Titration method for detecting polyvinyl sulfonate (PVS) in buffer solution

Abstract

This invention provides a method for detecting levels of polyanionic compounds that inhibit enzymes involved in PCR, which can disrupt efforts to monitor the purity of proteins obtained from cell cultures, such as biological products and biosimilars. [Solution] The level of polyvinyl sulfonate or other substances in a fluid such as a buffer solution is detected by complex titration or a titration-based technique.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 093,124 filed on 16 October 2020, U.S. Provisional Patent Application No. 63 / 144,744 filed on 2 February 2021, and U.S. Provisional Patent Application No. 63 / 251,465 filed on 1 October 2021, which are incorporated herein by reference as a whole.

[0002] This disclosure generally relates to the field of chemistry, and more specifically to the field of determining or titrating the concentration of compounds in fluids. [Background technology]

[0003] Biological agents and biosimilars have become an important therapeutic class for treating diseases and conditions in humans and other animals. These products, which include recombinant proteins such as antibodies and their fragments in various forms that retain binding ability, are typically obtained from cell cultures. Consequently, these therapeutic agents need to be purified from contaminants inherent to the cell culture process, such as unwanted and potentially toxic proteins, lipids, carbohydrates, and other small molecules associated with cell proliferation in the culture. Since therapeutic biological agents and biosimilars are gradually purified by isolating them from these culture contaminants, the therapeutic agents are stabilized in buffered solutions. Furthermore, as purification progresses, the level of purification needs to be monitored to ensure that the therapeutic agent maintains safety for use. One significant contaminant found when purifying therapeutic agents from cell cultures is fragments of host cell DNA. Residual amounts of host cell DNA may survive rigorous purification processes and remain as harmful contaminants in the purified therapeutic agent. Residual host cell DNA in protein preparations administered to animals, such as human patients, e.g., biological agents or biosimilars, may induce undesirable immune responses or increase the risk of cancer. Therefore, governments around the world impose restrictions on the concentration of host cell DNA in formulations intended for human administration. For example, the World Health Organization (WHO) and the European Union (EU) allow a maximum of 10 ng / dose of residual host cell DNA, while the U.S. Food and Drug Administration allows less than 100 pg / dose.

[0004] Given the low levels of host cell DNA tolerated in therapeutic formulations intended for human administration, a highly sensitive and accurate method is needed to determine the levels of host cell DNA in such formulations. One method for quantifying low levels of nucleic acids in a sample involves using polymerase chain reactions, such as monitoring nucleic acid levels in real time using qPCR. PCR is an enzyme-based technique that relies on the enzyme polymerase to amplify low levels of nucleic acids, making them easier to detect. [Overview of the project] [Means for solving the problem]

[0005] This disclosure provides a method for determining or titrating the level of polyanions in a sample, such as Good's buffer, including MES. The sample may include Good's buffer (e.g., raw materials or manufacturing lots of Good's buffer) and may further include therapeutic compounds such as biological agents or small molecules. An exemplary polyanion is polyvinyl sulfonate (i.e., PVS), which is often present in varying amounts in a sample of Good's buffer or biological agent at various stages of recovery and purification. These polyanions, particularly PVS, have been shown to inhibit various enzymes, including RNA and / or DNA enzymes such as polymerases. A method for detecting PVS levels in a sample is disclosed herein. The sample may include proteins such as biological agents, or other therapeutic compounds at various stages of production, recovery, or purification, such as small molecule therapeutics, or both. Methods known in the art have been unable to detect low levels of PVS in such samples, resulting in PVS contamination of therapeutic compounds such as biological agents. Such contamination can hinder approval for human use and the inhibitory effect of PVS can disrupt efforts to monitor other impurities in product formulations, such as host nucleic acids. Polyanions like PVS inhibit enzymes used in standard nucleic acid assays such as PCR, e.g., qPCR, which leads to inaccuracies in the measurement of contaminated host nucleic acids. A sensitive, accurate, and precise titration method is disclosed herein for measuring the level of PVS, a known inhibitor of RNA enzymes, in a sample (such as a protein sample) containing Good Buffers. The titration method according to this disclosure exhibits a dynamic detection range of 1.5 orders of magnitude, has high selectivity for PVS compared to MES, and provides a simple readout including an inflection point or equivalence point, which provides a simple pass / fail output for MES buffer lots, considering its use in monitoring nucleic acid contamination of host cells in protein samples such as biological samples. This method provides automated electrochemical or spectroscopic (e.g., colorimetric, photometric, fluorescence, Raman, or FTIR spectroscopy) endpoint detection probes at a reasonable cost, and also offers inexpensive embodiments that rely on standard manual titration configurations.

[0006] More specifically, the present disclosure relates to a titration method for detecting polyanionic enzyme inhibitors in a fluid, comprising: (a) contacting the fluid with a known amount of polycationic compound; (b) contacting the material from (a) with an indicator compound, wherein the indicator compound exhibits altered properties in its free form compared to its complex form if it forms a complex with the polycationic compound, and if no complex formation occurs, a sufficient amount of the indicator compound is added to detect the free form of the indicator compound; (c) repeating (a); and (d) detecting the free form of the indicator compound at a titration point, thereby detecting a polyanionic enzyme inhibitor. The indicator compound includes, but is not limited to, an anionic indicator compound, and may consist of such an indicator. In some embodiments, the fluid includes, is essentially, or consists of a buffer. It is assumed that (b) the addition of the indicator compound to the fluid may be carried out before, simultaneously with, or after the first repeating of (a), but if the indicator compound is added thereafter, it will be understood that the indicator compound is added before repeating (a). In some embodiments, multiple samples of the buffer are prepared, each buffer sample having a different concentration of the buffer compound, thereby generating a dilution series of the buffer. In some embodiments, the detection limit for polyvinyl sulfonate (PVS) is parts per 1.5 million of the buffer solution, parts per 0.25 million of the buffer solution, or 0.16 μg / mL of the buffer solution. In some embodiments, the detection limit for polyvinyl sulfonate (PVS) is parts per 1.5 million of the buffer compound, or parts per 0.25 million of the buffer compound. For example, automated methods such as those described herein can identify PVS at a detection limit of parts per 0.25 million of the buffer compound. In some embodiments, the titration endpoint is the point where the absorbance of the sample is midway between the initial absorbance of the sample and the steady-state absorbance, or the maximum value of the first derivative of the absorbance curve of the sample. In some embodiments, the released indicator compound is detected electrochemically or spectroscopically. In some embodiments, spectroscopic detection includes colorimetric detection, photometric detection, fluorescence detection, Raman, or FTIR spectroscopy.In some embodiments, the polyanionic enzyme inhibitor is polyvinyl sulfonate (PVS) or a derivative thereof. In some embodiments, the polyanionic enzyme inhibitor is polyvinyl sulfonate (PVS). 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). In some embodiments, multiple HDBr aliquots totaling at least 0.1% of the total fluid volume are added to the fluid. In some embodiments, the quaternary ammonium polymer is poly(diallyl)dimethylammonium chloride (pDADMAC).

[0007] Many suitable indicator compounds, such as anionic indicators, can be used in the embodiments herein. In some embodiments, the indicator compound is a dye, such as an azo dye. In some embodiments, the azo dye is eriocrom black T (ECBT), eriocrom blue black R (chalcone), or a sodium sulfonazo salt. In some embodiments, the azo dye is eriocrom black T (ECBT). In some embodiments, 0.8 to 1.7 μg of ECBT is added per 1 mL of fluid containing a known amount of polycationic compound. In some embodiments, the buffer is Good's buffer. In some embodiments, Good's buffer contains an ethanesulfonic acid derivative or a propanesulfonic acid derivative. In some embodiments, Good's buffer is MES, bis-trismethane, ADA, bis-trispropane, PIPES, ACES, MOPSO, coramine chloride, MOPS, BES, AMPB, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, CHES, CAPSO, AMP, CAPS, or CABS. Some embodiments of the method further include determining the concentration of the polyanionic enzyme inhibitor from the amount of polycationic compound required to titrate the polyanionic enzyme inhibitor. Some embodiments of the method further include comparing the results with those obtained on a standard curve of the polyanionic enzyme inhibitor to determine the concentration of the polyanionic enzyme inhibitor in the fluid. For example, the endpoint volumes of a series of calibration standards (e.g., 3 to 5 standards) for multiple polyanions (e.g., PVS) can be calculated, and a standard curve can be generated. The standard curve can be used to calculate the concentration of polyanions in a sample based on the endpoint values ​​of the sample. Some embodiments of this method further include performing a “limit test,” in which the endpoint volumes of a sample containing a blank (without polyanions such as PVS) and a specified limit concentration of polyanion (e.g., PVS) are calculated. The endpoint volumes of the sample can be determined, and an analytical “pass / fail” decision can be made based on whether the concentration of polyanions in the sample is within the specified limit.In some embodiments, the method is automated.

[0008] Another aspect of the present disclosure relates to an automated titration method for detecting polyanionic enzyme inhibitors in a fluid, comprising: (a) combining a fluid with an indicator compound, wherein the indicator compound exhibits altered properties in its free form compared to its complex form when it forms a complex with a polycationic compound, and if no complex formation occurs, a sufficient amount of the indicator compound is added to detect the free form of the indicator compound; (b) contacting the material from (a) with a known amount of the polycationic compound; (c) measuring the absorbance of the fluid containing the indicator compound and the polycationic compound using a titration instrument; and (d) automatically repeating (b) and (c), wherein the detection of the free form of the indicator compound is an automated titration method for detecting polyanionic enzyme inhibitors. It is assumed that (a) combining a fluid containing a polycationic compound with an indicator compound may be performed before, simultaneously with, or after the first repeating of (b), but it will be understood that if the indicator compound is subsequently combined, the indicator compound should be added before repeating (b). In some embodiments, the fluid contains, is essentially, or consists of a buffer. In some embodiments, the buffer is a Good buffer. In some embodiments, the method is performed in a titration apparatus. In some embodiments, the titration apparatus includes a pump, such as a syringe pump or an intelligent dosing drive, which is in fluid communication with the polycationic compound and the fluid. Suitable absorbance wavelengths for the methods and systems described herein may be selected based on the indicator compound used. For example, for the indicator compound of ECBT, wavelengths of 660–665 nm are preferred.

[0009] Some embodiments include automated titration systems for detecting polyanionic enzyme inhibitors in a fluid. The automated titration system may include a fluid delivery system, such as a pump. The automated titration system may be configured to automatically perform methods such as those described herein. For example, the automated titration system may include a titrator. For example, a titrator suitable for the methods and systems described herein is commercially available as part of Metrohm's TITRANDO series of instruments. Optionally, the titrator may include a pump for fluid communication (e.g., for fluid-communicating polycationic compounds with the fluid), such as a syringe pump or an intelligent dosing drive pump.

[0010] Other features and advantages of this disclosure will become apparent from the following detailed description, including the drawings. However, since various variations and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from the detailed description, please understand that the detailed description and specific examples, while illustrating embodiments, are provided merely as examples. [Brief explanation of the drawing]

[0011] [Figure 1] (a) Chemical structures of 2-(N-morpholino)-ethanesulfonic acid (i.e., MES) as an acid and MES sodium salt as a base. (b) Chemical reactions that yield compounds that may inhibit enzymes activated by nucleic acids such as RNA enzymes. Figure 1(b) is quoted from Smith, et al. J. Biol. Chem. 278:20934-20938 (2003). [Figure 2](a) Linear calibration curves were revealed for two different lots of polyvinyl sulfonate (PVS) standards obtained from Sigma-Aldrich and provided as a 30 wt% aqueous solution by varying the concentration of PVS from 0 to 1.0 ppm. It was found that the concentration of PVS varied significantly between lots. However, it is assumed that the concentration of a particular lot can be adjusted by diluting it to make it a suitable standard. (b) Titration curves using hexadimethrin bromide (HDBr) to titrate PVS were prepared over the range of PVS concentrations from 0 to 1.0 ppm. A linear range of approximately 1.5 orders of magnitude was found. [Figure 3] (a) Schematic diagram of the titration quantification of PVS with HDBr by spectroscopic detection of the endpoint. The reaction scheme shows the formation of a complex between PVS and HDBr caused by an inducible electrostatic interaction. At the titration endpoint, the indicator compound (nD-) binds to the charged site of the adjacent HDBr, causing a change in the absorbance properties. The blue circular "plus" and "minus" symbols in Figure 3(a) represent background salts in the solution. (b) Plot showing the development of absorbance in a blank sample solution (i.e., 50 mM borate buffer supplemented with EBT indicator compound, pH 8.5) measured with a benchtop absorbance spectrometer when HDBr is gradually titrated into the solution. As the concentration of HDBr increases, the absorbance of the indicator at 665 nm decreases, as the maximum absorbance shifts from approximately 630 nm to 593 nm. [Figure 4] Titration curves plotting normalized and volume-corrected absorbance at 665 nm for a series of PVS standard solutions. [Figure 5] (a) Plots of absorbance at 665 nm of solutions corrected for the amount of HDBr (titrate) for three different PVS standards prepared in an MES matrix blank. (b) Comparison of inflection points of titration curves between PVS standards prepared in 50 mM sodium borate (green triangles) and MES mixed with 50 mM sodium borate (black squares). [Figure 6]Comparison of inflection points of titration curves for PVS standards prepared in MES matrix blanks (black squares), MES negative control lots (blue diamonds), and MES lots that caused qPCR ineffective assays (lot number I; red circle). [Figure 7] Typical profile (black line) and corresponding first derivative (red line) for titration of a blank standard (100 mM carbonate buffer supplemented with 1.25 μg / mL EBT indicator) using 0.04 mg / mL HDBr. [Figure 8] (a) Plot of titration endpoint volume against the concentration of PVS added to 50 mM MES dissolved in 100 mM carbonate buffer. (b) Plot of titration endpoint volume against the concentration of PVS for a standard sample prepared in 100 mM carbonate buffer. [Figure 9] Representative titration curves of (A, B) PVS standard solutions prepared at 0(A) or 0.75(B) μg / mL in 100 mM carbonate buffer and (C, D) PVS (sample H in Table 2) added at 0(C) or 0.70(D) μg / mL to 50 mM MES prepared in 100 mM carbonate buffer. [Figure 10] The following plots show the titration endpoint volume against the concentration of PVS. A) is a plot of the titration endpoint volume against the concentration of PVS for a standard sample prepared in 100 mM carbonate buffer. B) is a plot of the titration endpoint volume against the concentration of PVS added to 50 mM MES sodium salt dissolved in 100 mM carbonate buffer. [Modes for carrying out the invention]

[0012] Polyanionic compounds such as poly(vinyl sulfonate) (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, ranging from parts per million, compared to buffering compounds such as MES. Such buffers are used in the production of therapeutic proteins, and the presence of these impurities in Good's buffers is a serious concern because PVS, in particular, is a potent polymerase inhibitor that can interfere with quantitative PCR (qPCR) DNA detection. Measuring host cell nucleic acids (e.g., DNA) in therapeutic protein formulations or other therapeutic compound formulations purified from cultures is routinely required to assess the safety of therapeutic drugs intended for administration to humans. A major challenge in using PCR-based techniques to detect and quantify host cell nucleic acids in therapeutic formulations is the presence of nucleic acid enzyme inhibitors in many buffers (e.g., Good's buffers) used to purify proteins, such as biological agents and biosimilars, from cell cultures. Given the speed, accuracy, and reproducibility of PCR-based methods for detecting and quantifying host cell nucleic acid levels in these formulations, identifying and removing inhibitors of nucleic acid enzymes involved in PCR, such as RNA enzymes, is a significant need in this field. Therefore, the presence of polyanionic compounds such as PVS in Good's buffers, such as MES, can interfere with qPCR detection of host cell DNA, potentially causing batches of therapeutic proteins or other therapeutic compounds to be unacceptable for human administration.

[0013] A titration method is disclosed herein that is based on the formation of a complex with an analyte (e.g., PVS) and a high molecular weight titrator with the opposite charge. This interaction results in an extremely high equilibrium bonding constant (K). a A solution is obtained, and the endpoint can be detected electrochemically or spectroscopically (e.g., by colorimetric, photometric, fluorescence, Raman, or FTIR spectroscopy). Figure 3 shows an overview of a detection scheme applicable to the titration of PVS using hexadimethrin bromide (HDBr), an exemplary titrator.

[0014] Nine commercially available MES buffer lots were obtained and analyzed using the titration method for detecting and measuring PVS disclosed herein. Comparative evaluations of these lots of MES buffer were performed using the disclosed titration method for detecting and measuring PVS and using qPCR. As the experimental data show, the method can sensitively detect low levels of PVS and can correctly and accurately detect the variation in PVS levels among lots. Such analysis reveals that a commercially available lot of MES buffer (lot number I) contains a significantly high level of PVS, which is consistent with the observation that the inhibition associated with buffers contaminated with host cell nucleic acids of biological samples varies among lots when using PCR (e.g., qPCR).

[0015] The data provided in the following examples demonstrate that the titration method for detecting and measuring PVS in samples using a polycationic compound such as hexadimethrine bromide (i.e., HDBr) has high selectivity for PVS compared to MES (K a,PVS >>K a,MES where K a represents the equilibrium binding constant for the complex formation reaction between the titrant (HDBr) and either PVS (K a,PVS ) or MES (K a,MES ). From the results disclosed herein, it is clear that the disclosed titration method is reproducible (accurate) and can detect low levels of polyanions, such as PVS, in good buffers such as MES at a quantification limit (i.e., LOQ) of approximately 100 -  200 ng / mL.

[0016] The procedures disclosed herein describe a polymer electrolyte titration technique for quantifying polyanions such as poly(vinyl sulfonate) (PVS) in a good buffer such as 2-(N-morpholino)-ethanesulfonic acid (MES) buffer. This methodology can also be extended to other good buffers (e.g., HEPES) manufactured from vinyl sulfonic acid. The mechanism underlying the detection of PVS is based on its binding to the polycationic species hexadimethrine bromide (HDBr). A schematic of the binding reaction is shown in Fig. 3a. This technique utilizes the high equilibrium binding constant (Ka) between PVS and HDBr, which is selective compared to the monoanion MES. In fact, the Ka between a polycation and a polyanion increases sharply with the number of charge sites (showing a positive correlation with the polymer molecular weight). At the end point of the titration, the excess HDBr binds to the anionic indicator compound eriochrome black T (ECBT), causing a shift in the ultraviolet-visible absorbance profile of the indicator (Fig. 3b). By tracking the progress of the titration at a single wavelength (i.e., 665 nm) and calculating, for example, the inflection point of the sigmoid curve obtained, as shown in Fig. 2, it becomes possible to associate with the concentration of PVS in the sample.

Example

[0017] Example 1 Materials and Methods Approximately 30% by weight of poly(vinyl sulfonic acid) (PVS) sodium salt was purchased from Sigma-Aldrich (#278424) and Alfa Chemistry (#ACM25053274), and diluted to prepare PVS standards of 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). Approximately 0.1 mM ethylenediaminetetraacetic acid (EDTA; MP Biomedicals #06133713) was added to this carbonate and bicarbonate buffer. 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (hexadimethrin 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 purified water to achieve a minimum resistivity of 18 MΩ-cm. A 100 mM MES hydrate solution was filtered through a 0.2 μm Posidyne® filter (surface area 2.8 cm²). 2 The PVS was removed by filtering with ) and the sample blank for Example 1 was obtained.

[0018] Assay buffers prepared according to the procedure described above yielded buffer A, containing 50 mM sodium borate adjusted to pH 8.5 with hydrochloric acid, and buffer B, containing 100 mM mixed sodium carbonate and bicarbonate, formulated to produce a pH 10.0 solution. An indicator compound, i.e., a dye solution, such as eriocrom black T (ECBT; 55 wt%), was used as the indicator compound. When the indicator compound was 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 and stored at 2–8°C as 1 mL aliquots in a 1.6 mL polypropylene microcentrifuge tube until use. The polycationic compound of the disclosed method is the titrator, and exemplary titration solutions were prepared using HDBr. This material was stored 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 titrant was prepared by diluting the 5 mg / mL HDBr solution 1:20 or 1:100 in 50 mM borate buffer supplemented with 0.1 mM EDTA. This solution was used as the titrant 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.

[0019] Standard preparation Assay standards were prepared using commercially available poly(vinyl sulfonate) (PVS) stock solution by serial dilution of the stock solution with water (Alfa Chemistry, 25% by wt, sodium salt, lot number A19X05191). Then, the PVS solutions shown in Table 1 were added to 50 mM borate buffer (supplemented with 0.1 mg / mL EDTA) to prepare standards of known PVS concentrations.

[0020] [Table 1]

[0021] The stock solution and standard solution were stored at 2-8°C.

[0022] Sample preparation A 100 mM solution of MES hydrate (lot numbers I and II) adjusted to pH 7.00 ± 0.05 was prepared as follows: 2.132 g of MES hydrate was dissolved in 95 mL of water, the pH was adjusted using an aqueous NaOH solution, and then the solution was diluted with water to a final volume of 100 mL. The pH was measured using a conventional pH meter. The solution was stored at 2-8°C.

[0023] Assay procedure Titration demonstration experiments were conducted using the simple procedure described below, but such experiments can be automated by using a photometric titration apparatus, thereby automating the steps described herein. The spectrometer's ultraviolet and visible light lamps were warmed for at least 20 minutes before use by turning on the spectrometer. Before each assay, the spectrometer was blanked with either a standard solution 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 in assay buffer. Samples were prepared by mixing 100 mM MES as an exemplary Good's buffer with the assay buffer. This step is performed because the exemplary ECBT indicator compound undergoes a color change at pH values ​​of 6–7, while pH values ​​above 7 are above the buffering range of MES. Therefore, MES was mixed with a basic buffer, i.e., A or B, as described above, to ensure that the ECBT indicator is deprotonated.

[0024] In the initial experiment, buffer A and MES were mixed in a 1:1 ratio. It is expected that assay performance will improve with more basic buffers (e.g., B and C) mixed with MES in different volume ratios.

[0025] The spectrometer was blanked once, and then 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 a final ECBT concentration of 25 μg / mL. Absorbance scans were obtained at all wavelengths. The standard / sample solution was titrated by adding small amounts (10-100 μL) of 0.050 mg / mL of HDBr solution to a cuvette, and the absorbance of the sample was measured while adding each amount of HDBr. The solutions were mixed using a 200 μL pipette, and the absorbance was measured after the solution had stood for approximately 1 minute. The amount of HDBr was gradually increased throughout the titration process. For example, if the absorbance profile changed dramatically at the beginning of the titration, a small amount (e.g., 10 μL) was added initially. If the change in absorbance was more significantly affected by dilution, a larger amount was added after the titration.

[0026] In some cases (e.g., solutions containing higher concentrations of PVS), a higher concentration of HDBr solution, 0.25 mg / mL, was used. The spectrophotometer was then blanked, and the preceding step of adding a small amount of dye solution to the standard / sample was repeated for each sample.

[0027] Data Analysis From the UV-Vis spectrum, the absorbance at 665 nm was plotted against the mass (μg) of added HDBr. The absorbance needed to be corrected for the change in solution volume due to dilution. This correction was achieved by multiplying the total solution volume (i.e., the original volume of the solution [1,000 mL] plus the cumulative volume of the added titration solution) by A665 nm.

[0028] Figures 4 and 5 summarize the evaluation results. Figure 4 shows the absorbance at 665 nm of the assay buffer solution, with volume corrected relative to the mass of the HDBr titrator added at three different PVS levels.

[0029] Figure 5a represents the absorbance at 665 nm of the volume-corrected solution with respect to the mass of the HDBr titrant for the MES matrix blank added at three different levels of PVS. Neither the 0 ppm PVS standard nor the sample blank (i.e., the MES blank) initially caused a decrease in A 665 until the addition of approximately 5.00 μg of HDBr to the solution, after which it stabilized. The remaining PVS standards and samples prepared by adding commercially supplied PVS to the solution required a greater amount of titrant to reach a steady-state absorbance. For example, the 7.5 ppm sample (Figure 5a) only stabilized at A 665 after adding more than 40 μg of HDBr. Overall, these data indicated a clear difference in the titration curves with respect to the amount of PVS in the sample solution (Figures 4 and 5a). Figure 5b summarizes this relationship by plotting the inflection points calculated for PVS standard solutions (green triangles) prepared in 50 mM borate buffer (pH 8.5) or MES (black squares) to which PVS was added and then mixed with 50 mM borate buffer (pH 8.5) to adjust the pH of the solution. The slopes of the two datasets are equivalent, indicating that the quantification of PVS was not hindered even in the presence of high-concentration (100 mM) MES. Furthermore, these data support that PVS was detected at low concentrations of 1.5 ppm (μg / mL) in 100 mM MES solution and 0.3 ppm in the assay buffer.

[0030] To further evaluate the performance of the titration procedure, MES from two separate lots was evaluated along with a PVS standard. A lot of MES hydrate that yielded invalid qPCR results in several products (Sample I) was compared to another MES sample with the minimum amount of PVS present by the qPCR assay (i.e., the same material used to generate the sample blank in Figure 5). The results of this evaluation, shown in Figure 6, indicated that MES Sample I contained a measurable amount of PVS, while the negative control MES material did not, making it indistinguishable from a matrix blank from which PVS had been removed. These results demonstrate that the disclosed methodology can accurately identify MES hydrate material with inadequate PVS levels. Furthermore, MES hydrate that does not contain PVS or contains intermediate levels of PVS that do not interfere with qPCR is distinguished from unsuitable MES material.

[0031] Example 2 Automated titration Automated titration of PVS standard solutions and MES sample solutions was performed using a Metrohm 907 Titrando apparatus equipped with an intelligent dosing drive (#2.800.0010) and a 20 mL volumetric dosing unit (#6.303.2200). 100 mL of the standard or sample solution was supplemented with 0.8–1.7 μg / mL of EBT indicator (e.g., by adding it to a 0.5–1.0 mg / mL EBT stock) immediately before titration. The sample was monotonically titrated with 50–150 μL of HDBr, and the resulting solution was assayed by stabilizing the signal from the photometric probe between dose increments. 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 of the first derivative of the titration curve.

[0032] A typical titration profile of the blank standard is shown in Figure 7 (black line), along with the corresponding first derivative (red line). The amount at which the maximum value of the first derivative occurs (i.e., approximately 0.55 mL in Figure 7) is V. TitrantThis corresponds to the titration endpoint and is used to determine the PVS concentration.

[0033] The pH of the sample solution affects the charge density of the anions in the PVS analyte, or indirectly protonates the indicator compound, causing a change in absorbance when it forms a complex with HDBr, resulting in monovalent anions (H2In - By forming a ) it plays an important role in measuring PVS. The experiment described above in Example 1 showed that mixing the prepared MES solution with an alkaline buffer is an effective method for ensuring the correct pH of the sample. The use of this method 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 rate of PVS added to the MES sample solution. In this evaluation, various concentrations of 10 ppm PVS stock solution were added to the MES hydrate sample solution (Sample H; see Table 2). When assayed by titration without adding further PVS to the sample, an endpoint volume indistinguishable from the blank standard was obtained with this material, indicating that the level of PVS was below the detection limit of this method.

[0034] Figure 8a shows the results of the add-and-recovery evaluation, plotting the titration endpoint volume against PVS concentrations at four different PVS levels (each assayed three times). For comparison, Figure 8b shows the results for a PVS standard prepared with 100 mM carbonate buffer alone. For both datasets, linear regression between titration endpoint volume and PVS concentration yielded similar slopes (0.99 and 0.95 mL / (μg / mL)), indicating a suitable linear coefficient of determination (R). 2 The result was 0.99. In addition, visual inspection of the representative titration curves shown in Figure 9 for the PVS standard (Figures 9A and 9B) and the added and recovered sample (Figures 9C and 9D) showed that the effect of low pH or the presence of 50 mM MES on the titration profile was not discernible. In summary, these results indicate that the low pH of the sample or the presence of 50 mM MES does not clearly affect the measured PVS level.

[0035] Throughout the titration procedure, the PVS content of several MES hydrate lots was evaluated by titrating 50 mM MES (dissolved in 100 mM carbonate buffer) with 0.10 mg / mL HDBr. The titration endpoints were compared to the results obtained in a series of PVS standard solutions. The results of these evaluations are shown in Table 2. Among these samples, one MES hydrate lot (Sample I) was the cause of qPCR assay failure for several batches of therapeutic proteins. In Sample I, the PVS level measured by titration was 71 ± 4 μg of PVS per gram of MES hydrate, which was significantly higher than the PVS levels measured in any of the other samples tested, supporting the usefulness of titration in screening MES material with inappropriate PVS levels. Note that different repeated titration procedures are shown for some MES hydrate lots (i.e., E.1 and E.2, F.1 and F.2, and H.1 and H.2). For example, E.1 represents a repetition based on a single iteration, while E.2 represents a repetition based on three iterations.

[0036] [Table 2]

[0037] Example 3 Comparison of detection methods Several methods for detecting and measuring polycations such as PVS in protein samples (e.g., biological samples) were evaluated. A turbidimetric detection method involving ion coordination related to the induced aggregation of counterions of polyionic reporters by PVS is a relatively simple method, but it could not reliably detect lots of MES buffer containing high levels of PVS. Another fluorescence-based method involving the direct detection of PVS aqueous solutions by excitation and detection of fluorescence was also relatively simple, but it proved impractical for PVS detection because PVS in solution does not fluoresce, while fluorescence associated with dried PVS samples was judged to be PVS-nonspecific artifacts related to the dried sample. Another fluorescence-based method involving the PVS-induced quenching of fluorescent reporter molecules was more complex and did not appear promising due to its limited ability to selectively detect PVS relative to MES. A method based on the physical properties of polyanions found in Good's buffer includes size exclusion chromatography (i.e., SEC-CAD) with charged particle 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, which involves polymer electrolyte complex formation and titration using UV-Vis absorbance detection. This method, including but not limited to the Good buffers shown in Table 3, proved to yield unexpectedly excellent results in providing accurate, precise, and sensitive detection and quantification of PVS in Good buffers. In addition to offering the advantages of accuracy, precision, and sensitivity, the titration method disclosed herein is a simple method that is neither complex nor costly.

[0038] [Table 3]

[0039] Example 4 Quantification of PVS in prepared MES sodium salt aqueous solution Aqueous solutions of MES sodium salt that are considerably more alkaline than the solution of MES hydrate conjugate acid (for example, pH values ​​of approximately 10.0 and 8.5 for 50 mM MES sodium salt and MES hydrate solution in 100 mM carbonate buffer, respectively) can also be adapted for PVS determination using a titration procedure similar to that shown in Example 2. Figure 10(B) plots the titration endpoints, photometrically determined at 660 nm, for a 50 mM MES sodium salt solution prepared in 100 mM carbonate buffer and to which a PVS standard was added. For comparison, Figure 10(A) plots the titration endpoint volume as a function of PVS concentration for a standard prepared in 100 mM carbonate buffer alone. For both datasets, similar slopes (1.04 and 1.09 mL / (μg / mL)) were obtained in the linear regression between titration endpoint volume and PVS concentration, and the linear coefficient of determination (R) was calculated. 2 The ratio was 1.00. Importantly, the magnitude of the slope of the recovery rates for both the MES hydrate sample and the MES sodium salt sample was within the range of typical experimental error for this method (i.e., there was no clear difference in the analytical reaction between the two sample types).

[0040] Each cited patent or other publication is expressly incorporated herein by reference, in whole or in part, as will be apparent to those skilled in the art from the context, and such incorporation effectively explains and discloses methodologies described in such publications, which may be used, for example, in connection with the information disclosed herein.

Claims

1. A titration method for detecting polyanionic enzyme inhibitors in a fluid, (a) bringing a fluid into contact with a known amount of polycationic compound; (b) Contacting the material of (a) with an indicator compound, wherein the indicator compound, when it forms a complex with a polycationic compound, exhibits altered properties in its free form compared to that form, and when no complex is formed, a sufficient amount of the indicator compound is added to detect the free form of the indicator compound; (c) Repeat (a); (d) The free form of the indicator compound is detected at the titration point, thereby detecting the polyanionic enzyme inhibitor. Methods that include...

2. The method according to claim 1, wherein the fluid includes or consists of a buffer solution.

3. The method according to claim 2, wherein a plurality of samples of the buffer are prepared, each buffer sample having a buffer compound of a different concentration, thereby generating a dilution series of the buffer.

4. The method according to any one of claims 1 to 3, wherein the detection limit for polyvinyl sulfonate (PVS) is parts per 1.5 million of the buffer solution, parts per 0.25 million of the buffer solution, or 0.16 μg / mL of the buffer solution.

5. The method according to any one of claims 1 to 4, wherein the endpoint of the titration is the point where the absorbance of the sample is midway between the absorbance of the initial sample and the absorbance of the steady state, or the maximum value of the first derivative of the absorbance curve of the sample.

6. The method according to any one of claims 1 to 5, wherein the free indicator compound is detected electrochemically or spectroscopically.

7. The method according to claim 6, wherein the detection by spectroscopic method includes colorimetric detection, photometric detection, fluorescence detection, Raman, or FTIR spectroscopy.

8. The method according to any one of claims 1 to 7, wherein the polyanionic enzyme inhibitor is polyvinyl sulfonate (PVS) or a derivative thereof.

9. The method according to claim 8, wherein the polyanionic enzyme inhibitor is polyvinyl sulfonate (PVS).

10. The method according to any one of claims 1 to 9, wherein the polycationic compound is a pH-independent polycationic compound or a pH-dependent polycationic compound.

11. The method according to claim 10, wherein the pH-independent polycationic compound is a quaternary ammonium polymer.

12. The method according to claim 10, wherein the pH-dependent polycationic compound is a polyamine.

13. The method according to claim 11, wherein the quaternary ammonium polymer is hexadimethrin bromide (HDBr), poly(diallyl)dimethylammonium chloride (pDADAMAC), or methyl glycol chitosan.

14. The method according to claim 11, wherein the quaternary ammonium polymer is hexadimethrin bromide (HDBr).

15. The method according to claim 14, wherein a plurality of HDBr aliquots totaling at least 0.1% of the total fluid volume are added to the fluid.

16. The method according to claim 11, wherein the quaternary ammonium polymer is poly(diallyl)dimethylammonium chloride (pDADAMAC).

17. The method according to any one of claims 1 to 16, wherein the indicator compound is a dye.

18. The method according to claim 17, wherein the dye is an azo dye.

19. The method according to claim 18, wherein the azo dye is eriocrom black T (ECBT), eriocrom blue black R (chalcone), or a sulfonazo sodium salt.

20. The method according to claim 19, wherein the azo dye is eriocrom black T (ECBT).

21. The method according to claim 20, wherein 0.8 to 1.7 μg of ECBT is added per 1 mL of the fluid containing a known amount of the polycationic compound.

22. The method according to any one of claims 2 to 21, wherein the buffer solution is a Good buffer solution.

23. The method according to claim 22, wherein the Good buffer solution comprises an ethanesulfonic acid derivative or a propanesulfonic acid derivative.

24. The method according to claim 22, wherein the Good buffer is MES, bis-trismethane, ADA, bis-trispropane, PIPES, ACES, MOPSO, coramine chloride, MOPS, BES, AMPB, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, CHES, CAPSO, AMP, CAPS, or CABS.

25. The method according to any one of claims 1 to 24, further comprising determining the concentration of the polyanionic enzyme inhibitor from the amount of polycationic compound required to titrate the polyanionic enzyme inhibitor.

26. The method according to claim 25, further comprising comparing the results with those obtained using a standard curve for the polyanionic enzyme inhibitor, thereby determining the concentration of the polyanionic enzyme inhibitor in the fluid.

27. The method according to any one of claims 1 to 26, which is automated.

28. An automated titration method for detecting polyanionic enzyme inhibitors in a fluid, (a) Combining a fluid with an indicator compound, wherein the indicator compound, when it forms a complex with a polycationic compound, exhibits altered properties in its free form compared to that form, and, when no complex is formed, a sufficient amount of the indicator compound is added to detect the free form of the indicator compound; (b) Contacting the material of (a) with a known amount of polycationic compound; (c) Using a titration apparatus, measure the absorbance of the fluid containing the indicator compound and the polycationic compound; (d)(b) and (c) are to be repeated automatically. A method for detecting the polyanionic enzyme inhibitor, comprising detecting the free form of the indicator compound.

29. The method according to claim 28, wherein the fluid includes or consists of a buffer solution.

30. The method according to claim 29, wherein the buffer solution is Good buffer solution.

31. The method according to claim 30, wherein the titration apparatus includes a pump, such as a syringe pump or an intelligent dosing drive, which is in fluid communication with the polycationic compound and the fluid.

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