Detection and removal of polyvinyl sulfonates from biomolecular compositions

Abstract

The present disclosure provides materials and methods for assessing the presence and levels of polyanionic compounds in a sample that inhibit PCR reactions, including assays for the presence and optionally amount of polyvinyl sulfonate, as well as materials and methods for reducing or removing such compounds from buffer and protein solutions using a variety of approaches, including titration-based techniques.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 093,120, filed on 16 October 2020, which is incorporated herein by reference in its entirety.

[0002] Integration by referencing electronically submitted documents The sequence listing, which is part of this disclosure, is submitted together with this specification as a text file. The name of the text file containing this sequence listing is "55057A_Seqlisting.txt", created on October 4, 2021, and has a size of 1,070 bytes. The subject of this sequence listing is incorporated herein by reference in its entirety.

[0003] This disclosure generally relates to the field of biomolecular purification, and more specifically to protein purification. [Background technology]

[0004] Currently, the potential of biologics and biosimilars is becoming a reality, and these therapeutic classes are further contributing to the available treatment options for diseases and conditions in humans and other animals. These products, including recombinant proteins, various forms of antibodies and their fragments that retain binding ability, and vaccines, are all expressed in host cells ranging from bacteria, yeast, mammalian cells, and a continuous range of cell lines. As seen in cell culture, these products are mixed with various contaminants, including fragments of host cell DNA. Furthermore, residual amounts of host cell DNA can survive rigorous purification processes and remain as harmful impurities in purified protein preparations such as biologics. Residual host cell DNA in protein preparations administered to animals, such as human patients, may induce undesirable immune responses or increase the risk of cancer. As a result, regulatory bodies worldwide impose limits on the concentration of host cell DNA in preparations intended for human administration. The World Health Organization (WHO) and the European Union (EU) allow residual host cell DNA up to 10 ng / dose, while the U.S. Food and Drug Administration allows less than 100 pg / dose. Accurate, precise, and highly sensitive methods for detecting and quantifying low levels of host cell DNA are necessary to ensure that purified protein formulations intended for administration fall below these thresholds. Furthermore, cell cultures used for the efficient production of these proteins contain impurities beyond host cell DNA. Some of these impurities, such as small molecule compounds, can have a direct adverse effect on biologics and biosimilars (i.e., target proteins) produced by these cells by inhibiting the transcription or translation of target proteins, inhibiting the activity of expressed target proteins, or interfering with efforts to measure or monitor target proteins during the purification process.

[0005] Several polyanionic compounds, typically found in living cells used for culture-based recombinant target protein expression, may be found as impurities in cell cultures. Furthermore, some of these polyanionic compounds found in buffers and cell culture media are known inhibitors of various enzymes. Polyvinyl sulfonates (PVS) are polyanionic compounds that are known inhibitors of several enzymes, including RNA enzymes and DNA polymerases. PVS is also known to be present in preparations of 2-(N-morpholino)-ethanesulfonic acid (MES), a common buffer in biotherapeutic processes and purification operations. PVS may also be (undesirably) present in other buffer systems that utilize vinyl sulfonic acid as a starting material, such as Good's buffer. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there is a continuous need in the art for methods to accurately quantify host cell DNA contained in protein preparations intended for administration to humans or other animals. Furthermore, there is a continuous need for methods to reduce or remove such impurities from such protein preparations intended for administration. [Means for solving the problem]

[0007] This disclosure provides methods for assaying polyanionic PCR inhibitors, such as polyvinyl sulfonate compounds. These compounds inhibit a variety of enzymes, including nucleic acid polymerases such as DNA polymerase. These compounds also inhibit engineered forms of DNA polymerase, which is currently dominant in PCR amplification, and this disclosure presents methods for detecting and quantifying these inhibitory compounds. This disclosure further provides methods for removing such compounds from buffers (e.g., MES and Good's buffers) and protein solutions. These methods generally represent significant advances in the handling of biologics and proteins by providing methods for monitoring the presence of a major class of PCR inhibitors that interfere with efforts to monitor the purity of biologics produced in cell cultures, where host DNA needs to be monitored to ensure the purity of the target protein sufficient for use as a therapeutic agent in humans and / or other animals.

[0008] In one embodiment, the Disclosure provides a method for quantifying a polyanionic PCR inhibitor in a sample, comprising: a) preparing a dilution series of the sample comprising: b) spiking each member of the dilution series with a fixed amount of template DNA identifiable from host cell DNA; c) performing a PCR assay on each member of the dilution series and on a fixed amount of template DNA in the absence of any sample; d) creating a standard curve for the polyanionic inhibitor; e) comparing the PCR assay results of the dilution series with the PCR assay results of a fixed amount of template DNA in the absence of any sample; and f) identifying the concentration of the polyanionic PCR inhibitor in the sample. In some embodiments, the concentration of the polyanionic PCR inhibitor in the sample is within a range determined by the concentrations of the polyanionic PCR inhibitor in the least diluted member of the dilution series that shows complete spike recovery and the most diluted member of the dilution series that does not show complete spike recovery. In some embodiments, the number of members in the dilution series is 5, 6, 7, 8, 9, 10, 12, 15, or 20, thereby narrowing the range of concentrations of the polyanionic PCR inhibitor in the sample to a range provided by assaying fewer members of the dilution series. In some embodiments, the constant amount of template DNA is at least 100 pg. In some embodiments, the polyanionic PCR inhibitor is a sulfone (sulfonate) compound, such as polyvinyl sulfonate. In some embodiments, polyvinyl sulfonate is the polyanionic PCR inhibitor used when creating a polyanionic inhibitor standard curve, and the concentration of the polyanionic PCR inhibitor in the sample is in units of polyvinyl sulfonate concentration equivalents. Some embodiments further include spiking one or more amounts of polyanionic PCR inhibitor, such as polyvinyl sulfonate, into a sample sufficiently diluted to recover amplification, demonstrating that the addition of the inhibitor impairs or causes loss of amplification recovery.

[0009] Other aspects of the present disclosure relate to a method for removing polyanionic PCR inhibitors (polyanionic impurities) from a buffer solution, comprising: a) preparing a buffer solution of an acidic buffer species, a basic buffer species, or a combination thereof; b) contacting the buffer solution with an anion exchange medium; and c) separating the buffer solution from the polyanionic impurities, thereby removing the polyanionic impurities from the buffer solution. Related aspects of the present disclosure provide a method for removing polyanionic PCR inhibitors (polyanionic impurities) from a buffer solution, comprising: a) preparing a buffer solution of an acidic buffer species, a basic buffer species, or a combination thereof; b) contacting the buffer solution with a mixed mode resin; and c) separating the buffer solution from the polyanionic impurities, thereby removing the polyanionic impurities from the buffer solution. In any of the above methods for removing polyanionic PCR inhibitors, the volume of buffer used in this method is not limited and can range from small analytical volumes in the range of milliliters to commercial-scale buffer preparations containing many liters. In some embodiments of any of the removal methods, the polyanionic impurity is a sulfone (sulfonate) compound, such as polyvinyl sulfonate. In some embodiments of any of the removal methods, the buffer solution is a Good's buffer solution, such as a 2-(N-morpholino)-ethanesulfonic acid (MES) buffer solution. In some embodiments of any of the removal methods, the buffer solution contains a buffer salt or an acid species of the buffer salt. Some embodiments of each removal method further include adding at least one modified compound to the buffer solution. In some embodiments of any of the removal methods, the modified compound is a non-buffered salt, an excipient, or both.

[0010] In some embodiments of the removal method involving an anion exchange medium, the anion exchange medium is a diethylaminoethyl modified matrix, a dimethylaminoethyl modified matrix, a dimethylaminopropyl modified matrix, a polyethyleneimine modified matrix, a quaternary polyethyleneimine modified matrix, a fully quaternary amine modified matrix, an anion exchange modified diatomaceous earth-containing deep filter, anion exchange membrane adsorbent, salt-resistant anion exchange membrane adsorbent, Macro-Prep 25Q, TSK-Gel Q, Poros Q, Q Sepharose Fast Flow, Q HyperD, Q Zirconia, Source 30Q, Fractogel EMD TMAE, Express-Ion Q, DEAE Sepharose Fast Flow, Poros 50 D, Fractogel EMD DEAE(M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, Toyopearl DEAE 650 M, Capto Q, Sartobind Q membrane absorbent, Posidyne charged membrane, Amberlite® (polyamine) modified matrix, deep filter containing anion exchange modified diatomaceous earth, anion exchange membrane adsorbent, salt-resistant anion exchange membrane adsorbent, Macro-Prep 25Q, TSK-Gel Q, Poros Q, Q Sepharose Fast Flow, Q HyperD, Q Zirconia, Source 30Q, Fractogel EMD TMAE, Express-Ion Q, DEAE Sepharose Fast Flow, Poros 50 D, Fractogel EMD DEAE(M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, Toyopearl DEAE 650 M, Capto Q, Sartobind Q membrane absorbent, Posidyne charged membrane, Amberlite® (iminodiacetic acid) modified matrix, Amberlite® Type I (trialkylbenzylammonium) modified matrix, Amberlite® TypeThe matrix is ​​either a dimethyl-2-hydroxyethylbenzylammonium II (I) modified matrix, a Dowex® (polyamine) modified matrix, a Dowex® (I) Type I (trimethylbenzylammonium) modified matrix, a Dowex® (I) Type II (dimethyl-2-hydroxyethylbenzylammonium) modified matrix, a Dowex® (mixed bed), a Capto® Adhere anion exchange multimodal medium, a Capto® Adhere anion exchange multimode, PPA Hypercel, HEA Hypercel, or a Duolite® (polyamine) modified matrix. In some embodiments of the removal method with a mixed mode resin, the mixed mode resin is a Capto® Adhere anion exchange multimode resin, a PPA Hypercel resin, or a HEA Hypercel resin. Any matrix known in the art, including but not limited to cellulose, agarose, Sepharose®, methacrylic polymers, ceramic scaffolds having polymerized hydrogels, and proprietary matrices, is suitable for use in removal methods.

[0011] Some embodiments of the removal method with an anion exchange medium provide an anion exchange medium that binds to up to 15 mg, 9 mg, or 3 mg of PVS per mL of the anion exchange medium. Some embodiments of the removal method with a mixed modal resin provide a mixed modal resin that binds to up to 15 mg, 9 mg, or 3 mg of PVS per mL of the mixed modal resin. In some embodiments of the removal method with an anion exchange medium, the anion exchange medium is a polycationic compound that is a titrator that forms a complex with the polyanionic impurity (analyte). In some embodiments, the anion exchange medium is a quaternary ammonium polymer. In some embodiments of the removal method with an anion exchange medium, the polycationic compound is added in an amount sufficient to reach at least the equivalence point when titrating the polyanionic impurity analyte. As used herein, the equivalence point is the point in titration where enough titrator is added to bind to all of the analyte, and is a synonym for titration point.

[0012] Further embodiments of the present disclosure relate to a method for removing polyanionic buffered impurities from a protein solution, comprising: a) adjusting the pH of the protein solution containing the anionic buffered impurities to a pH no less than 4 pH units below the isoelectric point of the protein; b) contacting the protein solution with an anion exchange medium; and c) separating the protein from the anionic buffered impurities. In a related embodiment, the present disclosure provides a method for removing polyanionic buffered impurities from a protein solution, comprising: a) adjusting the pH of the protein solution containing the anionic buffered impurities to a pH no less than 4 pH units below the isoelectric point of the protein; b) contacting the protein solution with a mixed mode resin; and c) separating the protein from the anionic buffered impurities. In some embodiments of any of these pH-based removal methods, the pH is adjusted to be no less than 2 pH units below the isoelectric point of the protein.In some embodiments of pH-based removal methods involving anion exchange media, the anion exchange media is a diethylaminoethyl modified matrix, a dimethylaminoethyl modified matrix, a dimethylaminopropyl modified matrix, a polyethyleneimine modified matrix, a quaternary polyethyleneimine modified matrix, a fully quaternary amine modified matrix, an anion exchange modified diatomaceous earth-containing deep filter, anion exchange membrane adsorbent, salt-resistant anion exchange membrane adsorbent, Macro-Prep 25Q, TSK-Gel Q, Poros Q, Q Sepharose Fast Flow, Q HyperD, Q Zirconia, Source 30Q, Fractogel EMD TMAE, Express-Ion Q, DEAE Sepharose Fast Flow, Poros 50 D, Fractogel EMD DEAE(M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, Toyopearl DEAE 650 M, Capto Q, Sartobind These include Q membrane absorbents, Posidyne charged membranes, Amberlite® (polyamine) modified matrices, Amberlite® (iminodiacetic acid) modified matrices, Amberlite® Type I (trialkylbenzylammonium) modified matrices, Amberlite® Type II (dimethyl-2-hydroxyethylbenzylammonium) modified matrices, Dowex® (polyamine) modified matrices, Dowex® Type I (trimethylbenzylammonium) modified matrices, Dowex® Type II (dimethyl-2-hydroxyethylbenzylammonium) modified matrices, Dowex® (mixed bed), Capto® Adhere anion exchange multimode, PPA Hypercel, HEA Hypercel, or Duolite® (polyamine) modified matrices. As described above, any matrix known in the art is suitable for use in the methods of this disclosure.In some embodiments of pH-based removal methods involving a mixed-mode resin, the mixed-mode resin is Capto® Adhere anion exchange multimode, PPA Hypercel, or HEA Hypercel.

[0013] Further embodiments of the present disclosure are titration methods for detecting a polyanionic enzyme inhibitor in a buffer solution, comprising: (a) contacting the buffer solution with a polycationic compound; (b) adding the polyanionic compound to the solution in (a), wherein the polyanionic compound exhibits a detectable change in properties when complexed with the polycationic compound compared to an uncomplexed polyanionic compound; (c) repeating steps (a) and (b) with various concentrations of the buffer solution or various concentrations of the polycationic compound; and (d) detecting the change in properties at the titration point, thereby detecting the polyanionic enzyme inhibitor. In some embodiments, the concentration of the buffer is varied to create a dilution series of the buffer. In some embodiments, the concentration of the polycationic compound is varied. In some embodiments, the polyanionic enzyme inhibitor is a polyvinyl sulfonate or a derivative thereof. In some embodiments, the polycationic compound is a pH-independent polycationic compound or a pH-dependent polycationic compound. In some embodiments, the pH-independent polycationic compound is a quaternary ammonium polymer. In some embodiments, the pH-dependent polycationic compound is a polyamine. In some embodiments, the quaternary ammonium polymer is hexadimethrin bromide (HDBr), poly(diallyl)dimethylammonium chloride (pDADMAC), or methyl glycol chitosan. In some embodiments, the quaternary ammonium polymer is hexadimethrin bromide (HDBr) or poly(diallyl)dimethylammonium chloride (pDADMAC). In some embodiments, the quaternary ammonium polymer is hexadimethrin bromide (HDBr).

[0014] In some embodiments, the polyanionic compound is a dye. In some embodiments, the dye is Eriochrome Black T (ECBT), Eriochrome Blue Black R (Calcon), or a sodium sulfonazo salt. In some embodiments, the dye is Eriochrome Black T (ECBT). In some embodiments, the buffer is a Good buffer. In some embodiments, the Good buffer comprises a polyethanesulfonic acid derivative or a polypropanesulfonic acid derivative. In some embodiments, the Good buffer is MES, bis-trismethane, ADA, bis-trispropane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, AMPB, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, CAPSO, AMP, CAPS, or CABS. In some embodiments, the uncomplexed polyanionic compound is detected using fluorescence or spectrophotometric methods. In some embodiments, the method further includes determining the concentration of the polyanionic enzyme inhibitor from the amount of polyanionic compound required to detect a detectable change in properties.

[0015] Other aspects of the present disclosure relate to a method for quantifying a polyanionic PCR inhibitor in a sample, comprising: (a) contacting a sample containing the polyanionic PCR inhibitor with at least one aliquot of a polycationic compound; (b) adding a polyanionic indicator dye in an amount sufficient to detect the free form of the dye; and (c) quantifying the polyanionic PCR inhibitor based on the amount of polycationic compound required to detect the free form of the polyanionic indicator dye. Related aspects of the present disclosure relate to a method for removing polyanionic impurities from a sample, comprising: (a) contacting a fluid containing the polyanionic impurities with a polycationic counterion; and (b) separating the fluid from the polyanionic impurities complexed with the polycationic counterion, thereby removing the polyanionic impurities from the fluid. In some embodiments, the polyanionic impurities are polyanionic PCR inhibitors. In some embodiments, the complexes of the polyanionic impurities with the polycationic counterions are removed by precipitation. In some embodiments, polycationic counterions are derivatized by binding to members of a binding pair or magnetic particles to facilitate the removal of complexes between polyanionic impurities and polycationic counterions from the fluid. Exemplary binding pairs include, but are not limited to, antigen / antibody pairs, biotin / streptavidin, magnetic particles / iron-containing materials, and polyhistidine / metal ions (e.g., nickel) pairs.

[0016] Other features and advantages of the disclosed subject matter will become apparent from the detailed description and drawings below, as well as from the claims. [Brief explanation of the drawing]

[0017] [Figure 1] DNA assay spike recovery inhibitor vs. PVS standard. [Figure 2] This is a dilution series spike recovery analysis compared to a standard curve to provide PVS concentrations. [Figure 3] This is spike recovery data for the MES fraction. [Figure 4]Spike recovery data for MES fractionation. [Figure 5] Spike recovery data for MES fractionation using AEX resin. [Figure 6] Assay results demonstrating that polyvinyl sulfonate (PVS) is a potent inhibitor of qPCR-mediated DNA assays. [Figure 7] Chromatography method for PVS spike challenge. [Figure 8] (a) Chemical structures of 2-(N-morpholino)-ethanesulfonic acid (i.e., MES), MES hydrate in acidic form, and sodium salt of MES in basic form. (b) Chemical reactions leading to compounds that can inhibit enzymes active on nucleic acids such as ribonucleases. Figure 1(b) is adapted from the figure of Smith et al., Journal of Biological Chemistry 2003;20934 - 20938. [Figure 9-1] (a) By varying the concentration of polyvinyl sulfonate (PVS) from 0 to 1.0 ppm, linear calibration curves for two different lots of PVS standard obtained from Sigma - Aldrich, provided as 30 wt% aqueous solutions, were revealed. It was found that the concentration of PVS varies significantly between lots. However, the concentration of a particular lot can be adjusted by dilution to function as an appropriate standard. [Figure 9-2] (b) Titration curves using hexadimethrine bromide (HDBr) for titrating PVS were constructed over the range of PVS concentrations from 0 to 1.0 ppm. An approximately 1.5 - digit linear range was found. [Figure 10](a) A schematic diagram of the quantification of PVS by titration with HDBr using spectroscopic endpoint detection. The reaction scheme shows complexation between PVS and HDBr driven by attractive electrostatic interactions. At the titration endpoint, the indicator compound (nD-) changes its absorbance characteristics upon association with adjacent HDBr charge sites. The "plus" and "minus" signs in the blue circles in Figure 3(a) indicate background salts in solution. (b) The plot shows the change in solution absorbance of a blank sample (i.e., 50 mM borate buffer supplemented with EBT indicator compound, pH 8.5) measured with a benchtop spectrophotometer as HDBr is titrated incrementally in solution. As the HDBr concentration increases, the indicator absorbance at 665 nm decreases, and the maximum absorbance shifts accordingly from approximately 630 to 593 nm. [Figure 11] This is a titration curve plotting the volume-corrected absorbance normalized at 665 nm for a series of PVS standard solutions. [Figure 12-1] (a) Plot of volume-corrected solution absorbance at 665 nm against the mass of HDBr (titrant) for three different PVS standards prepared on an MES matrix blank. [Figure 12-2] (b) Comparison of the inflection points of the titration curves of the PVS standard prepared in 50 mM sodium borate (green triangle) and the MES mixed with 50 mM sodium borate (black square). [Figure 13] This is a comparison of the inflection points of titration curves of PVS standards prepared in an MES matrix blank (black square), a negative control lot of MES (SLBT 8755; blue diamond shape), and an MES lot that caused an initial qPCR invalid assay (lot number I; red circle). [Figure 14] This is a typical titration profile of a blank standard (100 mM carbonate buffer supplemented with 1.25 μg / mL Eriochrome Black T (EBT) indicator dye) containing 0.04 mg / mL hexadimethrin bromide (HDBr; black trace) and its corresponding primary derivative (red trace). [Figure 15](a) A plot of titration endpoint volume against the concentration of PVS spiked in 50 mM MES dissolved in 100 mM carbonate buffer. (b) A plot of titration endpoint volume against the concentration of PVS for standard samples prepared in 100 mM carbonate buffer. [Figure 16] (A, B) PVS standard solutions prepared with 0 (A) or 0.75 (B) μg / mL in 100 mM carbonate buffer; and (C, D) titration curves for representative PVS spiked with 0 (C) or 0.70 (D) μg / mL in 50 mM MES (sample H in Table 10) prepared with 100 mM carbonate buffer. [Modes for carrying out the invention]

[0018] MES (2-(N-morpholino)-ethanesulfonic acid) buffer and other Good buffers are common buffers in biopharmaceutical processes, allowing for pH control around pH 6 (the pKa of MES is 6.15). MES synthesis involves Michael addition of a morpholine ring to a vinyl sulfonate. A common side reaction is oligomerization / polymerization of the vinyl sulfonate, forming polyanionic polyvinyl sulfonates. Polyvinyl sulfonates are potent inhibitors of quantitative polymerase chain reaction (qPCR) assays used to quantify residual host cell deoxyribonucleic acid (DNA), resulting in spike recovery failures and invalid test results in assay controls. The levels of PVS inhibiting DNA assays are far below the levels that raise safety concerns. However, the inability to determine host cell DNA content using effective spike recovery controls impacts lot characterization of purified proteins such as biopharmaceuticals, as well as the release and disposal of such lots.

[0019] This disclosure provides a method for assaying polyanionic compounds present in cell culture media or fluids derived therefrom that are sensitive to low levels of polyanionic compounds, such as mammalian cell culture media or fluids. This disclosure reveals that low levels of polyanions in cell culture fluids generally and particularly disrupt efforts to monitor the purification of proteins in biologics, increasing the time and cost required to obtain approval for therapeutic use. The method of this disclosure provides a simple and efficient approach to monitoring the reduction of polyanionic impurity concentrations to concentrations that are small enough to disappear or not present. The recent surge in commercial manufacturing processes for biologics, i.e., proteins such as therapeutic antibodies and antibody fragments, is putting increasing pressure on the industry to develop protein purification processes that efficiently yield high-yield pure protein products suitable for therapeutic formulation, and the method of this disclosure provides an answer that facilitates the simple and effective monitoring of polyanionic impurities, which are often found in cell culture fluids containing proteins of varying purities. The knowledge disclosed herein that polyanionic impurities are present in protein-containing fluids, e.g., solutions conventionally considered purified, has led to disclosed methods for reducing or removing polyanionic impurities from protein-containing fluids, e.g., solutions. This disclosure reveals that even in such fluids, polyanionic impurities may be found at levels that interfere with enzymes, e.g., DNA polymerase, which are often used to assay the purity of protein solutions. For example, qPCR is frequently used to monitor levels of host cell DNA in purification processes designed to obtain proteins from cell cultures, e.g., mammalian cell cultures. Given that even the presence of trace amounts of polyanionic impurities present in purification processes that interfere with efforts to monitor purity has been discovered, this disclosure provides methods for reducing or removing polyanionic impurities from protein solutions that are considered pure in the latest art.

[0020] Polyanionic compounds such as poly(vinyl sulfonic acid) (PVS) are polymeric impurities in Good's buffers, such as MES buffers. These polyanionic compounds, e.g., PVS, are present in such buffers at low levels, in the range of parts per million, compared to buffer compounds such as MES. The presence of these impurities in Good's buffers is a significant concern because such buffers are used in the production of therapeutic proteins, and these impurities, particularly PVS, are potent polymerase inhibitors that can interfere with quantitative PCR (qPCR) detection of DNA. Measurement of host cell nucleic acids (e.g., DNA) in therapeutic protein formulations purified from cultures is routinely required to assess the safety of therapeutic drugs intended for human administration. Therefore, the presence of polyanionic compounds such as PVS in Good's buffers, such as MES, may interfere with qPCR detection of host cell DNA, potentially causing batches of therapeutic proteins to fail to meet human administration acceptance criteria.

[0021] This specification discloses a titration method based on the complexation of an analyte (e.g., PVS) with a high molecular weight titrator that is oppositely charged. This interaction results in an extremely high equilibrium association constant (Ka), allowing the endpoint to be detected spectroscopically (e.g., colorimetrically or photometrically). Figure 10 shows an overview of a detection scheme applied to the titration of PVS using hexadimethrin bromide (HDBr), an exemplary titrator.

[0022] The methods disclosed herein include methods for verifying the accuracy of nucleic acid enzyme-based assays of host cell DNA as an impurity in protein formulations. For example, the methods disclosed herein are useful for verifying nucleic acid enzyme-based assays of host cell DNA, such as polymerase chain reaction (i.e., PCR). Exemplary PCR assays useful in the disclosed methods are quantitative PCR or qPCR, which provide a rapid, inexpensive, accurate, precise, and highly sensitive method for determining the amount of DNA in a sample. Therefore, preferred methods for verifying the concentration of host cell DNA impurities in protein fluids, solutions, preparations, or formulations include quantification of DNA in a sample, such as a cell culture sample using qPCR, and comparison with a standard curve of polyanionic PCR inhibitors to determine the concentration of the polyanionic PCR inhibitor in the protein fluid, solution, preparation, or formulation to verify the host cell DNA assay results. Relevant aspects of the disclosure provide methods for reducing or removing such inhibitors from cell culture fluids of varying purities, i.e., protein-containing fluids or solutions, and address the problem of nucleic acid enzyme inhibition by removing such inhibitors from buffers in which such proteins may be placed.

[0023] The methods disclosed herein are useful for reducing or removing one or more polyanionic compounds, such as polyanionic compounds found in cell cultures, e.g., mammalian cell cultures, or in buffers found in therapeutic formulations such as 2-(N-morpholino)-ethanesulfonic acid (MES) or Good's buffer. Exemplary groups of polyanionic compounds that can be reduced or removed according to the methods of this disclosure are sulfonate compounds, exemplified by polyvinyl sulfonate (i.e., polyethylene sulfonate). This disclosure aims to reduce or remove polyanionic compounds regardless of the size or range of size of the one or more polymers involved. Polyanionic impurities that can be removed using the methods of this disclosure also include polyoxometalates (i.e., POM), proteoglycans (storage), glycosaminoglycans (e.g., heparin, chondroitin sulfate, dextran sulfate), polyglutamates, polysaccharides, actin microfilaments and actin microtubules, polyvinyl sulfonates, polyacrylic acid, and inositol phosphate.

[0024] The method according to this disclosure uses an anion exchange medium to separate polyanionic impurities from a purified protein, such as a biologic. Any anion exchange medium known in the art may be used in the disclosed method, and may include, but is not limited to, weak basic groups such as diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), and dimethylaminopropyl (DMAP), or strong basic groups such as quaternary aminoethyl (Q), trimethylammonium ethyl (TMAE), and quaternary aminoethyl (QAE), and may be used in anion exchange. Exemplary anion exchange media include GE Healthcare Q-Sepharose FF®, Q-Sepharose BB®, Q-Sepharose XL®, Q-Sepharose HP®, Mini Q®, Mono Q, Mono P DEAE Sepharose FF®, Source® 15Q, Source® 30Q, Capto Q®, Streamline DEAE®, Streamline QXL®; Applied Biosystems Poros® HQ 10 and 20 μm self-pack, Poros® HQ 20 and 50 μm, Poros® PI 20 and 50 μm, Poros® D 50 μm; Tosohaas Toyopearl® DEAE 650S M and C, Super Q 650, QAE 550C; Pall Corporation DEAE HyperD®, Q Ceramic HyperD®, Mustang Q membrane absorbers include Merck KG2A Fractogel DMAE®, FractoPrep DEAE, FractoPrep TMAE, Fractogel EMD DEAE®, Fractogel EMD TMAE®, and Sartorious Sartobind Q® membrane absorbers.Any mixed-mode or multimodal media known in the art, including anion exchangers, including but not limited to Capto® Adhere Anion Exchange Multi Mode, PPA Hypercel, or HEA Hypercel media, can be used in the disclosed methods. Furthermore, the disclosed methods may include the use of polyanion-binding proteins such as α-synuclein, tRNA / rRNA methyltransferase, and / or low-molecular-weight heat shock proteins. In some preferred embodiments, Hybrid Purifier® is used as an anion exchange medium in addition to functioning as a deep filter. Viresolve prefilters (VPFs) are also preferred for use as an anion exchange medium.

[0025] For example, a method of this disclosure useful for verifying the accuracy of a host cell DNA assay of a cell culture sample may use any enzyme-based nucleic acid assay, such as a variant form of PCR. A preferred type of PCR for use in such a method is qPCR. PCR, including qPCR, is well-suited for the detection and quantification of DNA from cultured cells, such as host cell DNA found as an impurity in tissue culture fluid. The advantage of qPCR is its ability to detect and quantify the increase in fluorescence that occurs after each PCR run. To provide this ability, the forward and reverse primers are designed to be adjacent to the target DNA sequence of interest, and the target-specific probe is designed to hybridize to a complementary sequence between the two primers. The probe consists of an oligonucleotide sequence having a fluorophore molecule at its 5' end and a quencher molecule at its 3' end. When the fluorophore is in close proximity to the quencher, fluorescence is minimized. However, in the presence of the target sequence, the probe may anneal to the target sequence and subsequently cleave by the exonuclease activity of Taq polymerase. When the probe is cleaved as a result of the extension of the forward primer, the probe's fluorophores are no longer quenched, which leads to an increase in fluorescence as a direct result of the presence of the target DNA sequence. Fluorescence is monitored during each cycle of qPCR in the thermal cycle extension phase, and a threshold cycle is determined for each reaction. A threshold cycle is the cycle in which the fluorescence from a given reaction significantly exceeds the background fluorescence. The threshold cycle value is inversely proportional to the amount of starting DNA in the reaction. The threshold cycle value for each sample is compared to that from a standard curve, which allows for the quantification of samples with unknown amounts of DNA.

[0026] Any set of primers functional in qPCR, as readily determined by those skilled in the art, is suitable for use in the methods of this disclosure. An exemplary qPCR primer is derived from a repeating sequence specific to CHO cells, thereby specifically hybridizing. The targeted CHO cell-specific sequence is the following 68-nucleotide region: 5'-GAAATCGGGCTGCCTGAGTCCCGAGTGCGGGTGTGGTTTCAGAACCGCCGAAGTCGTTCGGGGATGGT-3' (SEQ ID NO: 1). The 5' end of this sequence has the same sequence as the forward primer, the 3' end of the sequence is the complement of the reverse primer, and the fluorophore-labeled probe targets the region between these sequences. The forward, reverse, and probe sequences are as follows: RepA forward primer: 5'-GAA ATC GGG CTG CCT GAG T-3' (SEQ ID NO: 2); RepA reverse primer: 5'-ACC ATC CCC GAA CGA CTT C-3' (SEQ ID NO: 3); and RepA probe: 5'-CC GAG TGC GGG TGT GGT TT-3' (SEQ ID NO: 4). The RepA probe contains a fluorophore group at the 5' end and a quencher group at the 3' end.

[0027] The qPCR assay for host cell DNA impurities was performed according to conventional procedures. After DNA extraction from the samples, qPCR reagents containing qPCR primers, a DNA polymerase such as a thermostable polymerase (e.g., Taq® DNA polymerase), and an appropriate amount of the necessary nucleoside triphosphates were added, as is known in the art. DNA spike controls were added to several samples in a form of DNA suitable for qPCR amplification. The amount of spike added to the spiked samples was 100 pg of CHO genomic DNA. Other samples remained unspiked. The difference in results between the spiked and unspiked samples allowed for the calculation of spike recovery. In other words, the spike recovery rate is given by [(result spiked at pg - result unspiked at pg) / amount of spike at pg] × 100.

[0028] Fluorescence can be measured from individual wells of a 96-well plate. Since this measurement is obtained before the reaction is complete at the end of 40 thermal cycles, it is possible to determine the extent of PCR occurring in real time. PCR is measured by monitoring the increase in fluorescence as a function of the number of cycles.

[0029] qPCR can be performed using instruments such as the QuantStudio 7 real-time qPCR instrument. Fluorescence can be monitored as a function of the number of cycles in which fluorescence emission signal detection occurs during the amplification extension phase. Normalized reporter signal (R n A threshold cycle value is generated for each run of each sample on the plate in each cycle. By comparing the threshold cycle value from each well with a standard curve (linear regression of threshold cycles versus log(input DNA mass in each reaction)), it becomes possible to interpolate unknown values.

[0030] The use of qPCR for nucleic acid assays is widespread, and as a result, there are now kits available to facilitate such assays. Any known protocol and any kit known in the art can be used in the method of this disclosure. An exemplary protocol is the TaqMan® qPCR protocol for the quantification of residual host cell DNA, described in Verardo et al., Biotechnol. Prog. 28:428-434 (2012), which is incorporated by reference in Example 1 and the relevant parts of this specification. An exemplary kit is the PrepSEQ® Residual DNA Sample Preparation Kit (Applied Biosystems®, Beverly, MA).

[0031] The methods disclosed herein, useful for confirming the presence and amount of host cell DNA impurities in protein-containing fluids, were developed to address the problem disclosed herein, where relatively low levels of polyanionic inhibitors of enzyme-based nucleic acid assays persist in protein-containing fluids during the purification process. Several embodiments of these methods achieve remarkable sensitivity while maintaining the ability to deliver accurate and precise results by serially diluting the sample and comparing the results to a standard curve. Samples, such as those from cell cultures, are serially diluted according to any scheme known in the art, provided that the dilution of each aliquot of the sample is known. A suitable dilution scheme is a constant 2:1 dilution, where an aliquot of the sample is diluted with an equal volume of a suitable solution, such as PCR buffer solution, to produce a 2:1 dilution. Then, the aliquot of this dilution itself is diluted 2:1, and so on. n This results in a series of dilutions in the range of :1, where n is the number of aliquots. Determining the actual number of aliquots of the diluted sample is within the scope of the art; typically, the number of aliquots is in the range of 4 to 10. The method of the present disclosure further intends to add or spike a control template DNA to monitor the amplification levels in the sample and its dilutions. The control template DNA or spike control is distinguishable from any host cell DNA impurities that may be present in the sample or its dilutions, and the spike control has PCR primer binding sites. The control template DNA or spike control may be added to the original dilution series of the sample, to isolated portions of each aliquot in the original dilution series, or to a second dilution series of the sample prepared in conjunction with the original dilution series.

[0032] The use of dilution series in the methods of this disclosure, designed to determine the presence or absence of host cell DNA impurities, may initially seem counterintuitive or counterproductive, because when a sample is diluted, any impurities in that sample are also diluted, potentially making detection and quantification difficult. However, the extremely high sensitivity of enzyme-based nucleic acid assays such as PCR (e.g., qPCR) overcomes such dilutions, allowing for the detection of even minute amounts of host cell DNA impurities. Furthermore, there are other reasons for including dilution series in the methods of this disclosure. Serial dilutions of a sample also serially dilute any inhibitors of the enzymes used in these highly sensitive enzyme-based nucleic acid assays, such as DNA polymerase. As described herein, the methods disclosed herein are, in part, based on the discovery of relatively low levels of polyanionic inhibitors of the enzymes used in nucleic acid assays, which will be referred to for convenience as polyanionic PCR inhibitors. When preparing a sample dilution series, a dilution series of any polyanionic PCR inhibitor is also prepared. This provides an opportunity to determine the level of sample dilution at which inhibition is released by DNA polymerase-mediated polymerization and enzyme-based amplification is resumed. Because a finite number of dilutions exist in this series, the results may result in a range of concentrations of the polyanionic PCR inhibitor, from the least diluted sample showing recovery or spike recovery of PCR activity to the most diluted sample still showing inhibition of PCR activity. Those skilled in the art can easily narrow or broaden the concentration range of the detected and quantified inhibitor by adding or subtracting aliquots from the dilution series. Those skilled in the art will also understand that a standard curve for polyanionic PCR inhibitors allows for conversion to actual concentrations of relative dilutions based on a standard curve constructed from serial dilutions of pure polyanionic PCR inhibitors subjected to enzyme-based nucleic acid assays of control template DNA (spike control) in the presence of the necessary reagents (e.g., TaqMan® Universal PCR Master Mix, Applied Biosystems), but in the absence of the sample or its dilution. The standard curve can identify the absolute concentration of the polyanionic PCR inhibitor at which a level of nucleic acid amplification was observed, and this can then be carried over to the results seen in the sample dilution series.This method is intended to generate a standard curve using any known polyanionic PCR inhibitor, and polyvinyl sulfonate (polyethylene sulfonate) is a preferred polyanionic PCR inhibitor suitable for use in constructing the standard curve. When PVS is used, the concentration is expressed in PVS equivalents. In many cases, the PVS equivalent is the actual concentration of PVS in the sample or its dilution, as the identity of polyanionic PCR inhibitors is known to be PVS.

[0033] The sample used in the method of this disclosure is either a cell culture fluid or a fluid derived from a cell culture fluid in a process for purifying proteins such as biologics and biosimilars. The sample is in any volume suitable for detecting impurities and can be obtained from an ongoing cell culture, a continuous effluent from a cell culture, or a batch of cell culture discharges. The sample can be obtained and processed without delay, or obtained from a holding tank, or maintained at a suitable temperature, typically 4°C.

[0034] In addition to methods for determining whether formulations of various purities containing proteins produced in cell cultures have host DNA impurities, this disclosure provides methods for reducing or removing impurities, based in part on the discovery that partially purified protein formulations may have levels of polyanionic PCR inhibitors that, while often at low levels in highly purified protein formulations, must be addressed to meet regulatory bodies involved in ensuring the quality of pharmaceutical formulations. Therefore, other aspects of this disclosure relate to methods for removing polyanionic PCR inhibitors, such as PVS, from protein-containing solutions. Upon being informed by the disclosure of the presence of relatively low levels of polyanionic PCR inhibitors in protein-containing solutions obtained from cell cultures and the purification processes of those proteins, those skilled in the art can contact the sample (or a dilution thereof) with any known anion exchange medium to bind to the polyanionic PCR inhibitors, resulting in their separation and removal from the protein-containing sample or dilution. To facilitate the reduction or removal efforts, the pH of the sample or dilution thereof is adjusted to be 2–4 pH units lower than the pI of the protein target or protein to be purified in the sample. In this pH range, the target protein does not have a net negative charge, but PVS exhibits its complete negative charge. As a result, PVS readily binds to anion exchange resins known in the art, but the target protein does not.

[0035] The purified proteins, such as recombinant proteins or polypeptides, may be homopolymers or heteropolymers and may be of scientific or commercial interest, including protein-based therapeutic agents. The biomolecules of interest (e.g., proteins such as biologics or biosimilars) include, among other things, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. The biomolecules of interest can be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins.” The expressed proteins may be produced intracellularly and secreted into culture media into which they can be recovered and / or collected. The term “isolated protein” or “isolated recombinant protein” refers to the polypeptide or protein of interest purified from proteins or polypeptides or other impurities that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. The biomolecules of interest include proteins that exert a therapeutic effect by binding to targets, particularly those listed below, including targets derived from them, related targets, and modifications thereof.

[0036] "Purification" means increasing the purity of the protein in a composition by removing (partially or completely) at least one type of impurity from the product. Protein recovery and purification are achieved by any downstream processes, particularly recovery operations, that produce a more "homogenous" protein composition that satisfies yield and product quality targets (e.g., reduced product-related impurities and increased product quality).

[0037] As used herein, the term “isolated” means (i) free from at least some other proteins or polynucleotides that are normally found with it; (ii) substantially free from other proteins or polynucleotides of the same source, such as the same species; (iii) isolated from at least about 50 percent of the polypeptides, polynucleotides, lipids, carbohydrates or other substances to which it is bound in nature; (iv) operably bound (by covalent or noncovalent interactions) to polypeptides or polynucleotides not to which it is bound in nature; or (v) not occurring in nature.

[0038] The target biomolecules (e.g., proteins) include "antigen-binding proteins." Antigen-binding proteins refer to proteins or polypeptides that contain an antigen-binding region or part that has affinity for another molecule (antigen) to which they bind. Antigen-binding proteins include antibodies, peptide bodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFv) and double-chain (bivalent) scFv), mutaines, multispecific proteins, and bispecific proteins.

[0039] scFv is a single-chain antibody fragment that possesses variable regions in the heavy and light chains of a linked antibody. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol.Immunotherapy (1997) 45:131-136. scFv retains the ability of the parent antibody to specifically interact with the target antigen.

[0040] The term "antibody" includes references to its antigen-binding region competing with intact antibodies for specific binding to both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass. Unless otherwise specified, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclone, heteroIgG, bispecific and their oligomers or antigen-binding fragments. Antibodies include IgG1, IgG2, IgG3, or IgG4 types. Also, Fab, Fab', F(ab')2, Fv, diabody, Fd, dAb, maxibody, single-chain antibody molecule, single-domain V H This also includes proteins having antigen-binding fragments or regions such as H, complementarity-determining region (CDR) fragments, scFv, diabodies, triabodies, and tetrabodies, as well as polypeptides containing at least a portion of immunoglobulins sufficient to confer specific antigen binding to a target polypeptide.

[0041] Furthermore, it also includes other antigen-binding proteins such as human, humanized, and human and humanized antibodies that do not produce a significantly harmful immune response when administered to humans.

[0042] Furthermore, modified proteins include proteins that are chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. Also included are proteins that further include one or more post-translational modifications that can be performed by cell modification systems, or modifications introduced ex vivo by enzymatic and / or chemical methods, or otherwise.

[0043] The terms "multispecific protein" and "multispecific antibody" are used herein to refer to proteins that have been recombinantly modified to simultaneously bind to and neutralize at least two different antigens or at least two different epitopes on the same antigen. For example, multispecific proteins may be modified in combination with targeted cytotoxic agents to target an immune effector to a tumor or infectious pathogen. Multispecific proteins include trispecific antibodies capable of binding to multiple targets, tetravalent bispecific antibodies, multispecific proteins without antibody components such as diabodies, triabodies, or tetrabodies, minibodies, and single-chain proteins. (Coloma, MJ et al., Nature Biotech. 15(1997) 159-163).

[0044] The most common and diverse group of multispecific proteins are those that bind to two antigens, and are referred to herein as “bispecificity,” “bispecificity constructs,” “bispecificity proteins,” and “bispecificity antibodies.” Bispecificity proteins can be classified into two broad categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like molecules retain Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell phagocytosis (ADCP), and the Fc region enhances solubility and stability and facilitates some purification operations. Non-IgG-like molecules are smaller and enhance tissue permeability (see Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol & Oncology 8:130-143, 2015; Spiess et al., Mol Immunol 67, 95-106, 2015; Williams et al., Chapter 41 Process Design for Bispecific Antibodies in Biopharmaceutical Processing Development, Design and Implementation of Manufacturing Processes, Jagschies et al. (eds.), 2018, pp. 837-855). Bispecific proteins have binding specificity to different antigens or several epitopes and may be used as a framework for additional components to increase the binding specificity of a molecule.

[0045] The forms of bispecific proteins, including bispecific antibodies, are constantly evolving, and include single-chain antibodies, quadromas, knob-in-holes, cross-MAb, bivariable domain IgG (DVD-IgG), IgG-single-chain Fv (scFv), scFv-CH3 KIH, dual-action Fab (DAF), half-body exchange, κλ-body, tandem scFv, scFv-Fc, diabody, single-chain diabody (scdiabody), scdiabody-CH3, triplebody, mini-antibody, minibody, TriBi minibody, tandem diabody, scdiabody-HAS, tandem scFv-toxin, biaffinity retargeting molecule (DART), nanobody, nanobody-HSA, and dock. Androck (DNL), Strand Exchange Modified Domain SEED Body, Triomab, Leucine Zipper (LUZ-Y), XmAb (Registered Trademark); Fab-Arm Exchange, DutaMab, DT-IgG, Charged Pair, Fcab, Orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-Ig4 (four functions), Fab-scFv, scFv-CH-CL-scFV, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc-diabody-Fc, diabody-Fc, intrabody, ImmTAC, HSABody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, bispecific T cell engager (BiTE®) and bispecific T cell engager with extended half-life (HLE BiTE®), hetero-Ig BiTE® (Fan above; Spiess above; Sedykh above; Seimetz et al., Cancer Treat Rev 36(6)458-67, 2010;This includes, but is not limited to, Shulka and Norman (Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottschalk editor, pp. 559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011). The target biomolecules (e.g., proteins) may include recombinant fusion proteins containing multimerization domains such as leucine zippers, coiled coils, and the Fc portion of immunoglobulins. It also includes proteins containing all or part of the amino acid sequence of a differentiation antigen (called a CD protein), or their ligands, or proteins substantially similar to either of these.

[0046] The target biomolecules (e.g., proteins such as biologics and biosimilars) include genetically modified receptors such as chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs), as well as other proteins containing antigen-binding molecules that interact with their target antigens. CARs can be modified to bind to antigens (such as cell surface antigens) by incorporating antigen-binding molecules that interact with their target antigens. Typically, CARs incorporate an antigen-binding domain (such as an scFv) in cooperation with one or more co-stimulatory ("signaling") domains and one or more activation domains.

[0047] In some embodiments, the biomolecule of interest may include colony-stimulating factors such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methoxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit® (epoetin alfa), and epoetin alfa. This also includes erythropoiesis-stimulating agents (ESAs) such as Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists and their molecules, variants, or analogs, and any of the biosimilars mentioned above.

[0048] In some embodiments, the biomolecules of interest may include one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factor, fibroblast growth factor, transform growth factor (TGF), insulin-like growth factor, bone-inducing factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factor (CSF), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptor, T cell receptor; neurotrophic factors, neurotrophins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, adresins, regulatory proteins, and proteins that specifically bind to immunoadhesins.

[0049] In some embodiments, the biomolecule of interest may bind to one or more of the following, either alone or in any combination: CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, CD174, but not limited to CD proteins; HER receptor family proteins including HER2, HER3, HER4; the EGF receptor EGFRvIII; cell adhesion molecules such as LFA-1, Mol, and p150. , 95, VLA-4, ICAM-1, VCAM, αv / β3 integrin, growth factors including but not limited to vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müller inhibitor, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors including aFGF and bFGF, epidermal growth factor (E) GF), Cripto, in particular transform growth factors (TGF) including TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5, insulin-like growth factors-I and-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone formation factors, insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding proteins, insulin and insulin-related proteins, coagulation and coagulation-related proteins, For example, plasminogen activators such as factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, urokinase, and tissue plasminogen activator ("t-PA"), bombazine, thrombin, thrombopoietin, thrombopoietin receptors, colony-stimulating factors (CSFs) including M-CSF, GM-CSF, and G-CSF, albumin, IgE, and other blood and serum proteins including but not limited to blood group antigens, such as the flk2 / flt3 receptor and obesity (OB) receptor.Receptors and receptor-related proteins, including growth hormone receptors and T cell receptors; neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain, and prorelaxin; interferons, including interferon-α, -β, and -γ; interleukins (IL), such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, and IL-12 / IL-23. IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP, IL1-α, IL-1β, AIDS enveloped virus antigen, viral antigens including but not limited to these, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and -β, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesoserine, RANTES (regulated on activation normally expressed and (secreted), mouse gonadotropin-related peptide, DNase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, disintegration-promoting factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP1-6, EPCAM, PSA, adresin, regulatory protein, immunoadhesin, antigen-binding protein, Somatropin, CTGF, CTLA4, Eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, Ganglioside GD2, Ganglioside GM2, BAFF, OPGL (RANKL), Myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, Hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, P-cadherin, NKG2D-1,PD-1 (Programmed Cell Death Protein 1) and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesoserine dsFv[PE38 conjugate], Legionella pneumophila Pneumophila (lly), gpA33, B7H3, IFNγ, interferon-γ-inducing protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphocyte neoplastic factor (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein Iib / IIIb (PAC-1), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any of the above bioactive fragments or variants.

[0050] In some embodiments, the biomolecule of interest is absiximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosuzumab, brentuximab vedotin, brodalumab, cantuzumab meltansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, This includes ipilimumab, reldelimumab, lumiliximab, ixekizumab, mapatuzumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelbequin, palivizumab, panitumumab, pembrolizumab, penitumumab, paxerizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, salglamostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, vizilizumab, boroxiximab, zanolimumab, zaltumumab, and any of the aforementioned biosimilars.

[0051] In some embodiments, the target biomolecule is blinatumomab, catumakisomab, erzumakisomab, solitomab, targomiR, lutikizumab (ABT981), vanucizumab (RG7221), memtolumab (ABT122), ozoralizumab (ATN103), furotuzumab (MGD006), pasotuxizumab (AMG112, MT112), lymphomun (FBTA05), (AT N-103), AMG211 (MT111, Medi-1565), AMG330, AMG420 (B1836909), AMG-110 (MT110), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, Indium-labeled IMP-205, xm 734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013(ACE910), RG7597(MEDH7945A), RG7 802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM-111, MM14 1. MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352 and their molecules or variants or analogs and any of the above biosimilars may be included.

[0052] The biomolecules of interest as disclosed herein encompass all of the foregoing and further include antibodies containing complementarity-determining regions (CDRs) 1, 2, 3, 4, 5, or 6 of any of the antibodies described above. Variants also include regions whose amino acid sequences are identical to or more than 70%, particularly 80%, more specifically 90%, even more specifically 95%, particularly 97%, even more specifically 98%, and even more specifically 99% of the reference amino acid sequence of the biomolecule of interest in protein form. This identity can be determined using various well-known and readily available amino acid sequence analysis software. Preferred software includes those implementing the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of sequence searching and sorting. Other algorithms may also be employed, particularly when speed is a critical consideration. In this regard, commonly used programs for DNA, RNA, and polypeptide alignment and homology matching include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waterman algorithm for execution on a massively parallel processor created by MasPar.

[0053] Chimeric antigen receptors incorporate one or more co-stimulatory (signal transduction) domains to enhance their efficacy. See U.S. Patents No. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (above), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).Preferred co-stimulatory domains include, among other sources, CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3(α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, and lymphocyte function-associated antigens. 1 (LFA-1, CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signal transduction lymphocyte activating molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IL-2R Beta, IL-2R Gamma, IL-7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-lc, ITGBl, CD29, ITGB2, CD18, LFA- 1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, 41-BB, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligands, or fragments or combinations thereof. The co-stimulatory domain may include one or more extracellular, transmembrane, and intracellular portions.

[0054] Due to the polymeric nature of anionic impurities such as PVS, these compounds can be removed by flocculation using charged particles, charged nanoparticles, cationic polymers, mixed-mode cationic polymers, smart polymers, etc. After precipitating the PVS, it can be removed by sedimentation or filtration using a flocculant. For example, an MES buffer containing PVS can be exposed to Clarisolve® mPAA (a cationic smart polymer), and then the polymer can be precipitated by applying a stimulus to remove it.

[0055] The following examples disclose functional embodiments of methods for detecting and removing high molecular weight polymeric impurities, such as polyvinyl sulfonate (PVS), from proteins and buffer solutions. In some cases, it is beneficial to reduce rather than completely remove PVS, and this reduction is sufficient to achieve satisfactory quality. In some cases, these methods are used to detect and remove anionic flocculants and residual anionic flocculants. PVS removal can be achieved using anion exchange media such as chromatography resins, ion exchange resins, depth filters, synthetic depth filters, charged filters, membrane chromatography devices, mixed-mode resins, and combinations thereof. [Examples]

[0056] Example 1 Determination of polyvinyl sulfonate Polyvinyl sulfonate concentrations were measured using a qPCR assay for DNA quantification and a dilution series to monitor DNA spike recovery. The qPCR DNA assay is described in the following paragraph.

[0057] The TaqMan® qPCR method for quantifying residual host cell DNA is described (Verardo et al., Biotechnol. Prog. 28:428-434 (2012), the relevant portion of which is incorporated herein). Briefly, all test samples were diluted in nuclease-free water to the desired protein concentration or volume as indicated, and digested with Proteinase K (Promega) at 60°C for 2–24 hours. DNA was extracted from the samples using a standard chaotropic salt (sodium iodide) and alcohol precipitation protocol. The extracted DNA pellet was resuspended in nuclease-free water, and the total volume of DNA recovered by qPCR total DNA analysis was measured using ABI QuantStudio 7 running SDS software (version 4.1). Primers were designed to amplify CHO cell-specific repeat DNA sequences, and specific probes were designed to anneal between them. Forward primer sequence: 5'GAA ATC GGG CTG CCT GAG T 3'(SEQ ID NO: 2); Reverse primer sequence: 5'ACC ATC CCC GAA CGA CTT C 3'(SEQ ID NO: 3) TaqMan® probe sequence: 5' <fam>CC GAG TGC GGG TGT GGT TT <tam>3' (SEQ ID NO: 4). The probe was labeled with the fluorescent reporter dye FAM (6-carboxyfluorescein) at its 5' end and with the quencher dye TAMRA (6-carboxytetramethylrhodamine; TAM) at its 3' end. Standard reaction cycle conditions were used (40 cycles of 2 minutes at 50°C, 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C). The reaction was carried out in a 96-well plate with a reaction volume of 50 μL using Taqman® Universal PCR Master Mix (Applied Biosystems). Analysis was performed using an automated baseline setting with relative thresholds set to fall within the exponential range of the amplification plot for each gene target. The level of standard curve fluorescence was correlated to the DNA concentration in the original sample using a calibration curve of known amounts of genomic DNA isolated from CHO host cells. The measured DNA amounts were converted to units of pg DNA per 1 mg of sample. All amounts were measured with two or three samples, and the mean was calculated.

[0058] Samples were analyzed for PVS by observing qPCR assay inhibition and PVS standard dilution series (see Figure 1). Using 100 pg of DNA spikes as a positive DNA control, acceptable spike recovery was 50%–150%. A negative control consisting of double-deionized water showed 100% recovery. DNA spike recovery was evaluated against PVS standards to determine the "minimum inhibitory concentration" and "maximum non-inhibitory concentration" (see Figure 2). The minimum inhibitory concentration is the lowest concentration of sample or PVS that shows DNA assay interference (i.e., unsuccessful spike recovery). This value essentially provides the "worst-case" inhibitor concentration for a given sample. The maximum non-inhibitory concentration is the highest concentration with acceptable DNA spike recovery and essentially provides the lowest measurable amount of PVS. For brevity, the average PVS concentration between the minimum inhibitory concentration and the maximum non-inhibitory concentration was used in the following analyses. In some evaluations, it may be important to use the worst-case concentration. The acceptable process range can be adjusted for the entire range of measurable concentrations, as shown in Example 2.

[0059] Example 2 Removal of polyvinyl sulfonate from buffer solution using an anion exchange membrane A 100 mM MES buffer solution with pH 6 was prepared using 21.51 g / L MES hydrate, and then titrated with 1 M sodium hydroxide. An anion exchange membrane (2.8 cm) was used. 2 A 0.2 μm charged nylon filter (Posidyne® filter) with the specified front surface area was washed with 10 mL of deionized (DI) water. A 100 mM MES solution was then passed through an AEX membrane (10 mL) and recovered. The anion exchange (AEX) flow-through pool and the MES buffer-loaded material (before AEX) were subjected to PVS quantification by DNA assay dilution series inhibition. As expected, no DNA spikes were recovered from the loaded material due to the presence of PVS (0%). The results are shown in Table 1. The volume transfer for DNA spike recovery was identical in all three replicates. Therefore, the PVS removal calculated for each replicate was identical. A 100-fold variation in PVS levels was observed in the MES lots used to characterize these AEX media. The effective membrane loading level depends on the incoming PVS concentration (i.e., PVS challenge on the membrane). The "PVS removal load range" is calculated by setting the lower load limit using the worst-case scenario (maximum observed MES PVS level) and the upper load limit using the best-case scenario (minimum observed MES PVS level) for a 100 mM MES buffer solution. The possible load range is very large and demonstrates that the sizing of the charged nylon film depends on the incoming PVS impurity level.

[0060] [Table 1]

[0061] The surface area of ​​porous media such as membranes is approximately proportional to the pore size. Table 2 estimates the preferred loading range for Posidyne filters with a smaller pore size of 0.1 μm, using the assumption that the surface area and binding capacity are twice that of a 0.2 μm Posidyne filter. These loading levels are expected to provide robust removal of PVS during typical buffer preparation operations.

[0062] [Table 2]

[0063] Example 3 Removal of polyvinyl sulfonate from buffer solution using a synthetic depth filter A 100 mM MES buffer solution at pH 6 was prepared using 21.51 g / L MES hydrate and then titrated with 1 M sodium hydroxide (named Runs #1 and #2). A second MES sample was prepared in the same manner except for the addition of 2.04 g / L sodium chloride (Targeting 35 mM NaCl, Runs #3 and #4). A synthetic anion exchange depth filter (2.5 cm) was used. 2 90 mL of DI water was passed through a Hybrid Purifier (registered trademark) having the specified front surface area. Then, a 100 mM MES solution was collected by passing it through an AEX synthetic depth filter (1800 mL). The AEX flow-through pool and the MES buffer-loaded material (before AEX synthetic depth filter) were subjected to PVS quantification by DNA assay dilution series inhibition. As expected, no DNA spikes were recovered from the loaded material (0%) due to the presence of PVS. The results are shown in Figures 3 and 4 and Table 3. DNA spike recovery was identical for both conditions and in both replicates, and no PVS breakthrough at the inhibition level was observed. The "PVS removal loading range" is calculated by setting the lower loading limit using the worst-case scenario (maximum observed MES PVS level) and the upper loading limit using the best-case scenario (minimum observed MES PVS level) for the 100 mM MES buffer solution.

[0064] [Table 3]

[0065] Example 4 Removal of polyvinyl sulfonate from buffer solution using a depth filter A 100 mM MES buffer solution at pH 6 was prepared using 21.51 g / L MES hydrate, and then titrated with 1 M sodium hydroxide. A positively charged depth filter (Viresolve pre-filter (VPF), 5 cm) was used. 2 10 mL of DI water was flowed over the front surface area. Then, a 100 mM MES solution was flowed through a depth filter (10 mL) and collected. As expected, no DNA spikes were recovered from the loaded material due to the presence of PVS (0%). The depth filter pool and the MES buffered loaded material were subjected to PVS quantification by DNA assay dilution series inhibition. The results are shown in Table 4.

[0066] [Table 4]

[0067] Example 5 Removal of polyvinyl sulfonate from buffer solution using anion exchange chromatography medium A 100 mM MES buffer at pH 6 was prepared using 21.51 g / L MES hydrate and then titrated with 1 M sodium hydroxide. 30 mL of DI water was passed through a positively charged anion exchange (AEX) resin (Q-Capto(trademark) ImpRes, 10 mL pre-packed column). The 100 mM MES solution was then passed through the column (5370 mL) and collected. The AEX pool, fraction, and MES buffer-loaded material were subjected to PVS quantification by DNA assay dilution series inhibition. The results are shown in Figure 5 and Table 5. As expected, no DNA spikes were recovered from the loaded material due to the presence of PVS (0%). In a second experiment to determine the maximum PVS binding capacity, 14 mL of DI water was passed through the same AEX resin (Q-Capto(trademark) ImpRes, 5 mL Hi-Screen Column). Next, a 100 mM MES solution was passed through a column (500 mL), the column was washed with 20 mL of DI water, and the bound components were eluted with 3 M sodium chloride and recovered. PVS was quantified in a dilution series, and the results are shown in Table 5.

[0068] [Table 5]

[0069] Example 6 Removal of polyvinyl sulfonate from protein-containing solutions using anion exchange media Several anion exchange (AEX) media were tested for flow-through removal of PVS from a solution containing a fusion protein (target protein) with an isoelectric point of 8.8. All media were run with DI water and then equilibrated to the test pH with 10 mL of the following buffers: 100 mM acetate pH 4.2, 25 mM Tris pH 7.4, and 25 mM Tris pH 8.0. The protein-containing solution (approximately 1 mg / mL of target protein) was conditioned with concentrated target buffers to achieve the target pH and buffer concentration shown in Table 6. The conditioned protein solution was then loaded onto a 30 mg protein / mL medium in flow-through mode. The flow-through pool was collected and subjected to DNA assay testing. As expected, the loaded material showed no DNA spike recovery (0%) due to the presence of PVS. At pH 4.2, no significant PVS removal was observed, resulting in failure of DNA spike recovery. While we do not wish to be bound by theory, this is likely due to stronger binding between the target protein and the polymer inhibitor, resulting from a higher net positive charge on the protein (at a much lower pH than the protein pI). PVS complexed with the target protein significantly reduced removal in flow-through mode. Significant PVS removal was observed at pH 7.4 and pH 8.0. Again, while we do not wish to be bound by theory, these results are likely due to a decrease in the net positive charge on the target protein. This effectively improves the availability of PVS bound to anion exchange media (i.e., not as strongly complexed with the target protein). The volume in this experiment was similar for charged membranes (Posidyne filters), pure AEX media (Q-Sepharose Fast Flow), and mixed-mode resins with AEX functionality (Capto® Adhere). PVS volume was similar in all cases tested with medium at approximately 1.5 μg / mL. These data demonstrate that when the pH is within 1-2 pH units of the protein's isoelectric point, an anion exchange medium or an anion exchange-containing medium separates PVS from the protein solution.

[0070] [Table 6]

[0071] Example 7 Determination of polyvinyl sulfonate bond capacity using multimodal chromatography media. In this experiment, to establish PVS binding capacity, PVS spikes were tested at levels far exceeding those expected in typical downstream purification platforms. Typical process conditions were tested with 100 mM MES buffer at pH 6 and 100 mM MES buffer containing 200 mM sodium chloride at pH 6. Furthermore, to represent worst-case buffer conditions, PVS capacity at higher sodium chloride concentrations (400 mM NaCl) was determined.

[0072] The PVS binding capacity of Capto® Adhere mixed-mode chromatography (MMC) resin (Cytiva) was determined on a laboratory scale. A 0.66 cm × 20 cm column (15.7 mL of resin) was challenged with three solutions spiked with a 30% PVS standard to achieve a PVS concentration of 1.5 mg / mL (Table 7). The column was tested according to the procedure outlined in Figure 7. MES buffers were prepared as described above to achieve the target buffer concentration and pH. After deionization (DI) of the column, the PVS spike buffer was loaded onto the column at 250 cm / hour. Fractions were collected every two column volumes (CV) over 60 CV. Then, the first seven fractions and pools of each buffer condition (7-15, 16-24, and 25-30) were tested to quantify the PVS levels. Each fraction or pool was measured in three replicates.

[0073] [Table 7]

[0074] Capto® Adhere mixed-mode resins support two binding modes using anion exchange and hydrophobic ligands. Increasing NaCl represents a decrease in anion exchange binding and an increase in hydrophobic interactions of PVS. To represent the worst-case buffering conditions, the PVS capacity at higher sodium chloride concentrations (400 mM NaCl) was determined. The results of the DNA qPCR spike recovery assay are shown in Table 8. Passing DNA spike recovery results indicated PVS concentrations acceptable for DNA quantification using conventional DNA assay procedures. PVS standard curves were used to estimate the PVS concentrations for pass / fail results. The PVS binding capacity of the resin was determined by the amount of PVS bound to the PVS breakthrough (first failing DNA spike recovery result) and is shown in Table 8. The binding capacity of the mixed-mode resin for the corresponding chromatographic fraction is also shown in Table 8 (second column). The 400 mM NaCl buffering condition represents the worst-case scenario where ionic interactions decrease with increasing salt concentration. This observation is consistent with the highly charged structure of polyvinyl sulfonate, in which each polymer repeating unit has a negative sulfonate group.

[0075] [Table 8]

[0076] DNA assay spike recovery was determined for the flow-through fraction. Acceptable fractions showed acceptable PVS clearance, while the first unacceptable sample determined the PVS binding capacity of the Capto(trademark) Adhere resin. For example, the standard condition sample (100 mM MES pH 6; 100 mM MES, 200 mM NaCl, pH 6) showed acceptable clearance for loadings up to fraction 7. Fraction 7 represented a PVS loading of 15 mg PVS / mL resin, and PVS loadings above this level caused consistent spike recovery interference (all pools from fractions 7 to 30 showed unacceptable spike recovery). Analysis under the worst-case 400 mM NaCl condition showed spike recovery interference in fraction 4. Therefore, the worst-case binding capacity was 9 mg PVS per mL of resin. Thus, for mixed-mode resins, a binding capacity of 9–15 mg PVS per mL of resin was observed.

[0077] Example 8 Titration method for the detection and removal of polyanions Nine lots of commercially available MES buffers were obtained and subjected to analysis using the titration method for detecting and measuring PVS disclosed herein. Comparative evaluation of these lots of MES buffers was performed using the disclosed titration method for detecting and measuring PVS and qPCR. As the experimental data demonstrate, this method enables highly sensitive detection of low levels of PVS and accurately and precisely detects lot-to-lot variability in PVS levels. Such analysis revealed a commercially available lot of MES buffer (lot number I) containing significantly high levels of PVS, which was consistent with the observation of lot-to-lot variability in buffer-related inhibition of host cell nucleic acid contamination in biological samples using PCR (e.g., qPCR).

[0078] The data provided in this example and Example 9 demonstrate that a titration method for detecting and measuring PVS in a sample using a polycationic compound such as hexadimethrin bromide (i.e., HDBr) is K a、PVS >>K a、MES Therefore, it has been established that it is highly selective for PVS than for MES. The results disclosed herein have shown that the disclosed titration method is reproducible (accurate), can detect low levels of polyanions, such as PVS, in Good buffers such as MES, and has a limit of quantification (i.e., LOQ) of approximately 100-200 ng / ml.

[0079] The protocol disclosed herein describes a polymer-electrolyte titration approach for quantifying polyanions, such as polyvinyl sulfonic acid (PVS), in Good's buffers, such as 2-(N-morpholino)ethanesulfonic acid (MES) buffers. This methodology is extendable to other Good's buffers produced from vinyl sulfonic acid (e.g., HEPES). The mechanism underlying PVS detection is based on binding with polycation species such as hexadimethrin bromide (HDBr). A schematic diagram of the binding reaction is shown in Figure 10a. This approach utilizes the high equilibrium association constant (Ka) between PVS and HDBr for selectivity to the monoanion MES. In fact, the Ka between polycations and polyanions increases rapidly with the number of charge sites (positively correlated with polymer molecular weight). At the titration endpoint, excess HDBr associates with the anionic indicator molecule Eriochrome Black T (ECBT), resulting in a shift in the indicator's UV-Vis absorbance profile (Figure 10b). The titration progress can be tracked at a single wavelength (i.e., 665 nm), and the concentration of PVS in the sample can be correlated, for example, by calculating the inflection point of the resulting sigmoid curve, as shown in Figure 9.

[0080] Materials and methods of Examples 8 and 9 In reagent preparation, assay buffers are prepared using conventional techniques, comprising buffer A containing 50 mM sodium borate adjusted to pH 8.5 with hydrochloric acid, and buffer B containing a combination of 100 mM sodium carbonate and bicarbonate formulated to produce a pH 10.0 solution. Polyanionic indicator compounds, such as Eriochrome Black T (ECBT; 55 wt%), function as indicator compounds or dye solutions. When the indicator compound is ECBT, solid aliquots of this material were stored at room temperature. To prepare an exemplary ECBT dye solution, 125 mg of ECBT was added to a 25 mL volumetric flask and the actual mass was recorded. The ECBT was dissolved in 25 mL of deionized (i.e., DI) water, dispensed into 1.6 mL or 5 mL polypropylene microcentrifuge tubes, and stored at 2–8°C until use. The polycationic compounds of the disclosed method are titrants, and exemplary titrant solutions are prepared using hexadimethrin bromide (HDBr). Store this material at 2–8°C. To prepare the solution, 18.7 mg of HDBr was weighed directly into a glass vial and dissolved in 3.74 mL of water to obtain a 5 mg / mL stock solution. Then, a 0.05 μg / mL HDBr titrator was prepared by diluting the 5 mg / mL HDBr solution in 50 mM borate buffer supplemented with 0.1 mM EDTA at a ratio of 1:20 or 1:100, respectively. This solution was used as the titrator for the assay method disclosed herein. The HDBr titrant was prepared as a 10 mL solution in a 15 mL polypropylene centrifuge tube and stored at 2–8°C.

[0081] For an experiment involving a titration method for detecting and removing biomolecules, approximately 30 wt% sodium poly(vinyl sulfonic acid) (PVS) was purchased from Sigma-Aldrich (#278424) and Alfa Chemistry (#ACM 25053274), diluted, and used to prepare PVS standards with known concentrations ranging from 0.1 to 20 μg / mL. A 50 mM borate buffer (pH 8.5) was prepared using conventional techniques. A 100 mM carbonate buffer (pH 10.0) was prepared from sodium carbonate (Sigma-Aldrich #223484) and sodium bicarbonate (Sigma-Aldrich #S6014). The carbonate buffer and bicarbonate buffer were supplemented with approximately 0.1 mM ethylenediaminetetraacetic acid (EDTA; MP Biomedicals #06133713). 1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide (hexadimethrine bromide; HDBr) was purchased from Sigma-Aldrich (107689) and Carbosynth (#FH165280). Eriochrome Black T (EBT or ECBT) was purchased from Sigma-Aldrich (#858390). All solutions were prepared using water purified to a minimum resistivity of 18 MΩ-cm. A 100 mM solution of MES hydrate was filtered through a 0.2 μm Posidyne® filter (surface area 2.8 cm 2 ) to remove PVS and functioned as a sample blank for the experiments disclosed herein.

[0082] Standard Preparation Using a commercially available poly(vinyl sulfonate) (PVS) stock solution, assay standards (Alfa Chemistry, 25 wt%, sodium salt, lot #A19X05191) were prepared by serially diluting the stock solution in water. The PVS solutions in Table 9 were then spiked into 30 mM borate buffer (supplemented with 0.1 mg / mL EDTA) to prepare standards with known PVS concentrations.

[0083]

Table 9

[0084] Stock and standard solutions were stored at 2-8°C.

[0085] Sample preparation A 100 mM solution of MES hydrate (lots #1 and #2) adjusted to pH 7.00 ± 0.05 was prepared as follows: 2.132 g of MES hydrate was dissolved in 95 mL of water, and the pH was adjusted using an aqueous NaOH solution. The pH was measured using a conventional pH meter. The solution was stored at 2–8°C.

[0086] Assay procedure Feasibility experiments for titration were performed using the simple protocol described below, but such experiments can be automated by using a photometric titrator to automate the steps described herein. The UV and visible lamps of the spectrometer were warmed for at least 20 minutes by turning on the spectrometer before use. The spectrometer was blanked before each assay using either a standard or a sample solution. The standard cell used in the disclosed assays was a 10 mm, 1.5 mL quartz cuvette. The standard consisted of PVS diluted with the assay buffer. Samples were prepared by mixing 100 mM MES with the assay buffer as an exemplary Good buffer. The exemplary ECBT indicator compound undergoes a color change over pH values ​​of 6–7, but pH values ​​above 7 are above the buffering range of MES, so this step is performed. Thus, as described above, MES was mixed with a basic buffer, i.e., A or B, to ensure that the ECBT indicator was deprotonated.

[0087] In the initial experiment, buffer A and MES were mixed in a 1:1 ratio. It is expected that a more basic buffer (e.g., B), mixed with MES in a different volume ratio, would improve assay performance.

[0088] The spectrometer was blanked, and a small amount of ECBT solution was added to the standard / sample. First, 995 μL of standard / sample was mixed with 5 μL of ECBT (5 mg / mL) to obtain a final ECBT concentration of 25 μg / mL. A full-wavelength absorbance scan was obtained. The standard / sample solution was titrated by adding small amounts (10–100 μL) of 0.050 mg / mL HDBr solution to a cuvette and measuring the sample absorbance between each HDBr addition. The solution was mixed using a 200 μL pipette, and after letting the solution stand for about 1 minute, the absorbance was measured. The volume of HDBr was gradually increased throughout the titration. For example, small amounts (e.g., 10 μL) were added first because the absorbance profile changed rapidly at the beginning of the titration. Larger volumes were added later in the titration if the absorbance change was more significantly affected by dilution. In some cases (e.g., for solutions with higher PVS concentrations), a more concentrated 0.25 mg / mL HDBr solution was used. Next, the spectrometer was blanked, and the aforementioned process of adding a small amount of indicator compound solution to the standard / sample was repeated for each sample.

[0089] Data Analysis From the UV-vis spectrum, the absorbance at 665 nm was plotted against the mass (μg) of added HDBr. To account for dilution, it was necessary to correct the absorbance for changes in solution volume, which was achieved by multiplying A665 nm by the total solution volume (i.e., the original volume of the solution [1,000 mL] + the cumulative volume of the added titrant solution).

[0090] Figures 11 and 12 summarize the evaluation results. Figure 4 shows the volume-corrected solution absorbance at 665 nm relative to the mass of the HDBr titrator for assay buffers spiked at three different PVS levels.

[0091] Figure 12a shows the volume-corrected solution absorbance at 665 nm relative to the mass of HDBr titrant for MES matrix blanks spiked at three different PVS levels. For both the 0 ppm PVS standard and the sample blank (i.e., the MES blank), the addition of the titrant is A 665 This caused a rapid initial drop, which stabilized after adding approximately 5.00 μg of HDBr to the solution. The remaining PVS standard samples, prepared by spiking commercially available PVS into solution, required larger amounts of titrator to reach steady-state absorbance. For example, the 7.5 ppm sample (Figure 12a) only stabilized after adding more than 40 μg of HDBr. 665 This was achieved. In summary, these data showed a clear difference in the titration curves (Figures 11 and 12a) related to the amount of PVS in the sample solution. Figure 12b summarizes this relationship by plotting the inflection points calculated for the PVS standard solution (green triangle) prepared in 50 mM borate buffer (pH 8.5) or for MES (black square) prepared by spiking PVS and then mixing it with 50 mM borate buffer (pH 8.5) to adjust the solution pH. The slopes of the two sets of data were similar, indicating that the presence of high concentrations (100 mM) of MES did not interfere with PVS quantification. Furthermore, these data support the detection of PVS at low concentrations of 1.5 ppm (μg / mL) in a 100 mM MES solution with 0.3 ppm in the assay buffer.

[0092] To further evaluate the performance of the titration procedure, two separate lots of MES were evaluated along with a PVS standard. A lot of MES hydrate that yielded invalid qPCR results for several products (Sample I) was compared to another MES sample with the minimum amount of PVS per qPCR assay (i.e., the same material used to create the sample blank in Figure 12a). The results of this evaluation, shown in Figure 13, show that MES Sample I has a measurable amount of PVS, but the negative control MES material does not, and the latter was indistinguishable from a PVS-depleted matrix blank. These results demonstrate that the disclosed methodology can accurately identify MES hydrate material with inadequate levels of PVS. Furthermore, MES hydrate material that contains no PVS or intermediate levels of PVS that would not interfere with qPCR is distinguished from inadequate MES material.

[0093] Example 9 automated titration Automated titration of PVS standards and MES sample solutions was performed using a Metrohm 907 Titrando instrument equipped with a discerning dispensing drive device (#2.800.0010) and a 20 mL dispensing unit (#6.303.2200). Immediately before titration, 100 mL of standard or sample solution was supplemented with 0.8–1.7 μg / mL of EBT indicator (e.g., by spiking in 0.5–1.0 mg / mL of EBT stock). The resulting solution was assayed by monotonic titration of the sample with HDBr in volume increments of 50 μL–150 μL. The titration progress was monitored by continuously measuring the absorbance of the sample solution at 660 nm using an immersion photometric probe (Optrode, #6.1115.000), and the titration endpoint was determined using the maximum dU / dV in the first derivative of the titration curve.

[0094] Automated PVS measurements were performed using a 907 Titrando (Metrohm) equipped with an immersion-type photometric probe capable of measuring solution absorbance at a wavelength of 660 nm. Titration was performed by gradually increasing the dosage of 0.05–0.15 mL of HDBr titrant. During each increment of titrant, the signal from the photometric probe was stabilized before administering the next volume of titrant. A representative titration profile of the blank standard is shown in Figure 14 (black trace) along with the corresponding first derivative (red trace). The volume at which the maximum value of the first derivative occurs (i.e., V in Figure 14) is shown. 滴定剤 (Approximately 0.55 mL) represents the endpoint of the titrant and is used to determine the PVS concentration.

[0095] The pH of the sample solution affects the anionic charge density of the PVS analyte, or indirectly affects the monovalent anion (H2In) through the protonation of the indicator compound. - By forming a complex with HDBr, it plays a crucial role in PVS measurement, and this complex does not undergo a change in absorbance. The experiment described above in Example 8 demonstrated that mixing the prepared MES solution with an alkaline buffer is a viable approach to ensure an appropriate sample pH. The use of this approach in automated titration experiments (i.e., by dissolving the MES sample in 50 mM MES in 100 mM carbonate buffer) was validated by evaluating the recovery of PVS spikes in the MES sample solution. For this evaluation, a 10 ppm PVS stock solution was spiked at various concentrations into the sample solution corresponding to a lot of MES hydrate (Sample H; see Table 10). When assayed by titration, this material produced an endpoint volume indistinguishable from the blank standard and showed a PVS level below the detection limit of the method.

[0096] The results of the spike recovery evaluation are shown in Figure 15a, which plots the titration endpoint volume against PVS concentration at four different PVS levels (each with 3 replicates in the assay). For comparison, the results for PVS standards prepared with 100 mM carbonate buffer alone are shown in Figure 15b. For both datasets, the linear regression between titration endpoint volume and PVS concentration yields a suitable linear coefficient of determination (R 2 Similar slopes (0.99 and 0.95 mL / (μg / mL)) were obtained at pH = 0.99. In particular, the size of the y-intercept of the spike recovery data (0.55 mL) was larger than that of the standard curve in Figure 15b (0.43 mL), which is likely due to the lower PVS level of the MES sample H. Furthermore, visual inspection of the representative titration curves shown in Figure 16 for the PVS standard (Figures 16A and 16B) and spike recovery samples (Figures 16C and 16D) did not show any perceptible effect of the low pH or the presence of 50 mM MES on the titration profile. In summary, these results do not indicate a significant effect of lower sample pH or the presence of 50 mM MES on the measured PVS levels.

[0097] During the development of the titration procedure, several MES hydrate lots were evaluated for PVS content by titration with 50 mM MES (dissolved in 100 mM carbonate buffer) using 0.10 mg / mL HDBr. The titration endpoints were compared to the results obtained for a series of PVS standard solutions. The results of these evaluations are shown in Table 10. Among these samples, there was an MES hydrate lot (Sample I) that caused qPCR assay failures for several therapeutic protein batches. Sample I had a PVS level of 71 ± 4 μg PVS per 1 g of MES hydrate, as measured by titration, which was significantly higher than the PVS levels measured for any of the other samples tested, supporting the usefulness of titration in screening MES materials with inadequate PVS levels.

[0098] [Table 10]

[0099] Example 11 Comparison of detection methods Several methods for detecting and measuring polycations such as PVS in protein samples (e.g., biological samples) were evaluated. An ion coordination method involving PVS-induced reporter aggregation using turbidity detection is a simple and less complex method, but it could not reliably detect MES buffer lots with high levels of PVS. A fluorescence-based method involving direct detection of aqueous PVS by fluorescence excitation and detection was another simple and less complex method, but it proved impractical for PVS detection. Other fluorescence-based methods involved more PVS-induced quenching of the fluorescent reporter molecule and did not show promise, likely due to their limited ability to selectively detect PVS compared to MES. A method based on the physical properties of polycations found in Good's buffer is size exclusion chromatography (i.e., SEC-CAD) using charged aerosol detection. This method was able to detect PVS in MES buffer, but it is considerably more complex than the other methods. Another ionic coordination method was evaluated and found to yield unexpectedly excellent results, providing accurate, precise, and highly sensitive detection and quantification of PVS in Good's buffers, including but not limited to the Good's buffers provided in Table 11, through complexation of a polymer electrolyte and titration using UV-Vis wavelength absorbance detection. In addition to offering advantages in accuracy, precision, and sensitivity, the titration method disclosed herein is a simple method with low complexity and cost.

[0100] [Table 11]

[0101] Each of the references cited herein is incorporated herein by reference, either in whole or in part, as is evident from the context of the citation.

[0102] Although the claimed subject matter has been described in detail, it should be understood that the foregoing description is intended to illustrate, not to limit, the scope of the claimed subject matter as defined by the attached claims. Other embodiments, advantages, and variations are within the scope of the following claims.< / tam> < / fam>

Claims

1. A method for quantifying polyanionic PCR inhibitors in a sample, a) Prepare a sample dilution series containing at least four members; b) Spike each member of the sample dilution series with a certain amount of template DNA that is identifiable from host cell DNA; c) To create a standard curve for polyanionic inhibitors, including serial dilutions of the polyanionic inhibitor in the absence of the sample; d) Spike a fixed amount of the template DNA into each sequential dilution of the polyanionic inhibitor standard curve; e) Perform a PCR assay against the template DNA for each member of the sample dilution series and each serial dilution of the polyanionic inhibitor; f) Comparing the PCR assay results of the dilution series of the sample with the PCR assay results of the polyanionic inhibitor standard curve; and g) Determine the concentration of the polyanionic PCR inhibitor in the sample. A method comprising, wherein the concentration of the polyanionic PCR inhibitor in the sample is within the range determined by the concentration of the polyanionic PCR inhibitor in the least diluted member of the dilution series of the sample that shows complete spike recovery of the template DNA and the least diluted member of the dilution series that does not show complete spike recovery of the template DNA.

2. The method according to claim 1, wherein the number of members in the dilution series is 5, 6, 7, 8, 9, 10, 12, 15, or 20, thereby narrowing the range of concentrations of the polyanionic PCR inhibitor in the sample compared to the range provided in claim 1.

3. The method according to any one of claims 1 to 2, wherein the certain amount of template DNA is at least 100 pg.

4. The method according to any one of claims 1 to 3, wherein the polyanionic PCR inhibitor is a sulfonate compound.

5. The method according to claim 4, wherein the sulfonate compound is polyvinyl sulfonate.

6. The method according to claim 5, wherein the polyvinyl sulfonate is a polyanionic PCR inhibitor used when creating a standard curve for polyanionic inhibitors, and the concentration of the polyanionic PCR inhibitor in the sample is in units of polyvinyl sulfonate concentration equivalent.

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