintain proper column chemistries and minimize bacterial growth. A buffer containing 2% benzyl alcohol, a bacteriostatic agent that helps to preserve the column chemistry, is used to store a Protein A affinity- column between antibody purification runs. After storage, the column undergoes a series of pre-equilibration steps intended to eliminate the beiLzyl alcohol prior to loading the protein containing mixture. Even so, analysis of the protein containing mixture for residual benzyl alcohol must be performed to show sufficient removal. Therefore, for accurate detection of the benzyl alcohol, the protein in the protein containing mixture must be removed to prevent interference with detection of the benzyl alcohol. Thus, a sample treatment mixture must be used to remove protein prior to assaying for the presence of benzyl alcohol using GC-MS. Materials

[0107] A capillary gas chromatography system (Agilent Technologies, Palo Alto,

CA) coupled with a mass spectroscopy detector (Agilent Technologies, Palo Alto, CA) (GC-MS) was used to measure concentrations of benzyl alcohol down to ng/μl levels. Methods

[0108] 2 ml samples of buffer containing 20 mg/ml of protein or water for injection (WFI) were aliquoted into 10 ml Teflon centrifuge tubes (Oak Ridge) and were either spiked or not spiked with benzyl alcohol. A series of benzyl alcohol spikes were made using commercially available benzyl alcohol standard (Ultra Scientific). A sample treatment mixture containing three chaotropic agents 4M urea, 4M guanidine hydrochloride, and 4M sodium iodide was then added to each of the samples. All samples were incubated overnight (approximately 14-16 hours) at 55° C. Following incubation, 1 ml of ethyl acetate (Sigma- Aldrich) containing 10 μg/ml pentadecane (Ultra Scientific) was added to the samples. The samples were then vigorously shaken for 2 hours at ambient temperature in order to extract the benzyl alcohol into the organic phase. The benzyl alcohol was concentrated two-fold during extraction. Phase separation was further facilitated by a five minute centrifugation at 2000 rpm in a bench top centrifuge. Following centrifugation, an aliquot of the ethyl acetate layer (300μl) was transferred to a GC sample vial (Agilent Technologies) and analyzed in an Agilent HP6890GC/MS. Sample analysis was completed as follows: A 30-meter DB-I MS column (J&W Scientific) was continuously perfused with He gas at 1 ml/minute. An aliquot of the sample (1 μ.1) was injected into an inlet and maintained at 270° C (splitless injection). The temperature of the CG oven starts at 40° C and ramps up to 100° C at 10° C/minute. The second ramp goes from 100° C to 300° C at 20° C /minute ramp. Total run time equals 21 minutes and each chemical elutes corresponding to its boiling point. Benzyl alcohol eluted first at 8.4 minutes (206° C) and the internal standard, pentadecane eluted at 14.5 minutes (270° C). The area under the curve (AUC) at 8.4 minutes (benzyl alcohol) was normalized to the area under the curve at 14.5 minutes (pentadecane) to generate a peak ratio value. The peak ratio was used to generate the standard curve. The benzyl alcohol concentration in the samples was interpolated from the standard curve, corrected for dilution and reported in nanograms (ng). See Figure 6 for a schematic of the GC-MS process.

[0109] Commercial benzyl alcohol (Ultra Scientific) was used to generate the standard curve. The benzyl alcohol was first diluted to 200 μg/ml. in ethyl acetate. The 200 μg/ml was further diluted to a final concentration of 100 μg/ml using the pentadecane standard solution (20 μg/ml pentadecane in ethyl acetate). The 100 μg/ml standard was serially diluted 1 to 2 in assay diluent (10 μg/ml pentadecane in ethyl acetate) using a Hamilton syringe and washing the syringe with, ethyl acetate between each dilution. Results

[OHO] Adequate recovery of benzyl alcohol prior to sample pre-treatment indicates that the method accurately measured benzyl alcohol without significant interference from protein in the sample. Samples with buffer containing 20 mg/ml of protein or WFI were spiked with 5, 10, 20, or 40 ng and percent recoveries were determined. As shown in Table 9, benzyl alcohol spikes at 5 ng or greater were recovered within 30% of the expected concentration. These results indicate that possible protein interference was decreased by the sample pretreatment mixture and that benzyl alcohol spike concentrations at 5 ng or greater were adequately recovered.

Table 9 Spike Recovery of Benzyl Alcohol

Example 7 - Evaluation of Sample Treatment Mixtures

[0111] Sample treatment mixtures were tested for their ability to reduce protein in samples with 100 mg/ml of protein in a citrate ^"buffer containing sucrose, glycine, and polysorbate-80. Samples (50μl at 100mg/ml) were treated with sample treatment mixtures as indicated in Table 10 and incubated overnight at 55° C. Following the 55°C overnight treatment, samples were chilled on ice and then centrifuged for 10 minutes at 16,000 x g to pellet any precipitated protein. For each sample, the supernatant was separated from the pellet and the pellet was resuspended in an equivalent volume to the amount of supernatant removed.

Table 10. Sample Treatment Mixtures

[0112] The efficiency of each treatment step was then qualitatively determined using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were loaded onto a 4-20% Nu-PAGE gel (Invitrogen, Carlsbad, CA)). The gels were overloaded with a higher concentration of protein normally required for silver stains in order to monitor subtle differences in the level of proteolysis of the protein. The proteins were separated using the Nu-PAGE system (Invitrogen, Carlsbad, CA) as recommended by the manufacturer and the protein was visualized using a silver staining kit (Owl Scientific, Woburn, MA). Results

[0113] A successful sample treatment is achieved if the protein in the sample is solubilized and thus no precipitation is observed using SDS-PAGE. As indicated by the protein precipitation in laτies 2-4, the samples treated with guanidine hydrochloride, buffered urea and the WAKO kit method, respectively, were ineffective. In contrast, as shown in lane 5 of Figure 7, the treatment mixture with three chaotropic agents (buffered urea, guanidine hydrochloride and sodium iodide) was effective in decreasing protein precipitation as indicted by no protein precipitation in the lane.

Claims

What is Claimed:

1. A method for decreasing protein interference in a protein-containing sample wherein the method comprises treating the sample with a sample treatment mixture comprising three denaturants.

2. The method of claim 1, wherein one of said three denaturants is selected from the group consisting of: guanidine hydrochloride and guanidine thiocyanate.

3. The method of claim 2, wherein one of said three denaturants is guanidine hydrochloride.

4. The method of claim 3, wherein the guanidine hydrochloride is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

5. The method of claim 1 , wherein one of said three denaturants is urea.

6. The method of claim 5, wherein the urea is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

7. The method of claim 1, wherein one of said three denaturants is sodium iodide.

8. The method of claim 7, wherein the sodium iodide is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

9. The method of claim 1, wherein the three denaturants in the sample treatment mixture comprise sodium iodide, guanidine hydrochloride, and urea.

10. The method of claim 1, wherein the sample treatment mixture also comprises a reducing agent.

11. The method of claim 10, wherein the reducing agent is dithiothreitol.

12. The method of claim 11, wherein the dithiotreitol is included in the sample treatment mixture at a concentration between about 1 mM and about 1 M.

13. A method for quantifying DNA in a protein-containing sample wherein the method comprises: pretreatmg the sample with a sample treatment mixture comprising three denaturants; digesting the protein in the sample; extracting the DNA from the sample; and detecting the extracted DNA.

14. The method of claim 13, wherein one of said three denarurants is selected from the group consisting of: guanidine hydrochloride and guanidine thiocyanate.

15. The method of claim 14 wherein one of said three denaturants is guanidine hydrochloride.

16. The method of claim 15, wherein the guanidine hydrochloride is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

17. The method of claim 13, wherein one of said three denaturants is urea.

18. The method of claim 17, wherein the urea is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

19. The method of claim 13, wherein one of said three denarurants is sodium iodide.

20. The method of claim 19, wherein the sodium iodide is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

21. The method of claim 13, wherein the denaturants in the sample treatment mixture comprise sodium iodide, guanidine hydrochloride, and urea.

22. The method of claim 13, wherein the sample treatment mixture also comprises a reducing agent.

23. The method of claim 22, wherein the reducing agent is dithiothreitol.

24. The method of claim 23, wherein the dithiotreitol is included in the sample treatment mixture at a concentration between about 1 mM and about 1 M.

25. The method of claim 13, wherein said digestion is performed using a protease solution.

26. The method of claim 25, wherein the protease solution comprises proteinase K.

27. The method of claim 13, wherein said extraction is performed using the WAKO DNA extraction kit.

28. The method of claim 13, wherein said detection includes labeling the DNA, immobilizing the DNA, and measuring the DNA.

29. The method of claim 13, wherein the detecting step comprises the Threshold™ detection system.

30. The method of claim 13, wherein the detecting step utilizes the polymerase chain reaction.

31. The method of claim 13, wherein the protein comprises an antibody.

32. The method of claim 13, wherein the protein comprises a non-antibody protein.

33. The method of claim 13, wherein the protein comprises a fusion protein.

34. The method of claim 13, wherein the protein in said sample is included at a concentration between about 0.1 mg/ml and about 200 mg/ml.

35. The method of claim 13, wherein the protein in said sample is included at a concentration between about 5 mg/ml and about 125 mg/ml.

36. The method of claim 13, wherein the protein comprises human serum albumin.

37. The method of claim 13, wherein the sample is obtained from the group consisting of:

(a) fermentation broth;

(b) conditioned cell culture medium;

(c) serum; and

(d) milk.

38. A method for quantifying DNA in a protein containing sample wherein the method comprises: treating the sample with a sample treatment mixture comprising: guanidine hydrochloride; urea; sodium iodide; and dithiotreitol digesting the protein in the sample with proteinase K; extracting the DNA from the sample using the WAKO DNA extraction kit; and detecting the extracted DNA using the Threshold™ detection system.

39. The method of claim 38, wherein the guanidine hydrochloride is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

40. The method of claim 38, wherein the urea is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

41. The method of claim 38, wherein the sodium iodide is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

42. The method of claim 38 wherein said proteinase K used for digesting the protein is included in said sample treatment mixture.

43. A method for detecting a small molecule in a protein-containing sample wherein the method comprises: pretreating the sample with a sample treatment mixture comprising three denaturants; digesting the protein in the sample; and detecting the small molecule.

44. The method of claim 43, wherein one of said three denaturants is selected from the group consisting of: guanidine hydrochloride and guanidine thiocyanate.

45. The method of claim 44 wherein one of said three denaturants is guanidine hydrochloride.

46. The method of claim 45, wherein the guanidine hydrochloride is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

47. The method of claim 43, wherein one of said three denaturants is urea.

48. The method of claim 47, wherein the urea is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

49. The method of claim 43, wherein one of said three denaturants is sodium iodide.

50. The method of claim 49, wherein the sodium iodide is included in the sample treatment mixture at a concentration between about 100 mM and about 10 M.

51. The method of claim 43, wherein the denaturants in the sample treatment mixture comprise sodium iodide, guanidine hydrochloride, and urea.

52. The method of claim 43, wherein the sample treatment mixture also comprises a reducing agent.

53. The method of claim 52, wherein the reducing agent is dithiothreitol.

54. The method of claim 53, wherein the dithiotreitol is included in the sample treatment mixture at a concentration between about 1 mM and about 1 M.

55. The method of claim 43, wherein said digestion is performed using a protease solution.

56. The method of claim 55, wherein the protease solution comprises proteinase K.

57. The method of claim 43, wherein said extraction is performed using the WAKO DNA extraction kit.

58. The method of claim 43, wherein said detection includes labeling the DNA, immobilizing the DNA, and measuring the DNA.

59. The method of claim 43, wherein the detecting step comprises the Threshold™ detection system.

60. The method of claim 43, wherein the detecting step utilizes the polymerase chain reaction.

61. The method of claim 43, wherein the protein comprises an antibody.

62. The method of claim 43, wherein the protein comprises a non-antibody protein.

63. The method of claim 43, wherein the protein comprises a fusion protein.

64. The method of claim 43, wherein the protein in said sample is included at a concentration between about 0.1 mg/ml and about 200 mg/ml.

65. The method of claim 43, wherein the protein in said sample is included at a concentration between about 5 mg/ml and about 125 mg/ml.

66. The method of claim 43, wherein the protein comprises human serum albumin.

67. The method of claim 43, wherein the sample is obtained from the group consisting of:

(a) fermentation broth;

(b) conditioned cell culture medium;

(c) serum; and

(d) milk.